Photocatalytic, Antimicrobial, and Cytotoxic Efficacy of Biogenic Silver Nanoparticles Fabricated by Bacillus amyloliquefaciens

Abstract

The biomass filtrate of the endophytic bacterial strain Bacillus amyloliquefaciens Fa.2 was utilized for the eco-friendly production of silver nanoparticles (Ag-NPs). The yellowish-brown color’s optical properties showed a maximum surface plasmon resonance at 415 nm. The morphological and elemental composition analysis reveals the formation of spherical shapes with sizes of 5–40 nm, and the Ag ion comprises the major component of the produced Ag-NPs. X-ray diffraction confirmed the crystalline structure, whereas dynamic light scattering reveals the high stability of synthesized Ag-NPs with a polydispersity index of 0.413 and a negative zeta potential value. The photocatalytic experiment showed the efficacy of Ag-NPs to degrade methylene blue with maximum percentages of 73.9 ± 0.5 and 87.4 ± 0.9% under sunshine and UV irradiation, respectively, compared with 39.8% under dark conditions after 210 min. Additionally, the reusability of Ag-NPs was still more active for the fifth run, with a percentage decrease of 11.6% compared with the first run. Interestingly, the biogenic Ag-NPs showed superior antimicrobial activity against different pathogenic Gram-negative bacteria (MIC = 6.25 µg mL⁻¹), Gram-positive bacteria (MIC = 12.5 µg mL⁻¹), and uni- and multicellular fungi (MIC = 12.5 µg mL⁻¹). Moreover, the biosynthesized Ag-NPs could target cancer cells (Pc3 and Mcf7) at low concentrations compared with normal cell (Vero) lines. The IC₅₀ of normal cells is 383.7 ± 4.1 µg mL⁻¹ compared with IC₅₀ Pc3 (2.5 ± 3.5 µg mL⁻¹) and McF7 (156.1 ± 6.8 µg mL⁻¹). Overall, the bacterially synthesized Ag-NPs showed multifunctional features to be used in environmental catalysis and biomedical applications.

1. Introduction

Nanotechnology is a multidisciplinary field that combines chemistry, bioengineering, physics, and biology to develop new substances called nanoparticles (NPs) with sizes less than 100 nm [1]. Recently, the unique features of newly synthesized substances have benefited many biological and biotechnological applications. These distinctive properties include thermal conductivity, small size, a high surface area-to-volume ratio, high reactivity, optical and magnetic properties, high mechanical strength, surface properties, chemical stability, and biocompatibility [2,3]. Moreover, global industrialization and the emergence of new strains that have resistance properties to most antibiotics are leading to the discovery of new materials for the treatment of these issues [4]. Among the several kinds of NPs used in medicine, environmental waste treatment, agriculture, electronics, cosmetics, disinfection, food preservation, and more are metallic NPs such as Ag, Pd, Fe, Pt, Au, Ti, Se, Cu, Al, Zn, and Ni [5,6]. Due to their extraordinary features and varied uses, silver nanoparticles (Ag-NPs) have gained more attention. They are characterized by their efficacy in inhibiting the microbial growth of different pathogenic strains, including bacteria, viruses, and fungi. Additionally, they show promise in environmental applications for the treatment of different wastes, including dyes. This finding, due to their tiny size and large surface area allows them to come into better contact with microbial cells, disrupt cell membranes, and inhibit processes essential for cell function [7]. Due to their high antimicrobial and anticancer efficacy, Ag-NPs can be used in the healthcare sector by adding them to materials such as textiles, medical apparatus, and wound dressings to prevent the growth and spread of pathogenic microbes [8,9]. Recently, Ag-NPs were recorded as anti-growth agents toward pathogenic microbes such as Bacillus subtilis, B. megaterium, Staphylococcus aureus, E. coli, Vibrio cholera, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella typhimurium, Candida spp., Aspergillus spp., Fusarium spp., and Penicillum spp. [10,11].

Ag-NPs have shown encouraging results in water purification and disinfection. Their potential ability to inhibit the multiplication of bacteria and other waterborne pathogens makes them beneficial for both water treatment and disease control [12]. Furthermore, Ag-NPs can degrade organic dyes present in wastewater because of their catalytic actions [13]. Ag-NPs are capable of degrading dye molecules in textile industry effluents and other such sites where pollution is caused by dye when exposed to direct sunlight or UV light [14]. Finally, Ag-NPs possess promise as nanocatalysts in numerous ecological applications. Their capacity to remove toxic materials holds promise for addressing environmental stability and promoting environmentally beneficial measures [15,16].

Methylene blue (MB) is widely employed as a synthetic dye in various industrial processes. It is poisonous and causes environmental concerns because it is persistent and can potentially negatively impact living organisms [15]. Among the many industries that use MB, the textile, printing, and dyeing industries are among the most popular users of this cationic dye. The disposal of these contaminants into the environment without treatment can cause poisonous effects on plants, aquatic ecosystems, and human and animal health [17]. There is an urgent need to treat these contaminants before discharge. The removal of dyes can be accomplished using a variety of treatment approaches that prioritize efficiency, cost-effectiveness, and environmental sustainability.

Therefore, the main challenge for the researchers is the discovery of new active substances that have multifunctional properties for wide applications [18]. In this investigation, eco-friendly synthesized Ag-NPs were used to treat environmental contaminants such as dyes (environmental applications), and they have biomedical activities, including killing the pathogenic microbes that may also be found in the wastewater and causing serious problems. In addition, they are used to treat cancer cells and reduce the implications of radioactive substances and toxic metals used in the treatment program.

Bacterial endophyte strains are the most promising microbial communities that have the potential to secrete active substances with varied applications [19]. Usually, the active metabolites secreted by endophytic strains exhibit greater activity or superior quality compared with metabolites secreted by the same bacterial strains when grown outside the plant [20]. Moreover, the metabolites produced by bacterial endophytes often resemble those secreted by their host medicinal plants, contributing to the variation in endophytic metabolites [21,22]. Hence, the metabolic diversity of endophytes can be exploited for the metabolic reduction of metal precursors into nanoparticles. This ability is likely attributable to the natural symbiotic relationship between endophytic bacterial strains and medicinal plants, which enhances the biocompatibility and potential therapeutic applications of the synthesized NPs. In the current investigation, Bacillus amyloliquefaciens Fa.2 isolated from healthy leaves of the medicinal plant, Fagonia mollis, was used to produce Ag-NPs with multifunctional activities. The optical properties, morphology, elemental composition, crystallinity, and stability of the formed Ag-NPs were examined. Finally, their wide range of applications including biomedical (antimicrobial and anticancer) and environmental pollutants treatment such as dyes were investigated.

2. Results and Discussion

2.1. Synthesis of Ag-NPs Utilizing Bacterial Metabolites

In this article, the cell-free filtrate (CFF) of the endophytic bacterial strain defined as Bacillus amyloliquefaciens was utilized as an eco-friendly and cost-effective method for investigating their efficacy in the biosynthesis of Ag-NPs. Due to the benefits of endophytic microbes, such as unique metabolite production, endophytic bacterial strains have attracted more interest in incorporation into NP manufacturing [10]. Endophytes, residing within plant tissues, possess the essential power to influence the development and growth of their host plants [23].

The limitations of traditional approaches of chemical and physical techniques, particularly their reliance on harsh chemicals and energy-intensive procedures, highlight the potential of endophytic bacteria for nanoparticle manufacturing becomes clear. These traditional methods can raise environmental and toxicity concerns [24]. An alternative that is both sustainable and kind to the environment is the bacterial production of NPs. Bacteria, being living organisms, have the innate ability to reduce metal ions and release biomolecules that contribute to the production of nanoparticles by acting as both reducing and stabilizing agents. This method of biological manufacturing makes the nanoparticles more biocompatible and reduces their environmental effect [25,26].

Endophytic bacteria are promising candidates for nanoparticle synthesis for several reasons. They are known to produce a wide variety of bioactive chemicals, and their cell-free filtrate is hypothesized to contain unique reducing and stabilizing agents that could facilitate the synthesis of Ag-NPs. Additionally, there is a rising need for sustainable NP production methods, and endophyte-mediated synthesis fits the bill [25]. Therefore, we predict the use of the endophytic bacterial strain Bacillus amyloliquefaciens will offer a confirmed approach for the synthesis of biocompatible Ag-NPs with improved antimicrobial, cytotoxic, and photocatalytic activities.

2.2. Characterization

2.2.1. Optical Analysis

The detection of the optical properties of fabricated Ag-NPs is a critical characterization step. Utilizing techniques such as ultraviolet–visible light spectroscopy (UV-Vis) to examine the absorption spectra, one can ascertain the surface plasmon resonance (SPR) of Ag-NPs. Absorption spectra can be used to understand how light interacts with nanoparticles (NPs), and surface plasmon resonance (SPR) can demonstrate the conduction electrons on the surface of the NPs vibrate collectively. The application of Ag-NPs in a wide variety of fields where their optical properties are significant, such as sensing and catalysis, is made possible by optical analysis, which not only assists us in comprehending the fundamental optical behavior of Ag-NPs but also enables us to utilize them [27]. In the current investigation, the color of the bacterial CFF was shifted to yellowish-brown as an indicator of Ag-NP formation. The absorption of this newly formed color was measured in the 200–800 nm range. As shown, the SPR for bacterially synthesized Ag-NPs was observed at a wavelength of 415 nm (Figure 1), which is characteristic of the absorption light of Ag-NPs. Multiple investigations have found that an SPR peak between 400 and 460 nm signifies the effective synthesis of Ag-NPs [7,28]. The shape of synthesized Ag-NPs can be predicted based on the SPR value. In a recent investigation, spherical shapes were typically associated with an SPR peak specifically located between 410 and 420 nm [29].

2.2.2. Morphological Properties

Detecting the morphological features of NPs is considered the main step for their characterization. Detailed information regarding the size, shape, and distribution of Ag-NPs can be obtained through morphological analysis, commonly carried out using techniques such as transmission electron microscopy (TEM). The potential impact of NPs on various applications is correlated with their physical structure, which was detected by TEM analysis. Here, the Ag-NPs formed using endophytic bacterial strain B. amyloliquefaciens Fa.2 were spherical shapes with sizes of 5–40 nm and an average diameter of 22.2 ± 8.3 nm (Figure 2A,B). Also, TEM analysis reveals that the formed Ag-NPs were well distributed without agglomeration or aggregation. In a similar investigation, spherical Ag-NPs were formed using an endophytic B. cereus strain with a size of 20–40 nm [30]. Also, endophytic bacterial strain B. siamensis C1 has the efficacy to produce Ag-NPs with spherical sizes of 25–50 nm and particle average diameter of 34.0 ± 3.0 [10].

The link between morphological characteristics and biological consequences makes detecting these parameters important. Surface area and reactivity are greatly affected by size, and the exposed facets are determined by form; together, these factors impact interactions with biological entities [31]. This finding agrees with what Kambale et al. found when they produced Ag-NPs with sizes of 115 nm, 47 nm, and 45 nm based on TEM analysis [32]. In their antibacterial assessment against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, the authors found that Ag-NPs with a size of 45 nm exhibited superior antibacterial activity compared with those with sizes of 47 and 115 nm, respectively. The enhanced antibacterial activity was attributable to the smaller size of the Ag-NPs and the varied capping agents used, which depend on the biological entities employed for fabrication. Additionally, spherical and rod-shaped Ag-NPs were synthesized and tested for their antibacterial activity against various Gram-positive and Gram-negative microorganisms [33]. Curiously, when compared with a rod shape, a spherical shape showed more efficacy against harmful bacterial strains.

2.2.3. Elementary Mapping of Bacterial-Based Ag-NPs

In this investigation, energy-dispersive X-ray spectroscopy (EDX) was utilized to detect the elemental composition of bacterially synthesized Ag-NPs. This analysis gives knowledge on elemental mapping, revealing the presence and relative abundance of varied ions, thus aiding in NP purity assessment. These data are vital to determine before incorporating the Ag-NPs in specific applications such as medical sectors and catalysis, where the composition is of the utmost importance. Figure 2C showed that the peaks at bending energies of 2.8, 3.0, and 3.2 KeV were identical for Ag, indicating the successful reduction of Ag⁺ to Ag⁰, as reported previously [10,34]. Similarly, the EDX profile of Ag-NPs formed using the CFF of B. amyloliquefaciens and B. subtilis showed their peaks at bending energies in the 2.8–3.2 KeV [34]. The EDX chart showed that the Ag peak represented by the major content of as-formed Ag-NPs with weight and atomic percentages of 62.7% and 41.6%, respectively (Figure 2C). Interestingly, the CFF of the endophytic strain B. amyloliquefaciens can reduce all metal precursors (AgNO₃) and produce Ag-NPs, as seen by the absence of the N peak. The scattering of capping agents that cover the produced Ag-NPs could be why other peaks such as C, O, Cl, and Ca occur [22,35]. These elements were found with varied weights and atomic percentages, as shown in Figure 2C.

2.2.4. Crystallographic Assessment

Understanding the crystal structure of biosynthesized Ag-NPs is crucial for fitting them to various biomedical, technological, and environmental applications. The XRD analysis shows the specific arrangement of atoms within the NPs, indicating their crystalline nature. XRD identifies crystal phases of the synthesized materials, which are influenced by the purity, stability, and uses of NPs. Here, the XRD pattern showed four Bragg’s reflection peaks of (111), (200), (220), and (311) related to 2θ° of 38.2°, 44.2°, 64.5°, and 77.4°, respectively (Figure 3). Based on these diffraction peaks, the synthesized Ag-NPs had a face-centered cubic (FCC) structure and crystalline nature according to the JCPDS-file No-04-0783 standard [36,37]. Similarly, the XRD of bacterially synthesized Ag-NPs showing Bragg’s peaks (111), (200), (220), and (311) at 2θ of 38.3°, 46.4°, 64.6°, and 77.6°, respectively, confirm the crystalline Ag structure [38]. The presence of additional peaks indicates the presence of some impurities that may originate from capping agents and media components. These results were compatible with EDX analysis. Furthermore, the average crystallite size of the formed Ag-NPs can be determined using XRD analysis, enabling the production of the desired NP properties. This investigation used the Debye–Scherrer equation to measure the crystal size based on Bragg’s reflection peak 111 [39]. The size of the crystallite of Ag-NPs obtained in the current investigation was 27 nm. The average crystallite sizes of Ag-NPs formed using the fungal strain Amylomyces rouxii was 27 nm, calculated using the Debye–Scherrer equation based on XRD analysis [40]. In addition, the average crystallite sizes of Ag-NPs formed using the endophytic bacterial strain B. cereus were 21.5 nm, as calculated by the equation of Debye–Scherrer [41].

2.2.5. Ag-NPs Stability Test

A critical aspect of Ag-NP characterization is understanding their stability and surface charge. Dynamic light scattering (DLS) and zeta potential (ζ) are the most useful techniques. This analysis uses scattering light fluctuation using particle Brownian motion to investigate the distribution and hydrodynamic size of colloidal fluid. Based on these obtained data, the stability and aggregation percentages of NPs in the liquid solution were explored [42]. According to Figure 4, the size of bacterially mediated Ag-NPs in the colloidal solution was 97.88 nm in diameter. The obtained Ag-NPs-DLS size was larger than the TEM and XRD sizes. These data were matched with different published investigations about the sizes of Ag-NPs according to TEM, XRD, and DLS analyses. For instance, the size of Ag-NPs formed using B. cereus was 75.16 nm based on DLS analysis, which is bigger than the sizes of TEM (6–51 nm) and sizes of XRD (10–55 nm) [36]. In addition, the sizes of the Ag-NPs produced by two different types of lichens, namely Xanthoria parietina and Flavopunctelia flaventior were 145 nm and 69 nm, according to DLS analysis. In contrast, its sizes ranged from 1–40 nm, as detected by TEM [43]. This discrepancy between the particle sizes seen in DLS and TEM may be attributable to coated agents on the surface of the Ag-NPs. In addition, DLS primarily evaluates NP hydrodynamic radius, while TEM provides images of the individual particles. In addition, DLS analyzes the NPs in a liquid solution and is hence affected by aggregation or accumulation [43,44,45].

The polydispersity index (PDI) of the Ag-NPs in the colloidal fluid was measured using DLS analysis, indicating their size distribution is monodisperse or polydisperse. Following Honary et al., particles were classified as polydisperse or monodisperse, depending on whether the PDI value was above or below 0.7 [46]. Here, the mono-dispersity nature of the bacterially produced Ag-NPs was confirmed by a PDI of 0.413. Similarly, the PDI value of Ag-NPs formed using endophytic actinomycetes Streptomyces antimycoticus strain L-1 was 0.5 [47].

Ag-NP surface charges are calculated via ζ analysis. A measure of the electrostatic repulsion between particles is the ζ, which is the electric potential at the shear plane. Because repulsion keeps particles from becoming too near to one another and prevents agglomeration, a larger ζ value suggests more stability [48]. The following details were provided as standards for categorizing the stability of NPs according to the ζ-analysis: particles that fall within the range of ±0–10 mV are highly unstable, while those falling within the range of ±10–20 mV are generally stable. When comparing the two, ±20–30 mV is deemed moderate, and values above ±30 mV are deemed extremely steady [49]. Figure 3 shows that the produced Ag-NPs are very stable since the ζ-value was −37.5 mV. This result was compatible with those reported that the plant-based Ag-NPs were highly stable due to the value obtained from ζ-analysis being −37.8 mV [50]. In addition, the bacterial Ag-NPs made from the intracellular extract of B. subtilis and B. megaterium were more stable (ζ values of −33.9 and −34.1 mV, respectively) than the ones made from the supernatant of the same strains of bacteria (ζ values of −21.9 and −18.1 mV, respectively), which demonstrated lower stability [11]. A negative charge due to ζ analysis may be attributable to the bioorganic substances in the bacterial extract and serve as a capping agent. Moreover, a high negative ζ value confirms that the particles repel each other and avoid agglomeration with time, ensuring excellent stability.

2.3. Environmental Investigation

Photocatalytic Activity

The potential catalytic and adsorption capabilities of Ag-NPs make them an intriguing candidate for studying the removal of methylene blue (MB) dye. One promising strategy for dealing with the problems caused by dye pollution is to use the catalytic potential of Ag-NPs because of their reactive nature and large surface area. Here, the potential activity of bacterially synthesized Ag-NPs to remove MB dye was investigated under the effect of sunlight, UV irradiation, and dark conditions. According to these data, the amount of catalyst and time incubated directly correlated with the efficiency with which Ag-NPs removed dyes when exposed to sunshine, ultraviolet light, and dark conditions. Mavaei et al. [51] found that the degradation activity of Ag-NPs for three distinct dyes, methylene blue, erythrosine B, and new fuchsine, was time-dependent, and these obtained data are consistent with their findings. An essential characteristic in dye decomposition is the size and surface area of the nanocatalyst; a smaller size means a larger surface area, which in turn means better dye decomposition [52,53]. Within the scope of this experiment, we anticipate a high level of catalytic activity for the removal of dye because of the smaller sizes of the obtained Ag-NPs. Data analysis showed that the dye removal percentages under dark incubation conditions were (2.1 ± 0.5% and 15.4 ± 0.5%) at an Ag-NP concentration of 10 µg and jumping to (10.7 ± 0.4% and 39.8 ± 0.3%) at a concentration of 50 µg after incubation periods of 10 and 210 min, respectively (Figure 5A). Interestingly, the percentage of dye decomposition was 6.03 ± 0.2% after 10 min and climbed to 24.7 ± 0.4% after 210 min when exposed to sunshine and a low quantity of Ag-NPs (10 µg). This is compared with the control group, which had percentages of 2.6 ± 0.1%/10 min and 8.7 ± 0.3%/210 min, respectively (Figure 5B). A dye clearance rate of 18.5 ± 0.6% after 10 min and 73.9 ± 0.5% after 210 min was observed at a high concentration of 50 µg of Ag-NPs catalyst (Figure 5B). Strangely, when compared with sunlight, the presence of UV-irradiation conditions accelerated the degradation of the dye. The data analysis demonstrated that under UV irradiation, MB decomposition reached 25.9% ± 0.1% and 26.6% ± 0.6% after 180 and 210 min of contact time at a concentration of 10 µg, respectively. At 50 µg, the decomposition increased to 78.1% ± 0.8% and 87.4% ± 1.0%, as shown in Figure 5C. Comparing the dye removal under different incubation conditions, it can be concluded that although there is dye removal by adsorption method in the dark incubation condition, it is significantly lower compared with removal in the presence of light irradiation. This means that the photocatalytic process has a significant role in dye degradation. In addition, the dye removal in the presence of UV light was better than that achieved under sunshine conditions.

The high decomposition of MB dye observed with increasing Ag-NP concentration can be attributed to either the presence of high adsorption sites on the surface of the NPs, which increased as the concentration of the NPs grew, or the generation of reactive oxygen species (ROS) that play a crucial part in the breakdown of the dye [54,55]. The strong electrostatic attraction between the positive charges of the dyes and the negative charges of the NPs may explain why high concentrations of NPs result in such efficient dye removal [56].

Based on the result analysis, MB decolorization was more effective when exposed to UV rather than other incubation conditions. The high release of electron-hole pairs (e⁻ and h⁺) following exposure to UV radiation, as compared with sunshine, may explain this discovery. These electron-hole pairs are formed due to exposure of NPs to light, which excites the electron found on the NPs’ surface and transmits it from the valence band (VB) to the conduction band (CB). Upon forming hole pairs, hydrogen peroxide radicals (^•OOH) and ^•O²⁻ occur when O₂ reacts with e⁻, while H₂O reacts with h⁺ to produce ^•OH (Figure 5D). When exposed to MB dye, these ROS radicals accelerate the dye’s breakdown [51,57]. The complete breakdown of dyes to CO₂, H₂O, and small ions is facilitated by the photocatalytic activity of hole pairs formed by exposure of nanomaterials to light [58,59].

The trapping assay method was used to explore the role of active species, including ^•OH, ^•O²⁻, and h⁺, in the breakdown of MB. In this method, MB breakdown was reduced by scavenging the ^•OH, ^•O²⁻, and h⁺ ions with isopropyl alcohol (IPA), benzoquinone (QB), and ethylenediaminetetraacetate (EDTA), respectively. Figure 6A shows that the trial in which no scavenger was used (control) resulted in dye degradation of 87.6 ± 1.5%. In contrast, adding BQ resulted in the greatest reduction to 21.01 ± 0.9%, followed by adding IPA and EDTA with percentages of 51.1 ± 1.8% and 75.1 ± 1.1%, respectively. This phenomenon proved that the photodecomposition of pollutants is primarily facilitated by the ^•O²⁻ followed by ^•OH and h⁺ reactive species. An analogous investigation examined the function of several active species in the photodegradation of 4-chlorophenol pollutants using the trapping assay method, which involves quenching ^•OH with methanol, scavenging h⁺ with ammonium oxalate, and overcoming ^•O²⁻ with oxygen reduction (deoxygenated solution) [51]. The authors reported that ^•O²⁻ plays an important role in the photodegradation of carcinogenic 4-chlorophenol pollutants, followed by ^•OH. A study conducted by Jose et al. examined the photodegradation of MB and indigo carmine, two organic dyes, using a graphene oxide sheet loaded with Ag-NPs [58]. They used EDTA as a h⁺ scavenger and IPA as a ^•OH scavenger. In contrast to the small reduction observed after adding IPA, the authors found that EDTA significantly reduced the photodegradation of organic colors. Hence, the author concluded that h⁺ is the primary active species in degrading MB and indigo carmine chemical dyes.

We conducted five cycles to examine the recyclability of produced Ag-NPs in degrading MB dye under ideal conditions (50 µg/50 mL of catalyst after 210 min of contact time with UV irradiation). Data showed that the degradation percentages of Ag-NPs after the first cycle was 86.9 ± 1.2% and decreased to percentages of 75.7 ± 1.1% with reduced percentages of 11.2% at the end of the fifth cycle (Figure 6B). Similarly, the photocatalytic efficacy of Ag-NPs for a breakdown of new fuchsine dye was reduced with percentages of 8% after four runs [51]. The comparison study showed the efficacy of green synthesized Ag-NPs in the degradation of MB compared with the current study, as illustrated in

Table 1. Comparison study of green synthesized Ag-NPs for degrading the environmental pollutant dyes with significant refers to recyclability test.

Synthesized by…	Shape and Size	MB Removal %			Recyclability with Decreasing Percentages	Ref.
		UV-Light	Sunlight	Dark
Pongamia pinnata	Spherical, 30–40 nm	-	64.4% after 120 min	-	-	[61]
Green tea	Spherical, 9–12 nm	-	73.9% after 120 min	-	-
Spirulina platensis	agglomerated	-	75% after 120 min	-	-
Bacillus subtilis SJ 15	Spherical, 10–36 nm	-	76.5% after 900 min	-	-	[62]
Cyanobacteria sp.	Spherical, 5–10 nm	18% after 240 min	-	-	-	[63]
Prangos ferulacea	Spherical, 79–200 nm	96.5% after 60 min	-	-	Fourth cycle with a decrease of 8%	[51]
Streptomyces tuirus	Spherical, 64 nm	11.3% after 360 min	71.3% after 360 min	-	-
Bacillus amyloliquefaciens	Spherical, 5–40 nm	87.4% after 210 min	73.9% after 210 min	39.8% after 210 min	Fifth cycle with a decrease % of 11.2%	Current study

- meaning not tested.

. There are two possible explanations for the observed decrease in efficiency during repeated cycles. First, the centrifugation process used for catalyst recovery might lead to the gradual loss of small catalyst particles. Second, the catalyst may have limited solubility in aqueous solutions, which could also contribute to the decline in efficiency [60].

2.4. Biomedical Investigations

2.4.1. Antimicrobial Activity

With the rise of antibiotic-resistant microbes, it is urgent to discover new active compounds to fight harmful microorganisms continuously. Nanotechnology offers this advantage by producing new active compounds and nanoparticles with unique characters and a wide range of potential uses. Ag-NPs exhibit significant potential in combating the emergence of antibiotic-resistant strains, paving the way for the development of new treatment protocols that rely on safe and biocompatible materials [1,64]. In this investigation, the agar well diffusion method was employed to investigate the antimicrobial potential of bacterially formed Ag-NPs against different pathogenic microbes. The results demonstrated that the antimicrobial activity peaked at high concentrations and declined with decreasing concentrations of Ag-NPs. This finding was consistent with previous investigations showing that the antimicrobial action of Ag-NPs was concentration-dependent [29,30,36].

Data analysis showing that the maximum clear zones due to Ag-NPs treatment was achieved at a concentration of 100 µg mL⁻¹ with zone of inhibitions (ZOIs) of 17 ± 0.9 for Bacillus subtilis, 17.3 ± 0.5 mm for Staphylococcus aureus, 21.4 ± 0.6 mm for Pseudomonas aeruginosa, 20.7 ± 0.5 mm for Escherichia coli, 17.7 ± 1.1 mm for Candida albicans, and 18.0 ± 0.9 mm for Aspergillus brasiliensis (Figure 7, and Figure S1 see supplementary data). At low concentrations, these ZOIs were reduced. For instance, the ZOIs for microbiological strains of B. subtilis, S. aureus, P. aeruginosa, E. coli, C. albicans, and A. brasiliensis were noted to be 11.7 ± 0.5 mm, 12.3 ± 0.5 mm, 16 ± 0.9 mm, 14.7 ± 1.03 mm, 12.8 ± 0.7 mm, and 12.6 ± 0.5 mm, respectively, at a concentration of 25 µg mL⁻¹. In a similar study, at a concentration of 200 µg mL⁻¹, the Ag-NPs produced by the callus water extract of Solanum incanum demonstrated their highest antimicrobial activity. These Ag-NPs inhibited the growth of S. aureus, B. subtilis, Klebsiella pneumoniae, E. coli, P. aeruginosa, and C. albicans, with ZOI values ranging from 19.8 to 24.2 mm. A concentration of 12.5 µg mL⁻¹ was found to be the minimum activity level obtained [65]. Furthermore, the greatest clear zones resulting from the treatment of S. aureus (MRSA) (30.1 ± 0.5 mm), Klebsiella pneumoniae (39.0 ± 0.1 mm), E. coli (31.04 ± 1.5 mm), and C. albicans (35.9 ± 0.3 mm) with Ag-NPs formed using B. subtilis were achieved at a concentration of 200 µg mL⁻¹ [66].

Determining the MIC value of Ag-NPs is of the utmost significance before their possible inclusion into biomedical applications. The MIC value is a measure of the minimum concentration of Ag-NPs that is necessary to inhibit the growth of bacteria. This value serves as an indicator of the effectiveness of these agents as antimicrobial agents. Bacterially synthesized Ag-NPs had MIC values of 12.5 µg mL⁻¹ for Gram-positive bacteria and eukaryotic organisms with ZOIs of 9–11 mm. In contrast, Gram-negative bacteria had MIC values of 6.25 µg mL⁻¹ with ZOIs of 12 ± 0.9 and 10.3 ± 0.5 mm for P. aeruginosa and E. coli (Figure 7). Similarly, the MIC value of Ag-NPs synthesized by supernatant or intracellular extract of bacterial strains of B. megaterium and B. subtilis was (2.8 ± 0.5 and 1.9 ± 0.3 µg mL⁻¹) and (5.1 ± 0.6 and 5.5 ± 0.001 µg mL⁻¹), respectively, against E. coli [11].

The bactericidal action of biosynthesized Ag-NPs could be attributable to different mechanisms, including the secretion of toxic ions (Ag⁺), production of reactive oxygen species (ROS), dysfunction of permeability function due to interaction with the cell membrane, and interaction with cellular macromolecules leading to inhibit their function. Upon entrance of Ag-NPs to microbial cells, toxic silver (Ag⁺) ions will be liberated. These toxic ions have the potential to attack the main microbial components such as proteins, DNA, ribosomes, mitochondria, cell membranes, and cell walls. Moreover, the accumulation of these ions enhances the production of reactive oxygen species (ROS), which increases oxidative stress and cellular components, such as lipids and proteins, damage [25]. In addition, enzymatic activity and various metabolic functions can be inactivated or impaired by oxidative stress. On the other hand, the interactions of Ag-NPs with cell walls and cytoplasmic membranes destroy their integrity and impair their selective permeability function [67]. The results showed that Ag-NPs targeted Gram-negative bacteria more effectively than Gram-positive ones. This activity could be linked to the structural difference in bacterial cell walls. Gram-positive bacteria, with their thicker peptidoglycan cell walls, may exhibit delayed uptake of Ag-NPs. In contrast, Ag-NPs can readily penetrate the thin peptidoglycan layers of Gram-negative bacteria, leading to the dysfunction of various intracellular components such as proteins, enzymes, nucleic acids, and amino acids [68]. Inside cells, Ag-NPs can bind with the -SH group of proteins, causing denaturation and loss of function. In addition, they bind with DNA to prevent its replication. The efflux pump mechanism that is used to remove the toxic substances is inhibited by interaction with Ag-NPs, leading to an increase in the susceptibility of cells to toxic ions. The synthesis of the ergosterol pathway can be blocked by Ag-NPs, which can rupture the sterol present in the Candida cell wall and cause cell death [69,70]. There are several possible explanations for why Ag-NPs are effective against multicellular fungi. One is the separation of toxic ions (Ag⁺) upon entrance, which harms macromolecules and cytoplasmic membrane function. Another is that the interaction of conidia with Ag-NPs inhibits their germination. Last, the breakdown of the selective permeability function disrupts the electron transport chain, destroying the mitochondria caused by high ROS production, also known as oxidative stress [71].

2.4.2. Anticancer Activity

In this investigation, the colorimetric MTT method was utilized to investigate the cytotoxic potential of bio-formed Ag-NPs toward kidney normal cell lines collected from African green monkey (Vero cells), human prostate cell cancer (Pc3), and human breast cell cancer (Mcf7). The analysis was achieved at concentrations of 1000 to 31.25 µg mL⁻¹ of Ag-NPs. Recently, the main challenge to overcome the drawbacks, including poor selectivity, unfavorable side effects, and drug resistance, of traditional cancer therapies has been discovering new substances that have cost-effectiveness and efficiency as well as being cell-targeted [72]. Nanoparticles formed using green approaches provide these advantages because of the unique characteristics that appear on the substances at the nanoscale. Interestingly, Ag-NPs have fascinating physical, chemical, and antiproliferative properties, enabling them to be successfully integrated into cancer therapy [73,74].

Analysis of variance showed a concentration-dependent effect of Ag-NP on cell viability. Interestingly, both normal and cancer cells exhibited increased viability at lower Ag-NP concentrations. This phenomenon aligns with various published investigations on the in vitro cytotoxic potential of green synthesized Ag-NPs [68,75]. As shown, the viability percentages of Vero, Pc3, and Mcf7 after treatment with 1000 µg mL⁻¹ were 7.7 ± 1.1, 3.5 ± 0.4, and 2.9 ± 0.1%, respectively. (Figure 8). These percentages were increased to (94.1 ± 0.7, 11.9 ± 1.5, and 30.7 ± 0.4%) and (99.9 ± 0.3, 85.7 ± 1.9, and 99.6 ± 1.9%) for the same previous sequence of cell lines at concentrations of 250 and 31.25 µg mL⁻¹, respectively. Accordingly, it can be concluded that the green synthesized Ag-NPs were more toxic toward human prostate cancer cell lines, followed by human breast cancer cell lines and normal Vero cell lines. Similarly, the toxicity of Ag-NPs against Mcf7 was higher compared with the toxicity toward normal Vero cells [76]. The author reported that the synthesized Ag-NPs have the potential to inhibit the growth and proliferation of cancer cell lines (Mcf7) with percentages of 40% at a concentration of 100 µg mL⁻¹ compared with zero toxicity against normal cell lines (Vero) at the same concentration. In addition, the plant-synthesized Ag-NPs using an aqueous extract of Allium saralicum have promising anticancer properties against human breast cancer cells (MDA-MB-231, SK-BR-3, Hs-281-T, and AU-565) compared with normal cell lines (HUVEC) with a dose-dependent effect [77]. The authors reported that the cell viability percentages of breast cancer cells were 21.8% for SK-BR-3, 30.2% for MDA-MB-231, 17.6% for AU-565, and 15.8% for Hs-281-T compared with viability percentages of 64.2% for normal cells HUVEC due to treatment with 500 µg mL⁻¹. These percentages were increased to 62.5, 68.9, 61.5, 60.0, and 82.4% for the same cell lines at a concentration of 125 µg mL⁻¹.

The concentration of Ag-NPs that reduce the viability of cell lines with percentages of 50% is designated as IC₅₀. Calculating this value for active compounds is important before incorporating them into medications. Herein, the IC₅₀ value of bacterially synthesized Ag-NPs was 383.7 ± 3.1 µg mL⁻¹ for normal Vero cell lines, and it was 2.5 ± 1.4 µg mL⁻¹ for Pc3 and 156.1 ± 6.9 µg mL⁻¹. Accordingly, it can be concluded that the synthesized Ag-NPs have the selectivity to target the cancer cells at low concentrations compared with normal cells. In addition, the obtained results confirmed the above finding about the formed Ag-NPs being more toxic against Pc3, followed by Mcf7 and Vero cells. Similarly, the IC50 values of formed Ag-NPs against Pc3, MDAMB-231, Mcf7, Cal-33, and HEK-293 were 30.8, 26.8, 9.1, 6.4, and 1.5 µg mL⁻¹, respectively [78]. In addition, the IC₅₀ of green fabricated Ag-NPs toward cancer cells SK-BR-3, AU565, MDA-MB-231, and Hs-28-T was 208, 200, 250, and 188 µg mL⁻¹, respectively [77].

One possible way that Ag-NPs could fight cancer is by triggering oxidative stress. This is because when Ag-NPs enter cells and react with biological macromolecules, they produce reactive oxygen species (ROS). An increase in ROS causes cell death by damaging proteins, nucleic acids, lipids, mitochondria, and other crucial macro- and micromolecules [79]. The production of various ROS such as ^•OH and ^•O²⁻ was confirmed as shown below (3.5. photocatalytic activity). The process of programmed cell death, or apoptosis, is another way in which Ag-NPs fight cancer. This process is critical for eliminating damaged or aberrant cells, such as cancer cells, through turnover. By blocking anti-apoptotic proteins or activating apoptotic pathways, certain nanoparticles, such as Ag, might cause cancer cells to die [80]. In addition, Ag-NPs hold promise for disrupting signaling pathways essential for cancer cell survival and growth. Ag-NPs can achieve this by blocking proteins and enzymes critical for these processes [81]. The immunomodulatory properties of some nanoparticles, such as Ag-NPs, influence the immune response to cancer cells, suppressing or eliminating these aberrant cells [82]. Endocytosis is a cellular uptake mechanism that causes Ag-NP accumulation in many cellular components. Consequently, this interaction disrupts cellular functions and organelles, ultimately causing harm to the cancer cells [83].

3. Materials and Methods

3.1. Bacterial Strain

The Ag-NPs were synthesized using cell-free filtrate of the endophytic bacterial strain Bacillus amyloliquefaciens Fa.2. This strain was isolated from the Fagonia mollis medicinal plant. The healthy plant shoots were obtained from Saint Katherine Protectorate, Wadi al-Zwatin, South Sinai, Egypt. The selected bacterial strain was identified using 16Sr RNA sequence analysis and loaded in Genbank with accession number KY555786 [84].

3.2. Ag-NPs Synthesis

Nutrient broth medium was inoculated with the endophytic bacterium Fa.2 and left to incubate at 35 ± 2 °C for 24 h. Centrifugation at 7000 rpm for 10 min was applied to the inoculated broth media after the incubation period to collect the bacterial cell pellets, which were washed twice using sterilized dH₂O before resuspended the obtained cells (10 g) into 100 mL sterilized dH₂O and incubated for 24 h in dark conditions. After that, centrifugation for the last mixture was achieved for 10 min at 7000 rpm to extract the supernatant (cell-free filtrate, CFF). A solution with a final concentration of 1 mM was prepared by dissolving 16.9 mg of metal precursor (AgNO₃) in 10 mL of dH₂O and then adding it to 90 mL of the collected CFF. The solution was to undergo stirring conditions for one hour at 150 rpm. During this step, the pH of the mixture was adjusted to 8 using NaOH, which was added drop-wise. After completing one hour, the solution was kept at room temperature overnight to confirm the complete reduction of AgNO₃ to produce Ag-NPs, which were detected by shifting color from colorless to yellowish-brown [85]. The new solution was evaporated in an air oven at 70 °C before the residue was collected and subjected to calcination at 200 °C for 2 h.

3.3. Characterization

3.3.1. Optical Properties

The alteration of the CFF to a yellowish-brown color indicates Ag-NP synthesis. The absorbance of the new color was analyzed spectroscopically (UV-Vis, JENWAY 6305) to identify its maximum surface plasmon resonance (SPR). We measured the absorbance of 2 mL of a yellowish-brown solution in a quartz cuvette at 200–800 nm. The CFF before the addition of metal precursor was blank.

3.3.2. Morphological Properties

The characterization of the size and morphology of Ag-NPs was accomplished by transmission electron microscopy (TEM, JEOL 1010, Tokyo, Japan). The NPs suspension was placed on a TEM grid of carbon/copper to achieve full adsorption. Blotting paper was used to remove excess solution from the grid before analysis gently.

3.3.3. Chemical Composition Analysis

To conduct an Energy-Dispersive X-ray (EDX) examination, which was used to map the elemental composition of the Ag-NPs that were generated, the JEOL, JSM6360LA, Tokyo, Japan, was utilized. This analytical technique makes it feasible to visualize and identify elements in the Ag-NPs.

3.3.4. Crystallographic Assessment

An X-ray diffraction pattern (XRD) was created with the assistance of a Philips X’ Pert Pro apparatus to analyze the generated Ag-NP crystalline structure. The X-ray diffraction (XRD) pattern encompassed 2θ values ranging from 5 to 80 degrees within the operational range when coupled with a Cu-Kα radiation source having a wavelength (λ) of 1.54 Å and a voltage of 40 kilovolts and a current of 30 milliamperes. During the XRD study, the Debye–Scherrer equation was applied to compute the average crystallite size of the Ag-NPs. This was performed to ensure that the results were accurate. There is a possibility that the equation could be expressed as follows:

$$D=\frac{0.9 \lambda }{\beta \cos (\theta )}$$

(1)

Here, the symbol D denotes the average size of the crystallites, the symbol 0.9 for Scherrer’s constant, the symbol λ for the wavelength of the radiation, which is equal to 1.54 Å, the symbol β for half of the highest intensity, and the symbol θ for Bragg’s angle.

3.3.5. Stability and Surface Charge

To assess the characteristics of Ag-NPs in the colloidal solution, a mix of dynamic light scattering (DLS) size distribution, stability determination, and surface charge analysis with Zeta potential (ζ) were utilized. The manufactured Ag-NPs were dissolved in extremely pure water to eliminate any potential interference for precise examination. The Malvern Zetasizer, a precise device manufactured by Nano-ZS of Malvern, UK, was used to ascertain the surface charge of the Ag-NPs.

3.4. Photocatalytic Activity

The photocatalytic experiment was conducted in the presence of sunlight and UV-light conditions compared with dye removal in dark conditions. The methylene blue (MB) solution containing various NP concentrations was exposed to natural sunlight for different interval times as mentioned below. For the experiment in the presence of UV light, the MB solution was incubated under a UV mercury lamp with a λmax > 253 nm and an intensity of 25 mW/cm². All experiments were achieved at room temperature.

Multiple concentrations of Ag-NPs (ranging from 10 to 50 µg with intervals of 10 µg) were added to a solution (50 mL) containing MB (10 mg L⁻¹) and left to react for different amounts of time (10, 30, 60, 90, 120, 150, 180, and 210 min). Under lighted circumstances with air bubble aeration, the nanocatalyst and MB were mixed to assess the degradation capacities of varying concentrations of Ag-NPs on MB. A group that did not obtain Ag-NPs in their MB solution served as the control. Every so often, about 2 mL of each treatment was removed and centrifuged at 1000 rpm for ten minutes. After collecting the clear supernatant, the M-ETCAL spectrophotometer (Cangkat Bukit Belah, 11920 Bayan Lepas, Penang, Malaysia) was used to measure the optical density at a wavelength (λmax) of 664 nm. The following equation was used to compute the percentages of color removal [22]:

$$\mathrm{D}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \%=\frac{\mathrm{A}-\mathrm{B}}{\mathrm{A}}\times 100$$

(2)

where A and B are the initial and final absorbances, respectively, at interval times.

In the current investigation, the trapping assay technique was utilized to detect the type of ROS produced during the photocatalysis process under optimum conditions (50 µg/50 mL of catalyst after 210 min of contact time with UV irradiation emission). This method used specific quenchers to neutralize the ROS produced, including hydroxyl radicals (^•OH), superoxide radicals (^•O²⁻), and holes (h⁺) that were utilized to decompose MB. In this method, 1 mM of isopropyl alcohol (IPA), benzoquinone (QB), or ethylenediaminetetraacetate (EDTA) was added to a reaction solution based on the catalytic experiment. These compounds quenched ^•OH, ^•O²⁻, and h⁺ radicals, respectively [86]. As specified, the absorbance of the solution reaction was measured at 664 nm after contact time to calculate degradation percentages. We used this comprehensive method to determine how ^•OH, ^•O²⁻, and h⁺ degraded MB in optimal conditions when exposed to bacterially synthesized Ag-NPs.

Moreover, the potential of Ag-NPs to be reused for several cycles was investigated by the meaning of the reusability experiment. In this test, the Ag-NPs were collected by centrifugation from the end of each cycle, rinsed thrice with dH₂O, and oven-dry at 70 °C before being added to the second cycle. The process was carried out for a total of four cycles.

3.5. Antimicrobial Activity

A variety of pathogenic microorganisms were chosen for this study to test the antimicrobial effectiveness of bacteria-based Ag-NPs. These included Gram-positive bacteria (Staphylococcus aureus ATCC6528 and Bacillus subtilis ATCC6533), Gram-negative bacteria (Pseudomonas aeruginosa ATCC9027 and Escherichia coli ATCC8739), eukaryotic strains of fungi (Candida albicans ATCC10231), and Aspergillus brasiliensis ATCC16404 as a multicellular fungal strain. The evaluation was conducted using the agar well diffusion technique.

In that order, the selected bacterial, yeast, and Aspergillus strains were first cultured on nutritional agar, sabouraud dextrose, and Capek’s Dox agar plates. The period incubation time varied between 24 h at 35 ± 2 °C for bacteria and yeast and 72 h at 28 ± 2 °C for A. brasiliensis. While the Aspergillus strain was cultivated on Capek’s Dox agar medium, the newly cultured bacterial and yeast strains were re-inoculated onto Muller–Hinton agar. 0.6 mm diameter wells were made on each agar plate using a sterile cork borer.

Various quantities of Ag-NPs from a concentration of 100 to 3.13 µg mL⁻¹ were added to the 100 µL wells. After half an hour in the fridge, the plates were incubated at 35 ± 2 °C for 24 h. A solvent system treated a control well (DMSO, >99.7%, Merck, Darmstadt, Germany). Clear zones surrounding each well were surveyed using the millimeter scale as part of the data-gathering process. The well’s distinct zone with the lowest concentration of green-produced Ag-NPs was used to determine the minimum inhibitory concentration (MIC) value. We experimented three times to ensure the results could be trusted.

3.6. Anticancer Activity

The cytotoxic efficacy of bacterially formed Ag-NPs was assessed against normal cell lines (Vero cells) and cancer cells, namely Pc3 (prostate cancer cells) and Mcf7 (breast cancer cells) at a concentration of 1000 to 31.25 µg mL⁻¹. These cells were obtained from the Holding Company for Biological Products and Vaccines (VACSERA) in Cairo, Egypt. The assessment was achieved using the MTT assay method after 24 h of incubation. The collected cells were inoculated in 96-well plate tissue culture (100 µL (1 × 10⁵) per well) followed by incubation in a CO₂ incubator for 24 h at 37 °C. Once the monolayer sheet of the cells was formed, it was washed with washing media and incubated with maintenance media (RPMI with 2% serum) for 48 h and treated with Ag-NP concentrations. The RPMI media without NPs served as a control. After the incubation period, the excess RPMI media was discarded, and 50 µL of MTT solution was added to each well. The solution was mixed well under shaking conditions before being incubated for 4 h to metabolize the MTT. After that, the formazan crystal (formed from the metabolization of MTT solution) was dissolved in 10% DMSO and shaken well for 30 min in dark conditions. The optical density (OD) of the formed color was monitored at 570 nm using an ELIZA plate reader [87]. The viability of the treated cells was calculated using the following equation:

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\%=\frac{\mathrm{A}}{\mathrm{B}}\times 100$$

(3)

3.7. Statistical Data Analysis

Statistical analysis was carried out on these observed data by utilizing SPSS version 18 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was performed to compare samples before Tukey’s multiple comparison test.

4. Conclusions

The endophytic bacterial strain identified as Bacillus amyloliquefaciens was used to form Ag-NPs with encouraging biomedical and biotechnological results. The characterization methods have verified that the successfully produced spherical-shaped, uniformly sized, with an average size of 21.17 nm, crystalline Ag-NPs showed maximum SPR at 415 nm. The formed Ag-NPs exhibit potential antimicrobial and anticancer agents, highlighted by their concentration-dependent manner. In addition, Ag-NPs showed promising catalytic activity by superior breaking down of MB dye under sunshine or UV radiation. The results show these eco-friendly Ag-NPs have promising applications in different fields, such as nanotechnology, medicine, and ecology. When taken as a whole, the study opens the way to eco-friendly production methods and functional characterization of Ag-NPs for a wide range of environmentally friendly uses.