Mechanistic evaluation of antibacterial and photocatalytic properties of biosynthesized silver nanoparticles against pathogenic bacteria and synthetic dyes

Abstract

Hazardous materials and bacteria are known to cause life-threatening issues to people with compromised immune systems. Another global threat of manufacturing industries; synthetic dyes are one of the key factors of water pollution; it is difficult to treat this wastewater in an efficient and economical way. Thus, there is an urgent need for developing new antibacterial and photocatalytic agents to combat harmful microorganisms and biohazardous compounds (dyes). Therefore, in present research work, biosynthesized silver nanoparticles (AgNPs) were evaluated as a novel antibacterial and photocatalytic agent. The cyanobacterium (Anabaena variabilis) cell-free extract was used to synthesis AgNPs by following the optimized conditions, which included a 1:9 cell extract to 1 mM AgNO₂ ratio, pH 7.4, 30 °C, and characterize by UV-visible spectroscopy. Pathogenic bacteria like Gram-positive Staphylococcus aureus, Gram-negative including Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa were used to evaluate the antibacterial properties of AgNPs. The minimum inhibitory concentrations (MICs) were found 25 ± 2.7 µg/mL for S. aureus, 12.5 ± 1.7 µg/mL for K. pneumoniae, 12.5 ± 1.7 µg/mL for E. coli, and 6.25 ± 1.3 µg/mL for P. aeruginosa. Transmission electron microscopic study confirms the physical disruption of AgNPs treated bacteria, while propidium iodide staining based fluorescence microscopy, and flow cytometric study confirm the qualitative and quantitative antibacterial activity of AgNPs. Additionally, biosynthesized AgNPs were evaluated for photocatalytic degradation in methylene blue (MB) and acridine orange (AO). After 120 min of AgNPs treatment, MB degradation efficiency was observed 83.78 ± 1.21 with a degradation rate constant 0.0035 min^− 1, while AO, percentage degradation efficiency was observed 92.24 ± 1.32 with a degradation rate constant 0.0164 min^− 1.

Introduction

Nanoparticles are a unique class of materials that have a wide range of uses not only in the field of health but also in the fields of energy sciences, material sciences, biotechnology, and other industries [1–3]. The synthesis of nanoparticles involves a variety of physical and chemical techniques that call for advanced equipment to maintain high temperatures, vacuums, and chemical additives [4]. The usage of toxic substances which adhere to the synthesized nanoparticles has increased potential biological risks to the environment for the current advancements in chemical methods for nanoparticle synthesis [5]. The Sustainable Development Goals (SDGs) are problems the United Nations has divided into 17 categories to confront planetary threats. Science and technology were extensively employed as instruments to guarantee the execution of these courses. Accordingly, sustainability science searches for methods by which the scientific community could contribute to the resolution of issues about sustainable development [6]. It has been recognized that nanotechnology is an essential instrument for providing creative and effective strategies for accomplishing the SDGs. We developed green approach-based silver nanoparticles in this work, which could help us achieve SDG-3: good health and well-being as a top priority on guaranteeing healthy lifestyles and promoting well-being for individuals of all ages [7]. Green nanotechnology encourages the use of biological raw materials and eco-friendly agents as sustainable reagents for nanomaterial production to address these problems [8]. Biologically synthesized nanoparticles are biocompatible because they use fewer toxic substances and produce fewer hazardous byproducts that harm the environment and living systems [9]. Hence, researchers are mainly focusing on the biosynthesis of AgNPs using plants, microorganisms, and cyanobacteria, due to cost-effectiveness, eco-friendly nature has opened a new era for safe nano-biotechnology [10–13]. Recently, metal nanoparticles have been focused on in medical field as an antimicrobial as well as anticancer agents [14, 15]. It is well known that human tissues can endure trauma, cancer, and other certain microbial diseases during a person’s lifetime; especially bacterial infection, which is one of the most detrimental effects on humans. As a result, finding new ways to treat microbial diseases is critically important. Many studies have been done to improve, primarily biomaterials and scaffolds for microbial treatment [13]. However, there is a clear correlation between the insertion of foreign materials or devices into the human body and a higher risk of microbial infection [16]. For instance, even a relatively small number of microbes like bacteria, found in operating rooms, are lethal [17]. Implant failure and patient suffering may result from these microbial infections, which can develop months or years after surgical intervention [17]. Some microbes become resistant to all their target sites of antibiotics like protein translational, transcriptional machinery, cell wall, and DNA replication [18] Antibacterial materials that are both long-lasting and effective are badly needed, as the impact of antibiotic-resistant microbial species continues to grow, due to this reason in the present research work we selected four microbes including S. aureus (Gram-positive) K. pneumonia, E. coli, P. aeruginosa (Gram-negative). Additionally, hazardous materials are another well-known cause life-threatening problems to people as well as the aquatic system. The aquatic system is important to humans because it provides a major supply of water for agriculture, animal husbandry, and pisciculture. However, the direct release of several pollutants, particularly synthetic dyes (methylene blue and acridine orange) and other organic waste, which are extensively utilized in hospitals, laboratories, food products, textiles, and other industries, is causing significant harm to the aquatic environment [19–21]. The uncontrolled discharge of waste into the environment is concerning because these dyes and their transformation products are mutagenic, carcinogenic, and poisonous. In order to improve aquatic life and attain acceptable drinking water quality, these contaminants must also be treated for ground and water surface contamination [22]. Methylene blue and acridine, these dyes are not biodegradable, due to this reason, in the present research work, these dyes were chosen, additionally, these dyes have drawn all attention because of the combined effect on natural issues and human wellbeing through drinking water. Basically, MB is counter ion containing an organic chloride salt, with 3, 7-bis (dimethyl-amino) phenothiazine-5-ium. One of the most widely used dyes in industries for printing and coloring silk, cotton, and other textile materials, as well as wood and paper, industries [23]. The uncontrolled discharge of this waste into the aquatic system is a remarkable source of aquatic pollution due to the loss of aquatic life. An excessive amount of the MB may cause several diseases like respiratory and digestive system problems, as well as nausea, vomiting, and profuse sweating [24]. So, degradation of waste MB is needed for better aquatic and ecological survival. Additionally, another model dye, Acridine orange (AO) is also uncontrolled release into the aquatic system. AO is a heterocyclic dye that contains nitrogen atoms and is used extensively in the leather and textile industries, including lithography, paper ink, leather, and printing and dyeing [25]. This dye is widely used in biological science, especially in staining and toxicological research, to investigate the suggested amino-acridine mutagenic potential [26]. Uncontrolled discharge of this colored effluent into the environment is a notable cause of eutrophication, water pollution, and aquatic system disruption [27]. The removal or destruction of these pollutant dyes before to their discharge into the environment and wastewater treatment is so important in life. There are several ways to get rid of this waste, including ion exchange [28], UV radiation [29], floccution [30] ozonization [31] adsorption [32], photochemical process of electrochemical destruction [33] etc.; however, because of the excessive use of chemicals, these methods are costly and result in secondary pollutants. Therefore, it has become difficult for environmental biologists to find a quick and easy way to degrade these organic wastes (dyes). To degrade these organic wastes other metal nanoparticles like Gold and palladium have been used to effectively decompose these waste [34, 35]. AgNPs synthesis is significant compared to palladium and gold because of its low cost, strong photostability, abundance, eco-friendliness, and shape and size-based catalytic efficacy [36, 37]. In the present study, several experiments, including the serial dilution method, transmission electron microscopy, fluorescence microscopy, and flow cytometric study, were used to assess the antibacterial activity of biosynthesized AgNPs against pathogenic bacteria. Additionally photocatalytic activity of the biosynthesized AgNPs was assessed in synthetic dyes, including methylene blue, acridine orange, under visible light irradiation, and the percentage of dye degradation and rate constants were reported for these model dyes.

Materials and methods

Chemicals, reagents and microbial cultures

Acridine orange (AO) and methylene blue (MB) were bought from Merck (Dr. Annie Besant Road, Worli, Mumbai, Maharashtra) and Hi-Media (Swastik Disha Business Park, via Vadhani Industrial Estate, L.B.S. Marg, Mumbai, Maharashtra), both in India. Sigma-Aldrich (Spruce Street, St. Louis, MO 63103, USA) provided the silver nitrate (AgNO₂). The Indian Agricultural Research Institute (IARI), located in New Delhi, provided the Anabaena variabilis. We purchased the bacterial strains Pseudomonas aeruginosa (MTCC-2453) and Staphylococcus aureus (MTCC-2409) from the Institute of Microbial Technology’s Microbial Type Culture Collection (MTCC) in Chandigarh, India. Escherichia coli (MCC-2412) was procured from the National Centre for Microbial Resources (NCMR), Pune, India, and Klebsiella pneumoniae (KJ938546) was obtained from Amity University, Noida, India. A single colony was taken out from the primary culture and resuspended in a freshly made nutrient broth to prepare a concentration of 1 × 10⁶ cells/mL. In these experiments, all glass wares were rinsed with sterilized double-distilled water (ddH₂O) before use.

Synthesis and characterization of AgNPs

To synthesize AgNPs, 90 mL of silver nitrate (AgNO₂) solution (1 mM) and 10 mL of Anabaena variable cell extract were combined, and the mixture was incubated at 30 °C for up to 24 h. Following the conclusion of the reaction period, a UV-visible spectrophotometer (Labtronics, USA) verified the synthesis of AgNPs. An AgNO₂ solution was utilized as a control in this experiment, and a spectrum was acquired between 300 and 700 nm.

The size and shape of synthesized AgNPs were examined using transmission electron microscopy (TEM), and their elemental content and purity were ascertained using scanning electron microscopy (SEM) in conjunction with energy-dispersive X-ray spectroscopy (EDX). Utilizing Fourier-transform infrared spectroscopy (FTIR), the functional groups of biomolecules in the cell extract and on the surface of the AgNPs were identified. The Debye-Scherrer equation was also used to do X-ray diffraction (XRD) examination to determine the particle size and crystalline nature of AgNPs. These characterizations have been thoroughly performed and mentioned in our previous publication [38].

Applications of biosynthesized AgNPs

Investigation of antibacterial activity of AgNPs

The antibacterial activity of biosynthesized AgNPs was assessed using the serial dilution technique followed by standard procedure advised by the Clinical and Laboratory Standards Institute (CLSI) [39]. After harvesting the test organisms, including both Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, their turbidity was adjusted using the McFarland standard of 0.5. A 96-well plate was filled with 100 µL of bacterial cultures (about 2.5 × 10³ cells/mL) with various doses of AgNPs in a final volume of 100 µL per well. Experimental plates were incubated at 37 °C for 24 h, after completion of incubation time at 600 nm, the turbidity of microbial growth was measured. The minimum inhibitory concentration (MIC) of AgNPs was considered the lowest concentration at which no visible bacterial growth.

Transmission electron microscopic study of bacterial cells

To evaluate the AgNPs treated morphological alterations of microbial cells, previously published protocol was used [40]. Silver nanoparticles (AgNPs) at their corresponding minimum inhibitory concentrations (MICs) were added to bacterial cell suspensions (1 × 10⁶ cells/mL) and incubated for three hours at 37 °C. Following incubation, samples were centrifuged for five minutes at 3000 rpm after being rinsed three times with new phosphate-buffered saline (PBS), obtained, pellets were fixed at 4 °C using 2.5% glutaraldehyde (v/v). Fixed samples were again rinsed with PBS and then dried with CO₂ at a critical point. A thin layer of gold-palladium (20–30 nm) was applied to the dried samples, and a transmission electron microscope (TEM; ZEISS, Oberkochen, Germany) was used for analysis. Untreated bacterial cells (no AgNPs) were used as controls.

Fluorescence microscopic study of bacterial cells

To evaluate the qualitative impact of AgNPs on bacteria, freshly grown and diluted suspensions (1 × 10⁶ cells/mL) were used. AgNPs were added to the test samples at ½ MIC, MIC, and 2×MIC doses, and they were then continuously shaken at 120 rpm for three hours at 37 °C. After incubation samples were washed with PBS and incubated with 50 µg/mL propidium iodide (PI) solution at 25 °C for 15 min in the dark. Eclipse-80 fluorescent microscope was used with a 530/590 nm excitation/emission filter pair [41].

Flow cytometric study of bacterial cells

To evaluate the quantitative impact of AgNPs on membrane permeability of bacteria, propidium iodide (PI) uptake experiments were performed following a previously published protocol [40]. Bacterial cells were grown, diluted to 1 × 10⁶ cells/mL, and incubated for 3 h at 37 °C with shaking at 120 rpm in the presence of AgNPs at concentrations corresponding to ½ MIC, MIC, and 2×MIC. To get rid of any remaining nanoparticles, the samples were rinsed three times with phosphate-buffered saline (PBS) after incubation. Following staining with a filtered propidium iodide (PI) solution (50 µg/mL), the bacterial suspensions were left to incubate for 15 min at 25 °C in the dark. A fluorescence-activated cell sorter (BD Biosciences, San Jose, CA, USA) was used for flow cytometric analysis to ascertain the proportion of PI-positive cells, which indicate impaired membrane integrity. Bacterial cells treated with PI served as reference control, whereas untreated cells served as the blank control. For each sample, a total of 10,000 events were recorded to ensure statistical reliability.

Investigation of the photocatalytic activity of AgNPs

The photocatalytic activity of biosynthesized AgNPs was assessed in synthetic dyes, such as MB and AO, using by a previously published protocol with partial modification [42]. To perform these experiments, 10 mg of AgNPs were separately mixed into 100 mL of aqueous solution of 10 mg/L of respective dyes. Before light irradiation, the subsequent solution was ultrasonically treated for 10 min, then, magnetic stirrer was used to ensure the mixing for 1 h in the dark. Subsequently, the sample was illuminated under visible light at different time intervals up to 120 min. After light irradiation treatment, 4 mL of the sample was spun at 15,000 rpm for 5 min to separate the photocatalyst particles. The resulting supernatant was then filtered through a 0.22 μm nylon 66 membrane filter. The maximal absorption spectra of the filtrate were subsequently recorded at specific wavelengths by using a UV-visible spectrophotometer (Labtronics, USA). A respective dye solution without AgNPs under visible light illumination was used as a control (blank), while deionised water was used as a reference. The percentage of degradation of dye was calculated by using the following formula.

1

Here, C₀ stands for the sample concentration at adsorption equilibrium, while C indicates the sample’s residual concentration during different illumination times.

Moreover, the reaction kinetics of dyes were articulated by first-order kinetics equation.

2

The degradation kinetics were studied using a pseudo-first-order model, in which t is the irradiation time (minutes), C₀ is the sample’s initial concentration, C is the concentration at a certain time point, and k (min⁻¹) is the degradation rate constant. The concentrations of the samples used in the studies were kept within the optical range where the Lambert-Beer rule applies, which is significant. Best-fit linear plots of ln (C₀/C) vs. t were plotted to ascertain the rate constant.

Statistical analysis

Analysis of variance and one-way ANOVA were used in the statistical analyses, which were performed using GraphPad Prism version 8.01 (GraphPad Software, San Diego, CA, United States). The mean ± standard error was used to compare the group data to the untreated control. A statistically significant p-value was defined as less than 0.05.

Results and discussion

Biosynthesis of AgNPs

The initial synthesis of AgNPs using A. variabilis was observed visually through a color change in the reaction mixture from colorless to reddish-brown. Further the color observation was confirmed by UV-visible spectrophotometry, which revealed a characteristic surface plasmon resonance peak at 464 nm, (Fig. 1). X-ray diffraction (XRD) and transmission electron microscopy (TEM) revealed that the synthesized AgNPs were spherical, uniformly distributed, and crystalline, with a size range of 11–15 nm, these characterization data have been reported in our previous study [38]. In the present study a prokaryotic photosynthetic filamentous cyanobacterium Anabaena variabilis was chosen as the model strain for AgNPs production because of its rapid growth, low nutritional cost, and environmental friendliness. Compared to plant-based synthesis, biosynthesis of AgNPs using A. variabilis extract has several benefits, like this cyanobacterial strain has a relatively short life cycle, is easy to culture under laboratory conditions, and is considered a well-established model organism in microbial research. A key feature of A. variabilis is the presence of phycobilin proteins, which facilitate the reduction of AgNO₂ to AgNPs. The biosynthesis process occurs when the cyanobacteria interact with Ag⁺ ions in the AgNO₂ solution, reducing them to elemental silver (Ag⁰) through the action of various biomolecules produced during cellular metabolism. The cell-free extract of A. variabilis contains a rich collection of bioactive compounds that not only reduce silver ions to their insoluble form but also help in the formation and stabilization of silver nanocrystals [43].

Fig. 1.

UV Visible spectrum of Anabaena variabilis derived AgNPs

Biosynthesized AgNPs induced the antibacterial activity

The antibacterial properties of biosynthesized AgNPs were evaluated against pathogenic bacteria, such as Gram-positive Staphylococcus aureus, and Gram-negative Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentrations (MIC) of AgNPs against these bacteria were found to be 25 ± 2.7 µg/mL for S. aureus, 12.5 ± 1.7 µg/mL for K. pneumoniae, 12.5 ± 1.7 µg/mL for E. coli, and 6.25 ± 1.3 µg/mL for P. aeruginosa respectively.

Biosynthesized AgNPs induced the cell disruption in bacteria

In the bacterial morphological disruptions experiments, pathogenic bacteria including (Gram-positive) Staphylococcus aureus and (Gram negative) Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa were treated with their respective minimum inhibitory concentrations (MICs) of AgNPs. AgNPs-treated bacterial cells and control TEM results were shown in Figs. 2, 4 and 6, and 8. AgNPs treated cells showed significant damage the cell wall and pore in their plasma membrane, while the untreated (control) cell showed that the cell membrane structures are intact.

Fig. 2.

Antibacterial activities of biosynthesized AgNPs (a) Control S. aureus with scale bar 500 nm (b) AgNPs treated S. aureus with scale bar 500 nm. (All TEM images were captured without AgNPs treated (control) and with their respective MIC doses of AgNPs)

Fig. 4.

Antibacterial activities of biosynthesized AgNPs (a) Control K. pneumonia with scale bar 500 nm (b) AgNPs treated K. pneumonia with scale bar 500 nm. (All TEM images were captured without AgNPs treated (control) and with their respective MIC doses of AgNPs)

Fig. 6.

Antibacterial activities of biosynthesized AgNPs a Control E. coli with scale bar 500 nm b AgNPs treated E. coli with scale bar 200 nm. (All TEM images were captured without AgNPs treated (control) and with their respective MIC doses of AgNPs)

Fig. 8.

Antibacterial activities of biosynthesized AgNPs a Control P. aeruginosa with scale bar 500 nm b AgNPs treated P. aeruginosa with scale bar 200 nm. (All TEM images were captured without AgNPs treated (control) and with their respective MIC doses of AgNPs)

Biosynthesized AgNPs induced the PI uptake in bacteria

The effect of AgNPs on the integrity of bacterial cell membranes was evaluated quantitatively and qualitatively with propidium iodide (PI) by using flow cytometry and fluorescence microscopy respectively. Basically, PI is a membrane-impermeable fluorescent dye that binds to nucleic acids and emits a red glow when viewed under a fluorescence microscope, serving as an indicator of membrane damage. PI can enter the to the cells with disrupted membranes, because PI cannot enter the healthy cells with normal membrane. In the present study we found that AgNPs treated cells showed the bacterial membranes disruption in a concentration-dependent manner, although control cells without AgNPs treated nearly did not exhibit the fluorescence (Figs. 3a, 5a and 7a, and 9a).

Fig. 3.

Cell membrane permeability of S. aureus was measured with propidium iodide (PI). The control represents the S. aureus cells without the treatment of AgNPs (a) All the fluorescence microscopic images of S. aureus including control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC were captured with a scale bar of 200 µm. (b) Flow cytometric analysis of S. aureus cells including control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC. (c) Histogram analysis shows the percentage of PI-positive in S. aureus cells. The data are presented as ± SD. (n = 3, * p < 0.05) are considered significant

Fig. 5.

Cell membrane permeability of K. pneumonia was measured with propidium iodide (PI). The control represents the K. pneumonia cells without the treatment of AgNPs (a) All the fluorescence microscopic images of K. pneumonia including control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC were captured with a scale bar of 200 µm. (b) Flow cytometric analysis of K. pneumonia cells including control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC. (c) Histogram analysis shows the percentage of PI-positive in K. pneumonia cells. The data are presented as ± SD. (n = 3, * p < 0.05) are considered significant.

Fig. 7.

Cell membrane permeability of E. coli was measured with propidium iodide (PI). The control represents the E. coli cells without the treatment of AgNPs a All the fluorescence microscopic images of E. coli inluding control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC were captured with a scale bar of 200 μm. b Flow cytometric analysis of E. coli cells inluding control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC. c Histogram analysis shows the percentage of PI-positive in E. coli cells. The data are presented as ± SD. (n = 3, * p < 0.05) are considered significant

Fig. 9.

Cell membrane permeability of P. aeruginosa was measured with propidium iodide (PI). The control represents the P. aeruginosa cells without the treatment of AgNPs a All the fluorescence microscopic images of P. aeruginosa including control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC were captured with a scale bar of 200 μm. . b Flow cytometric analysis of P. aeruginosa cells including control and treated with AgNPs at the concentrations of ½ MIC, 1MIC, and 2MIC. c Histogram analysis shows the percentage of PI-positive in P. aeruginosa cells. The data are presented as ± SD. (n = 3, * p < 0.05) are considered significant

During the quantitative flow cytometric analysis, AgNPs were used at ½ MIC, MIC, and 2×MIC concentrations to assess PI uptake in all bacterial strains. In the Gram-positive bacterium Staphylococcus aureus, PI-positive cells were observed at 38 ± 2.7%, 54 ± 3.3%, and 58 ± 3.9% for ½ MIC, MIC, and 2×MIC treatments, respectively. Among the Gram-negative bacteria, Klebsiella pneumoniae exhibited PI uptake of 16.7 ± 1.3%, 44 ± 2.9%, and 70 ± 4.1%; Escherichia coli showed 9.3 ± 1.2%, 36 ± 2.8%, and 62.9 ± 4.0%; while Pseudomonas aeruginosa demonstrated the highest uptake with 27.5 ± 1.8%, 77.6 ± 4.2%, and 88.4 ± 4.8% at the respective concentrations. These results indicate a dose-dependent increase in membrane permeability across all tested bacterial strains, with Gram-negative species generally showing greater susceptibility at higher AgNPs concentrations (Figs. 3bc, 5bc, 7bc, and 9bc). In the present study, the data illustrating the antibacterial properties of AgNPs against both Gram-positive and Gram-negative bacteria were truly remarkable. In flow cytometry data, a significant percentage of PI-positive cells were found in bacterial cells with damaged membranes. Based on the qualitative and quantitative data generated, we can say AgNPs showed outstanding antibacterial activities against pathogenic bacteria. Antibacterial activity of biosynthesized AgNPs has been studied by many other researchers. A research team synthesized AgNPs using fungus Trichoderma viride extract and reported the MIC 30, and 80 µg/mL against E. coli, S. aureus respectively [44]. Another research team synthesized AgNPs from Streptomyces xinghaiensis and reported the MIC 16, and 64 µg/mL against P. aeruginosa, and E. coli, while 256 µg/mL were observed against both strain K. pneumoniae, and S. aureus [45]. While another group of researchers synthesized AgNPs using aqueous leaf extract of Eucalyptus globulus and reported the MIC 27 and 36 µg/mL against P. aeruginosa and E. coli [46]. Based on the previous data we can say in the present study A. variabilis-derived AgNPs showed the much better MIC values against all experimental bacteria than previous study.

Proposed antibacterial mechanism of AgNPs

In humans, silver ions (Ag⁺) demonstrate minimal or insignificant toxicity, however they are recognized for their toxicity to microorganisms, including bacteria, viruses, fungus, and other species [47]. Because of their beneficial properties, AgNPs are getting more attention in scientific fields for a broad range of applications against microbes. The best things of AgNPs are their size, and shape can be controlled during biosynthesis, and their biochemical properties can be adjusted as per the needs of the applications [48]. Therefore, AgNPs have been extensively studied as a potent nanoweapon for microbial elimination as well as in cancer disease. In addition to their intrinsic antimicrobial as well as anticancer action, AgNPs can also have the properties of ionic silver, which is generated when Ag⁺ spontaneously releases from the nanoparticle surface. AgNPs release Ag⁺ ions when they bind with oxygen as well as H₂O₂ [49, 50]. The precise mechanism of antimicrobial activities remains unprecise despite wide research on Ag⁺ and AgNPs. However, the most probable mechanism of AgNPs is categorized into three parts.

• (i)Direct disruption of cellular activities via interfering with the bacterial cell membrane, resulting in compromised membrane integrity and cellular leakage.
• (ii)Interaction with intracellular biomolecules, such as proteins and DNA, which can impair vital cellular functions and inhibit replication and metabolism.
• (iii)Free radicals and reactive oxygen species (ROS) are produced during metal-induced oxidative stress, which damages lipids, proteins, and nucleic acids and ultimately results in cell death.

• (i)Disruption of the cell membrane.

  The electrostatic attraction between Ag⁺ ions in nanoparticles and the negatively charged microbial surface facilitates adherence to the cell wall and membrane, hence commencing the interaction between silver species and the positively charged microbial cells [51]. The interaction between silver species and microbial cell surfaces can alter the zeta potential, reflecting changes in surface charge. This charge disruption may lead to a reduction in respiratory potential, increased membrane permeability, and membrane depolarization, collectively contributing to cellular dysfunction and eventual cell death [52]. Finally, irretrievable cell damage and ultimately, cell death result from a complete disruption of membrane integrity [52, 53]. AgNPs quickly interact with the bacterial cell wall, resulting in many pits and significant peripheral damage to the bacterial surface [54]. In the present study, AgNPs-treated TEM data confirm the alteration in the morphology of bacterial cells. AgNPs treated bacterial cells showed the notable disruption characteristics and irregular pits in their cell walls, while the control cells displayed normal cell walls with a uniform, regular cytoplasmic cell content. These findings are consistence with earlier research on the impact of AgNPs on bacterial cells [55]. Different entrance mechanisms for AgNPs have been hypothesized based on the creation of bacteria’s cell wall and membrane. Ag⁺ and AgNPs have lower antibacterial activity against Gram-positive bacteria due to their thicker peptidoglycan (PGN) layer [56]. The attachment and capture of silver ions in Gram-negative bacteria are facilitated by the negatively charged lipopolysaccharide (LPS) layer [57]. In the present study, images of S. aureus showed a small amount of AgNPs entered the cells and led to intracellular damages, which might be caused by the bonding of nanoparticles with PGN and LPS layers of the bacterial cell wall. Interestingly, Gram-positive bacteria’s thick PGN layer served as a stronger barrier against Ag⁺ and AgNPs than Gram-negative bacteria’s LPS [15]. AgNPs may bind with proteins that adhere to bacterial membranes and cell walls, forming toxic complexes that alter the bacteria’s physicochemical properties. In bacteria, the respiratory electron transport chain, protein secretion, and lipid biosynthesis can be completely inhibited when silver ions rapidly interact with sulfhydryl groups (R-SH) on the cell membrane. This occurs through the replacement of the terminal hydrogen atom, forming a stable S-Ag bond that disrupts essential cellular functions [58, 59]. In the present research work, Staphylococcus aureus (Gram-positive), Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa (Gram-negative) demonstrated nearly the same result in the AgNPs-treated cells.

• (ii)Disruptions of intracellular proteins and DNA.

  The initial level of Ag+/AgNPs-induced microbial membrane damage is crucial for the subsequent cellular penetration, where the nanoparticles might damage essential cellular functions [60]. The crucial step in the concept of antibacterial action is the release of silver ions (Ag⁺) from the AgNPs; these ions can freely diffuse, bind to biomolecules, and change how proteins and nucleic acids function [61, 62]. Another proposed mechanism reported when AgNPs bind to relaxed bacterial DNA, its condensed form is induced, leading to DNA replication is completely stopped [56]. Furthermore, AgNPs have been shown to cause DNA denaturation and degradation. The role of sulfur in the fundamental metabolic route suggests that silver ions from released AgNPs interact with thiol groups of nucleoid-associated proteins. These findings show that chromatin-regulating proteins have changed, altering DNA replication and mRNA transcription [63]. Yan et al. used a proteomic and bioinformatic method to investigate the effect of silver-regulated and silver-binding proteins on the Gram-negative bacterium P. aeruginosa [63]. Yan et al. found silver-binding proteins such as chaperonin, elongation factor Tu, flagellin, electron-transfer flavoprotein subunit alpha, and an unidentified protein (PA3309). In addition, several silver-regulated proteins-such as, cytochrome c oxidase, ATP synthase subunit C and porin-D were also affected. These proteins are primarily involved in crucial cellular functions, including membrane stability, pore formation, flagellum assembly, and metal ion transport. Notably, several silver-binding proteins were found to interact with both Ag⁺ ions and silver nanoparticles (AgNPs), supporting the hypothesis that AgNPs exert their antimicrobial effect at least in partially through the controlled release of silver ions. Another study has reported that after exposing the AgNPs on E. coli cells various proteome interactions changed [64]. According to a research group, enzymes like tryptophanase and alcohol dehydrogenase are reported for 65% of the AgNPs interacting proteins, while reaming 35% correspond to chaperones, membrane porins, and peptide-binding proteins. After protein, bacterial ribosomal unit is another important target of AgNPs. A group of researchers found that the Ag⁺ of AgNPs has a significant impact on several ribosomal subunit proteins, including the S₂ protein. These findings indicated that reduced S2 protein caused by Ag⁺ impairs ribosome function and, subsequently, affect protein synthesis [65]. This effect of protein synthesis led to inhibit the key cellular enzymes, like fructose-bisphosphate aldolase-essential for glycolysis and gluconeogenesis succinyl-CoA synthetase, a critical enzyme in the citric acid cycle. As a result, the ATP in the cells is gradually depleted, which causes starvation, and, eventually, leads to microbial cell death.

• (iii) Generation of reactive oxide species.

  The generation of reactive oxygen species (ROS) by AgNPs is a significant aspect of metal-induced antibacterial properties [66, 67]. Oxidative stress and the generation of ROS are the most predicted mechanisms for the toxicity of AgNPs [68]. The surface-generated free radicals of AgNPs interact with the bacterial cell membrane, oxidizing unsaturated fatty acids. This oxidative damage degrades membrane integrity and fluidity, affecting cellular function and finally leading to cell death [69]. Long et al. investigated the morphology of E. coli cells by treating with AgNPs both with and without 1mM GSH, and found that bacteria given additional GSH were smooth, undamaged, and closely matched the control samples that were not treated. This observation suggested that oxidative stress were cause of the cell structure collapse in AgNPs-treated cells [70]. The present study findings demonstrated a significant correlation between AgNPs and ROS formation because AgNPs-treated cells showed collapse and damage to the membrane’s integrity. These results support the idea that oxidative stress and reactive oxygen species (ROS) cause several kinds of biological effects, such as membrane rupture and the initiation of cell death pathways [71]. Additionally, ROS generated by Ag⁺ ions can deactivate membrane-bound enzymes, leading to disruptions in the proton motive force and impairments in the electron transport chain [72]. Significantly, damaging the electron transport chain ultimately affects ATP generation, which in turn affects other vital cell functions. Elevated ROS levels have been demonstrated to damage a variety of cellular machinery systems, including DNA, proteins, and other macromolecules, as well as rupture the cell membrane and impede respiratory enzymes [73, 74]. All ROS-related possible antibacterial mechanisms of AgNPs have been shown in Fig. 10.

Fig. 10.

Possible antibacterial mechanism of biosynthesized AgNPs

Biosynthesized AgNPs induced the photocatalytic Activity in synthetic dyes

Photocatalytic activities of the biosynthesized AgNPs were investigated for the degradation of synthetic dyes, including MB and AO. The absorption band intensity gradually decreased, which indicates the degradation of the MB dye with a good catalytic activity of AgNPs. The maximum absorption spectra were obtained at 664 nm, shown in Fig. 11b. The percentage degradation efficiency was observed 83.78 ± 1.21% in AgNPs (Fig. 11b; Table 1). The MB degradation rate constant was also evaluated, found 0.0035 min^− 1 in AgNPs (Fig. 11c; Table 1). Photocatalytic activity of biosynthesized AgNPs in AO observed that absorption band intensity gradually decreased, with increasing a time interval shown in Fig. 12b. The percentage degradation efficiency was observed 92.24 ± 1.32% in AgNPs shown in Fig. 12b; Table 1. The AO degradation rate constant was also evaluated, found 0.0164, in AgNPs (Fig. 12c; Table 1). The degradation efficiency was found better in AO as compare than in MB. This could be due to the external factors that influenced the degradation. The external factors include light sources and the sample of pH. The nearly linear graphs of the samples data confirm that the photodegradation of the test samples using the catalyst follows pseudo-first order kinetics shown in Figs. 11d and 12d. One important thing here the photocatalytic system has long-term stability. In the present work, AgNPs were involved in the degradation of the synthetic dyes up to 120 min before the nanoparticles were removed and regenerated (heated to ambient temperature and time), and the process was repeated up to five cycles. The result found that the AgNPs possessed the remarkable photostability regarding catalytic degradation of these samples with less than 10% decrease from the initial step of the photodegradation process (data not shown). Photocatalytic degradation methods must be eco- friendly, and cost-effective, to degrade the toxic compounds from the aquatic system [75]. The dyes targeted in these studies are recalcitrant organic compounds, resistant to biological degradation; their presence in water poses a serious threat to aquatic life. Therefore, photocatalytic degradation of such dyes is not only a significant environmental challenge but also serves as a useful model for treating effluents and organic pollutants. In the absence of biosynthesized AgNPs, these dyes remain nearly unchanged over time under identical conditions. Visible light is more effective compared to other irradiation techniques for dye degradation, as previously reported by a research group [76]. In photocatalytic degradations, except for light irradiations, the nature of AgNPs also matters; several studies have found that the size, shape, crystallographic nature, and structures of AgNPs have a significant impact on photocatalytic activity [76, 77]. During visible light exposure, photons strike the nanoparticles present in the colloidal mixture, exciting electrons on their surface to higher energy states [78]. The dissolved oxygen molecules in the reaction medium accept the excited electrons from the surface of the nanoparticles and are subsequently converted into reactive oxygen species, such as superoxide anion radicals (O₂⁻·). These highly reactive radicals attack the complex organic dye molecules, breaking them down into simpler compounds [79, 80].

Fig. 11.

a Chemical structure of Methylene blue, b Degradation of methylene blue (MB) dye under visible light irradiation using biosynthesized AgNPs, c Plot of MB photodegradation versus irradiation time, as measured by optical absorption, d Plot of ln (C₀/C) versus irradiation time for degradation kinetics of MB using biosynthesized AgNPs. The linear nature of the plot indicates a pseudo-first-order reaction

Table 1.

Percentage degradation and rate constant of synthetic dyes after biosynthesized AgNPs treatment

Synthetic dyes	Percentage degradation of synthetic dyes after 120 min of AgNPs treatment	Rate Constant (min− 1) AgNPs
Methylene Blue	83.78 ± 1.21	0.0035
Acridine Orange	92.24 ± 1.32	0.0164

Fig. 12.

a Chemical structure of Acridine orange, b Degradation of Acridine orange (AO) dye under visible light irradiation using biosynthesized AgNPs, c Plot of AO photodegradation versus irradiation time, as measured by optical absorption, d Plot of ln (C₀/C) versus irradiation time for degradation kinetics of AO using biosynthesized AgNPs. The linear nature of the plot indicates a pseudo-first-order reaction

Photocatalytic mechanism of AgNPs

A group of researchers proposed a mechanism in which, if the photon energy is equal to or greater than the band gap of AgNPs, the photocatalytic activity initiates when a photoexcited electron migrates from the filled valence band to the conduction band of AgNPs under visible light irradiation [57]. This process generates a hole in the valence band, forming an electron-hole (e⁻/h⁺) pair, where the excited electron acts as a strong reducing agent, while the hole serves as a powerful oxidizing agent [81]. These photocatalytic reactions initiate when exposed to visible light energy equivalent to or greater than the band gap of nanoparticles [82]. The higher band gap of the nonradiative recombination of electron and hole pairs leads to an increase in photocatalytic activity [83]. Water molecules adsorbed on AgNPs surfaces are oxidized by valence band holes, resulting in extremely reactive hydroxyl radicals (·OH). Electrons in the conduction band are captured by dissolved oxygen, resulting in superoxide anion radicals (O₂^−·). These radicals not only participate in oxidation events, but they also help to suppress the electron-hole recombination, ensuring that the AgNPs maintain the neutral charge. The superoxide radicals further react with protons (H⁺) to form intermediate species, which eventually generate hydrogen peroxide (H₂O₂). This hydrogen peroxide subsequently decomposes into additional hydroxyl radicals. These ·OH radicals attack dye molecules by breaking aromatic rings, cleaving azo bonds, and hydroxylating aromatic structures, ultimately leading to the formation of CO₂, H₂O, SO₄²⁻, NO₂⁻, and NH₄⁺ ions [21, 84]. The comparatively high band gap of the nanoparticles reduces nonradiative recombination of electron-hole pairs, which improves photocatalytic activity toward dye degradation. Hydroxyl radicals (·OH) are produced by the oxidation of water molecules adsorbed on AgNPs surfaces. Molecular oxygen captures electrons from the conduction band, converting them into superoxide anion radicals (O₂⁻·). These superoxide radicals not only participate in future oxidation events, but they also prevent electron-hole recombination, ensuring charge neutrality within the nanoparticles [85]. The suggested mechanism underlying the photocatalytic degradation mediated by AgNPs is illustrated in Fig. 13. The novelty of the present study highlights the role of biomolecules involved in the synthesis of green nanoparticles, enabling eco-friendly nanotechnology suitable for large-scale and sustainable applications, and making nanoparticle synthesis accessible in low-resource laboratories and industries. Intrinsic stabilization through biological capping agents, biological systems offer natural shape-directing agents, based on this evidence we can say green nanoparticles considered as next-generation nanomaterials. Significance of the present study addresses antibacterial activity by offering proposed mechanistic insight into non-antibiotic antibacterial strategies less prone to resistance development. Additionally, this study advances the field by shifting biosynthesized AgNPs from phenomenological observations to mechanistic understanding, thereby enabling predictive, mechanism-guided development of next-generation antibacterial and photocatalytic nanomaterials with high efficacy and environmental compatibility. This study bridges biomedical and environmental remediation research, positioning biosynthesized AgNPs as versatile materials for hospital infection control and wastewater treatment.

Fig. 13.

Possible photocatalytic mechanism of biosynthesized AgNPs for the degradation of synthetic dyes

Conclusion

Conclusive remarks: In the present research work, biosynthesized AgNPs were utilized to evaluate the antibacterial and photocatalytic activities against Gram-positive as well as Gram-negative bacteria and synthetic dyes. TEM results confirm the physical damage, while fluorescence microscopy and flow cytometry data showed the qualitative and quantitative confirmation of antibacterial properties of AgNPs. Based on the antibacterial data, biosynthesized AgNPs may be used in therapeutic nanomedicine formulations against bacterial disease in the future. However, before using AgNPs in biological applications, preclinical in vivo research is required. Future research will focus on clarifying the precise mechanisms underlying the specific pathways that underpin to improvement of the antibacterial properties of AgNPs. Additionally photocatalytic activities of the biosynthesized AgNPs showed the remarkable degradation of MB and AO dyes. Based on the photocatalytic data, we can say this green synthesis approaches offer several valuable advantages and offers an effective, eco-friendly, and cost-efficient approach for bioremediation in the future. Overall, we can say this green nanotechnology is not only used in nano-medicine formulations but also has important significance in fields of photocatalysis such as the textile sector and wastewater treatment plants.

Author contributions

Conceptualization, TF methodology, IA formal analysis, IA, TF investigation IA, TF, resources, IA data curation, IA, TF; writing-original draft preparation, IA; writing-review and editing, IA, TF; visualization, IA, TF; supervision, TF; project administration, TF; funding acquisition, TF. All authors have read and agreed to the published version of the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors.

Data availability

The data supporting this article is shown in Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. The datasets analyzed in the present study are available from the corresponding author upon reasonable request.

Declarations

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Humans and animals rights

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Informed consent

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Competing interests

The authors declare no competing interests.