The potato dextrose agar (PDA), yeast extract, peptone, nutrient agar, nutrient broth, Mueller–Hinton agar (MHA), sucrose, glucose, and lactose were purchased from HiMedia (Thane, Maharashtra, India). Chemicals such as ammonium sulfate, magnesium sulfate heptahydrate, dipotassium phosphate, methylene blue, sodium borohydride, sodium hydroxide, hydrochloric acid, copper sulfate, ferric chloride, sulfuric acid, sodium chloride, and barium chloride were purchased from Merck, India. AgNO₃ was purchased from Sigma-Aldrich, Bangalore, India. All the chemicals used in this study are analytical grade.

The fungal culture H. lixii GGRK4 was obtained from the laboratory storage facility. One mycelium disk (8 mm diameter) of the culture was transferred on a PDA medium containing a Petri dish and incubated at 30°C for 3–5 days. The PDA slant was used for sub-culturing and stored at 4°C for further use.

In a 250-mL Erlenmeyer flask, 50 mL of optimized CX broth medium (see Supplementary Table S1 for composition) was inoculated with three mycelium disks (8 mm diameter) and incubated at 150 rpm and 30°C for 3 days. The cell-free culture filtrate (CFS) was separated by filtering fungal biomass using Whatman No. 1 filter paper, which was further clarified by centrifugation at 10,000 rpm and 4°C for 15 min. For the mycosynthesis process, a reaction mixture containing 1.75 mL of cleared CFS and 20 mL of 2 mM AgNO₃ solution was incubated at room temperature under the incidence of light for 12 h. A brown color was recorded in the spectrum at a range of 200–800 nm using a UV-vis spectrophotometer (Spectro-UV8 MRC, United Kingdom).

Various process parameters for the mycosynthesis of AgNPs by H. lixii GGRK4 were optimized using a single-factor screening method. The physicochemical parameters such as AgNO₃ concentration, CFS volume, reaction time, temperature, and pH were screened. Initially, five different media (Supplementary Table S1) were screened for the most suitable fungal culture for the biosynthesis of AgNPs. The effect of temperature (25°C–35°C) on the growth of the fungal culture filtrate supernatant was assessed for AgNP mycosynthesis. The effect of the concentration ranges of AgNO₃ (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mM) was studied for the mycosynthesis of AgNPs. Furthermore, the effect of different volumes of CFS on AgNP production was assessed. The effect of the reaction time on the AgNP mycosynthesis process was studied by synthesizing the reaction mixture at varying incubation time ranges (2, 4, 6, 8, 10, 14, and 20 h). All sets of tests were examined continuously by a control condition containing silver nitrate solution and the cell-free culture filtrate.

Statistical optimization was performed to obtain the optimum parameters for the AgNP mycosynthesis. For this, the optimization model, i.e., the CCD, was obtained using Design-Expert software (DOE ver. 13.0). Three independent input parameters at five levels are given in Supplementary Table S2. To find the most important factors for the highest yield, a total of 20 experimental matrices (Table 1) were obtained. The substantial correlation between all the variables was estimated using ANOVA and 3D surface response. Finally, the final experiment was conducted using the predicted ideal conditions of the model (silver nitrate concentration of 3.411 mM, volume of 1.931 mL of CFS, and a reaction period of 3 h and 21 min) to validate the model.

The antibacterial activity of AgNPs was tested using the agar disk diffusion method against Gram-positive (Staphylococcus aureus MTCC 96 and Bacillus pumilus MTCC 121) and Gram-negative (Escherichia coli MTCC 43 and Pseudomonas aeruginosa MTCC 424) test organisms. A measure of 100 µL of bacterial culture was spread uniformly on MHA media. The sterile disks were immersed in different concentrations (100, 200, 300, 400, and 500 μL/mL) of AgNP solution. Afterward, the immersed disks were placed on Petri plates and incubated for 24 h at 37°C. The zone of inhibition was measured by taking the readings in triplicate.

The dye degradation efficacy of AgNPs against methylene blue (MB) was determined by performing a reaction. First, 20 mL of 5 mg/mL MB was taken in a reaction tube and mixed with 30 µg AgNPs and 200 µL sodium borohydride (NaBH₄). A control sample was also prepared by the addition of 20 mL of 5 mg/mL MB dye and 30 µg NaBH_4. The tubes were then observed for color change at regular intervals of time and the absorbance at 662 nm using a UV–visible spectrophotometer.

The percentage of degradation was calculated using the following formula: Degradation  % = A b s 0 − A b s t A b s 0 × 100 , (4)

where Abs₀ is the initial concentration and Abs_t is the final concentration.

The ability of the biosynthesized AgNPs to undergo biotransformation was evaluated through the transformation of p-nitrophenol into p-aminophenol. In brief, 1 mL of freshly prepared NaBH₄ solution (30 mM) was mixed with 150 μL of p-nitrophenol (2 mM). Finally, 10 μL of biosynthesized nanoparticles were added, and 3 mL of reaction volume was maintained using distilled water. The biotransformation was performed at room temperature and was monitored using a UV-vis spectrophotometer (Spectro-UV8 MRC, United Kingdom). At varied time intervals (0–180 min), the absorption peak at 400 nm decreased, while the peak at 300 nm increased.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used to assess the ability of AgNPs to scavenge free radicals. In brief, 200 µM DPPH solution was prepared in ethanol and used as the substrate. The reaction mixture contained 2.5 mL of AgNPs having different concentrations (50, 100, 150, 250, and 500 μg/mL) and 1 mL of DPPH solution. The reaction sets were gently shaken and incubated under dark conditions for up to 30 min at room temperature, and the absorbance was measured at 518 nm using a UV-vis spectrophotometer (Spectro-UV8 MRC, United Kingdom). The percentage scavenging activity was measured by comparing the absorbance to that of the control (which solely contained DPPH). Ascorbic acid solution was used as the positive control. The test was carried out in triplicate. The percentage of inhibition could be determined by using following formula: %  Scavenging activity = A b s c o n t r o l − A b s s a m p l e A b s c o n t r o l × 100 . (5)

The IC₅₀ values were determined using GraphPad Prism software (Aykul and Martinez-Hackert, 2016). First, we evaluated the % scavenging activity data taken at different concentrations of the biosynthesized AgNPs and the standard (ascorbic acid). To obtain the IC₅₀ values, we fitted the % scavenging activity as a function for fit spline analysis using the non-linear regression algorithm for log (concentration) versus normalized response implemented in GraphPad.

All investigations were performed in triplicate, and data were interpreted as the mean ± SD calculated using GraphPad Prism (ver 8) software. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test, and p < 0.05 was regarded as significant. The XRD analysis was performed using OriginPro 2021 software.

In the present study, biological approaches are preferred for reasons such as their convenient accessibility, inexpensiveness, and ecological properties (Ahmed et al., 2022). The color shift from pale milky to dark brown verified the reduction of silver nitrate into AgNPs in the fungal culture filtrate supernatant. The conversion of Ag⁺ to Ag⁰ is mostly caused by the presence of biomolecules in the culture filtrate supernatant of the fungus, including amino acids, polysaccharides, enzymes/proteins, and vitamins. Mukherjee et al. (2001) demonstrated and reported that the surface of fungi played a significant role in trapping Ag⁺ and successively reducing it into nanoparticles by extracellular enzymes secreted by the fungal system. Several other studies reported that the nitrate reductase activity of the isolates had a potent ability to synthesize NPs, which supports the hypothesis of the enzymatic reduction of AgNO₃ into AgNPs (Saifuddin et al., 2009; Hamedi et al., 2014; Ottoni et al., 2017). The significant participation of these bioactive molecules has been further assessed by FTIR analysis (see Section 3.3.2). The FTIR investigation revealed the connections between silver and biologically active molecules that play an important role in the formation and constancy of NPs as capping agents. The carboxylic acid group obtained from the amino acid of proteins has strong connections among the NPs due to the negative charge (Neethu et al., 2018; Ibrahim et al., 2021). A measure of 2 mM silver nitrate solution was introduced to the fungal culture filtrate of H. lixii GGRK4 at 30°C in the presence of white light under static conditions to synthesize silver nanoparticles. The initial confirmation of the synthesis of AgNPs was the color change of the reaction mixture from colorless to dark brown (Figure 1A). The presence of a band at a surface plasmon resonance (SPR) of 464 nm of AgNPs in the UV–visible spectrum indicated their synthesis (Figure 1B). The SPR concept describes the excitation of electrons in the conductive band surrounding silver particles. The absorption band observed at 464 nm closely matches the value reported by previous studies (Elgorban et al., 2016; Ottoni et al., 2017; Neethu et al., 2018). However, spectroscopic analysis of the silver nitrate solution in the absence of the culture filtrate supernatant revealed two bands at 222 and 240 nm. Additionally, it was shown that no synthesis of fungal biomass occurred, and that the mycosynthesis of AgNPs exclusively occurred extracellularly.

The medium composition has an impact on metabolite production and microbial growth, which are crucial processes in the biological synthesis of AgNPs (Shankar et al., 2020). In the present study, the CX medium containing lactose and peptone maintained healthy fungal growth and encouraged the production of extracellularly synthesized AgNPs (Figure 2A). In a related study, researchers produced AgNPs by the cultivation of F. oxysporum in 10 distinct medium sources (Birla et al., 2013). A study reported that glucose was used as a carbon source for the production of AgNPs using Aspergillus species (Zomorodian et al., 2016). Another study investigated various media, such as Czapek Dox (CZAPEK), Richard medium (RM), glucose yeast extract peptone (GYP), potato dextrose broth (PDB), protease production (PP) media, and Sabouraud’s dextrose (SB), for the enhanced synthesis of AgNPs (Saxena et al., 2016). Various concentrations of AgNO₃ ranging from 0.5 to 5 mM were used to determine to best suitable concentration. It was found that 2 mM of AgNO₃ reported the optimum concentration for the maximum synthesis of AgNPs (Figure 2B). In the best possible reaction circumstances, the amount of culture filtrate has a considerable and important impact on the yield of AgNPs. The results show that as the volume of the culture filtrate supernatant increases, the synthesis of AgNPs increases (Figure 2C). This is because it now requires more protein or an enzyme to convert AgNO₃ into AgNPs. Higher amounts of “reducing agents” (enzymes) in the broth medium result in the greater production of AgNPs (Singh et al., 2013). Several concentrations of AgNO₃ were used for the production of AgNPs using a fungal culture filtrate of Aspergillus oryzae (Phanjom and Ahmed, 2017). A similar type of experiment was conducted by Omran et al. (2018). Similarly, many researchers conducted experiments with different concentrations of dry biomass of culture with optimum concentrations of AgNO₃ and reported that increasing the amount of dry biomass of culture improved the synthesis of AgNPs (Saxena et al., 2016; Omran et al., 2018). pH of the media plays a crucial role in the maintenance and production of microbial metabolites, and in our study, the highest biosynthesis of AgNPs was found at pH 5.02. Omran et al. (2018) reported that the highest amount of AgNPs were synthesized at pH 7.0. It was also observed that a lower synthesis of AgNPs was obtained when increasing pH due to the denaturation of the enzymes, which can lead to a further decrease in Ag⁺ reduction. Additionally, temperature is another crucial factor that affects the proliferation and production of metabolites by fungi. Our study reports that the maximum amount of AgNPs were synthesized at 30°C, followed by 25°C (Figure 2D). Similarly, thermophilic mold Humicola sp. produced AgNPs at 50°C (Syed et al., 2013). Furthermore, similar experiments were performed using Humicola sp. to synthesize gadolinium oxide nanoparticles at 50°C (Khan et al., 2014). The present study demonstrated that the maximum AgNP synthesis was at 14 h of reaction time (Figure 2E). The yield of biosynthesized AgNPs was the maximum under light conditions (Figure 2F). Similar observations were obtained by researchers, and they concluded that the presence of light encouraged Klebsiella pneumoniae to produce more AgNPs (Mokhtari et al., 2009). For the first time, it has been documented that Penicillium polonicum ARA10, an endophytic fungus, produces AgNPs in the presence of light (Neethu et al., 2018). In addition, a similar investigation was performed using laser light during the AgNP synthesis (Zomorodian et al., 2016). In contrast, another study observed the formation of gadolinium oxide nanoparticles in the dark (Khan et al., 2014).

The biosynthesized AgNPs showed significant antibacterial activity against all tested organisms, i.e., Gram-positive (S. aureus MTCC 96 and B. pumilus MTCC 121) and Gram-negative (E. coli MTCC 43 and P. aeruginosa MTCC 424) bacteria. Table 4 shows that the highest antibacterial activity was obtained against S. aureus MTCC 96, followed by E. coli MTCC 43. Among the different AgNP concentrations, 300 μL/mL showed the maximum zone of inhibition (17 mm) against S. aureus MTCC 96 (Figure 7). On the other hand, the highest zone of inhibition with 11.1 mm and 9 mm was observed against P. aeruginosa MTCC 424 and B. pumilus MTCC 121, respectively (Table 4). The possible reason may be that Ag ⁺ ions attract and associate together with the bacterial cell-membrane, resulting in the modifications in the bacterial structure with major cell membrane damage. Moreover, it could be due to the inactivation or loss of bacterial enzymes due to the significant involvement of Ag ⁺ ions with the sulfhydryl group (-SH) of the bacterial enzyme. Hence, a possible mechanism of antibacterial activity is shown in Figure 8. Several recent studies reported and supported the antibacterial activity of synthesized AgNPs (Przemieniecki et al., 2022; Tripathi and Goshisht, 2022; Ivankovic et al., 2023).

The DPPH method was performed for evaluating the free radical scavenging activity of mycosynthesized AgNPs using CFS of H. lixii GGRK4. DPPH is a well-known stable free radical and can receive hydrogen and electrons from the donors, followed by change in color from purple to yellow (Rajkumar and Sundar, 2022). These investigations were performed in triplicate, and the results are reported as the mean value. In the present study, 50–500 μg/mL of AgNPs revealed a scavenging percentage of 32.34–78.30% in response to DPPH. As shown in Figure 9A, the AgNPs showed the highest inhibition efficiency of 78.30%, and ascorbic acid exhibited 69.74% of free radical scavenging activity at 500 μg/mL. The outputs make it evident that the DPPH radical scavenging capability of mycosynthesized AgNPs was concentration-dependent and improved with an increase in AgNP concentration from 50 to 500 μg/mL (Figure 9A). This antioxidant action might be attributed to capping components found in the culture filtrate and on metal surfaces (Ahmed et al., 2023).

The results of the IC₅₀ value of AgNPs and ascorbic acid against DPPH free radicals are shown in Figure 9B. Compared to the standard, AgNPs had the lowest IC₅₀ value (121.74 ± 3.44 μg/mL) with the highest % scavenging activity. The standard has the lowest % scavenging activity with the highest IC₅₀ value of 190.55 ± 6.39 μg/mL. The ANOVA study revealed significant p-values with 0.0001. The lower IC₅₀ value indicates the greater potency for the antioxidant activity of the biosynthesized AgNPs.

The biotransformation activity of AgNPs was significantly observed. The decrease in the intensity at 400 nm and increase in the intensity at 300 nm indicate the bioconversion of p-nitro to p-amino (Figure 9C). Similar findings have been reported while using gold and platinum nanoparticles in combination with NaBH₄ (Wunder et al., 2010; Das et al., 2012). Halligudra et al. (2022) reported that Fe₃O₄–MoS₂ nanoflowers cause a significant reduction in the p-nitrophenol compound (Halligudra et al., 2022). Similarly, Zhang et al. experimented with the cellulose/silver-based composite material, demonstrating efficient degradability potential against p-nitrophenol (Zhang et al., 2023).

Methylene blue was significantly degraded in the presence of AgNPs (serving as a catalyst). First, the absorption spectrum of MB dye was documented at 662 nm. After 20 min of treatment, the absorption almost completely disappeared, signifying the complete degradation of the MB chromophore due to the dissolution of its aromatic ring. In a reaction mixture, 20 mL MB dye (5 mg/L) reacted with 200 µL NaBH₄ (reducing agent), along with 30 µg AgNPs, resulting in 92.75% (residue 7.25%) degradation of the dye, which was extremely high compared to the reaction without the AgNP catalyst, which was 30.6% (residue 69.4%) in 20 min (Figure 9D). The biosynthesized AgNPs exhibited superior catalytic activity to others, showing efficient activity in converting methylene blue at 0.131 min⁻¹ at a dose of 30 μg/mL. Similar results were observed by several researchers (Kumar and Bharadvaja, 2022; Mechouche et al., 2022; Nguyen et al., 2022; Gokul Eswaran et al., 2023; Shaban et al., 2024). The following steps describe the mechanism of the reduction process: (i) adsorption of the dye– BH 4 ¯ complex by AgNPs; (ii) the reaction of Ag with dye molecules; (iii) the transfer of electron (e⁻) from BH 4 ¯ to dye molecules via AgNPs; and (iv) the conversion of colorful dye (–N=N–) into the monochrome amine (–NH₂=H₂N–) components. A similar mechanism of dye degradation was also reported by Wang et al. (2022), Wan et al. (2021), and Cherian et al. (2023). The probable mechanism for MB dye degradation is shown in Figure 10. First, the MB dye elements are adsorbed on the surface of AgNPs. Furthermore, when NaBH₄, which serves as a reducing agent, was introduced into the reaction system, the AgNPs immediately absorbed it and reduced the reductive properties significantly. The electrons (e⁻) generated from nucleophilic NaBH₄ oxidize the dye. As a result, MB dye was easily adsorbed by electrons and generated amine groups by causing a redox reaction (Saha et al., 2017; Wang et al., 2022).