Extracellular Mycogenic Synthesis of Silver Nanoparticles Using Indigenous White-Rot Fungi Ganoderma lucidum and Pleurotus ostreatus Characterization and Antimicrobial Potential

Nanotechnology enables the design and investigation of materials whose physicochemical behaviour differs from that of their bulk counterparts. Among metallic nanomaterials, silver nanoparticles (AgNPs) have attracted sustained attention because of their optical properties, high surface-area-to-volume ratio and broad antimicrobial activity. These properties have encouraged investigation of AgNPs in antimicrobial surfaces, coatings, packaging, environmental technologies, biosensing and biomedical applications [1,2].

Conventional physical and chemical routes can provide control over nanoparticle formation, but may involve substantial energy requirements, elevated temperatures or pressures, and synthetic reducing or stabilizing agents. Biological synthesis has consequently been investigated as an alternative in which microorganisms, plants or biological extracts provide reducing and capping components under comparatively mild aqueous conditions [1,3]. Extracellular synthesis is especially attractive because nanoparticles formed in the reaction medium are potentially easier to recover than particles accumulated intracellularly [4–6].

Fungi are promising biological nanofactories because they can generate substantial biomass and secrete diverse extracellular biomolecules. Proteins, enzymes, peptides, polysaccharides, amino acids and secondary metabolites may contribute to reduction of Ag? to Ag? and subsequently remain associated with the nanoparticle surface as a biological capping layer. Recent reviews emphasize that variability in fungal secretomes is an important determinant of synthesis kinetics, surface chemistry, stability and reproducibility [2,3].

White-rot fungi are particularly interesting because their ecological and physiological adaptation to lignocellulosic substrates involves extensive extracellular oxidative enzyme systems. Laccases, peroxidases, reductases and other secreted metabolites may provide chemically active environments capable of interacting with metal ions. Earlier work has demonstrated extracellular AgNP synthesis using several fungi, including Fusarium oxysporum, Aspergillus fumigatus and white-rot fungal culture filtrates [4–7]. Gudikandula et al. reported extracellular AgNP synthesis using white-rot fungi, with plasmon peaks around 419–421 nm and TEM-confirmed particles in the 15–25 nm range, illustrating the relevance of white-rot fungi to mycogenic nanomaterial production [4].

The antimicrobial behaviour of AgNPs is generally attributed to several interacting processes rather than to a single mechanism. These include adsorption to bacterial cell envelopes, disruption of membrane integrity, release of Ag? ions, oxidative stress and reactive oxygen species generation, inhibition of respiratory enzymes and interaction with proteins and nucleic acids [8–10]. The magnitude of activity can vary with particle size, morphology, surface chemistry, aggregation state, Ag? release and the test organism [1,8,9].

The present work examines two indigenous white-rot fungal isolates, Ganoderma lucidum and Pleurotus ostreatus, collected in Warangal, Telangana, as extracellular platforms for AgNP synthesis. The experimental workflow integrates fungal isolation and cultivation, preparation of cell-free mycelial extract, reaction with AgNO?, visual monitoring, UV–Visible spectroscopy, FTIR analysis and preliminary antibacterial evaluation.

1.1 Objectives

• To isolate and establish indigenous white-rot fungal cultures provisionally identified as Ganoderma lucidum and Pleurotus ostreatus.

• To prepare cell-free mycelial extracts and assess their ability to reduce Ag? ions extracellularly.

• To monitor AgNP formation by visual colour change and UV–Visible spectroscopy.

• To examine the functional groups associated with the synthesized nanoparticle preparations using FTIR spectroscopy.

• To evaluate preliminary antibacterial activity against selected Gram-negative and Gram-positive bacteria using agar well diffusion.

2. MATERIALS AND METHODS

2.1 Collection and preliminary identification of fungi

Indigenous white-rot fungal fruiting bodies were collected from hardwood-associated material in Warangal, Telangana, India. Two isolates were designated Ganoderma sp. and Pleurotus sp. and are referred to in this manuscript as Ganoderma lucidum and Pleurotus ostreatus based on preliminary macromorphological identification. Characters considered included growth habit, attachment, pileus morphology, colour, lamellae or pore-bearing surface, stipe characteristics and spore-associated features.

Figure 1. Representative fruiting-body material used for fungal isolation and culture establishment: (A) Ganoderma lucidum fruiting body; (B) Pleurotus ostreatus fruiting body.

2.2 Establishment and maintenance of pure cultures

Approximately 10 mg of internal fungal tissue was aseptically excised from fruiting bodies and transferred to malt extract agar (MEA) containing malt extract (30.0 g L?¹) and mycological peptone (5.0 g L?¹), with the pH adjusted to approximately 5.5. Plates were sealed with Parafilm and incubated at room temperature. Emerging colonies were subcultured to obtain pure cultures and maintained on MEA slants at 4 °C.

Figure 2. Representative Ganoderma lucidum growth during culture establishment

Figure 3. Representative culture plates showing development of fungal mycelia used for subsequent biomass production:  Pleurotus ostreatus culture.

2.3 Biomass production and preparation of cell-free mycelial extract

Established fungal cultures were propagated in malt extract broth and incubated on an orbital shaker at 180 rpm for three days. Biomass was recovered by filtration and washed repeatedly with deionized water to reduce residual medium components. The washed biomass was suspended in 100 mL deionized water and maintained under shaking conditions for 72 h to facilitate release of extracellular biomolecules. The suspension was then filtered through Whatman No. 1 filter paper to obtain the cell-free mycelial extract used for AgNP synthesis. This extracellular approach is consistent with established fungal biosynthesis strategies in which secreted proteins and other metabolites participate in Ag? reduction and nanoparticle stabilization [4–7].

Figure 4. Representative culture tubes used for maintenance of the fungal isolates: (A)G. lucidum and (B) P. ostreatus fungal cultures maintained on laboratory medium.

2.4 Extracellular synthesis of AgNPs

The cell-free mycelial extract was mixed with aqueous AgNO? to obtain a final concentration of 1 mM. Reaction mixtures were incubated at 180 rpm for 48 h. An AgNO?-only control lacking fungal extract was maintained under comparable conditions. Formation of AgNPs was monitored initially through visible colour development. The change from pale yellow to reddish-brown was interpreted as preliminary evidence of colloidal silver formation, consistent with the optical behaviour reported for biologically synthesized AgNPs [4–7].

Figure 5. Schematic workflow for extracellular mycogenic synthesis of AgNPs using fungal biomass, aqueous extraction, AgNO? reduction and formation of colloidal silver nanoparticles.

2.5 UV–Visible spectroscopy

UV–Visible spectroscopy was used to evaluate the optical response of the synthesized preparations. The reported λmax and absorbance values were read from the supplied plotted spectrum because the original instrument-exported data file was not available. Accordingly, the values are reported as approximate figure-derived measurements and should be replaced by the instrument-generated numerical values before submission to a journal. Surface plasmon resonance (SPR) in the visible region is a commonly used preliminary indicator of AgNP formation and is influenced by particle size, morphology, aggregation and the local refractive environment [1,4].

2.6 FTIR spectroscopy

FTIR spectroscopy was used to identify functional groups associated with biomolecules retained on or interacting with the AgNP preparations. The supplied spectra were obtained using a Bruker instrument. Peak positions were interpreted approximately from the plotted spectra; exact numerical peak maxima should be obtained from the original instrument report. FTIR assignments were therefore treated as tentative rather than definitive, particularly in regions where multiple biomolecular functional groups can overlap [4,7].

2.7 Antibacterial activity

Antibacterial activity was evaluated by agar well diffusion against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis. The study record contains inhibition-zone values for Ganoderma-derived AgNPs, Pleurotus-derived AgNPs, fungal culture filtrate and a standard antibiotic. Agar diffusion is an established screening method, but the magnitude of an inhibition zone is influenced by diffusion, inoculum, medium composition, incubation conditions and agent concentration [11].

2.8 Statistical treatment and data-quality considerations

Descriptive comparisons were calculated from the supplied single observations. The difference between the two UV–Visible peak absorbances was calculated as 1.66 − 0.92 = 0.74 absorbance units, and the relative difference was approximately 80.4% when the Pleurotus value was used as the reference.

3. Results

3.1 Isolation and culture establishment

The collected fruiting-body material yielded two fungal isolates that were successfully established on malt extract medium. The supplied photographs show active mycelial growth and subsequent maintenance cultures. Successful establishment of stable cultures provided the biomass required for preparation of cell-free extracts and subsequent extracellular AgNP synthesis (Figures 1–4).

3.2 Visual evidence of AgNP formation

Following addition of fungal cell-free extracts to 1 mM AgNO?, the reaction mixtures developed a reddish-brown colour within approximately 48 h, whereas the AgNO? control did not show the same colour transition (Figure 9). Such colour development is a widely reported preliminary observation during AgNP formation and is associated with the collective optical response of nanoscale silver particles [4–7]. Although colour change alone does not establish particle size or morphology, its occurrence together with the observed SPR band provides convergent preliminary evidence for nanoparticle formation.

Figure 9. Representative reaction flasks showing the fungal extracts and the visible colour development observed during extracellular AgNP synthesis.

3.3 UV–Visible spectral characteristics

Both preparations exhibited a broad optical maximum centred at approximately 450 nm (Figure 6). The Ganoderma-derived preparation showed a substantially higher peak absorbance than the Pleurotus-derived preparation. The absolute difference was 0.74 absorbance units, corresponding to approximately 80.4% higher absorbance relative to the Pleurotus preparation. This observation should not be interpreted as an 80.4% higher nanoparticle yield because absorbance is affected by particle concentration, size distribution, morphology, aggregation, path length and the refractive environment [1,4].

Figure 6. UV–Visible absorption spectra of AgNP preparations synthesized using Ganoderma lucidum and Pleurotus ostreatus mycelial extracts. Both preparations show a broad maximum near 450 nm, with higher absorbance for the Ganoderma-derived preparation.

3.4 FTIR characteristics

Both spectra contained features in the C–H, amide/carbonyl, deformation and carbohydrate-associated regions (Figures 7 and 8). A prominent feature near approximately 1380 cm?¹ occurred in both preparations. The similarity suggests that fungal-derived organic molecules remained associated with the nanoparticle preparations. However, because exact peak maxima and appropriate fungal-extract controls were not supplied, these assignments remain tentative and should not be presented as proof of a specific enzyme or metabolite.

Figure 7. FTIR spectrum of the Ganoderma-derived AgNP preparation, showing prominent mid-infrared absorption features associated with fungal-derived organic functionalities.

Figure 8. FTIR spectrum of the Pleurotus-derived AgNP preparation, showing characteristic absorption features in regions associated with fungal proteins, carbohydrates and other organic constituents.

3.5 Antibacterial activity

Both fungal AgNP preparations inhibited all four test organisms in the supplied agar-well dataset. Pleurotus-derived AgNPs produced the largest inhibition zone observed among the AgNP treatments against K. pneumoniae (10 mm), followed by 8 mm against E. coli. Ganoderma-derived AgNPs produced 8 mm against S. aureus and 6 mm against E. coli. Against B. subtilis, Pleurotus and Ganoderma preparations produced 6 and 5 mm, respectively. The culture filtrate alone produced much smaller zones (0.3–1.2 mm), whereas the standard antibiotic produced substantially larger zones (12–20 mm).

Figure 9: Antimicrobial activity of Ganoderma and Pleurotus-derived AgNPs