Dry fungus-mediated gold bio-nanocomposite synthesis: an efficient green and sustainable heterogeneous catalyst for selective nitro reduction

This dry fungus-reinforced nanoparticle synthesis is the first proficient plan of action to replace the use of hazardous chemical, physical, and other bio-methods. Here, we report the synthesis of gold bio-nanocomposites (GBNCs) via immobilization of dry biomass prepared with conventional and lyophilization methods. Under atmospheric conditions, dried Aspergillus trinidadensis VM ST01′ OL587588 functions as a reducing and capping agent in water without any solvent or buffer interference. The use of dried biomass provides additional benefits for the synthesis of GBNCs, such as short synthesis time (24 h; 36 h with wet biomass) without incubation, better shelf life (more than 18 months), improved catalytic activity, intact morphology, etc. The generated GBNCs were characterized by various analytical techniques and were found to have a roughly spherical shape with a mono-dispersed diameter of approximately 25 nm, as determined with high-resolution transmission electron microscopy. The influence of stirring and biomass concentration on the kinetics was also studied for the GBNC fabrication process. Optimized stoichiometric results have shown 3.5 × 10¹⁵ gold atoms per milligram of dried biomass prepared by both methods. The crystalline nature and surface charge of GBNCs were analyzed by powder X-ray diffraction and zeta potential studies, respectively. FT-IR studies have shown the participation of various biomass functional groups in forming GBNCs. The surface morphology of GBNCs was investigated by scanning electron microscopy. A comparative thermal stability of dried biomass and GBNCs was evaluated by thermo-gravimetric analysis, with a large difference in residual mass. Here, GBNCs have been shown to be a truly potent heterogeneous catalyst for the reduction of nitrobenzene in water using sodium borohydride with yields up to 95% isolation. The industrial suitability of GBNCs has been established with their broad operational pH (4–10) and temperature (25 °C–80 °C) ranges, reusability (more than 10 cycles), storage stability (more than 18 months), and successful scale-up investigations (up to 5 gm).

GRAPHICAL ABSTRACT

GRAPHICAL ABSTRACT | A novel, user-friendly, and economic approach for the synthesis of gold bio-nanocomposite as a potential heterogeneous catalyst for nitro reduction was investigated, leading to a green and sustainable development in industrial catalysis.

1 Introduction

Amines are a crucial chemical in a range of industries, including fine chemicals, agrochemicals, polymers, and pharmaceuticals (Yan et al., 2024). Most of these amines are synthesized by the reduction of corresponding nitro compounds (Li et al., 2021). However, conventional methods for reducing nitro compounds often rely on hazardous chemicals and stringent reaction conditions, raising concerns about safety and environmental impact (Chakraborty et al., 2024). In recent years, there has been a notable shift toward the development of more environmentally friendly approaches to nitro reduction (Binupriya et al., 2010; Li et al., 2021). One promising avenue involves harnessing biologically derived materials such as fungi in conjunction with metal nanoparticles as catalysts (Kumar et al., 2023). Fungi possess a diverse array of enzymes capable of catalyzing various redox reactions, including the reduction of nitro groups (Menon et al., 2017). Additionally, gold nanoparticles exhibit exceptional catalytic activity in a wide range of chemical processes due to their unique geometric and electrical properties (Shen et al., 2017; Bhambure et al., 2009). By combining gold nanoparticles with fungi in nitro reduction processes, a promising opportunity arises for more sustainable and efficient synthesis methods (Binupriya et al., 2010). This hybrid approach leverages the bio-catalytic activity of fungi and the catalytic capabilities of gold nanoparticles, enabling nitro group reduction under mild reaction conditions (Vala, 2015). Yunbo Li has reported the gold nanoparticle films facilitated by thiourea for catalytic reduction of nitrophenol in water (Xia et al., 2024). However, this method requires a two-step process to develop this heterogeneous catalyst. It also uses chemical methods for membrane-based capping of gold nanoparticles. Chih-Chien Chu and team demonstrated the reduction of nitroaromatics by gold nanoparticles doped on porous silicon fabricated using metal-assisted chemical etching (Liang et al., 2023). Here, the use of two-step silicon-based friction makes it less environmentally friendly. Bastien Léger and group established catalytic reduction of 4-nitrophenol with gold nanoparticles stabilized by large-ring cyclodextrins (Noël et al., 2020). This process involves two steps to prepare the heterogeneous catalyst. The use of cyclodextrins makes this technique expensive.

Our research aims to investigate the feasibility of utilizing gold nanoparticles alongside fungi-mediated nitro reduction as a novel and environmentally friendly method for synthesizing various amines (Morgan and Aboshanab, 2024). The key to the success of this strategy lies in the synthesis of gold nanoparticles and the selection of suitable fungi. In several experiments on bio-inspired nanoparticle synthesis, the researchers used wet biomass cells as the precursor (Daigger et al., 1982). The presence of water in biomass encourages microbial growth and enzymatic degradation, affecting the culture’s shelf life (Radojči et al., 2021). In this case, a dry biomass is anticipated for better performance. However, most drying methods, such as microwave drying, oven drying, and ultrasound drying, may result in ruptured cell walls and the formation of large channels (Parniakov et al., 2022). The lyophilization technique is widely accepted in several industries, including biotechnology, food manufacturing, food preservation, and pharmaceuticals, as it causes no damage to the proteins and microstructure of the dried biomass (Meng and Wang, 2020). During lyophilization, water present in the biological material is removed in two steps: freezing (resulting in volume expansion due to water-ice transformation) and conventional vacuum drying (enhancing the pore structure of biomass) (Guo et al., 2023). Consequently, the lyophilized biomass contains high-quality porous structures, providing additional active sites for reactions (Antaby et al., 2021). Moreover, the integrity of the biomass is retained due to the low temperature of the process and the absence of liquid water in the mass. Additionally, lyophilized biomass is easy to handle, store, and transport, as it is space-saving, lighter, and poses a lower contamination risk (Deshmukh et al., 2023a; Seju et al., 2024). As a comparative investigation and an economical approach, the biomass was dried using conventional vacuum drying.

In this context, here we report the use of dried fungal biomass as a precursor for gold bio-nano composite (GBNC) synthesis, with the benefits of (a) one-pot synthesis; (b) short reaction time; (c) bulk preparation of GBNCs; (d) formation of a firm powder with stable shelf life; (e) ready-to-use material; (f) effective reaction sites; (g) no use of buffer and incubator. The prepared GBNCs were shown to be an efficient heterogeneous green and sustainable catalyst for highly efficient reduction of nitro group compounds (Figure 1).

Figure 1

Figure 1. Unique characteristics of dry fungus-mediated gold bio-nanocomposites as a heterogeneous catalyst.

2 Experimental

2.1 Materials and methods

The solvents, that is, ethyl acetate, acetonitrile, and water, were manufactured by Rankem and purchased from a local vendor. Nitrobenzene (99% pure) and aniline (99% pure) (manufactured by Sigma-Aldrich and Rankem) were also purchased from local vendors and used without additional purification. Sigma-Aldrich and Rankem provided the gold chloride, sodium borohydride, and hydrochloric acid with high purity. For the Bruker AVANCE III 500 MHz (AV 500) multi-nuclei solution NMR spectrometer, ¹H and ¹³C NMR spectra were recorded in CDCl₃ or DMSO-d6 (Cambridge Isotope Laboratories, Inc., United States) at room temperature. TMS served as the internal reference. To determine the % conversion of the products, high-performance liquid chromatography (HPLC) on a SHIMADZU Prominence–I, LC-2030 Plus with a reverse-phase C₁₈ column (250 mm × 2.5 mm) and mobile phase ACN/H₂O (70:30; flow rate 1 mL/min) with a PDA detector was used. Product purification was done using a normal glass column (600 mm × 30 mm) with hexane:ethyl acetate used as the mobile phase with different polarity ratios. Kiesel gel 60 15 F254 aluminum sheets (Merck 1.05554) were utilized for the analysis, utilizing thin-layer chromatography, and ninhydrin was used as a staining agent to detect amines under the UV light. The gold nanoparticles’ zeta potential was calculated using Zetasizer Ver. 7.13 (Malvern Panalytical, United Kingdom). The Thermo Scientific-Orbitrap Elite with electrospray ionization was used for high-resolution liquid chromatography-mass spectroscopy (LC-MS).

2.2 Preparation of dry biomass of Aspergillus trinidadensis VM ST01′ OL587588 using lyophilization

Aspergillus trinidadensis VM ST01′ OL587588 strain was cultured in potato dextrose broth using granulated media made up of 200 g/L potato infusion and 20 g/L dextrose. To maintain stock culture under sterile conditions at 0 °C, 100 mL of potato dextrose agar slant was created every month using three Petri plates with a length and thickness of 55 mm and 0.12 mm, respectively (3.9 g in 100 mL). Pre-culture was done by inoculating four loops (5 mm in diameter) into sterile medium (500 mL) and incubating for 48 h at 36 °C in an orbital shaker at 160 rpm. Based on our earlier process optimization (Deshmukh et al., 2023a), the mature biomass was harvested after 48 h and washed using sterile water till the media was removed. The washed biomass was then kept in a deep freezer for 6 h at −18 °C. Afterward, the frozen biomass (−15 °C) underwent vacuum lyophilization at −60 °C for 24 h. The lyophilized biomass was crushed into powder using a mortar and pestle and stored in airtight vials. Gloves, masks, and safety goggles were mandatory throughout the experiment.

2.3 Preparation of dry biomass of Aspergillus trinidadensis VM ST01′ OL587588 using conventional vacuum drying

Biomass was grown and washed following the process mentioned above. A 5-g sample of washed wet biomass was then kept in a 100-mL round-bottom flask (RBF) for 24 h at room temperature. The obtained dried biomass was crushed into powder using a mortar and pestle and stored in airtight vials. Gloves, masks, and safety goggles were mandatory throughout the experiment.

2.4 Synthesis of gold bio-nanocomposites (GBNCs) via immobilization of dried biomass

There are many reports on the use of wet biomass for the synthesis of metal nanoparticles (Eid et al., 2023; Krishnan et al., 2021; Misbah et al., 2025); however, this is the first report on the use of dried biomass. Dried biomass (prepared either by lyophilization or a conventional method) of 50 mg was suspended in 25 mL of distilled water with 1 mM AuCl₃. This dried yeast cell suspension and gold chloride were stirred for 24 h at room temperature (∼36 °C) and 300 rpm. After completion of 24 h, the amount of gold in the residual supernatant was measured using UV. The same concentration of the gold precursor (1 mM AuCl₃) was added to the distilled water in the control experiment and stirred under the same circumstances. The obtained GBNCs were separated from the reaction mixture by simple filtration. Solid GBNC residues were then dispersed in distilled water, and the procedure was repeated to yield GBNCs free of unreacted gold ions. Then, the obtained pure GBNCs were dried under atmospheric conditions.

The process was repeated with different dried biomass powder quantities (1 mg, 2 mg, 3 mg, and 4 mg per mL) with fixed Au (III) ion concentration (1 mM). The uptake of gold in the samples was analyzed by UV–visible spectroscopy. The quantity of gold uptake by the immobilized fungal cells was calculated using a mass balance. The dry weight of GBNCs was calculated, and the amount of gold uptake and cellular shape were analyzed to establish the ideal cellular concentration. The kinetics of gold accumulation were examined, utilizing the entire supernatant at regular intervals, and the remaining gold was then quantified. The ratio of the number of cells to the amount of gold removed for each amount of dried biomass powder was used to calculate the number of gold atoms inside each cell. The number of yeast cells in a 2 mg/mL cell solution was calculated using a Hemocytometer Cell Counter. An average of 2.6 × 10³ cells per mL was discovered to be present, and 3.51 × 10¹⁷ Au atoms of gold chloride were added to the mixture. The cells’ ability to absorb gold was found to be 7.8 × 10¹⁹ gold atoms per gram of fungi. Later on, the stirring time (12 h, 24 h, and 48 h) was optimized with 2 mg/mL dried biomass and the same gold concentration. Among all the studied stirring times, 24 h showed the maximum gold uptake. Finally, the process was optimized under different temperatures (30 °C, 36 °C, and 42 °C) with 24 h stirring, 2 mg/mL dried biomass, and the same gold concentration. Here, a reaction temperature of 36 °C resulted in the maximum gold uptake.

2.5 Characterization methods

The UV-1900/Vis Spectrophotometer by SHIMADZU was utilized to measure the distinctive surface plasmon resonance peak of the gold nanoparticles (GBNC). A PerkinElmer Optima 5300 DV instrument was used to measure the amount of gold extracted from the aqueous solution using immobilized biomass by inductively coupled plasma optical emission spectrometry. Optical emission at λ/nm (267.59), intrinsic to the gold element, was recorded to determine the residual content of gold in the reaction supernatants. A Zetasizer was used to measure the hydrodynamic diameter (Dh), polydispersity index (PDI), and zeta potential of the GBNC. Three sets of these characterization experiments were carried out, and the corresponding mean values and standard deviations were given for each set of results. The GBNCs were analyzed morphologically with a field emission scanning electron microscope (FESEM) (Make: Zeis, Model: Sigma 300), and their high-resolution particle size was verified with field emission transmission electron microscopy (FETEM) (Make: JEOL, Model: 2100F) device. A Bruker Discover D8 diffractometer with a powder X-ray diffraction (powder XRD) detector was used to analyze the phase identity and crystalline nature of the GBNCs. Cu (Kα) radiation (λ/Å = 1.54) was used at room temperature within the 2θ range of 30° to 100°. Using an Eco-ATR Bruker ALPHA II spectrometer, Fourier transform infrared spectroscopy (FT-IR) was used to examine the surface chemistry of the GBNCs at a resolution of 4 cm⁻¹. The spectrometer was used to measure the v_max/cm⁻¹ range of 600–4,000. The thermal breakdown behavior of the GBNC was examined using thermal gravimetric analysis (NETZSCH STA 449F3) in a nitrogen atmosphere and a heating rate of 5 °C per minute. Al₂O₃ was used as a reference sample. The conversion of nitrobenzene, the model substrate, to aniline was observed with C18 column chromatography and a mobile phase of ACN/Water (70/30) at a pressure of 0.50 mL/min. A photodiode array (PDA) detector was employed for detection through HPLC on a SHIMADZU Prominence–I, LC – 2030 Plus apparatus.

2.6 Storage, pH, and temperature stability of GBNCs

The purified solid GBNCs were immobilized in solutions with different pH levels (i.e., 2, 4, 6, 8, 10, and 12). The variation of pH was done by altering the pH of double-distilled water with either hydrochloric acid or sodium hydroxide. After the dispersion of gold nanoparticles for 12 h in the appropriate pH solutions, the samples were examined by UV–Vis spectroscopy. Additionally, after a year of storage at 4 °C, the long-term stability of the better-distributed GBNCs was also assessed by UV–Vis. To verify the temperature stability of GBNCs, it was suspended in distilled water, incubated for 2 h, and examined by UV–Vis.

2.7 General process for the reduction of nitrobenzene by GBNCs

Nitrobenzene was used as a model substrate to perform the heterogeneous catalytic reaction by GBNCs (Scheme 1). GBNCs (10 mg) (0.01 mmol% Au) were immobilized with 4 mL of distilled water in a 25-mL round-bottom flask to form a stable suspension. Subsequently, nitrobenzene (99%) (0.5 mM, 0.69 g) was added with constant stirring at room temperature. Then the reaction mixture was cooled at 0 °C–4 °C, and NaBH₄ (0.25 mM) was added slowly. The ice bath was removed after 5 min, and the reaction mixture was allowed to stir at room temperature. The reaction was monitored by thin-layer chromatography and ninhydrin staining. Once the reaction was completed, the product was extracted thrice with ethyl acetate (5 mL), and the organic layer was dried over anhydrous sodium sulfate and further concentrated. The same process was used to build the substrate scope for GBNCs, except for the change in reaction time.

Scheme 1

Scheme 1. Sustainable reduction of nitrobenzene as a model substrate.

3 Results and discussion

3.1 Impact of dried biomass quantity on nanofabrication and catalytic activity of GBNCs

Different amounts of dried biomass (ranging from 1 mg/mL to 3 mg/mL) with fixed Au (III) concentration (1 mM) were used in preliminary tests on the biosynthesis of GBNCs. Additionally, similar reactions were run at intermediate biomass concentrations of 0.5 mg/mL, 1.5 mg/mL, and 2.5 mg/mL. According to quantitative analysis of the reaction supernatants, the biomass concentration of 2.0 mg/mL was found to be optimal for producing GBNCs in the reaction supernatants. Reactions with higher biomass quantity (2.5 mg/mL and 3.0 mg/mL) exhibit lower UV bands, indicating less production of GBNCs (Figure 2).

• Research on the catalytic activities of all these prepared catalysts has additionally demonstrated that GBNCs produced using a biomass concentration of 2 mg/mL exhibit the highest catalytic efficiency (Table 1). Additional experiments were carried out to determine the hydrodynamic diameter, zeta potential, and polydispersity index of the resultant GBNCs (Table 6). These results confirmed that the 2 mg/mL dried biomass amount is the best for GBNCs fabrication. The spherical GBNCs generated with 2.0 mg/mL dried biomass were more evenly disseminated, according to HR-TEM (Supplementary Figure S1). Therefore, this biomass concentration was used for the remaining optimization studies. Upon reviewing our previous attempts at GNP synthesis utilizing wet biomass, it becomes clear that employing dry biomass leads to a reduction in the quantity requirement. This increased productivity may be attributed to the greater organic content, which acts as a reducing agent, found in the dormant cells. It is notable that, apart from lyophilization, alternative methods of biomass drying, such as vacuum drying and sun drying, facilitated the procedure involved in generating gold nanoparticles and contributed to the reduction reaction, yielding comparable results

Figure 2

Figure 2. Effect of dried biomass quantity on nanoparticle synthesis under identical reaction conditions. (A) UV spectrophotometer and (B) visual examination.

Table 1

Table 1. Synthesis of GBNCs using different quantities of dried biomass vs their catalytic efficiency.

3.2 Time optimization of nanofabrication and catalytic activity of GBNCs

GBNCs were synthesized with 2.0 mg/mL dried biomass of 48 h culture with fixed Au (III) concentration (1 mM), stirred for different time durations at room temperature (∼25 °C). The quantitative analysis of the reaction mixture showed production of the highest amount of GBNCs within 24 h (Figure 3). As reported in our earlier report, the same amount of wet fungus took 72 h to synthesize gold nanoparticles with good catalytic activity. This comparison shows the effectiveness of dried biomass over wet biomass for the synthesis of GBNCs. Their diminished catalytic effect provided additional evidence that this GBNC prepared with 24 h of stirring has the highest catalytic efficiency (Table 2). Hence, the 24-h period was deemed the optimally efficient stirring time for subsequent investigations. The catalytic activity of GBNCs prepared from dried biomass is almost double with respect to wet biomass.

Figure 3

Figure 3. UV–Vis spectra of GBNCs prepared by lyophilizing biomass at different stirring times.

Table 2

Table 2. Synthesis of GBNCs using different stirring times vs their catalytic efficiency.

3.3 pH and solvent stability of GBNCs

The UV–Vis spectra of GBNCs prepared under optimized conditions and immobilized for 12 h in solutions with different pH (i.e., 2, 4, 6, 8, 10, and 12) indicate their stability across all tested pH levels, corroborating their visual appearance. (Figure 4).

Figure 4

Figure 4. (A) UV–Vis spectra of GBNCs under different pH and solvents. (B) Visual inspection of the fresh samples.

The stability and catalytic efficiency of the prepared GBNCs were also evaluated through UV–Vis spectroscopy, revealing that dispersing them in a pH 7.4 solution notably enhanced the monodispersing of the gold nanoparticles and nitro reduction efficiency. Water, ethanol, and DMSO were employed as alternative solvents for dispersing the GBNCs. All three studied solvents were able to show uniform dispersion of GBNCs with catalyst morphology. This study shows that apart from water as a green solvent, GBNCs can also be used in organic solvents.

3.4 Storage stability

The durability of the biomass was assessed by immersing the dried GBNCs, which contained gold nanoparticles, in 3 mL of distilled water and monitoring it using UV–Vis spectroscopy for a period of 1 year (Figure 5; Table 3). Absorbance measurements were taken at 550 nm. Subsequently, the same sample underwent sonication for 1 h at 60 °C, and the absorbance of the sonicated sample was compared to the original (Figure 5B), highlighting differences in intensity. The GBNCs settled spontaneously without the need for a centrifuge when the sample was allowed to settle for 1 h, and no absorbance was found in the water layer. (Figure 5C). Remarkably, GBNCs were found to be extremely stable under all tested conditions. In addition, the HPLC analysis, as shown in Figure 6, indicated the intact catalytic nature of the GBNCs even after 18 months of storage.

Figure 5

Figure 5. UV–Vis spectra and visual inspection of GBNCs: (A) dispersed in water after 1 year, (B) sonicated for 1 h at 60 °C, and (C) sample left for 1 h.

Table 3

Table 3. Reduction of nitrobenzene using 1 year-stored and sonicated GBNCs.

Figure 6

Figure 6. HPLC analysis for the reduction of nitrobenzene using GBNCs in the presence of NaBH₄; Panel I: Aniline (standard); Panel II: Reduction of nitrobenzene using a catalyst stored for 18 months.

3.5 Reusability, leaching

Reusability investigations demonstrated that even after the 11th cycle (refer to Figure 7A), a conversion rate up to 92% was maintained, with results consistent with those of other gold-supported catalysts. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis indicated that only trace amounts of gold (0.251 ppm, 1.2%) were detected in the reaction supernatants following the 11th catalytic cycle. After this cycle, examination via FESEM revealed the presence of intact fungal cells (GBNCs) on the recovered catalyst, as depicted in Figure 7B.

Figure 7

Figure 7. Research on the potential for catalysts to be reused in converting nitrobenzene to aniline; (A) Reusability up to 11th cycle; (B) SEM image after 11th cycle; (C) ICP-OES before 1st cycle and (D) ICP-OES after 11th cycle.

The HR-TEM analysis of GBNCs after the 6th and 11th cycles clearly shows that the particle size and distribution in biomass remain unaffected (Figure 8). These findings confirm that fungal cells with in-situ-produced gold nanoparticles serve as an effective support structure for sustainable catalysis. To perform the leaching study, GBNCs were stirred at 60 °C for 24 h and filtered. Reduction of nitrobenzene by the filtrate did not show any trace of aniline under the optimized conditions (Supplementary Figure S2), while the residual GBNCs gave the final reduced product, aniline. This study shows the leaching stability of GBNCs.

Figure 8

Figure 8. HR-TEM of GBNCs after the 6th and 11th reuse cycles.

3.6 GBNCs mediated nitrobenzene catalytic reduction

The optimization of various parameters for the reduction of nitrobenzene with NaBH₄ as a source of hydride was performed for a fixed 2 h reaction time using GBNCs. It is observed that the increase in NaBH₄ from 0.5 mmol to 2.0 mmol, with fixed 0.2 mmol GBNCs, accelerates the conversion rate, but further excessive addition of NaBH₄ creates pressure inside the reactor. To avoid such inconvenience, the amount of Au was increased to 0.5 mmol, 1.0 mmol, and 1.5 mmol, and the amount of NaBH₄ was varied, respectively. Subsequently, a 100% conversion was obtained with 1.0 mmol Au and 1.5 mmol NaBH₄ with an isolated yield of 99.97% aniline. The reduction of nitrobenzene to aniline using the dry GBNCs/NaBH₄ was investigated by HPLC chromatogram (Supplementary Figure S3). These results provide additional evidence for the notion that gold nanoparticles aid in electron transfer between the electron donors (NaBH₄) and acceptor (nitrobenzene) via the electron relay mechanism (Meng and Wang, 2020).

Aniline synthesis was not detected in the control experiments, encompassing: i) NaBH₄ in isolation; ii) GBNCs alone; iii) simple dried biomass under identical reaction conditions (Table 4). The joint effectiveness of NaBH₄ and GNBCs in reducing nitrobenzene was explored by adjusting their individual compositions. The optimization of the reaction time for nitrobenzene reduction was conducted by assessing the percentage conversion at various time intervals, as shown in Table 5. Thus, the optimal reaction time was determined to be 2 h.

Table 4

Table 4. Finding an efficient mole ratio of GBNCs/NaBH₄.

Table 5

Table 5. Reduction of nitrobenzene at different reaction times.

3.7 Characterization of GBNCs

An initial confirmation for the formation of GBNCs was done with a UV–Vis study (Supplementary Figure S4). As per the earlier report, GBNCs with a desired size of ∼25 nm were confirmed by an absorption maximum at 548 nm (Figures 2, 3) (Eid et al., 2023).

3.8 Size analysis

Subsequent confirmation was obtained through FETEM imaging, which supported this assumption and yielded an average particle size of 25 nm, as shown in Figure 9.

Figure 9

Figure 9. FETEM images (A) at lower magnification; (B) at higher magnification; (C) size distribution of GNBCs; (D) selected area electron diffusion (SAED) pattern of GBNCs.

3.9 FT-IR analysis

The probable biochemistry involved in synthesizing and stabilizing the GBNCs was investigated by comparative FT-IR analysis of dried biomass and GBNCs. FT-IR analysis was used to investigate the potential surface chemistry involved in the stability of GBNCs (Figure 10A). The identification of probable functional groups involved in bio-reduction and the subsequent capping of GBNCs was also made possible by FT-IR analysis. The N-H stretching of the major amines in the protein molecules was shown by a large band in the FT-IR spectra at 3,256 cm⁻¹ and 2,919 cm⁻¹ (Hossan et al., 2023). The amide I, amide II, and amide III bands of peptide units of polypeptide proteins, respectively, were seen as bands at 1,643 cm⁻¹ and 1,547 cm⁻¹ (Le Ouay et al., 2023). The symmetric stretching of the carboxylate (COO-) groups produced a band at 1,377 cm⁻¹ (Naseem et al., 2023). A noticeable band was seen at 1,023 cm⁻¹, indicating the C-N stretching vibrations that may have been caused by the proteins’ aliphatic amines (Roseline et al., 2023). The GBNC creation is shown by the development of peaks at 2,361 cm⁻¹, 1,261 cm⁻¹, 795 cm⁻¹, and 666 cm⁻¹ (Figure 10B) (Deshmukh et al., 2023b). Following reduction, these bands were completely absent, indicating that amino acids like cysteine and methionine may have been involved in capping the GBNCs that had been produced. It has been revealed that peptide/protein molecules cap stable GBNCs produced by green nanoparticle synthesis using microbial extracts.

Figure 10

Figure 10. FT-IR analysis of (A) dried Aspergillus trinidadensis (control) and (B) dried GBNCs.

3.10 FESEM analysis

Visual inspection of the dried powder of GBNCs showed the presence of pink coloration, which is due to gold nanoparticles (Figure 11A). FESEM images, as shown in Figures 11B–D, were used to investigate the surface morphology of Au (III)-treated Aspergillus trinidadensis fungi. The FESEM image of the dry biomass confirms the existence of spherical gold nanoparticles within the dormant fungal body. The gold nanoparticles obtained through both lyophilized drying and sun drying exhibit similar morphologies.

Figure 11

Figure 11. (A) Visual inspection of GBNCs; SEM images of (B) Dry biomass; (C) lyophilized biomass-based GNP; (D) sun dried biomass-based GBNCs.

3.11 X-ray diffraction analysis

The powder XRD spectra of pristine lyophilized biomass of Aspergillus and biomass-supported GNPs are shown in Figures 12A,B. The powder XRD pattern exhibits sharp peaks over the whole spectrum of 2θ values, from 9° to 78°. The existence of crystalline gold nanoparticles was established by comparing our powder XRD spectrum to the literature (Abuzeid et al., 2023). The strong peaks at 2θ values of 38.17°, 44.37°, 64.63°, and 77.53° are associated with gold nanoparticles (111), (200), (220), and (311) (Abuzeid et al., 2023). The GBNCs created in this study were shown to be stable for at least 1 year at 4 °C, providing unchanged absorption bands and no sign of GNP agglomeration throughout this time. It is evident from the XRD pattern that the synthesized GBNCs are crystalline.

Figure 12

Figure 12. Powder XRD analysis: (A) dried biomass (control) and (B) GBNCs.

3.12 Size and surface morphology analysis by zeta potential

The zeta potential of the GBNCs produced for each reaction ranged from −7.25 to −10.80 mV, and it was very stable (Table 6). A suitable control experiment that employed heat-killed biomass in place of dried biomass likewise produced fewer GBNCs. Therefore, dried biomass mediated was used to make polydispersed GBNCs. Additionally, the effects of biomass quantity, that is, 1.0 mg/mL, 2.0 mg/mL, and 3.0 mg/mL, were tested against the fixed gold precursor, that is, 1 mM Au (III). These effects were studied for the resulting GBNCs’ hydrodynamic diameter, polydispersity index, and zeta potential.

Table 6

Table 6. Influence of biomass content on the biogenesis of GBNCs.

The formation of spherical GBNCs with particle Dh ranging from 48 nm to 214 nm was observed. The zeta potential of the GBNCs produced for each reaction ranged from −11 to −7 mV and was very stable (Table 6). The results of the experiments demonstrated that producing GBNCs at 1 mM Au (III) and a biomass concentration of 2.0 mg/ML was successful (Table 6). If the reduction of Au (III) to Au⁰ is caused by an enzyme, the rise in biomass levels must promote the manufacture of GBNCs. On the other hand, the biosynthesis of GBNCs was not enhanced by further raising the dried biomass content at a given concentration of Au (III) chloride.

The effect of the hydride source and reduction reaction conditions was also investigated by zeta potential analysis (Table 7). A large change is observed by an increase in zeta potential and Dh after sodium borohydride treatment. This confirms the binding of hydride ions on the gold particle surface before transferring to the nitro substrate. There is a recovery in zeta potential and Dh of recovered GBNCs, which shows the reversible hydride transfer, making it a reusable catalyst.

Table 7

Table 7. Influence of reduction reaction on GBNCs.

3.13 Thermal gravimetric analysis and differential scanning calorimetry

The thermal properties of prepared GBNCs and the effect of gold nanoparticles on biomass were studied by using thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis. The thermal degradation study of GBNCs prepared by lyophilized and conventional vacuum-dried biomass also confirms the capping of the nanoparticle by biomass, which decomposes in the range of 300 °C–500 °C. The decomposition behavior of both the biomass prepared by the conventional method (control) and its GBNCs is shown by the TGA curve (Figure 13). The TGA and DSC spectra of lyophilized biomass and its GBNCs are shown in Supplementary Figure S8. Here, as accepted about residual mass, a little over 25% (0.81 mg) for reference was still present, while 45% (1.33 mg) of the GBNCs remained following the complete heating cycle. This is most likely due to the thermal stability caused by the addition of gold, which operates as supporting spots and minimizes biomass decomposition. The GBNCs showed major weight loss when heated to approximately 300 °C; however, the sample biomass (control) showed major weight loss above 225 °C, which could be regarded as the beginning of mass loss.

Figure 13

Figure 13. Thermal gravimetric analysis of dried biomass and GBNCs.

Initial weight loss in GBNCs (9.50%) and control biomass (6.25%) could be due to loss of water trapped inside the samples. The main weight loss regions for biomass (control; 80.28%) and GBNCs (62.20%) were from 225 °C to 500 °C and from 300 °C to 500 °C, respectively. Compared to the reference samples, the peak temperature for functionalized nanofabrication increased from 150 °C to 300 °C. This shows the thermal stability of GBNCs induced by gold nanoparticles.

Any phase transition changes that might occur were then recorded by DSC, for instance, at the melting points of biomass after they have been functionalized with gold nanoparticles. Both kinds of nanofabrication in this instance showed conventional DSC curves. According to Figure 14, Tg curves were noticed at 50 °C in both biosynthesized GBNCs and biomass. Due to the Tg of biomass present in both samples, an exothermic peak was visible for GBNCs at 121 °C, and a significant endothermic peak was visible for the reference sample at 157 °C. It was therefore reasonable to assume that among GBNCs and biomass, nanoparticles helped to reduce the heat generated throughout analysis. An exothermic peak for GBNCs observed at 121 °C shifted to a significant endothermic peak for the reference sample at 157 °C. The biomass (control) had a peak with weak intensity, but the peak for GBNCs was relatively much stronger because of the Au fabrication. In biomass (control) and GBNCs, the normalized peak heat needed to enable those transitions was 320 mW/mg and −75 mW/mg, respectively. The area under curves revealed that the predicted enthalpies for these samples were 130.34 J/g and −05.46 J/g for GBNCs and biomass (control) samples, respectively. More heat was required to allow a similar transition in GBNCs because the nanoparticles utilized for functionalization absorbed extra heat. The functionalization of biomass using GBNCs gave extra thermal stability in terms of absorbed heat and degradation rate, as confirmed by TGA and DSC. Based on this study for numerous samples, it should be noted that TGA and DSC values were fairly repeatable.

Figure 14

Figure 14. Differential scanning calorimetry of dried biomass and GBNCs.

3.14 Mechanistic investigation

Metal nanoparticles participate in two distinct routes during the reduction of nitroarenes to aryl amines. The first pathway entails direct involvement of intermediates such as nitroso benzene and phenyl hydroxyl amine, while the second pathway involves intermediates such as azoxybenzene, azobenzene, and diazobenzene (Figure 15) (Yang et al., 2021).

Figure 15

Figure 15. Mechanistic pathway for utilizing GBNC/NaBH₄ to reduce nitrobenzene to aniline.

The GBNC-mediated reduction of the nitrobenzene reaction mixture was subjected to GC-MS analysis before completion in order to investigate the mechanism. Confirming the presence of (Z)-1,2-diphenyldiazene oxide (azoxybenzene) and (E)-1,2-diphenyldiazene (azobenzene), as illustrated in Supplementary Figure S5, validates that the reaction follows an indirect (condensation) pathway. GBNC (0.0001 mol Au) and NaBH₄ (1.5 mmol, 1.5 equiv.) were used to accomplish the maximum conversion to up to 96% (Z)-1, 2-diphenyldiazene oxide (87% yield) in a 2 h reaction (Supplementary Table S1; NMR Supplementary Figures S6, S7). A similar mechanism was found when gold nanoparticles and CeO₂ were used as a supported catalyst for the hydrogenation of nitroarenes (Wani et al., 2021).

4 Conclusion

The first report on an efficient, green, and sustainable one-pot protocol has been established using dried biomass of Aspergillus trinidadensis as an effective microbe for biosynthesis of GBNCs. Dried biomass gave a faster process than wet biomass to form more stable GBNCs. It has rendered a more economical process by the use of simple water at room temperature with normal staining instead of using a buffer and an incubator. As a comparative investigation, dried biomass was prepared by conventional vacuum drying and lyophilization. Both forms of biomass have shown almost equivalent efficiency for the synthesis of GBNCs. The GBNC morphologies were completely characterized with UV–Vis, SEM, HR-TEM, powder XRD, FT-IR, and zeta potential analysis. Prepared GBNCs were extremely stable when dispersed in different pH solutions (2–12). The application of a prepared catalyst has been demonstrated with the reduction of nitrobenzene. GBNCs prepared under different conditions were used for the catalytic study. GBNCs prepared by 48-h culture age with 2 mg/mL dried biomass under 24-h stirring at 36 °C, and dispersion in water showed the highest reduction efficiency. Aspergillus trinidadensis VM ST01′ OL587588 was advantageous because it was simple to handle during downstream processing and large-scale manufacturing. As they release a significant quantity of extracellular redox enzymes, these fungi may be a superior scale-up choice. Importantly, it would be helpful to have a non-pathogenic biological system that generates metal nanoparticles for commercialization reasons. In this study, fungi-derived dried biocatalysts serve as ideal scaffolding for this goal. The field of nanoparticles biosynthesized using dried biomass is very new. Therefore, the GBNCs produced through the biosynthetic approach have enormous promise for a variety of uses. The commercial potency of the GBNCs for their industrial applications has been well demonstrated by their storage stability, reusability, leaching study, and broad pH and temperature ranges. As GBNCs have a very minute quantity of gold with respect to biomass, the prepared catalyst is economically viable. Its bulk production is possible with the use of ordinary stirrers and shakers, which shows its commercial potency. As an application of GBNCs, it has the capability to scale the process for selective nitro reduction up to production scale.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Ethics statement

The manuscript presents research on animals that do not require ethical approval for their study.

Author contributions

AD: Writing – review and editing, Writing – original draft, Validation, Data curation, Formal Analysis, Methodology, Visualization. RB: Formal Analysis, Visualization, Writing – review and editing. HM: Writing – original draft, Writing – review and editing, Software, Resources, Data curation. PP: Writing – review and editing, Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – original draft.

Funding

The authors declare that financial support was received for the research and/or publication of this article. The authors would like to acknowledge the financial support of the Gujarat State Biotechnology Mission (GSBTM, Government of Gujarat), SHODH and GSBTM proect grant ID KSEV49.

Acknowledgements

The authors are also thankful to Bishnupada Mandal, Shubham Kumar, and Aviti Katare of IITG for providing support in FESEM and FETEM analysis. The authors are also thankful to Abhisek Dadhaniya and Changa University for thermal analysis.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Generative AI was used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fctls.2025.1690697/full#supplementary-material

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