Introduction

Nanoparticles (NPs) are usually defined as nanomaterials in which all dimensions are in a range less than 100 nm and present a high surface-to-volume ratio, which provides their unique properties compared to materials that are already in bulk1,2,3. NPs have wide applications in different fields areas as food4, agriculture5, medicine, pharmacy, photocatalysis, and cosmetics among others6,7. Conventional methods for synthesizing nanoparticles often involve high energy consumption and the use of chemical reagents, which can be potentially harmful to the environment and human health8. However, in the last few years, green NPs have emerged as an eco-friendly, simple and economically feasible alternative since they can be produced in large volumes and at reasonable cost, under low temperature and pressure and absence of organic solvents9. These NPs are synthesized using a variety of microorganisms, including bacteria, filamentous fungi, yeast, algae, and plants10,11,12,13. In particular, filamentous fungi are interesting microorganisms for this purpose due to their active metabolism and ability to secrete numerous proteins involved in nutrient acquisition, environmental sensing and signaling, cell wall construction, and remodeling under stressful conditions14,15. Thus, fungal NPs biosynthesis is more efficient than other microorganisms by their ability to produce a large amount of biomass and a variety of bioactive metabolites as reducing agents, and besides tolerate and accumulate metals in their tissues16. Additionally, NPs derived from fungi offer the advantage of forming coating biomolecules, providing stability and potentially contributing to various biological activities17.

The biosynthesis of metal nanoparticles derived from fungi has been described either as an intracellular or as extracellular process. During intracellular synthesis, nanoparticles are formed and localized in the cytoplasm, cell wall or cell membrane18. In contrast, during extracellular synthesis, fungal mycelium in an aqueous solution under certain conditions of pH, temperature and agitation synthesize nanoparticles in contact with metal ions19,20,21. Ascomycetes, fungi including Alternaria, Aspergillus, Fusarium, and Trichoderma species have been reported, because of their ability in the extracellular biosynthesis of nanoparticles22,23,24,25. Particularly, Trichoderma is a rhizospheric fungus frequently used in agriculture as bioinoculants and biocontrol agents due to an active metabolism and secretion of hydrolytic enzymes as proteases, chitinases, glucanases26, as well as, secondary metabolites, plant growth regulators and antibiotics27. Mycosynthesis of NPs using Trichoderma derivatives has gained significant attention as it is considered a green method that is simple, fast, non-toxic, and environmentally friendly, This approach allows for the control of the size and shape of extracellularly synthesized NPs. Additionally, it has been found, that the large amounts of proteins and biologically active secondary metabolites naturally secreted by these species contribute to the NPs production, providing clear synergistic support for their functionality28. Some Trichoderma species have been useful for metallic biosynthesis of nanoparticles, as example, zinc oxide was obtained from T. longibranchiatum, copper oxide from T. virens and T. asperellum, and silver from T. citrinoviride and T. harzianum29. Moreover, multiple oxide nanoparticles have also been obtained from a single strain of T. harzianum30.

Meanwhile, numerous studies describe the secretome of T. harzianum related to plant growth promotion, stress tolerance, and interactions with roots31,32. However, the composition of the fungal secretome involved in the nanoparticle biosynthesis process has been scarcely elucidated. Moreover, little information about biomolecules involved as capping of NPs, has been reported. In this context, the aim of this work was to study the process of biosynthesis of nanoparticles through a proteomic approach analyzing the free cell supernatant of culture medium and aqueous filtrate derived after cultivation of T. harzianum. As validation of extracellular protein function as reducer agent, metallic ZnO and FeO NPs were successfully synthesized from zinc and ferrous sulphate salts and characterized according to their structure, size distribution, and morphology through TEM, EDS, DLS, TGA and XRD. Additionally, the presence of other organic biomolecules linked to the NPs was analyzed through FTIR. This is the first study identifying proteins involved in the biosynthesis process of metallic nanoparticles from T. harzianum. Our results demonstrated the successful formation of two types of metallic NPs whose morphology differs from those reported in the literature.

Results and discussion

Electrophoresis of extracellular protein profile from T. harzianum

SDS‒PAGE of total proteins secreted by the fungus on culture media (CM) and aqueous filtrate (AF) after electrophoretic separation, revealed a wide profile of polypeptides in supernatants from AF. As shown in Fig. 1 in CM a single conspicuous band of approximately 70 kDa was evidenced, meanwhile in AF three main bands of higher intensity with approximately 10, 25 and 70 kDa, were observed. As expected in control culture media bands were absent.

Fig. 1
figure 1

Representative steps illustrating the process of nanoparticle biosynthesis from T. harzianum and SDS-PAGE electrophoresis protein profile of secreted proteins after fungal growth in liquid culture media (CM) or in aqueous filtrate (AF). Red arrows indicate main bands. M: molecular weight marker Precision Plus Protein™ Dual Xtra Prestained Protein Standards #1,610,377.

Full size image

Secretome of T. harzianum grown on culture media and aqueous filtrate solution

According to the criteria used for the analysis of the proteomic profile of each sample, approximately 99 proteins (at least two peptides per protein) were determined in the CM, and 304 proteins in the AF. A comparison of both secretomes revealed that the number of proteins shared between the two samples accounted for only 9% of the total proteins. Therefore, among the total proteins identified in the secretome of the fungus cultivated in CM, 71 proteins (72%) were exclusive to this secretome, and 28 (28%) was shared with the secretome of the fungus maintained in AF. Consequently, the remaining 276 proteins were exclusively secreted in the AF, representing 91% of the secretome.

Functional classification of proteins secreted by T. harzianum

The classification of the proteins secreted by the fungus in the CM revealed that 68.6% are involved in biological processes (BP) and that 83.9% are involved in metabolic functions (MF). The functional classification of the Gene Ontology (GO) terms for BP indicated that proteins more abundant were related to primary metabolism (44), cellular metabolism (29), organonitrogen compound metabolic processes (28), small molecule metabolic processes (25) and biosynthetic processes (23), among others (Fig. 2). Additionally, the proteins were related to various MFs: hydrolase activity (36), small molecule binding (25), protein binding (22), and organic cyclic compound binding (21). The analysis revealed that gene clustering was notable for its involvement in fundamental metabolic functions, essential for microorganism growth and development. These clusters include genes such as, carbohydrate esterase and glycoside hydrolase, which have hydrolase activity, class II peroxidase and carbohydrate-binding module family proteins that are related to catabolic processes, and glycoside hydrolase and carbohydrate-binding proteins, which are associated with organonitrogen compound metabolic processes.

Among the proteins present in the aqueous filtrate, 71.9% were assigned to BP, and 86.8% to MF. According to the analysis, the BPs included primary metabolic (131) and cellular (71) processes, as well as organonitrogen compound metabolic processes (73) and catabolic processes (62). Notably, among the MFs of the identified proteins, those related to hydrolase (113), transferase (27), catalytic activity acting on a protein (35) and stress response (8), were identified (Fig. 2). This analysis revealed that, in addition to having clusters of proteins related to developmental processes and primary metabolism, one cluster stands out for grouping genes associated with stress response and stimuli, such as peroxidase and catalase-peroxidase. It also includes proteins with catalytic activity that acts on other proteins, such as thioredoxin, extracellular metalloproteinase, and glutathione hydrolase (Table 1). These proteins were highlighted for their activities and potential roles in NP biosynthesis. Few researches link the activity of redox enzymes in the synthesis of NPs, as they can reduce metallic ions to their reduced metallic form, resulting in the production of NPs33. In this sense, NADPH and NADH reductases are key enzymes for NP synthesis34,35. In this work, an oxidoreductase with NADPH dehydrogenase activity and NADP binding ability was identified. Additionally, other redox enzymes, including thioredoxin, aldehyde reductase, aldo/keto reductase, GMC oxidoreductase, oxidoreductase, fumarate reductase, ketoreductase, class II peroxidase, catalase-peroxidase, and catalase, have been identified. Recently, Xue et al. (2024)36 related thioredoxin to the biogenic synthesis of SeNPs through the thioredoxin/thioredoxin reductase pathway by exposing Aureobasidium melanogenum to different doses of sodium selenite. Other enzymes, such as oxidoreductases, are also involved in NP synthesis. In this way, a purified sulfite oxidoreductase purified to homogeneity from Fusarium oxysporum was efficiently used in a cell-free system for producing gold nanoparticles37. In addition, when proteases produced from the biomass of Actinobacter spp were incubated with an aqueous solution of chloroauric acid in the presence of bovine serum albumin (BSA), anisotropic AuNPs of various dimensions under different experimental conditions were obtained38.

Fig. 2
figure 2

Bioinformatics analysis of the proteins present in the CM and AF of T. harzianum conducted within the GO category using the DAVID tool and level 2 specificity for each category. The percentages of the involved genes by function are presented relative to the total number of genes within each category.

Full size image
Table 1 List of proteins potentially related to the biogenic synthesis of NPs by T. harzianum. Access numbers from UniProt, descriptions, and functional annotations for proteins determined using the ‘Functional annotation clustering’ tool in DAVID. The categories defined by GO are biological process (BP) and molecular function (MF).
Full size table

Biosynthesized nanoparticles and characterization

From the aqueous filtrate of T. harzianum, ZnO and FeONPs were successfully synthesized. The size and morphology of the ZnONPs determined via TEM images revealed an average size of 660 ± 109 nm, with a bouquet-like morphology and clustering in pairs (Fig. 3a). This unusual morphology of ZnONPs was also reported by Huang et al. (2009)39 when a peptide chain derived from hydrolyzed spider silks was used in NPs biosynthesis.

The FeONPs were 18 ± 7 nm in size and had variable morphologies, including cubic, spherical, and elongated shapes all of them grouped (Fig. 3b). Additionally, they exhibited magnetic properties. Although, this is the first work in which this particularly ferrous nanoparticle morphology is reported using Trichoderma, similar nanoparticles were biosynthesized using plant extracts40 or Aspergillus flavus41.

The atomic composition of the ZnONPs determined through EDS analysis revealed high percentages of Zn (> 39%) and O (> 37%) in the analyzed samples (Fig. 3c), whereas the EDS of FeO NPs showed high percentages of Fe (> 67%) and O (> 13%) (Fig. 3d). In both cases, Cu and other metals were detected as interference in small proportions since the grid material used for sample mounting is composed of copper.

DLS analysis indicated that the average hydrodynamic radius of the ZnONPs was 373.1 nm, with PdI = 0.06 and an average Z diameter of 385.7 ± 59.95 nm. The distribution profile was monomodal, indicating low size variability and good physicochemical stability (Fig. 4a). FeONPs had an average hydrodynamic radius of 454.6 nm, with a PdI of 0.54 and an average Z diameter of 493.7 ± 128.1 nm. In this case, a bimodal curve is observed, which may be due to the presence of different particle sizes, sedimentation or agglomeration of the NPs (Fig. 4b).

Thermogravimetric analysis of the ZnONPs from 25 ºC to 900 °C under a nitrogen atmosphere revealed a weight loss of 2 to 5%, which can be related to the evaporation of adsorbed water, starting at 50 °C and reaching approximately 10% weight loss at 400 °C. Above 700 °C, the composition change was complete, and a constant weight loss was reached at temperatures up to 900 °C (Fig. 4c). Concerning the thermal degradation of FeONPs, a weight loss range was demonstrated starting at 50 °C and reaching approximately 5% weight loss at 400 °C, which can be related to the evaporation of adsorbed water. The composition changes were completed at 600 °C, when a steady weight loss was reached up to 900 °C (Fig. 4d).

XRD analysis confirmed the crystalline nature of the NPs, with peaks corresponding to zincite and magnetite in the case of ZnONPs and FeONPs, respectively. As shown in Fig. 4e, for the ZnONPs, strong absorption peaks coincident at angles of 31.6°, 34.3°, 36.2°, 47.3° and 56.4° were observed, consistent with other studies of biogenic ZnONPs obtained from the leaf extract of Sesbania grandiflora42. In the case of FeONPs, the absorbent peaks coincide at angles of 18.44°, 33.20°, 35.64°, 43.1°, 49.44° and 57.1° (Fig. 4f), a pattern that matches with non biogenic nanoparticles reported by Novoselova (2021)43.

FTIR analysis of the biosynthesized NPs identified functional groups present that might behave as capping agents. In the case of the ZnO NPs (Fig. 4g), a peak at approximately 460–500 nm corresponds to the Zn‒O stretching vibration41, whereas in the spectrum of the FeO NPs (Fig. 4h), the peak at 420–550 nm is attributed to the Fe‒O bond vibration44. In the spectra of both types of NPs, a peak is observed at approximately 3400 nm, corresponding to the OH stretching vibration, due to the adsorption of water on the NP surface45. Additionally, there are several peaks in the 2000–2500 nm range, corresponding to the residual background signal in the spectra of aqueous samples, and a signal at approximately 1000 nm corresponding to simple bonds of organic matter such as carbohydrates. Additionally, weaker peaks can be observed at approximately 1520–1700 nm, associated with amide groups, which could suggest the involvement of extracellular proteins in the synthesis of nanoparticles46.

In conclusion, FTIR indicates that NPs produced by T. harzianum carry a layer of proteins and organic compounds from the fungus. This occurs because during biosynthesis, the production and stabilization of the NPs occur simultaneously. Besides, being part of the synthesis, the reducing agent can also contribute to their stabilization, avoiding the addition of other substances used in chemical or physical synthesis. The biomolecules present in the aqueous filtrate adsorb onto the surface of the NPs, forming an outer layer that promotes their stabilization and, in some cases, enhances their biological action and biocompatibility47.

Fig. 3
figure 3

Characterization of ZnONPs (a, c) and FeONPs (b, d) biogenically synthesized with T. harzianum strain LPSc 1503. (a, b) Transmission electron microscopy (TEM) (c, d) Coupled to energy dispersive X-ray spectroscopy (EDS) analysis.

Full size image
Fig. 4
figure 4

Characterization of ZnONPs and FeONPs biogenically synthesized with T. harzianum strain LPSc 1503. (a, b) Dynamic light scattering (DLS, n = 3), (c, d) Thermogravimetric analysis (TGA), (e, f) X-ray diffraction (XDR) analysis, and (g, h) Fourier transform infrared spectroscopy (FTIR) analysis.

Full size image

Conclusions

In this study, we characterized the secretome from a strain of T. harzianum grown on the culture media and also of the aqueous filtrate used as a solution for the biosynthesis of metal nanoparticles. Although, few proteins related to primary metabolism were identified in the culture media, a large number of proteins associated with stress response and cellular communication were identified in the aqueous filtrate, such as oxidoreductases and peroxidases. In this sense, only a few works have reported a plethora of enzymes as agents that intervene in the reduction of metal salts to obtain NPs. As validation of protein functionality two types of nanoparticles ZnO and FeO, were successfully synthesized and identified, as zincite and magnetite respectively, which have not been previously reported for T. harzianum. Further, FTIR studies demonstrated some functional groups such as carbohydrates, amides, and proteins attached to NPs that probably act as capping agents. This work contributes to understanding the key role of low molecular mass proteins secreted by the fungus intervening in the process of biogenesis of metallic NPs, opening new possibilities and strategies for their application in biotechnology, given that this fungus and its biosynthetic process is economical, simple and harmless to humans and the environment.

Materials and methods

Fungal strain and growth conditions

Trichoderma harzianum strain LPSc 1503 from INBIOTEC (Mar del Plata, Argentina) was deposited in the culture collection of the Institute of Botany “Carlos Spegazzini” (La Plata, Argentina). To obtain fungal biomass for nanoparticle biosynthesis, two agar discs of 5 mm of mycelia grown on potato dextrose agar (PDA) at 24 °C after 7 days were transferred to Erlenmeyer flasks containing 50 ml of culture broth, according to Consolo et al. (2020)29. The cultures were incubated on an orbital shaker at 150 rpm and 24 °C for 5 days. After growth, approximately 1 g of mycelia was harvested and washed with sterile double distilled water to remove the components of the biomass and culture media, and immediately transferred to flasks containing 50 ml of sterile double distilled water for 72 h, on an orbital shaker at 150 rpm and 24 °C. After incubation, the fungal biomass was filtered and discarded, and the aqueous filtrate was stored. All the experiments were carried out in duplicates.

SDS‒PAGE analysis

After the incubation period, supernatants of culture media (CM) in which T. harzianum was grown for 5 days, and the aqueous filtrate (AF) in which the fungal mycelia was submerged for 72 h, were analyzed. Quantitative determination of soluble proteins was carried out through Lowry method48. Then, 30 µg of the protein sample was run in 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel (SDS‒PAGE)49. Electrophoresis was carried out in a vertical-cube Bio-Rad Mini Protean System Cell at 110 V running for 120 min in 1.5 M Tris-glycine buffer (pH 8.3). To observe the band profile, the gels were incubated in a solution of 0.25% (w/v) Coomassie brilliant blue R 250 under stirring, washed with an aqueous solution of 45% (v/v) ethanol and 10% (v/v) acetic acid 15% (v/v) for 30 min, and then submerged in distilled water. For proteomic analysis, samples were prepared as described above and briefly electrophoresed (1-cm-long into the resolving gel). Then, gels were stained with colloidal Coomassie Blue G-250, and the entire bands were excised from the gel for their analysis by mass spectrometry and protein identification at Unidad de Espectrometría de Masa at Instituto de Biologia Molecular y Celular (UEM-IBR), Rosario, Argentina (https://ibr-conicet.gov.ar/areas_de_personal/espectrometria-de-masa/).

Secretomic analysis by LC MS/MS

Peptide separations were carried out on a nano-high-performance liquid chromatography (nano HPLC) Ultimate3000 (Thermo Scientific) using an EASY-Spray ES903 nanocolumn (50 cm × 50 μm ID, PepMap RSLC C18). The mobile phase flow rate was 300 nL/min, and 0.1% formic acid in water (solvent A), 0.1% formic acid and 100% acetonitrile (solvent B) were used. The gradient profile was set as follows: 4–30% solvent B for 114 min, 30–80% solvent B for 14 min and 80% solvent B for 2 min. Two microliters of each sample were injected. MS analysis was performed via a Q Exactive HF mass spectrometer (Thermo Scientific). For ionization, 1,9 kV liquid junction voltage and 250 °C capillary temperature were used. The full-scan method employed, m/z 375–1600 mass selection, an Orbitrap resolution of 120,000 (at m/z 200), a target automatic gain control (AGC) value of 3e6, and a maximum injection time of 100 ms. After the survey scan, the 20 most intense precursor ions were selected for MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 27 eV, and MS/MS scans with a dynamic first mass were acquired. The AGC target was 5e5, the resolution was 30,000 (at m/z 200), the intensity threshold was 4.0e4, the isolation window was 1.4 m/z units, and the maximum IT was 200 ms. Charge state screening was enabled to reject unassigned, singly charged, and equal or more than seven protonated ions. A dynamic exclusion time of 15 s was used to discriminate against previously selected ions. Mass spectra data were analyzed with Mascot (Matrix Science) via standardized workflows. The mass spectra files were searched against the database of T. harzianum (Hypocrea lixii) from UniProt (UP000034112). The precursor and fragment mass tolerances were set to 10 ppm and 0.02 Da, respectively, allowing 1 missed cleavage, carbamidomethylation of cysteines as a fixed modification, and acetylation of the N-terminus and oxidation of methionine as variable modifications. The identified peptides were filtered via the Percolator algorithm with a q-value threshold of 0.01.

Gene ontology analysis of secreted proteins

The identified proteins were analyzed via the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). For functional term analysis GO terms were conducted with the DAVID Gene Functional Classification Tool38, and classified into BP and MF domains and used the “2” category with the default T. harzianum background. The ‘functional annotation clustering’ tool was subsequently employed. In this way, the proteins were grouped into terms with similar biological significance, so that they share similar genes. In the functional annotation clustering analysis, the lowest classification stringency, to obtain broader groups with a greater number of clusters was used.

Validation of secretomic activity on biosynthesis of nanoparticles

The synthesis of zinc and ferrous NPs was carried out in 100 ml flasks containing, 50 ml aqueous filtrate adding ZnSO4·7H2O or FeSO4·7H2O salts at a final concentration of 100 mM adjusting pH to 11 with NaOH. The reaction was carried out in dark conditions at 40 °C, stirring vigorously and 250 rpm. Simultaneously, a positive control composed of the culture aqueous filtrate without metallic salt, as well as, its respective negative control, 100 mM of metal salt in deionized water, were tested. The formation of ferrous nanoparticles was observed by their color change from light to dark brown and zinc NPs as a precipitation of a white fine powder. After that, NPs were separated by centrifugation (5,000 rpm for 20 min), washed twice with double distilled water, and then lyophilized. The NPs were stored in polypropylene tubes under dark conditions at room temperature. Both metallic NPs were further validated using different physicochemical techniques.

Physicochemical characterization of metallic nanoparticles

Transmission electron microscopy - energy dispersive X-ray spectroscopy (TEM-EDS)

The biosynthesized NPs were dispersed with deionized water under ultrasound, deposited on a copper grid with a Lacey Carbon Film coating and observed under a transmission electron microscope equipped with an Oxford X-MAX 65 T EDS (JEOL JEM-2100 Plus, Tokyo, Japan) at Servicio Centralizado de Grandes Instrumentos (SECEGRIN), Santa Fe, Argentina. Observations were made in the HRTEM and HAADF modes, and images and X-ray spectra were obtained at an accelerating voltage of 200 kV.

Dynamic light scattering (DLS)

Zinc and ferrous NPs size and distribution were measured in a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire WR14 1XZ, United Kingdom). All measurements were carried out in triplicate with a temperature equilibration time of 1 min at 25 °C with an angle of 90 °C. The data processing mode was set to a high multimodal resolution.

Thermogravimetric assay (TGA)

To determine the mass of the NPs exposed to different temperature conditions, TGA was carried out in a Q500 V20 instrument 13 Build 39 (TA Instruments, New Castle, Delaware, United States). Approximately 10 mg of each sample were processed, placed under a N2 atmosphere to avoid thermo-oxidation, and heated at a rate of 5 °C from room temperature to 900 °C.

Fourier transform infrared spectroscopy (FTIR)

The presence of surface capping biomolecules bound to the NPs was determined with a Nicolet 6700 instrument with an ATR Smart Orbit accessory (Thermo Scientific, Waltham, Massachusetts, United States) at INTEMA (CONICET-UNMdP). The diffuse reflectance method (DRIFT) was used to obtain FTIR spectra. Sixty-four scans were performed in the wavenumber range between 4000 and 600 cm− 1.

X-ray diffraction (XRD)

The samples were investigated via powder X-ray diffraction (XRD) patterns. The XRD data of the solids were recorded via an X’Pert Pro PANalytical X-ray diffractometer (Malvern Panalytical, United Kingdom) at INTEMA (CONICET-UNMdP) in the 2 theta (2θ) range of 10–80° with a time per step of 1 s and a step size of 0.02 degrees. The voltage was set at 40 kV, and the current was set at 30 mA via a filtered Cu Kα source (λ = 0,1542 nm).