Green Synthesis of Silver Nanoparticles Using a Biosurfactant from Bacillus cereus UCP 1615 as Stabilizing Agent and Its Application as an Antifungal Agent

Silver nanoparticles have great potential in a wide range of applications. Therefore, the purpose of this work was to synthesize, in a simple and green way, via the Tollens method, silver nanoparticles (AgNPs), using as a stabilizer the biosurfactant produced by Bacillus cereus UCP 1615 cultivated in a low-cost medium, with waste frying oil as a substrate. The obtained nanoparticles were identified and morphologically characterized using ultraviolet/visible (UV/vis) spectroscopy, scanning electron microscopy (SEM), and zeta potential. The maximum UV/vis absorption was observed at 400 nm for newly formed silver nanoparticles, while, for silver nanoparticles stored for 120 days, the peak was observed at 430 nm. SEM micrographs confirmed the formation of nanoparticles, with predominantly spherical structures. The average size of the formed nanoparticles was estimated to be 20 nm. The presence of the biosurfactant promoted stability, as a zeta potential of −23.4 mV was observed. The antimicrobial potential of AgNPs was evaluated at different concentrations against three pathogenic fungi (Aspergillus niger, Penicillium fellutanum, and Cladosporium cladosporioides). No less than 100% and 85% inhibitions of P. fellutanum and A. niger growth were observed, respectively, at the AgNP concentration of 16.50 μg/mL in potato dextrose agar medium. These results suggest the potential use of the biosurfactant as a stabilizer for silver nanoparticles and its application as an antimicrobial agent.


Introduction
Nanotechnology has been growing due to the large number of effective applications in various sectors, ranging from traditional chemical techniques to medicinal and environmental technologies [1]. It is well accepted that a particle is nano if its diameter is from 1 to 100 nanometers (billionths of a meter), including in this range nanoparticles (NPs) considered especially practical and efficient which are traditionally synthesized by chemical means [2]. In particular, the size of silver nanoparticles (AgNPs) can be adjusted according to the desired application, as in the case of those prepared for drug delivery, which are larger than 100 nm to accommodate the amount of drug to be delivered [3].

Microorganism
The bacterium Bacillus cereus UCP 1615 obtained from the culture bank of the Catholic University of Pernambuco (Recife, Brazil) was used as the biosurfactant-producing microorganism. This strain was previously isolated from environmental water samples contaminated with petroleum byproducts spilled from ships (port area) in the Atlantic Ocean in the state of Pernambuco, Brazil.
The biosurfactant was extracted and isolated from cell-free broth, obtained by removing the cells via centrifugation (Heraeus Megafuge 16R Centrifuge, Thermo Scientific, Osterode am Harz, Germany) of the fermented broth at 5000× g for 30 min. A 6.0 M HCl solution was added to the supernatant to adjust the pH to 2.0, followed by the addition of a 2:1 (v/v) CHCl 3 /CH 3 OH solution. After vigorous manual stirring for 15 min and phase separation, the organic phase was removed, and the operation was repeated twice more. The organic phases were combined, and the isolated biosurfactant was concentrated on a rotary evaporator (Q344B, Quimis, Diadema, Brazil).

Synthesis of Silver Nanoparticles
All reagents were analytical grade and were used without further purification. The technique used was adapted from Le at al. [5]. First, 1.7 g (1.0 × 10 −2 mol) of silver nitrate (AgNO 3 ) was dissolved in 100 mL of deionized water. The AgNO 3 solution was then precipitated with 0.62 g (1.55 × 10 −2 mol) of sodium hydroxide. The precipitate obtained, composed of Ag 2 O, was filtered and dissolved in 100 mL of 0.4% (w/w) aqueous ammonia (NH 3 ) (2.3 × 10 −2 mol) until a clear solution of silver-ammonium complex, (Ag(NH 3 ) 2 ) + (aqueous), was formed. Then, 2.5 g (8.9 × 10 −3 mol) of the isolated biosurfactant (BS) was added dropwise to the complex, and the resulting solution was gently stirred for 2 h at room temperature until a homogeneous mixture was obtained. Finally, 2 g (1.11 × 10 −2 mol) of glucose was added to the mixture at room temperature with gentle agitation.
The reduction of the silver complex solution (in quartz glass) was started with ultraviolet (UV) light irradiation. UV treatment was carried out for 8 h under vigorous stirring without heating. A UV lamp (λ = 365 nm, 35 W) was used as a light source to stimulate the reduction process. After 8 h of irradiation, a clear dispersion of AgNPs stabilized with biosurfactant was obtained. The synthesis of silver NPs was successfully conducted with a final silver concentration around 1%. The dispersion obtained, containing biosurfactant-complexed silver nanoparticles (BS-AgNPs), was stored at 4 • C for further experiments.

Properties and Characteristics of Silver Nanaoparticles
The absorption property of the BS-AgNP dispersion was analyzed using ultraviolet/visible (UV/vis) spectroscopy (Digital Spectrophotometer SP-22, Biospectro, Shanghai, China), at time zero and after 120 days of storage, in the wavelength range between 320 and 700 nm. On the other hand, the zeta potential and, consequently, the stability of the dispersed nanoparticles were determined using a Zeta-Meter 4.0 (Zeta-Meter Inc., Staunton, VA, USA).
Preliminary analysis of the morphology and size of the synthesized silver nanoparticles was performed using a scanning electron microscope (JSM-5600, JEOL, Tokyo, Japan) using an accelerating voltage of 30 kV. Before analysis, a small aliquot of the nanoparticle suspension was deposited on the surface of a carbon ribbon, dried at room temperature, and subsequently coated by a thin layer of platinum to make the samples conductive.

Determination of Nanoparticles' Antifungal Activity
Three species of fungi (Aspergillus niger UFPEDA 5117, Penicillium fellutanum UFPEDA 6472, and Cladosporium cladosporioides UFPEDA 6703), which are well-known plant or human pathogens, were obtained from the Collection of Cultures of Microorganisms of the Department of Antibiotics of the Federal University of Pernambuco (UFPEDA), Recife, Brazil.
The in vitro assay of antifungal activity was performed in potato dextrose agar (PDA) fungal growth medium with added dispersion of synthesized silver nanoparticles. Here, 200 µL of the dispersion was added in order to obtain final concentrations of silver nanoparticles of about 1.65, 8.25, and 16.50 µg/L per Petri dish. After preparing the plates, agar plugs from each fungus were inoculated in the center of them and incubated at 30 • C until mycelial growth on the control dish reached the edge of the Petri dish, and the size of the inhibition halo was recorded [17].
The radial growth of fungal mycelium was measured and recorded after the incubation period of the fungus in culture media with different concentrations of BS-AgNPs. The following equation was used to calculate the inhibition rate (%) [18]: where R is the radial growth of fungal mycelium in the control plate, and r is the radial growth of fungal mycelium in the plate containing BS-AgNP.

Statistical Analysis
The data were analyzed statistically using the one-way procedure in the Statistica ® version 7.0 software package (Statsoft Inc., Tulsa, OK, USA), followed by one-way linear analysis of variance (ANOVA). The results were expressed as the mean ± standard deviation (SD) determined from triplicate experiments.

Characterization of Silver Nanoparticles
The biosurfactant was successfully produced by Bacillus cereus UCP 1615 in mineral medium supplemented with residual frying oil, which makes the process more feasible, in addition to contributing to a reduction in input costs [16]. Because fermentation took place in 48 h at room temperature (28 • C), this method would be attractive for industrial use.
The synthesis of nanoparticles using the biosurfactant as a stabilizer was revealed, according to Tyagi et al. [18], by the change in the color of the solution (Figure 1), and confirmed in Figure 2 by the presence of a maximum UV/vis absorption peak characteristic of silver nanoparticles, between 400 and 450 nm, corresponding to localized surface plasmon resonance (LSPR) [18].
NPs made of coin metals have their unique optical properties arising from their specific behavior under light irradiation, i.e., the appearance of LSPR [19]. Garcia [20] highlighted that the main characteristic of LSPR in NPs is that the oscillation of excited electrons is limited by the volume of the NPs. Thus, the UV/visible spectral signature serves to assess the presence, uniformity, and size of AgNPs stabilized with the biosurfactant.
The plasmonic absorption peak, resulting from the LSPR characteristic of NPs, was observed in the newly synthesized (0 days) BS-AgNPs close to 400 nm, consistently with a previous study carried out on AgNPs stabilized with a lipopeptide from Bacillus subtilis grown in sunflower oil [6]. The diameter of the colloidal silver nanoparticles was found to be approximately 20 nm in the study with B. subtilis. Spherical silver NPs have the LSPR absorption light band normally close to 400 nm [21]; therefore, it is possible to infer that BS-AgNPs could actually have this spherical shape and a size predominantly close to 20 nm. The peak observed after 120 days was close to 440 nm, which, in turn, corroborates the observations made for AgNP stabilized with a rhamnolipid produced by Pseudomonas aeruginosa that exhibited the characteristic peak at 428 nm and particle size around 160 nm [21].  NPs made of coin metals have their unique optical properties arising from their specific behavior under light irradiation, i.e., the appearance of LSPR [19]. Garcia [20] highlighted that the main characteristic of LSPR in NPs is that the oscillation of excited electrons is limited by the volume of the NPs. Thus, the UV/visible spectral signature serves to assess the presence, uniformity, and size of AgNPs stabilized with the biosurfactant.
The plasmonic absorption peak, resulting from the LSPR characteristic of NPs, was observed in the newly synthesized (0 days) BS-AgNPs close to 400 nm, consistently with a previous study carried out on AgNPs stabilized with a lipopeptide from Bacillus subtilis grown in sunflower oil [6]. The diameter of the colloidal silver nanoparticles was found to be approximately 20 nm in the study with B. subtilis. Spherical silver NPs have the LSPR absorption light band normally close to 400 nm [21]; therefore, it is possible to infer that BS-AgNPs could actually have this spherical shape and a size predominantly close to 20 nm. The peak observed after 120 days was close to 440 nm, which, in turn, corroborates the observations made for AgNP stabilized with a rhamnolipid produced by Pseudomonas   NPs made of coin metals have their unique optical properties arising from their specific behavior under light irradiation, i.e., the appearance of LSPR [19]. Garcia [20] highlighted that the main characteristic of LSPR in NPs is that the oscillation of excited electrons is limited by the volume of the NPs. Thus, the UV/visible spectral signature serves to assess the presence, uniformity, and size of AgNPs stabilized with the biosurfactant.
The plasmonic absorption peak, resulting from the LSPR characteristic of NPs, was observed in the newly synthesized (0 days) BS-AgNPs close to 400 nm, consistently with a previous study carried out on AgNPs stabilized with a lipopeptide from Bacillus subtilis grown in sunflower oil [6]. The diameter of the colloidal silver nanoparticles was found to be approximately 20 nm in the study with B. subtilis. Spherical silver NPs have the LSPR absorption light band normally close to 400 nm [21]; therefore, it is possible to infer that BS-AgNPs could actually have this spherical shape and a size predominantly close to 20 nm. The peak observed after 120 days was close to 440 nm, which, in turn, corroborates the observations made for AgNP stabilized with a rhamnolipid produced by Pseudomonas It can be noted that the peak was broad and had a strong tail, indicating a wide distribution of nanoparticle size, requiring studies to adapt the technique, such as the synthesis time and the amount of added metallic particles, to obtain a well-defined peak and, consequently, a solution with majority presence of particles of a single size [22].
The shift of the maximum absorption peak of BS-AgNPs from 400 to 440 nm after 120 days of storage shown in Figure 2 was indicative of an increase in the size of the nanoparticle diameter. Such a particle enlargement may be an example of the Ostwald ripening phenomenon, based on which small particles in solution lose stability and reorganize into larger particles so as to achieve greater thermodynamic stability, causing a reduction in the surface-to-volume ratio [23].
Scanning electron microscopy (SEM) was used to assess the morphology of nanoparticles (Figure 3), as well as to measure their actual diameter. thesis time and the amount of added metallic particles, to obtain a well-defined peak and, consequently, a solution with majority presence of particles of a single size [22].
The shift of the maximum absorption peak of BS-AgNPs from 400 to 440 nm after 120 days of storage shown in Figure 2 was indicative of an increase in the size of the nanoparticle diameter. Such a particle enlargement may be an example of the Ostwald ripening phenomenon, based on which small particles in solution lose stability and reorganize into larger particles so as to achieve greater thermodynamic stability, causing a reduction in the surface-to-volume ratio [23].
Scanning electron microscopy (SEM) was used to assess the morphology of nanoparticles (Figure 3), as well as to measure their actual diameter. Observing the BS-AgNP solution at a 5000× magnification ( Figure 3A), points with NP agglomeration can be seen, which hindered the characterization of particle size; however, when the magnification was enlarged to 12,000×, non-aggregated and rounded AgNPs with a diameter of approximately 160 nm could be observed (indicative arrow in Figure 3B). This means that particles were not completely separated from each other, exhibited a high surface area, and formed clusters [24].
The SEM images obtained by Elakkiya et al. [21] for AgNP synthesized with a rhamnolipid biosurfactant revealed high-density structures with diameter in the range of 30-150 nm and a random distribution. The rounded shape corroborates the fact that silver NPs have the LSPR absorption light band normally close to 400 nm [19].
The colloidal stability of NPs was indirectly checked through the zeta potential. This parameter reached a value quite far from zero (−23.4 mV ± 1.4), which highlights a relatively strong electrostatic repulsion among particles, resulting in low chance of aggregation or precipitation.
In the work published by Nehal and Singh [24], a lipopeptide-rich cell-free extract, obtained by Bacillus paramycoides fermentation in a medium supplemented with olive oil, was added to a solution of AgNO3 and sodium borohydride (NaBH4). At the end of the reaction, stable nanosuspensions were generated with a zeta potential peak of −38.7 mV. Elakkiya et al. [21], using a similar methodology, but with a P. aeruginosa rhamnolipid, reported a value of −31.6 mV. Lastly, in another study, also carried out with a P. aeruginosa rhamnolipid, but where AgNPs were formed via reaction with an AgNO3 solution at pH Observing the BS-AgNP solution at a 5000× magnification ( Figure 3A), points with NP agglomeration can be seen, which hindered the characterization of particle size; however, when the magnification was enlarged to 12,000×, non-aggregated and rounded AgNPs with a diameter of approximately 160 nm could be observed (indicative arrow in Figure 3B). This means that particles were not completely separated from each other, exhibited a high surface area, and formed clusters [24].
The SEM images obtained by Elakkiya et al. [21] for AgNP synthesized with a rhamnolipid biosurfactant revealed high-density structures with diameter in the range of 30-150 nm and a random distribution. The rounded shape corroborates the fact that silver NPs have the LSPR absorption light band normally close to 400 nm [19].
The colloidal stability of NPs was indirectly checked through the zeta potential. This parameter reached a value quite far from zero (−23.4 mV ± 1.4), which highlights a relatively strong electrostatic repulsion among particles, resulting in low chance of aggregation or precipitation.
In the work published by Nehal and Singh [24], a lipopeptide-rich cell-free extract, obtained by Bacillus paramycoides fermentation in a medium supplemented with olive oil, was added to a solution of AgNO 3 and sodium borohydride (NaBH 4 ). At the end of the reaction, stable nanosuspensions were generated with a zeta potential peak of −38.7 mV. Elakkiya et al. [21], using a similar methodology, but with a P. aeruginosa rhamnolipid, reported a value of −31.6 mV. Lastly, in another study, also carried out with a P. aeruginosa rhamnolipid, but where AgNPs were formed via reaction with an AgNO 3 solution at pH 12, NP stability was greatly enhanced by an increase in zeta potential (in absolute value) from −23.8 to −56.3 mV in the presence of the biosurfactant [25].

Antifungal Analysis
Many filamentous fungi that are plant and human pathogens can cause devastating damage to agriculture, as well as potentially fatal diseases in humans [26,27]. In agriculture, they cause a reduction in crop yield, loss in germination, development of discoloration and deformation due to plant diseases, and biochemical changes in the seed. In humans, damage to health is often associated with the action of these microorganisms either directly or through the ingestion of toxic chemical compounds and mycotoxins produced by them [28].
For this reason, the fungicidal potential of silver nanoparticles against fungi of genera known for their pathogenicity to humans and plants has been evaluated in the present work. Table 1 shows the inhibition rate (%) of Aspergillus niger, Cladosporium cladosporioides, and Penicillium fellutanum growth promoted by the addition of BS-AgNPs at different concentrations to the growth medium. Table 1. Inhibition rate (%) caused by biosurfactant-complexed silver nanoparticles (BS-AgNPs) at different concentrations on Aspergillus niger, Cladosporium cladosporioides, and Penicillium fellutanum growth in potato dextrose agar (PDA) medium. Data expressed as the mean ± SD of triplicate determinations.

BS-AgNP Concentration (µg/L) Inhibition Rate (%)
A. niger C. cladosporioides P. fellutanum The inhibition of P. fellutanum growth stood out for being close to 100% at all BS-AgNP concentrations tested, with complete inhibition at 16.50 µg/L. Among the fungi selected for the study, the lowest growth inhibition rates, between 41.48% and 66.67%, were observed for C. cladosporioides, when compared to growth in a medium without BS-AgNP. The inhibition rate of A. niger growth achieved 85.78% at the highest concentration of BS-AgNP and 74.20% at the lowest one. In general, a higher concentration of nanoparticles in the growth medium led to a higher growth inhibition rate against all fungi. The inhibition halos caused by the addition of BS-AgNP at the highest concentration are illustrated in Figure 4.
Many filamentous fungi that are plant and human pathogens can cause devastating damage to agriculture, as well as potentially fatal diseases in humans [26,27]. In agriculture, they cause a reduction in crop yield, loss in germination, development of discoloration and deformation due to plant diseases, and biochemical changes in the seed. In humans, damage to health is often associated with the action of these microorganisms either directly or through the ingestion of toxic chemical compounds and mycotoxins produced by them [28].
For this reason, the fungicidal potential of silver nanoparticles against fungi of genera known for their pathogenicity to humans and plants has been evaluated in the present work. Table 1 shows the inhibition rate (%) of Aspergillus niger, Cladosporium cladosporioides, and Penicillium fellutanum growth promoted by the addition of BS-AgNPs at different concentrations to the growth medium. The inhibition of P. fellutanum growth stood out for being close to 100% at all BS-AgNP concentrations tested, with complete inhibition at 16.50 µ g/L. Among the fungi selected for the study, the lowest growth inhibition rates, between 41.48% and 66.67%, were observed for C. cladosporioides, when compared to growth in a medium without BS-AgNP. The inhibition rate of A. niger growth achieved 85.78% at the highest concentration of BS-AgNP and 74.20% at the lowest one. In general, a higher concentration of nanoparticles in the growth medium led to a higher growth inhibition rate against all fungi. The inhibition halos caused by the addition of BS-AgNP at the highest concentration are illustrated in Figure 4. A study on growth inhibition caused by silver nanoparticles synthesized by the green method against the phytopathogens Alternaria solani, Corynespora cassiicola, and Fusarium A study on growth inhibition caused by silver nanoparticles synthesized by the green method against the phytopathogens Alternaria solani, Corynespora cassiicola, and Fusarium spp. revealed inhibition rates above 90% for all fungi in PDA medium at the highest concentration of nanoparticles (25 µg/mL) [18]. AgNPs biosynthesized through the mediation of the endolytic bacterium Pseudomonas poae showed strong antifungal activity against the pathogen Fusarium graminearum, with inhibition rates above 50% at concentrations between 10 and 20 µg/mL, which was attributed, at least partially, to inhibition of spore germination, growth of the germ tube, mycotoxin production, and cell membrane damage [29].
Another study on AgNPs ecologically synthesized using luteolin tetraphosphate as a reducing and stabilizing agent showed that a smaller diameter of NPs in the range of 9-21 nm led to a higher antimicrobial activity against plant pathogens [30].
The results described in the literature corroborate the data obtained in this work, thus indicating the potential of nanoparticles as antifungal agents, with the additional advantage of having been prepared via a green method using the biosurfactant as a biocompatible stabilizing agent.

Conclusions
The present study was successful in the green synthesis, through the Tollens method, of silver nanoparticles (BS-AgNPs), using as a stabilizing agent the biosurfactant produced by Bacillus cereus UCP 1615, in a low-cost medium supplemented with waste frying oil. BS-AgNPs were shown to be stable for over 120 days, even if an increase in their size was observed over time. In this preliminary evaluation, the AgNPs appeared to be spherical and uniform, with such a small diameter being compatible with several biotechnological applications. The nanoparticles exhibited dose-dependent antifungal activity against pathogens that harm agriculture and human health. Therefore, it can be concluded that biosurfactants produced by green synthesis have potential for use as ecofriendly nanoparticle stabilizers.