Novel static magnetic field effects on green chemistry biosynthesis of silver nanoparticles in Saccharomyces cerevisiae

The bacteriocidal properties of silver nanoparticles (AgNPs) depend on their average diameter (toxicity increases with decreasing diameter). In the present work, we describe novel green chemistry biosynthesis of AgNPs from AgNO3 added to cell-free culture medium of baker’s yeast, Saccharomyces cerevisiae, yielding nanoparticles in the range 11–25 nm. However, when yeast was grown in a moderate static magnetic field, AgNPs obtained from the resulting cell-free culture medium, were significantly smaller (2–12 nm) than those obtained without magnetic field. These latter nanoparticles were highly crystalline, stable and near-uniform shape. Furthermore, the antibacterial activity of AgNPs obtained from static magnetic fields were greater than those from control cultures. Static magnetic fields show a promising ability to generate biocidal nanoparticles via this novel green chemistry approach.

www.nature.com/scientificreports/ and interact with charged species such as ions and proteins 13,14 . The interactions between biology and magnetic fields remain somewhat mysterious. However, studies have shown profound effects of SMF on mice including oxidative stress and weight loss 15 . Investigation of the effect of SMF on cultured mammalian and microbial cells also indicated an increase in oxidative stress 16 . A recent study demonstrated that baker's yeast, Saccharomyces cerevisiae, experiences oxidative stress and profound reduction in growth rate in the presence of weak SMF 17 .
Here, we explore a novel green chemistry approach to biosynthesis of AgNPs using cell-free supernatants of S. cerevisiae cultures grown in the presence and absence of SMF. When silver nitrate was added to cell-free culture supernatants, biosynthesis of AgNPs resulted. Interestingly, supernatants of cultures grown in the presence of SMF produced notably smaller AgNPs which were more bacteriocidal. To our knowledge, this is the first report on the effects of SMF on the biosynthesis of metal nanoparticles.

Results and discussion
In the present study, S. cerevisiae culture was used for the synthesis of AgNPs. The color of S. cerevisiae control supernatant changed from light yellow to brown with the addition of 1 mM aqueous solution of silver nitrate after 24 h of incubation at 30 °C (Fig. 1a inset). When the S. cerevisiae culture that had been exposed to SMF was treated with 1 mM AgNO 3 , the color of the reaction medium gradually intensified with increased incubation period time as shown in Fig. 1b, it changed from yellow to reddish brown (12 h at 30 °C) and then dark brown (24 h at 30 °C). These color changes were consistent with synthesis of nanoparticles. Previously, it has been reported that the reduction of Ag + into AgNPs can be seen very clearly when the color of the solution changes from yellow to brown due to the excitation of surface plasmon resonance (SPR) 18,19 .
To follow the process of Ag reduction, mixtures were scanned in a UV-Vis spectrophotometer at 6 h, 12 h, and 24 h (Fig. 1). Absorbance maxima at 420 and 450 nm for AgNPs SMF and AgNPs control , respectively, were observed and intensity increased with incubation time. This intensity increase was consistent with reduction of silver ions and an increase in the concentration of AgNPs 20 . It seems that nucleation was initiated more rapidly and the formation of AgNPs occurred more quickly by using a yeast filtrate from cultures exposed to SMF as opposed to the filtrates from control cultures (no SMF). The absorption band at around 265 nm, has previously been attributed to aromatic residues in proteins 21 . It is believed that metabolic products of the yeast are mainly responsible for the reduction of Ag + ions.
Media from yeast cultures grown in the presence and absence of SMF produced small AgNPs on the addition of AgNO 3 while blank medium (no yeast cells) did not show any nanoparticle synthesis. When analyzed by TEM, AgNPs were in the diameter ranges 11-25 nm and 2-12 nm for media from cultures grown in the absence and presence of SMF, respectively (Fig. 2a, b, b' , b''). The nanoparticles were mostly spherical in both cases but those from SMF-exposed cultures seemed to be more uniform (Fig. 2b' ,b''). All nanoparticles were well-separated and no agglomeration was noticed. Histograms of average diameters showed quite narrow size distributions of the synthesized AgNPs without and under magnetic field (Fig. 2c,d). The nanoparticles were highly crystalline as shown by selected-area electron diffraction (SAED) patterns (Fig. 2e,f).
Niknejad et al. 22 reported that biosynthesis of AgNPs by S. cerevisiae resulted in mainly spherical and polydispersed nanoparticles with diameters in the range 5-20 nm. Korbekandi et al. 23 made AgNPs from dried culture of S. cerevisiae with a spherical shape and diameter range of 2-20 nm. In our study, the diameters of the obtained AgNPs were smaller than in these reported and also than those synthesized from Candida utilis (20-80 nm) 24 . To the best of our knowledge, there are no previous reports on using cell-free cultures or SMF to modify biosynthesis of nanoparticles in yeast cultures.
Moreover, the zeta potential is an important indicator of the surface charge of synthesized nanoparticles in colloidal dispersion. In Fig. 2g,h, the surface charge of AgNPs CONTROL and AgNPs SMF were around -13.2 mV and -10 mV, respectively. These results indicate the stability of the silver nanoparticles, which are highly polydispersed due to their negative charge 25 .  www.nature.com/scientificreports/ Concentric rings with intermittent bright dots were evident in SAED, indicating a high degree of crystallinity (Fig. 2e,f). The lattice constant calculated from this pattern was a = 4.087 Å, which agrees well with the reported value of 4.086 Å 18 . A fringe spacing of 0.23 nm was measured between lattice planes and is in accordance with the (1 1 1) lattice spacing of face-centered cubic (fcc) silver (d1 1 1 = 0.2359 nm). Average particle diameters of AgNPs were calculated from X-ray diffraction using the Debye-Scherrer equation 26 as 18 nm (control) and 6 nm (SMF), respectively (Fig. 2a,b). These values agree well with those from TEM (Fig. 2c,d).
Crystallinity of the biosynthesized nanoparticles was evaluated using X-ray diffraction (XRD). Patterns presented in Fig. 3 show several Bragg reflections with various 2θ values depending on the AgNPs batches. Nevertheless, both AgNPs formulations exhibited three diffraction peaks (marked with asterisks in Fig. 3a  www.nature.com/scientificreports/ face-centered cubic lattice structure of metallic silver (JCPDS-ICDD files No 04-0783). This confirms the successful synthesis of AgNPs and establishes their crystalline nature 27,28 . Similar diffraction patterns were observed with AgNPs biosynthesized using other yeast and plant extracts [29][30][31][32] . These data confirmed the purity of the synthesized nanoparticles. Elemental composition on the surface of the biosynthesized nanoparticles was determined using EDX spectroscopy. In general, the optical absorption peak of silver appears at around 3 keV due to SPR 33,34 . As shown in Fig. 3c,d, EDX analysis revealed the presence of silver in the nanoparticles formed from SMF-treated cultures and those of controls. Silver peaks were the most prominent for all the samples, but no nitrogen peaks were evident in the EDX patterns of the AgNPs formulations. This indicates that no ions from AgNO 3 were present in AgNPs, consistent with complete reduction of silver ions as indicated by UV-Vis spectroscopy and visual observation. In addition to silver peaks, EDX revealed the presence of carbon and copper atoms on the surface of the biosynthesized AgNPs that may be due to the TEM grid composition. We also noticed that these silver nanoparticles were accompanied by different trace impurities magnesium (Mg) and silicon (Si). It is known that organisms such as yeasts, fungi, and plant metabolites have previously been reported to act not only as reducing agents but also as capping agents for AgNPs 35,36 . Herein, it could be hypothesized that the extracellular synthesis of AgNPs occurs due to the adsorption of silver ions on the surface of metabolic products (enzymes, polysaccharides, compound derived magnesium or silicon, etc.) present in the culture supernatant of S. cerevisiae, which reduce the Ag + to AgNPs. While, the effects of SMF on the synthesis of nanoparticles have not yet been elucidated, it is believed that SMF creates wave cavitation through the liquid that may enhance the decomposition of biomolecules and liberate free radicals, which then act as reducing agents.
Antibiotic resistance of human pathogens is an important public health concern. Nanoparticles may provide a viable alternative to conventional antibiotics 37 . The biosynthesized AgNPs, in our study, showed effective inhibition of bacterial growth (Table 1 and Fig. 4). Their respective minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 25 µg/mL and 70 µg/mL for AgNPs SMF ; 40 µg/mL and 90 µg/mL for AgNPs CONTROL for E. coli. While, S. aureus showed the respective value of MIC and MBC: 15 µg/ mL and 50 µg/mL for AgNPs SMF ; 20 µg/mL and 70 µg/mL for AgNPs CONTROL These results suggest that the AgNPs prepared from cultures grown in SMF displayed good to excellent inhibitory effects as antibacterial agents, due to their smaller size. Generally, the difference in the peptidoglycan layer of the bacterial cell between Gram ( +) and Gram (-) bacteria plays a crucial role in the inhibitory activity [38][39][40] . In addition, the negative surface charge and low size of AgNPs are found to be effective factors enhancing antibacterial activity 41 .
Smaller AgNPs show the highest antibacterial effect and are more cytotoxic when compared to larger nanoparticle diameters. This may be due to easier absorption and/or a larger functional surface area 42,43 . It has been reported that the concentration and small size of AgNPs play important roles in antimicrobial activity by easier www.nature.com/scientificreports/ diffusion, penetration into bacterial cell membranes and growth inhibition 44,45 . In addition, the AgNPs induce the generation of reactive oxygen species inside bacterial cells that damage the membrane leading to cell death 46 . Antimicrobial agents that are effective against free-living bacterial cells, are sometimes ineffective against the same species growing in a biofilm state. AgNPs have previously been shown to be potentially important candidates against biofilms 47,48 . AgNPs prepared in this study showed good ability to inhibit biofilm formation. Tested microorganisms were grown in microliter plate wells with/without AgNPs to form a biofilm for 24 h. The treatment of cell-free filtrate (positive control) showed no significant decrease in biofilm formation (Fig. 5). An Table 1. Antibacterial activity of biosynthesized silver nanoparticles (AgNPs) at different concentrations (125, 250, and 500 µg/mL) against E. coli and S. aureus. Data represent mean ± SD (n = 3).  www.nature.com/scientificreports/ effective antibiofilm activity of both AgNPs against Gram (-) and Gram ( +) bacteria was observed, which may be due to the easy diffusion of AgNPs inside cells damaging the biofilm metabolic formation 49 . It is evident that the Ag-NPs SMF exhibited greater antibiofilm activity than the Ag-NPs control . This difference in inhibitory activity of both AgNPs can be explained by several factors, including efficacy in antimicrobial activity, physical and chemical properties such as affinity between the AgNPs and biofilms 50 . Goswami et al. 51 previously investigated AgNPs-mediated biofilm eradication and they found an inhibition of 89% for S. aureus and 75% for E. coli at 15 mg/mL. Similarly, Barapatre et al. 52 reported a significant decrease of biofilm formation at (64 µg/mL) of 70% and 30% for Gram (-) bacteria and Gram ( +) bacteria, respectively. In addition, Rolim et al. 53 demonstrated a potential antibiofilm activity of biologically synthesized AgNPs against S. aureus and E. coli, which agree with our findings. This supports application of AgNPs from yeast culture supernatants as biofilm-disrupting agents.
The present study demonstrates the efficiency of yeast (S. cerevisiae) cultures for green chemistry biosynthesis of bacteriocidal nanoparticles. Employing SMF promoted rapid formation of AgNPs with smaller particle diameter which were more highly crystalline than those from control cultures. Biosynthesized AgNPs formed under both conditions displayed pronounced antimicrobial and antibiofilm activities with two model pathogenic microorganisms. We hypothesize that yeast cells stressed by SMF secrete reductases and/or reducing agents in response to oxidative stress 17 . These likely promote more efficient reduction of silver forming AgNPs. SMF provides a promising and completely novel route to green chemistry biosynthesis of commercially important nanoparticles.

Materials and methods
Growth and treatment of yeast cells. S. cerevisiae (strain BY4741. Genotype: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 obtained from the Euroscarf collection; http:// www. euros carf. de) were cultured in YPD broth (1% bacto-yeast extract, 2% bacto-peptone, 2% glucose). The cultures were exposed to a parallel and globally homogeneous magnetic induction of 250 mT and SMF was generated by a pair of cylindrical coils (diameter 20 cm, length 13 cm; Beaudouin, Paris, France). The terrestrial magnetic field at the experimental location was 0.075 mT. Control cultures were prepared identically but with no SMF. Cell-free supernatants were obtained from each culture by centrifugation (10,000g) and 1 mM AgNO 3 was added to this supernatant. The supernatants were maintained in the dark at room temperature for 24 h. AgNPs were collected by sequential centrifugation.
Characterization of nanoparticles. UV-visible absorbance spectral analysis. The biosynthesized Ag-NPs were preliminarily detected by visual observation of the color change of cell filtrate after treatment with www.nature.com/scientificreports/ silver nitrate (1 mM). Characterization of the synthesized Ag-NPs was further carried out using scanning absorbance spectra (200-800 nm) of the mixture using a UV-Visible spectrophotometer (Biowave II (England)) with a resolution of 1 nm.
Transmission electron microscopy (TEM). The transmission electron microscopy (TEM, JEOL JEM-1010) operated at 200 kV accelerating voltage was performed on the biosynthesized AgNPs. 200 kV accelerating voltage was used to investigate the process of formation of AgNPs, and to study their sizes and shapes. Samples for TEM were prepared by drop-coating the AgNPs suspensions onto carbon-coated copper grids. Micrographs were obtained using an EM 10C ZEISS ® (Germany) TEz.
Energy dispersive X-ray (EDX). EDX analysis was also performed by energy-dispersive spectroscopy (EDS) using INCA Energy TEM 200 with analysis software (JEOL) for identifying the elemental composition of the biosynthesised AgNPs.
X-ray diffraction analysis (XRD). Films of nanoparticle sizes and their crystalline structure were examined using an X-ray diffractometer (Bruker D8 ADVANCE). The operating voltage of 40 kV and current of 30 mA were used with Cu kα radiation of 0.1541 nm wavelength, in the 2θ range 10°-80° angle.
Zeta potential. The zeta potential measurement was used to determine the surface potential of the synthesized AgNPs in water was analyzed by Malvern Nano-Zetasizer ZS (Malvern, UK).
Antimicrobial properties of nanoparticles. Antibacterial activity of AgNPs was evaluated against, E. coli and S. aureus using the agar-well diffusion and broth micro dilutions method 1 . Approximately 10 7 colony forming units (CFU/mL) of each strain were inoculated in Muller-Hinton agar (MHA) plates and then 50 μl of AgNPs solution with different concentrations (125, 250, and 500 µg/mL) was added to wells which were bored using a core borer of 6 mm diameter. 1 mM silver nitrate solution and ciprofloxacin antibiotic were used as controls. The plates were incubated at 37 °C for 24 h. After incubation, the maximum zone of inhibition was observed and measured for analysis against each type of test. Moreover, stock solutions of tested AgNPs were prepared by serial twofold dilution to obtain a serial concentration ranging from 10 to 100 µg/mL. An aliquot of working suspension (100µL) was added to a sterile 96-well plate containing (100 µL) of tested stock solutions. Plates were incubated for 24 h at 37 °C. According to the Lehtinen et al. 54 method, the minimum inhibitory concentration (MIC) was defined as the lowest concentration of the tested compounds that completely inhibited microbial growth and the minimum bactericidal concentration (MBC) were recorded as the lowest concentration giving no visible microorganisms. The standard error was calculated using three experimental replicates. Antibiofilm activity of AgNPs with S. aureus and E. coli was tested in order to determine ability to inhibit biofilm formation using 96-well microtiter plate method 55 . Individual wells of the sterile microliter plate were filled with 180 μL of MH broth, inoculated with 10 μL of overnight grown culture of each strain and 10 μL of AgNPs solution at various concentrations (25,50,75, and 100 µg). The microliter plates were incubated for 24 h at 37 °C. Only sterile Muller Hinton Broth was used as the blank. After incubation, content of each well was gently removed and washed with 0.2 mL of phosphate buffered saline (PBS, pH 7.2) three times, to remove free-floating 'planktonic' cells. The remaining biofilms in the wells were mixed with 2% sodium acetate (SigmaAldrich S2889) and stained with 0.1% crystal violet. Then, the excess dye was washed with deionized water and the wells were kept for drying. After drying, 200 μL of 95% (v/v) ethanol was added to the wells. After incubation, content of each well was gently removed and washed with 0.2 mL of PBS (pH 7.2) three times, to remove free-floating 'planktonic' bacteria. Biofilms formed by adherent 'sessile' organisms in plate walls were fixed with sodium acetate (2%, w/v) and stained with crystal violet dye (0.1%, w/v). Excess stain was rinsed off by thorough washing with sterilized Millipore water and plates were kept for drying. After drying, 200 μL of 95% (v/v) ethanol was added to the wells.
The absorbance was measured at 620 nm on an ELISA reader (Multiskan® EX, Thermo Scientific, Finland) and values obtained were considered as an index of bacteria adhering to the surface of well wall for developing biofilms. The percentage of biofilm inhibition was calculated using the following equation: Statistical analysis. All antibacterial and antibiofilm activities were carried out in three independent determinations and all data are presented as mean ± standard deviation. The significance of difference between mean values and comparison among groups was performed by using the one-way ANOVA test followed by Tuckey's test with SPSS software version 25. Values of p < 0.05 was considered statistically significant.

Conclusion
Media from cultures of S. cerevisiae were prepared by removing yeast cells and proved capable of synthesizing AgNPs from AgNO 3 without additional chemical agents. This use of yeast culture medium provides a novel, scalable and green chemistry route to produce biocidal AgNPs with narrow size-ranges, crystalline shape, and spherical form. However, medium from cells grown in the presence of moderate SMF biosynthesized significantly smaller AgNPs with more potent antibacterial and antibiofilm activities. AgNPs produced in this way were % Inhibition of biofilm = OD of control − OD of treatment OD of treatment × 100. www.nature.com/scientificreports/ physically characterized by a variety of methods and found to be of excellent quality. Our approach provides a novel green chemistry route to synthesis of metallic nanoparticles with important biomedical and commercial applications. We speculate that chemical reducing agents are secreted into culture medium more abundantly from cells treated with SMF leading to efficient reduction of silver to AgNPs.