Green synthesis of silver chloride nanoparticles using Rhodotorula Mucilaginosa

The biosynthesis of silver chloride nanoparticles (AgCl NPs) is presented in this paper. Silver chloride nanoparticles were synthesized using fungi culture from Rhodotorula Mucilaginosa and aqueous AgNO3 solution, as precursor. The plasmon resonance of the nanoparticles containing solution has shown through UV-visible spectrophotometry an absorbance peak at about 437 nm. Scanning Electron Microscopy, Energy Dispersive Spectroscopy, and X-ray Diffraction analyses confirmed the presence of spherical silver chloride nanoparticles with a face centered cubic crystal structure and an average particle size of 25 nm. Silver chloride nanoparticles have been shown to be able to inhibit the growth of different microorganisms, including bacteria and fungi, which would make them suitable for antimicrobial applications. Introduction The development of materials and structures at nanoscale dimensions has gained a huge interest in the nanomaterials and nano-technology research fields. One of the most important properties of metallic nanoparticles is their antimicrobial activity. Eco-friendly methods concerning nanomaterials synthesis present a substantial importance for biological applications, mainly due to nontoxic substances and environmentally friendly procedures employed [1]. Green synthesis of silver chloride nanoparticles has been reported to be mediated by different kinds of organisms, from bacteria to plant extracts. Cell-free culture supernatant of Streptomyces strain, Klebsiella planticola, biomass of Bacillus subtilis, leaf extract of Cissus quadrangularis, aqueous extract of Sargassum plagiophyllum, extract from needles of Pinus densiflora, Prunus persica L. outer peel extract are just a few examples of organisms able to synthesize AgCl NPs [2]-[8]. Weili Hu et al. have presented the synthesis of silver chloride nanoparticles under ambient conditions in nanoporous bacterial cellulose membranes as nanoreactors. It has been demonstrated that the synthesized silver chloride nanoparticles exhibited high hydrophilic ability and a strong antimicrobial activity against Staphylococcus aureus and Escherichia coli bacteria [9]. The antibacterial effect of biosynthesized AgCl NPs investigated against Escherichia coli was found to be dose-dependent [6]. The biosynthesized silver chloride nanoparticles exhibited besides the antimicrobial activity, cytotoxicity activity against HeLa and SiHa cancer cell lines [2]. Powder Metallurgy and Advanced Materials – RoPM&AM 2017 Materials Research Forum LLC Materials Research Proceedings 8 (2018) 28-34 doi: http://dx.doi.org/10.21741/9781945291999-4 29 M. Sophocleous and J. K. Atkinson have described in their review the significant development of Ag/AgCl screen printed sensors [10]. Moreover, Ag/AgCl NPs are examined for further applications of nanoparticles as a plasmonic photocatalyst [6]. The reports which have shown the importance of the AgCl NPs application, from sensors, catalysts, to antimicrobial activity, have led to the research results presented herein. Even though several methods to obtain nanoparticles are currently developed, the green methods have captured the interest of researchers due to their lack of toxicity. In the present work, the green synthesis of silver chloride nanoparticles is described. This is, to the best of our knowledge, the first report for green synthesis of AgCl NPs mediated by Rhodotorula Mucilaginosa. Materials and Methods 1. Fungi and culture conditions The fungi Rhodotorula Mucilaginosa used in this study were provided from Soroka University Medical Center from Beersheva, Israel. The fungi were cultivated in the solid media, Sabouraud agar supplied by Scharlau Chemicals, and incubated at 35 °C for 48 h. 2. Biosynthesis of AgCl nanoparticles using Rhodotorula Mucilaginosa In order to synthesize the silver chloride nanoparticles, 1μl of bacterial strains were freshly inoculated in test tubes containing 15 ml of growth medium, namely Brain Heart Infusion from Sigma Aldrich. The liquid media contained beef heart (infusion from 250 g), 5 g/L; calf brains (infusion from 200 g), 12.5 g/L; disodium hydrogen phosphate, 2.5 g/L; D(+)-glucose, 2 g/L; peptone, 10 g/L; sodium chloride, 5 g/L. The liquid culture was kept in a thermostat at 35 °C for 24 h, followed by centrifugation at 4000 rpm for 30 min. The supernatant and biomass were tested in parallel. In the first situation 5 ml of supernatant was used, while for the second one the biomass was kept with the addition of 5 ml of distilled water. The next step was similar by adding the 5-ml culture over 40 ml Ag NO3 precursor solution, at 1 mM, 2 mM and 3 mM concentration, respectively. The culture, supernatant and biomass + distilled water and precursors were kept as control. The samples were kept in a thermostat set at 35 °C, for 48 h. 3. Characterization of AgNPs Ultraviolet-visible spectral analysis was carried-out by using Jasco V-630 spectro-photometer. The UV-visible spectra were measured in the range 200-600 nm with a wavelength step size of 1.5 nm. For morphological characteristics and chemical composition, a JSM 7400f scanning electron microscope (SEM) with a platform for Energy Dispersive Spectroscopy (EDS) was used. The silver chloride nanoparticles colloid was dropped on a copper grid and coated with a platinum thin film. After this the samples were mounted on a double sided adhesive carbon-tape. The acceleration voltage was fixed to 10kV. The crystalline nature of silver chloride nanoparticles was analyzed by XRD using a Philips PW 1050/70 X-ray powder diffractometer with graphite monochromator using CuKα1 (λ =1,54Å), at a voltage of 40 kV, a current of 28 mA, in the scan range 10÷80 °, in Bragg-Brentano geometry. Results and Discussion After incubation in the thermostat for 48 h at 35 °C, the final color of the colloid, containing biomass of Rhodotorula Mucilaginosa, changed from light yellow to light brown. The change in color is an indication for the formation of nanoparticles. In Fig.1 the obvious difference in color can be observed, depending on the precursor concentration. The samples with supernatant and the control have remained unchanged. Furthermore, considering the optical indication (changing the color), the UV-visible absorption spectra of the colloidal solutions with nanoparticles was measured (shown in Fig. 2). Powder Metallurgy and Advanced Materials – RoPM&AM 2017 Materials Research Forum LLC Materials Research Proceedings 8 (2018) 28-34 doi: http://dx.doi.org/10.21741/9781945291999-4 30 Fig. 1. Image of colloidal nanoparticles synthesized in the presence of Rhodotorula Mucilaginosa. 200 300 400 500 600 0.2 0.4 0.6 0.8 1.0 1.2 Ab so rb an ce (a .u .)


Introduction
The development of materials and structures at nanoscale dimensions has gained a huge interest in the nanomaterials and nano-technology research fields.One of the most important properties of metallic nanoparticles is their antimicrobial activity.
Eco-friendly methods concerning nanomaterials synthesis present a substantial importance for biological applications, mainly due to nontoxic substances and environmentally friendly procedures employed [1].
Green synthesis of silver chloride nanoparticles has been reported to be mediated by different kinds of organisms, from bacteria to plant extracts.Cell-free culture supernatant of Streptomyces strain, Klebsiella planticola, biomass of Bacillus subtilis, leaf extract of Cissus quadrangularis, aqueous extract of Sargassum plagiophyllum, extract from needles of Pinus densiflora, Prunus persica L. outer peel extract are just a few examples of organisms able to synthesize AgCl NPs [2]- [8].
Weili Hu et al. have presented the synthesis of silver chloride nanoparticles under ambient conditions in nanoporous bacterial cellulose membranes as nanoreactors.It has been demonstrated that the synthesized silver chloride nanoparticles exhibited high hydrophilic ability and a strong antimicrobial activity against Staphylococcus aureus and Escherichia coli bacteria [9].The antibacterial effect of biosynthesized AgCl NPs investigated against Escherichia coli was found to be dose-dependent [6].The biosynthesized silver chloride nanoparticles exhibited besides the antimicrobial activity, cytotoxicity activity against HeLa and SiHa cancer cell lines [2].
M. Sophocleous and J. K. Atkinson have described in their review the significant development of Ag/AgCl screen printed sensors [10].Moreover, Ag/AgCl NPs are examined for further applications of nanoparticles as a plasmonic photocatalyst [6].
The reports which have shown the importance of the AgCl NPs application, from sensors, catalysts, to antimicrobial activity, have led to the research results presented herein.Even though several methods to obtain nanoparticles are currently developed, the green methods have captured the interest of researchers due to their lack of toxicity.In the present work, the green synthesis of silver chloride nanoparticles is described.This is, to the best of our knowledge, the first report for green synthesis of AgCl NPs mediated by Rhodotorula Mucilaginosa.

Materials and Methods 1. Fungi and culture conditions
The fungi Rhodotorula Mucilaginosa used in this study were provided from Soroka University Medical Center from Beersheva, Israel.The fungi were cultivated in the solid media, Sabouraud agar supplied by Scharlau Chemicals, and incubated at 35 °C for 48 h.

Biosynthesis of AgCl nanoparticles using Rhodotorula Mucilaginosa
In order to synthesize the silver chloride nanoparticles, 1μl of bacterial strains were freshly inoculated in test tubes containing 15 ml of growth medium, namely Brain Heart Infusion from Sigma Aldrich.The liquid media contained beef heart (infusion from 250 g), 5 g/L; calf brains (infusion from 200 g), 12.5 g/L; disodium hydrogen phosphate, 2.5 g/L; D(+)-glucose, 2 g/L; peptone, 10 g/L; sodium chloride, 5 g/L.The liquid culture was kept in a thermostat at 35 °C for 24 h, followed by centrifugation at 4000 rpm for 30 min.The supernatant and biomass were tested in parallel.In the first situation 5 ml of supernatant was used, while for the second one the biomass was kept with the addition of 5 ml of distilled water.The next step was similar by adding the 5-ml culture over 40 ml Ag NO 3 precursor solution, at 1 mM, 2 mM and 3 mM concentration, respectively.The culture, supernatant and biomass + distilled water and precursors were kept as control.The samples were kept in a thermostat set at 35 °C, for 48 h.

Characterization of AgNPs
Ultraviolet-visible spectral analysis was carried-out by using Jasco V-630 spectro-photometer.The UV-visible spectra were measured in the range 200-600 nm with a wavelength step size of 1.5 nm.
For morphological characteristics and chemical composition, a JSM 7400f scanning electron microscope (SEM) with a platform for Energy Dispersive Spectroscopy (EDS) was used.The silver chloride nanoparticles colloid was dropped on a copper grid and coated with a platinum thin film.After this the samples were mounted on a double sided adhesive carbon-tape.The acceleration voltage was fixed to 10kV.
The crystalline nature of silver chloride nanoparticles was analyzed by XRD using a Philips PW 1050/70 X-ray powder diffractometer with graphite monochromator using CuK α1 (λ =1,54Å), at a voltage of 40 kV, a current of 28 mA, in the scan range 10÷80 °, in Bragg-Brentano geometry.

Results and Discussion
After incubation in the thermostat for 48 h at 35 °C, the final color of the colloid, containing biomass of Rhodotorula Mucilaginosa, changed from light yellow to light brown.The change in color is an indication for the formation of nanoparticles.In Fig. 1 the obvious difference in color can be observed, depending on the precursor concentration.The samples with supernatant and the control have remained unchanged.Furthermore, considering the optical indication (changing the color), the UV-visible absorption spectra of the colloidal solutions with nanoparticles was measured (shown in Fig. 2).Fig. 2 corresponds to the UV-visible absorption spectrum of the solution containing AgCl NPs synthesized using biomass of Rhodotorula Mucilaginosa fungi.The spectra showed the maximum absorbance at 437 nm for samples with concentration 3 mM AgNO 3. Several reports have provided similar results concerning the absorption spectrum.The reports have confirmed that peaks around 440 nm coincide to the plasmon resonance of silver chloride nanoparticles [4], [5].
The crystalline nature of synthesized nanoparticles, obtained from X-ray diffraction, is confirmed by the diffraction peaks shown in Fig. 3, which correspond to the (111), (200), and (220) planes of the face centered cubic structure of AgCl crystal (Fig. 3) [11].
An explanation for the presence of silver chloride nanoparticles can be based on the interaction between silver nitrate and bacteria, which was previously grown in Brain Heart Infusion Broth media containing sodium chloride.The SEM analysis (Fig. 4) confirmed the presence of nano-scaled particles and also showed the spherical shape of them.As can be seen on the SEM micrographs, the silver chloride nanoparticles have dimensions smaller than 25 nm.The particle size/antimicrobial efficacy relation was reported previously, smaller particles, due to their much larger surface area, are expected to exhibit a much higher antibacterial efficacy.
The EDS spectra presented in Fig. 5 show the presence of the principal elements, namely Ag and Cl.The EDS analysis also revealed others elements which can be found on the samples due to the preparation stages (copper grid, carbon tape, thin film of platinum).In table 1, one can observe that silver chloride nanoparticles can be synthesized by different approaches, most of them starting from the AgNO 3 like precursor.The difference consists in the type of stabilizer and surfactant.
The spherical shape is predominant in the case of silver chloride nanoparticles.Very closely to silver nanoparticles, as shown by the authors in [13], AgCl NPs present significant anti-microbial activity against Escherichia coli, Candida albicans, Staphylococcus Aureus.
The antimicrobial effect of colloidal silver chloride nanoparticles has been tested against different microorganisms, including fungi and bacteria.The results will be used for further investigation to determine the synergism of the samples in the presence of different antibiotics, how is shown in the paper "Characterization and antimicrobial activity of silver nanoparticles, biosynthesized using Bacillus species" [13].It is important to emphasize that the precursor used was AgNO 3 , a reason for which we have to take into account that the recipe for growth media used for the microorganisms is very important for the final results.For example, the Brain Heart Infusion used for growing the present fungi, because it contains sodium chloride, may be the main factor which has led to the silver chloride nanoparticles formation by reducing the precursor AgNO 3 in the presence of biomass of Rhodotorula Mucilaginosa.

Summary
In the present work, silver chloride nanoparticles were synthesized via bio-reduction.It has been proved that the aqueous enzymatic extract of Rhodotorula Mucilaginosa fungi is able to synthesize silver chloride nanoparticles.It has been found that the silver chloride nanoparticles bioreduced by Rhodotorula Mucilaginosa decrease in size with increasing precursor concentration.The low-cost synthesis method is strengthened by the environmentally friendly steps of the procedure.

Table 1 .
Characteristics and applications of AgCl NPs synthesized through different methods.