Antimicrobial activity of Eco-friendly silver ...

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Nov 22, 2015 - Nat. Mater 3:482-488. 10. Mohanpuria P, Nisha K, Rana NK, Yadav SK, et al. (2008) ... Venkataraman A, et al. (2008) Extracellular biosynthesis ...
WORLD JOURNAL OF EXPERIMENTAL BIOSCIENCES

Vol. 3, No. 2: 118-122 (2015). ISSN: 2313-3937

Research article

Antimicrobial

activity

of

Eco-friendly

silver

nanoparticles synthesized by Saprolegnia ferax Rana H. Al-Shammari1*, Shaimaa N. Mizil1 and Huda Z. Majeed1

ABSTRACT An eco-friendly method to synthesis silver nanoparticles using (fungus) Saprolegnia ferax was investigated in current study. The formation of nano silver was detected by using UV-visible spectroscopy. The absorption was around 430-450 nm. To determine shape and size of partials, atomic force microscope (AFM) and Scanning electron microscope (SEM). The particle was irregular polygon in shape with diameters were between 1.0 to 130 nm and the average of the nanoparticls diameter was (63.3 nm). The obtained nanoparticls showed antimicrobial activity. The most effective concentration of the paricls was 50 µg/µl against Escherichia coli, Pseudomonas and 40 µg/µl against Candida albicans. Keywords: Escherichia coli, Pseudomonas, Silver nanoparticles, Saprolegnia ferax.

Citation: Al-Shammari RH, Mizil SN, Majeed HZ. (2015) Antimicrobial activity of Eco-friendly silver nanoparticles synthesized by Saprolegnia ferax. World J Exp Biosci 3: 118-122. Received September 20, 2015; Accepted October 27, 2015; Published November 22, 2015.

INTRODUCTION Fungi have higher productivity when used in nanoparticles biosynthesis due to their higher protein secretion [8,10]. Several reports have been reported to demonstrate the biosynthesis of metal nanoparticles using fungi, including Fusarium acuminatum [11], Fusarium semitectum [12], Verticillium spp. [8], Penicillium citrinum [13] and Aspergillus oryzae [14]. Recently, nanoparticles have been used as antimicrobial agent due to the increasing of bacterial resistance [15]. Silver has been used as antimicrobial agent mainly as ion Ag+ and also as different systems that release silver ions in different concentrations [16,17]. Silver nanoparticles used as an antimicrobial agent because it

Nanotechnology is the application of science and technology at the molecular level [1,2] and recently become one of the most active research fields in technology and engineering [3]. There is a need to use eco-friendly nanoparticles that do not produce toxic materials in the synthesis protocol and this is called ”Green chemistry” [4,5,1]. Biological methods for synthesis of materials are a novel idea compared with traditional synthetic methods [6]. Microorganisms such as bacteria, viruses and fungi produce inorganic materials either intra or extra-cellular and they can reduce the metal ions [7,8]. It is used as eco-friendly method for synthesis of nano-materials [9,10].

*Correspondence: [email protected]. Department of Biology, College of Science, AL- Mustansiriyah University, Baghdad, Iraq. Full list of author information is available at the end of the article 68 Copyright: © 2015, Al-Shammari RH et al., This is an open-access article distributed under the terms of the Creative Commons

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Attribution License, which permits unrestricted use, distribution, and reproduction in any site, provided the original author and source are credited.

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Al-Shammari RH, et al. (2015) World Journal of Experimental Biosciences. Vol. 3, No. 2: 118-122. is very toxic to fungi, bacteria and viruses due to their size and the large surface area of silver nanoparticles results a great activity [18,19]. The present study aims to demonstrate method of biosynthesizing of silver nanoparticles using fungus, determine the size and shape of the synthesized silver nanoparticles, investigate the antimicrobial activity of the synthesized silver nanoparticles against Gram negative bacteria (E. coli and Pseudomonas) and yeast (Candida albicans). The present study also showed the time dependent formation of silver nanoparticles using fungi isolated from water employing UV–visible spectrophotometer as the first indicator for silver nanoparticle biosynthesis. Size and morphology of nanoparticles were shown by employing Atomic Force Microscope (AFM) and Scanning electron microscope (SEM).

MATERIALS AND METHODS Samples collection Water samples were collected From Al-Jaderria region in Baghdad in sterilized plastic screw cap bottles with capacity (1.5 L). The bottle was opened under water surface with 10 cm water column depth and closed under water (half filled with water) then transferred to the laboratory to isolate Saprolegnia ferax. Some clinical samples such as E. coli, pseudomonas sp. and Candida albicans were gifted from the laboratory of University of Baghdad.

centrifugation at 4500 rpm for 15 min. The mycelia pellet was washed thrice with sterile deionized water. The washed mycelia (1%w/v) were treated with 1 mM of (AgNO3) the mixtures incubated at 25 °C in darkness at 200 rpm for 3 days. A control experiment containing only 0.001 M of silver nitrate solution was also performed. All experiments were carried out in triplicates and samples were drawn everyday throughout the days of incubation. In this process, silver nanoparticles were produced through reduction of the silver ions to metallic silver. The biosynthesis was confirmed by UV-VIS, AFM, SEM techniques.

Characterization of nanoparticles UV-vis spectroscopy The formation of silver nanoparticles was routinely monitored by visual inspection of the solution as well as by measuring the UV-Visible spectra of the solution by periodic sampling of aliquots (2 ml) of the aqueous component. The UV-Vis spectroscopy measurements were recorded on a Shimadzu 10 UV Visible spectroscopy (japan) operated at a resolution of 1 nm.

Atomic Force Microscopy (AFM) A thin film of the sample was prepared on a glass slide by dropping 100 μl of the sample on the slide, and was allowed to dry for 5 min. The slides were then scanned with the AFM [25].

Scanning electron microscope (SEM)

Isolation and identification of S. ferax

The colloidal solution were centrifuged for 15 min at 4000 rpm the supernatant again centrifuged (25 and 900 rpm) for 30 min the pellets were dissolved in 0.1 ml of distill water then placed on cover slip and dried by air and the sample coated with gold the images of silver nanoparticles obtained using scanning electron microscope [26]. These tests were done at Nanotechnology Center at University of Technology in Baghdad. The study was

Fungus was isolated from water samples that collected from Tigress river in Baghdad. The fungal isolates were characterized by colony characteristics and microscopic appearance. On potato dextrose agar (PDA) medium colonies arise after 7 days at 25 OC. Baiting method which adapted by previous study [20] was used to collect Saprolegnia by using sesame seeds Sesamum indicum which considered the most efficient bait [21]. Bottles were shaken gently and 1 ml of chloramphenicol solution was added to inhibit bacterial contamination in the culture, five sesame seeds (boiled for few minutes) were added to the petri dishes and incubated at 20 OC for 24 h, then each seed transferred into new petri dish containing (10 ml sterilized distilled water with chloramphenicol 100 M/ml and three sesame seeds). Saprolegnia were identified according to the classification keys of fungi [22, 23].

conducted following approval from the animal ethics committee of Biology Department, College of Science, AL-

Mustansiriyah University.

Antibacterial activity of silver nanoparticles Well diffusion method was performed to investigate the antimicrobial activity of silver nanoparticles. Few colonies from overnight culture of E. coli and Pseudomonas were transferred to 2 ml of normal saline to prepare the bacterial suspension and adjusted to 0.5 McFarland turbidity that is equal to 1.5×108 CFU/ml. The bacterial suspension was inoculated into nutrient agar plates using a sterile cotton swab. The plates were left to dry for 5 min. nine mm in diameter wells have been done on nutrient agar medium and swabbed with bacteria using cotton swabs. Hundred microgram of dried AgNO3 in 100 µl of distilled water was prepared, 20, 30, 40 and 50 µl of dispersed solution was put in the well to get concentration of 20, 30, 40 and 50 µg, respectively. Cephalexin (1 mg/l) was used as a control. The diameter

Nanoparticle Synthesis The method used in the preparation of biomass for biosynthesis of silver nanoparticles from the fungus is described by previous study [24] with few modifications, involved the aerobic culturing of fungus in 500 ml bottles containing 250 ml of GC broth medium composed of (0.5 % glucose and 0.4 % casein hydrolysate) and incubated at 20 oC for 72 h under continuous mixing condition by a magnetic stirrer at 200 rpm, the mycelia and culture broth were separated by

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Al-Shammari RH, et al. (2015) World Journal of Experimental Biosciences. Vol. 3, No. 2: 118-122. of inhibition zone around the wells was measured in millimeter. The same procedure has been done to investigate the antimicrobial activity of silver nanoparticles against C. albicans using Ketoconazole (1 mg/l) as a control. The cut off value of sever nanoparticles against both bacteria and yeast was 15 µg/µl.

RESULTS AND DISCUSSION After 24 h of the reaction between S. ferax biomass and aqueous solution of AgNO3 led to change the color of the mixture to yellowish brown this indicating of silver nanoparticles formation. The change of color from pale yellow to yellowish brown due to the excitation of surface plasmon vibrations in the silver nanoparticles. Control was prepared without adding AgNO3. This showed no change in color under the same condition (Fig. 1).

Fig 2. UV-Visible spectra of silver nanoparticles formed after 24 h.

Silver nanoparticles were biosynthesized in different sizes by three isolates of S. ferax the size was mesured

Fig 3. AFM picture shows silver nanoparticles with maximum tip height (130 nm).

Fig 1. Tubes contain Saprolegnia ferax biomass(A)before and(B) after 24hrs of exposure to Ag+ ions.

The UV-VIS spectrum of silver nanoparticles produced by S. ferax showed an absorption peak at around 430450 nm (Fig. 2), this is an indicator of silver nanoparticles formation, it is reported that the absorption band at 265 nm due to the excitation of the electron in tyrosine and tryptophan residue in the proteins [27]. The protein that found in the fungal biomass plays a role in the synthesis of nanoparticles [28].

by using AFM the diameter starting from 1 to 130 nm and the average of the silver nanoparticles diameter was 63.3 nm (Fig. 4).

Atomic Force Microscopy (AFM) AFM is an instrument to mesure the matter at the nanoscale level and suitable for charecterizing nanoparticles. It is capabile of giving a three dimension visualization and information about size, surface texture, morphology and roughness. AFM gives a wide rang of particle size from 1 nm to 8 μm [29]. Fig 3 shows three dimension image for silver nanoparticles. The maximum tip offered by this instrument is very height (130 nm).

Fig 4. Diameters, volumes and cumulation of prepared silver nanoparticles.

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Al-Shammari RH, et al. (2015) World Journal of Experimental Biosciences. Vol. 3, No. 2: 118-122. 13.5, 17.5 mm and in E. coli were 10.3, 10.5, 11, 12.5 mm and in case of C. albicans were 15.3, 16.3, 18.3, 18.2 using the concentrations 20, 30, 40, 50 µg/µl, respectively (Fig 6). This results agreed with the results of previous study [30], it reported that the biosynthesized silver nanoparticles by Streptomyces hygroscopicus significantly inhibited the growth of medically important pathogenic of Grampositive bacteria (Enterococcus faecalis and Bacillus subtilis), Gram negative bacteria (E. coli and Salmonella typhimurium) and yeast (C. albicans). Silver nanoparticles is a killing agent against Gram posetive, Gram negetive bacteria and fungus including antibiotic resistant bacteria [31,32] and this action is belong to the large surface of silver nanoparticles that give a larg area to contact with cell membrane and easly penetrate the bacteria [33] the other theory is that the nanosilver attack the respiratory chain in bacterial cell and some researchers reported that the antibacterial activity may belong to interact the nanosilver with sulphur containing protein in microbial membrane [34]. The present study concluded that the silver nanoparticles have been successfully synthesized by the fungus S. ferax and it have high antimicrobial activity. The size of silver nanoparticles was ranged from 1.0 to 130 nm and the average of diameter was 63.3 nm, this diameter is suitable for penetration the bacterial wall.

SEM analysis The image obtained by SEM shown in (Fig 5) it is seen that the silver nanoparticles are irregular polygon shape and the diameter was about 50-60 nm.

Fig 5. SEM of silver nanoparticles synthesized by treating fungal cell filtrates with 1 mM AgNO3 solution.

Antibacterial activity of silver nanoparticles Antimicrobial activities of silver nanoparticles have been observed using well diffusion method against E. coli, pseudomonas and C. albicans. Inhibition zone was determined by measuring the diameter of bacterial clearance after 24 h using cephalexin as a control and Ketoconazole in case of fungus. The means diameter of inhibition zones in case of pseudomonas were 12.2, 13,

Conflict of interest The authors declare that they have no conflict of interests.

Fig 6. Antimicrobial activity of nanosilver particles against Pseudomonas (A), E.coli (B), C. albicans (C). a, Cephalexin 1 mg/l for bacteria and Ketoconazole (1 mg/l) for C. albicans; b, 20 µg/µl; c, 30 µg/µl; d, 40 µg/µl; e, 50 µg/µl.

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Author affiliation: 1. Department of Biology, Collage of Science, ALMustansiriyah University, Baghdad, Iraq.

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