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Extracellular Biosynthesis of Silver Nanoparticles

by Bacteria Alcaligenes Faecalis with Highly

Efficient Anti-Microbial Property Bahig El-Deeb

Faculty of Science, Taif University, P.O.Box:888 Al-Haweiah, Taif, Saudi Arabia Botany Department, Faculty of Science, sohag University, Sohag, Egypt.

Nassar Y. Mostafa Faculty of Science, Taif University, P.O.Box:888 Al-Haweiah, Taif, Saudi Arabia

Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt Abdulla Altalhi

Faculty of Science, Taif University, P.O.Box:888 Al-Haweiah, Taif, Saudi Arabia Youssif Gherbawy

Faculty of Science, Taif University, P.O.Box:888 Al-Haweiah, Taif, Saudi Arabia Botany Department, Faculty of Science, sohag University, Sohag, Egypt.

ABSTRACT

In this study, the biosynthesis of silver nanoparticles using

supernatant of Alcaligenes faecalis was described. UV–

Vis absorption spectra showed the reduction of silver ions

to silver nanoparticles with a surface Plasmon resonance

(SPR) band centered at 455 nm. The TEM analysis

revealed the formation of spherical, and monodispersed

nanoparticles with an average size of 11 nm. The silver

nanoparticles were stabilized by the capping of protein

present in the culture supernatant as revealed by FT-IR

spectral analysis. Synthesized nanoparticles showed

efficacy on antibacterial property against clinically

isolated multi-drug resistant (MDR) microorganisms. This

study also demonstrates that nanoparticles synthesis could

be controlled by varying the parameters such as

temperature and pH. It is suggested that biogenic synthesis

of nanoparticles have wide-application in medicine and

physical chemistry and it can produce with eco-friendly,

easy downstream processing and rapid scale-up

processing.

Keywords -Silver nanoparticles, Alcaligenes faecalis,

TEM. X-ray, Antimicrobial, Pathogenic

1. INTRODUCTION

In recent years, Nanotechnology has attracted a great

interest due to its expected impact on many areas such as

energy, medicine, electronics, and space industries. The

development of new materials with nanometer size,

including nanoparticles, nanotubes, nanowires, etc., is the

major activity. Nanoparticles with their unique properties

in chemistry, optics, electronics, and magnetic have led to

an increasing interest in their synthesis. Nanoparticles

have been synthesized by various physical and chemical

processes; however, some chemical methods cannot avoid

the use of toxic chemicals in the synthesis process [1].

Biosynthesis methods, employing microorganisms, have

emerged as a simple, clean and viable alternative to

chemical methods. So far, many microbes, including,

actinomycetes, bacteria, fungi and yeasts have been

successfully used for generating nanostructured mineral

crystals and metallic nanoparticles [2], [3], [4], [5], [6], [7],

[8]. However, these methods using biomass of microbes

are slow [9] with times ranging from one to several days

[4], compared to the procedures using microbial

supernatants for nanoparticle synthesis which are more

rapid [10]. The approach on using culture supernatants

from different bacteria, yeasts, fungi and actinomycetes

for the synthesis of silver nanoparticles is well

documented [11], [12], [13]. Nevertheless, the number of

microbial culture supernatants, evaluated so far for their

ability to induce nanoparticles, is limited and needs to be

extended to include microbes from various habitats.

Therefore, To our knowledge, this is the first report

showing such rapid extracellular synthesis of silver

nanoparticles by Alcaligenes faecalis isolated from desert

habitat at high altitude sites.

2. MATERIALS AND METHODS

2.1 Chemicals

AgNO3 was obtained from Sigma–Aldrich, USA. All other

chemicals were purchased from Merck, Germany. Freshly

prepared doubly distilled water was used throughout the

experimental work.

2.2. Isolation and Characterization of

Bacteria from the Silver Enriched Soil

Soil samples from metal-rich dump sites near industrial

city, (Lat 99o44’ and 79o45’ E), Taif province, North

West of Saudi Arabia, were used as inoculums.

Enrichment was performed in shaker incubators at 30°C in

Erlenmeyer flasks containing universal Mueller-Hinton

Broth medium (g/L) (17.50 Peptone, 2.00 Beef infusion

solids, 1.50 Starch). After 48 h growth, the cell

suspensions were serially diluted and plated onto Mueller-

Hinton Agar media. The plates were incubated at 30°C for

24 hr. The colonies obtained were further subcultured on

Mueller-Hinton Agar supplemented with 1.0 mM AgNO3

(Sigma) and incubated at 30°C for 72 hr.

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2.3. Screening of Bacterial Strains for Silver

Nanoparticle Synthesis

Silver resistant strains were grown up in test tubes

containing 10 ml nutrient medium in shaker incubators at

28 °C. After 24–48 hr incubation, the biomass was

separated from the medium by centrifugation (10,000 rpm,

10 min) and washed three times in sterile distilled water to

remove any nutrient media that might interact with the

silver ions. The biomass was resuspended in 10 ml

distilled water and the pH adjusted to 6.5 with 0.2 M

NaOH. Silver nitrate was added to give an overall Ag-

concentration of 1.0 mM. In Separate experiment,

supernatant was also tested for synthesis of silver

nanoparticles by mixed with 1.0 mM AgNO3. The mixture

was left for a further 24–72 h in a shaker incubator at

30 °C. The reduction of silver ions and synthesis of silver

nanoparticles were followed by the colorless of AgNO3

changed to brownish color.

2.4. Genotypic Characterization of the AgNPs

Synthesizing Strain

The morphological and physiological characterization of

the selected isolates was carried out by biochemical tests

using the Bergeys Manual of Determinative Bacteriology

[14]. Further characterization of isolate was done by

means of 16S rRNA gene analyses. The genomic DNA of

the isolate was extracted according to standard method.

The 16S ribosomal RNA gene was amplified by using the

PCR method with Taq DNA polymerase and primers 27F

(51 AGT TTG ATC CTG GCT CAG 31) and 1492 R(51

ACG GCT ACC TTG TTA CGA CTT 31). The conditions

for thermal cycling were as follows: denaturation of the

target DNA at 94 °C for four minutes, followed by 30

cycles at 94 °C for one minute, primer annealing at 52 °C

for one minute, and primer extension at 72 °C for one

minute. At the end of the cycling, the reaction mixture was

held at 72 °C for 10 min and then cooled to 4 °C. The PCR

product obtained was sequenced by an automated

sequencer (Genetic Analyser 3130, Applied Biosystems,

USA). The sequence was compared for similarity with the

reference species of bacteria contained in genomic

database banks, using the NCBI BLAST available at

http://www.ncbinlmnih.gov/.

2.5. Biosynthesis of Silver Nanoparticles

Synthesis of AgNPs was carried out according to the

method described previously [2], [4]. Briefly, bacteria

were grown in a 500 mL Erlenmeyer flask that contained

Mueller-Hinton Broth. The flasks were incubated for 21 h

in a shaker set at 120 rpm and 37 ◦C. After the incubation

period, the culture was centrifuged at 10,000 rpm and the

supernatant used for the synthesis of AgNPs. Three test

tubes, the first containing AgNO3 (Sigma, USA, 99.9%

pure) without the supernatant, the second containing only

the media and the third containing the supernatant and

AgNO3 solution at a concentration of 1mM were incubated

for 24 h. The extracellular synthesis of AgNPs was

monitored by visual inspection of the test-tubes for a

change in the color of the culture medium from a clear,

light-yellow to brown, and by measurement of the peak

exhibited by AgNPs in the UV–vis spectra.

2.6. Effect of Temperature and pH on

Nanoparticle Synthesis

To obtain optimum conditions for maximum synthesis of

nanoparticles, 1 mM of AgNO3 was added to the

supernatant and incubated at various temperatures (10-

60◦C) and pH conditions (3–10). The pH of the incubation

mixtures was adjusted using 1M HCl and 1M NaOH

solutions.

2.7. Characterization of Silver Nanoparticles

The biologically synthesized silver nanoparticles using the

cell free supernatant were characterized by UV-Vis

spectroscopy (Perkin Elmer, Lambda 25) instrument

scanning in the range of 200-900 nm, at a resolution of

1nm. All Samples were prepared by centrifuging an

aliquot of culture supernatant (1.5 ml) at 10,000 rpm for

10 min and diluted 10-fold for all experiments involving

measurement of UV–vis spectra. Cell free supernatant

without addition of silver nitrate was used as a control

throughout the experiment.

Samples for transmission electron microscopy (TEM)

analysis were prepared on carbon-coated copper TEM

grids. Studies of size, morphology and composition of the

nanoparticles were performed by means of transmission

electron microscopy (TEM) operated at 120 kV

accelerating voltage (JTEM-1230, Japan, JEOL) with

selected area electron diffraction (SAED). Finally, the

obtained images were processed using the software ImageJ.

ImageJ developed at the National Institutes of Health

(NIH), USA is a Java-based public domain image

processing and analysis program [15].

FTIR measurements were carried out using attenuated

total reflection Fourier transform infrared (ATR-FTIR)

Spectrometer (Bruker, Germany, Alpha-P). The

instrument was configured with ATR sample cell

including a diamond crystal with a scanning depth up to 2

micrometers. Sample powders were applied to the surface

of the crystal then locked in place with a “clutch-type”

lever before measuring transmittance. Each of the spectra

was collected in the range 4,000-400 cm-1

at 2 cm-1

resolution. Comparing with the conventional transmission

mode, the present technique is faster sampling without

preparation, excellent reproducibility and simpler to use.

X-ray diffraction (XRD) patterns was obtained using an

automated diffractometer (Philips type: PW1840), at a step

size of 0.02◦, scanning rate of 2◦ in 2θ/min., and a 2θ

range from 10◦ to 70◦. Indexing of the powder patterns and

least squares fitting of the unit cell parameters was

possible using the software X’Pert High Score Plus204.

2.8. Antibacterial Test 2.8.1. Disc Diffusion Method

The antibacterial assays were performed by standard disc

diffusion method as described by Yoshida et al. [16].

Escherichia coli, Pseudomonas aeruginosa,

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Staphylococcus aureus and Bacillus cereus were used as a

representative of gram-negative and positive strains as

well as multidrug resistant pathogenic bacteria obtained

from King Fasial Hospital at Taif, KSA. Mueller-Hinton

medium was used to cultivate bacteria. The media was

autoclaved and cooled. The media was poured in the petri

dishes and kept for 30 min for solidification. After 30

minutes the fresh overnight cultures of inoculum (100 μl)

of four different cultures were spread on to solidified

nutrient agar plates. After solidification, sterilized stainless

cylinders (6 mm internal diameter and 10 mm high) were

equidistantly placed open end up on each plate. Filter

sterilized silver nanoparticles suspension (100 mL) was

added to the cylinder. After incubation under suitable

conditions and time, the diameter of a round inhibition

zone against the indicator strains was measured with

calipers.

2.8.2. CFU Measurement

E. coli and / or Bacillus cereus was used for colony

forming units (CFU) measurements on the solid medium

plate. Samples treated with different concentrations of Ag

nanoparticles (0, 0.2 mM, 0.5 mM, 1 mM) were spread on

nutrient agar plates. These samples were diluted at 109

folds to get the better colonies. After incubation at 37 ◦C

for 24 h, the numbers of CFU were counted.

3. RESULTS AND DISCUSSION

3.1. Identification and Characterization of

Bacterial Strain

Among the tested organisms, G 10 showed rapid synthesis

of silver nanoparticles when compared to other isolates

and then the isolate was characterized according to the

method described in Bergy’s manual of bacteriology [14].

The strain shows the characteristic features of Gram

negative bacteria, rod shaped bacteria. Optimization

studies in the isolate G 10 revealed that the strain has

maximum growth at 37 ◦C and at neutral pH. The strain

grew well in the temperature range between 30 ◦C and 40

◦C and in the pH range 5.0–9.0. The biochemical level

identification of the isolate showed that the strain belongs

to genus Alcaligenes. In addition, the strain was

characterized by 16S rDNA technique. The sequences

were deposited in GenBank (NCBI) with accession

number KC857621. The obtained 16S rRNA sequence

was compared to the Gen Bank database in the National

Center for Biotechnology Information (NCBI) using the

BLAST program. Based on physiological, biochemical

characterization and 16S rDNA sequence analysis the

isolate was identified as Alcaligenes faecalis

3.2. Characterization of AgNPs Synthesized

by Alcaligenes Faecalis Supernatant

3.2.1. Visualation of Color

In this work, a study on extracellular biosynthesis of

AgNPs by bacterial supernatant was described. Visual

observation of the culture supernatant incubation with

silver nitrate at room temperature showed a color change

from light yellow to yellowish brown whereas no color

change could be demonstrated in culture supernatant

without silver nitrate or media with AgNO3 alone (Fig. 1).

Fig. 1 Photograph of samples containing; (1) silver nitrate

without supernatant, (2) supernatant without silver nitrate

after 24 hr (no color change) , and (3) supernatant with

AgNO3 after 1 hr and (4) after 24 hr.

In harmony with a previous report, the color change from

light yellow to yellowish brown, in the reaction mixture

due to the excitation of surface plasmon resonance (SPR)

for the biosynthesized AgNPs by the reduction of AgNO3

[4]. The UV–visible spectra for the aqueous AgNO3-

culture supernatant mixture and AgNO3 alone were

recorded. A peak at 445 nm corresponds to the

characteristic wavelength of AgNPs (Fig. 2). In agreement

with previous reports, the absorption peak at 455 nm is

probably due to the excitation of longitudinal plasmon

vibrations and formation of quasi-linear superstructures of

nanoparticles [4, 6].

3.2.2. Effect of temperature and pH on nanoparticle

synthesis Effect of temperature

Fig. 3 shows the effect of temperature on the biosynthesis

of silver nanoparticles by bacterial supernatant.

300 400 500 600 700 800 900 1000

455 nm

Abso

rban

ce (

a.u.)

Wavelength (nm) Fig. 2 UV–Vis absorption spectra of silver nanoparticles

produced at neutral pH with the bacteria Alcaligenes

faecalis supernatant after 24 hr.

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At an AgNO3 concentration of 1mM and a pH of 8.0, it

was apparent that increasing the temperature of the

reaction, up to 60 ◦C, results in an increase in the rate of

synthesis of AgNPs. The enhanced rate of synthesis of

AgNPs might be the direct result of the effect of

temperature on an reducing agents present in the culture

supernatant of A. faecalis. The results show that the

optimum temperatures for cell growth and silver

accumulation are different. The rate of formation of

AgNPs was related to the incubation temperature of the

reaction mixture, with increased temperature levels

allowing particle growth at a higher rate. These results are

in agreement with earlier reports that the enhanced rate of

synthesis of AgNPs might be the direct result of the effect

of temperature on a key enzyme present in the culture

supernatant [24].

3.2.3 Effect of pH

The present study involved a regular analysis of pH-

dependent changes in the reaction mixture for synthesis of

AgNPs. In this reaction, the concentration of AgNO3 was

maintained at 1mM and the reaction temperature at 60 ◦C.

When pH was increased from 3.0 to 10, maximum

synthesis was observed at pH 10.0 as evidenced by UV–

vis spectroscopy (Fig. 4). Therefore, the present study

shows that the optimum pH for synthesis of AgNPs is 10.0.

This is also in agreement with earlier reports that addition

of an alkaline ion is necessary to carry out the reduction

reaction of metal ions [24]. The effect of pH on

nanoparticle synthesis has been explained previously in

the case of platinum nanoparticles synthesis [17].

3.2.4. TEM analysis

For the transmission electron microscopy (TEM) study, a

drop of the silver nanoparticles solution synthesized by

treating silver nitrate solution with bacterial supernatant

was deposited onto a TEM grid which was coated with

carbon support film. After drying, this grid was imaged

using TEM. Figure 5 shows a representative TEM image,

with an size distribution on its right side. The TEM image

and their size distribution showed that the particles were

spherical, monodispersed with average diameter (11 ± 2.9

nm). The presence of spherical, mondispersed and small

particles in TEM image is in accordance with the UV–Vis

spectral study [4].

Fig. 3 (a) UV-Vis absorption spectra recorded for the different temperatures, (b) Photograph of samples, 1-2 the blank

reactions and 3-7 reactions done at 10ºC; 25ºC; 37ºC; 40ºC and 60ºC respectively

.

Fig. 4 (a) UV-Vis absorption spectra recorded for the different pH, (b) Photograph of samples, 1-2 the blank reactions

and 3-8 reactions done pH, at 3.0; 5.0; 7.0; 8.0; 9.0 and 10.0 respectively.

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(a)

(b)

Fig. 5 (a) TEM image of the silver nanoparticles (scale bar:

50 nm) produced by the reaction of 1 mM aqueous AgNO3

solution with bacteria A. faecalis supernatant at pH 7; (b)

its particle size distributions

The corresponding selected area electron diffraction

(SAED) pattern of silver particles is show in Fig. 6. The

SAED pattern was carried out on polycrystalline show

only some spots of diffraction distributed on concentric

circles. The rings patterns with plane distances 2.37Å,

2.05Å, 1.44Å, 1.23Å and 1.02Å Å are consistent with the

plane families (111), (200), (311), (220) and (400) of pure

face-centered cubic (fcc) silver structure. This was

confirmed by X-ray diffraction (XRD) analysis of the

freeze dried AgNPs as shown in Fig. 7. It can be seen from

the Figure that all the diffraction peaks corresponding to

(111), (200) and (220) planes have been indicated that are

characteristic of crystalline fcc silver (JCPDS, File No. 00-

001-1164).

Fig. 6 Selected area electron diffraction (SAED) of silver

nanoparticles showing the characteristic crystal planes of

elemental silver.

20 30 40 50 60 70

(220)(200)

(111)

2

Fig. 7 Representative XRD pattern of silver nanoparticles

synthesized by the reaction of 1 mM aqueous AgNO3

solution with bacteria A. faecalis supernatant at pH 8.

3.2.5. ATR-FTIR analysis

ATR-FTIR measurement was carried out to recognize

possible interactions between silver ions and protein

molecules which could responsible for capping and

stabilization of Ag nanoparticles. Medina-Ramirez et al.

reported that the involvement of intermolecular forces may

prevent the nanoparticles from aggregation by the

formation of hydrophobic–hydrophilic interactions [18].

Fig. 8 shows the FTIR spectrum of the freeze-dried

powder of silver nanoparticles formed after incubation of

bacterial supernatant with the aqueous AgNO3 for 24 hrs.

FT-IR analysis revealed intense bands at 3268, 3067, 2960,

2878, 1633, 1538, 1450, 1395, 1119 and 699 cm-1

(Fig. 8).

The bands at 3268 cm-1

and 1633 cm-1

could be attributed

to free N-H and C-C vibrations, respectively, which

corresponds to heterocyclic compounds like proteins [19].

This serves as support for proteins present in the bacterial

supernatant as capping agents for the biosynthesized

AgNPs [20], [21], [22]. Furthermore, the FTIR spectrum

revealed two bands at 1630 and 1543 cm-1

corresponding

to the amide I and II bands of proteins, respectively. The

Amide I band is primarily a C-O stretching mode and the

Amide II band is a combination of N-H in-plane bending

and C-N stretching. The more-complex Amide III band is

located near 1395 cm-1

. It has been reported that proteins

may bind to the nanoparticles either with the free amine

groups or cysteine residues and cap the nanoparticles [4],

[22], [24], [25]. Based on these earlier reports, in the

present study we speculate that the proteins present in the

bacterial supernatant capped and stabilized the AgNPs.

3.3. Antimicrobial activity against pathogenic

bacteria

3.3.1. Disc Diffusion test of AgNPs

The biologically synthesized AgNPs showed excellent

antimicrobial activity against clinically isolated Multi drug

resistant human pathogens bacteria such as E.coli, P.

aureginosa, S. aureus and B. cerus by disc diffusion

method. The mean inhibitory zone of the three replicates

of diameter was measured tabulated (Table 1).

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4000 3500 3000 2500 2000 1500 1000 500

699

452

529

615

921935

994

1035

1082

1119

1198

2878

cm-1

1331

1450

1395

1280

3067

2960

1243

3268 2

937

1538

1633

Fig. 8 ATR-FTIR spectra of freeze-dried silver nanoparticles formed after 24 hr.

The antibacterial activity of AgNPs increased with

increase the concentration AgNPs. Furthermore, silver

nanoparticles were fairly toxic to E.coli while they showed

a moderate toxicity against P. aeruginosa, and S. aureus.

and B. cereus. Silver nitrate was also tested as blank

control and the results indicate that silver nitrate has no

effect on tested strains at low concentration. However, 2

mM of silver nitrate has slight inhibitory effect test strains

(Table 1). A similar result was also reported that the

antibacterial activity of AgNPs against E. coli, P.

aeruginosa, and S. aureus. and B. cereus [26], [27]. This

result proved that AgNPs might be used as an

antimicrobial agent. In addition, AgNPs exhibited high

toxicity against pathogenic bacteria as compared with the

standard antibiotics like erythromycin, and Ampicillin

(Table 1). Sarkar et al. [28] reported that for E. coli

(ATCC 10536) and S. aureus (ML 422), AgNPs

demonstrated greater bactericidal efficiency compared to

penicillin [27], [28].

Table. 1 Zone of Inhibition of Antibacterial Test

Co

nc.

(m

M) Zone of inhibition (mm)

E.coli

Pseudomon

as

aeruginosa

Bacillus

cereus

Staphyloco

ccus

aureus

NPs Ag+ NPs Ag

+ NPs Ag

+ NPs Ag

+

0.2 3.2 nil 2.0 nil 2.2 nil 1.5 nil

0.5 5.2 0.2 4.2 nil 2.8 0.6 2.8 nil

1.0 8.8 0.4 5.9 0.2 5.4 0.8 5.8 0.8

2.0 12.5 1.2 9.2 0.8 9.8 1.3 9.9 1.8

Er nil nil 3.4 nil

Amp nil nil 3.8 0.6

NPs; silver nanoparticles, Ag+; silver nitrate, Er;

erythromycin. Amp; Ampicillin, nil, no inhibition zone.

3.3.2. Antimicrobial test by the estimation of Colony

Forming Units (CFU)

Fig. 9 shows the plot of number of bacterial colonies

(E.coli or B. cereus) grown on nutrient agar plates in

presence of different concentration of silver nanoparticles.

The results indicated that in both tested bacteria, the

colony forming unite (CFU) reduced significantly with the

increase of Ag nanoparticles as compared with control.

However the inhibitory effect of silver nanoparticles was

more pronounced on gram negative bacteria (E. coli) than

gram positive bacteria (B. cereus).

These results are in agreement with the previously results

obtained by Sondi et al. [26] who studied the effect of

silver nanoparticles on gram negative and positive bacteria

by colony forming unit (CFU) and growth curve at

different concentrations of silver nanoparticles. Their

studies showed a significant reduction of bacterial

population and their growth pattern with increasing the

concentration of silver nanoparticles. A number of

possible mechanisms are suggested for the antibacterial

activity of AgNPs. Silver ions have been known to bind

with the negatively charged bacterial cell wall resulting in

the rupture and consequent denaturation of proteins which

leads to cell death [29].

The synthesized AgNPs with smaller size can act

drastically on cell membrane and further interact with

DNA and DNA loses its replication ability [30]. Other

proposed mechanisms include the AgNPs causing

exhaustion of intracellular ATP by rupture of plasma

membrane or by blocking respiration in association with

oxygen and sulfhydryl (SH) groups on the cell wall to

form RSSR bonds thereby leading to cell death [31].

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Figure 9: Antimicrobial characterization by CFU of E.coli

and B. cereus as a function of AgNPs concentrations on

agar plates

4. CONCLUSION

The present study is an attempt for the extracellular

synthesis of silver nanoparticles from bacteria isolated

from metal-rich dump sites which was identified as

Alcaligenes faecalis. The silver nanoparticles synthesized

by the supernatant of A.faecalis were characterized by

UV–vis spectroscopy, TEM, and X-ray. The average

particle size of nanoparticle was found to be 11 nm. The

synthesized silver nanoparticles show potential

antimicrobial activity against pathogenic bacteria E.coli, P.

aureginosa, S.aureus and B.cereus using the well diffusion

method and Colony Forming Units (CFU). This study

would therefore lead to an easy procedure for producing

silver nanoparticles with the added advantage of less time

consuming, environmental friendly and cost effective

approach.

ACKNOWLEDGEMENT

This work was supported by a grant (Contract No. 1866-

433-1) from Taif University, Kingdom of Saudi Arabia.

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