Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms

doi:10.4236/msa.2010.11001

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Materials Sciences and Applications, 2010, 1, 1-7

doi:10.4236/msa.2010.11001 Published Online April 2010 (http://www.SciRP.org/journal/msa)

Synthesis of Crystalline Ag Nanoparticles (AgNPs)

from Microorganisms

Seema Sharma1, Naheed Ahmad2, Anuradha Prakash3, Vidya Nand Singh4, Ashok Kumar Ghosh3,

Bodh Raj Mehta4

1Department of Physics, A.N College, Patna, India; 2University Department of Botany, Patna University, Patna, India; 3Department

of Water Management, A.N College, Patna, India; 4Thin Film Laboratory, Department of Physics, Indian Institute of Technology,

New Delhi, India.

Email: [email protected]

Received January 26th, 2010; revised February 5th, 2010; accepted February 9th, 2010.

ABSTRACT

Bacteria obtained from the isolates of the biodiversity of riverine coast of Ganga identified as Bacillus Koriensis, when

challenged with silver nitrate solution accumulated silver nanoparticles on the surface of its cell wall. These nanoparti-

cles showed an absorption peak at 438 nm in UV-visible spectrum corresponding to the plasmon resonance of AgNPs.

The transmission electron micrographs of nanoparticles in aqueous solution showed the production of reasonably

monodisperse AgNPs (average particle size: 9.92 ± 1.311 nm) by the bacteria. X-ray diffraction spectrum of the

nanoparticles confirmed the formation of metallic silver.

Keywords: Crystal Structure, Nanostructures, Metal, AgNPs

1. Introduction

Nanostructured materials are being viewed as the future

material and for various diverse applications in areas

such as biomedical science, optics, mechanics, magnetics

catalysts, biosensors and energy science [1-3]. However

most synthesis techniques employed involve huge inputs

in terms of capital and energy. In the era of growing

awareness about increasing pollution and global warming,

developing new methods of synthesis of nanomaterials

with green technologies is a challenge. Biodiversity is the

best resource for biotechnological innovations as organic

materials of nanometer dimensions form the basis of life.

This diversity of life especially microorganisms is being

used as ecofriendly nanofactories for bioproduction and

synthesis of different compounds of nanometer size [4-6].

The concept of exploiting locally available species with

potential to synthesize extracellular nanoparticles is a

novel concept for bioprospecting. It provides valuable

leads for new product development and new applications

of biological species that have not been studied earlier.

The metal microbe interaction has made the material

scientist aware of the immense potential of the microorg-

anisms as ecofriendly nanofactories [6-8]. Biological sy-

nthesis of such nanomaterials has gained significant in-

terest due to the use of mild experimental conditions of

temperature, pH and pressure. If harnessed to their full

potential, biological synthesis could present extra advan-

tages over chemical methods such as higher productivity

and lower cost. One new dimension in the metal-microbial

interaction has emerged in the last few years, is that the syn-

thesis of metallic nanoparticles have been reported from bac-

teria, yeast, fungi, and other biological sources [9-16].

Bacteria are ubiquitous and constantly exposed to str-

essful situations; hence they develop diverse ways to res-

ist and survive. When exposed to metals or other toxic

substances beyond a certain level they develop many me-

chanisms such as efflux, alteration in the solubility and

toxicity by change in redox state of the metalion, extrac-

ellular complexation or precipitation of metals and the

lack of specific metal transport systems [17-19]. The

exact mechanism leading to the formation of reduction to

silver ions and formation of nanoparticles is not fully

understood. However, it is known that the tolerance of

bacterial cells and formation of mineral particles is very

much dependant on the composition of the growth envi-

ronment.

Various workers have exploited various microorgan-

isms for nanosynthesis. Bacteria as for instance Bacillus

subtilis 168 was reported to reduce Au+ to nanoscale di-

mensions [20,21].Other bacterial strains like Pseudomo-

nas stutzeri Ag 259 were found to be silver resistant and

were able to produce nanosize silver [22].Other workers

have used the fungi like Fusarium oxysporum and Veri-

Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms

cillium to produce Magnetite, Silica and Titania [23-25].

Klebsiella aerogens was manipulated to produce Cds

nanoparticles extracellularly. In addition to gold and sil-

ver nanoparticles synthesis of semiconductors like CdS,

ZnS and PbS have been obtained from bioorganisms.

Clostridium thermoaceticum precipitates was observed

to precipitate CdS at the cell surface as well as the me-

dium from CdCl2 in presence of cystenine hydrochloride

in the growth media [26]. It was also reported that the

monodispersity of silver/gold nanoparticles produced eit-

her intra or extracellularly by these bioorganisms is not

very high or inferior to those obtained by the conven-

tional chemical methods [27].

Ag Nanoparticles (AgNPs) have received considerable

attention due to their attractive physicochemical proper-

ties.The surface plasmon resonance and large effective

scattering cross section of individual AgNPs make them

ideal candidates for molecular labelling [28]. The exist-

ing physical methods for AgNPs synthesis, such as gas

condensation and irradiation by ultraviolet or gradiation,

are usually associated with a low production rate and

high expense. Further, the largescale synthesis of silver

nanomaterial suffers from issues such as polydispersity

and stability, especially if the reduction is carried out in

aqueous media. Therefore the extracellular biological sy-

nthesis of AgNPs could be an attractive and ecologically

friendly alternative method for the preparation of large

quantities because it offers the advantage of easy downst-

ream processing. Moreover, bacteria are easy to handle

and can be manipulated genetically without much diffic-

ulty. Considering these advantages, a bacterial system

could prove to be an excellent alternative for the extrace-

llular synthesis of AgNPs. Ionic silver is highly toxic to

most bacterial cells and has long been used as a potent

bactericidal agent [29]. However, several silver-resistant

bacterial strains have been reported and even shown to

accumulate AgNPs in their periplasmic space [4,14,15].

In this paper, we explore the local biodiversity for co-

mmercially valuable biological and genetic resources, as

they offer great opportunity for searching more environ-

ment friendly biomolecules as an answer to cleaner tech-

nologies for sustainable development. Biosynthesis of

nanomaterials is carried out from the microbial diversity

obtained from the state of India, Bihar (rich fertile allu-

vial Gangetic plain). Bacillus species has been used to

synthesize silver nanoparticles which was isolated from

the Gangetic riverine belt was seen to synthesize silver

nanoparticles extracellularly. This is the first report when

the microbial diversity of the Gangetic plain has been

tapped for nanosynthesis. The extracellular synthesis off-

ers a great advantage over an intracellular process of

synthesis from the application point of view. Since the

nanoparticles formed inside the biomass would have re-

quired additional step of processing for release of the

nanoparticles from the biomass by ultrasound treatment

or by reaction with suitable detergents. The extracellular

synthesis of nanoparticle makes it possible to harness and

immobilize/deposit onto desired solid support for the use

of different practical purposes.

2. Experimental Details

2.1 Culture

Soil samples were collected from the banks of Ganges at

various places in Patna, capital of Bihar during an ex-

haustive screening programme undertaken in our labora-

tory to isolate microorganisms capable of synthesizing

metal-based nanoparticles.The intracellular silver accu-

mulation was negligible (< 1%). However, extracellular

precipitation of black silver was observed with no loss in

the viability of cells. The bacteria selected for this study

was gram positive and rod shaped, forming chain like co-

lonies and was identified as Bacillus koriensis (Figure 1).

The selection was based on the criteria that this bacte-

rium was tolerating and reducing heavy metals.

2.2 Production of Silver Nanoparticles

Serial dilutions were made and plated on nutrient agar pl-

ates for culture of the bacteria. The plates were incubated,

after the requisite period the colonies were picked up and

pure cultures were isolated. The cultures were then che-

cked for their silver resistant properties by spot inocula-

tion with increasing concentrations of AgNO3.The se-

lected culture Bacillus Koriensis sp. was then inoculated

in at 0.5% level in 2 l Erlenmeyer flasks containing MG-

YP (Malt extract 1%, glucose 1%, yeast extract 0.3%,

Peptone 0.5%) media and incubated at room temperature

till the absorbance of the culture was between 0.8-0.9 and

the media was observed to be turbid. Colour changes

were observed both before and after exposure to sunlight.

Silver in form of AgNO3 from 1 mM to 8 mM was then

Figure 1. Photograph of Bacillus koriensis sp

Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms3

added and left for further incubation.The culture super-

natant that was obtained at different time intervals after

the addition of aqueous AgNO3 solution was centrifuged

at 3000 rpm for 5 min prior to harvesting bacterial cells;

the supernatant was filtered through a 0.22-mm filter,

and centrifuged again at 8000 rpm for 15 min to precipi-

tate AgNPs. While the Bacillus Koriensis sp. incubated

with deionized water (positive control) retained its origi-

nal colour, the silver nitrate treated organism turned dark

red after 24 h due to the deposition of silver nanoparti-

cles.This colour is primarily due to the surface plasmon

resonance of deposited silver nanoparticles. In case of

negative control (silver nitrate solution alone), no change

in colour was observed even after 10 days.

2.3 Characterization of AgNPs

2.3.1 UV–Visible Spectral Analysis

The optical absorbance of silver nano particles suspended

in distilled water was recorded on UV VIS spectrophoto-

meter (Systronics 2202 double beam model) from wave-

lengths 200-800 nm. The formation and quality of com-

pounds were checked by XRD technique.

2.3.2 X-Ray Diffraction Analysis

The X-ray diffraction (XRD) pattern measurements of

drop-coated films AgNPs on glass substrate were recor-

ded in a wide range of Bragg angles 2 at a scanning rate

of 20min-1, carried out on a Philips PW 1830 instrument

that was operated at a voltage of 40 kV and a current of

30 mA with Cu Kα radiation (1.5405Å).

2.3.3 TEM and Electron Diffraction Analysis

High Resolution Transmission Electron Microscopy (HR-

TEM) was performed by TECHNAI G20-STWIN (200 KV)

machine with a line resolution 2.32 (in angstrom). These

images were taken by drop coating AgNPs on a carbon-

coated copper grid. Energy Dispersive Absorption Spec-

troscopy photograph of AgNPs were carried out by the

HRTEM equipment as mentioned above.

3. Results and Discussions

Figure 2(a) shows the characteristic Surface Plasmon

Resonance (SPR) peak for AgNPs [30] obtained from

Baccilus koriensis, which was absent in all appropriate

simultaneously run controls. The AgNP production was

not detected even in a closely related genus of the same

family of E. coli, which indicates that the phenomenon

could be specific to silver-resistant Baccilus koriensis.

Production optimization of AgNPs was performed with

respect to concentrations of Ag+ ions, which ranged from

1 to 8 mM. It clearly showed an increase in the intensity

of the SPR up to 4 mM concentration, and an intensity

decrease at higher concentrations. The results clearly

indicated that 44 mM concentration of Ag+ ions was

most appropriate for the synthesis of AgNPs from Bac-

cilus koriensis. While no absorption band was observed

250 450 650 850

Wav e le ng th(n m)

Absorbanc

Absorbance

250 450 650 850

Wavelength (nm)

(a)

-0.5

0.5

1.5

2.5

3.5

200 300 400 500600 700 800

Wavelength (nm)

15 mins

30mins

10day

1month

(b)

Figure 2. UV/Vis spectra recorded from the culture super-

natant that shows the production of AgNPs (a) after 24 h of

reaction; (b) stability on storage of AgNPs

in both controls (positive and negative), a characteristic

surface Plasmon absorption band at 438 nm was ob-

served at 6 h that attained the maximum intensity after 24

h. After 24 h of incubation, no change in intensity at 438

nm was observed indicating complete reduction of silver

ions. The Plasmon bands are broad with an absorption

tail in the longer wavelengths, which could be in princi-

ple due to the size distribution of the particles [31]. Since

the varying intensity of the plasmon resonance depends

on the cluster size, the number of particles cannot be re-

lated linearly to the absorbance intensities [31].

Nanoparticles find various applications in electronics,

optoelectronics etc. in some of these applications it is a

requirement that the particles are stable in certain organic

solvents. Hence, the stability of the culture in organic

solvents such as ethanol, acetone and diethyl ether was

determined. It was observed that the Ag solution was

soluble in ethanol and no shift in absorbance maxima

was observed when the absorbance measurements of the

silver nanoparticles suspended in ethanol were taken.

However, the particles did not disperse uniformly in

acetone and diethyl ether and precipitated. The stability

of the synthesized silver nanoparticles was studied by

Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms

measuring its intensity over a period of 1 month in room

temperature. A narrow peak at ∼280 nm can be attributed

to the presence of contaminating proteins in the sample.

No significant change in the intensity was observed

which proved its stability over a period of 1 month indi-

cating that the coating polymer is not allowing the ag-

glomeration of the particles (Figure 2(b)). This may be

described by the fact that bacterial cells to protect itself

from the toxic environment of the silver takes advantage

of the detoxification mechanism by precipitation of silver

to elemental silver, following the interaction of metal

with chemical reactive group located at the bacterial sur-

face or its translocation into the cell. These sites nulcle-

ate deposition of metal chemical precipitate, thus leading

to formation of more metals as chemical precipitate and

lead to the formation of mineralized crystalline particles.

Such a response results in detoxification of Ag+ because

AgO is less toxic. The silver cation (Ag+) is a highly re-

active chemical structure which binds strongly to elec-

tron donor groups containing sulfur, oxygen, or nitrogen

and these bindings with biomolecules like protein could

restrict the size of the particle [32]. Whatever the mecha-

nism, the production of silver based crystalline particles

seems to be connected with the ability of bacterial cell to

survive in an environment that would be highly toxic to

other bacteria.

The bacteria were found to be reducing the silver extr-

acellularly. The biosynthesis and reduction of metal was

indicated by change in colour. The colour changed from

golden yellow which turned to tea brown on addition of

AgNO3 after 24 hours (Figure 3).There was perceptible

reduction in time of biosynthesis when the bacteria in log

phase with AgNO3 was exposed to sunlight.

X-ray diffraction (XRD) pattern (Figure 4) shows in-

tense Bragg’s reflections that can be indexed on the basis

of the fcc structure of silver [33]. The XRD pattern thus

obtained clearly shows [111], [200], [220], and [311]

planes, and exhibit that the synthesized AgNPs by the

Bacillus koriensis were crystalline in nature. The diffrac-

tion peaks were found to be broad around their bases ind-

icating that the silver particles are in nanosizes. The peak

broadening at half maximum intensity of the X-ray dif-

fraction lines is due to a reduction in crystallite size, flat-

tening and micro-strains within the diffracting domains.

Scherrer’s equation for broadening resulting from a

small crystalline size, the mean, effective or apparent di-

mension of the crystalline composing the powder is

Phkl = k / 1/2 cos

where  and  have their usual meaning,  is the breadth

of the pure diffraction profile in radians on 2 scale and

k is a constant approximately equal to unity and related

both to the crystalline shape and to the way in which  is

defined. The best possible value of k has been estimated

as 0.89. The particle sizes of all the samples in our study

(a)

(b)

Figure 3. Photographs showing change in colour after add-

ing AgNO3. (a) after 6hrs; (b) after 24hrs

Figure 4. X-ray diffractogram of silver nanoparticles: (a)

angular and background corrected raw data with structure

factors of Ag represented by vertical bars

have been estimated by using the above Scherrer’s equa-

tion and was found to be ~10nm for the strongest peak.

Transmission electron microscopy (TEM) images of

AgNPs that were synthesized by Bacillus koriensis, indi-

cated that the nanoparticles were in the size range of ~10

Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms

nm (Figures 5(a) and (b)). Selected area electron dif-

fraction (SAED) spots that corresponded to the (from

inside to outside of the central ring) [111], [200], [220],

[311] and [222] planes of the face-centered cubic (fcc)

structure of elemental silver [21] are clearly seen in Figure

5(c). HRT-EM image (Figure 5(d)) shows the d spacing

of 2.02Å, which matches with the [200] plane of AgNPs.

to understand the biochemical and molecular mechanism

of the synthesis of the nanoparticles by the cell filtrate in

order to achieve better control over size and polydisper-

sity of the nanoparticles.

4. Conclusions

Biosorption mechanism of metal ions by microorganisms

includes ion exchange, precipitation and complexation.

Reduction and surface accumulation of metals may be a

process by which microorganisms protect themselves

from the toxic effects of metallic ions. This study shows

the biosorption of silver in the form of nanoparticles by

the Bacillus koriensis. These nanoparticles that can be

attributed to surface binding of stabilizing materials secr-

eted by the Bacteria.

Figure 6 shows the Energy Dispersive Absorption Sp-

ectroscopy photograph of AgNPs. All the peaks of Ag

are observed and are assigned. Peaks for Cu and C are fr-

om the grid used and the peaks for S, P and N correspond

to the protein capping over the AgNPs. It is reported ear-

lier that proteins can bind to nanoparticles either through

free amine groups or cysteine residues in the proteins [34,

35] and via the electrostatic attraction of negatively cha-

rged carboxylate groups in enzymes present in the cell

wall of mycelia [36] and therefore, stabilization of the

silver nanoparticles by protein is a possibility. The amide

linkages between amino acid residues in proteins give ri-

se to the well-known signatures in the infrared region of

the electromagnetic spectrum and have been shown by

the FTIR spectrum [37]. In future, it would be important

In this study, extracellular synthesis of AgNPs has be-

en shown from silver-resistant Bacillus k sp. XRD analy-

sis showed that nanoparticles were crystalline and metal-

lic in nature, respectively. HRTEM analysis showed that

most of the particles were spherical in shape with size

~10 nm. The extracellular bacterial synthesis of AgNPs

(a) (b)

Figure 5. (a) and (b) Transmission electron microscopy images of AgNPs from Bacillus koriensis at different magnifications;

(c) Selected area electron diffraction showing the characteristic crystal planes of elemental silver; (d) HRTEM image showing

characteristic d spacing for the [200] plane

Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms

Figure 6. Energy dispersive absorption spectroscopy photograph of AgNPs

has many advantages and might be an excellent means of

developing an ecologically friendly protocol. The bacte-

rium was highly resistant to silver cations. Thus it can be

safely said that metal microbe interactions have an im-

portant role in many biotechnological applications inclu-

ding biomineralization. So, today it seems to be a cheap

and viable method for the production of ecofriendly nan-

oparticles.

5. Acknowledgements

The authors wish to acknowledge University Grants Co-

mmission, New Delhi, India for the financial support

under the major research project scheme.

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