INTRODUCTION:

In recent years the field of nanobiotechnology have witnessed an increase in interest towards the noble nanoparticles and their biological effects and applications. These include bottom-up and molecular self-assembly, biological effects of naked nanoparticles and nano-safety, drug encapsulation and nanotherapeutics, and novel nanoparticles for use in microscopy, imaging and diagnostics. However, it is only recently found that microorganisms have been explored as potential biosource¹ for the synthesis of metallic nanoparticles such as cadmium sulfide, gold and silver². It was found that when Klebsiella aerogenes exposed to cadmium ions resulted in intracellular formation of CdS particles in the range of 20–200 nm³. Even fungal species Verticillium sp. and Fusarium oxysporum, was exposed to gold and silver ions, reduced the metal ion rapidly and formed respective metallic nanoparticles. Pseudomonas stutzeri AG259, a strain isolated from a silver mine, when placed in a concentrated solution of silver nitrate produced silver nano particles⁴ .Many fungi which exhibit the characteristic property, of producing nitrate reductase are capable of reducing Au (III) or Ag (I)⁵.

Besides these extracellular enzymes, several naphthoquinones⁶and anthraquinones⁷ with excellent redox properties, were reported in F. Oxysporum that could act as electron shuttle in metal reductions⁸ .However the mechanistic reduction of metal ions occurs in the fungal case probably either by reductase action or by electron shuttle quinones or both⁹ The exotic properties of nanoparticles have been exploited in applications such as optoelectronics^10,11 single-electron transistors (SETs) and light emitters^12,13 nonlinear optical devices¹⁴. Currently research interest has been developed in exploring the use of metal nanoparticles – such as gold, silver and their alloys – for diagnostic and therapeutic imaging and moreover recent literature reports suggest that silver nano particles also have good bactericidalactivity^15,16. Considering all these phenomenon, a detailed study was carried out for the extracellular biosynthesis of silver nanoparticles using Aspergillus niger which is a prospective candidate for this purpose. As it is purely extracellular process the burden involved in the downstream processing is reduced.It belongs to family of the fungal biomass that had exhibited very good binding capacity for uranium¹⁷. The present study includes kinetics of synthesis, spectroscopic and microscopic characterization of the silver nanoparticles.

MATERIALS AND METHODS:

Aspergillus niger strain was obtained from National Chemical Laboratory, Pune and the fungal inoculates were prepared in the culture medium containing KH₂PO₄ (7 g/l), K₂HPO₄ (2 g/l), MgSO₄.7H₂O (0.1 g/l), (NH₄)₂SO₄(1 g/l), Yeast extract (0.6 g/l), Glucose (10 g/l) at 37 C in Petri plates. The fungal growth was carried out in flasks containing the medium and incubated in orbital shaker at 25 C agitated at 150 rpm. The biomass was harvested after 72 h of growth by sieving through a plastic sieve followed by extensive washing with distilled water to remove any medium component from biomass.

Silver reduction and characterization:

Method 1:

20gm biomass (fresh weight) was brought into contact with 200 ml Milli Q (MQ) distilled water for 72 h at 25 C in Erlenmeyer flasks and agitated. Cell filtrate was obtained by passing through Whatmann filter paper 1. Silver nitrate (1mM final concentration) was mixed with 50 ml of cell filtrate in 250 ml Erlenmeyer flask. It was then agitated in the dark at 25 C. Another flask containing only the biomass without the silver ion was used as control.

Method 2:

20 gm biomass (fresh weight) was suspended in 200 ml MQ distilled water. The AgNO₃ solution (10^-3 M) was added to the Erlenmeyer flask and the reaction was carried out in the dark. Periodically, aliquots of the reaction solution were removed and the absorptions were measured using a UV-Vis spectrophotometer.

The silver nanoparticles were characterized using a 25 kV scanning electron microscope (SEM).

Determination of optimal glucose concentration:

The fungal biomass was grown in culture media containing varying concentration of glucose other components remaining same. Glucose concentrations used were 2.75 g/l, 3 g/l, 3.5 g/l, 4 g/l, 4.5 g/l, and 5 g/l. The concentration of the silver nanoparticles was correlated to the absorbance at 415-420 nm in UV-Vis Spectrophotometer. The data showing the absorbance at different wavelengths is shown in Figure 1.

Figure 1: UV-Vis spectra recorded as a function of wavelength for various glucose concentrations of an aqueous solution of 10^-3 M AgNO₃with the fungal filtrate. Maximum absorbance is observed at around 440 nm which is due to the surface plasmon resonance of silver nanoparticles. The maximum yield was observed for glucose concentration 2.75 g/l which is the optimum value.

Determination of electron shuttling compounds:

In order to determine the water soluble quinones that might function as electron shuttling compounds, the filtrate obtained by filtering the biomass in MQ water was subjected to High performance Thin Layer Chromatography (HPTLC). The mobile system Ethyl acetate- Methanol- Water (100:13.5:10) used to mobilize pigments on a Silica Gel 60 plate. The filtrate was adjusted to pH 3 using 1 M HCl and 2μL samples were spotted on silica gel plate and observed under UV light at 254 nm.

RESULTS AND DISCUSSION:

The Erlenmeyer flasks were pale yellow in color before the addition of silver ions but changed to yellowish brownish color on completion of reaction with silver ions for 48 h. The appearance of yellowish brown color in the solution containing biomass confirmed the presence of silver nanoparticles¹⁸. The UV-Vis spectra recorded from the A. niger culture flasks at different reaction times is presented in Figure 2. The data in the figure shows that the optimal concentration of glucose for maximum yield of the silver nanoparticles is 2.75 g/l.

(a)

(b)

Figure 2: Cell filtrate (72 h) of A. niger with silver ion (1 mM): (a) at the beginning of the reaction and (b) after 48 h of reaction.

The solution was extremely stable, with no evidence of flocculation of the particles even several weeks after reaction. The strong surface plasmon resonance centered at 415–420 nm clearly shows the presence of silver nanoparticles. The increase in intensity could be due to increasing number of nanoparticles formed as a result of reduction of silver ions present in the aqueous solution. The fact that silver nanoparticles peak remained close to 420 nm even after 72 h of incubation indicates that the particles were well dispersed in the solution and there was not much aggregation. Monodispersity is an important characteristic of the nanoparticles. Gold nanoparticles with very good monodispersity have been reported by Ahmad et al¹⁹ using Thermonospora sp. The plot at different glucose substrate concentrations over different time intervals showed that the yield of silver nanoparticles reached maximum at 2.75 g/l glucose concentration. The absorption band at 265 nm was attributed to aromatic amino acids of proteins. It is well known that the absorption band at ca. 265 nm arises due to electronic excitations in tryptophan and tyrosine residues in the proteins¹⁸.This observation indicates the release of proteins into solution by A. niger and suggests a possible mechanism for the reduction of the metal ions present in the solution.

In the second method when the biomass was immersed in Milli Q (MQ) water and only the fungal filtrate was added to 10^-3 M AgNO₃ solution, the initially colorless aqueous solution changed to a pale yellowish-brown (Figure 3) within 48 h of reaction clearly indicating that the reduction of the ions occurs extra cellular through reducing agents released into the solution by A. niger as it shows the UV-Vis spectra for the strain. Figures 4 and 5 show the SEM micrographs of the silver nanoparticles produced by A. niger (Method 1) at magnifications x2500 and x9500 respectively. The same micrograph was obtained with Method 2 also (data not shown).

Figure 3: SEM micrograph of silver nanoparticles produced by A. niger strain at x2500 magnification.

Figure 4: SEM micrograph of silver nanoparticles produced by A. niger strain at x 9500 magnification.

Figure-5: Acidified filtrate (immobilized as a spot in silica plate) under the UV emitting blue color florescence confirming the presence of quinones

The HPTLC (Chromatography of Thin Layer) analysis of the filtrate obtained by filtering the biomass in MQ water on silica gel 60 plates using Ethyl acetate- Methanol- Water (100:13.5:10) showed a spot with R_f value of 0.65 corresponding to 2-acetyl-3,8- dihydroxy-6-methoxy anthraquinone or its isomers as reported by Nelson 2005.

In Fluorescence test for Quinones The filtrate from A. niger was acidified with concentrated sulphuric acid and observed under UV light. Blue color fluorescence was observed which is characteristic of quinines.

CONCLUSIONS:

Thus the extracellular biosynthesis of silver nanoparticles from A. niger has been demonstrated by our study. The silver nanoparticles thus synthesized were stable in solution with no evidence of flocculation even several weeks after synthesis. This stability is very important for the application of silver nanoparticles in biological platform. The mechanism of the biosynthesis of the silver nanoparticles from A. niger was also investigated and our preliminary data shows that quinones act as electron shuttling compounds for the biological reduction of the silver nitrate to silver nanoparticles.

Competing Interests:

We declare that there are no financial or non-financial competing interests.

ACKNOWLEDGEMENT:

We express our heartfelt thanks to Dr. K.N. Somasekaran, Dean, School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, India for his extensive guidance and support throughout the work.

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Received on 15.01.2010 Modified on 27.02.2010

Asian J. Research Chem. 4(1): January 2011; Page 31-34