Investigation of Culture Conditions for Biosynthesis of Silver Nanoparticles Using Aspergillus fumigatus

One of the most important criteria of nanotechnology is that of the development of clean, nontoxic and environmentally acceptable ‘‘green chemistry’’ procedures, involving organisms ranging from bacteria to fungi and even plants (Bhattacharya et al., 2005; Sastry et al., 2004). The interactions between microorganisms and metals have been well documented and the ability of microorganisms to extract and/or accumulate metals is already employed in biotechnological processes such as bioleaching and bioremediation (Gericke et al., 2006).
Synthesis of nanoparticles employing microorganisms has attracted great interest due to their unusual optical (Krolikowska et al., 2003), chemical (Kumar et al., 2003), photoelectrochemical (Chandrasekharan et al., 2000) and electronic properties (Peto et al., 2002), which enable the synthesis of nanoparticles of different chemical compositions, well-defined sizes and distinct morphologies (Bhattacharya et al., 2005). It is well known that many organisms can synthesize inorganic materials either intra- or extracellularly (Dickson, 1999; Kroger et al., 1999; Mandal et al., 2006; Mann, 1996; Oliver et al., 1995; Mann, 1993; Simkiss et al., 1989; Lovley et al., 1987).
The use of fungi in the synthesis of nanoparticles is a relatively recent addition to the list of microorganisms possessing nanoparticle biosynthesis “ability”. Application of fungi to produce nanoparticles is potentially exciting because of their ability to secrete large amounts of enzymes. Some of the microorganisms, which have been widely used for synthesizing of silver nanoparticles include microorganisms such as, Verticillium sp., Fusarium oxysporum, Pseudomonas stutzeri AG259 (Kuber et al., 2006; Sastry et al., 2003) and Aspergillus fumigatus (Bhainsa and D’Souza, 2006). Except for F. oxysporum and A. fumigatus, biosynthesis of silver nanoparticles by the abovementioned microorganisms is intracellular. It is possible to release intracellular silver and gold nanoparticles via ultrasound treatment of the biomass-nanoparticles composite or via reaction with suitable detergents. Nonetheless, it would be far more practical if the metal ions exposed to the fungus could be reduced outside the fungal biomass, leading to the formation of metal nanoparticles in solution. (Mandal et al., 2006) Therefore in this study A. fumigatus was used to reduce silver ions extracellularly. According to the preliminary study of Bhainsa and D’Souza (2006), this fungus is a good candidate for rapid biosynthesis of silver nanoparticles. In this research, the effects of glucose concentration, pH and number of spores on the size and morphology of silver nanoprticles were investigated using UV- visible spectrophotometry and transmission electron microscopy (TEM).
In order to prepare the required biomass for the biosynthesis of silver nanoparticles, spores of A. fumigatus UB260o which had originally been isolated from the Bidestan Distillery and Food Products, Qazvin, Iran for biodecolorization of distillery spent wash (Pazouki et al., 2006; Pazouki et al., 2005) were maintained on potato dextrose agar (PDA) slants for 5 days. Twenty experiments were designed using the central composite design (CCD) at five levels (Montgomery, 2005) to investigate the effects of culture media conditions on the biosynthesis of silver nanoparticles. This design has the advantage of using a minimum number of trials. The parameters studied, were glucose concentration, initial pH and the number of spores in liquid culture media. These parameters were selected due to their influence on morphology and metabolite production (Pazouki and Panda, 2000). Hence 20 liquid media containing (g/l) KH2PO4 7.0, K2HPO4 2.0, MgSO4·7H2O 0.1, (NH4)2SO4 1.0, yeast extract 0.6, different glucose concentrations and pH were prepared using CCD (Table 1). Then, the respective numbers of spores were inoculated and the fungus was grown aerobically. The flasks were incubated at 25ºC on an orbital shaker (Sm-920, Borhan Pouya Co., Iran) with agitation at 160 rpm. After 72 h of cultivation, the resulting biomasses in the flasks were harvested using a sterilized sieve and washed extensively with distilled water to remove medium components. The obtained biomass in each run of the experiment as it was produced, was added to 100 ml of Milli-Q deionized water and incubated at 30°C for 72 h on an orbital shaker, with shaking at 130 rpm. After the incubation period, for the purpose of the synthesis of silver nanoparticles, AgNO3 (1 mM) was mixed with the biomass filtrate that was obtained by passing it through Whatman filter paper no. 42,  in a 250 ml Erlenmeyer flask . The resulting mixture was incubated at 25ºC in the dark, with agitation at 130 rpm (Bhainsa and D’Souza, 2006).
In order to observe the effect of biomass on synthesized silver nanoparticles, silver nitrate was added to the samples under the same conditions but, containing biomass. Whereas the rapid synthesis of silver nanoparticles is possible using A. fumigatus, the UV spectra of samples were obtained using UV-visible spectrophotometer (CECIL 30E 9500, England) after 4 h in order to compare the intensities of peaks related to silver nanoparticles. The sample of biomass filtrate which had the desirable UV spectrum with regard to the silver nanoparticles was used for electron transmission microscopy (TEM). A drop of the sample was placed on the carbon coated copper grid of the microscope (CEM902A, Zeiss, Germany).
Reduction of silver ions was reflected in the changing color of the cell filtrate, from pale yellow to a brown shade as shown in Figure 1. Due to the completion of the reaction, the intensity of the brownish color increased. In addition, the presence and production of silver nanoparticles were confirmed by UV-visible spectrophotometry, whereby light absorption at approximately 420 nm was attributed to to the surface plasmon resonance of the silver nanoparticles .
Desirable UV spectra were obtained by samples with high absorption intensity due to high levels of reduced silver ions or smooth curves due to better size distributions (Khosravi et al., 2007). As denoted in Figure 2, the sample from run 19 had the highest intensity of light absorption at 420 nm but the shape of the UV spectrum of the sample from run 15 was much smoother.
Figure 3 shows the UV spectrum of the samples from runs 15 and 19 containing biomass. The UV spectrum of samples containing biomass did not show admissible absorption intensity at 420 nm as compared to the samples containing cell filtrate, indicating low concentration of silver nanoparticles in the media. The color of media did not change significantly either, instead the color of the biomass surfaces changed to a brownish red after adding silver nitrate. It shows that the synthesized silver nanoparticles had accumulated on the cell wall. Hence, the benefits of external biosynthesis of silver nanoparticles could not be appreciated because the detachment of nanoparticles would be a difficult and extra process.
Figure 4 shows the TEM micrograph of silver nanoparticles synthesized according to the culture conditions of runs 15 and 19. As seen in this Figure, the morphology of nanoparticles is almost spherical. The sizes the nanoparticles in the sample of run 19 ranged from 7- to 35 nm and the sizes of nanoparticles in the sample from run 15 was in the range of 16-50 nm. Moreover, there was some aggregation between the silver nanoparticles synthesized in the sample from run 15.
     Table 2 includes the cell dry mass produced during each run, indicating the effects of culture conditions on the amount of cells produced. In addition, it was observed that these parameters affected the morphology and even the color of the fungal cells. Therefore, the chosen culture variables including glucose concentration, initial pH and number of spores were required to be considered in order to obtain desirable nanoparticles. According to the obtained results, the optimum culture conditions of A. fumigatus for biosynthesis of silver nanoparticles included a glucose concentration of 16 g/l, pH of 4.5 and spore concentration of 1.5×107/l.
     Application of biological systems for synthesis of silver nanoparticles has already been reported (Sastry et al., 2003; Klaus-Jeorger et al., 2001).  However, the exact mechanism leading to the formation of silver nanoparticles by all these organisms is yet to be elucidated. Ahmad et al. (2003) have reported that certain NADH dependent reductases of F. oxysporum were involved in the reduction of silver ions. Also, Duran et al. (2005) have studied several strains of the fungus F. oxysporum and have reported that the reduction of the metal ions occurs by a nitrate-dependent reductase and a shuttle quinone extracellular process. One reason for aggregation of sample 15 could be the use of a fewer number of spores as compared with that of sample 19. Therefore, the spore inoculum concentration affects the synthesized nanoparticles.
There are a few reports on the effects of culture conditions on the biosynthesis of metal nanoparticles. Mohammadian et al. (2007) have investigated the effects of growth conditions on the biosynthesis of silver nanoparticles by F. oxysporum. They have reported that consistency in the fungus growth conditions including cultivation temperature, carbon source concentration and shaking frequency is not a determining factor affecting the rate of nanoparticles biosynthesis. However, there is no report on the effects of the numbers of spores on biosynthesis of metal nanoparticles. In this study,  some of the fungus culture variables including initial pH, glucose concentration and spore number  have been found to affect the morphology and amount of fungal biomass produced and also the size and morphology of synthesized silver nanoparticles.

Acknowledgments

This study was performed at the Materials and Energy Research Center and financially supported by the Graduate Studies Office, Karaj, Tehran.