The success of reducing silver to nano-sized particles helps to make the element highly effective, making it more in demand for several use in the industry of medicine and technology. Biosynthetic methods using microorganisms are ecofriendly in nature (Sastry et al., 2003) and it has been investigated as an alternative to chemical and physical ones. Biosynthesis of silver nanoparticles has significant levels of antibacterial activity even in the presence of low silver nanoparticle concentrations. Silver is safe for human cells when it is used in small concentrations but that concentration is lethal for bacteria and viruses (Sharma and Yngard, 2009). The intra- or extracellular methods depends on the place where the nanoparticles or nanostructures are created on the microorganisms (Mann, 1996). Fungi were found to be highly efficient for the synthesis of silver nanoparticles. The silver nanoparticles synthesized from fungi have become a challenge for the nano-biologists which possess good antimicrobial activity against various multi drug resistant pathogens (Saravanan and Nanda, 2010). Colletotrichum sp. or Aspergillus fumigates were reported to have extracellular synthesis of nanosilver. Mukherjee et. al. proposed a biosynthesis of silver nanoparticles with Vercillum by trapping the silver ions on the fungal surface then the released enzymes of the fungi reduced the silver ions to nano size. Duran et. al. described the extracellular synthesis of silver nanopartilces from Fusarium oxysporum strain of fungus by the presence of hydrogenase enzyme was observed to have exceptional redox properties, acting as an electron shuttle for reduction of metal ions. It is known that a large number of organisms, both unicellular and multicellular, are able to produce inorganic nanomaterials, either intracellularly or extracellularly (Bruins et al, 2000; Beveridge et al, 1977; Mukharjee et al, 2002 and Ahmad et al, 2003). The present study focused on the synthesis of silver nanoparticles from Penicillium chrysogenum. . As Penicillium spp are well known for their antibiotic compounds, the study also comprises the antimicrobial activity of silver nanoparticles against bacterial pathogens.
Extracellular Biosynthesis of Ag Nanoparticles using Penicillium chrysogenum:
The Sabouraud Dextrose media plate was prepared, exposed in air for 5 minutes and incubated for 3-5 days. The isolated Penicillium culture was grown aerobically in liquid medium containing KH2PO4, K2HPO4, MgSO4. H2O, (NH4)2SO4, Yeast extract and glucose (Ahmad et al, 2003). The flask was inoculated and incubated on orbital shaker at 25°C and agitated at 150 rpm. The biomass was harvested after 72 hrs of growth by filtration using ordinary filter paper. The biomass was followed by extensive washing with distilled water to remove all possible medium components from the biomass. Typically 20gm of biomass (fresh weight) was brought in contact with 200ml of Milli- Q deionized water for 72h at 25°C in an Erlenmeyer flask and agitated for further 72 hours. After the incubation, the cell filtrate was subjected to AgNO3 for synthesis of silver nanoparticles. At different time intervals the absorbance was measured by UV - visible spectrophotometer at a resolution of range 200-800 nm after 24h onwards to 72h of treatment with AgNO3. The cell filtrates containing nanoparticles were treated against the pathogens for checking their antimicrobial activity and the formation of silver nanoparticles.
Intracellular Biosynthesis of Ag Nanoparticles using Penicillium chrysogenum:
The fungal culture was grown aerobically in same liquid medium and incubated on orbital shaker at 25°C and agitated at 150 rpm up to72 hrs. The biomass was harvested by filtration followed by extensive washing with distilled water. Typically 20gm of biomass (fresh weight) was brought in contact with 200ml of Milli- Q deionized water in an Erlenmeyer flask and followed by sonication. Sonication was done at 100% amplitude by using 30 mm probe for 15 min. The suspension was centrifuged at 12,000 rpm for 10 min at 25°C. The cell filtrate was subjected to AgNO3 for intracellular synthesis of silver nanoparticles. At different time intervals the absorbance was measured by UV-visible spectrophotometer at a resolution of range 200-800 nm.
Antimicrobial Activity Test:
Disc diffusion method and well diffusion method was used for checking the antimicrobial activity silver nanoparticles against bacterial pathogens and also to confirm the dose dependant concentration. The zone of inhibition was measured and compared with the control in its raw form and with the antibiotic disc to confirm their activity ability.
RESULTS AND DISCUSSION:
Extra and intracellular biosynthesis of silver nanoparticles from Penicillium chrysogenum
The fungal biomass was harvested after 72 hrs of growth by filtration using ordinary filter paper followed by extensive washing. Typically 20gm of biomass (fresh weight) was agitated for 72 hours at 25°C in 200ml of Milli- Q deionized water and the color was completely white. The Penicillium chrysogenum biomass when subjected to AgNO3, the reaction was started after six hours and the color of the solution turned to yellowish brown, indicating the formation of AgNPs. Similarly a typically 20gm of biomass was sonicated at 100% amplitude by using 30 mm probe for 15 min. The suspension was centrifuged at 12,000 rpm for 10 min at 25°C. The cell filtrate was subjected to AgNO3 for intracellular synthesis of silver nanoparticles (Sastry et al, 2003). The cell filtrate when subjected to AgNO3, the reaction was started after four hours and the color of the solution turned to yellowish brown, indicating the formation of AgNPs. It is well studied by several researchers' investigation that microorganisms have been explored as potential bio-factories for synthesis of metallic nanoparticles such as cadmium sulfide, gold and silver (Sastry et al, 2003; Tillmann, 2004; and Ahamad, 2005) and the AgNPs exhibit a yellowish brown color in water, arising from excitation of surface plasmon vibrations in the metal nanoparticles. The AgNPs were characterized by UV-vis spectrophotometry. The formation and stability of the reduced AgNPs in the colloidal solution was monitored by using UV-vis spectral analysis. The UV-vis spectra recorded at different time intervals of reaction were plotted in figure-6 and the curves a, b, c, d, and e correspond to the readings at different time intervals like 6, 12, 24, 48 and 72 hours, respectively and the peak was noted around 420 nm. It is observed from the spectra that the silver surface plasmon resonance band occurs at 420 nm.
Antimicrobial Activity Test
Disc diffusion method and well diffusion method was used for checking the antimicrobial activity of extracellular and intracellular silver nanoparticles against Bacillus cereus, Staphylococcus aureus, E. coli and Pseudomonas aeruginosa. The extracellular and intracellular silver nanoparticles were treated against all the pathogens and zone of inhibition measured and compared with antibiotic penicillin G. The dose dependant concentration confirm that 15µl showed the zone of inhibition was more in Bacillus cereus followed by E. coli, and Pseudomonas aeruginosa. But in all the cases the pathogens did not respond to the antibiotic disc penicillin G. But there is not much difference about extracellular and intracellular silver nanoparticles (Table-1). When Staphylococcus aureus was treated with extracellular nanoparticles the zone of inhibition measured more (18mm) than compared to intracellular silver nanoparticles. Nanda and Saravanan (2009, 2010) also proved that silver nanoparticles synthesized extracellularly showed positive response against multidrug resistant pathogens. Our findings correlating with others findings showed good result when treated with extracellular silver nanoparticles (Table-1). The maximum zone of inhibition was recorded by Staphylococcus aureus (18mm) followed by E. coli (15mm), and Pseudomonas aeruginosa (10mm) and the least Bacillus cereus (8mm) (Sondi and Salopek-Sondi, 2004). We have not studied the mechanism perhaps the silver nanoparticles do not respond to Bacillus cereus. Marcato et al, (2003) showed that silver nanoparticles, like its bulk counterpart, are an effective antimicrobial agent against various pathogenic microorganisms. Though various chemical and biochemical methods are being explored for silver nanoparticles production, microbes are very much effective in this process. New enzymatic approaches using bacteria and fungi in the synthesis of nanoparticles both intra- and extra cellularly have been expected to play a key role in many conventional and emerging technologies.
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