Indian Journal of Biotechnology Vol 11, January 2012, pp 72-76
Extracellular biosynthesis of silver nanoparticles by culture supernatant of Pseudomonas aeruginosa P Jeevan1*, K Ramya1 and A Edith Rena2 1
PG and Research Department of Microbiology and 2PG and Research Department of Biotechnology J J College of Arts and Science, Pudukkottai 622 404, India Received 29 January 2010; revised 10 February 2011; accepted 18 March 2011
The development of reliable, eco-friendly processes for the synthesis of nanoscale materials is an important aspect of nanotechnology. Silver bionanoparticles have been known to have inhibitory and bactericidal effects. In the present study, authors report the extracellular synthesis of nanoparticles of silver by reduction of aqueous Ag+ ions with the culture supernatant of Pseudomonas aeruginosa. It was found that aqueous Ag+ ions in solution when exposed to P. aeruginosa get reduced, thereby leading to the formation of silver nanoparticles. The formation of silver nanoparticles was confirmed by the change in colour of the culture filtrate from yellow to reddish brown after the addition of silver nitrate. The morphology and uniformity of silver nanoparticles were investigated by UV-Vis spectroscopy, X-ray diffraction and scanning electron microscope (SEM). The interaction between protein and silver nanoparticles was analyzed using FTIR. The process of reduction was extracellular, which makes it an easier method for the synthesis of silver nanoparticles. These biosynthesized silver nanoparticles were also evaluated for their antimicrobial activities against Escherichia coli and Vibrio cholerae. Keywords: FTIR, Pseudomonas aeruginosa, SEM, silver nanoparticles, X-ray diffraction
Introduction For the synthesis of nanoparticles, a number of chemical methods exist in the literature1. In these protocols, toxic chemicals are used, which have been a matter of great concern for environmental reasons. Consequently, researchers in the field of nanoscale material synthesis and assembly have been eagerly looking at biological systems for an alternative. The metal microbe interactions have an important role in several biotechnological applications, including the fields of bioremediation, biomineralization, bioleaching and microbial corrosion2,3. Recently, the utilization of biological systems has emerged as a novel method for the synthesis of metal nanoparticles. It is well known that many microbes, both unicellular and multicellular, produce inorganic materials either intra or extracellularly4. The microorganisms, such as, bacteria, yeast and fungi, play an important role in the remediation of toxic metals through reduction of metal ions and act as interesting nanofactories5. These microbes are extremely good candidates in the synthesis of cadmium, gold and silver nanoparticles (Ag-NPs)6-8. —————— *Author for correspondence: Tel.: +91-4322-260 103; Mobile: +91-9791886609 E-mail: [email protected]
Studies in antibacterial materials containing various natural and inorganic substances have been intensified recently9,10. Metal nanoparticles (Me-NPs), which have a high specific surface area and a high fraction of surface atoms, have been studied extensively because of their unique physicochemical characteristics including optical, electronic and magnetic properties as well as catalytic and antimicrobial activittes11. Among Me-NPs, Ag-NPs have been known to have inhibitory and bactericidal effects10. It can be expected that the high specific surface area and high fraction of surface atoms of Ag-NPs would lead to high antimicrobial activity as compared with bulk silver metal. In recent years, resistance to commercially available antimicrobial agents by pathogenic bacteria and fungi has become a serious problem12. Microbes, such as, bacteria, molds, yeasts and viruses, in living environment are often pathogenic and cause severe infections in human beings. Therefore, there is a pressing need to search for new antimicrobial agents from natural and inorganic substances9,10. Among inorganic antimicrobial agents, silver has been employed most widely since ancient times to fight infections13-17. For this reason, the present work
JEEVAN et al: BIOSYNTHESIS OF SILVER NANOPARTICLES
has been focused on the development of an extracellular biosynthesis of Ag-NPs using culture supernatant of Pseudomonas aeruginosa and the evaluation of their antimicrobial activity against human pathogenic bacteria, Escherichia coli and Vibrio cholerae. Materials and Methods Source of Microorganism
The bacterium, P. aeruginosa was obtained from Culture Collection Centre, CAS in Botany, University of Madras, Tamil Nadu, India. The culture was grown on Nutrient agar (Himedia, Mumbai) slants at 37°C for 24 h and maintained at 4° C in a refrigerator. Synthesis of Silver Nanoparticles
Nutrient broth was prepared, sterilized and inoculated with a fresh growth of test strain P. aeruginosa. The cultured flasks were incubated at 37°C for 72 h in an orbital shaker at 150 rpm. After the incubation period, the culture was centrifuged at 12,000 rpm for 5 min and the supernatant was used for the synthesis of silver nanoparticles (AgNPs). The supernatant of P. aeruginosa culture was separately added to the reaction vessels containing silver nitrate at a concentration of 0.1 g/L. The reaction between these supernatant and silver ions was carried out in bright conditions for 72 h. The bioreduction of the silver ions in the solution was monitored by sampling the aqueous solution (2 mL) and measuring the absorption spectrum of the solution using (Beckman–Du-50) UV-Visible spectrophotometer at a resolution of 1 nm.
The formation of silver nanoparticles was checked by X-ray diffraction (XRD) using an X-ray diffractometer (Philips PW 1710). The supernatant treated with silver nitrate was evaporated to dryness under sunlight. The air dried biomass was analyzed. The full widths at half maximum (FWHM) values of X-ray diffractions were used to calculate particle size using the Debye-Sherrer formula. Determination of Antimicrobial Activity
The Silver nanoparticles synthesized from P. aeruginosa were tested for antimicrobial activity by well-diffusion method against pathogenic microorganisms E. coli and V. cholerae. The pure cultures of organisms were subcultured on Muller-Hinton broth at 35°C on a rotary shaker at 200 rpm. Wells of 6 mm diameter were made on MullerHinton agar plates using gel puncture. Using sterile cotton swabs, each strain was swabbed uniformly onto the individual plates. 20 µL (0.002 mg) of the sample of water as control, liquid culture filtrate, silver nitrate and silver nanoparticle was loaded into the well using a micropipette. After incubation at 35°C for 18 h, the different levels of zone of inhibition were measured. Results Characterization of Synthesized Silver Nanoparticles
Characterization of Silver Nnanoparticles
In the present study, extracellular biosynthesis of silver nanoparticles by the culture supernatant of P. aeruginosa was studied. Visual observations showed a change of colour in silver nitrate solution from yellow to brown (Fig. 1), whereas no colour
After 4 h of incubation of the above mixture, the preliminary detection of silver nanoparticles was carried out by visual observation of color change of the culture filtrate. These samples were later subjected to optical measurements, which were carried out by using a UV-Vis spectrophotometer (Beckman–Du-50) and scanning the spectra between 200-800 nm at the resolution of 1 nm. The interaction between protein and silver nanoparticles was analysed by Fourier transform infrared (FTIR) analysis. The FTIR spectrum of the dried sample was recorded on Perklin Elmer instrument in the range of 450 to 4000 cm-1 at a resolution of 4 cm-1. A scanning electron microscope (JEOL, Japan, JFC-1600) was used to record the micrograph images of synthesized silver nanoparticles.
Fig. 1—Conical flasks containing P. aeruginosa culture supernatant in aqueous AgNO3 solution: (A) At the beginning of reaction showing no colour change; & (B) After 72 h of reaction showing brown colour.
INDIAN J BIOTECHNOL, JANUARY 2012
change was observed in the culture supernatant without silver nitrate or in media with silver nitrate alone. The appearance of a yellowish brown colour in silver nitrate treated culture supernatant suggested the formation of silver nanoparticles6. A similar observation was made by Duran et al18 in the biosynthesis of Ag-NPs by Fusarium oxysporum strain by extracellular process. The brown colour of the medium could be due to the excitation of surface plasmon vibration of AG-NPs8. The exact mechanism of biosynthesis of Ag-NPs is not known. However, it has been hypothesized that silver ions required the NADPH-dependent nitrate reductase enzyme for their reduction, which was secreted by the bacteria in its extracellular environment19. The use of this enzyme has previously been demonstrated in the in vitro synthesis of silver nanoparticle under anaerobic conditions. Nitrate reductase is known to shuttle electron from nitrate to the metal group. Thus, these results substantiate the role of nitrate reductase enzyme in the biosynthesis of silver nanoparticles20. The synthesized Ag-NPS were characterized by UV-Vis spectroscopy. In the UV-Vis absorption spectrum, a strong, broad peak located between 420 and 430 nm was observed (Fig. 2). Observation of this peak, assigned to a surface Plasmon, is well documented for various metal nanoparticles with sizes ranging from 2-100 nm7. X-ray diffraction (XRD) was carried out to confirm the crystalline nature of the particles and the XRD pattern obtained is shown in Fig. 3. The XRD
Fig. 2—Absorption spectrum of silver nanoparticles synthesized by the culture supernatant of P. aeruginosa (420 nm)
pattern shows four intense peaks in the whole spectrum of 2Ø values ranging from 20-80. A comparison of the XRD spectrum with the standard confirmed that the silver particles formed in the present study were in the form of nano-crystals, as evident from the peaks at 2Ø values of 39.01, 46.48, 64.69 and 77.62 corresponding to (111), (200), (220) and (311), respectively for silver. Fig. 4 shows a representative SEM image recorded from the drop coated film of the silver nanoparticles synthesized in the present study. The particle size ranges from 20-100 nm and possesses an average size of 50 nm. The results obtained from the SEM
Fig. 3—Representative XRD pattern of silver nanoparticles formed after reaction of culture supernatant with AgNO3 (1×10−3 M) for 72 h. JCPDS (Joint Committee on Powder Diffraction Standards) - File No.: 04- 0783.
Fig. 4—SEM micrograph of silver nanoparticles formed after reaction of culture supernatant with AgNO3 (1×10−3 M) for 72 h (particles at higher resolution shown by scale bar of 50 nm)
JEEVAN et al: BIOSYNTHESIS OF SILVER NANOPARTICLES
image gave the clear shape and size of the AgNPs produced from the P. aeruginosa. The diameter of the AgNPs in the solution was found to be in the range of 20–100 nm. FTIR measurements were carried out to identify possible interaction between silver salts and protein molecules, which could account for the reduction of silver ions and stabilization of silver nanoparticles formed after 72 h (Fig. 5). The amide linkages between aminoacid residues in proteins give rise to the well known signatures in the infrared region of the electromagnetic spectrum. The bands seen at 3226.39 cm-1 and 2935.57cm-1 were assigned to the stretching vibrations of primary and secondary amines, respectively. The bands seen at 1387.41 cm-1 and 1048.05 cm-1 corresponds to –C-N stretching vibrations, while the band at 1457.34 cm-1 is characteristic of amine and amino-methyl stretching groups. The band seen at 1639.16 cm-1 is characteristic of -C═O carbonyl groups and -C═Cstretching. The overall observation confirms the presence of protein in samples of silver nanoparticles. It has also been reported earlier that protein can bind to nanoparticles either through their free amine groups or cysteine residues21. Therefore, stabilization of silver nanoparticles by proteins is a clear possibility.
Antimicrobial Activity of Silver Nanoparticle
The antimicrobial activity of silver nanoparticles was investigated against pathogenic organisms, viz., E. coli and V. cholerae using well-diffusion method (Fig. 6). The highest antimicrobial activity was observed against E. coli (18 mm) followed by V. cholerae (17.5 mm). The lower activity was found in wells poured with silver nitrate alone. Sondi and Salopeak-Sondi22 studied the antibacterial acivity of AgNPs against E. coli and concluded that it might be used as an antimicrobial agent. Shahverdi et al16 also opined that the AgNPs had inhibitory effect on Staphylococcus aureus and E. coli. The susceptibility
Fig. 6—Antimicrobial activity of silver nanoparticles against E. coli (A) and V.cholerae (B) bacterial strains shown by well-diffusion method. (1. Sterile water (Control), 2. Liquid culture filtrate, 3. Silver nitrate, & 4. Silver nanoparticle)
Fig. 5—FTIR spectra recorded from powder of silver nanoparticles synthesized using P. aeruginosa
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of E. coli and V. cholerae to AgNPs has been confirmed by earlier work done by Song et al23 who opined that the susceptibility of E. coli and V. cholerae is due to the inhibition of bacterial cell wall synthesis. The present study focuses on E. coli and V. cholerae because they are found to be highly pathogenic to human beings and show resistance to a wide range of broad-spectrum antibiotics. In the present study, authors reported the extracellular biosynthesis AgNPs using the bacterium P. aeruginosa. The AgNPs were found bioactive showing inhibitory effect on important human pathogens, E. coli and V. cholerae. It is also clear that the bacterium P. aeruginosa can be used to synthesize bioactive nanoparticles efficiently using inexpensive substances in an eco-friendly and non-toxic manner.
Acknowledgment The authors extend their thanks to the management of University of Madras, Guindy Campus, Chennai, India, for providing the facilities to do the research work in the Department of CAS in Botany.
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