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ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES SYNTHESIZED BY FUSARIUM OXYSPORUM STRAIN

P.D. Marcato1*, G.I.H. De Souza2, O.L. Alves3, E. Esposito2 , N. Durn 1,2
2 1 Biological Chemistry Laboratory, Universidade Estadual de Campinas-SP Biological Chemistry and Biotechnology Laboratory, Center Environmental Sciences, Universidade de Mogi das Cruzes 3 Solid State Chemistry Laboratory, Instituto de Qumica, Universidade Estadual de Campinas SP

Abstract. Microorganisms (bacteria, yeast and fungi) play an important role in toxic metals remediation
through reduction of metal ions, this was considered interesting as nanofactories. Recently, it was found that aqueous chloroaurate ions may be reduced extracellularly using Fusarium oxysporum, to generate extremely stable gold or silver nanoparticles in water. These particles can be incorporated in materials and cloth becoming them sterile. The sterile materials are important in hospital, where often wounds are contaminated with microorganisms, for example, fungi and bacteria, in particular, Staphylococcus aureus. A new generation of dressing incorporating antimicrobial agents like silver was development to reduce or prevent infections. Extracellular production of silver nanoparticles by F. oxysporum strain and its effect bactericide in cotton and silk cloth against S. aureus were studied in this work. In the silver reduction, approximately 10 g of F. oxysporum biomass was taken in a conical flask containing 100 mL of distilled water, kept for 72 h at 28 oC and then the aqueous solution components were separated by filtration. To this solution, AgNO3 (10-3 M) was added and kept for several hours at 28oC. In order to incorporate silver nanoparticles in the cloth, these were immersed in the filtrate fungal, centrifuged and dried. The reduction of metal ions occur by a nitrate-dependent reductase and quinone extracellular process. Antibacterial activity was observed when silver nanoparticles were incorporated in cotton cloth. However, in silk cloth this activity was not observed demonstrating a low incorporation of the silver nanoparticles due possibly to the size of the silk pores. This work demonstrates the possible use of biological synthesized silver nanoparticles and its incorporation in cloths leading them to sterilization. Moreover, these particles could have innumerable applications, in different areas as receptors, catalysis, biolabelling and others.

Keywords: Nanoparticles, biosynthesis, Fusarium oxysporum, antibacterial activity.

1. Introduction
The Microorganisms such as bacteria, yeast and now fungi play an important role in remediation of toxic metals through reduction of the metal ions, this was considered interesting as nanofactories very recently (Fortin and Beveridge, 2000). Using this dissimilatory properties of fungi, the biosynthesis of inorganic nanomaterials using eukaryotic organisms such as fungi may be used to grow nanoparticles of gold (Mukherjee et al., 2001a) and silver (Mukherjee et al., 2001b) intracellularly in Verticillium fungal cells (Sastry et al., 2003). Recently, it was found that aqueous chloroaurate ions may be reduced extracellularly using the fungus F. oxysporum, to generate extremely stable gold or silver nanoparticles in water (Ahmad et al., 2003). The sterile cloth and materials are important in hospital, for example, where often wounds are contaminated with microorganisms, in particular fungi and bacteria, like Staphylococcus aureus. Thus, to reduce or prevent infections, various antibacterial disinfections techniques have been developed for all types of textiles. Recently, several antibacterial agents of textiles based on metal salt solutions (CuSO4 or ZnSO4) have been developed

Priscyla Daniely Marcato. Address: Biological Chemistry Laboratory, Unicamp, C.P. 6154, Campinas, CEP 13083-970, SP/ Brazil E-mail: priscyla@iqm.unicamp.br

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(Lee et al., 2003). Moreover, a new generation of dressing incorporating antimicrobial agents like silver and iodine have been studied (Schaller et al., 2004).

2. Materials and Methods

The F. oxysporum strain used was 07 SD strain from ESALQ-USP Genetic and Molecular Biology Laboratory-Piracicaba, S.P., Brazil. The fungal inoculated were prepared in a malt extract 2% and an yeast extract 0.5% at 28o C in Petri plates. The liquid fungal growth was carried out in the presence of an yeast extract 0.5% at 28oC for 6 days. The biomass was filtrated and resuspended in sterile water. 2.1. Silver reduction Approximately 10 g of F. oxysporum biomass was taken in a conical flask containing 100 mL of distilled water, kept for 72 h at 28oC and then the aqueous solution components were separated by filtration. To this solution (liquid fungal), AgNO3 (10-3 M) was added and kept for several hours at 28oC. Periodically, aliquots of the reaction solution were removed and the absorptions were measured in a UV-Vis spectrophotometer (Agilent 8453 - diode array).

2.2. Silver nanoparticles-incorporating The cloth was immersed in the filtrate fungal and centrifuged (3500 rpm) for 15 minutes and dryed after that.

2.3. Antibacterial evolution The antibacterial proprieties were evaluation against Staphylococcus aureus, a Gram-positive bacterium. The cloths were placed on germ containing agar plates, inoculated with S. aureus and then incubated in an agar media. Inoculum concentration was 1.3-1.6 105/mL and 0.5% non-ionic agent. The cloths were coated with carbon under vacuum and their scanning electron micrographs were obtained using a Jeol (JSM-6360LV) scanning electron microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) at a voltage of 20 kV.

3. Results and Discussion

The Erlenmeyer flasks with the F. oxysporum biomass were a pale yellow color before the addition of Ag+ ions and this change to a brownish color on completion of the reaction with Ag+ ions for 28 h. The appearance of a yellowish-brown color in solution containing the biomass was a clear indication of the formation of silver nanoparticles in the reaction mixture (Sastry et al., 1998). The UV-Vis spectrum of the F. oxysporum reaction vessels at different times of reaction is presented in Fig.1.

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Fig. 1: UV-Vis spectrum recorded as a function of time of reaction in an aqueous solution of 10-3 M AgNO3 with the liquid fungal.

The SEM micrograph (fig. 2A) shows silver nanoparticles aggregates. In this micrograph observed spherical nanoparticles in the size range 20-50 nm. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by a capping agent. In The EDS spectrum (fig. 2B) observed a strong signals from the silver atoms in the nanoparticles and weaker signals from N, P and S atoms. These weaker signals were provenients from proteins/enzymes present in the wall of the fungus.

Fig. 2: A) SEM micrograph of the silver nanoparticles, B) EDS spectrum of the silver nanoparticles

These silver nanoparticles were incorporation in cotton and silk cloths. Antibacterial activity was observed when silver nanoparticles were incorporated in cotton cloth like shows fig. 3 and 4. In the control cotton cloth (fig. 3) was observed the presence of the bacteria. But, in the cotton cloth with silver nanoparticles were not observed (fig. 4). In the other hand, in the silk cloth activity was not observed demonstrating a low incorporation of the silver nanoparticles due possibly the size of the pores of the silk. The incorporation of the silver nanoparticles in the cloths were verified by EDS. In the silk cloth was not verificated the incorporation, however, this incorporation was verified in cotton cloth as shows the fig. 5.

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Fig. 3: SEM micrograph of the cotton fiber without silver nanoparticles incorporation (controlled).

Fig. 4: SEM micrograph from cotton fiber with silver nanoparticles incorporation.

Fig. 5: EDS spectrum of the cotton cloth with silver nanoparticles. The inset shows the SEM micrograph of the cotton fibers.

4. Conclusion
Silver nanoparticles synthesized by F. oxysporum strain incorporated in cotton cloth exhibition antibacterial activity against S. aureus. This study demonstrated the possible of the use of biological synthesized silver nanoparticles incorporated in materials with the objective to become them steriles. These particles could have innumerable applications, such as non-linear optics, spectrally selective coating for solar energy absorption and intercalation material for electrical batteries and others.

5. References
Ahmad, A., Mukherjee, P., Senapati, S., Mandal, D., Khan, M.I., Kumar, R., Sastry, M. (2003). Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids Surf. B. 28, 313.

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Fortin, D., Beveridge, T.J. (2000). Mechanistic routes towards biomineral surface development. In: Biomineralisation: From Biology to Biotechnology and Medical Application (E. Baeuerlein Ed.), Wiley-VCH, Verlag, Germany, 294. Lee, H.J., Yeo, S.Y., Jeong, S.H. (2003). Antibacterial effect of nanosized silver colloidal solution on textile fabrics. J. Mater. Sci. 38, 2199
a

Mukherjee, P., Ahmad, A., Mandal, D., Senapati, S., Sainkar, S.R., Khan, M.I., Ramani, R., Parischa, R., Ajaykumar, P.V., Alam, M., Sastry, M., Kumar, R. (2001). Bioreduction of AuCl4- ions by the fungus, Verticillium sp. and surface trapping of the gold nanoparticles formed. Angew. Chem. Int. Ed. 40, 3585.

Mukherjee, P., Ahmad, A., Mandal, D., Senapati, S., Sainkar, S.R., Khan, M.I., Parischa, R., Ajayakumar, P.V., Alam, M., Kumar, R., Sastry M. (2001). Fungus-mediated synthesis of silver nanoparticles and their immobilization in the mycelial matrix: A novel biological approach to nanoparticle synthesis. Nano Lett. 1, 515.

Sastry, M., Patil, V., Sainkar, S.R. (1998). Electrostatically controlled diffusion of carboxylic acid derivatized silver colloidal particles in thermally evaporated fatty amine films. J. Phys. Chem. B 102, 1404. Sastry, M., Ahmad, A., Islam, N.I., Kumar, R. (2003). Biosynthesis of metal nanoparticles using fungi and actinomycete. Current Sci. 85, 162. Schaller, M., Laude, J., Bodewaldt, H., Hamm, G., Korting, H.C. (2004). Toxicity and antimicrobial activity of a hydrocolloid dressing containing silver particles in an ex vivo model of cutaneous infection. Skin Pharm. Physiology 17, 31.

Acknowledgments
Supports from Brazilian Network of Nanobiotechnology, CNPq/MCT and FAPESP are acknowledged. We acknowledge Dr. Fernando de Oliveira from NCA-UMC for the UV-Vis analyses.