Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains
- Nelson Durán1, 2Email author,
- Priscyla D Marcato†1,
- Oswaldo L Alves†3,
- Gabriel IH De Souza†2 and
- Elisa Esposito†2
© Durán et al; licensee BioMed Central Ltd. 2005
Received: 11 January 2005
Accepted: 13 July 2005
Published: 13 July 2005
Extracellular production of metal nanoparticles by several strains of the fungus Fusarium oxysporum was carried out. It was found that aqueous silver ions when exposed to several Fusarium oxysporum strains are reduced in solution, thereby leading to the formation of silver hydrosol. The silver nanoparticles were in the range of 20–50 nm in dimensions. The reduction of the metal ions occurs by a nitrate-dependent reductase and a shuttle quinone extracellular process. The potentialities of this nanotechnological design based in fugal biosynthesis of nanoparticles for several technical applications are important, including their high potential as antibacterial material.
The dissimilatory ferric reductase, which are found in bacteria are an essential part of the iron cycles  and are essentially intracellular, but one extracellular one was isolated from Mycobacterium paratuberculosis . Another possible mechanism could be active in this process since it was discovered that some bacteria reduce Fe3+ oxides by producing and secreting small, diffusible redox compounds that can serve as electron shuttle between the microbe and the insoluble iron substrate . The role of excreted compounds in extracellular electron transfer was recently reviewed .
The presence of hydrogenase in fungus as Fusarium oxysporum was demonstrated with washed cell suspensions that had been grown aerobically and anaerobically in a medium with glucose and salts amended with nitrate . The nitrate reductase was apparently essential for ferric iron reduction . Many fungi that exhibit these characteristic properties, in general, are capable of reducing Au (III) or Ag (I) . Besides these extracellular enzymes, several naphthoquinones [8–10] and anthraquinones  with excellent redox properties, were reported in F. oxysporum that could be act as electron shuttle in metal reductions .
Although it is known that 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 . Using these dissimilatory properties of fungi, the biosynthesis of inorganic nanomaterials using eukaryotic organisms such as fungi may be used to grow nanoparticles of gold  and silver  intracellularly in Verticillium fungal cells . 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 . Other process, which was described in the literature, was related to produce silver nanoparticles through oligopeptides catalysis, precipitating the particles with several forms (hexagonal, spherical and triangular) . However, in the fungal reduction of Ag ions led colloidal suspension, differently that in the oligopeptides case. Then the mechanistic aspects are still an open question, however this process occur in the fungal case probably either by reductase action or by electron shuttle quinones or both. Our aims in this research are to compare different strains of F. oxysporum in order to understand if the efficiency of the reduction of silver ions is related to a reductase or quinone action.
Results and Discussion
Even though gold/silver nanoparticles have been synthesized using prokaryotes such as bacteria [24, 25] and eukaryotes such as fungi [13, 14], the nanoparticles grow intracellularly, except in the case of a recent report in which F. oxysporum was used. In that case the nanoparticles grew extracellularly . In our case, all the F. oxysporum strains studied exhibited silver nanoparticle production capacity, however, depending on the reductase/electron shuttle relationships under these conditions. Biologically synthesized silver nanoparticles could have many applications, in areas such as non-linear optics, spectrally selective coating for solar energy absorption and intercalation materials for electrical batteries, as optical receptors, catalysis in chemical reactions, biolabelling , and as antibacterials capacity .
The F. oxysporum strains used were the following: O6 SD, 07 SD, 534, 9114 and 91248 from ESALQ-USP Genetic and Molecular Biology Laboratory-Piracicaba, S.P., Brazil. The fungal inoculates were prepared in a malt extract 2% and yeast extract 0.5% at 28°C in Petri plates. The liquid fungal growth was carried out in the presence of yeast extract 0.5% at 28°C for 6 days. The biomass was filtrated and resuspended in sterile water.
Silver reduction and its characterization
Method A: In the silver reduction, the methodology described previously was followed . Briefly, approximately 10 g of F. oxysporum biomass was taken in a conical flask containing 100 mL of distilled water. 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 (Agilent 8453 – diode array).
Method B: Another test was also carried out as following: 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°C 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 28°C.
The silver nanoparticles were characterized by scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) at a voltage of 20 kV (Jeol – JSM-6360LV) and previously coated with gold under vacuum.
Determination of the electron-shuttling compounds
Release of electron-shuttling compounds was followed the methodology described previously : In order to determine the water-soluble quinones that might function as an electron shuttle, cultures were filtered 4–6 weeks, and the filtrate adjusted to pH 3 with HCl 1 M. The acidified solution was then passed through a column with ion exchange resin (Amberlite®) for absorption of the pigments. Compounds were removed from the column by elution with acetone, the acetone removed using a Buchi rotary evaporation and the aqueous phase extracted 3 times with ethyl acetate. All ethyl acetate extractions were combined and reduced using the rotary evaporator. After that, 2 μL samples were repeatedly spotted on a Silica gel 60 plate until a spot was visible under UV light at 254 nm. Samples were resolved using a chloroform-methanol-acetic acid (195:5:1) and benzene-nitromethane-acetic acid (75:25:2) system designed to mobilize polar pigments. Plates were air dried, and spots visualized under UV light .
Nitrate reductase assay
Nitrate reduction was demonstrated in the same medium (Method A and B) of the same growth broth of F. oxysporum with the addition of 0.1% of KNO3 . The nitrate reductase test was made after 2 days by fluorometric method . Briefly, 100 μL fungal filtrate and 200 μL of dionized water. To this, 10 μL of freshly prepared 2,3-diaminonaphtalene (DAN) (0.05 mg/mL in 1 M HCl) is added and mixed immediately. After 10 min incubation at 20°C, the reaction was stopped with 5 μL of 0.1 M NaOH. The intensity of the fluorescent signal produced by the product was maximized by the addition of base. The 2,3-diaminonapthotriazole formation was measured using a Perkin-Elmer (LS-55) luminescence spectrophotometer with and excitation wavelength at 375 nm and the emission band measured at 550 nm .
Determination of the tryptophan/tyrosine residues
Presence of tryptophan/tyrosine residues in proteins release in the fungal filtrated was analyzed by fluorescence . The fluorescence measurements were carried out on a Perkin-Elmer (LS-55) luminescence spectrophotometer. The exitation wavelength was 260 nm, close to maximal optical transitions of the tryptophan and tyrosine.
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 support.
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