Silver nitrate (PubChem CID: 24,470); sulphanilamide (PubChem CID: 5333); N-(1-naphthyl) ethylene diamine dihydrochloride (PubChem CID: 15,106); potassium nitrate (PubChem CID: 24,434); propanol (PubChem CID: 3776).
A batch of 20 different filamentous fungal strains (identified as: IPT825, IPT827, IPT829, IPT849, IPT853, IPT859, IPT868, IPT856, IPT1005, IPT1008, IPT1009, IPT1010, IPT1011, IPT1012, IPT1013, IPT1014, IPT1015, IPT1016, IPT1017, and IPT1018), previously isolated from sugar cane plantation soil, was supplied by Instituto de Pesquisa Tecnológica do Estado de São Paulo (IPT, São Paulo, Brazil). All strains were maintained on malt extract agar (MEA; 20 g/L malt extract, 20 g/L glucose, 1 g/L peptone and 15 g/L agar) as stock cultures at 4 °C. These were maintained by regular subculturing.
Biosynthesis of silver nanoparticles
All 20 strains were screened for the biosynthesis of AgNP through the process hereby described. From the stock cultures grown in MEA, a 6 mm diameter disk from the peripheral area of the colony was transferred into a new Petri dish containing malt-glucose-yeast and peptone (MGYP) medium (3.0 g/L malt extract, 10.0 g/L glucose, 3.0 g/L yeast extract, 5.0 g/L peptone and 15 g/L agar) and incubated for 7 days at 30 °C in the dark. Fungal biomass was obtained by inoculating 5 culture disks (6 mm diameter) of each strain in Erlenmeyer flasks containing 100 mL of MGYP broth (3.0 g/L malt extract, 10.0 g/L glucose, 3.0 g/L yeast extract, and 5.0 g/L peptone). Cultures were incubated in an orbital shaker (Quimis, Brazil), for 120 h at 30 °C and 200 rpm. Biomass was then harvested by filtration through Whatman filter paper Grade 3 and was washed three times with sterile distilled water. Wet fungal mycelia (10 g) were suspended in 100 mL of sterilised distilled water and incubated at 30 °C with agitation (200 rpm) for 72 h. After this period, cell-free filtrate was collected by filtration through Whatman filter paper Grade 3. Suspensions were filtrated through a 0.22 µm filter (Millipore) and treated with a silver nitrate (AgNO3) solution (1 mM), followed by incubation at 30 °C with agitation (200 rpm), for 120 h in the dark.
UV–vis absorption spectra (UV–vis)
UV–vis is a widespread method of detection of AgNP (Chan and Don 2012). When bioreduction of AgNP occurred, a change in colour was observed in the AgNO3 solution, which turned from yellow into brown. This effect has been reported as an indicator of surface plasmon resonance (SPR) of AgNP (Chan and Don 2013). The position of the plasmonic band detected on the solutions of metallic nanoparticles is dependent on several parameters such as: size, shape, and polydispersity of particles. And, the more the narrow is the band, the bigger is the uniformity index of distribution according to AgNPs size (Becaro et al. 2015). Even though, there was no monitoring of the increase in absorbance until its maximum value, it has been described in the literature that the incubation period used in this study allows for the detection of maximum absorbance, implying the maximum concentration of synthesised AgNPs (Muthukrishnan et al. 2015). The UV–visible spectra of this solution was then recorded on UV–Vis Hitachi U-2000 spectrophotometer (Hitachi, Japan) in a range between 200 and 800 nm.
Transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDX)
The size and shape of the synthesized AgNP were also determined by transmission electron microscopy (TEM) as described by Singhal et al. (2011). A JEOL electronic microscope (model JEM-2100) operated at 200 kV was used for TEM analysis. The average nanoparticle sizes were measured by counting approximately 100 nanoparticles in different regions of each sample, which were then used for the construction of histograms and determination of the average size of the nanoparticles. Energy dispersive X-ray spectroscopy (EDX), model JEOL-JSM 5410 LV (JEOL, USA). To prepare each sample, AgNP were sonicated for 5 min, and a drop of a diluted sample was placed onto a carbon-coated copper grid for analysis.
X-ray diffraction analysis (XRD)
X-ray diffraction was carried out using a Rigaku, Miniflex II diffractometer (Rigaku, Brazil), equipped with Cu Kα (0.15406 nm) at 40 kV and 30 mA. The diffractograms were recorded over the range 20–90 angles. Lyophilized nanoparticles were placed on a glass grid containing silicon substrate for XRD analysis.
Size and distribution analysis
For the aqueous suspension containing the AgNP, previously filtered through a 0.22 μm filter, the size distribution and average size of the synthesized AgNP were determined by Dynamic Light Scattering (DLS), Zetasizer Nano ZS90 (Malvern Instruments, UK).
Nitrate reductase activity
Nitrate reductase activity in the fungal filtrate was assayed by determining the presence of the extracellular enzyme according to the procedure described by Hamedi et al. (2013). Succinctly, the cell filtrate (5 mL) was mixed with an assay medium (30 mM KNO3 and 5% propanol in 0.1 M phosphate buffer pH 7.5) in a 1:1 (v/v) proportion and incubated at 30 °C, in the absence of light for 1 h. A sulphanilamide (SA) solution and a N-(1-naphthyl) ethylene diamine dihydrochloride (NEED) solution were added to the mixture. The released nitrites on the assay medium then reacted with the SA and NEED solutions and converted into a pink azodye. The absorbance of the resultant pink solutions was measured by UV–visible spectrophotometry, at 540 nm. The enzyme activity of the fungal cell-free filtrate was determined based on the increase in nitrite content of the solution over 1 h and expressed as nmol nitrite/h mL.
Antibacterial assay of silver nanoparticles
The antimicrobial activities against the Gram-positive bacteria: Staphylococcus aureus IPT246; and the Gram-negative bacteria: Escherichia coli IPT245, and Pseudomonas aeruginosa IPT322, were determined by agar plate well diffusion assay. Bacteria were cultured in Mueller–Hinton agar (MHA; 2.0 g/L beef extract, 17.5 g/L casein hydrolysate, 1.5 g/L starch, and 17 g/L agar), MHA, applying 100 μL of an initial inocullum (106 CFU/mL) of each strain in the agarised media and uniformly spreading. Subsequently, 100 μL of a AgNP solution at 1.0, 5.0, 10, 50, and 100 μg/mL concentrations were added into 3 mm diameter wells, cut out in the centre of the plate, and incubated at 37 °C for 24 h. Streptomycin solutions (100 μL) were used, in the same concentration values, as positive control, and water as negative control. After incubation, the zones of inhibition were measured. The assays were performed in triplicate.