Journal of Microscopy and Ultrastructure 3 (2015) 29–37 Contents lists available at ScienceDirect Journal of Microscopy and Ultrastructure jo urnal homepage: www.elsevier.com/locate/jmau Original Article Ultrastructures of silver nanoparticles biosynthesized using endophytic fungi ∗ Lamabam Sophiya Devi, S.R. Joshi Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong 793 022, Meghalaya, India a r t i c l e i n f o a b s t r a c t Article history: Three endophytic fungi Aspergillus tamarii PFL2, Aspergillus niger PFR6 and Penicllium Received 12 August 2014 ochrochloron PFR8 isolated from an ethno-medicinal plant Potentilla fulgens L. were used for Received in revised form 6 October 2014 the biosynthesis of silver nanoparticles. Scanning and transmission electron microscopic Accepted 14 October 2014 analysis were performed to study the structural morphology of the biosynthesized silver Available online 28 October 2014 nanoparticles. The electron microscopy study revealed the formation of spherical nano- sized silver particles with different sizes. The nanoparticles synthesized using the fungus A. Keywords: tamarii PFL2 was found to have the smallest average particle size (3.5 ± 3 nm) as compared Electron microscopy to the nanoparticles biosynthesized using other two fungi A. niger PFR6 and P. ochrochloron Endophytic fungi PFR8 which produced average particle sizes of 8.7 ± 6 nm and 7.7 ± 4.3 nm, respectively. Silver nanoparticles Crystalline The energy dispersive X-ray spectroscopy (EDS) technique in conjunction with scanning electron microscopy was used for the elemental analysis of the nanoparticles. The selected area diffraction pattern recorded from single particle in the aggregates of nanoparticles revealed that the silver particles are crystalline in nature. © 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved. 1. Introduction surface defects [1–3]. They have tremendous applications in the area of catalysis, opto-electronics, diagnostic bio- Nanoparticles are the clusters of atoms in the size range logical probes, display devices and photo electrochemical of 1–100 nm. In this size range, materials often develop applications due to their unique size-dependent optical, useful attributes that are distinct from the properties of electrical and magnetic properties [4–9]. the bulk material. Metal particles in the nanometre size There has been a rapid increase in microbes that are exhibit unique physical properties that are different from resistant to conventionally used antibiotics [10]. This has both the ion and the bulk material. Their uniqueness arises resulted in an inevitable and urgent need for develop- specifically from higher surface to volume ratio which ment of novel antimicrobial agents. It has been known results in increased catalytic activity due to morphologies since ancient times that silver and its compounds are with highly active facets; hence, the nanosize materi- effective antimicrobial agents [11–13]. Compared with als are more advantageous than their bulk materials. The other metals, silver exhibits higher toxicity to broad spec- enhanced reactivity of nanomaterials can also be attributed trum of microorganisms while it exhibits lower toxicity to their large number of edges, corners, and high-energy to mammalian cells [14]. Silver ion has been known to be effective against a broad range of microorganisms includ- ing antibiotic-resistant strains [15]. Silver nanoparticles ∗ with higher surface to volume ratio compared to common Corresponding author. Tel.: +91 9436102171; fax: +91 3642550076. E-mail address: [email protected] (S.R. Joshi). metallic silver have shown better antimicrobial activity. http://dx.doi.org/10.1016/j.jmau.2014.10.004 2213-879X/© 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved. 30 L.S. Devi, S.R. Joshi / Journal of Microscopy and Ultrastructure 3 (2015) 29–37 Fig. 1. Reproductive structures of the endophytic fungal isolates as seen under compound microscope: (a) PFL2, (b) PFR6 and (c) PFR8. Most importantly silver nanoparticles are also non-toxic effectively reacting with them leading to the inhibition to the mammalian cells at low concentrations [16]. Sil- of enzyme functions [18,19]. The nanoparticles bind to ver nanoparticles have been known for a long time but proteins and DNA and damage them by inhibiting replica- have not been given due attention [17]. Nanoparticles tion. Thus the silver nanoparticles interrupt the respiratory can disturb functions of cell membranes such as per- chain and cell division leading to cell death [20–22]. In meability and respiration. The silver nanoparticles get addition, complex action mechanisms of metals decrease attached to the cell membrane and also penetrate inside the probability of bacteria developing resistance to them the bacteria. Inside the bacterial cells silver nanoparti- [23]. Thus, one of the promising approaches for overcom- cles can disturb the functions of sulfur-containing proteins ing antibiotic resistance of microorganisms is the use of and phosphorus-containing compounds such as DNA by silver nanoparticles. Fig. 2. Phylogenetic relationships between the three endophytic fungi and the ITS sequences of closely related fungal strains retrieved from NCBI GenBank. L.S. Devi, S.R. Joshi / Journal of Microscopy and Ultrastructure 3 (2015) 29–37 31 Fig. 3. UV–vis absorption spectrum at different time intervals for silver nanoparticles biosynthesized using (a) Aspergillus niger PFR6, (b) Penicillium ochrochloron PFR8 and (c) Aspergillus tamarii PFL2. Owing to the fact that the endophytic fungi provide with 70% alcohol for 1 min. The final rinsing was done a broad variety of bioactive secondary metabolites with with sterile distilled water and blot dried on sterile filter unique structures they could be the explored for their abil- paper. The excess water was dried and the surface sterilized ity to biosynthesis of silver nanoparticles to developed explants were then inoculated onto PDA plates. The plates an efficient environment friendly process. Few studies on were periodically observed for fungal growth. The fungus the biosynthesis of silver nanoparticles using endophytic growing out from the plant explants was then sub-cultured fungi like Amylomyces rouxii, Penicillium sp., P. janthinel- in PDA plates. Three morphologically distinct endophytic lum, Pestalotia sp., Aspergillus clavatus, A. concius, Epicoccum fungi isolated from an ethno-medicinal plant P. fulgens L. nigrum and Phomopsis sp. have shown the proficiency of were used for the biosynthesis of silver nanoparticles (Fig. 1 the endophytic fungi to synthesize silver nanoparticles a–c). possessing antimicrobial activity [24–31]. In the present The fungal isolates were characterized based on the study, an attempt has been made to investigate endophytic analysis of the phylogenetic relationship of the iso- fungi associated with an ethnomedicinal plant Potentilla lates using internal transcribed spacer (ITS) region gene fulgens L. for their ability to synthesize silver nanopar- sequences. Amplification of ITS (ITS1, 5.8S and ITS2) region ticles and the ultrastructure and size distribution of the using primers ITS1 5 -TCCGTAGGTGAACCTGCGG-3 and biosynthesized nanoparticles were studied using electron ITS4 5 -TCCTCCGCTTATTGATATGC-3 [33] were performed microscopy techniques. using a 9700 Gold thermal cycler (Applied Biosystems, UK) under the following conditions: initial denaturation at ◦ ◦ 94 C for 1 min, annealing temperature at 52 C for 1 min 2. Materials and methods ◦ and extension at 72 C for 1 min with an initial dena- turation and final extension for 5 and 10 min at 94 and 2.1. Isolation and characterization of endophytic fungi ◦ 72 C, respectively. For sequencing, the amplified ITS prod- ucts were purified using QIAquick Gel Extraction Spin Kit The endophytic fungi were isolated according to the (QIAGEN, Germany). The purified PCR products were bi- method described by Strobel et al. [32] with minor mod- directionally sequenced using the forward and reverse ifications. The plant material was gently rinsed in running primers. Sequencing of the ITS gene products were per- water to remove any adhering dirt. The samples were then formed using the Big DyeTerminator v3.1 Cycle Sequencing cut into 2 mm blocks and were surface sterilized with 70% Kit (Applied Biosystems, USA). Sequencing of ITS region ethanol for 1 min. Then the samples were soaked in 4% of fungal rDNA resulted in approximately 564 bp long sodium hypochlorite solution for 3 min, and then rinsed 32 L.S. Devi, S.R. Joshi / Journal of Microscopy and Ultrastructure 3 (2015) 29–37 Fig. 4. SEM micrographs of the biosynthesized silver nanoparticles using endophytic fungi (a) Aspergillus tamarii PFL2, (b) Aspergillus niger PFR6 and (c) Penicillium ochrochloron PFR8. nucleotide sequences. The nucleotide sequences were used flask and agitated again at 120 rpm. After the incubation, for the identification of the fungal isolates. Molecular Evo- the cell filtrate was obtained by filtering it through What- lutionary Genetics Analysis (MEGA version 5) software was man filter paper No. 1. The filtrates were treated with 1 mM used for phylogenetic analyses [34]. The closest homo- silver nitrate (Sigma Aldrich) solution in an Erlenmeyer logues to the sequences were selected and the multiple flask and incubated at room temperature in dark. Control sequence alignments were carried out using the ClustalW containing cell-free