Biosynthesis of silver Nanoparticles from marine sponge Callysspongia diffusa associated-P . fluorescens BCPBMS-1

New applications of nanomaterials are rapidly emerging. The synthesis of nanoparticles is a cornerstone of nanotechnology. Microbial cells are highlyorganized units, regarding morphology and metabolic pathways, capable of synthesizing well size-calibrated and well-structured particles. Furthermore, biogenic nanoparticles often are water-soluble and biocompatible, which is essential for many applications. Molecular identification of a novel strain P. fluorescens BCPBMS-1 from sponge Callysspongia diffusa (Mandapam Coast) through 16s rRNA ribotyping (Gen bank accession number: HQ907732). The silver nanoparticles were analyzed by UV-Visible spectroscopy. Their chemical composition was determined by FT-IR spectroscopy. SEM observationrevealed that silver nano particles are having spherical shape. The antibacterial activities of silver nanoparticles were screened against common human pathogen Escherichia coli, Proteus mirabilis, Salmonella typhi, Salmonella paratyphi, Vibrio cholerae, Klebsiella oxytoca, Klebsiella pneumoniae and Staphylococcus aureus. Among these 5mm antibacterial activity was observed with E. coli, 4mm with P. mirabilis and S. typhi, 3mm activity was observed with S. paratyphi. These results suggest that Ag nanoparticles can be used as effective growth inhibitors in various microorganisms, making them applicable to diverse medical devices and antimicrobial control systems.


INTRODUCTION
Marine microbial biotechnology has opened up unexpected new horizons for finding novel organism for trapping their potential resources .Oceans account for more than 70% of the earth's surface and the microorganisms growing in marine environments are metabolically and physiologically diverse from the terrestrial organisms (Takizawa et al., 1993).
Ag-NPs (silver nanoparticles) are used in hygienic products including water Purification systems, linings of washing machine, dishwashers, refrigerators, and toilet seats (Rai et al., 2009) Thus, silver ions have been used in many kinds of formulations (Sondi & Salopek-Sondi, 2004), and recently it was shown that hybrids of silver nanoparticles with amphiphilic hyper branched macromolecules exhibit effective antimicrobial surface coating (Ymonier et al., 2002).One of the most studied aspects of nanotechnology nowadays is their ability to offer the opportunity to fight microbial infections via synthesis of nanoparticles.The mechanism of prevention of bacterial growth by antibiotics is quite different from the mechanisms by which nanoparticles inhibit microbial growth.Therefore, nanoparticles have the potential to serve as an alternative to antibiotics and to control microbial infections.
Chemical synthesis of nanoparticles leads to presence of traces of toxic chemical adsorbed on the surface which is undesirable in the medical applications of nanoparticles.Biosynthesis of silver nanoparticles provides an alternative to chemical and physical methods as it is cost effective, environment friendly, and it does not involve use of high pressure, energy, temperature and toxic chemicals.Currently, there is a growing need to development of environmentally safe processes for synthesis of nanoparticle that do not use toxic chemicals in the synthesis protocol.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 & Beveridge, 2000).
The biosynthesis of silver nanoparticles of different sizes, ranging from 1-70nm, and shapes, including spherical, triangular and hexagonal has been conducted using bacteria, fungi, plant extracts.The mechanisms for the bioreduction of silver by bacteria involve reducing and other proteins in which sulphur and carboxylate group from cell wall (Sathyavathi et al., 2010;Bhainsa & De'Souza, 2006).
Various salts of silver and their derivatives are used as antimicrobial agents (Russell & Hugo, 1994).Recent studies have reported that nanosized silver particles exhibit antimicrobial properties (Petica et al., 2008) Silver has been recognized to have inhibitory action on microbes (Rai et al., 2009).

Isolation of Pseudomonas sp. from the marine sponge Callyspongia diffusa
The sponge was collected from Mandapam.It is situated (79˚8´E,9˚17´N) on a narrow tongue of land projecting from the southern part of the east coast of india.To the north of this penisular extension is in the Palk Bay and to the south the Gulf of Mannar.
The sponge sample soon after collection was transferred to a sterile polyethylene bag and transported at 4ºC to the laboratory for the isolation of associated microbes.On reaching the laboratory, the invertebrate was brought to room temperature and cut aseptically into small pieces (2 × 2 cm) using a sterile scissors .The pieces were freed from adhering particles by vortexing twice for 20 sec.with 2 ml of sterile seawater.The seawater was decanted, which was once again replaced with sterile seawater with continued vortexing between washings.Finally, sample in sterile seawater was homogenized using sterilized mortar and pestle in a Laminar flow chamber.The homogenate was serially diluted up to 10 −6 dilutions and then spread plated on King b agar (Peptone 20.0g, Dipotassium hydrogen phosphate1.5g,Magnesium sulfate 1.5g, Agar 10.0g, 50% Aged Sea water-1000ml, pH 7.2 +/-0.2).The plates were incubated at room temperature for 24-48 hrs.

Identification of Pseudomonas sp by 16S r RNA partial sequencing
The genomic DNA extracted from the marine sponge associated Pseudomonas sp was PCR amplified for 16S rRNA genes using the universal bacterial primers Eubac27F (5'-AGA GTT TGA TCG TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3').This primer combination amplified a 1500bp 16S rDNA fragment.
The PCR product was purified using the QIAGEN PCR purification kit for Sequencing and further analysis.The partial 16S r RNA gene sequencing was done using Perkin Elmer applied biosystems and ABI Prism software was used to allign the sequence and compared sequences were retrieved by the queries generated by BLAST of Gen Bank Database.Phylogenetic analysis was performed with the MEGA 4.0 program (Molecular Evolutionary Genetics Analysis, Version 4.0).

UV-Vis Spectra analysis
About 50 mL aqueous solution of 1 mM silver nitrate (AgNO3) was treated with 50 mL of P.fluorescens supernatant solution in a 250 mL Erlenmeyer flask (pH adjusted to 8.5).The whole mixture was put into a shaker at 40°C (200 rpm) for 5 days and maintained in the dark.Control experiments were conducted with un inoculated media, to check for the role of bacteria in the synthesis of nanoparticles.The reduction of Ag + ions was monitored by measuring the UV-Vis spectrum of the reaction medium after 72 hrs.UV-Vis spectral analysis was done by using UV-Vis spectrophotometer UV-2450 (Shimadzu).The nanoparticles were separated from the reaction mixture by centrifugation at 10,000 rpm for 10 minutes at 4°C (Saifuddin et al., 2009).

FT-IR analysis of silver nanoparticles
The supernatant containing silver nanoparticles was lyophilized.It was subjected to FTIR analysis.The interaction between protein and silver nanoparticles were analysed by Fourier transform infrared analyzer (Perklin Elmer).

SEM analysis of silver Nanoparticles
The lyophilized samples were for SEM.Scanning Electron Microscopic (SEM) analysis was done by using JEOL JSM-6360 SEM machine.

Antibacterial activity of silver Nanoparticles
Antibacterial assay was done by an agar-well diffusion method in aerobic condition.Bacterial pathogens such as Escherichia coli, Proteus mirabilis, Salmonella typhi, Salmonella paratyphi, Vibrio cholerae, Klebsiella oxytoca, Klebsiella pneumoniae and Staphylococcus aureus were spreaded on Muller Hinton agar plates.Then wells were made and 50 µl of silver nanoparticles was inoculated.Antagonistic activity was detected after an incubation of 24-48 hrs at 35°C.The presence of zone of clearance on agar plates was used as an indicator for the antibacterial activity.50 µl of distilled water was used as control.

RESULTS
In the present study Phylogenetic tree revealed that P. fluorescens BCPBMS-1 (bioactive compound producing bacteria) which was isolated from marine sponge Callyspongia diffusa was closely related to P. fluorescens CIAH-Pf-196-16s strain (Fig. 1).
The tree Topologies were evaluated by bootstrap analyses based on 1,000 replicates and phylogenetic trees were inferred using the neighbour-joining method and submitted to NCBI Gen Bank (accession number: 1428145 HQ907732).
The appearance of a brown colour in solution containing the supernatant is a clear indication of the formation of silver nanoparticles in the reaction mixture (Fig. 2).The colour of the solution is due to the excitation of surface Plasmon vibrations (essentially the vibration of the group conduction electrons) in the silver nanoparticles.The colour intensity of the cell filtrate with AgNO 3 was sustained even after 5 days incubation, which indicated that the particles were well dispersed in the solution, and there was no obvious aggregation.The spectra recorded from the P. fluorescens BCPBMS-1 reaction vessel at different wavelength were reported.The strong surface Plasmon resonance centered at ca. 350-400 nm.Increased in the peak was observed at 400 nm.The spectra clearly show the increase in intensity of silver solution indicating the formation of silver nanoparticles in the solution.
FT-IR is a powerful tool for identifying types of chemical bonds in a molecule by producing an infrared absorption spectrum that is like a molecular "fingerprint".FT-IR analysis was carried out to identify the possible bio-molecules and cell-metal ions interaction responsible for formation and stabilization of silver nanoparticles.
In the present investigation strong stretching bonds O-H occurred 3434 cm -1 .There is a very strong C-H stretching vibration occurred at the wave number 2958 cm -1 .The strong medium Methyl group has occurred at 2923 cm -1 and 2852 cm -1 .The medium strong 2427 cm -1 shows the O-H.The wave number 2362 cm -1 , 2344 cm -1 2092 cm -1 and 1717 cm -1 shows the C=O stretching vibration.The medium stretching N -H band has occurred at 1654 cm-1 , 1647 cm -1 , 1630 cm -1 .1637 cm -1 showed C=O carboxyl group and -C=C stretching .1458cm -1 shows amine and amino methyl stretching.The wave number 1438 cm -1 , 1384 cm -1 showed the medium strong C-N stretching.A medium strong stretching vibration occurred at the wave number 1245 cm -1 .A medium weak stretching occurred at the wave number 1082 cm -1 .A medium strong stretching C-O band has occurred at 919 cm -1 .The wave number 555 cm -1 showed the strong medium rocking vibration represents C-Cl bond (Fig. 3).This SEM micrograph showed that 10µm size of nanoparticles synthesized from P. fluorescens BCPBMS-1 (Fig. 4).The SEM micrographs of nanoparticle obtained in the filtrate showed that Ag-NPs are spherical shaped, well distributed without aggregation in solution.In the present investigation among the tested pathogens antibactreial activity was observed with E. coli , P. mirabilis, S. typhi, S. paratyphi,.P. mirabilis.Whereas it was absence with S. aureus and V. cholera.Among these 5mm antibacterial activity was observed with E. coli, 4mm with P. mirabilis and S. typhi, 3mm activity was observed with S. paratyphi (Figs. 5,6 &7).