INTRODUCTION

Nanoparticles are being viewed as the fundamental building blocks of nanotechnology, since they are the starting point for preparing many nano materials and devices. Nano biotechnology represents an economic alternative for chemical and physical methods of nano particles formation. These methods of synthesis can be divided on intracellular and extracellular. The nanoparticle synthesised by biological method has tremendous effect and application when compared to that of the physical and chemical method ofsynthesis. The actinomycetes are the gram positive bacteria which are well known for their antibiotic production which are belonging to unicellular micro-organisms. The Streptomyces spp. are the more commonly used among the various kinds of actinomycetes. The actinomycetes are the micro-organism which is sharing both the characters of fungi and bacteria.

The silver nanoparticle is produced by using biological synthesis method. Biosynthesis of nanoparticles is attracting many researchers because it is rapid, easy, cost effective and environmental friendly. The biological application of nanoparticles is a rapidly developing area of nanotechnology that raises new possibilities in the diagnosis and treatment.The biological applications of silver nanoparticles (Ag NPs) have been widely studied; antimicrobial properties in particular. The strong toxicity of silver against wide range of micro organisms is well known and silver nanoparticles showed to be promising antimicrobial materials. The biosynthesis of silver and gold NPs has been performed earlier using microorganisms primarily bacteria and eukaryotic organisms such as fungi and higher plants.There have been several reports on the synthesis of Silver NPs using different species of bacteria such asBacillus, Lactobacillus, Proteus, Staphylococcusaureus, Streptomyces.Klebsiella, Corynenobacterium, Geobacter, Brevibacterium, Sinorhizobium, Pseudomonas .sp.forvarious applications includingpharmaceutical and biological process. The antimicrobial and antifungal testing techniques including agar well diffusion, agar disc diffusion and tube dilution method. The Minimum Inhibitory Concentration (MIC) test for bacterial cells and fungal spore germination also has to be done.

SUMMARY OF INVENTION

This project work summarise that the actinomycetes is efficiently isolated from the marine soil sample by changing the composition of the Starch Casein Nitrate agar media. The isolated actinomycetes was successfully identified by using 16s rRNA sequencing method. The silver nanoparticle was synthesised from silver nitrate by Streptomyces pseudogriseolusand characterised by UV spectrophotometer, FTIR and TEM analysis. The anti microbial test was performed and confirmed that silver nanoparticle has the ability to inhibit the growth of the micro organism.

BACKGROUND OF INVENTION

The work in this project reveals that isolation, identification, synthesis and characterisation of silver nanoparticle from actinomycetes and its application of silver nanoparticle in anti microbial activity against various pathogens. The actinomycetes were isolated from the marine soil sample. The isolation of the actinomycetes is done with the starch casein nitrate agar media and the isolated culture was identified by 16s rRNA technology. Thus the isolated actinomycetes is confirmed as Streptomyces pseudogriseolusby 16s rRNA technology. The Streptomyces pseudogriseolusrwas used for the synthesis of the silver nanoparticle. The different concentration of the silver nitrate solution is added along with the Streptomyces pseudogriseolusand the solution was incubated at 37°C for 6 days in the shaking incubator at 150 rpm. After 6 days of incubation the colour change was observed in the conical flask. The change in colour obtained was brown from colourless solution. The initial confirmation was done with UV Spectrophotometer by having the wavelength at 422nm. A further confirmation for the synthesised silver nanoparticle was done by Fourier Transform Infrared Spectroscopy and Transmission Electron Microscopy. From the above mentioned characterisation methods, the particle produced was confirmed as silver nanoparticle by its size and the morphology. The synthesised and confirmed silver nanoparticle was used in the application of anti-microbial activity against various pathogens like Staphylococcusctureusandsalmonella. The media composition was changed slightly from the standard composition even though we got the effective growth of the actinomycetes.

OBJECTIVE OF INVENTION

• Isolation and identification of the actinomycetes by 16s rRNA sequencing.
• Synthesis of the silver nanoparticles by biological method.
• Characterization of silver nanoparticle.
• Anti-microbial activity of silver nanoparticle.

METHODOLOGY

SILVER NANOPARTICLE PRODUCTION

The silver nano particles are produced by biological synthesis method. The biomass was inoculated under standard condition kept for incubation for certain hours. After incubation AgNp was obtained.

Collection of Soil Sample

The soil sample was obtained from the coastal area, Parangipettai, India. The collected soil sample was air dried for about 5 days. The soil sample was washed twice with double deionised distilled water. The obtained soil sample was serially diluted for the isolation of actinomycetes. From the serial dilution the dilution rate taken for the actinomycetes was 10"2and 10"3. The serially diluted soil sample was grown in Starch Casein Nitrate Agar media for its growth and proliferation.

STARCH CASEIN NITRATE AGAR COMPOSITION

• Starch - 10 g
• Casein - 0.2 g
• Dipotassium hydrogen phosphate - 1.5 g
• Potassium Nitrate - 1.5 g
• Sodium Chloride - 2 g
• Magnesium sulphate - 0.02 g
• Calcium Carbonate - 0.05 g
• Agar - 20 g
• Distilled water -1000 ml

Culture preparation

The isolated actinomycetes were grown in starch casein nitrate broth media. A loop full of grown actinomycetes from the conical flask was taken and poured into plate supplemented with cyclohexamide and naldixic acid in the laminar air flow chamber to avoid the contamination. This was incubated at 37°C for 4 days in the incubator. Starch Casein Nitrate agar plates were made and the actinomycetes was streaked to it and kept in incubator at 37°C for 4 days. The growth of the organism was observed. This acts as the primary screening process for nanoparticles production.

IDENTIFICATION PROCEDURE

DNA isolation method

Growth from mature slant culture of the actinomycetes were inoculated aseptically into 250 ml Erlenmeyer flasks each containing 30 ml of Nutrient broth medium (beef extract 2 g/1, yeast extract 2 g/1, peptone 5 g/1 and sodium chloride 8 g/1, pH 7.1 after sterilization) and incubated in a rotary shaker at 30°C for 2 days at 180 rpm. Cultures were centrifuged at 10,000 rpm for 10 minutes. The 0.1 g of mycelium was transferred in to sterile porcelain dish and crushed with liquid nitrogen. The crushed mycelium was transferred into fresh tube containing 500 ul of TE buffer supplemented with lysozyme (20 mg/ml). The tube was incubated at 37°C for 30 minutes.

Biomass preparation of Streptomycespseudogriseolus

Streptomyces pseudogriseolus were grown in 500 ml Erlenmeyer flask containing 100 ml sterile Starch Casein Nitrate broth supplemented with cyclohexamide and naldixic acid 100 ug/ml on shaker at 200 rpm at 37°C for four days. The flasks were removed from the shaker and kept at steady condition at 5 to 10°C, so that mycelial bio mass get settled. The supernatant was discarded and 100 ml of sterile distilled water was added for washing the cells. The flasks were kept at 5 to 10°C for 30 minutes to settle the biomass. Supernatant was discarded slowly, 100 ml of sterile distilled water was added and repeated for three times. The mycelial mass was then separated from the sterile distilled water by centrifugation at 1500 rpm for 20 minutes; mycelial pellets were weighed and used for synthesis of silver nanoparticles.

Exposure of biomass to metal solutions

Five grams of actinomycetal wet biomass was exposed to 50ml of an sterilized aqueous solution of metals at various dilutions as given above in 250 ml Erlenmeyer flasks and the flasks were kept on shaker at 200 rpm and 37°C for 6 days.

Inoculation into different concentration of silver nitrate solution

The concentration of Silver Nitrate solution at which silver nanoparticle produced was detected. For this IMm stock Silver Nitrate solution viz 8.0mM, 1.6mM, 2mM were taken in Erlenmeyer flask. To each flask 5 ml of culture was added. This was kept for incubation for 6 days in dark condition. The UV spectrophotometer reading was taken at 400-450 nm. The peak at which maximum absorbance was observed. From this, the required concentration of Silver Nitrate for Silver nano particle production was detected. The silver nano particle production in the culture was also observed by UV-VIS Spectroscopy and in which part Silver Nano particle synthesized was detected by observing the colour change.

Synthesis of silver nano particle

The culture obtained by centrifugation was inoculated in 100 ml of 10"3 M aqueous Silver Nitrate in 250 ml Erlenmeyer flask. In 100 ml sterile distilled water, the culture alone was inoculated. This acts as the control. Both were kept for incubation at 37°C for 6 days. After 6 days, the change in colour from white to brown was observed.

RESULTS AND DISCUSSION

The isolated actinomycetes were identified as Streptomyces pseudogriseolus by 16s rRNA sequencing.

The flask was almost colourless before the addition of Silver nitrate and the colour changed to brown colour on the completion of the reaction. Thus, it was evident that the cultures produce enzymes that reduce silver nitrate to Silver ions. Colour reduction clearly indicates that the reduction of ions occurs through intracellular method.The silver nanoparticles exhibited striking colour light yellow to brown, due to excitation of surface Plasmon vibration in the particles and thus provided a convenient means of visually determining their presence in the reaction which was detected using UV Spectrophotometer.

Figure 1 Synthesized Silver Nanoparticles by actinomycetes at 6th day of incubation.

UV SPECTROPHOTOMETER ANALYSIS

The primary detection for synthesis of nanoparticles is done by UV Spectroscopy. A surface Plasmon peak located at interval of 400-420 nm was observed for the AgNp production from Streptomyces pseudogriseolus. (Figure 4) shows the absorption spectra of Silver nanoparticle produced by Streptomyces pseudogriseolus.

Figure 2. UV Spectrophotometer analysis

FTIR ANALYSIS

Fourier transform infra red spectroscopy demonstrated that the chemical reduction of nanoparticles synthesized was non homogenous.

Figure 3: FTIR analysis of silver nanoparticle produced by Streptomyces pseudogriseolus.

TRANSMISSION ELECTRON MICROSCOPY

Figure 4. TEM analysis of silver nanoparticles produced by Streptomyces pseudogriseolus..

The image showed the variable morphology of the nanoparticles. The aggregate size of the nanoparticles was observed as 29 to 583 nm.

ANTI MICROBIAL ACTIVITY OF SILVER NANOPARTICLE

Silver nanoparticle exhibit antibiotic properties against various pathogens. The produced silver nanoparticle is treated with the various bacteria includes Staphylococcus aureusand salmonella typhi. The antibacterial activity was done by well diffusion method. The culture was treated with water, silver nanoparticle and a standard (gentamycin). The zone of inhibition was noted that the silver nanoparticle shown the maximum inhibition for the bacteria.

Figure 5. Antibacterial activity of silver nitrate, silver nanoparticle and culture against pathogen

The antibiotic disc gentamycin 10 meg was used as positive control and distilled water of 20}il was used as negative control. The silver nitrate, AgNps and culture of 20ul was added separately to the plates and the activity was observed. The formation of clear zone (restricted bacterial growth) around the cavity is an indication of antibacterial activity .The size of metal nanoparticles ensures that a significantly large surface area of the particles is in contact with the bacterial species. The synthesized silver nanoparticle shows an effective antibacterial activity against pathogens of gram positive and gram negative bacteria.

From this work, we conclude that marine soil sample has the capability of reducing the silver nanoparticle into uniform size. The present investigation indicates intracellular synthesis of highly stable silver nanoparticles by biotransformation using various species of marine sample. The synthesised silver nanoparticles are having the ability to inhibit the growth of micro organism. The media composition was modified slightly from the standard composition even though we got the effective growth of the actinomycetes. This simple procedure for the biosynthesis of silver nanoparticles formStreptomyces pseudogriseolus has several advantages such as cost-effectiveness and compatibility for biomedical applications.

MERITS OF THE INVENTION

• Biological synthesis of silver nanoparticle is more effective when compared to physical and chemical methods.

• Silver nanoparticle shows the effective results against various microbes.

• Even by modifying the composition of starch casein nitrate agar media there was effective growth of actinomycetes.

• The bioavailability and the biocompatibility of silver nanoparticle is more when compared to other metals.

• Though silver nanoparticles are cytotoxic but they have tremendous applications in the field of high sensitivity bimolecular detection and diagnostics, antimicrobials and therapeutics, catalysis and micro-electronics.

DEMERITS OF THE INVENTION

• The production of silver nanoparticle is very cost effective.
• Silver nanoparticle is toxic substance.
• The chance of contamination of culture during its growth is more.

CLAIMS

1. Actinomycetes isolated from the marine soil sample shown the good activity when compared to the other soil sample.

2. Streptomyces pseudogriseolus comes under the Actinomycetes family, it shown the good reduction against silver ions, this organism is having the enormous amount of capability to reduce the silver when compare to other organism.

3. Streptomyces pseudogriseolus has synthesised the silver in a remarkable manner so, that during the characterisation it has reduced the metals and it exhibits in a nano size. (

4. Characterisation studies revealed that this organism is having the capability to reduce and synthesise the silver. Bioactive compounds shown the good result during the characterisation and antimicrobial study.