In Situ Production of Silver Bio Nanoparticles from Marine Biosurfactant Bacteria and Evaluation of Its Antibacterial Activity

BIOSURFACTANTS FROM MICRORGANISMS Biosurfactants are the surface-active molecules synthesized by microorganisms. With the advantage of environmental compatibility, the demand for biosurfactants has been steadily increasing and may eventually replace their chemically synthesized counterparts. Marine biosurfactants produced by some marine microorganisms have been paid more attention, particularly for the antimicrobial activity against various microbes in medicinal field. In this study, the screening of biosurfactant-producing marine microorganisms, the determination of biosurfactant activity as well as the recovery of marine biosurfactant and the antibacterial activity of the biosurfactant were done. The uses of silver nanoparticles with marine biosurfactants for the antibacterial activity also discussed. The marine Serratia sp. had the ability to produce the biosurfactant and shows high amount of antibacterial activity against some pathogenic bacteria.


I. INTRODUCTION
Marine environment is the largest habitat as compared to other habitat in the biosphere. About 70% of the earth surface is covered by salt water. It is believed that the life is originated first from ocean. Biosurfactants are amphiphilic agents which, by accumulating at interface between immiscible phases, can reduce surface and interfacial tensions produced by bacteria, fungi and yeast, which either adhere to the cell surfaces or are excreted extracellular in the growth medium. They belong to various classes including glycolipids, lipopeptides, fatty acids, phospholipids, neutral lipids and lipopolysaccharides. The best biosurfactants can lessen the surface tension of water from 72 to 30 Nm− 1 . The large surface-to-volume ratio enables many bacterial species to produce several types of structurally diverse surfaceactive compounds, which having antimicrobial, antiviral, haemolytic, and anti tumour activity [1]. During the last decades, there has been a growing interest in isolating microorganisms that produce surface active molecules with good surfactant characteristics such as low critical micelle concentration (CMC) and high emulsification activity, simultaneously presenting low toxicity and good biodegradability. Generally the type and amount of the biosurfactants, produced by microbes depends on the producer organism, factors like carbon and nitrogen, trace element, temperature. They are also classified into two different categories on the basis of their molecular weight: Low molecular weight biosurfactants are lipopeptides, glycolipids and phospholipids (having lower surface and interfacial tension) and high molecular weight biosurfactants are polymeric and particulate surfactants (more efficient as emulsion stabilizing agents) [2].
The silver nanoparticles coated textiles with enhanced functionalities, such as antibacterial, antistatic and UV protection, are greatly appreciated by a more discerning and demanding consumer market for high-value-added products. Silver nanoparticles can be exploited in medicine for burn treatment, dental materials, coating stainless steel materials, textile fabrics, water treatment, sunscreen lotions, etc. and possess low toxicity to human cells, high thermal stability and low volatility [3]. Antibacterial finishes are applied to gauge cloth for the major reasons is to control the spreading of disease through microorganism and avoid the danger of injury-induced infections.

Isolation of biosurfactant producing bacteria:
The marine water sample was spread on nutrient agar plate by using the serial dilution process. The dilutions of 10 -3 , 10 -4 , 10 -5 , 10 -6 dilutions were spread on the nutrient agar plate with salt. Then it had been incubated for 28 hours. After incubation individual colonies had been grown in different morphology.

Screening of biosurfactant producing bacteria: Cetyltrimethyl ammonium bromide (CTAB) methylene blue agar method
The bacterial isolates were inoculated on to MSM agar plates supplemented with 2 % (w/v) glucose, 0.25% w/v yeast extract and containing 200 mg l -1 cetyltrimethylammonium bromide (CTAB), a cationic surfactant and 5 mg l -1 methylene blue. The plates were incubated at 30°C for24-48 h to observe dark blue halo. The clear zones are due to accumulation of methylene blue around the colonies of cells producing anionic surfactant.

Determination of the Emulsification Index
A mixture of 2 mL supernatant and 3 mL kerosene (or diesel) was vertically stirred for 2 min and the height of the emulsion layer was measured after 24 hr to determine the emulsification index. The equation used to determine the emulsification index (E 24 (%)) is as follows: The E 24 (%) = (height of emulsion layer/ height of total solution) *100%

Blood haemolysis test:
Blood agar haemolysis method was used to screen biosurfactant producing strain. The method was based on the fact that biosurfactants were able to haemolysis the red blood cell present in blood. The selected isolates were incubated on blood agar plates incubated for 24-48 hours at 37 • C. The zone of haemolysis indicated the production of biosurfactant.

Biochemical characterization of isolated bacteria: Extraction of biosurfactant
The culture was centrifuged at 5000rpm at 4°C for 30 minutes to obtain the supernatant. To the supernatant, equal volumes of chloroform: methanol in the ratio 2:1 was added and the mixture was acid to pH 2.0 using 6 N HCl and kept overnight for evaporation. Finally white colour precipitation was seen between the two liquid layers i.e., supernatant and chloroform: methanol. The same was then collected and lyophilized and characterized using FTIR.

Characterization of biosurfactant by FTIR analysis
For FTIR measurements, the Ag nanoparticles solution was centrifuged at 10,000 rpm for 30 min. The pellet was washed three times with 20 ml of de-ionized water to get rid of the free proteins/ enzymes that are not capping the silver nanoparticles. The samples were dried and grinded with KBr pellets and analyzed on a Shimadzu IR-IR Affinity1 model in the diffuse reflectance mode operating at a resolution of 4 cm−1.

GC-MS (Gas Chromatography Mass Spectrometry)
GC-MS technique was used in the study to identify the components present in the sample. GC-MS analysis of this sample was performed using a Perkin Elmer GC Clarus 500 systems and chromatography interfaced to a silica capillary column (30mx 1µl was Mdf. Composed of 100% Dimethyl poly siloxane) for GC-MS detection. An electron ionization energy system with ionization energy of 70eV was used. Helium gas (99.999%) was used as the carrier gas at a constant flow rate of 1ml/min and an injection volume of 2µl was employed (split ratio of 10:1). Injector temperature was 250 ∘ c. The oven temperature was programmed from 110 ∘ c (isothermal for 2 mins) with an increase of 10 ∘ c/min to 200 ∘ c, then 5 ∘ c/min to 280 ∘ c ending with 9min isothermal at 280 ∘ c. Mass spectra were taken at 70eV, a scan interval of 0.5 seconds and fragments from 45 to 450 Da. Total GC running time was 36mins. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a Turbomass Version 5.2.0. Compound identification was obtained by comparing the retention times with those of authentic compounds and the spectral date obtained from library data of the corresponding compounds. The given sample was extracted by ethyl acetate and analysed in GC-MS for different components.

Estimation of biosurfactant
The biosurfactant activity had been estimated by using the orcinol method. The rhamnose had been prepared for 100 mg and it had estimated by using our sample, water and orcinol reagent. Then it would keep at 80 o C for 10 mins in the water bath. Then the nm had been identified in the UVspectrophotometer. The precipitated silver nano particles were isolated by centrifugation at 15,000 rpm. This procedure results in the production of silver nano sized particle. The particles where then sonicated in 10ml.

Synthesis of Silver Nanoparicles:
The synthesis of silver nano particles in the water in oil micro emulsion phase was performed by the addition of synthesized two reverse micelles in the presence of reducing agent.The synthesis involves mixing up to 0.5 m/l aqueous silver nitrate solution, 3.0g of biosurfacatant, 1.5g of butanol together and stirred thoroughly at room temperature until homogeneous reverse micelles were formed.

Characterization of silver nanoparticles: UV-VIS spectra analysis
The bio reduction of Ag+ ions in solution was monitored in optimal measurement, which carried out by using UV-Visible spectrophotometer and scanning the spectra between 200-800 nm at the reduction of 1mn.

EDAX (ENERGY DISPENSIVE X RAY SPECTRA ANALYSIS)
EDX pattern shows the crystalline and elemental composition of silver nanoparticles synthesized from the biosurfactant producing organism. The strong signal in the silver region was observed at 3 keV for silver nanoparticles due to the surface Plasmon resonance.A weak signal from "O" is recorded it may due to the presence of organic moieties from the enzymes or proteins in the extract.

SEM (Scanning Electron Microscope)
The SEM image showing the high intensity of silver nanoparticles synthesized by biosurfactant producing organism further confirmed the development of silver nanostructures. SEM provided further insight into the morphology and size details of the silver nanoparticles. SEM analysis showed the particle size of about 100 nm as well the crystal structure of the nanoparticles. The silver nanoparticles synthesized via green route are highly toxic to multidrug resistant bacteria hence has a great potential in Biomedical applications.

Antimicrobial activity
The well diffusion method had been used for the antimicrobial activity studies. The Muller Hinton Agar had been prepared. The sample supernatant had been added in the wells. Incubate the plates at 37 ∘ c for 24 hours. After incubation the zone of inhibition had been formed and the zones had been measured in millimeters using the scale.

Isolation of Bacteria
Five strains observed different colony morphology were streaked on nutrient agar plate to obtain pure culture and they were taken for further study. Out of five, isolates 5 produced pink coloured non diffusible pigment producing colonies and the colony morphology.

Staining characters
All the five bacterial isolates reviewed in this study were stained by Gram"s stain. All the five isolates were Gram negative. The morphology details of the bacteria.

Isolation of biosurfactant producing organisms
5 Bacterial isolates were screened as a biosurfactant producer and then the bacterial isolates were identified based on biochemical tests and Bergy"s Manual classifications. The isolates are as followed,  [4] reported that the selected isolates were performed with different screening test to check the biosurfactant production ability and to find the efficient biosurfactant producer by CTAB plate assay

Determination of emulsification index
The height of the emulsion layer was measured after 24 hr to determine the sample emulsification index. The equation used to determine the emulsification index (E 24 (%)) is as follows E 24 (%) = (height of emulsion layer/ height of total solution) *100%

Blood haemolysis test
In the blood haemolysis test, the organism had get haemolysed in the blood agar medium. The blood agar had get haemolysed by Serratia sp. cell free supernatant. Dhail and Jasuja [5] studied that haemolytic activity assay, and emulsification activity measurement was used to screen the biosurfactant producer.

Estimation of biosurfactant
The surface tension of the culture medium decreased to a maximum extent of 26.5 mN/m after 72 h of incubation. Furthermore, the CMC of the crude biosurfactant produced by Serratia sp. was estimated at 60.13 mg/l.

Extraction and characterization of biosufactant
The biosurfactant was extracted by acidification method from cell free supernatant of Serratia sp. This was used for FTIR and GCMS studies.

FTIR analysis of biosurfactant
The functional group of the partially purified biosurfactant was investigated for the chemical nature of the biosurfactant using FTIR. The IR spectrum was collected in the range of 4000 -428 cm -  The UV-Visible absorbance spectrum of silver nanoparticles which the peak hbeen formed in 350mm

EDAX result
Determine the elemental composition of synthesized nanoparticles EDAX analysis was performed. In the EDAX spectra of Ag Nanoparticles, the most intense peak around 3 key corresponds to binding energies of Ag. Hence it was confirmed the formation of AgNPs. The remaining elements were due to presence of biosurfactant producing organism serratia sp. Silver Nano crystallites display an optical absorption band peak at approximately 3 keV, which is typical of the absorption of metallic silver Nano crystals due to surface Plasmon resonance. Corresponding band peak was obtained in same AgNPs synthesized from the organism serratia sp. EDS confirmed the Presence of the signal characteristic of elemental silver. Silver Nano crystallites display an optical absorption band peak at approximately 3 keV, which is typical of the absorption of metallic silver Nano crystals due to surface Plasmon resonance [9].

SEM Result
SEM analysis was carried out to understand the topology of Ag nanoparticles, which showed the synthesis of mono dispersed spherical Ag nanoparticles. In the family of electron microscopy techniques the sample is exposed to a high energy focused beam of electrons. In scanning electron microscopy (SEM) the interaction of the beam with the particle surface are scanned over the sample and measured as secondary electrons (most common), or backscattered electrons or X-ray photons. Due to the high depth of field in SEM a three dimensional appearance can be obtained. The sample needs to be conductively coated with gold or graphite and maintained under ultrahigh vacuum in order not to have the secondary electrons interact with gas molecules. The substrate is typically a filter membrane or a conducting grid.

Antimicrobial activity of biosurfactant by well diffusion method
The antimicrobial activity of organism serratia sp. which was synthesized by silver nanoparticles was studied in different concentrations (5, 25, 50, 100 and 250) against the organism E.coli (gram negative bacteria). The antimicrobial potential of the organism were assessed in terms of Zone of inhibition of bacterial growth [10]. A number of studies exist which reports the Antimicrobial activity of extracellular synthesized biosurfactant silver nanoparticles isolated from various bacterial strains [11].

IV. CONCLUSION
The Serratia sp. was isolated from the marine water which had been collected from marina beach, Chennai, Tamilnadu. The isolated organism was screened for the production of biosurfactant. The screening process had been used by CTAB method, blood haemolysis method, emulsification activity. Then the silver nanoparticles get synthesized by using the organism serratia sp. and the wavelength had been measured in the graphical representation in the UV-visible spectrophotometer. Then the characterization process had been done by using the methods of FTIR, SEM, GCMS and EDAX. These methods clearly show about the compounds present in the organisms and it shows the graphical representation.