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Phycosynthesis of silver nanoparticles using Spirulina platensis as apotential source of antibacterial agent

INTRODUCTION

Nanoscience is currently a fast growing niche and nano-technology is at the cutting edge of

this rapidly evolving area (Mandal et al., 2006). Nano-technology collectively describes

technology and science involving nano-scale particles (nanoparticles) that increases the scope

of investigating and regulating the interplay at cell level between synthetic materials and

biological systems (Du et al., 2007). It can be employed as an efficient tool to explore the

finest process in biological processes (Sondi & Salopak, Sondi, 2004) and biomedical

sciences (Hutten et al., 2004).

The word “nano” is used to indicate one billionth of a meter or 10-9. The term nano

technology was coined by Norio Taniguchi a, researcher at the University of Tokyo, Japan.

Nanotechnology is field that is burgeoning day by day making an impact in all spheres of

human-life (Vaidyanathanh et al., 2009). Besides this, NPs play an indispensable role in drug

delivery, diagnostics, imaging, sensing, gene delivery, artificial implants and tissue

engineering (Marones et al., 2004). In the current context, importance is being given to the

fabrication of a wide range of nano-materials for developing environmentally benign nano-

technologies in material synthesis (Bhattacharya et al., 2005).

One of the major developments in nanotechnology is the production and application of nano

particles in biology. New methods to produce nanoparticles are constantly being studied and

developed. The enormous interest in the bio-synthesis of NPs is due to their unusual optical

(Krotiknowska et al., 2003), chemical (Kumar et al., 2003), Photochemical (Chandrasekharan

et al., 2000), electronic (Peto et al., 2002) and magnetic (Watson et al., 1999), properties,

NPS are either newly created via nanotechnology or are present naturally over the earths crust

or in the environment caused by weathering of Au deposits. The metal formed by evaporation

is coupled with minerals and been deposited rapidly from saline ground water.

Nanotechnology has been defined as a technology that mainly consist of the process of

separation, consolidation and deformation of materials by one atom or molecule (Taniguchi,

1974).

The micro-organisms, such as bacteria, yeast and fungi play an important role in remediation

of toxic metals through reduction of the metal ions; this was considered interesting as

nanofactory, using the dissimilatory properties of fungi and hence these biological systems

have been extensively used for the rapid and eco-friendly biosynthesis of metal nanoparticles

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(Bhattacharya and Gupta, 2005). Uni and multicellular organisms have shown immense

potential for the synthesis of nanoparticles, as they can synthesize inorganic materials either

intracellularly or extracellularly. More recent and detailed investigations into the use of

microbes in the synthesis of different metal nanoparticles include bacteria for gold

(Beveridge and Murray, 1980; Southam and Beveridge, 1996; Fortin and Beveridge, 2000),

silver (Joerger et al., 2000; Klaus et al,.1998), ZnS (Labrenz et al., 2000), magnetite (Lovley

et al., 1987,1999, 2001; Nair and Pradeep, 2002) CdS (Smith et al., 1998; Philipse and Maas,

2002), iron sulfide (Watson et al., 1999, 2000), yeast for PbS (Kowshik et al., 2002a), CdS

(Kowshik et al., 2002b), and silver (Kowshik et al., 2003), and algae for gold (Robinson et

al., 1997).

Recently the development of resistant or even multi resistant pathogens has become a major

problem for instance Staphylococcus aureus resistance to methicillin and Candida albicans

resistance to fluconazole have to be mentioned (Schaller et al., 2004) on the other hand, the

introduction of newly devised wound dressing has been a major breakthrough in the

management of wound or infections. In order to prevent or reduce infection a new generation

of dressing incorporating antimicrobial agents like silver was developed (Yin et al., 1999).

It is well known that silver ions and silver based compounds are highly toxic to micro-

organisms. Thus silver ions have been used in many kinds of formations (Sondi et al., 2004)

and recently it was shown that hybrids of silver nanoparticles with amphiphilic hyper

branched macro molecules exhibit effective antimicrobial surface coating (Aymonier et al.,

2002). Nanometer sized silver particles synthesized by inert gas condensation or co-

condensation techniques showed anti bacterial activity against E.coli at low concentrations.In

addition it was observed a relationship between the antibacterial properties and the total

surface area of the nanoparticles. Smaller particles with a larger surface area were more

efficient in the antibacterial activity tests (Baker et al., 2005).

Silver is a non toxic safe inorganic antibacterial agent used for centuries and is capable of

killing about 650 types of diseases causing micro-organisms (Jeorg et al., 2005). Silver has

been described or being “oligodynamic” because of its ability to exert a bactericidal effect at

minute concentrations (Percivala et al., 2005). It has a significant potential for a wide range

of biological applications such as antifungal agent, antibacterial agents for antibiotic resistant

bacteria, preventing infections, healing wounds and anti inflammatory (Taylor et al., 2005).

Silver ions and its compounds are highly toxic to micro-organisms exhibiting strong biocidal

effects on many species of bacteria but have a low toxicity towards animal cells. Therefore

silver ions, being antibacterial component, are employed information of dental resin

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composites, bone cement, ion exchange fibers and coatings for medical devices (Panacek et

al., 2006: Alt et al.,2004). Compounds of silver such as silver, nitrate and silver sulfadiazine

are used to prevent bacterial growth in drinking water, sterilization and burn care. It is

economical to consolidate silver in polymers, composites, fabrics and catheters for anti

bacterial functionality (Oloffs et al., 1944, Li et al., 2005, Zhang et al., 2004, Baker et al.,

2005).

Your running shoes, socks and even computer keyboard may be impregnated with silver

nanoparticles that can kill some bacteria keep you swelling sweet and preventing the spread

of infection among computer users. Researchers in India point out that silver nanoparticles

are not only antibacterial against so called Gram Positive bacteria, such as resistant strains

Staphylococcus aureus and Streprococcus pneumoniae but also against Gram Negative

Escherichia coli and Pseudomonas aeruginosa (Pattabi, 2010).

Scientists are reporting development and successful lab tests of “killer paper” a material

intended for use as a new food packaging material that helps preserve foods by fighting that

bacteria that cause spoilage. The silver coated paper showed potent antibacterial activity

against E.coli and S.aureus, two causes of bacterial food poisoning killing all of the bacteria

in just three hours. (Langmuir, 2011).

The use of nanoparticles derived from noble metals has spread too many areas including

jewelry, medical fields, electronics, water treatment and sport utilities thus improving the

longevity and comfort in human life. The application of nanoparticles as delivery vehicles for

bactericidal agents represents a new paradigm in the design of antibacterial therapeutics.

(Vijayaraghavan and Nalini, 2010). Major consumer goods manufactures like LG and

Samsung already produce household items that silver nanoparticles. These products include

nano-silver lined refrigerators air conditioners and washing machines (www.azom.com,

2011).

Other current applications for silver nanoparticles impregnated materials include:

*Toy

*Baby pacifiers

*Clothing

*Food Storage Containers

*Face Masks

*HEPA Filters

*Laundry Detergent

Medical application

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Other potential applications for silver nanoparticles include:

*Diagnostic biomedical optical imaging

*Dressing and Bandages

*Biological implants

(Like heart valves)

BACTERIAL INFECTION

The recent years infections caused by bacteria resistant to multiple antibiotics have been a

significant problem. Escherichia coli is the head of the large bacterial family,Entero-

bactericidal, the enteric bacteria, which are facultatively anaerobic Gram-negative rods that

live in the intestinal tracts of animals in health and disease (Todar, 2011).It is an extremely

reversible opportunistic pathogen (Cheesbrough, 2000) causes septic-mias and can infect the

gall bladder, meningers, surgical would skin lesions and the lungs especially in debilitate and

immuno-deficit patients (Black, 1996).

Staphylococcus aureus is a well armed virulent pathogen that is currently the most common

cause of infections in hospitalized patients (Archer, 1998). S. aureus is a facultatively

anaerobic gram positive, which causes food poisons and usually grows on the usual

membranes and skin. It is also found in the gastrointestinal and urinary tracts of warm-

blooded animals. Also cause boils, abscesses, wound infection, pneumonia, toxic shock

syndrome and other diseases (Cheesbrough, 2000).

Bacillus subtilis is not a human pathogen. It may contaminate food but rarely causes food

poisioning. It produces the proteolytic enzyme subtilisin and spores can survive the extreme

heat during cooking. B.subtilis is responsible for causing ropiness, a sticky, stringy,

consistency caused by bacterial production of long chain polysaccharides in spoiled bread

dough (Ryan and Ray, 2004).

Bacillus fusiformis was described originally as occurring in cases of hospital gangrene. It is

more common at present in the form of Vincent’s angina, an antiflammatory condition of the

throat (McConnell, 2010). The organism has been observed in ulcero-membranous angina,

hospital gangrene, noma, appendicitis, diphtheria, forted bronchitis, gangrenous laryngitis,

pyorrhea, alveolaris, brain abscess and in the healthy mouth (Peters, 1911).

The pathogenicity of 13 strains of Bacillus licheniformis was studied in immunodepressed

mice. (Agerholm et al., 1997) There are several reports in the literature of human infections

with Bacillus licheniformis (Farar, 1963; Banerjee et al 1988; Logan 1988) however these

occurred in immunosuppresed individuals or following trauma (Tabbara and Tarabay, 1979).

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Due to its ubiquitous presence as spores in soil and dust, B.licheniformis is widely known as a

contaminant of food (Norris et al, 1990).

Several studies revealed S.platensis or its extract could show physiological benefits as

antioxidants, anti-microbial, antiinflammatory, antiviral or anti-fungal (Carreri et al., 2001,

Mendes et al., 2003, Pinero, 2001, Subhashim et al., 2004). The problem of microbial

resistance is growing and the outlook for the use of antimicrobial drugs in the future is still

uncertain. The majority of clinically used anti-microbial drugs have various draw back in

terms of toxicity, efficacy, cost and their frequent use has led to the emergence of resistant

strains.

Thus there is an urgent need to develop alternative biodegradable agents, which could be free

from side effects. This search prompted the exploration of natural Algal product that could be

exploited as biodegradable, more systematic and non-toxic anti-microbial agent with

microbial toxic properties. There are few reports present regarding the anti-microbial activity

of Spirulina against some pathogenic bacteria and fungi (Boussiba and Richmond 1978,

Ozdemir et al., 2004, Mendiola et al., 2007, Santyo et al., 2006) and anti-bacterial effect of

silver nanoparticles (Morones et al., 2005, Feng et al., 2000, Sharma et al., 2009,Kim et al.,

2007, Duran et al.,2007). Thus anti microbial effects of silver (Ag) ion or salts are well

known, but the effects of Ag nanoparticles on micro-organisms and anti-microbial

mechanism have not been revealed clearly. But no report is available regarding effectiveness

of antimicrobial activity of silver nanoparticles produced by Spirulina platensis. Therefore,

the present work entitled, “Phycosynthesis of silver nanoparticles using Spirulina platensis

as a potential source of antibacterial agent.” is chosen for the present study.

PRELIMINARY WORK ON SPIRULINA PLATENSIS AGAINST

PATHOGENIC BACTERIA

Preliminary work of silver nanoparticles synthesized using living and non-living stains of

Spirulina platensis and its antibacterial activity against some pathogenic bacteria such as E.

coli, B. fusiformis etc have already done. Among non-living strains of S. platensis of

Jalmahal Lake, Jaipur was found to show synthesis of AgNPs and its antibacterial activity

against E.coli as compare to other non-living strains (Ramgarh and Rajkot strain). Living

culture of Spirulina platensis also shows synthesis of silver nanoparticles and its antibacterial

activity. Silver nanoparticles synthesized by Spirulina platensis were monitored and

characterized by UV-vis spectroscopy and FTIR.

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REVIEW OF LITERATURE

CHARACTRISATION OF SYNTHESIZED SILVER NANOPARTICLES USING SPIRULINA

& OTHER ALGAE

Alga is diverse group in plant kingdom that is being explored for application in

nanotechnology. Besides the production of NPs algae are also being explored for determining

its mutational value, efficacy in bio-diesel improvement as well as its vast potential for

therapeutic application (Sinha et al., 2009).

Interaction of single cell protein of Spirulina platensis with aqueous silver nitrate (AgNO3)

and chloroauric acid (HAuCl4) was investigated for the synthesis of Ag, Au and Au core Ag

shell nanoparticles (Govindaraju et al., 2008). High resolution transmission electron

microscopy showed formation of nanoparticles in the range of 7-16 (Silver), 6-10 (Gold) and

17 – 25 nm (bimetallic 50:50 ratio). XRD analysis of the silver and gold particles confirmed

the formation of metallic silver and gold. Fourier transform infrared spectroscopic (FTIR)

measurements revealed the fact that the protein is the possible bimolecular responsible for the

reduction and capping of the biosynthesized nanoparticles. In another attempt biological

synthesis of silver, gold and Ag shell-Au core nanoparticles using single cell protein

Spirulina platensis and seaweed Sargassum wightii have been achieved. (Singaravelu et al.,

2007). Tsibakhashvili et al (2010), reported synthesis silver nanoparticles using blue green

alga S. platensis and actinobacteria-Streptomyces spp. The samples have been characterized

by UV-Visible spectrophotometry and XRD.

The formation of extracellular silver nanoparticles by photoautotrophic cyanobacterium

Plectonema boryanum had been described (Langke et al., 2007). The reaction products were

analyzed using transmission electron microscopy (TEM) and X-ray photoelectron

spectroscopy (XPS). Ali et al (2011), reports the extracellular biosynthesis of silver

nanoparticles using marine cyanobacterium, Oscillatoria willei NTDM01 which reduces

silver ions and stabilizes the silver nanoparticles by a secreted protein. The silver nitrate

solution incubated with washed marine cyanobacteria changed to a yellow color from 72h

onwards, indicating the formation of silver nanoparticles. The characteristics of the protein

shell at 265nm were observed in Ultra violet spectrum for the silver nanoparticles in solution.

While Fourier Transform Infra Red (FTIR) confirmed the presence of a protein shell which

are responsible for the nanoparticles biosynthesis. Scanning Electron Microscopy (SEM)

studies showed that the formation of agglomerated silver nanoparticles due to the capping

agent in the range of 100 - 200nm. EDS spectrum of the silver nanoparticles was confirmed

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the presence of elemental silver signal in high percentage. Apart from Eco-friendliness and

easy availability and low cost cyanobacterial biomass production will be more advantageous

when compared to other classes of micro organism. Extracellular synthesis of silver

nanoparticles by brown seaweed, Sargassum wightti was characterized by means of UV-Vis

spectroscopy, FTIR, XRD and HR-TEM. Antibacterial studies were carried out using the

bacterial isolated from the infected silkworm. The recorded antibacterial effect of silver

nanoparticles was found more potent when compared to the chemically synthesized silver

nanoparticles and it is expected to be bio compatible. (Govindaraju et al, 2009). Bunghez et

al (2010) obtained silver nanoparticles from AgNO3 using red algae (Porphyridium

purpureum). The red algae contain the red pigment-phycobilins, responsible for red color and

for the strong absorption in visible spectrum. The properties and structure of silver

nanoparticles have been put into evidence by means of: Fourier transform infrared

spectroscopy-FTIR, optical microscopy, X-ray fluorescence spectrometry-EDXRF.

Venkatpurwar and Pokharkar (2011) synthesize silver nanoparticles through green route

using sulfated polysaccharide isolated from marine red algae, Porphyra vietnamensis. FTIR

spectra revealed the involvement polysaccharide for reduction of silver nitrate. The dose

dependent effect of synthesized silver nanoparticles revealed strong anti bacterial activity

against gram negative bacteria as compared to gram positive bacteria.

ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES AGAINST BACTERIA

The antibacterial property of silver has been known for thousands of years with the ancient

Greeks cooking from silver pots and the old adage’ born with a silver spoon in his mouth’

referring to more than just wealth. Eating with a silver spoon was known to be more hygienic.

Manufacturing entire objects from pure silver is prohibitively expensive for consumer items

but research has found that impregnating other materials with silver nanoparticles is a

practical way to exploit the germ fighting properties of silver (www.azom.com, 2011).

Application of silver nanoparticles can prevent accumulation of bacterial contaminants from

accumulating in the further protecting the wearer from other infections. The success of silver

nanoparticles against bacterial growth is due to damage of plasma membrane or bacterial

enzymes. This results in morphological distortion of the bacterial cells, leading to impairment

of bacterial metabolism and escape of cytoplasmic substance to the surroundings. The coating

of medical instruments is one recent silver nanoparticles medical application. The treated

instruments were observed to have a powerful bacterial action against Klebsiella pneumoniae,

Bacillus anthracis sterne and Bacillus subtilis for 1 hour and 30 minutes. Staphylococcus

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aureus and Acinetobacter baylyi were contained for 5 hours. (www.silvernanoparticles.info,

2010).

The antibacterial activity of silver nanoparticles synthesized by Streptomyces species is

proving against the multi drug resistant bacteria of Gram positive (Staphylococcus aureus, S.

epidermidis and Gram negative strains (E. coli, S. typhi, Pseudomonas aeruginosa,

Klebsiella pneumoniae, Proteus vulgaris) (Shirley et al., 2010). Silver nanoparticles

synthesized by Fusarium oxysporum strain incorporated in cotton cloth exhibit antibacterial

activity against Staphylococcus aureus to become them steriles. (Marcato et al., 2010).The

AgNPs were evaluated for their anti-microbial activities against different pathogenic

organisms. The most sensitive antimicrobial activity has been observed against methicillin

resistant S. aureus followed by methicillin resistant S. epidermidis and Streptococcus

pyogenes whereas only moderate anti-microbial activity was seen against Salmonella typhi

and S. pneumoniae (Nanda and Saravanan, 2009).

Inhalation of silver nanoparticles results in “Miraculous” protection against pneumonia

caused by Pseudomonas aeruginosa, according to a study conducted by researchers from the

Washington University School of Medicine and the University of Akron, Ohio and presented

at the 105th national Conference of the American Thoracic Society (Gutierrez, 2002).

Agile silver nanoparticle is a pure liquid silver suspension product which can achieve over

99.9% effect in inhibiting growth of numerous kinds of bacteria, including multiple drug

resistance bacteria and fungi (Lok et al, 2007). Agile silver nanoparticles can inhibit the

growth of E. coli, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter

baumanii which are silver ion resistant and multiple-drug resistance.

ANTIBACTERIAL ACTIVITY OF SPIRULINA PLATENSIS & OTHER ALGAE

Algal organisms are rich source of structurally novel and biologically active metabolites.

These induce antibiotics which in laboratory tests inhibited bacteria and fungi that incite

diseases of humans (Kulik, 1995). Various strains of cyanobacteria are known to produce

intracellular and extracellular metabolites with diverse biological activites such as antialgal,

antibacterial, antifungal and antiviral activity (Noaman et al, 2004). The crude extracts of

Spirulina platensis were tested for their antimicrobial activities against different species of

human pathogenic Bacteria [(Klebsiella pneumoniae (NCIM 2063), Proteus vulgaris (NCIM

2027), Pseudomonas aeruginosa (NCIM 2076) and Escherichia coli (NCIM 2065),

Salmonella typhi (NCIM 2080),] by the agar-solid diffusion method ( Mala et al., 2009).

Ozedmir et al (2004) tested S. platensis in vitro for their antimicrobial activity against four

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Gram positive, six Gram negative bacteria and Candida albicans ATCC 1023. In another

attempt , 3 cyanobacteria (Anabaena oryzae, Tolypothrix ceytonica and Spirulina platensis

and 2 green ( Chlorella pyrenoidosa and Scenedesmus quadrifolia ) were tested in

compliance with the agar well diffusion method for their antibacterial and antifungal agent

production on various organisms that incite diseases of humans and plants (E. coli, Bacillus

subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus flavus, Penicillium

herquei, Fusarium moniliforme, Helminthosporium species, Alternaria brassicae,

Saccharomyces cerevisiae, C. albicans) (Rania et al, 2008). Antibacterial activity of

methanolic extracts from 32 macroalgae (13 Chlorophyta and 19 Phaeophyta) from the

Atlantic and Mediterranean coast of Moracco were evaluated for the production of

antibacterial compounds against E. coli, S. aureus, Enterococcus faecalis, Klebsiella

pnumeuniae and E. faecalis. (Ibtissum etal., 2009). Khairy et al (2010) examine 3 blue green

algal species (Anabaena flos aquae (L.) Bory, Anabaena variabilis (Kutzing) and

Oscillotoria angustissima (West and West) against 8 Gram+ve and Gram-ve bacteria (B.

subtilis 1020, B. cereus, S. aureus, Streptococcus faecalis (G+ve), E. coli 1357,

Pseudomonas aeruginosa, Aeromonas hydrophila and Vibrio fluvialis Antimicrobial

activity of cyanobacteria isolated from the mangrove forest of Sundarbans have shown

Minimum inhibitory concentrations (MIC) of the extracts against S. aureus, E. coli, B.

subtilis, P. aeruginosa, and multiple drug-resistant clinical isolates ranged between 0.25-0.5

mg/ml.( Pramanik et al., 2011).

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OBJECTIVES

1. Isolation, identification and purification of Spirulina platensis for synthesis of silver

nanoparticles.

2. Purification of pathogenic strains of isolated bacteria, available in Microbiology Lab.

3. Screening of living and non living strains of Spirulina platensis for biosynthesis

of AgNPs.

4. Characterization of AgNPs of most potent strains of Spirulina platensis.

5. Antimicrobial activity & MIC of biosynthesized strains of AgNPs against pathogenic

bacteria.

METHODOLOGY

Criteria of Selection

ALGAL SPECIES (Spirulina platensis)

Spirulina is a photoautotrophic prokaryote found in a variety of environment e.g., soil, sand,

marshes, brackish, seawater and freshwater. It is abundantly available throughout the year.

TARGET SPECIES (Pathogenic bacteria)

The pathogenic bacteria included in the present study will be E. coli, Staphylococcus aureus,

Bacillus subtilis, Bacillus fusiformis and Bacillus licheniformis.

ISOLATION, PURIFICATION AND CULTIVATION OF SPIRULINA PLATENSIS

Spirulina platensis will be isolated from Dayalbagh region and cultivated in the CFTRI

medium. Before experimentation the biomass will be washed thrice in deionized water to

remove unwanted materials (Govindaraju et al., 2008)

MEDIA PREPARATION

The media used for culturing S. platensis is CFTRI medium (4.5 g sodium bi carbonate, 0.5,

Di-Potassium, 1.5 Sodium nitrate, 1 potassium sulphate, 1.0 sodium chloride, 0.2 magnesium

sulphate, 0.04 Calcium Chloride, 0.01 Iron sulphate.) The media used for culturing the

organisms would be Nutrient Agar Media (NAM) (5 g Peptone, 3 g beef extract and 5 g NaCl

in a litre solution) for S. aureus, E. coli, B. subtilis B. fusiformis and B. licheniformis.

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A. Isolation of Spirulina platensis

Spirulina platensis culture will be obtained from Microbiology lab, Department of Botany,

Dayalbagh Educational Institute, Dayalbagh, Agra. The culture would be picked up from the

stock culture with the help of needle and transferred to petriplates and culture tube containing

CFTRI medium and incubated at 280C for 30 days with (600 – 1600 lux) with a continous

light, 12 hrs per day (Sony et al., 2006). Identification will be done using morphological

variation, studies and taxonomical approaches according to Anagnostidis and Komarek, 1998

and Desikachary, 1959.

B. Isolation of Microbial Cultures

Microbial cultures (E. coli, S. aureus, B. subtilis, B. fusiformis and B. licheniformis ) used

will be obtained from the Microbiology lab., Department of Botany, Dayalbagh Educational

Institute, Dayalbagh, Agra. Bacterial cultures would be picked up from the stock cultures

with the help of inoculation needle and transferred to the petridishes containing NAM

medium directly and incubated at 370C for 24 – 48 hours. In a petridish when bacterial

colonies appeared on NAM medium, it would be transferred to other dishes or slants for

experiment. Confirmation of bacterial culture would be carried out using “Bergey’s Manual

of Systemic Bacteriology” (Claus and Berkly, 1986).

C. Screening of different strains of Spirulina platensis for biosynthesis of AgNPs.

Silver nitrate will be purchased from A.B. Company, Agra, India. Before experimentation,

the algal biomass will be washed twice in distilled water to remove the unwanted materials.

Silver nanoparticles formations will be carried out by taking 1 g of wet biomass in a 250 ml

Erlenmeyer flask with 10-3 M aqueous AgNO3 for different times (2, 4, 7, 8 days) and

incubated at room temperature. The pH will be checked during the course of reaction and it

was found to be 5.6 (Tsibakhashvili, 2010). Non living strains of Spirulina platensis will be

collected from Jalmahal (Jaipur), Ramgarh (Jaipur) and Rajkot (Gujrat) from Pushpa

Srivastava. Silver nanoparticles synthesis using non living culture of Spirulina platensis will

be carried out by taking 5 mg of Spirulina powder in 50 ml of aqueous AgNO3 solution of 10-

3 molar concentration. (Govindaraju et al., 2008).The time of addition of extract into the

aqueous AgNO3 solution will be considered as the start of the reaction. Under continuous

stirring conditions, after 10 min., the light yellow color of AgNO3 solution will gradually

changed to brownish yellow color indicates the formation of silver nanoparticles. The

principle of preparation of silver nanoparticles by using microorganism is a bioreduction

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process; the silver ions are reduced by the extracellular reductase enzymes produced by the

microorganisms to silver metal in nanometer range (Fu et al.,2006 ).The reaction involved in

this process is shown in Fig-I.

Fig-I: Reaction showing preparation of silver nanoparticles.

It is concluded from protein assay of microorganisms that the preparation of silver

nanoparticles is an NADH-dependent reductase. The enzyme reductase gains electrons from

NADH and oxidizes it to NAD+. The enzyme is then oxidized by the simultaneous reduction

of Silver ions forming silver metal in nanoform. In some cases a nitrate-dependent reductase

is responsible for the bioreduction process whereas in the case of rapid extracellular

synthesis of nanoparticles the reduction happens within few minutes, therefore a complex

electron shuttle materials may be involved in the biosynthesis process (Moghadham et al.,

2010). Screening of different strains will be done on the basis of intensity of color change

during the course of experiment.

D. Characterization of most potent strains of Spirulina platensis

The bio-reduction of AgNO3 ions in solution will be monitored by periodic sampling of

aliquots (0.1 ml) of aqueous component and measuring UV-vis spectra of the solution. The

nanoparticles will be characterized and confirmed by FTIR, XRD and HR-TEM analysis

(Chandran et al., 2006)

E. Anti-microbial assay

(1) Agar well diffusion method: The silver nanoparticles synthesized using Spirulina

platensis would be tested for anti microbial activity by agar well diffusion method against

pathogenic bacteria. E. coli, S. aureus, B. subtilis, B. fusiformis and B. licheniformis will be

sub-cultured on nutrient agar medium. Wells of 10 mm diameter will be made on nutrient

agar plates using gel puncture. Each strain was swabbed uniformly onto the individual plates

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using sterile cotton swabs. Using a micropipette different concentration of the sample of nano

particles solution (10 µl, 20 µl and 50 µl) will be poured onto each well on all plates. After

incubation of 370C for 24 hours, the different levels of zone of inhibition of bacteriae will be

measured. (Govindaraju et al, 2010).

(2) MIC (Minimal Inhibitory Concentration): This concentration was found with lowest

possible zone of inhibition. The paper discs in different concentrations were placed on the

surface of medium containing bacterial culture. The zone of inhibition will be measured at

the diameter and recorded as the MIC.

IMPORTANCE OF WORK

Elemental silver occurs naturally. It is considered non-toxic, non-allergic, is not cumulative

and is not known to harm either wildlife or the environment. Antibiotic drugs can be used to

kill the pathogens attacked by silver nanoparticles but bacteria and viruses are becoming

increasingly resistant to drug therapies. Silver nanoparticles kill all types of fungal infections,

bacteria and viruses, including antibiotic resistant strains. No drug based antibiotic is

effective on all types of bacteria. Additionally, research to date has shown that bacteria have

been unable to develop any immunity to silver.

Spirulina platensis (SP), a blue-green alga (photosynthesizing cyanobacterium) possesses

diverse biological activity due to high content of highly valuable proteins, indispensable

amino acids, vitamins, beta-carotene and other pigments, mineral substances, indispensable

fatty acids and polysaccharides. Spirulina is gaining more attention in the field of medical

science because of its nutraceutical and pharmaceutical importance. It has been demonstrated

that small amounts of Spirulina reduced HIV-1 replication while higher concentration totally

stopped its reproduction.

Thus, the Spirulina mediated synthesis of Ag nanoparticles and their antibacterial activity

communicated in this work gains its importance in its medical application. The present study

is likely to explore the unexploited bioefficacy of Spirulina synthesized silver nanoparticles

as antibacterial agent. The proposed work is likely to add following information to the

present state of knowledge to the area of relevance:

*Outcome of new nano phyco product possessing antibacterial activity.

*Establishment of bioactive constituents responsible for bioefficacies.

*Provides scope for further research leading to structure of nanodrug reactivity relation.

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