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Rapid Synthesis of Silver Nanoparticles by a Marine-derived Fungus Aspergillus Niger and their

Antimicrobial Potentials

A.K.Vala1*, S. Shah2

1-DepartmentofPhysics,BhavnagarUniversity,SardarVallabhbhaiPatelCampus,Bhavnagar,India2-DepartmentofMarineandCoastalStudies,MaduraiKamrajUniversity,Madurai,India

(*)Correspondingauthor:anjana_vala@yahoo.co.in(Received:12 Aug. 2012 and Accepted: 10 Oct. 2012)

Abstract:Recently, biosynthesis of nanoparticles has received attention due to an increasing need of developing rapid, simple and ecofriendly protocol. Pathogenicity of some of the organisms and lengthy reaction are the drawbacks involved with biosynthesis. We describe a simple protocol for rapid synthesis of silver nanoparticles through biological route using a marine-derived fungus Aspergillus niger. Silver nanoparticles biosynthesis could be achieved within 3 minutes (which otherwise generally takes about 24h) by altering pH of the reaction mixture. Silver nanoparticles biosynthesized at different pH have been observed to have antimicrobial potentials against four test bacteria (viz. Bacillus megaterium, Proteus vulgaris, Staphylococcus aureus and Shigella sonnei). Further, combined effect of Gentamicin and biosynthesized silver nanoparticles and effect of culture condition (pH) on antimicrobial effect have also been studied. Based on the findings it is concluded that the present study provides a solution to the drawbacks involved in biosynthesis of silver nanoparticles, an ecofriendly approach. It is envisaged that the biosynthesized silver nanoparticles alone, or their combination can be potentially used as effective agents against pathogens. Keywords: Antibacterial activity, Biosynthesis, Filamentous fungi, Silver nanoparticles, Microbial growth, Environmental preservation.

Int. J. Nanosci. Nanotechnol., Vol. 8, No. 4, Dec. 2012, pp. 197-206

1.INTRODUCTION

Over the years, development of resistance tocommercially available antimicrobial agents bypathogenic microorganisms has been a pressing problem[1,2].Consequently,developmentofneweffectivebactericidesbecomesimperative.Silversaltsandelementalsilverarewidelyusedasantimicrobial agents. Silver attacks a broad rangeof targets inmicrobes, hence, theorganismsneedtodeveloparangeofmutationssimultaneouslytoprotect themselves.Therefore, it’s implausible formicrobestodevelopresistanceagainstsilver[3,4].

However, silver salts and bulk silver face certainlimitationsfortheirmedicalapplications,theformermaypossessquickanduncontrolledsilver releasewhilethelatterisasluggishandinefficientreleasingsystem.Silverinthenanoparticleform,nothavingany such issue, has better prospects for medicalapplications [5]. This is oneof the reasons for agreatdemandofsilvernanoparticlesinrecentyears.However,thephysico-chemicalprotocolsgenerallyappliedforsynthesisofsilvernanoparticlessufferfrom one or the other limitations like high cost, use oftoxicchemicals,etc.Recently,biosynthesisofnanoparticleshasreceived

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attention due to an increasing need of developingrapid, simple and ecofriendly protocol. There hasbeen a growing number of reports on harnessingmicroorganisms for biosynthesis of nanoparticles [6-10]. However, compared to their terrestrialcounterparts, marine microorganisms are givenless attention despite the fact that researches onnanobiotechnology in future may progressivelydepend more on marine microbes having thecapabilitytogrowunderextremeconditions[11-13].Major drawback involved in biological synthesisof nanoparticles is pathogenicity of some of the organismsandlengthyreactionasthetimerequiredto complete the reaction generally ranges from 24-120h[9].In thepresent investigation,wedescribe a simpleprotocol for rapidsynthesisofnarrowlydispersedsilvernanoparticles(AgNPs)byalteringthereactioncondition.Furthermore,antibacterialactivityoftheas biosynthesizedAgNPs was tested against twoGram negative and two Gram positive bacterialstrains. The AgNPs synthesized at pH 10 werefoundtobethemosteffectiveagainsttestbacteria.The present investigation is the first-ever reporton effect of physico-chemical conditions onbiosynthesisandantimicrobialefficiencyofAgNPsbyamarine-derivedfungus.Advantages of usingAspergillus niger include (i)easy cultivation (ii) high yield (iii) inexpensivemedium requirement iv) it can be obtained as abyproduct from industries and (v) A. niger is generally regarded to be safe, therefore, can beeasilyaccepted[14,15].

2.MATERIALSANDMETHODS

2.1.Organism

A marine-derived fungus Aspergillus niger wasselectedforthepresentstudy.ThetestisolatewasisolatedfromwatersofBhavnagarcoast (Lat.21º45’ N and Long. 72º14’ E), Gulf of Khambhat,West Coast of India. The isolate was grown andmaintainedonpotatodextroseagar(PDA)medium[16]andstoredat4ºCuntiluse.Themediumwasprepared inagedseawateranddistilledwaterataratioof3:1[17].

Onemlinoculum(sporesuspensionapproximately106/ml)wasinoculatedin250mlPotatoDextrosemedium (prepared in 75% ‘aged’ seawater).The inoculated flasks were incubated at roomtemperature for 4 days. After incubation period,fungal biomass was separated from the mediumby filteration and extensivelywashedwith steriledistilledwater.

2.2.Biosynthesissilvernanoparticles

Approximately5g fungalbiomasswaschallengedwith 1.0mM silver nitrate at different pH (3-10).TheAgNO3 solutions were prepared in sterilizeddeionized water. The inoculated flasks wereincubatedat270Cfor72hunder staticcondition.Negative and positive controls (without thesilver nitrate, only biomass and without biomassonly silver nitrate) were also run along with theexperimentalflasks.One ml sample from each flask was withdrawnat different time intervals and the spectra wererecorded at a resolution of 1 nm using UV–visible spectrophotometer (Elico BL-198). Theexperimentswerecarriedoutintriplicates.Transmission electronmicroscopic (TEM) imagesof biosynthesized silver nanoparticles (mountedoncarbon-coatedcoppergrids) wereobtainedonJEOL (Model GEM 200) transmission electronmicroscopeoperatedat200kV.

2.3.Estimationoftotalextracellularprotein

After72hofincubation,thebiomasswasseparatedfromtheflasksbyfilterationandestimationoftotalextracellularproteincontentwascarriedoutusingbicinchonicacid(BCA)method[18].

2.4.Antimicrobialactivityofthebiosynthesizedsilvernanoparticles

Antimicrobial activity of silver nanoparticlesbiosynthesizedatpH5,8,9,and10wasexaminedagainst two Gram positive (Bacillus megaterium andStaphylococcus aureus)andtwoGramnegative(Proteus vulgaris and Shigella sonnei) bacteria. Diskdiffusionmethodandbacterialgrowthkineticstudieswerecarriedouttoinvestigatetheeffectin vitro. Synergistic antibacterial activity combined

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withantibiotictogetherwiththeeffectofpH,werealsostudied.

(A) Disk diffusion method was used to assaybiosynthesized nanoparticles for bactericidalactivityagainstteststrainsonMueller-Hintonagarplates.In this method, Mueller-Hinton agar plates werepreparedforeachbacterialteststrain.Eachplatewas prepared by aseptically pouring 100μl ofbacterialsuspension(approaximately108CFU/ml)alongwith20mlofsterilemoltenMueller-Hintonagar into the sterile petri plates. The plates wereallowedtosolidifyandmarkedaccordingly.Small filter discs approximately of the diameter6mm were sterilized. Silver nanoparticlessynthesizedatpH5,8,9and10wereusedtochecktheirantimicrobialactivity.Each plate was divided into 3 parts and thesterilizeddiscsimpregnatedwith40μlrespectivesilvernanoparticleswereasepticallyplacedonthecentre of each compartment of the plate. Similar experimentswerecarriedoutwith1mMAgNO3 as wellasSuspension(fungalcellextract)fromflaskwithoutsilvernitrate.Afterincubationat370Cfor24 hours the zones of inhibition were measured.Theassayswereperformedintriplicate[19].

Synergisticantibacterialactivitycombinedwithantibiotic:

CombinationofsilvernanoparticleswithantibioticGentamicin,was investigated against test bacteriausingthediskdiffusionmethod.Standard Gentamicin disks (10 µg/disk) wereused as positive control, and Gentamicin disksimpregnatedwith silvernanoparticles (10μl)wereplaced onto the Mueller-Hinton agar mediuminoculatedwithtestorganisms.Suspension(Fungalcellextract) fromflaskwithoutsilvernitratewasusedasthenegativecontrol.These plates were then incubated at 37°C for 24hours. Similar experiments were carried out withonly silver nanoparticles. After incubation, thezonesofinhibitionweremeasured.Theassayswereperformed in triplicates and average values arereportedhere.The increase in fold area [20] was assessed by

calculating the mean surface area of the inhibition zone of Gentamicin and Gentamicin+Silvernanoparticles.ThefoldincreaseareaofdifferenttestorganismsforGentamicinandforGentamicin+Silvernanoparticleswascalculatedbytheequation(B2–A2)/A2,whereA andBwere zones of inhibitionforGentamicin,Gentamicin+Silvernanoparticles,respectively.

(B)Growth curves of test bacteria exposed todifferentconcentrationsofsilvernanoparticlesTo examine the growth curves of bacterialtest strains exposed to biosynthesized silvernanoparticles,Mueller-Hintonbrothwithdifferentquantityofsilvernanoparticles(0,50,100,and150μl) was used. Respective flasks were inoculatedand incubated in a shaking incubator at 370C for24h.Growthcurvesofbacterialcellcultureswereobtained throughrepeatedmeasuresof theopticaldensity(O.D.)at600nmatregulartimeinterval.

Effect of pHon antimicrobial activity of silvernanoparticles:Antibacterial testing of the selected silvernanoparticleswasalsocarriedoutatdifferentpH(pH6,6.5,7.4,8.0and8.5)usingdiskdiffusiontest.

3.RESULTSANDDISCUSSION

3.1.Biosynthesisofsilvernanoparticles

Silver nanoparticles are produced by variousphysico-chemicalmethods. Recently, biosyntheticroute also has been attempted. Generally,biosynthesisinvolvingmicrobesistimeconsuming,taking 24-120 h [9]. In ourmethod, to overcomethisproblemwehavedevelopedasimpleprotocolfor rapid synthesis of AgNPs. Biosynthesis ofAgNPs was carried out using marine-derivedfungus Aspergillus niger, isolated fromwaters ofBhavnagar coast,West coast of India, in the pHrange of 3-10 (Plate 1). Whenthetestfungalbiomasswaschallengedwith1mMAgNO3 atdifferentpH,all the testflasks inthepHrange3-7showedgradualchange incolorofreactionmixturefromcolorlesstodeepbrownish

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pinkwithin24h,indicatingbiosynthesisofAgNPs.Achangeincolorwasobservedwithin1minuteofexposuretofungalbiomasstotestAgNO3 solution atpH10.IncaseofpH8and9colorchangewasobservedwithin2minutes.Hence,pHinthealkalinerangesupportsupportedrapidbiosynthesisofsilvernanoparticles. The negative as well as positivecontrol did not show the characteristic change incolor.Biosynthesis boosted with increasing pHreached the maximum at pH 10. The proteinsinvolved in nanoparticlesbiosynthesismay bindwithsilveratthiolregions(–SH)forminga–S–Agbond,aclearindicationofwhichaidstheconversionofAg+toAg0.Inaddition,thealkalineion(OH-) is verymuchrequiredforthereductionofmetalions.AthigherpH,efficiencyoftheenzymesinvolvedinsynthesisofsilvernanoparticlesincreases[21].The present findings help tackling the drawbacksinvolved with process of biosynthesis ofnanoparticles, which could otherwise be reliableandecofriendly,becauseasalreadymentioned,A. niger is generally regarded to be safe andAgNPsynthesisbythisorganisminthepresentstudydoesnoteventake5minutes,whichisaremarkabletraitforlargescale,commerciallyviableprocess.

(*pH 8.9 dilution 1:4; pH 10 dilution 1:8)

Figure1:UV spectra of biosynthesized silver nanoparticles at different pH*

CharacterizationofSilverNanoparticles

Figure 1 shows uv-vis absorbance spectra ofAg-NPs from different pH range at different timeintervals.Inthespectra, asinglesurfaceplasmonresonance(SPR)isobservedwhichoriginatesfromelementalsilver[22,23].

 

 

 

    

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Figure2:TEM images of silver nanoparticles biosynthesized by marine-derived A. niger at (a) pH 5 and (b) ph 10

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When transmission electronicmicroscopic images(Figure 2) were examined it was found that theparticles were spherical in all cases.As reportedearlier[13],inthepreviousstudyusingthesametestorganism, theparticleswerefound tobesphericalwith size range of 5-26nm, except in the case ofsamplesofpH8,9and10.Here,theparticleswerespherical in the size range of 1.7-20 nm. Moreuniformitywasobservedinsizeoftheparticleswithincreasing pH. Smaller size of biosynthesized particles withincreaseinpHcouldbeduetoincreaseddynamicsoftheionsandmorenucleationregionsformedduetotheavailabilityof–OHions.TheconversionofAg+toAg0increasesfollowedbyincreaseinthekineticsofthedepositionofthesilveratoms[24].Possible mechanisms for synthesis of nanoparticles byfungihavebeenproposed,however,sofarnosinglegeneralizedmechanismhasbeenidentified[25]. Mukherjee et al. (2001) [6] suggested thatcellwallandcellwallsugarsplayimportantrolein reduction of metal ions.A number of studieshave suggested role of protein in nanoparticlesformation. Bansal et al. (2004) [26] observedthat fungus secreted proteins that were capableof extracellularly hydrolyzing compound withzirconium ions and hence, aided in zirconiumnanoparticle formation. In the succeeding studies with silica and titaniananoparticles it was confirmed [27]. Bharde etal. (2006) [28] suggested role of protein fromextracellularextractsofVerticilliumsp.inhydrolysiaof[Fe(CN)6]

3and[Fe(CN)6]4.BhainsaandD’Souza

(2006) [29] reported thatproteinaceouscompoundinfungalbiomasshadamajorroleinformationofmetalnanoparticles.Ingleetal.(2008)[30] reportedinvolvement of NADH-dependent enzyme inreductionofmetalionsanddemonstratedpresenceof nitrate reductase in fungal cellfilterate.Takingthe importance of fungal proteins in biosynthesis of nanoparticlesintoconsideration,totalextracellularproteinfromthefunguswasestimatedanditwasfoundthatextracellularproteincontentwashigherwhenthefunguswaschallengedwithsilvernitrate(Figure 3). These data corroborate with earlierfindings thathad suggested the roleofproteins inbiosynthesis of nanoparticles by fungi.

Figure3:Extracellular proteins produced by A. niger at different pH

Antimicrobial activity of the biosynthesizedsilvernanoparticles

The silver nanoparticles biosynthesized by A. niger atpH5,8,9and10wereselected for theirantibacterial activity against four bacteria: twoGram positive viz. Bacillus megaterium and Staphylococcus aureus and two Gram negative Proteus vulgaris andShigella sonnei.Discdiffusionmethod and bacterial growth kinetic studies werecarried out to study the effect in vitro. Effect ofpH on antimicrobial activity of Ag-NP also wasexamined.

(A)Discdiffusionmethod

When disc diffusion test was carried out, noinhibitionzonewasobservedwhen fungalculturefiltratewasused,similarobservationwasrecordedin case of silver nitrate also for all test bacteria.Inhibitionzonesofdifferentdiameter(fordifferentbacteria)wereobservedwhenbiosynthesizedsilvernanoparticles were used. Hence, biosynthesizedsilvernanoparticlesexertedactivityagainstalltestbacteria(Table1;Plate2).The present findings about antibacterial activityof silver nanoparticles are similar to the report ofGuzmánet al., (2009) [31]. Antibacterial activityofbiosynthesizedsilvernanoparticlesinthepresentstudyarefoundtobehigher thanthatreportedby

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Veerasamyetal.,(2011)[32].AntibacterialactivityreportedbyNithyaetal.,(2011)[10] wasmorethanobserved in the present study. However, strain tostrainvariationisalogicalhappening. Bhimbaetal.,(2011)[33]alsoreportedantibacterialactivityofsilver nanoparticles synthesized byHypocrea lixii isolatedfrommangrovesedimentsoil.Interestingly,particlesbiosynthesizedatdifferentpHshoweddistinctactivityateachpH.Invariably,highestanti-bacterialactivitywasimpartedbysilvernanoparticles synthesized at pH 10. In general,silver nanoparticles synthesized at pH 5 showedleastactivitywithanexceptionof S. sonnei.Thereason for this could be variation in particle size[34].Paletal.,(2007)[3]demonstratedthatsilvernanoparticlesundergoashape-dependentinteractionwith the Gram-negative organismE. coli. Shukla etal(2012)[35] reportedantibacterialactivityofagar-basedsilvernanoparticlesandnanocompositefilmagainstBacillus pumilis.

Synergisticantibacterialactivitycombinedwithantibiotic

CombinationofsilvernanoparticleswithantibioticGentamicin,was investigated against test bacteria

using the disk diffusion method. As maximuminhibitionzoneforalltestbacteriawasfoundwithsilver nanoparticles biosynthesized at pH 10, itwasselectedforstudying itseffectwithantibioticGentamycin. Gentamicin was selected for thestudy because, as an aminoglycoside antibiotic,gentamicin is of unquestionable practical interest and is effective against a large number of GramnegativeandGrampositivebacteria.Themodeofgentamicinactioninvolvesdisruptionofribosomalsynthesisofprotein.Gentamicinisamainantibioticused to treat severe purulent infection, especiallythat causedbya resistantGram-negativebacteria.Being a broad spectrum antibiotic, gentamicin isoften used for treatment of patientswithmixedinfection and also in the case of unidentifiedinfectingagent[36].When tested for sensitivity against antibioticGentamicin, P. vulgaris was the most sensitivefollowed by Staphylococcus aureus and Bacillus metagerium. S. sonnei, compared to other testorganisms,showedlesssensitivity(Table2;Plate3).When biosynthesized silver nanoparticles wereimpregnated on the antibiotic Gentamicin disc,and tested for antibacterial activity, an increase

 

 

 

    

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Table 1 : Zone of Inhibition (mm) in absence and presence of Silver nanoparticles (40 μl) against ١ test bacteria ٢

٣ ٤ ٥ ٦ ٧ ٨ ٩ ١٠ ١١ ١٢

(1: Ag particles synthesized at pH 5, 2: fungal culture filtrate without AgNO3, 3: AgNO3, 4: Ag particles ١٣ synthesized at pH 8, 5: Ag particles synthesized at pH 9, 6: Ag particles synthesized at pH 10) ١٤ ١٥

١٦

١٧

١٨

١٩

٢٠

٢١

٢٢

٢٣

٢٤

٢٥

٢٦

Sr. no Bacteria Staphylococcus

aureusBacillus

megaterium Proteusvulgaris

Shigella sonnei

1 10 10 8 15 2 0 0 0 0 3 0 0 0 0 4 10 10 8 13 5 11 15 8 13 6 12 12 10 16

Table1:Zone of Inhibition (mm) in absence and presence of Sliver nanoparticles (40ml) against test bacteria

Table2:Zone of Inhibition (mm) for Gentamicin (10mg/disk) in absence and presence of silver nanoparticles

 

 

 

    

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Table 2 : Zone of Inhibition (mm) for Gentamicin (10µg/disk) in absence and presence of silver ١ nanoparticles ٢ ٣

٤

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٦

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٨

(1: Gentamicin, 2: Gentamicin + Ag particles synthesized at pH 10 (10μl), 3 : increase in fold area ) ٩ ١٠

١١

١٢

١٣

١٤

١٥

١٦

١٧

١٨

١٩

٢٠

٢١

٢٢

٢٣

٢٤

Sr. no Bacteria B. megaterium S. aureus P. vulgaris S. sonnei

1 25 33 40 20 2 36 40 44 20 3 1.0736 0.4692 0.21 0

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in fold areawas observed in all cases, exceptS. sonnei.Amongalltestbacteria,S. sonneiexhibitedleast sensitivity towards gentamycin, the suppliedsilvernanoparticlescouldhavebeeninsufficientforexertingadditiveeffect.EnhancementinantibacterialactivityonE .coli andStaphylococcus aureus hasbeenreportedbySingh

et al., (2011 ) [37]whenCefazolin is conjugatedwithsilvernanoparticles.Fayaz et al., (2010) [38] also observed thatantibacterial activities of ampicillin, kanamycin,erythromycin,andchloramphenicolwereincreasedinthepresenceofAgnanoparticles.An increase the antifungal effects of drugs

Figure4:Absorbance indicating bacterial growth in presence of different concentration of biosynthesized silver nanoparticles

 

 

 

    

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fluconazole and griseofulvin in presence of Agnanoparticles has been recorded by Noorbakhshetal.,(2011)[39]. Gajbhiyeetal.,(2009)[8]alsoobservedenhancedantifungalactivityoffluconazoleinpresenceofsilvernanoparticlesagainst the testfungi.

(B) Bacterial growth kinetics in presence ofbiosynthesizedsilvernanoparticles

To examine the bacterial growth kinetics in thepresenceofbiosynthesizedsilvernanoparticles,testbacteriaweregrownin100mlofMuellerHintonbroth supplementedwith different doses of silvernanoparticles. It was found that the nanoparticlesexerted growth delay at all test concentrations(Figure 4).When a comparison is made with disc diffusiontestdataitwasfoundthattheeffectswerestrongerwhen the test bacteriaweregrownon agarmediathanthatgrowninbroth.Thiscouldlikelybeduetochangesinexposuredoseovertheexperimentsduetonanoparticles’aggregationandsedimentationinbroth,whileinagarthereisacontinuedpresenceofparticlesonthesolidsurface.Cultural condition for bacterial growth alsois an important criterion when antimicrobialtesting is being carried out. For this obviousreason, antibacterial testing of the selected silvernanoparticleswas also carried out at different pH(pH6,6.5,7.4,8.0and8.5).Figure5showsthedataofinhibitionzoneforthetestmicrobeswhengrownatdifferentpHinpresenceofbiosynthesizedsilvernanoparticles.MaximumsensitivityofStaphylococcus aureuswasobservedatpH8.5,thiscouldbeduetoanincreasein antibiotic uptake and hence killing of the cell[40].Bacillus megateriumwasfoundtobethemostsusceptible at pH 8.Highest antibacterial activityof silver nanoparticles against Proteus vulgaris wasfoundtobeatpH7.4,atotherpHtheactivityremained the same. Among all test bacteria, S. sonnei was themost sensitive. Highest inhibitionzonewas observed forS. sonnei when the pH ofthemediumwas6followedbypH6.5,7.4,8and8.5. Hence, in the acidic condition, the organismwasmoresensitive towardsexposure to thesilvernanoparticles.

4.CONCLUSION

Rapid biosynthesis of silver nanoparticles usingmarine-derivedfungusAspergillus nigerisachievedbyalteringthepHofthereactionmixture.AlkalinepH range led to rapid biosynthesis.Antibacterialefficiencyoftheassynthesizedsilvernanoparticlesaloneandincombinationwithantibioticgentamicinwas examined against Gram negative and Grampositive bacteria.The biosynthesized particles arefoundtobepromisingantibacterialagents.EffectofpHonantibacterialactivityofsilvernanoparticleswasalsostudiedthat indicatedmoresensitivityoforganisms in acidic condition. The present studyis thefirst-ever reporton rapid synthesisof silvernanoparticles by marine-derived fungus. Thesefindingsaresuggestiveofpossibleexploitationofthetestfungusforecofriendlyaswellascommerciallyviablebiosynthesisofsilvernanoparticles.

 

 

 

    

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Figure5: In hibition Zone (mm) for silver nano-particles against test bacteria grown at different pH

ACNOWLEDGEMENTS:

Thanks are due to the Council of Scientific andIndustrialResearch(CSIR),NewDelhi,forfinancialsupportsanctionedunderScientists’PoolSchemetoAKV.Dr.D.SrivastavaisgratefullyacknowledgedforTEManalysis.

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