Analysis of antimicrobial silver nanoparticles synthesized by coastal strains of Escherichia coli...

Analysis of antimicrobial silver nanoparticlessynthesized by coastal strains of Escherichia coliand Aspergillus niger

Kandasamy Kathiresan, Nabeel M. Alikunhi, SriMahibala Pathmanaban,Asmathunisha Nabikhan, and Saravanakumar Kandasamy

Abstract: The present study investigated the extracellular biosynthesis of antimicrobial silver nanoparticles by Escherichiacoli AUCAS 112 and Aspergillus niger AUCAS 237 derived from coastal mangrove sediment of southeast India. Both mi-crobial species were able to produce silver nanoparticles, as confirmed by X-ray diffraction spectrum. The nanoparticlessynthesized were mostly spherical, ranging in size from 5 to 20 nm for E. coli and from 5 to 35 nm for A. niger, as evi-dent by transmission electron microscopy. Fourier transform spectroscopy revealed prominent peaks corresponding toamides I and II, indicating the presence of a protein for stabilizing the nanoparticles. Electrophoretic analysis revealed thepresence of a prominent protein band with a molecular mass of 45 kDa for E. coli and 70 kDa for A. niger. The silvernanoparticles inhibited certain clinical pathogens, with antibacterial activity being more distinct than antifungal activity.The antimicrobial activity of E. coli was more pronounced than that of A. niger and was enhanced with the addition ofpolyvinyl alcohol as a stabilizing agent. This work highlighted the possibility of using microbes of coastal origin for syn-thesis of antimicrobial silver nanoparticles.

Key words: antimicrobial activity, eukaryote, polyvinyl alcohol, prokaryote, silver nanoparticles.

Resume : Cette etude visait a examiner la synthese extracellulaire de nanoparticules d’argent antimicrobiennes par Esche-richia coli AUCAS 112 et Aspergillus niger AUCAS 237 issus de sediments de mangrove cotiere du sud-est de l’Inde.Les deux especes microbiennes pouvaient produire des nanoparticules d’argent, tel que confirme le spectre de diffractionde rayons-X. Les nanoparticules synthetisees etaient principalement spheriques, dont la taille allait de 5 a 20 nm chezE. coli et de 5 a 35 nm chez A. niger, selon l’analyse par microscopie electronique par transmission. La spectroscopie atransformee de Fourier a revele la presence de pics proeminents correspondant aux amides I et II, indiquant la presenced’une proteine qui stabiliserait les nanoparticules. L’analyse en electrophorese a revele la presence d’une proteine majeured’un poids moleculaire de 45 kDa chez E. coli et de 70 kDa chez A. niger. Les nanoparticules d’argent inhibaient certainspathogenes cliniques. L’activite antibacterienne etait plus nette que l’activite antifongique. L’activite antimicrobienne etaittres prononcee chez E. coli et A. niger, et elle etait accrue lorsque l’alcool polyvinylique etait ajoute comme agent de sta-bilisation. Ce travail met en evidence la possibilite d’utiliser des microbes d’origine cotiere pour synthetiser des nanoparti-cules d’argent antimicrobiennes.

Mots-cles : activite antimicrobienne, eucaryote, alcool polyvinylique, procaryote, nanoparticules d’argent.

IntroductionBiosynthetic methods employing microorganisms or plant

extracts have emerged as simple and viable alternatives tochemical and physical methods for synthesis of nanopar-ticles (Kathiresan et al. 2009). Biological synthesis wouldhave greater commercial viability if the nanoparticles couldbe synthesized more rapidly, at a larger scale, and withgreater stability. The use of microorganisms can potentiallyeliminate the problem of environmental contamination,therefore, microorganisms are continuing to be investigated

in the synthesis of metal nanoparticles (Ahmad et al. 2003).However, marine microbes remain relatively unexplored.

The biological application of nanoparticles is a rapidly de-veloping area of nanotechnology in the production of novelmaterials and devises. Among the various nanoparticles, sil-ver nanoparticles have received much deserved attention be-cause of their effective antimicrobial properties and lowtoxicity toward mammalian cells. Silver nanoparticles withtheir unique chemical and physical properties show betterantimicrobial properties (Rai et al. 2009) than other antimi-crobial compounds that are generally being used. Silvernanoparticles have become one of the most commonly usednanomaterials in consumer products (104 out of 502 nano-products surveyed) (Maynard and Michelson 2006). It isknown that a free silver ion (Ag+) is highly toxic to a widevariety of organisms including bacteria (Slawson et al.1992). However, little work has been done to evaluate theinhibitory activity of silver nanoparticles against microbialpathogens.

Silver nanoparticles are reportedly protected by polymers,

Received 17 July 2010. Revision received 113 October 2010.Accepted 20 October 2010. Published on the NRC ResearchPress Web site at cjm.nrc.ca on 7 December 2010.

K. Kathiresan,1 N.M. Alikunhi, S. Pathmanaban,A. Nabikhan, and S. Kandasamy. Centre of advanced study inMarine Biology, Annamalai University, Parangipettai, 608502Tamil Nadu, India.

1Corresponding author (e-mail: [email protected]).

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Can. J. Microbiol. 56: 1050–1059 (2010) doi:10.1139/W10-094 Published by NRC Research Press

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like polyvinyl sulphonate and polyvinyl alcohol (PVA)(Zhou et al. 1999; Chou and Ren 2000; Monti et al. 2004;Vasilev et al. 2010). PVA is considered a good host materialfor metal because of its excellent thermostability and chem-ical resistance (Fussell et al. 2005; Porel et al. 2005; Lili etal. 2007). In addition, owing to its water solubility, the sil-ver nanoparticles can be easily prepared in aqueous mediumand the preparation is virtually nontoxic. A polymer compo-site composed of silver nanoparticles and PVA is interestingin terms of its easy manipulation, low cost, and controllableparticle size (Giannetti et al. 1986). PVA is frequently usedas a stabilizer for silver nanoparticles because of its opticalclarity (Badr and Mahmoud 2006). However, studies on thesynthesis of polymer composites and their application arevery much lacking. Keeping these facts in mind, the presentstudy was designed to determine the potential of Aspergillusniger and Escherichia coli in extracellular biosynthesis ofantimicrobial silver nanoparticles, stabilized with and with-out PVA.

Materials and methods

ChemicalsAll analytical reagents and media components were pur-

chased from Hi-media (Mumbai, India) and Sigma Chemi-cals (St. Louis, Missouri, USA).

Isolation of microbes and biomass productionAspergillus niger AUCAS 237 and E. coli AUCAS 112

were isolated from soil samples from mangrove roots ob-tained from the Vellar estuary (11829’N, 79846’E) in thesoutheast coast of India.

To prepare fungal biomass, A. niger was grown aerobi-cally in a liquid medium containing 7 g potassium dihydro-gen phosphate, 2 g dipotassium hydrogen phosphate, 2 gmagnesium sulphate, 1 g ammonium sulphate, 0.6 g yeastextract, and 10 g glucose in l L of 50% seawater. The flaskswere inoculated and incubated on an orbital shaker at 25 8Cand agitated at 150 r/min.

To prepare bacterial biomass, E. coli was grown in mini-mal medium containing 5 g glucose, 6 g disodium hydrogenphosphate, 3 g potassium dihydrogen phosphate, 1 g ammo-nium chloride, 0.5 g sodium chloride, 0.12 g magnesiumsulphate, and 0.01 g calcium chloride in l L of 50% sea-water. After 72 h of growth, the microbial biomass was har-vested by sieving using Whatman No. 1 filter paper,followed by extensive washing 3 times with 10 mL ofMilli-Q deionized water. The dry mass of the biomass was3.5 g for A. niger and 2.3 g for E. coli.

Synthesis of silver nanoparticlesFresh biomass of bacterium (20 mg) and fungus (20 mg)

was mixed separately with 200 mL of Milli-Q deionizedwater in separate Erlenmeyer flasks and agitated for 72 h at25 8C on a shaker. After the incubation, the cell-free filtratewas obtained by passing the mixture through Whatman filterpaper No. 1. For synthesis of silver nanoparticles, 45 mL of1 mmol/L silver nitrate (AgNO3) was mixed with 50 mL offiltrate in a 250 mL Erlenmeyer flask and agitated at 25 8Cin dark. A control (without the AgNO3 but only microbial

filtrate) was also run at the same time as the experimentalflasks. The optical density was taken at different wave-lengths ranging from 300 to 700 nm using a UV-visiblespectrophotometer (Elico, Chennai) to find out the wave-length at which the adsorption peak occurred.

Effect of time duration on formation of silvernanoparticles

The microbial filtrate containing silver ions was analyzedfor colour intensity at 420 nm at different periods of incuba-tion (0 min, 10 min, 20 min, 30 min, 40 min, 50 min,60 min, 2 h, 3 h, 4 h, 6 h, 24 h, and 48 h).

Characterization of silver nanoparticlesThe nanoparticles synthesized after 4 h of incubation with

microbial filtrate were analyzed by X-ray diffraction (XRD)using a Cu–Ka radiation source in a powder diffractometer(PANalytical X’pert PRO model X-ray diffractometer) onfilms of the suspension drop-coated onto glass substrates onthe instrument operating at a voltage of 50 kV and a currentof 30 mA. This was followed by Fourier transform infrared(FTIR) spectroscopy measurement and transmission electronmicroscopy (TEM) analysis.

For FTIR spectroscopy measurement, the followingmethod was followed. The nanoparticles synthesized after4 h of incubation were centrifuged at 10 000 r/min (8000g)for 15 min, and the pellet was redispersed in sterile distilledwater to remove any unadsorbed biological molecules. Cen-trifugation and redispersion in sterile distilled water was re-peated 3 more times to ensure better separation of freeentities from the metal nanoparticles. The purified pelletswere then dried and the powder was subjected to FTIR spec-troscopy measurement (Paragon 500, Perkin Elmer RX1spectrophotometer) in the diffuse reflectance mode at a res-olution of 4/cm in KBr pellets. After 4 h of incubation, thereaction mixture was used to form a film of silver nanopar-ticles on carbon-coated copper TEM grids (Electron micro-scopy sciences, Hatfield, Pennsylvania, 19440) and wasanalyzed under TEM (JOEL, JEM 100 SX) at a voltage of120 kV.

Partial purification and PAGE analysisA 1 mmol/L concentration of AgNO3 was mixed with

50 mL of cell-free filtrate of A. niger and E. coli in a250 mL Erlenmeyer flask and agitated. After 24 h, the reac-tion mixture was precipitated by using solid ammonium sul-phate to 80% saturation. The pellet obtained aftercentrifugation (5000g for 5 min) was dissolved in 0.05 mol/L phosphate buffer (pH 8.0). The concentrated protein ob-tained was dialyzed overnight against 0.05 mol/L phosphatebuffer (pH 8.0) to remove salts. The protein samples wereanalyzed by sodium dodecyl sulphate – polyacrylamide gelelectrophoresis (SDS–PAGE) according to Laemmli (1970)to check the purity and to determine the molecular mass ofthe purified protein by comparing it with a protein standardhaving a molecular mass ranging from 10 to 250 kDa.

Preparation of silver nanoparticles with PVA asstabilizer

The nanocomposite consisting of nanoparticles and PVAwas prepared by following the method of Lili et al. (2007)

Kathiresan et al. 1051

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with slight modification. In this method, 0.8 g of PVA wasdissolved in 100 mL of distilled water at 80 8C by vigo-rously stirring to form a homogeneous solution. A 20 mLaqueous suspension of silver nanoparticles was prepared us-ing E. coli and A. niger and then added to the above PVAsolution. This mixture was then stirred in a flask for about10 min and then purged with nitrogen. A fresh aqueous 5 �10–3 mol/L sodium borohydride solution was prepared andintroduced drop by drop into the PVA–AgNO3 solution.The solution was then stirred for 15 min under inert atmos-phere at a room temperature of 25 ± 2 8C. Silver nanopar-ticles were also similarly prepared in the absence of PVA.

Antimicrobial activityThe antimicrobial assay was done by a log reduction test,

which has been proven to be of greater use in assessing anti-microbial efficacy (Gallant-Behm et al. 2005). In thismethod, bacterial cultures (Pseudomonas aeruginosaRMMC 126, Staphylococcus aureus RMMC 131, Listeriamonocytogenes RMMC 146, Micrococcus luteus RMMC135, and Klebsiella pneumoniae RMMC 128) and fungalcultures (Alternaria alternata RMMC 218, Penicillium itali-cum RMMC 208, Fusarium equiseti RMMC 214, and Can-dida albicans RMMC 205; RMMC is Raja Muthaih medicalcentre) were diluted in 20% Muller–Hinton broth to a con-centration of 1 � 106 CFU (colony forming units)/mL. Ninemillilitres each of a silver nanoparticle suspension preparedfrom culture filtrates of A. niger and E. coli with and with-out PVA were transferred into widemouthed tubes andplaced in a water bath at 22 ± 2 8C for approximately20 min. A control (saline solution without silver nanopar-ticle suspension) was maintained similarly. As well, a solu-tion of 9 mL of 0.8% PVA and 9 mL of AgNO3 (68% ofconcentrated AgNO3 and 32% distilled water, which isequivalent to the metal concentration in a nanoparticle sus-pension of A. niger and E. coli) was also separately main-tained. A 10 mL culture suspension of each of the bacteriaand fungi (1 � 106 CFU/mL) was added midway betweenthe center and the edge of the surface with the tip of the pip-ette slightly immersed in the test suspension. Thirty, 60, and90 s after addition of the suspension, a 1.0 mL aliquot of

this mixture was transferred to 9.0 mL of Letheen broth,used to neutralize the residual antimicrobial activity of theproduct. The number of viable microorganisms in the brothwas determined by the pour-plate method in replicate (n =3) on Muller–Hinton agar and was incubated for 48 h. Sur-vival numbers of bacteria and fungi were obtained using astandard plate count procedure using Muller–Hinton me-dium, and the log reduction value was calculated as the dif-ference between the log numbers of microbes surviving onthe control and the test.

Results

Synthesis of silver nanoparticlesThe microbial filtrates exhibited a colour change when in-

cubated with AgNO3 in the dark. The color of the reactionmixture changed to an intense brown after 4 h of incubation.The control without AgNO3 did not show any change in thecolor of the microbial filtrates. The absorption spectrum ofthe microbial filtrates revealed the peak value at 420 nm(Fig. 1). The colour intensity increased with incubation timeup to 4 h and then started to decline (Fig. 2). The nanopar-ticle synthesis in terms of colour intensity was higher in thefiltrate of E. coli than A. niger.

Characterization of silver nanoparticlesTo study the nature of nanoparticles produced by micro-

bial filtrates, XRD was carried out. The XRD exhibited in-tense peaks (Fig. 3) in the whole spectrum of 2q valueranging from 20 to 80, and this pattern was similar Braggpeaks of silver nanocrystals.

The TE micrograph, as shown in Fig. 4, revealed that thenanoparticles produced were generally spherical in shapeand ranged in size from 5 to 20 nm for E. coli and from 5to 35 nm for A. niger.

The nanoparticles were then analyzed for FTIR spectra asrepresented in Fig. 5. For E. coli, prominent peaks appearedat 2743, 2514, 2013, 1765, 1631, 1343, 923, 713, and493 per cm, and for A. niger, at 2381, 1741, 1651, 1553,1221, 754, and 491 per cm. The peaks corresponded toamides I, II, and III; aromatic rings; geminal methyl and

Fig. 1. Absorbance spectra for filtrates of Escherichia coli and Aspergillus niger with and without silver nitrate (AgNO3).

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Fig. 2. Effect of incubation period on the absorbance at 420 nm for culture filtrates of Escherichia coli and Aspergillus niger with silvernitrate.

Fig. 3. X-ray diffraction pattern of silver nanoparticles synthesizedin filtrates of Escherichia coli (A) and Aspergillus niger (B).

Fig. 4. Transmission electron micrograph of silver particles in fil-trates of Escherichia coli (A) and Aspergillus niger (B) (scale bar =50 nm).



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ether linkages, commonly present in filtrates of both mi-crobes.

SDS–PAGE of proteinThe molecular mass of the protein present in microbial

filtrates, estimated by SDS–PAGE, is depicted in Fig. 6.The prominent band obtained for E. coli was of 45 kDa andthat of A. niger was 70 kDa.

Antimicrobial activityAntimicrobial activity of silver nanoparticles synthesized

by A. niger and E. coli with or without PVA is shown inTable 1. The nanoparticles treated with PVA exhibitedhigher antimicrobial activity against all the test pathogens

than those not treated. The greatest antibacterial activitywas observed against K. pneumoniae by nanoparticles syn-thesized by E. coli and stabilized with PVA, and the lowestwas against M. luteus by nanoparticles produced by E. coliwithout PVA (Table 1). The greatest antifungal activity wasobserved against A. alternata by nanoparticles synthesizedby A. niger and stabilized with PVA, and the lowest was ob-served against P. italicum by nanoparticles synthesized byA. niger without PVA (Table 1). In general, antibacterial ac-tivity was more pronounced than antifungal activity.

DiscussionNanoparticles are being viewed as fundamental building

blocks of nanotechnology. The use of microorganisms in

Fig. 5. Fourier transform infrared spectrum of silver nanoparticles synthesized in filtrates of Escherichia coli (A) and Aspergillus niger (B).



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the synthesis of nanoparticles is an exciting area of researchwith considerable potential for development of nanoparticleswith unique characteristics. Bacteria, fungi, and yeasts have

shown an ability to reduce metal ions to form metallic nano-particles (Mandal et al. 2006). However, the microbes frommarine environments are relatively less exploited but morepotent than their terrestrial counterpart, as they thrive in ex-treme environmental conditions (Kathiresan and Selvam2005). Hence the present work was conducted using 2widely distributed strains of E. coli and A. niger from amangrove environment. The results revealed that bothstrains could reduce silver ions and produce silver nanopar-ticles. This was evident by the change in colour intensityafter 4 h of incubation of microbial filtrates with AgNO3, aswas also observed by others (Kowshik et al. 2003; Sadowskiet al. 2008; Kathiresan et al. 2009). There was a colorchange in the filtrates of A. niger and E. coli immediatelyafter the addition of AgNO3, which indicates the rapid re-duction of silver nanoparticles by E. coli and A. niger. Gen-erally, other physical and chemical processes of synthesis ofnanoparticles are also fast, and the method of synthesisbeing reported in this study could be comparable to theseother processes as well as with various other studies whichused biological sources (Kathiresan et al. 2009; Bhainsa andD’Souza 2006; Ahmad et al. 2003).

The UV–VIS spectrum is one of the most important andeasiest techniques to verify the formation of metal nanopar-ticles provided that surface plasmon resonance exists for themetal (Brause et al. 2002; Ahmad et al. 2003). The absorb-ance peak was at 420 nm in both E. coli and A. niger(Fig. 1), a characteristic of silver nanoparticles (Petit et al.1993; Kong and Jang 2006), which indicated the presenceof silver nanoparticles in the reaction mixture. Because ofthe excitation of plasma resonances or interband transitions,some metallic nanoparticle dispersions exhibit unique bandsand (or) peaks (Creighton and Eadon 1991).

Among microorganisms, prokaryotic bacteria have pri-marily drawn due attention (Mandal et al. 2006). Synthesisof metal nanoparticles by eukaryotic organisms was initiatedby Mukherjee et al. (2001a, 2001b) by using Verticillium sp.They demonstrated that the shift from bacteria to fungi hasthe added advantage of a simpler means of processing andhandling of the biomass. There are many papers concernedwith nanoparticle synthesis by fungi (Mukherjee et al.2001a, 2001b; Sastry et al. 2003; Ahmad et al. 2003;

Table 1. Antimicrobial activity of silver nanoparticles synthesized by Escherichia coli andAspergillus niger with and without polyvinyl alcohol (PVA) against clinical bacterial andfungal pathogens.

E. coli A. niger

Species +PVA –PVA +PVA –PVA AgNO3 PVA

BacteriaPseudomonas aeruginosa 3.1 2.6 2.4 2.2Klebsiella pneumoniae 3.8 3.4 3.3 2.9 1.1 0.3Staphylococcus aureus 2.5 2.3 2.2 2.2 1.3 0.2Listeria monocytogenes 2.2 2.2 2.1 2.1 1.2 0.4Micrococcus luteus 2.1 1.9 2.3 2.2 0.9 0.4

FungiAlternaria alternata 1.5 1.4 1.6 1.5 0.5 0.2Candida albicans 1.3 1.2 1.3 1.1 0.3 0.4Penicillium italicum 0.8 0.7 0.6 0.6 0.7 0.1Fusarium equiseti 1.4 1.2 1.1 0.9 0.4 0.2

Fig. 6. SDS–PAGE patterns of purified protein of Escherichia coliand Aspergillus niger filtrates added with silver nitrate (SDS–PAGE carried out using 10% polyacrylamide gel containing 0.1%SDS, and stained with 0.1% Coomassie Brilliant Blue R-250 afterelectrophoresis). Lanes: MARKER, molecular mass marker; ECOL,purified protein extract of E. coli with silver nitrate; AN, purifiedprotein extract of A. niger with silver nitrate.



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Kowshik et al. 2003; Bhainsa and D’Souza 2006; Vignesh-waran et al. 2007; Sanghi and Verma 2009; Nithya andRagunathan 2009); however, there are very few investiga-tions on nanoparticle synthesis by marine microbes. Our lab-oratory has reported extracellular synthesis of silvernanoparticles by the marine fungus Penicillium fellutanum(Kathiresan et al. 2009). The present study proved thatE. coli was more potent than A. niger in the synthesis of sil-ver nanoparticles, as indicated by the absorbance spectra(Figs. 1, 2, and 3).

The characteristic feature of silver nanoparticles synthe-sized by E. coli and A. niger was analyzed by using XRDspectra. The intense peaks observed in the spectra are inagreement with Bragg peaks corresponding to the (111),(200), (220), (311), and (222) sets of lattice planes of silvernanocrystals (Lu et al. 2003; Bhainsa and D’Souza 2006).This further confirmed that the nanoparticles synthesized byE. coli and A. niger were present in the form of silver nano-crystals.

The shape and size of silver nanoparticles was confirmedby TEM. The micrograph (Fig. 4) showed nanoparticleswith variable shape, most of them present were spherical innature and more scattered. The size of silver nanoparticlesranged from 5 to 20 nm for E. coli and from 5 to 35 nm forA. niger, and these size ranges fall closer to those producedby other microorganisms (Ahmad et al. 2003; Chandran etal. 2006; Kathiresan et al. 2009).

FTIR spectroscopy measurement was carried out to iden-tify the possible biomolecules responsible for capping andefficient stabilization of the silver nanoparticles synthesizedby E. coli and A. niger (Fig. 5). The prominent peaks corre-sponded to amides I and II regions that are characteristic ofproteins and (or) enzymes that are responsible for the reduc-tion of metal ions for synthesis of metal nanoparticles(Mukherjee et al. 2001a, 2001b, 2002; Ahmad et al. 2003).It is well known that proteins can bind to silver nanopar-ticles through either free amine groups or cysteine residuesin the proteins (Gole et al. 2001) and, therefore, stabilizationof silver nanoparticles by the surface-bound proteins waspossible during their synthesis by microbial filtrates. Therole of proteins in plant extracts as reducing and cappingagents forming stable and shape-controlled silver nanopar-ticles has been reported (Sharma et al. 2009). Similarly, inthe present study, proteins were present in both microbialsuspensions studied, which was confirmed by SDS–PAGEanalysis (Fig. 6). In support of this observation, Kathiresanet al. (2009) found a band corresponding to the enzyme ni-trate reductase while using Pencillium fellutanum for silvernanoparticle synthesis. Similarly, Ahmad et al. (2003) re-ported that a certain NADH-dependent reductase is involvedin reduction of silver ions in the case of Fusarium oxyspo-rum. There are also reports on reductases (Anil Kumar etal. 2007) and polysaccharides (Huang and Yang 2004) asfactors involved in biosynthesis and stabilization of thenanoparticles, respectively. Hydrogenase, an extracellularenzyme, has excellent redox property and can act as an elec-tron shuttle in metal reduction (Duran et al. 2005). Electronshuttles or other reducing agents (e.g., hydroquinones) re-leased by microorganisms are capable of reducing ions tonanoparticles (Baker and Tatum 1998). Our hypothesis isthat several factors together determine the nanoparticle syn-

thesis; however, the exact reaction mechanism leading to theformation of silver nanoparticles by microbes is yet to beelucidated.

Silver ions and silver-based compounds are highly toxicto microorganisms (Slawson et al. 1992), showing a strongbiocidal effect against many species of bacteria (Spadaro etal. 1974). Silver and its salts have been used medically andin controlling bacteria and other organisms in water (Liau etal. 1997). However, Ag+ ions or salts have only limited use-fulness as antimicrobial agents because of the interfering ef-fects of salts and the discontinuous release of inadequateconcentration of Ag+ ions from the metal. In contrast, theselimitations can be overcome by using silver nanoparticles,since silver nanoparticles are highly reactive because of theirlarge surface area to volume ratio, which provides a moreeffective means for antibacterial activity (Baker et al. 2005).

The results showed that silver nanoparticles synthesizedby E. coli and A. niger inhibited the growth of clinicalpathogenic bacteria (especially gram-negative bacteria) andfungi. This antimicrobial activity varied with among thepathogenic microbes tested. The nanoparticles produced byE. coli were more efficient than those produced by A. niger(Table 1). This may be due to variation in the particle sizeand shape of the nanoparticles produced (Brunner et al.2006). The nanoparticles show greater inhibition againstgram-negative bacteria than gram-positive bacteria (Jain andPradeep 2005; Son et al. 2004; Li et al. 2005; Lok et al.2006; Pal et al. 2007). This differential activity can be at-tributed to cell wall structure. Gram-negative bacteria con-tain a lipopolysaccharide layer at the exterior, followed by athin layer (7–8 nm) of peptidoglycan (Madigan andMartinko 2005). Lipopolysaccharides are not as rigid as pep-tidoglycan because of the covalent linkage between the lipidand polysaccharide. Lipopolysaccharides contain a negativecharge (Salton et al. 1996) and attract the weak, positivelycharged silver nanoparticles (Sui et al. 2006). On the otherhand, gram-positive bacteria are principally composed of athick layer (20–80 nm) of peptidoglycan, consisting of linearpolysaccharide chains cross-linked by short peptides to forma 3-dimensional rigid structure (Baron 1996). The rigid andextended cross-linking not only endows the cell wall withfewer anchoring sites for the nanoparticles, but also makesit difficult to penetrate.

The exact mechanisms of the antimicrobial effect of silvernanoparticles are still not known. However, silver works in anumber of ways to disrupt critical functions in a micro-organism and several hypotheses have been proposed (Duranet al. 2010). A number of studies suggest that silver ions re-act with –SH groups of proteins (Liau et al. 1997; Feng etal. 2000) and play an essential role in bacterial inactivation(Morones et al. 2005). Micromolar levels of silver ions havebeen reported to uncouple respiratory electron transportfrom oxidative phosphorylation, which inhibits respiratorychain enzymes or interferes with membrane permeability toprotons and phosphate (Feng et al. 2000). The silver nano-particles also affect the membrane of the microbial cells,which may lead to a significant increase in the permeabilityand affects membrane transport (Sondi and Salopek-Sondi2004). Feng et al. (2000) suggested that the presence of sil-ver ions and sulfur in the electron-dense granules observedafter silver ion treatment in the cytoplasm of bacterial cells



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suggests an interaction with nucleic acids that probably re-sults in impairment of DNA replication. The binding of sil-ver to bacterial DNA may also inhibit a number ofimportant transport processes, such as phosphate and succi-nate uptake, and can interact with cellular oxidation proc-esses as well as the respiratory chain (Vermeiren et al.2002). The positive charge on a silver ion is another and im-portant factor for its antibacterial nature, through electro-static interaction between the negatively charged cellmembrane of the microorganisms and positively chargednanoparticles (Dragieva et al. 1999; Hamouda et al. 2001;Dibrov et al. 2002). It is proposed that the electrostatic forcemight be an additional cause for the interaction of the nano-particles with the bacteria (Morones et al. 2005). Hence, themicrobial inhibitory effect of silver nanoparticles is probablythe sum of distinct mechanisms of action.

In the present study, antimicrobial activities of silvernanoparticles synthesized by E. coli and A. niger were en-hanced with the addition of PVA (Table 1). PVA is a non-toxic, water soluble, biocompatible, and biodegradablesynthetic polymer. Since polymers prevent agglomerationand precipitation of the particles, they are frequently usedas particle stabilizers in chemical synthesis of metal col-loids. PVA could be considered a good host material formetal because of its excellent thermostability and chemicalresistance (Lili et al. 2007). Also nanocomposites of silvernanoparticles and PVA are intriguing because of their easymanipulation, low cost, and controllable particle size(Giannetti et al. 1986). The enhancement of antimicrobialactivity by Ag–PVA nanocomposites by chemical reductionwas reported by Lili et al. (2007). Studies have demon-strated that the distribution of silver nanoparticles protectedwith the polymer chain of PVA is uniform (Silva et al.2008; Lili et al. 2007). PVA acts as precursor, reductant,and stabilizer of silver nanoparticles and thus enhances theantimicrobial activity of the nanoparticles in the presentstudy.

The present study revealed that marine strains of the fun-gus A. niger and the bacterium E. coli could be able to syn-thesis silver nanoparticles extracellularly. The microbialculture filtrates catalyzed the formation of the nanoparticlesbecause of presence of various proteins that are involved inthe reduction of silver ions. The nanoparticles also showedremarkable inhibition against clinical pathogens. The presentstudy also proved the efficacy of PVA in augmenting anti-microbial activity through its ability to bind to the nanopar-ticles. However, further detailed studies are required todelineate the exact mechanism of inhibition at the molecularlevel.

AcknowledgementsThe authors are thankful to T. Balasubramanian, Director

of this Centre and authorities of Annamalai University, forproviding access to all facilities we used for this study.

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Analysis of antimicrobial silver nanoparticles synthesized by coastal strains of Escherichia coli...

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