Bioresource Technology 107 (2012) 295–300

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Bioresource Technology

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Synthesis and characterization of agar-based silver nanoparticles andnanocomposite film with antibacterial applications

Mahendra K. Shukla, Ravindra Pal Singh, C.R.K. Reddy ⇑, Bhavanath JhaDiscipline of Marine Biotechnology and Ecology, CSIR – Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

a r t i c l e i n f o

Article history:Received 26 September 2011Received in revised form 23 November 2011Accepted 24 November 2011Available online 11 December 2011

Keywords:AgarBacillus pumilusSilver nanoparticleTEM

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.11.092

⇑ Corresponding author. Tel.: +91 278 256 5801x67562.

E-mail addresses: crkcsmcri@gmail.com, crkcsmcri(C.R.K. Reddy).

a b s t r a c t

This study describes the synthesis and characterization of silver nanoparticles and nanocomposite mate-rial using agar extracted from the red alga Gracilaria dura. Characterization of silver nanoparticles wascarried out based on UV–Vis spectroscopy (421 nm), transmission electron microscopy, EDX, SAED andXRD analysis. The thermal stability of agar/silver nanocomposite film determined by TGA and DSC anal-ysis showed distinct patterns when compared with their raw material (agar and AgNO3). The TEM find-ings revealed that the silver nanoparticles synthesized were spherical in shape, 6 nm in size with uniformdispersal. The synthesized nanoparticles had the great bactericidal activity with reduction of 99.9% ofbacteria over the control value. The time required for synthesis of silver nanoparticles was found to betemperature dependent and higher the temperature less the time for nanoparticles formation. DSC andXRD showed approximately the same crystalline index (CIDSC 0.73).

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The nanoparticles research in recent times has been gainingconsiderable importance in the area of biology, medicine, and elec-tronics owing to their unique particle size and shape dependentphysical, chemical and biological properties (Sun et al., 2008; Koet al., 2007). The inherent characteristics of nanoparticles such ashigh surface to volume ratios and quantum confinement result inmaterials that are qualitatively different from their bulk counter-parts (EI-Sayed, 2001) and make them suitable for wide range ofbiomedical applications including drug targeting, drug delivery,cell imaging, labeling experiments and biosensors (Farokhzadet al., 2006). Nowadays, pharmaceuticals based on nanoparticlesof polymers, metals or metal oxides, liposomes, micelles or dendri-mers are being actively considered for combating various diseasesincluding cancer (Farokhzad et al., 2006) and bacterial pathogens(Morones et al., 2005). Elemental silver and silver salts have beenextensively employed as antimicrobial agents in curative and pre-ventive health care from time immemorial, even before the adventof synthetically manufactured organic medicines such as penicillin(Williams and Bardsley, 1999). At present, silver nanoparticles asan antimicrobial agent is gaining greater demand in medical appli-cations as antibiotic-resistant bacterial strains are of growing con-cern in public health care (Goldmann et al., 1996; Chastre, 2008).

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@yahoo.com, crk@csmcri.org

For wound healing, the medical appliances based on silver havebeen proven as an effective tool for retarding and preventing thebacterial infections. The previous studies have also dealt with anti-fungal activity (Kvitek et al., 2009) and toxicity of silver nanoparti-cles against higher organisms (Panacek et al., 2009).

There is a continuous effort being directed to synthesize novelsilver nanoparticles containing products having both antibacterialproperties and new functional attributes (Huang et al., 2004; Bal-ogh et al., 2001). Also new synthesis strategies have been continu-ously evolved to prepare nanoparticles with newer application. Inthe past two decades, various types of methods have been em-ployed for the synthesis of Ag and Au nanoparticles through reduc-tion of their respective salts with chemical reducing agents, such ascitric acid, borohydride and organic compound N,N-dimethyl-formamide (Plyuto et al., 1999; Wang et al., 2002; Pastoriza-Santosand Liz-Marzan, 2000; Guari et al., 2003). The synthesis of metalnanoparticles using aforementioned reducing agents is a straightforward resulting into their derivatization with biomolecules andthus, has been extensively studied while metal nanoparticlesderivatized with polysaccharides has largely been overlooked (Ply-uto et al., 1999; Wang et al., 2002; Guari et al., 2003). The polysac-charide-nanomaterial integration has recently emerged as anexciting research opportunity. Recently, there has been a reporton the synthesis of silver nanoparticles using polysaccharide fromPorphyra vietnamensis (Venkatpurwar and Pokharkar, 2011). Goleand his coworkers used glucose as a reducing agent for synthesiz-ing gold nanoparticles entrapped in the thermally evaporated fattyamine film (Gole et al., 2001). Raveendran et al. (2003) used b-D-glucose as a reducing agent and starch as a capping agent to

296 M.K. Shukla et al. / Bioresource Technology 107 (2012) 295–300

prepare starch/silver nanocomposite film. In another similar studychitosan and heparin were used as reducing and stabilizing agentsfor the synthesis of Au and Ag nanoparticles, respectively (Huanget al., 2004).

The studies on biogenic synthesis of silver nanoparticles arescanty (Suresh et al., 2010). There is no report on the exploitationof agar as a reducing agent for silver nanoparticle formation fol-lowed by investigations on its nanocomposite for in vitro antibac-terial property. Therefore, the present study demonstrate the useof native agar extracted from red seaweed Gracilaria dura as reduc-ing agent of silver for the synthesis of silver nanoparticles and sub-sequently entrapped these nanoparticles in the agar to make agar/silver nanocomposite film and evaluated its potential for antibac-terial applications.

2. Methods

2.1. Synthesis of silver nanoparticles

The agar polysaccharide was extracted from red alga G. durausing the method reported earlier (Meena et al., 2007). For the syn-thesis of silver nanoparticles, agar powder were dissolved in deion-ized water under constant stirring condition (200 rpm) to obtainthe solution of different concentration (1, 3 and 5 mg ml�1) andAgNO3 was added in the obtained solutions to get its final concen-tration 2.5 and 5.0 mM. The reaction mixtures were incubated forthe time period of 1, 4 and 48 h at 25 �C, 60 �C and 100 �C in darkcondition at constant stirring. The pH of the reaction mixtureswere adjusted towards slightly acidic (pH 6). Thereafter, obtainedsolutions were centrifuged at 5000�g for 20 min to get a clearsolution of silver nanoparticles. Furthermore, to make agar/silvernanocomposite, 1.5 g agar was added into 100 ml of silver nano-particles solution followed by heating in microwave oven for 2–3 min. Subsequently, the viscous solution of agar/silver nanocom-posite was poured into a plastic Petri plates in appropriate quanti-ties and dried at room temperature. The dried mass can be peeledoff as a thin film which was used in the further experiments.

2.2. Characterization of agar/silver nanocomposite

The dark brown color of the silver nanoparticles was observedby digital camera. The synthesis of silver nanoparticles was moni-tored by UV–Vis-NIR scanning spectrophotometer (UV-3101 PC,Shimadzu, Japan) over the range of 250–700 nm. For transmissionelectron microscopy (TEM), a drop of colloidal solution consistingof silver nanoparticles was dispensed directly onto a carbon coatedcopper grid and allowed to dry completely in a vacuum desiccator.The images of thus, prepared samples were obtained using a JEOLtransmission electron microscope (Model JEM 2100, Japan)equipped with an EDX attachment at 200 kV.

2.3. X-ray diffraction analysis and atomic force microscopy

X-ray diffraction (XRD) was performed according to (Singhet al., 2011). X-ray powder diffractometer (Philips X’pert MPD,The Netherlands) instrument equipped with a PW3123/00 curvedNi-filtered CuKa (k = 1.54056 Å) radiation generated at 40 kV and30 mA with liquid nitrogen cooled solid-state germanium detectorto study the physical properties of agar/silver nanocomposite pow-der using slow scan in different ranges of two-theta angles (2–80�).The agar/silver nanocomposite film were dried and ground to makea powder and mounted on a quartz substrate and intensity peaksof diffracted X-rays were continuously recorded with scan steptime 1 s at 25 �C.

Crystallinity index (CIxrd) was calculated from the area undercrystalline peaks normalized with corresponding to total scatteringarea (Ricou et al., 2005).

CIxrd ¼X

Acrystal=X

Acrystal þX

Aamorphous

� �

AFM for the bacterial sample prepared according to (Sureshet al., 2010). Briefly, the glass coverslip were dipped into 0.5% gel-atin (Sigma–Aldrich) solution in deionized water and dried over-night in oven at 60 �C. Control or silver nanoparticle-treatedbacterial cultures were washed and suspended in deionized water.A drop of bacterial suspension was applied to the gelatin-coatedsurface, allowed to stand for 10 min, rinsed in deionized water toremove unattached cells and air dried for AFM examination andimaging. AFM analysis was carried out with NT-MDT companybased (Moscow, Russia) – Ntegra Aura model. Semi-contact modeSPM NSG 01tip was used to determine the surface roughness whileimages were scanned in the probe mode. Images were developedon the Nova P9 software. Cantilever of the instrument is made ofSilicon Nitride with Gold coated covering. The tip used had a radiusof curvature of approximately 10 nm and the natural frequency forthe cantilever was 300 kHz.

2.4. Antibacterial assay of agar/silver nanocomposite

Agar/silver nanocomposite films were cut as a disc of 8.0 mmdiameter and washed with deionized water subsequently driedin a vacuum oven and 200 ll suspensions of bacterial isolates werespread on separate Petri plates. Thereafter, agar/silver nanocom-posite disc was placed in the middle of the plate and incubatedat 30 �C for 4 days. The presence of clear inhibiting zone thatformed around the disc was observed and captured by a digitalcamera. The average diameter of the inhibition zone surroundingthe disc was measured to assess toxicity. The culture Petri plateswith agar/silver nanocomposite were taken as a test and withoutagar/silver nanocomposite considered as a control.

2.5. Bactericidal effect of silver nanocomposite

To examine the bactericidal effect of silver nanoparticles theovernight culture of Bacillus pumilus (accession numberHQ318731) having approximately 28 � 106/ml CFU was added inthe 50 ml of Zobell marine broth (Himedia, India). Different con-centrations (50, 100 and 500 lM) of nanoparticles were added indifferent flasks along with control (without adding nanoparticle).The bacterial suspensions were then incubated in orbital shakingat 30 �C for 24 h. Thereafter, 1 � 103 and 1 � 105 dilution of100 ll bacterial suspensions from all the test and control werespread on the Zobell marine agar and incubated for 24 h to allowCFU counts. Data was the mean of at least four independentdeterminations.

2.6. Thermal gravimetric analysis (TGA) and differential scanningcalorimetric (DSC) analysis

Five milligrams of dried Agar, AgNO3 and agar/silver nanocom-posite were used for the TGA and DSC experiment. All TGA (25–600 �C) and DSC thermograms (0–500 �C) were obtained undernitrogen atmosphere at the rate of 10 �C min�1 and their respectivegraphs were plotted with percentage weight loss and heat flow v/stemperature, respectively. TG and DSC analysis of agar/silver nano-composite were carried with Mettler Toledo TGA/SDTA System(Greifensee, Switzerland). The activation energy (Ea) was calcu-lated with Arrhenius equation while enthalpy of transition (DH)and crystallinity (CIDSC) were calculated with following equation(Khanna and Kuhn, 1997).

M.K. Shukla et al. / Bioresource Technology 107 (2012) 295–300 297

Ea ¼ �2:303RT log10ðk=AÞ

where R is the universal gas constant, A is the frequency factor forthe reaction, T is the temperature (K, 288 �C) and k is the reactionrate coefficient. Value was calculated for the nth order of thereaction.

DHtotal ¼ KA

where DH is the enthalpy of transition, K is the calorimetric con-stant and A is area under the curve.

CIDSC ¼ DHNet=Htotal

where DHNet is differenced between heat of crystallization andmelting.

3. Result and discussion

Silver nanoparticles were formed by the reduction of Ag+ intoAg� with the addition of agar (5 mg ml�1) to the solution of5 mM AgNO3. The colorless solution of AgNO3 turned into darkbrownish yellow color indicating the formation of silver nanopar-ticles. The formation of silver nanoparticles was monitored byUV–Vis absorption spectra at 250–700 nm where an intense bandwas clearly detected at 421 nm. This band was identified as a ‘‘sur-face Plasmon resonance band’’ and ascribed to the excitation offree electrons in the nanoparticles. The UV–Vis spectrum showsno absorption peak for the AgNO3 solution or agar separately.The shape of the band was symmetrical suggesting uniform dis-persal of spherical shape nanoparticles (Travan et al., 2009).

The effect of concentration of both polymer and silver nitrate onsynthesis of silver nanoparticles was investigated (Fig. 1A). Thechanges in UV–Vis absorption peaks indicated that the size anddispersion of silver nanoparticle were affected by both the concen-tration of agar (which functions as a controller of nucleation aswell as a stabilizer) and AgNO3. The highest Plasmon peaks wererecorded for AgNO3 at 5 mM in the presence of 5 mg ml�1 agar.

Fig. 1. Optimization of (A) concentration of agar and silver nitrate for the silver nanopar25 �C, 60 �C and 100 �C at (B) 1 h (C) 4 h and (D) 48 h time period.

The boiling temperature played an important role in the reduc-tion of silver ion (Pillai and Kamat, 2004). Approximately 1, 4 and48 h was required for nanoparticle formation at 100 �C, 60 �C and25 �C, respectively (Fig. 1B–D). Thus, it is evident from this exper-iment that higher the temperature lower the time required for thenanoparticles formation.

The silver nanoparticle size, morphology and distribution wasanalyzed by TEM. The size of the nanoparticles reported to affectthe antimicrobial properties (Panacek et al., 2006). Conglomerationof silver nanoparticles was observed in samples synthesized at60 �C and 100 �C. Interestingly, the reaction carried out at roomtemperature showed no clustering of silver nanoparticles as con-firmed by TEM analysis. The silver nanoparticles synthesized atroom temperature were spherical in shape and also separated fromeach other. The average silver nanoparticles size as measured fromTEM images was 6.0 ± 2 nm while Travan and coworkers preparednanoparticles using chitlac as reducing agent with an average par-ticle size of 33.6 ± 7 nm (Travan et al., 2009). Kemp and coworkersdemonstrates the synthesis of silver nanoparticles with an averageparticle size of 7 ± 3 nm using hyaluronan as reducing agent (Kempet al., 2009).

The EDX spectrum obtained for nanoparticles showed the stron-gest peak detected at �3 eV confirming the presence of elementalsilver in nanoparticles (Magudapathy et al., 2001) while N, O and Catoms from the polymer are also detected with relatively weakerintensity. The Cu signal originates from the carbon-coated coppergrid.

The selected area electron diffraction (SAED) pattern showedcrystalline and face centered cubic structure of silver nanoparti-cles. The hexagonal nature of the diffraction spots indicates thatthe particles were highly oriented (111), with the top normal tothe electron beam. The spots could be indexed based on a face-cen-tered-cubic (fcc) structure of silver (Suresh et al., 2010). Also, thelattice fringes with a distance of 2.44 Å observed in HR-TEM whichsupports the crystalline nature of the silver nanoparticles (Jainet al., 2011). The ED rings were relatively broad because of the

ticles synthesis and effect of temperature on the synthesis of silver nanoparticles at

Fig. 2. TGA (A) and DSC (B) thermogram of agar/silver nanocomposite.

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polycrystalline nature and the small sizes of the particles (Lin et al.,2003).

XRD pattern of agar/silver nanocomposite powder showedcharacteristics peaks at 2h values of 38.203, 44.0, 64.54 and 77.0corresponding to 111, 200, 220 and 311 planes for silver crystal,respectively. These Bragg reflections in the 2h range agree with val-ues reported for silver nanocrystals (Kumar et al., 2007). The XRDpattern shows several amorphous peaks due to amorphous natureof agar (as a co-polymer of galactose and 3, 6 anhydro galactose)and it is difficult to interpret broad amorphous peaks of severalamorphous compounds in X-ray scattering profile (Shimazuet al., 2000).

TGA was carried out dynamically between weight loss v/s tem-peratures and experimental result showed 3.1% and 34.3% degrada-tion of silver and 9.0%, 38.7% and 13.4% degradation of agar duringfirst, second and third step, respectively. However, 7.23%, 25.71%,21.94% and 3.5% degradation of agar/silver nanocomposite was ob-served during first, second, third and fourth step, respectively(Fig. 2A). The first step degradation in agar and agar/silver nanocom-

Fig. 3. Bactericidal effect of silver nanoparticles using its different concentrations (A) coninhibition zone formed by agar/silver nanocomposite disc on the growth of Bacillus pum

posite observed more than AgNO3 due to more bound water mole-cules to agar. The complete degradation of silver has occurred insecond phase while agar/silver nanocomposite occurred in foursteps. The four steps degradation of agar/silver nanocomposite re-vealed more stability than their raw materials. Crystallization pro-cess is an exothermic process and DSC analysis showed asignificant thermal transition of silver, agar and agar/silver nano-composite into a crystalline state. DSC thermogram showed distinctexothermic peaks of agar and agar/silver nanocomposite with crys-tallization temperature (Tc) 71.61 �C (onset temperature 96.71 �C)and (Tc) 107.73 �C (onset temperature 99.16 �C) accompanied with1850.44 and 2353.43 mJ latent energy, respectively (Fig. 2B) whilein the AgNO3 only two exothermic peaks were observed with (TC1)214.56 �C (onset temperature 211.95 �C) and TC2 439.7 �C (onsettemperature 424.18 �C) accompanied with 318.78 and 4315.36 mJlatent energy. The endothermic peaks (Tm) of agar and agar/silvernanocomposite were found at 256.54 �C (onset temperature252.47 �C) with 414.22 mJ latent energy and 181.66 �C (onset tem-perature 167.05 �C) with 3200 mJ latent energy, respectively. The

trol (B) 1 lM (C) 3 lM and (D) 5 lM solution of silver nanoparticles (E) Diameter ofilus.

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activation energy (nth order of reaction) of exothermic transitionswere 66.25 ± 0.31 and 50.47 ± 0.34 kJ mol�1. It was found quite lessfrom 512.10 ± 2 and 171.6 ± 1.22 kJ mol�1 for endothermic transi-tion of agar and agar/silver nanocomposite, respectively. Howeveractivation energy (nth order of reaction) of exothermic transitionsof AgNO3 was 649.33 ± 19.84 and 454.47 ± 2.82 kJ mol�1. Two exo-thermic peaks of silver nitrate transform into one revealing the for-mation of agar/silver nanocomposite. The data obtained from TGAand DSC confirmed that these are the best tools for identificationof nanocomposite as their raw materials give different patterns fordegradation and crystallization.

The antibacterial activity of agar/silver nanocomposite wereanalyzed by disc diffusion and bacterial killing kinetics assay. The25 mm clear inhibitory zone appeared around agar/silver nano-composite disc after incubation for 24 h, suggesting that agar/silvernanocomposite showed completely inhibited bacterial growth(Fig. 3E). Furthermore, the silver nanoparticles showed a remark-able bactericidal effect against B. pumilus (accession numberHQ318731) with very fast killing kinetics as comparison to control(Fig. 3). The number of viable bacterial cells was found to be80 � 107/ml CFU count in control while 80, 30 and 5 � 104/ml CFUcount were calculated after 24 h incubation of bacterial cells with50, 100 and 500 lM of silver nanoparticles, respectively. Thus, sil-ver nanoparticles were found to be toxic and thus reduced the CFUcount up to 99.9% over the control value. AFM is suitable tool forthe investigation of cell membrane morphology and surface struc-ture of the bacterial cell (Mortensen et al., 2009). AFM image ofcontrol and nanoparticles treated bacterial sample showed distinctvariation in outer surface morphology. Although the mechanism ofthe interaction between silver nanoparticles and the constituent ofthe outer membrane of bacterial cell is largely unknown, it appearsthat silver nanoparticles may be interacting with the bacterial cellwall, causing structural changes, dissipation of the proton motiveforce leading to eventual cell death (Sondi and Branka, 2004). Thisstudy clearly demonstrates that the bactericidal effect depends onthe concentration of the agar/silver nanocomposite. Therefore,agar/silver nanocomposite not only retards the bacterial growthbut also killed the bacteria at minimum level of 99.93% reductionwhich is in line with the previous report for chitlac/silver nano-composite (Travan et al., 2009). Ivan and coworkers reported silvernanoparticles showing antibacterial effect in the solid agar mediabut growth delay activity against Escherichia coli in broth media(Sondi and Branka, 2004). As the high CFU applied in the presentstudy are hardly ever found in real-biological systems, thus, thisnanocomposite could be a potential candidate in reducing bacterialgrowth in various biocidal materials.

4. Conclusions

In conclusion, this study describes the synthesis of silver nano-particles using agar. Formations of silver nanoparticles were con-firmed by UV–Vis spectroscopy, TEM-EDX, SAED and XRD. Thenanoparticles formed were small in size (6 nm) and well dispersed.The method described in this study for synthesis of silver nanopar-ticles is green as compared to chemical routes. The antibacterial ef-fect of silver nanoparticles showed greater bactericidal effect(99.9%) on the bacterium B. pumilus (accession numberHQ318731). The silver nanocomposite film with proven antibacte-rial property may find applications in food preservation and wounddressing.

Acknowledgements

The financial support received from the Council of Scientific andIndustrial Research (RSP 0016), New Delhi is gratefully acknowl-

edged. Mr. Ravindra Pal Singh gratefully acknowledges the CSIR,New Delhi for awarding the Senior Research Fellowship. Theauthors are also grateful to Dr. Divesh N. Srivastava for assistanceand interpretation of TEM results and Dr. Paul for allowing us touse some analytical instruments for data reported in this study.We also thank two anonymous referees for their valuable and con-structive comments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2011.11.092.

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