714 DOI: 10.1021/la1036162 Langmuir 2011, 27(2), 714–719Published on Web 12/13/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Bacterial Kinetics-Controlled Shape-Directed Biosynthesis of SilverNanoplates Using Morganella psychrotolerans

Rajesh Ramanathan, Anthony P. O’Mullane, Rasesh Y. Parikh, Peter M. Smooker,Suresh K. Bhargava,* and Vipul Bansal*

School of Applied Sciences, RMIT University, GPO Box 2476 V, Melbourne, VIC 3001, Australia

Received September 9, 2010. Revised Manuscript Received November 21, 2010

We show for the first time that by controlling the growth kinetics of Morganella psychrotolerans, a silver-resistantpsychrophilic bacterium, the shape anisotropy of silver nanoparticles can be achieved. This is particularly importantconsidering that there has been no report that demonstrates a control over shape of Ag nanoparticles by controlling thegrowth kinetics of bacteria during biological synthesis. Additionally, we have for the first time performed electro-chemistry experiments on bacterial cells after exposing them to Agþ ions, which provide significant new insights aboutmechanistic aspects ofAg reduction by bacteria. The possibility to achieve nanoparticle shape control by using a “green”biosynthesis approach is expected to open up new exciting avenues for eco-friendly, large-scale, and economically viableshape-controlled synthesis of nanomaterials.

Metal nanoparticles, particularly Au and Ag, have gainedsignificant interest in the past decade because of their uniquephysicochemical properties with applications in diverse areasincluding molecular diagnostics,1 catalysis,2 electronics,3 drugdelivery,4 sensing,5 and surface-enhanced Raman scattering.6 Todate, the majority of research has been focused on the synthesis ofisotropic (i.e., spherical or quasi-spherical) metal nanoparticles,with the development of physical and chemical methods to achieveexcellent control over size distribution.7-11 Recently, the impor-tance of nanoparticle shape anisotropy has been realized with anemphasis to explore the correlation between different shapes andtheir physicochemical properties.2,9-11 A range of shapes includ-ing cubes,10 prisms,11 and rods12 can now be regularly synthesizedusing chemical methods. However, some of the limitations gen-erally associated with physical and chemical synthesis approaches

include the use of eco-toxic solvents, harsh reaction conditions,scale-up issues, and formation of harmful byproducts.13,14 There-fore, more recently there has been an increasing focus to developnontoxic, environmentally benign “green” approaches for thesynthesis of metal nanoparticles.10,13,14

To this end, our group and others have previously demonstratedthat a wide array of biological entities including bacteria,10,14,15

fungi,13,16 plants,17-19 algae,20 and yeast21 can be utilized for eco-friendly biosynthesis of metal, metal oxide, and metal sulfidenanoparticles. Among different biological entities, the use ofbacteria is preferred for nanoparticle biosynthesis due to extra-cellular nanoparticle production and ease of culturing, manipula-tion, and downstream processing.10,14 Importantly, most of thebiosynthesis efforts exploring microorganisms for nanoparticlessynthesis have hitherto led to formation of isotropic nanoparti-cles, with only limited reports on shape-controlled biosynthesis ofAunanoprisms using plant extracts such asAloe vera17 and lemongrass.18 Similarly, one of the studies has also demonstratedbiosynthesis of Ag nanoplates using extracts of unicellular algaChlorella vulgaris at room temperature.22 Notably, these reportsare either on plant or algal extracts-based synthesis of metalnanoprisms and nanoplates (and not the whole plant/algae assuch), wherein ketones and aldehydes (Au)18 and amino acids

*Corresponding authors. E-mail: vipul.bansal@rmit.edu.au (V.B.), suresh.bhargava@rmit.edu.au (S.K.B.). Phone: þ61 3 99252121. Fax: þ61 399253747.(1) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536–1540.(2) (a) Bansal, V.; Li, V.; O’Mullane, A. P.; Bhargava, S. K. Cryst. Eng.

Commun. 2010, 12, 4280-4286. (b) Bansal, V.; O'Mullane, A. P.; Bhargava, S. K.Electrochem. Commun. 2009, 11, 1639–1642. (c) O'Mullane, A. P.; Ippolito, S. J.;Sabri, Y. M.; Bansal, V.; Bhargava, S. K. Langmuir 2009, 25, 3845–3852.(3) Schmid, G.; Corain, B. Eur. J. Inorg. Chem. 2003, 17, 3081–3098.(4) Kang, B.; Mackey, M. A.; El-Sayed, M. A. J. Am. Chem. Soc. 2010, 132,

1517–1519.(5) Sawant, P. D.; Sabri, Y.M.; Ippolito, S. J.; Bansal, V.; Bhargava, S. K.Phys.

Chem. Chem. Phys. 2009, 11, 2374–2378.(6) Plowman, B.; Ippolito, S. J.; Bansal, V.; Sabri, Y. M.; O’Mullane, A. P.;

Bhargava, S. K. Chem. Commun. 2009, 5039–5041.(7) Grzelczak, M.; Perez-Juste, J.; Mulvaney, P.; Liz-Marzan, L. M.Chem. Soc.

Rev. 2008, 37, 1783–1791.(8) Lu, X.; Rycenga, M.; Skrabalak, S. E.; Wiley, B.; Xia, Y. Annu. Rev. Phys.

Chem. 2009, 60, 167–192.(9) Millstone, J. E.; Hurst, S. J.;M�etraux, G. S.; Cutler, J. I.;Mirkin, C. A.Small

2009, 5, 646–664.(10) Thakkar, K. N.; Mhatre, S. S.; Parikh, R. Y. Nanomed. Nanotechnol. Biol.

Med. 2010, 6, 257–262.(11) Xia, Y.; Xiong, Y.; Lim, B.; Skrabalak, S. E. Angew. Chem., Int. Ed. 2009,

48, 60–103.(12) Murphy, C. J.; Gole, A. M.; Hunyadi, S. E.; Orendorff, C. J. Inorg. Chem.

2006, 45, 7544.(13) Ahmad, A.; Mukherjee, P.; Senapati, S.; Mandal, D.; Khan, M. I.; Kumar,

R.; Sastry, M. Colloids Surf., B 2003, 28, 313–318.(14) Parikh, R. Y.; Singh, S.; Prasad, B. L. V.; Patole, M. S.; Sastry, M.;

Shouche, Y. S. ChemBioChem 2008, 9, 1415–1422.

(15) Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C. Proc. Nat. Acad. Sci. U.S.A.1999, 96, 13611–13614.

(16) (a) Bansal, V.; Syed, A.; Bhargava, S. K.; Ahmad, A.; Sastry, M. Langmuir2007, 23, 4993–4998. (b) Bansal, V.; Poddar, P.; Ahmad, A.; Sastry, M. J. Am. Chem.Soc. 2006, 128, 11958–11963. (c) Bharde, A.; Rautaray, D.; Bansal, V.; Ahmad, A.;Sarkar, I.; Yusuf, S. M.; Sanyal, M.; Sastry, M. Small 2006, 2, 135–141. (d) Bansal, V.;Sanyal, A.; Rautaray, D.; Ahmad, A.; Sastry, M. Adv. Mater. 2005, 17, 889–892. (e)Bansal, V.; Rautaray, D.; Bharde, A.; Ahire, K.; Sanyal, A.; Ahmad, A.; Sastry, M.J. Mater. Chem. 2005, 15, 2583–2589. (f) Bansal, V.; Rautaray, D.; Ahmad, A.; Sastry,M. J. Mater. Chem. 2004, 14, 3303–3305.

(17) Chandran, S. P.; Chaudhary, M.; Pasricha, R.; Ahmad, A.; Sastry, M.Biotechnol. Prog. 2006, 22, 577–583.

(18) Shankar, S. S.; Rai, A.; Ankamwar, B.; Singh, A.; Ahmad, A.; Sastry, M.Nature Mater. 2004, 3, 482–488.

(19) Huang, J.; Wang, W.; Lin, L.; Li, Q.; Lin, W.; Li, M.; Mann, S. Chem.;Asian J. 2009, 4, 1050–1054.

(20) Singaravelu, G.; Arockiamary, J. S.; Kumar, V. G.; Govindaraju, K.Colloids Surf., B 2007, 57, 97–101.

(21) Dameron, C. T.; Reese, R. N.; Mehra, R. K.; Kortan, A. R.; Carroll, P. J.;Steigerwald, M. L.; Brus, L. E.; Winge, D. R. Nature 1989, 338, 596–597.

(22) Xie, J.; Lee, J. Y.; Wang, D. I. C.; Ting, Y. P. ACS Nano 2007, 1, 429–439.

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(Ag)22 were identified to be responsible for shape control. There-fore, if we consider plant extract-based nanomaterials synthesis aspseudobiological, there is per se no report on shape-directedextracellular biological synthesis of anisotropic metal nanoparti-cles by directly involving a microorganism. The propensity ofmicroorganisms to almost always synthesize spherical nanoma-terials in the presence of metal ions is most probably due to theirfocus on energy minimization, where it is easiest to create thespherical shape.16

Notably, in the particular context of Ag nanoparticles bio-synthesis by bacteria, ionic silver (Agþ) is well-known to be toxicto bacteria.14,15,23 Previously, a silver-resistant bacterium Pseu-domonas stutzeri AG25915,23 was discovered from silver mines,which had the unique capability to reduce Agþ ions into Agnanoparticles and accumulate themwithin its cell to minimize theAgþ ion toxicity. It is notable that although silver-resistantbacterium P. stutzeri was able to synthesize a mixture of Agnanospheres and nanoplates intracellularly by reducing Agþ ionswithin the bacterial matrix, extracellular biosynthesis of Agnanoplates could not be achieved. Following this, there havebeen a few efforts that utilized different bacteria for the synthesisof Ag nanoparticles.10,14,15,23 However, to the best of the authors’knowledge, none of these reports have hitherto been able toachieve shape-controlled biosynthesis of silver nanoplates orother anisotropic Ag nanostructures. This is most possiblybecausemost of the previous studies in this field hadonly reportedthe outcomesof exposure ofAgþ ions to bacteria, withoutmakingany deliberate efforts to control the bacterial growth kinetics toachieve shape control. To this end, a recent study in which theeffect of temperature on the size of Ag nanoparticles formedduring a fungus-mediated biosynthesis process was investigated isparticularly notable.24 It is also notable that controlling reactionkinetics and altering reaction parameters via photochemical,microwave, and ultrasound-assisted techniques are known toachieve anisotropic growth in conventional chemical synthesis.7-11

However, contributing factors for biological synthesis are notwellunderstood. In the current study, we envisage that control overbacterial kinetics by controlling bacterial growth temperaturemight provide a facile tool to control the shape of nanomaterialssynthesized using biological approaches.

To demonstrate this proof-of-concept, in this study, we haveutilizedMorganella psychrotolerans as amodel organism, which isa close relative of silver resistant bacteria Morganella morganii.M. morganii RP42 strain was recently reported for its specificitytoward sustaining high concentrations of Agþ ions via extracel-lular synthesis of spherical Ag nanoparticles.14 In the currentstudy, M. psychrotolerans has been chosen as a model organismfor controlling shape anisotropy of Ag nanoparticles due to itstolerance for lower temperature (psychros: cold) and its capabilityto grow at a wider temperature range of typically 0-30 �C with20 �C as the optimum temperature.25 SinceM. morganii was pre-viously found to be capable of spherical Ag nanoparticles syn-thesis, we hypothesized that its close relative M. psychrotoleransmight also be able to produce Ag nanoparticles, thus making thisstudy feasible.

In an effort to synthesize anisotropic Ag nanoparticles via a“green” biological route, we herein demonstrate for the first timethat by controlling the growth kinetics of M. psychrotolerans bygrowing it at different temperatures (away from the reported

optimal temperature of 20 �C) in the presence of Agþ ions, anincrease in the formation of Ag nanoplates can be achieved. Thisis particularly important as there has been no report thatdemonstrates a control over shape of Ag nanoparticles using abacteria-mediated biological approach under extracellular condi-tions. Additionally, we have performed linear sweep voltammetry(LSV) experiments on bacterial cells after exposing them to Agþ

ions, which provide interesting insights about the Ag nanoparti-cles formation mechanism by M. psychrotolerans.

Results and Discussion

The optimum temperature for the growth and physiologicalactivities of the bacteriumMorganella psychrotolerans is 20 �C.25Although deviation of its growth environment from 20 �C tohigher temperatures (e.g., 25 �C) might lead to a faster bacterialgrowth and replication, such higher temperatures are expected toadversely affect its physiological activities. Conversely, deviationof M. psychrotolerans growth environment toward lower tem-peratures (e.g., 15 and 4 �C)will result in reduced bacterial growthas well as alteration of its physiological activities. To understandthe influence of bacterial growth and its physiological activity onAg biosynthesis capability of M. psychrotolerans, bacteria weregrown at four different temperatures (25, 20, 15, and 4 �C),followed by incubation with 5 mM AgNO3 in LB broth withoutNaCl in the absence of light. Following the reactions, all foursolutions changed in color from pale yellow (the color of growthmediumwith bacteria) tomuddy green over a period of time, thusindicating formation of silver nanoparticles at all the four growthtemperatures. In parallel control experiments, 5 mM AgNO3

solutions were prepared in the bacterial growth medium (LBbroth without NaCl) and incubated without bacteria. No colorchange was observed in the growth medium over the course ofcontrol experiments, which affirms that reduction of Agþ ions toAg nanoparticles is due to bacterial activity and not due to thebacterial growth medium.

UV-vis absorbance spectroscopywas employed tounderstandthe time-dependent kinetics of Ag nanoparticles biosynthesis byM.psychrotolerans at different temperatures (Figure 1). As showninFigure 1b, at anoptimumgrowth temperature of 20 �C, a broadsurfaceplasmon resonance (SPR) bandapparent at ca. 350-530nmappears within 2 h of reaction, which increases in intensity withtime for at least up to 24 h. The SPR feature in this range is typicalof Ag nanoparticles,14 and its increased intensity over a 24 hperiod can be assigned to the continuous biosynthesis of Agnanoparticles by bacteria.

When M. psychrotolerans was grown at a higher temperature(25 �C) with respect to the optimum, Ag SPR signature at ca.350-530 nmwere observed as early as 3 h, followedbyan increasein intensity of this SPR feature with time (Figure 1a). Interestinglyat 25 �C, an additional SPR feature at ca. 650-950 nm wasobserved that extends well into the near-infrared (NIR) region ofthe spectra and increases in intensity with the reaction time. SuchNIRSPR features are characteristic of either aggregation ofmetalnanoparticles with time,26 or formation of anisotropic nano-structures in the solution,9 or a combination of both.18

To understand the effect of lower than optimum growthtemperature on Ag nanoparticles synthesis, when M. psychroto-lerans was grown at 15 �C, the bacterial activity toward Agnanoparticles biosynthesis was found to be considerably reduced,as is evident from a weak yet detectable Ag SPR feature at ca.350-530 nmobserved only after 12 h of reaction and a prominent(23) Slawson, R.M.; Trevors, J. T.; Lee, H.Arch.Microbiol. 1992, 158, 398–404.

(24) Fayaz, A.M.; Balaji, K.; Kalaichelvan, P. T.; Venkatesan,R.Colloids Surf.,B 2009, 74, 123–126.(25) Emborg, J.; Dalgaard, P.; Ahrens, P. Int. J. Syst. Evol. Microbiol. 2006, 56,

2473–2479.(26) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996,

382, 607–609.

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SPR feature seen after 5 days of reaction (Figure 1c). With thereaction time, an increase in intensity of the Ag SPR signatureaccompanied by the development of a prominent SPR feature intheNIRregionwasobserved.This is similar to the reactionat 25 �C,wherein deviation from optimal growth temperature of 20 �Cleads to development of NIR features in the spectra.

A further reduction in reaction temperature to 4 �C consider-ably slowed down the bacterial growth as well as rate of Agnanoparticles biosynthesis, thereby leading to the appearance ofa prominent Ag SPR feature only after 14 days of reaction(Figure 1d). At 4 �C, the very broad nature of the UV-visspectrum after 14 days of reaction along with a significantlyhigher absorbance in the NIR region in comparison with that inthe visible region is particularly notable. In fact, comparison ofhighest timepoint spectra at reactions performedat 25, 15, and4 �C(compare highest intensity curves in Figure 1a,c,d) suggests thatthe relative intensity of the NIR SPR features with respect tovisible SPR features follow a temperature-dependent trend,wherein NIR absorbance is most predominant at 4 �C and theleast at 25 �C. A comparison of time-dependent kinetics of Agnanoparticles synthesis by M. psychrotolerans at four differenttemperatures also clearly suggests that the rate ofAg nanoparticlebiosynthesis follows a temperature-dependent trend, with fastestAgþ ions reduction observed at the highest temperature (25 �C;Figure 1a) and the slowest at the lowest temperature employed inthis study (4 �C; Figure 1d). This is expected as bacterialphysiological activity, growth, and multiplication will be con-siderably slowed down at lower temperatures; thus, a lowernumber of bacterial biosynthesis “nanofactories”will be availableto reduce Agþ ions into Ag0 nanoparticles.27

Ag nanoparticles biosynthesized using M. psychrotolerans atdifferent temperatures were further characterized using transmis-sion electron microscopy (TEM), as shown in Figure 2. At the

optimum growth temperature of 20 �C, predominantly sphericalAgnanoparticles of ca. 2-5 nmdiameter alongwith relatively fewnanoplates of 100-150 nm edge length were observed duringTEM imaging (Figure 2b). The synthesis of spherical Ag nano-particles at the optimum growth temperature of bacteria cor-roborates well with previous biosynthesis studies, wherein the useof differentmicroorganismshas hithertobeen reported to result inspherical Ag nanoparticles.10

However, in contrast to previous biosynthesis studies, whenM.psychrotoleransbacteriawas used in this study for biosynthesisof Ag nanoparticles at temperatures different from its optimumgrowth temperature, formation of Ag nanoplates was observed(Figure 2a,c,d). For instance, at 25 �C, which is 5 �C higher thanthe optimum growth temperature of bacteria, a mixture oftriangular and hexagonal nanoplates along with spherical nano-particles was obtained (Figure 2a). Similarly, at 15 �C, which is5 �C lower than the optimum growth temperature, again amixtureof nanoplates and spherical particles was obtained (Figure 2c).Further reduction in bacterial physiological activity and growthby reducing its growth temperature to 4 �C results in a significantincrease in the number of nanoplates, whereas only a relativelysmaller proportion of spherical nanoparticles were formed(Figure 2d). It is however notable that although the proportionof spherical Ag particles formed at 4 �C is lower than thatobserved at other temperatures; the spherical particles formedat 4 �C are larger in size (ca. 70-100 nm). This indicates that inaddition to the NIR absorbance from Ag nanoplates scatteringfrom larger size particles formed at 4 �C might have alsocontributed toward the broad SPR feature obtained at 4 �C(Figure 1d). A higher magnification TEM image of nanoplatessynthesized by bacteria at 4 �C revealed stacking and bucklingfaults that are typical of thin nanoplates (inset, Figure 2d).28 Thebiosynthesis of Ag nanoplates byM. psychrotolerans at tempera-tures away from its optimum growth temperature is particularlyinteresting as there has so far been no reports on tailoring the nano-particles shape by controlling bacterial growth kinetics. Typically,M. psychrotolerans was found to synthesize Ag nanoplates with

Figure 1. UV-vis absorbance spectra demonstrating the time-dependent kinetics of biogenic Ag nanoparticles biosynthesis byM. psychrotolerans at (a) 25, (b) 20, (c) 15, and (d) 4 �C.

Figure 2. TEM images of biogenic Ag nanoparticles biosynthe-sized by M. psychrotolerans at (a) 25, (b) 20, (c) 15, and (d) 4 �Cafter 20 h, 24 h, 5 days, and 15 days of reaction, respectively.

(27) Nedwell, D. B.; Rutter, M. Appl. Environ. Microbiol. 1994, 60, 1984–1992.(28) Germain, V.; Li, J.; Ingert, D.; Wang, Z. L.; Pileni, M. P. J. Phys. Chem. B

2003, 107, 8717–8720.

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50-150 nm edge length at 25 and 15 �C; however, biosynthesis at4 �C resulted in larger nanoplates with 150-450 nm edge length.The nanoplates obtained through biosynthesis often showedtruncated edges similar to that observed in Ag nanoprismssynthesized by a photoinduced method.29

X-ray diffraction (XRD) patterns of the drop-coated films ofbiogenic Ag nanoparticles synthesized by M. psychrotolerans atdifferent temperatures were similar and showedwell-definedBraggreflections corresponding to {111}, {200}, {220}, {311}, and {222}planes, which could be indexed based on the face-centered cubic(fcc) lattice structure of crystalline Ag (Figure 3).14 XRD analysisthus confirms that biogenic Ag nanoparticles are highly crystallinein nature. Since Ag nanoplates are well-known to be rich in the{111} plane, the ratios of the intensity of {111} to {200} peakswereplotted as a function of different temperatures at which bacterialsynthesis ofAg nanoparticleswas performed (inset, Figure 3). It isclear from the inset in Figure 3 that Ag nanoparticles formed bybacteria at its optimumgrowth temperature of 20 �Care least richin the {111} phase. Conversely, the intensity of the {111} peakincreases while increasing or decreasing the growth temperaturefrom theoptimumvalue,with the highest intensity observed at 4 �C.XRD analysis therefore corroborates well with UV-vis spectros-copy and TEM results, which exhibit similar trends.

To gain an understanding of the mechanism for Agþ ionreduction by the bacteria M. psychrotolerans, a systematic timedependence study on Ag nanoparticles biosynthesis was per-formed at 25 �C using linear sweep voltammetry (LSV). Toascertain thatLSVexperiments provide selective information aboutAg species associated only with bacteria (and not about free Agspecies in solution), prior to LSV experiments, any residual freeAgþ ions outside bacteria were removed by centrifugation andwashing bacterial cells after their growth in the presence of Agþ

ions for various time points. Figure 4 shows the LSV performedon a GC electrode immobilized withM. psychrotolerans that hadbeen exposed to Agþ ions for various time periods (0-72 h). Itshould be noted that uponholding the potential at-0.20V vsAg/AgCl the electroreduction of Agþ ions to Ag0 occurs. It is evidentfrom Figure 4 that at initial time points (up to 4 h) only oneoxidation peak associated with the oxidation of metallic Agoccurs. This can be attributed to the electroreduction of Agþ

ions taken up by bacteria at the initial time points. At later stages

of bacterial incubation with Agþ ions, it is predicted that bacteriawill start forming Ag nanoparticles from previously uptaken Agþ

ions. When LSV is performed on bacterial samples from 12 honward, two signatures corresponding to Ag oxidation can beclearly seen. In the LSVs after 12 h, the peak at the less positivepotential can be assigned to the oxidation of metallic silvergenerated through the electroreduction of Agþ ions within thebacteria, while the peak toward more positive potential can beattributed to the oxidation of Ag nanoparticles formed withinbacteria. This was confirmed by running an experiment fromopen-circuit potential (0.02 V, where no electroreduction of Agþ

occurs) to more positive values where only the peak at the morepositive potential was observed (Figure S1, Supporting In-formation). This indicates that silver ions from the solution areinitially taken up by the bacterial cells, which after associationwith bacteria may get bound to proteins and/or other biomacro-molecules within the bacteria wherein they undergo reduction,and later released out of the cells extracellularly.

The final outcome observed from the exposure of bacteriaM. psychrotolerans to Agþ ions is the extracellular appearance ofAg nanoparticles, and it is also ascertained that bacteria aresomehow involved in the biosynthesis process.However, it remainsan open and challenging question whether Ag nanoparticles areproduced extracellularly (in solution, outside the bacteria) bysome of the proteins secreted by bacteria in solution or Agþ ionsare initially taken up by the bacteria, followed by their reductionto Ag0 nanoparticles, before they are released out of bacteria inthe growthmedium. A previous study involving bacteria from thesame genus Morganella had predicted that Ag nanoparticles aremost probably produced extracellularly during biosynthesis.14 Inthe current study, when we exposed 5 mM Agþ ions to extra-cellular proteins secreted by M. psychrotolerans for 24 h atdifferent temperatures, the extracellular proteins were found toreduce Agþ ions to form Ag nanoparticles; however, the rate ofnanoparticles formationwas significantly slower, and only 1-2 nmAg nanospheres without any shape control could be obtained(Supporting Information, Figure S2). This clearly suggests that inaddition to extracellular proteins bacterial physiology also plays asignificant role toward the synthesis and shape control of Agnanoparticles. Additionally, LSV studies performed in this studyclearly indicate that biosynthesis of Ag nanoparticles by bacteriais not as simple as previously predicted, and a mere direct role ofextracellular bacterial proteins in reduction of Agþ ions, withoutinvolving bacterial machinery is rather questionable. To the bestof the authors’ knowledge, biosynthesis studies in the past havenot hitherto explored the potential of electrochemistry to studymetal ions uptake and their reduction by microorganisms. Theinteresting outcomes of the LSV experiments obtained from this

Figure 3. XRD patterns of biogenic Ag nanoparticles biosynthe-sized byM. psychrotolerans: (a) 25, (b) 20, (c) 15, and (d) 4 �C after20 h, 24 h, 5 days, and 15 days of reaction, respectively. Inset showsthe intensity ratio of {111} to {200} Bragg reflections for Agnanoparticles formed by bacteria at different temperatures.

Figure 4. Linear sweepvoltammogramsof thebacteriaM.psychro-tolerans after their exposure to Agþ ions at 25 �C for up to 72 h.

(29) Jin, R.; Cao, Y.; Mirkin, C. A.; Kelly, K. L.; Schatz, G. C.; Zheng, J. G.Science 2001, 294, 1901–1903.

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study suggest that detailed electrochemistry experiments in thefuture might provide some vital information regarding mecha-nisms of metal ion trafficking and its association with metal ionresistance in microorganisms.

Notably,M.psychrotolerans is in fact a silver-resistant bacteria,which was confirmed by investigating the presence of a genehomologue of the putative silver binding gene (silE) in thisbacteria (PCR data not shown for brevity). The silE gene isknown to encode a periplasmic silver binding protein (silE) thatplays a major role in Agþ ion uptake by providing histidine sitesas primary candidates for Agþ ions binding in silver-resistantbacteria.30 A potential mechanism for extracellular Ag nanopar-ticle biosynthesis byM. psychrotolerans is presented in Scheme 1.We believe that on exposure to Agþ ions the silE protein-basedsilver-binding machinery of bacterium gets activated, therebyleading to the cellular uptake of Agþ ions (step 1), as wassuggested previously in the case of another silver-resistant bacte-rium Salmonella.30 What happens after uptake of Agþ ions byM. psychrotolerans is rather interesting (steps 2-6), as it has pre-viously been arguably postulated that inmost of the heavy-metal-resistant bacteria metal resistance is achieved via initial uptake ofmetal ions followed by energy-dependent “pumping out” of theseions usingmembrane proteins.31Conversely, this is not the case inmercury-resistant bacteria, wherein the presence of a mercuricreductase enzyme has been established to reduce Hg2þ ions toHg0. However, the presence of a silver reductase enzyme in silver-resistant bacteria has not yet been established. Our LSV studiesstrongly indicate the reduction of Agþ ions to Ag0 nanoparticleswithin bacteria, and therefore a unique possibility of the existenceof silver reductase enzyme in biological systems cannot be com-pletely ignored. Our future endeavors will explore this excitingpossibility. At this stage, we believe that after uptake of Agþ ionsby bacteria they are presented to the Ag reduction machinery inbacteria (step 2), wherein biomolecules (silver reductase?) synthe-sized by silver reduction machinery bind to Agþ ions (step 3),thereby reducing Agþ ions to Ag0 nuclei or seed nanoparticles(step 4). The Ag seed particles undergo growth and assemblywithin bacterial cells, leading to spherical or platelike Ag nano-particles (step 5), which are thereafter released from the cell usinga cellular efflux system (step 6). Thebacterial efflux system involvedin extracellular release of Ag nanoparticles is most possibly similar

to the ATPases and chemiosmotic membrane potential-dependentmembrane cation/proton antiporter proteins, as was previouslyreported for the efflux of metal ions.31

Although interesting, it is not absolutely clear at this stage howa shift from optimal growth temperature of M. psychrotoleransled to increased formation of anisotropic Ag nanoplates incomparison to Ag nanospheres. It was, however, suggested in arecent review by Xia et al. that for seed nanoparticles to achieve ahigher energy structure (such as nanoplates) a kinetically con-trolled growth pathway is almost essential during chemicalsynthesis.11 Notably, to achieve a kinetically controlled growthpathway, it was found important to substantially reduce the rateof precursor reduction, which could be achieved by using weakreducing agents. In our opinion, a similar kinetics-controlledmechanism involving slow reduction of metal ions by the bacte-riumM. psychrotolerans takes place when the reaction is deviatedfrom the optimal bacterial growth temperature of 20 �C. A shiftaway from optimum growth conditions will slow down thebacterial physiological processes (including metal ions uptakeand reduction capability), which is probably the most importantfactor that directs the growth toward shape anisotropy. More-over, proteins and other biomolecules expected to be involved inthe Ag nanoparticles formation process will act as weak reducingagents in comparison to chemical reducing agents, thus facilitat-ing anisotropic growth of Ag nanoplates.

Conclusion

We have demonstrated for the first time that by controlling thegrowth kinetics of a silver-resistant bacteriaMorganella psychro-tolerans at different temperatures shape anisotropy of Ag nano-particles can be controlled. The possibility to achieve nanoparticleshape control by using a “green” biosynthesis approach wouldopen up new exciting avenues for eco-friendly shape-controlledsynthesis of suchmaterials. This proof-of-concept can in future beextended to other nanomaterials, wherein a combination ofreaction parameters influencing microbial growth kinetics willbe utilized to achieve a higher degree of shape control duringbiological synthesis. Electrochemical measurements for the firsttime revealed an interesting potential mechanism for Ag nano-particles formation by M. psychrotolerans, as well as suggestingthe possibility of a silver reductase enzyme in silver-resistantmicroorganisms. Identification of the biomacromolecules in-volved in the silver reduction machinery and its association withsilver resistance inM. psychrotolerans are aspects currently under

Scheme 1. Schematic Representation of the Potential Mechanism for Extracellular Ag Nanoparticles Biosynthesis by Silver-Resistant BacteriumM. psychrotoleransa

aThe process involves uptake of Agþ ions by bacterium on its exposure to Agþ ions (step 1), followed by presentation of Agþ ions to bacterial silverreductionmachinery (step 2),wherein biomolecules synthesizedby silver reductionmachinery bind toAgþ ions (step 3), thereby reducingAgþ ions toAg0

nuclei or seed nanoparticles (step 4). The Ag seed particles undergo growth and assembly within bacterial cell leading to spherical or platelike Agnanoparticles (step 5), which are thereafter released from the cell using a cellular efflux system (step 6).

(30) Gupta, A.; Matsui, K.; Lo, J.-F.; Silver, S. Nature Med. 1999, 5, 183–188.(31) (a) Silver, S.; Phung, L. T.Annu. Rev. Microbiol. 1996, 50, 753. (b) Silver, S.;

Phung, L. T. J. Ind. Microbiol. Biotechnol. 2005, 32, 587–605.

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investigation in our group, which will advance this challengingconcept toward fundamental biological understanding of silverresistance.

Methods

Synthesis of Ag Nanoplates. The bacteria Morganella psy-chrotolerans was grown in Luria-Bertani (LB) broth withoutaddedNaCl [1%peptone, 0.5%yeast extract, pH6.8 in deionizedwater] at 15, 20, and 25 �C for 24 h and at 4 �C for 5 days undershaking conditions, followed by addition of 5 mM equivalent ofAgNO3.After additionofAgNO3, the reactionswere incubated indark for up to 20 h, 24 h, 5 days, and 14 days at 25, 20, 15, and4 �C, respectively, under shaking conditions. After the reaction,bacteria were removed by centrifugation, and the colored super-natant containing ahomogeneous suspensionofAgnanoparticleswas collected at different time points for further analysis. Incontrol experiments, 5 mM AgNO3 solution was added tobacterial growth medium (without bacteria) and incubated indark as performed in all the test experiments. In control experi-ments, the solution color did not change during the course ofreaction, therebynegating the possibility ofAgþ ions reduction bygrowth media.

Nanoparticle Characterization. The homogeneous colloidalsolutions obtained after removal of bacterial cells at different timepoints were characterized by UV-vis absorbance spectroscopyusing Cary 50 bio-spectrophotometer. The samples for transmis-sion electron microscopy (TEM) were prepared by drop-coatingthe solution on to a carbon-coated copper grid, followed by TEMmeasurements using a JEOL 1010 TEM instrument operated atan accelerating voltage of 100 kV. XRD measurements of filmsdrop-casted on glass slide were carried out on a Bruker AXSX-ray diffraction system operated at a voltage of 40 kV andcurrent of 40 mA with Cu KR radiation. For linear sweepvoltammetry (LSV) measurements, 5 μL of solutions containingan equal number of bacteria M. psychrotolerans that had been

exposed toAgþ ions for various time periods (0-72 h) were drop-casted onto a glassy carbon (GC) electrode surface and allowed todry at room temperature. LSV experiments were conducted byusing a CH Instruments (CHI 760C) electrochemical analyzer. Amodified 3 mm GC (BAS) with the bacterial sample was used asthe working electrode, which, prior to modification, was polishedwith an aqueous 0.3 μm alumina slurry on a polishing cloth(Microcloth, Buehler), sonicated indeionizedwater for 5min, anddried with a flow of nitrogen gas. An Ag/AgCl (3 M KCl)reference and Pt wire counter electrode were used. Electrochemi-cal experiments were commenced after degassing the electrolytesolutions with nitrogen for 10 min prior to any measurement.

Determination of silE Gene in M. psychrotolerans. Thepresence of silE gene inM. psychrotoleranswas confirmed using astandard polymerase chain reaction (PCR) protocol previouslyused by Parikh et al. in a similar bacteriaM. morganii RP42.14

Acknowledgment.R.R. thanks theCommonwealthofAustraliafor an Australian Postgraduate Award toward his PhD at RMITUniversity. V.B. acknowledges the Australian Research Council(ARC), Commonwealth of Australia, for the award of an APDFellowship and research support through the ARC Discovery(DP0988099; DP110105125), Linkage (LP100200859), and LIEF(LE0989615) grant schemes. Support ofRMITUniversity toV.B.through the award of Seed Grants, Incentive Capital Funding,and Emerging Researcher Grant is also acknowledged. V.B. alsoacknowledges the support of Ian Potter Foundation to establish aMultimode Spectrophotometry Facility at RMIT University,which was used in this study.

Supporting Information Available: (S1) Control electro-chemistry experiments and (S2) UV-vis absorbance spectra ofAg nanoparticles synthesis by extracellular bacterial proteins inthe absence of bacteria. This material is available free of chargevia the Internet at http://pubs.acs.org.