Bacterial Kinetics-Controlled Shape-Directed Biosynthesis of Silver

pubs.acs.org/Langmuir © 2010 American Chemical Society

Bacterial Kinetics-Controlled Shape-Directed Biosynthesis of Silver Nanoplates 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-resistant psychrophilic bacterium, the shape anisotropy of silver nanoparticles can be achieved. This is particularly important considering that there has been no report that demonstrates a control over shape of Ag nanoparticles by controlling the growth kinetics of bacteria during biological synthesis. Additionally, we have for the first time performed electrochemistry experiments on bacterial cells after exposing them to Agþ ions, which provide significant new insights about mechanistic aspects of Ag 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 viable shape-controlled synthesis of nanomaterials.

Metal nanoparticles, particularly Au and Ag, have gained significant interest in the past decade because of their unique physicochemical properties with applications in diverse areas including molecular diagnostics,1 catalysis,2 electronics,3 drug delivery,4 sensing,5 and surface-enhanced Raman scattering.6 To date, the majority of research has been focused on the synthesis of isotropic (i.e., spherical or quasi-spherical) metal nanoparticles, with the development of physical and chemical methods to achieve excellent control over size distribution.7-11 Recently, the importance of nanoparticle shape anisotropy has been realized with an emphasis to explore the correlation between different shapes and their physicochemical properties.2,9-11 A range of shapes including cubes,10 prisms,11 and rods12 can now be regularly synthesized using chemical methods. However, some of the limitations generally associated with physical and chemical synthesis approaches *Corresponding authors. E-mail: [email protected] (V.B.), suresh. [email protected] (S.K.B.). Phone: þ61 3 99252121. Fax: þ61 3 99253747. (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.; Metraux, 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.

714 DOI: 10.1021/la1036162

include the use of eco-toxic solvents, harsh reaction conditions, scale-up issues, and formation of harmful byproducts.13,14 Therefore, more recently there has been an increasing focus to develop nontoxic, environmentally benign “green” approaches for the synthesis of metal nanoparticles.10,13,14 To this end, our group and others have previously demonstrated that 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 ecofriendly biosynthesis of metal, metal oxide, and metal sulfide nanoparticles. Among different biological entities, the use of bacteria is preferred for nanoparticle biosynthesis due to extracellular nanoparticle production and ease of culturing, manipulation, and downstream processing.10,14 Importantly, most of the biosynthesis efforts exploring microorganisms for nanoparticles synthesis have hitherto led to formation of isotropic nanoparticles, with only limited reports on shape-controlled biosynthesis of Au nanoprisms using plant extracts such as Aloe vera17 and lemon grass.18 Similarly, one of the studies has also demonstrated biosynthesis of Ag nanoplates using extracts of unicellular alga Chlorella vulgaris at room temperature.22 Notably, these reports are either on plant or algal extracts-based synthesis of metal nanoprisms and nanoplates (and not the whole plant/algae as such), wherein ketones and aldehydes (Au)18 and amino acids (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. Langmuir 2007, 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.

Published on Web 12/13/2010

Langmuir 2011, 27(2), 714–719

Ramanathan et al.

(Ag)22 were identified to be responsible for shape control. Therefore, if we consider plant extract-based nanomaterials synthesis as pseudobiological, there is per se no report on shape-directed extracellular biological synthesis of anisotropic metal nanoparticles by directly involving a microorganism. The propensity of microorganisms to almost always synthesize spherical nanomaterials in the presence of metal ions is most probably due to their focus on energy minimization, where it is easiest to create the spherical shape.16 Notably, in the particular context of Ag nanoparticles biosynthesis by bacteria, ionic silver (Agþ) is well-known to be toxic to bacteria.14,15,23 Previously, a silver-resistant bacterium Pseudomonas stutzeri AG25915,23 was discovered from silver mines, which had the unique capability to reduce Agþ ions into Ag nanoparticles and accumulate them within its cell to minimize the Agþ ion toxicity. It is notable that although silver-resistant bacterium P. stutzeri was able to synthesize a mixture of Ag nanospheres and nanoplates intracellularly by reducing Agþ ions within the bacterial matrix, extracellular biosynthesis of Ag nanoplates could not be achieved. Following this, there have been a few efforts that utilized different bacteria for the synthesis of Ag nanoparticles.10,14,15,23 However, to the best of the authors’ knowledge, none of these reports have hitherto been able to achieve shape-controlled biosynthesis of silver nanoplates or other anisotropic Ag nanostructures. This is most possibly because most of the previous studies in this field had only reported the outcomes of exposure of Agþ ions to bacteria, without making any deliberate efforts to control the bacterial growth kinetics to achieve shape control. To this end, a recent study in which the effect of temperature on the size of Ag nanoparticles formed during a fungus-mediated biosynthesis process was investigated is particularly notable.24 It is also notable that controlling reaction kinetics and altering reaction parameters via photochemical, microwave, and ultrasound-assisted techniques are known to achieve anisotropic growth in conventional chemical synthesis.7-11 However, contributing factors for biological synthesis are not well understood. In the current study, we envisage that control over bacterial kinetics by controlling bacterial growth temperature might provide a facile tool to control the shape of nanomaterials synthesized using biological approaches. To demonstrate this proof-of-concept, in this study, we have utilized Morganella psychrotolerans as a model organism, which is a close relative of silver resistant bacteria Morganella morganii. M. morganii RP42 strain was recently reported for its specificity toward sustaining high concentrations of Agþ ions via extracellular synthesis of spherical Ag nanoparticles.14 In the current study, M. psychrotolerans has been chosen as a model organism for controlling shape anisotropy of Ag nanoparticles due to its tolerance for lower temperature (psychros: cold) and its capability to grow at a wider temperature range of typically 0-30 °C with 20 °C as the optimum temperature.25 Since M. morganii was previously found to be capable of spherical Ag nanoparticles synthesis, we hypothesized that its close relative M. psychrotolerans might also be able to produce Ag nanoparticles, thus making this study feasible. In an effort to synthesize anisotropic Ag nanoparticles via a “green” biological route, we herein demonstrate for the first time that by controlling the growth kinetics of M. psychrotolerans by growing it at different temperatures (away from the reported (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.

Langmuir 2011, 27(2), 714–719

Article

optimal temperature of 20 °C) in the presence of Agþ ions, an increase in the formation of Ag nanoplates can be achieved. This is particularly important as there has been no report that demonstrates a control over shape of Ag nanoparticles using a bacteria-mediated biological approach under extracellular conditions. 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 nanoparticles formation mechanism by M. psychrotolerans.

Results and Discussion The optimum temperature for the growth and physiological activities of the bacterium Morganella psychrotolerans is 20 °C.25 Although deviation of its growth environment from 20 °C to higher temperatures (e.g., 25 °C) might lead to a faster bacterial growth and replication, such higher temperatures are expected to adversely affect its physiological activities. Conversely, deviation of M. psychrotolerans growth environment toward lower temperatures (e.g., 15 and 4 °C) will result in reduced bacterial growth as well as alteration of its physiological activities. To understand the influence of bacterial growth and its physiological activity on Ag biosynthesis capability of M. psychrotolerans, bacteria were grown at four different temperatures (25, 20, 15, and 4 °C), followed by incubation with 5 mM AgNO3 in LB broth without NaCl in the absence of light. Following the reactions, all four solutions changed in color from pale yellow (the color of growth medium with bacteria) to muddy green over a period of time, thus indicating formation of silver nanoparticles at all the four growth temperatures. In parallel control experiments, 5 mM AgNO3 solutions were prepared in the bacterial growth medium (LB broth without NaCl) and incubated without bacteria. No color change was observed in the growth medium over the course of control experiments, which affirms that reduction of Agþ ions to Ag nanoparticles is due to bacterial activity and not due to the bacterial growth medium. UV-vis absorbance spectroscopy was employed to understand the time-dependent kinetics of Ag nanoparticles biosynthesis by M. psychrotolerans at different temperatures (Figure 1). As shown in Figure 1b, at an optimum growth temperature of 20 °C, a broad surface plasmon resonance (SPR) band apparent at ca. 350-530 nm appears within 2 h of reaction, which increases in intensity with time for at least up to 24 h. The SPR feature in this range is typical of Ag nanoparticles,14 and its increased intensity over a 24 h period can be assigned to the continuous biosynthesis of Ag nanoparticles 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 nm were observed as early as 3 h, followed by an increase in intensity of this SPR feature with time (Figure 1a). Interestingly at 25 °C, an additional SPR feature at ca. 650-950 nm was observed that extends well into the near-infrared (NIR) region of the spectra and increases in intensity with the reaction time. Such NIR SPR features are characteristic of either aggregation of metal nanoparticles with time,26 or formation of anisotropic nanostructures in the solution,9 or a combination of both.18 To understand the effect of lower than optimum growth temperature on Ag nanoparticles synthesis, when M. psychrotolerans was grown at 15 °C, the bacterial activity toward Ag nanoparticles biosynthesis was found to be considerably reduced, as is evident from a weak yet detectable Ag SPR feature at ca. 350-530 nm observed only after 12 h of reaction and a prominent (26) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607–609.

DOI: 10.1021/la1036162

715

Article

Ramanathan et al.

Figure 2. TEM images of biogenic Ag nanoparticles biosynthesized by M. psychrotolerans at (a) 25, (b) 20, (c) 15, and (d) 4 °C after 20 h, 24 h, 5 days, and 15 days of reaction, respectively.

Figure 1. UV-vis absorbance spectra demonstrating the timedependent kinetics of biogenic Ag nanoparticles biosynthesis by M. psychrotolerans at (a) 25, (b) 20, (c) 15, and (d) 4 °C.

SPR feature seen after 5 days of reaction (Figure 1c). With the reaction time, an increase in intensity of the Ag SPR signature accompanied by the development of a prominent SPR feature in the NIR region was observed. This is similar to the reaction at 25 °C, wherein deviation from optimal growth temperature of 20 °C leads to development of NIR features in the spectra. A further reduction in reaction temperature to 4 °C considerably slowed down the bacterial growth as well as rate of Ag nanoparticles biosynthesis, thereby leading to the appearance of a prominent Ag SPR feature only after 14 days of reaction (Figure 1d). At 4 °C, the very broad nature of the UV-vis spectrum after 14 days of reaction along with a significantly higher absorbance in the NIR region in comparison with that in the visible region is particularly notable. In fact, comparison of highest time point spectra at reactions performed at 25, 15, and 4 °C (compare highest intensity curves in Figure 1a,c,d) suggests that the relative intensity of the NIR SPR features with respect to visible SPR features follow a temperature-dependent trend, wherein NIR absorbance is most predominant at 4 °C and the least at 25 °C. A comparison of time-dependent kinetics of Ag nanoparticles synthesis by M. psychrotolerans at four different temperatures also clearly suggests that the rate of Ag nanoparticle biosynthesis follows a temperature-dependent trend, with fastest Agþ ions reduction observed at the highest temperature (25 °C; Figure 1a) and the slowest at the lowest temperature employed in this study (4 °C; Figure 1d). This is expected as bacterial physiological activity, growth, and multiplication will be considerably slowed down at lower temperatures; thus, a lower number of bacterial biosynthesis “nanofactories” will be available to reduce Agþ ions into Ag0 nanoparticles.27 Ag nanoparticles biosynthesized using M. psychrotolerans at different temperatures were further characterized using transmission electron microscopy (TEM), as shown in Figure 2. At the (27) Nedwell, D. B.; Rutter, M. Appl. Environ. Microbiol. 1994, 60, 1984–1992.

716 DOI: 10.1021/la1036162

optimum growth temperature of 20 °C, predominantly spherical Ag nanoparticles of ca. 2-5 nm diameter along with relatively few nanoplates of 100-150 nm edge length were observed during TEM imaging (Figure 2b). The synthesis of spherical Ag nanoparticles at the optimum growth temperature of bacteria corroborates well with previous biosynthesis studies, wherein the use of different microorganisms has hitherto been reported to result in spherical Ag nanoparticles.10 However, in contrast to previous biosynthesis studies, when M. psychrotolerans bacteria was used in this study for biosynthesis of Ag nanoparticles at temperatures different from its optimum growth temperature, formation of Ag nanoplates was observed (Figure 2a,c,d). For instance, at 25 °C, which is 5 °C higher than the optimum growth temperature of bacteria, a mixture of triangular and hexagonal nanoplates along with spherical nanoparticles was obtained (Figure 2a). Similarly, at 15 °C, which is 5 °C lower than the optimum growth temperature, again a mixture of nanoplates and spherical particles was obtained (Figure 2c). Further reduction in bacterial physiological activity and growth by reducing its growth temperature to 4 °C results in a significant increase in the number of nanoplates, whereas only a relatively smaller proportion of spherical nanoparticles were formed (Figure 2d). It is however notable that although the proportion of spherical Ag particles formed at 4 °C is lower than that observed at other temperatures; the spherical particles formed at 4 °C are larger in size (ca. 70-100 nm). This indicates that in addition to the NIR absorbance from Ag nanoplates scattering from larger size particles formed at 4 °C might have also contributed toward the broad SPR feature obtained at 4 °C (Figure 1d). A higher magnification TEM image of nanoplates synthesized by bacteria at 4 °C revealed stacking and buckling faults that are typical of thin nanoplates (inset, Figure 2d).28 The biosynthesis of Ag nanoplates by M. psychrotolerans at temperatures away from its optimum growth temperature is particularly interesting as there has so far been no reports on tailoring the nanoparticles shape by controlling bacterial growth kinetics. Typically, M. psychrotolerans was found to synthesize Ag nanoplates with (28) Germain, V.; Li, J.; Ingert, D.; Wang, Z. L.; Pileni, M. P. J. Phys. Chem. B 2003, 107, 8717–8720.

Langmuir 2011, 27(2), 714–719

Ramanathan et al.

Figure 3. XRD patterns of biogenic Ag nanoparticles biosynthe-

sized by M. psychrotolerans: (a) 25, (b) 20, (c) 15, and (d) 4 °C after 20 h, 24 h, 5 days, and 15 days of reaction, respectively. Inset shows the intensity ratio of {111} to {200} Bragg reflections for Ag nanoparticles formed by bacteria at different temperatures.

50-150 nm edge length at 25 and 15 °C; however, biosynthesis at 4 °C resulted in larger nanoplates with 150-450 nm edge length. The nanoplates obtained through biosynthesis often showed truncated edges similar to that observed in Ag nanoprisms synthesized by a photoinduced method.29 X-ray diffraction (XRD) patterns of the drop-coated films of biogenic Ag nanoparticles synthesized by M. psychrotolerans at different temperatures were similar and showed well-defined Bragg reflections 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 analysis thus confirms that biogenic Ag nanoparticles are highly crystalline in nature. Since Ag nanoplates are well-known to be rich in the {111} plane, the ratios of the intensity of {111} to {200} peaks were plotted as a function of different temperatures at which bacterial synthesis of Ag nanoparticles was performed (inset, Figure 3). It is clear from the inset in Figure 3 that Ag nanoparticles formed by bacteria at its optimum growth temperature of 20 °C are least rich in the {111} phase. Conversely, the intensity of the {111} peak increases while increasing or decreasing the growth temperature from the optimum value, with the highest intensity observed at 4 °C. XRD analysis therefore corroborates well with UV-vis spectroscopy and TEM results, which exhibit similar trends. To gain an understanding of the mechanism for Agþ ion reduction by the bacteria M. psychrotolerans, a systematic time dependence study on Ag nanoparticles biosynthesis was performed at 25 °C using linear sweep voltammetry (LSV). To ascertain that LSV experiments provide selective information about Ag species associated only with bacteria (and not about free Ag species in solution), prior to LSV experiments, any residual free Agþ ions outside bacteria were removed by centrifugation and washing bacterial cells after their growth in the presence of Agþ ions for various time points. Figure 4 shows the LSV performed on a GC electrode immobilized with M. psychrotolerans that had been exposed to Agþ ions for various time periods (0-72 h). It should be noted that upon holding the potential at -0.20 V vs Ag/ AgCl the electroreduction of Agþ ions to Ag0 occurs. It is evident from Figure 4 that at initial time points (up to 4 h) only one oxidation peak associated with the oxidation of metallic Ag occurs. This can be attributed to the electroreduction of Agþ ions taken up by bacteria at the initial time points. At later stages (29) Jin, R.; Cao, Y.; Mirkin, C. A.; Kelly, K. L.; Schatz, G. C.; Zheng, J. G. Science 2001, 294, 1901–1903.

Langmuir 2011, 27(2), 714–719

Article

Figure 4. Linear sweep voltammograms of the bacteria M. psychrotolerans after their exposure to Agþ ions at 25 °C for up to 72 h.

of bacterial incubation with Agþ ions, it is predicted that bacteria will start forming Ag nanoparticles from previously uptaken Agþ ions. When LSV is performed on bacterial samples from 12 h onward, two signatures corresponding to Ag oxidation can be clearly seen. In the LSVs after 12 h, the peak at the less positive potential can be assigned to the oxidation of metallic silver generated through the electroreduction of Agþ ions within the bacteria, while the peak toward more positive potential can be attributed to the oxidation of Ag nanoparticles formed within bacteria. This was confirmed by running an experiment from open-circuit potential (0.02 V, where no electroreduction of Agþ occurs) to more positive values where only the peak at the more positive potential was observed (Figure S1, Supporting Information). This indicates that silver ions from the solution are initially taken up by the bacterial cells, which after association with bacteria may get bound to proteins and/or other biomacromolecules within the bacteria wherein they undergo reduction, and later released out of the cells extracellularly. The final outcome observed from the exposure of bacteria M. psychrotolerans to Agþ ions is the extracellular appearance of Ag nanoparticles, and it is also ascertained that bacteria are somehow involved in the biosynthesis process. However, it remains an open and challenging question whether Ag nanoparticles are produced extracellularly (in solution, outside the bacteria) by some of the proteins secreted by bacteria in solution or Agþ ions are initially taken up by the bacteria, followed by their reduction to Ag0 nanoparticles, before they are released out of bacteria in the growth medium. A previous study involving bacteria from the same genus Morganella had predicted that Ag nanoparticles are most probably produced extracellularly during biosynthesis.14 In the current study, when we exposed 5 mM Agþ ions to extracellular proteins secreted by M. psychrotolerans for 24 h at different temperatures, the extracellular proteins were found to reduce Agþ ions to form Ag nanoparticles; however, the rate of nanoparticles formation was significantly slower, and only 1-2 nm Ag nanospheres without any shape control could be obtained (Supporting Information, Figure S2). This clearly suggests that in addition to extracellular proteins bacterial physiology also plays a significant role toward the synthesis and shape control of Ag nanoparticles. Additionally, LSV studies performed in this study clearly indicate that biosynthesis of Ag nanoparticles by bacteria is not as simple as previously predicted, and a mere direct role of extracellular bacterial proteins in reduction of Agþ ions, without involving bacterial machinery is rather questionable. To the best of the authors’ knowledge, biosynthesis studies in the past have not hitherto explored the potential of electrochemistry to study metal ions uptake and their reduction by microorganisms. The interesting outcomes of the LSV experiments obtained from this DOI: 10.1021/la1036162

717

Article

Ramanathan et al.

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

a The process involves uptake of Agþ ions by bacterium on its exposure to Agþ ions (step 1), followed by presentation of Agþ ions to bacterial silver reduction machinery (step 2), wherein biomolecules synthesized 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 assembly within bacterial cell leading to spherical or platelike Ag nanoparticles (step 5), which are thereafter released from the cell using a cellular efflux system (step 6).

study suggest that detailed electrochemistry experiments in the future might provide some vital information regarding mechanisms of metal ion trafficking and its association with metal ion resistance in microorganisms. Notably, M. psychrotolerans is in fact a silver-resistant bacteria, which was confirmed by investigating the presence of a gene homologue of the putative silver binding gene (silE) in this bacteria (PCR data not shown for brevity). The silE gene is known to encode a periplasmic silver binding protein (silE) that plays a major role in Agþ ion uptake by providing histidine sites as primary candidates for Agþ ions binding in silver-resistant bacteria.30 A potential mechanism for extracellular Ag nanoparticle biosynthesis by M. psychrotolerans is presented in Scheme 1. We believe that on exposure to Agþ ions the silE protein-based silver-binding machinery of bacterium gets activated, thereby leading to the cellular uptake of Agþ ions (step 1), as was suggested previously in the case of another silver-resistant bacterium Salmonella.30 What happens after uptake of Agþ ions by M. psychrotolerans is rather interesting (steps 2-6), as it has previously been arguably postulated that in most of the heavy-metalresistant bacteria metal resistance is achieved via initial uptake of metal ions followed by energy-dependent “pumping out” of these ions using membrane proteins.31 Conversely, this is not the case in mercury-resistant bacteria, wherein the presence of a mercuric reductase enzyme has been established to reduce Hg2þ ions to Hg0. However, the presence of a silver reductase enzyme in silverresistant bacteria has not yet been established. Our LSV studies strongly indicate the reduction of Agþ ions to Ag0 nanoparticles within bacteria, and therefore a unique possibility of the existence of silver reductase enzyme in biological systems cannot be completely ignored. Our future endeavors will explore this exciting possibility. At this stage, we believe that after uptake of Agþ ions by bacteria they are presented to the Ag reduction machinery in bacteria (step 2), wherein biomolecules (silver reductase?) synthesized 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 assembly within bacterial cells, leading to spherical or platelike Ag nanoparticles (step 5), which are thereafter released from the cell using a cellular efflux system (step 6). The bacterial efflux system involved in extracellular release of Ag nanoparticles is most possibly similar (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.

718 DOI: 10.1021/la1036162

to the ATPases and chemiosmotic membrane potential-dependent membrane cation/proton antiporter proteins, as was previously reported for the efflux of metal ions.31 Although interesting, it is not absolutely clear at this stage how a shift from optimal growth temperature of M. psychrotolerans led to increased formation of anisotropic Ag nanoplates in comparison to Ag nanospheres. It was, however, suggested in a recent review by Xia et al. that for seed nanoparticles to achieve a higher energy structure (such as nanoplates) a kinetically controlled growth pathway is almost essential during chemical synthesis.11 Notably, to achieve a kinetically controlled growth pathway, it was found important to substantially reduce the rate of precursor reduction, which could be achieved by using weak reducing agents. In our opinion, a similar kinetics-controlled mechanism involving slow reduction of metal ions by the bacterium M. psychrotolerans takes place when the reaction is deviated from the optimal bacterial growth temperature of 20 °C. A shift away from optimum growth conditions will slow down the bacterial physiological processes (including metal ions uptake and reduction capability), which is probably the most important factor that directs the growth toward shape anisotropy. Moreover, proteins and other biomolecules expected to be involved in the Ag nanoparticles formation process will act as weak reducing agents in comparison to chemical reducing agents, thus facilitating anisotropic growth of Ag nanoplates.

Conclusion We have demonstrated for the first time that by controlling the growth kinetics of a silver-resistant bacteria Morganella psychrotolerans at different temperatures shape anisotropy of Ag nanoparticles can be controlled. The possibility to achieve nanoparticle shape control by using a “green” biosynthesis approach would open up new exciting avenues for eco-friendly shape-controlled synthesis of such materials. This proof-of-concept can in future be extended to other nanomaterials, wherein a combination of reaction parameters influencing microbial growth kinetics will be utilized to achieve a higher degree of shape control during biological synthesis. Electrochemical measurements for the first time revealed an interesting potential mechanism for Ag nanoparticles formation by M. psychrotolerans, as well as suggesting the possibility of a silver reductase enzyme in silver-resistant microorganisms. Identification of the biomacromolecules involved in the silver reduction machinery and its association with silver resistance in M. psychrotolerans are aspects currently under Langmuir 2011, 27(2), 714–719

Ramanathan et al.

Article

investigation in our group, which will advance this challenging concept toward fundamental biological understanding of silver resistance.

Methods Synthesis of Ag Nanoplates. The bacteria Morganella psychrotolerans was grown in Luria-Bertani (LB) broth without added NaCl [1% peptone, 0.5% yeast extract, pH 6.8 in deionized water] at 15, 20, and 25 °C for 24 h and at 4 °C for 5 days under shaking conditions, followed by addition of 5 mM equivalent of AgNO3. After addition of AgNO3, the reactions were incubated in dark for up to 20 h, 24 h, 5 days, and 14 days at 25, 20, 15, and 4 °C, respectively, under shaking conditions. After the reaction, bacteria were removed by centrifugation, and the colored supernatant containing a homogeneous suspension of Ag nanoparticles was collected at different time points for further analysis. In control experiments, 5 mM AgNO3 solution was added to bacterial growth medium (without bacteria) and incubated in dark as performed in all the test experiments. In control experiments, the solution color did not change during the course of reaction, thereby negating the possibility of Agþ ions reduction by growth media. Nanoparticle Characterization. The homogeneous colloidal solutions obtained after removal of bacterial cells at different time points were characterized by UV-vis absorbance spectroscopy using Cary 50 bio-spectrophotometer. The samples for transmission electron microscopy (TEM) were prepared by drop-coating the solution on to a carbon-coated copper grid, followed by TEM measurements using a JEOL 1010 TEM instrument operated at an accelerating voltage of 100 kV. XRD measurements of films drop-casted on glass slide were carried out on a Bruker AXS X-ray diffraction system operated at a voltage of 40 kV and current of 40 mA with Cu KR radiation. For linear sweep voltammetry (LSV) measurements, 5 μL of solutions containing an equal number of bacteria M. psychrotolerans that had been

Langmuir 2011, 27(2), 714–719

exposed to Agþ ions for various time periods (0-72 h) were dropcasted onto a glassy carbon (GC) electrode surface and allowed to dry at room temperature. LSV experiments were conducted by using a CH Instruments (CHI 760C) electrochemical analyzer. A modified 3 mm GC (BAS) with the bacterial sample was used as the working electrode, which, prior to modification, was polished with an aqueous 0.3 μm alumina slurry on a polishing cloth (Microcloth, Buehler), sonicated in deionized water for 5 min, and dried with a flow of nitrogen gas. An Ag/AgCl (3 M KCl) reference and Pt wire counter electrode were used. Electrochemical experiments were commenced after degassing the electrolyte solutions with nitrogen for 10 min prior to any measurement. Determination of silE Gene in M. psychrotolerans. The presence of silE gene in M. psychrotolerans was confirmed using a standard polymerase chain reaction (PCR) protocol previously used by Parikh et al. in a similar bacteria M. morganii RP42.14

Acknowledgment. R.R. thanks the Commonwealth of Australia for an Australian Postgraduate Award toward his PhD at RMIT University. V.B. acknowledges the Australian Research Council (ARC), Commonwealth of Australia, for the award of an APD Fellowship and research support through the ARC Discovery (DP0988099; DP110105125), Linkage (LP100200859), and LIEF (LE0989615) grant schemes. Support of RMIT University to V.B. through the award of Seed Grants, Incentive Capital Funding, and Emerging Researcher Grant is also acknowledged. V.B. also acknowledges the support of Ian Potter Foundation to establish a Multimode Spectrophotometry Facility at RMIT University, which was used in this study. Supporting Information Available: (S1) Control electrochemistry experiments and (S2) UV-vis absorbance spectra of Ag nanoparticles synthesis by extracellular bacterial proteins in the absence of bacteria. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la1036162

719