Characterization and Antimicrobial Properties of Silver and Silver

Apr 8, 2012 - Synthesis of silver nanoparticles by cell-free extract (CFE) of Pseudomonas aeruginosa M6 isolated from a mangrove ecosystem was ...
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Characterization and Antimicrobial Properties of Silver and Silver Oxide Nanoparticles Synthesized by Cell-Free Extract of a MangroveAssociated Pseudomonas aeruginosa M6 Using Two Different Thermal Treatments Seenivasan Boopathi, Selvaraj Gopinath, Thangavelu Boopathi, Vadivel Balamurugan, Radhakrishnan Rajeshkumar, and Muthuraman Sundararaman* Department of Marine Biotechnology, National Facility for Marine Cyanobacteria, Bharathidasan University, Tiruchirappalli−620 024, Tamilnadu, India S Supporting Information *

ABSTRACT: Synthesis of silver nanoparticles by cell-free extract (CFE) of Pseudomonas aeruginosa M6 isolated from a mangrove ecosystem was demonstrated using two physical methods, namely, boiling (conventional thermal treatment (CTT)) and microwave treatment (MWT) at pH 9. X-ray diffraction (XRD) analysis revealed the presence of smaller (10.4 nm), pure silver nanoparticles synthesized via CTT (C-NPs) and larger silver oxide nanoparticles in majority with negligible concentration of pure silver particles by MWT. Transmission electron microscopy (TEM) analysis showed that C-NPs are spherical in shape. Atomic force microscopy (AFM) analysis also confirmed the presence of large-sized, aggregated nanoparticles synthesized via MWT (M-NPs). Electrophoresis indicated the size and charge-based mobility in agarose gel (0.4%), wherein the C-NPs moved faster than M-NPs, because of their relatively smaller size. The zeta potential value of C-NPs and M-NPs was found to be −30.1 mV and −23.1 mV, respectively. Fourier transform infrared (FT-IR) results revealed that both C-NPs and M-NPs were capped with proteins, but with different conformations. Furthermore, TEM analysis of bacterial cells exposed to aqueous silver nitrate showed the presence of spherical silver nanoparticles accumulated in periplasmic space, indicating the possible involvement of periplasmic nitrate reductase in this process. In addition, both C-NPs and M-NPs have also shown good antibacterial and anticandidal activities. Thus, marine Pseudomonas aeruginosa M6 can be a potential source for the synthesis of silver nanoparticles. nanoparticles was investigated.6 Recently, microwave treatment (MWT) has evolved as one of the methods in the biosynthesis of nanoparticles.7 A few other studies involve some conventional thermal treatment (CTT), especially boiling, to promote the biosynthesis of nanoparticles.8,9 These methods could be used to obtain size-controlled nanoparticles. Hence, comparison of these physical conditions (CTT and MWT) in the biosynthesis process will provide insight into how they influence or enhance the synthesis process, which was not undertaken earlier. Currently, electrophoresis is increasingly exploited for the separation and characterization of nanoparticles based on size, shape, and capped molecules.10 However, such a characterization approach has not been utilized to understand the nature of biologically synthesized nanoparticles. The mangrove ecosystem is one of the marine ecosystems, which extends between the land and sea in tropical and subtropical regions of the Earth. This ecosystem acts as a nursery for many marine organisms and also provides social and ecological functions.11 Mangrove-associated bacteria efficiently recycle the available nutrients in sediments including mineralization of heavy metals.12,13 They play a major role in this

1. INTRODUCTION Nanotechnology, which involves nanoparticles, expands its scope to many fields, thanks to the unique features of the nanosized particles. Because of the feasibility of manipulating the basic structure and composition of the nanoparticles, they have obtained strong scientific scope for application in multiple fields. It was previously very difficult to target the drug across blood−brain barrier into the brain specifically. Recently, with the advent of nanotechnology, such a task was accomplished without cytotoxic effect.1 There are several physical and chemical methods available to synthesize nanoparticles. However, these demand hazardous chemicals, toxic byproducts, and high energy consumption.2 Instead, biological synthesis of nanoparticles that is eco-friendly and based on a “bottom-up” approach is receiving wider acceptance nowadays. There are many reports available on potential biological sources (from unicellular to multicellular organisms) in synthesizing various nanoparticles. Furthermore, the size and shape of the nanoparticles are critical factors in determining the physical properties, namely optical, thermal, and electrical.3 Thus, controlling the size and shape of the nanoparticles is a contemporary challenge. This could be controlled using a biological approach by adjusting physical parameters (under which conditions the synthesis is achieved) such as pH, temperature, and reaction time.4,5 For instance, the effect of temperature, as well as the effect of the concentration of leaf broth and chloroplatinate ion, on the size of platinum © 2012 American Chemical Society

Received: Revised: Accepted: Published: 5976

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valuable ecosystem.14 Hence, these bacteria could also be utilized and analyzed for nanoparticle synthesis. Among bacteria, some species of the genus Pseudomonas were also reported to synthesize silver and gold nanoparticles.3,15,16 Initially, Pseudomonas strutzeri was reported to synthesize silver nanoparticles intracellularly.15 Recently, Pseudomonas aeruginosa PA01 was shown to synthesize gold nanoparticles both intracellularly and extracellularly.16,17 Extracellular synthesis of silver nanoparticles was demonstrated using culture supernatant of Pseudomonas aeruginosa in which nitrate reductase was suspected to reduce the silver ion and rhamnolipid as a capping agent.18 Considering the nanoparticle-synthesizing ability of these Pseudomonas spp., any of them could be investigated for the rapid synthesis using the aforesaid thermal (physical) treatment methods such as CTT and MWT. There are many reports available on biosynthesised silver nanoparticles and their antimicrobial activity.19,20 However, there is no comparative study available on such antimicrobial activity by thermally manipulated biosynthesized nanoparticles. Thus, the present study aims to (i) investigate the synthesis of nanoparticles from cell-free extract (CFE) of Pseudomonas aeruginosa M6 isolated from a mangrove ecosystem using CTT and MWT toward achieving rapidness; (ii) characterize these nanoparticles and compare the two treatment methods; (iii) separate and characterize the synthesized nanoparticles by agarose gel electrophoresis; (iv) study the response of live cells of Pseudomonas aeruginosa M6 exposed to 1 mM AgNO3 in aqueous medium; (iv) analyze the effect of preheating and preautoclaving of CFE on the synthesis of silver nanoparticles; and (v) investigate the antimicrobial potentials of both nanoparticles synthesized via CTT (C-NPs) and nanoparticles synthesized via MWT (M-NPs).

Labsonic U-2000, B. Braun, Melsungen, Germany) with a duty cycle of 0.3 s (output of 200 W and 20 kHz). After that, the extract was centrifuged at 10 000 rpm for 15 min. Supernatant was refrigerated for further use. Initially, 100 μL of CFE of Pseudomonas aeruginosa M6 was added to 100 mL of 1 mmol/L silver nitrate solution at room temperature under fluorescence light (27.03 E m−2 s−1). After confirming the nanoparticle synthesis, the optimum pH was determined to be 9, by carrying out the same reaction under three different pH conditions such as 5, 7, and 9 (see Figure S1 in the Supporting Information). Henceforth, all reaction mixtures were adjusted to pH 9. To hasten the synthesis that was slower at room temperature, two physical methods of heating, such as CTT and MWT, were applied to the same reaction mixture. For CTT, a water bath maintained at 100 °C was utilized, and the reaction was allowed to continue for 2 h. Initial confirmation of silver nanoparticle synthesis was made and monitored every 30 min by recording ultraviolet−visible-light (UV−vis) spectra of samples withdrawn from the reaction mixture in Spectrophotometer 119 (Systronics, India) with an appropriate scanning range. Similarly, for MWT, the same reaction mixture (as described for the CTT method) was kept in a domestic microwave oven (LG, South Africa) for irradiation at 900 W, with 15 repetitive cycles (20 s on and 20 s off) of exposure to avoid overheating and subsequently analyzed for nanoparticle synthesis using UV−vis spectral reading as described above for every 3 cycles, along with measuring the reaction temperature. 2.2. Thermal Induction of CFE for Nanoparticle Synthesis under CTT Conditions. The effect of preheated reducing source (CFE) on nanoparticle synthesis was investigated to analyze the thermal stability. CFE was preheated in two ways. First, CFE was kept at 80 °C (in water bath) for 15 min. After cooling to room temperature, the CFE was added to a silver nitrate solution and kept under CTT conditions for 2 h and the UV−vis spectrum was taken as described earlier. Second, the CFE was preautoclaved (at 121 °C and 1 atm for 30 min), added to the silver nitrate solution after bringing down to room temperature, and subsequently processed as mentioned previously. The initial-day and seventh-day UV−vis spectra were recorded for both treatments, along with untreated CFE. 2.3. Protein Quantification and Qualitative Analysis of Nitrate Reductase in CFE. The protein content in CFE was estimated, in which bovine serum albumin (BSA) was used as a standard.22 To analyze (qualitatively) the presence of nitrate reductase in CFE, the method described earlier was adopted.23 As the enzyme substrate, 25 mmol/L potassium phosphate buffer with 10 mmol/L potassium nitrate and 0.05 mmol/L ethylene diamine tetraacetic acid, pH 7.3, was prepared. Then, 1.8 mL of substrate solution was taken with 100 μL of CFE as the enzyme source, and 100 μL of 200 mmol/L reduced β-nicotinamide adenine dinucleotide was added. The reaction mixture was subsequently incubated at 30 °C for 2 min. The reaction was terminated immediately by adding 1 mL of 58 mmol/L sulfanilamide solution in 3 mol/L of HCl and vortexed. Then, 1 mL of 0.77 mmol/L N-(1-naphthyl) ethylenediamine dihydrochloride solution was added and vortexed. After 2 min of incubation, absorbance was read at 540 nm in a spectrophotometer. Duplicate samples were used for this analysis (N = 2), which were obtained from independent processing. The result was presented as mean ± standard error. 2.4. Characterization of Silver Nanoparticles. After synthesis of nanoparticles (UV−vis spectral analysis), they were obtained by drying and collecting them in powder form.

2. MATERIALS AND METHODS 2.1. Synthesis of Silver Nanoparticles Using CFE of Pseudomonas aeruginosa M6 by CTT and MWT. The gram-negative bacterium, Pseudomonas aeruginosa M6, was isolated from the Pitchavaram mangrove forest (11° 24′ 57″ N, 79° 48′ 24″ E) in Tamil Nadu, India. This bacterium showed luxuriant growth in Zobell marine agar (Himedia, India) plate, and then it was identified by analyzing its biochemical characteristics and confirmed by using selective media, Pseudomonas HiVeg agar (HiMedia, India). Bacterial identity was further confirmed by amplification and sequencing of 16S rRNA gene. Amplification of the gene from bacterial DNA was done using forward primer (63f) 5′ CAG GCC TAA CAC ATG CAA GTC 3′ (63f) and reverse primer (1387r) 5′ GGG CGG WGT GTA CAA GGC 3′.21 The amplified product was then sequenced. This sequence was compared with those from NCBI nucleotide database using BLASTn and confirmed as Pseudomonas aeruginosa (having 99% similarity). The sequence then was deposited in GenBank (http://www.ncbi.nlm.nih.gov/), and the accession number is JF682387. The bacterium was cultivated in Luria−Bertani (LB) broth containing tryptone (10 g L−1), yeast extract (5 g L−1), and NaCl (10 g L−1) for experimental purposes. It was grown for 6 days at 37 °C to prepare CFE. The bacterial biomass was harvested by centrifugation at 10 000 rpm for 20 min. The harvested biomass then was washed thoroughly with sterile distilled water thrice to remove the salt content from the bacterial mass. A bacterial pellet with a mass of 1 g (wet weight) was added to 5 mL of sterile distilled water. It was then sonicated for 3 min using an ultrasonic homogenizer (Model 5977

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diameter), wells were made in the culture plates and 70 μL of the freshly synthesized nanoparticles (both C-NPs and M-NPs) was loaded. The plates then were incubated at 37 °C for 24 h and, after incubation, the zone of inhibition was measured. In addition, the minimal inhibitory concentration (MIC) was determined for both C-NPs and M-NPs, which is the lowest concentration of particles inhibiting the visible growth of the test organisms. The strains M. smegmatis and C. albicans were used for this study to find the inhibitory concentration range of the nanoparticles (C-NPs and M-NPs). Wherein 10 μL/mL of midlog culture (1 × 106 cells/mL) were seeded with test compound serially diluted (100, 250, and 400 μL/mL) and made into a total volume of 1 mL with nutrient broth and sabouraud dextrose broth for bacteria and yeast, respectively, and incubated at 37 °C for 24 h with gentle agitation. After 24 h of incubation, the antimicrobial activity of silver nanoparticles was determined by spreading 100 μL of test solution into respective agar plates and allowed further incubation for 37 °C for 24−48 h.26,27 All assays were done in duplicate and confirmed once again qualitatively.

To find the functional groups of biological origin (attached to nanoparticles), FT-IR spectral analysis was carried out with a FT-IR spectrometer using KBr (Perkin−Elmer, USA) in the range of 4000−400 cm−1 at a resolution of 4 cm−1. Furthermore, the nanoparticles (powder) were characterized after obtaining the X-ray diffraction (XRD) pattern from X’Pert Pro (PANalytical, USA) operated at 40 kV and a current of 30 mA with Cu Kα radiation (λ = 1.54 Å). The data obtained were processed using PowderX software.24 Crystalline size was calculated from the full-width at half-maximum (fwhm) of the diffraction peaks, using the Debye−Scherrer formula. M-NPs were visualized using atomic force microscopy (AFM) (Model Agilent 5500, Agilent, USA) and compared. Spin-coating of M-NPs on a clean glass substrate was done for visualizing nanoparticles in AFM. Electrophoretic separation of C-NPs and M-NPs was also carried out to observe the size- and charge-based characteristics. The zeta potential of the nanoparticles (for both C-NPs and M-NPs) was determined at 25 °C by dynamic light scattering (DLS) analysis, using Zetasizer Nano ZS90 (Malvern, U.K.). For this, the nanoparticles were drawn as concentrated solution (instead of obtaining as powder) after synthesis. Then, these two solutions (having equal optical density at 410 nm) were loaded in agarose gel (0.4%) and run at 100 V in 0.5× TBE (Tris-borate−EDTA) buffer having pH ∼9 for 30 min. In addition, TEM analysis of the synthesized nanoparticles was accomplished using Morgagni 268D (Fei Company, The Netherlands) at an operating voltage of 80 kV. The diameter of each nanoparticle was measured using the software MeasureIt (Olympus Soft Imaging Solutions Pvt. Ltd., Singapore) from the TEM image. 2.5. Bioaccumulation Study by Transmission Electron Microscopy (TEM). An overnight culture of Pseudomonas aeruginosa M6 was inoculated in fresh LB broth in the presence of silver nitrate (1 mmol/L) and incubated at 37 °C for 24 h. The culture then was centrifuged at 10 000 rpm for 15 min at 4 °C. The pellet was washed twice with distilled water, resuspended in 10−1 M phosphate buffer (pH 7.4), and again centrifuged at 10 000 rpm for 15 min at 4 °C. A fixative solution with buffer was added at a ratio of 3:7 to the pellet after discarding the supernatant. The pellet then was resuspended and fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in buffer for 2 h at 4 °C. The mixture was again centrifuged at 10 000 rpm for 10 min at 4 °C and the supernatant was discarded. The pellet was added with phosphate buffer and post-fixed in 1% OsO4 for 2 h at 4 °C. The samples were dehydrated in an ascending grade of acetone, then infiltrated and embedded in Araldite CY 212 (TAAB, U.K.). Sections of 1 μm thickness were obtained with an ultramicrotome, mounted onto the glass slides, stained with aqueous Toluidine Blue, and observed under a light microscope for gross observation of the area and quality of the fixation. Finally, thin sections of gray−silver color interference (70−80 nm) were cut and mounted onto 300-mesh copper grids. Sections were stained with alcoholic uranyl acetate and alkaline lead citrate, washed gently with distilled water, and observed under TEM (Morgagni 268D), as described previously. 2.6. Antimicrobial Activity Assays. The antimicrobial test was carried out primarily by the agar well diffusion method.25 Both bacterial and opportunistic pathogenic yeast inoculums were prepared from 18 h grown cultures (∼104−106 cells/mL). Petri dishes containing the bacterial and yeast inoculums (as lawn) on nutrient agar and sabouraud dextrose agar, respectively, were used for the study. Using a cork borer (6 mm

3. RESULTS AND DISCUSSION In the present study, the rapid synthesis of silver nanoparticles was demonstrated using CFE of mangrove-associated bacterium Pseudomonas aeruginosa M6 under two different physical conditions (viz, CTT and MWT) than the temperature synthesis. A marine fungus, Penicillium fellutanum, isolated from mangrove sediment was reported for their ability to synthesize silver nanoparticles.28 Although Pseudomonas aeruginosa was used to synthesize silver nanoparticles (extracellularly), this is the first report to demonstrate the rapid synthesis (within 10 min by MWT) of silver nanoparticles using CFE of a mangroveassociated Pseudomonas aeruginosa. 3.1. Characterization of Nanoparticles. During optimization, nanoparticle synthesis was found to be directly proportional to the pH of the solution (see Figure S1 in the Supporting Information). Hence, among the three pH values tested (5, 7, and 9), pH 9 induced the highest level of nanoparticle synthesis. During this preliminary analysis, it was found that a distinct peak appeared between 400−450 nm with a color change to yellowish brown under all pH conditions after only 24 h, indicating a slower rate of nanoparticle synthesis. However, executing the same reaction under both MWT and CTT has resulted in more rapid synthesis than under roomtemperature conditions (see Figures 1A and 1B). In CTTmediated synthesis, surface plasmon resonance (SPR) band occurred at 410 nm (Figure 1B) and increased in intensity with increasing reaction time of the experiment. In MWT, the SPR band occurred at 416 nm (Figure 1A), which has started distinctly from the ninth cycle of the experiment (within 10 min) and had a steady increase in intensity like the CTT method. The synthesis of nanoparticles and a change in the temperature during MWT have shared different trends (see Figure 5B, presented later in this paper). The temperature was found to be 94 °C at the 3rd cycle and reached a maximum of 104 °C at the 6th cycle. The temperature then was found to be stable at 104 °C until the end of the reaction. However, from the 3rd cycle to the 9th cycle, there is no considerable change in the rate of synthesis. However, from the 9th cycle to the 15th cycle, a sudden exponential hike in the synthesis was observed. These trends were observed every time while applying MWT for nanoparticle synthesis. On the other hand, in CTTmediated synthesis, a distinct narrow peak was observed after 5978

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Figure 1. UV−vis spectra showing the synthesis of (A) nanoparticles synthesized via MWT (M-NPs) and (B) nanoparticles synthesized via CTT (C-NPs).

Figure 2. UV−vis spectra of (A) silver nanoparticles synthesized by CFE (labeled as Extr-alone), CFE preheated at 80 °C for 30 min (labeled as Extr-80 °C), and CFE autoclaved (labeled as Extr-Autocl) on the initial day and (B) silver nanoparticles synthesized after seven days by CTT.

60 min of exposure in contrast to MWT-mediated synthesis, showing a broad peak. Broadening is generally due to the bigger size of the synthesized nanoparticles.29 In support of this, the size difference is also distinct in XRD results. In the present study, the MWT-mediated method has shown more rapid synthesis than the CTT method. This might have resulted from the concordant effect of vibrations (dielectric) of molecules and the temperature hike in the reaction mixture.30 Further, CFE of Pseudomonas aeruginosa M6 prepared by sonication has been used in this study and has shown efficient synthesis of silver nanoparticles. A similar venture was recently made which demonstrated the synthesis of triangular and hexagonal platinum nanoparticles, using sonicated CFE of a fungus Fusarium oxysporum.31 In addition, the present study demonstrates the increased synthesis of nanoparticles by preheated CFE (80 °C) when compared with untreated CFE, which indicates the thermostable or thermally inducible nature of reducing biological factor(s) involved (Figure 2A). Though preautoclaving has initially shown increased synthesis (Figure 2A) among all treatments, later it was found to decline on the seventh day, compared to all others (Figure 2B). These results convey that (i) the available nucleation site and/or reducing factor(s) is thermally inducible up to a certain temperature, which might be due to affirmative conformational change(s); (ii) possible degradation of certain metabolites resulted in large quantities of thermostable/thermally inducible reducing factor(s); and (iii) the factor(s) has apparently become unstable by preautoclaving CFE. Besides, the enzyme assay has shown the activity of nitrate reductase (0.124 ± 0.008 (A540)) in CFE and protein estimation has determined the concentration of protein to be 24 ± 5.2 μg/mL. Each UV−vis spectrum has confirmed the presence of proteins in CFE by showing a peak above 260 nm.32 Hence, it is perceived that the protein or enzyme or the active peptide with a specific functional motif (during preheating and preautoclaving) has been involved in the

nanoparticle synthesis process. There are many reports available on the involvement of enzyme nitrate reductase in the synthesis of silver nanoparticles.4,33 FT-IR spectra of C-NPs and M-NPs are depicted in Figure 3. The band at 1637.47 cm−1 (CTT) corresponds to amide I proteins,34 and the band at 1596.98 cm−1 (MWT) could be assigned to amide II.35 These results imply that the proteins or enzymes could have bound to nanoparticles and have given the stability.36 Similarly, the band at 1384.37 cm−1 (CTT) represents the stretching of the C−N bond and at 1358.97 cm−1 (MWT) corresponds to the geminal methyl group with a shift in the pattern.34 The XRD patterns of both CTT- and MWT-mediated methods have been shown in Figure 4. In MWT-mediated synthesis, the major peak (2θ = 32.5°) is perfectly matched with orthorhombic Ag2O3 (420), and other two are matched with reflections of hexagonal Ag2O ((105) at 2θ = 28° and (309) at 2θ = 57°) lattice planes, having a size of 42 nm, 295 and 112 nm, respectively. In addition, a small number of pure Ag particles (2θ = 38°), 12 nm in size, were also observed. These pure Ag particles would have been synthesized during the initial minutes of MWT, while the temperature was low, but ceased later. This wide range of size distribution is also reflected in the AFM image (see Figure 5A). The increased intensity of Ag2O3 and Ag2O may be due to the microwave-enhanced reaction with dissolved oxygen present in the mixture during the synthesis process. This could be controlled by prior flushing of reaction mixture with inert gases like argon to remove dissolved oxygen/air37 or by adding oxygen-scavenging compounds, which requires further analysis. In addition, changing the exposure time and intensity of microwaves can also aid in the suppression of Ag2O. However, new applications of silver oxides and silver−silver oxide composites have also been explored in different fields.37,38 In contrast, the 100% intensity 5979

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Figure 4. XRD patterns of M-NPs (top) and C-NPs (bottom). Figure 3. Fourier transform infrared (FT-IR) spectra of M-NPs (top) and C-NPs (bottom).

mixtures as such and concentrated solutions of C-NPs are both used for gel electrophoresis). The concentrated solution of C-NPs precipitates immediately after the addition of cetyl trimethylammonium bromide (CTAB), which is a cationic surfactant, but no precipitation was found with sodium dodecyl sulfate (SDS), an anionic surfactant, indicating that CTAB neutralizes the negative surface charge of C-NPs (see Figure 9). M-NPs have a zeta potential of −23.1 mV (Figure 8), which causes the instability and precipitation of particles during electrophoresis, consequently affecting the mobility of these particles to form a streaked band starting from the well along with particles aggregated in the well itself (see Figure 7). The aggregation of nanoparticles synthesized by MWT was also shown in AFM analysis. Apart from aggregation, the nonsmooth surface of M-NPs shown in AFM image is another reason that causes hindrance in the electrophoretic path (see Figure 5A). Another important reason for such improper mobility of M-NPs is their wide size range, which is evident from the AFM images and XRD results. Hence, C-NPs moved farther from the well than M-NPs. MWT might have probably interfered (by degrading or altering some key biomolecules involved in capping and bioreduction) with the normal process of capping and synthesis that occurred during CTT. In an earlier study, microwave exposure was used to suppress the enzymatic activity in reducing the source of plant origin in which flavonoids were found to be responsible for the synthesis.7 Such suppression would result in changes in capped biomolecules, which, in turn, affects the charge and stability of the nanoparticles. This is also supported by the FT-IR results (Figure 3), where a distinct variation and shifting of absorbance values are observed between CTT and MWT. In general, the

peak corresponds to a pure silver crystal peak (2θ = 38.4°), which is present along with other standard peaks of pure Ag in the case of C-NPs. Their Bragg reflections correspond to lattice planes, namely, (111), (200), (220), and (311). The size of the major peak (2θ = 38.4°) is found to be 10.4 nm, and this is also reflected in the histogram obtained from TEM images showing synthesized C-NPs (Figure 6D). The average of nanoparticles, calculated from TEM images (Figures 6A−C), is 14.6 ± 1 (in a format of mean ± standard error). Apart from the major peak at 2θ = 38.4° (10.4 nm) in the XRD graph, other three peaks showed slightly bigger-sized C-NPs (above 30 nm) which would have influenced the average nanoparticle size found in TEM fields, but differing slightly. Moreover, the XRD peak represents the size of majorly synthesized nanoparticles, but it does not represent the average. The TEM micrographs of C-NPs have shown only spherical nanoparticles. Furthermore, the difference in the size of the nanoparticles synthesized using these two methods has also shown distinct variation in electrophoretic mobility (Figure 7). Analysis of the zeta potential has revealed the negatively charged nature of the nanoparticles (see Figure 8). C-NPs have an average value of −30.1 mV. This clearly indicates the uniformly capped nature of surfaces, which is due to which discrete band forms in the agarose gel and moves from the negative end to the positive end. This result indirectly conveys that CTT has supported uniform capping of particles, which, in turn, provides stability. This might be due to uniform and constant temperature (100 °C) transmission inside the solution. They were found to be stable up to 4 months, without any change in color and intensity (reaction 5980

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Figure 5. (A) AFM microscopy (4.25 μm × 4.25 μm, presented in the form of height h × weight w) of M-NPs; (B) cyclewise changes in nanoparticle synthesis (absorbance at 416 nm) and reaction temperature during MWT.

Figure 6. (A-C) Transmission electron microscopy (TEM) images of C-NPs; (D) histogram showing the size distribution of C-NPs shown in panels A−C.

mobility of the nanoparticles is affected by factors such as size, shape, agarose concentration, attached biological molecules or polymer (their size and nature), and field strength.10 3.2. Bioaccumulation of Nanoparticles. This is the first study to report the bioaccumulation of silver nanoparticles in the periplasmic space of Pseudomonas aeruginosa while exposed to silver nitrate (see Figure 10A). Since the ability to bioaccumulate toxic heavy metals is a crucial factor in the bioremediation of heavy-metal-contaminated systems,39 this bacterium could be a potential candidate in such ventures if studied further at higher concentrations and also with other heavy metals. Previously, such periplasmic accumulation (at an

Figure 7. Agarose (0.4%) gel with lanes labeled as “B” (to indicate C-NPs) and “M” (to indicate M-NPs). 5981

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Figure 10B. It is also suspected that accumulation of nanoparticles in periplasmic space might be due to the presence of metal-ion-reducing enzymes.43 Some of the earlier reports encourage this suspicion by indicating the presence of nitrate reductase in the periplasmic space of Pseudomonas aeruginosa44 and the involvement of this enzyme in silver nanoparticle synthesis.33 Nitrate ions stimulate the activity of nitrate reductase, which reduces ionic silver (Ag3+) to metallic silver (Ag0), possibly in combination with electron shuttling.4,45 Furthermore, the nitrate reductase activity might also contribute to the synthesis of spherical-shaped silver nanoparticles.46 The present study also supports this demonstration by showing the activity of nitrate reductase in the CFE. However, the possible activity of other nitrate reductases (such as cytoplasmic- and membrane-bound compounds) in the synthesis process cannot be restricted, because of the preparation of the extract from whole cells. Furthermore, the metal ions also adhere to the cell surface (see Figure 10A), most probably through passive biosorption, which is obviously due to the negatively charged cell surface formed by the presence of lipopolysaccharides (a characteristic feature of gram-negative cell surface). In a similar study, the electrostatic interaction between Ag ions and negatively charged fungal cell surface moieties was found to be the first step in the process of bioaccumulation.47 Astonishingly, their shapes are spherical in both cases (live cells and CFE-aided synthesis), unlike previous observations in the same genus, where different shapes of particles were reported while exposing live cells to silver nitrate.15 This observation promotes the suspicion that a common factor or a set of common factors would have been involved in nucleation and reduction processes, although one environment (live cell) is under control metabolically and another (biomass as in CFE aided) is uncontrollable. This can be established in future investigations. The present study has also demonstrated the potential of a marine bacterium to be utilized as a source for rapid nanoparticle synthesis. 3.3. Antimicrobial Activity. Recently, antimicrobial properties of silver nanoparticles synthesized (extracellularly) by Pseudomonas aeruginosa BS161R were reported, relative to a wide range of bacterial and yeast pathogens.18 In contrast, the present study has demonstrated the antimicrobial properties of silver nanoparticles synthesized by cellular extract of P. aeruginosa M6 using two different thermal treatments (CTT and MWT). The antimicrobial assay was performed against bacteria including MRSA and M. smegmatis, as well as opportunistic yeast pathogens, viz, Candida albicans and C. glabrata. Both silver nanoparticles (C-NPs and M-NPs) showed excellent antibacterial activity against all tested bacterial strains at the volume of 70 μL/well (see Table 1 and Figure 11). The maximum zone of inhibition was observed against M. smegmatis (20.5 ± 0 for M-NPs and 20 ± 0 for C-NPs). Interestingly, both types of nanoparticles has exhibited good inhibition against MRSA (8 ± 0 for M-NPs and 9 ± 0 for C-NPs); however, few resistant colonies appeared in the zone of inhibition around the well loaded with C-NPs (Figure 11). There is no significant growth inhibition by both C-NPs and M-NPs against C. albicans and C. glabrata. However, there was an observable static effect found around the C-NPs loaded well against C. albicans alone. M-NPs are comprised of polydisperse (from small to bigger) particles as shown in earlier results; however, C-NPs are comprised of smaller-sized particles. Since the bigger particles of M-NPs were trapped at the edge of the agar well (Figure 11), only small-sized particles have diffused

Figure 8. Zeta potential distribution of M-NPs (top) and C-NPs (bottom).

Figure 9. Concentrated solution of C-NPs in centrifuge tubes reacted with CTAB (left), without any reaction (center), and reacted with SDS (right).

exposure concentration of 50 mmol/L silver nitrate) was observed in Pseudomonas stutzeri AG259, isolated from a silver mine.15 Similarly, the accumulation of silver nanoparticles was also found in the periplasmic space of Bacillus sp.40 In the present study, the accumulation describes the resistance mechanism of this marine isolate, Pseudomonas aeruginosa M6. There are many possible means by which a bacterium acquires resistance. In view of this, periplasmic metal-binding proteins (such as SilE, reported in some other gram-negative bacteria) can also aid in such an accumulation process, which actually are involved in providing metal resistance.41 In addition, other mechanisms are also involved in developing resistance, namely, biosorption efflux systems and alteration of solubility and toxicity via reduction or oxidation.42 With the knowledge from previous studies, the probable mechanisms are depicted in 5982

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Figure 10. (A) TEM image of a Pseudomonas aeruginosa M6 cell after exposure to aqueous AgNO3 at the concentration of 0.001 mol/L; the straight arrows show silver nanoparticles synthesized inside periplasmic space, the wavy arrow shows the cell-wall-adsorbed Ag ions, and the arrowhead shows the synthesized silver nanoparticles adhering on the cell surface. (B) Sketch of a hypothetical model showing mechanisms involved in the nanoparticle synthesis: (1) adsorption of silver ions to cell wall, (2) intake of Ag ions into periplasmic space, (3) transfer of silver to cytoplasm, (4) efflux of silver again to periplasmic space, and (5) synthesis of silver nanoparticles in periplasmic space, with the dashed arrow indicating the parts of the bacterial cell.

Table 1. Antimicrobial Activity of Nanoparticlesa

antimicrobial activities was also observed between C-NPs and M-NPs, especially against Candida albicans (see Table 1), indicating that the observed activities were not completely similar. There is no inhibition found against tested microbes in control (AgNO3 and CFE) treatments, but, a slight static effect was observed against three of them via AgNO3 control of both CTT and MWT (Table 1). To determine MIC, both particles were tested against the reference bacterial strain such as M. smegmatis and yeast pathogen C. albicans. At the concentration of 100 μL/mL, neither of the particles inhibited the growth; however, in higher volumes (i.e., 250 and 400 μL/mL), there was a complete cidal activity found against both bacteria and opportunistic yeast pathogens. Thus, these results have shown that a volume of ≥250 μL/mL is optimal to exhibit good activity against tested organisms. Silver has the greater affinity to bind the sulfur and phosphorus molecules; as a result, biological macromolecules such as membrane-bound proteins and DNA of the cells would be affected primarily.48,49 In the present study, the zeta potentials of synthesized silver nanoparticles were −30.1 (C-NPs) and −23.1 (M-NPs), which could be responsible for the observed antimicrobial activity and also the difference in their activity (especially against S. aureus, Candida albicans, and MRSA) (see Table 1). The relationship between microbial cell surface characteristics and their interaction with charged silver

Zone of Inhibition (mm) organism

M

B

AgNO3 control CFE control E. coli P. aeruginosa M. smegmatis S. aureus MRSA C. albicans C. glabrata

−b − 10.5 ± 1.5 9 ± 0.5 20.5 ± 0 6.25 ± 0.75 8±0 + −

−b − 11 ± 2 9 ± 0.5 20 ± 0 5±1 9±0 ++ −

Values represent mean ± SEM. “+/++” = static inhibition; “−” = no inhibition. “M” = M-NPs; “B” = C-NPs. bSlight static inhibition is observed against P. aeruginosa, M. smegmatis, and MRSA. a

through agar and have shown antimicrobial activity against the tested microbes. Hence, this result of antimicrobial activity by M-NPs was not an actual result, but rather a partial one. Since the source of the synthesis (CFE of P. aeruginosa M6) is the same, the capped biomolecules might be more or less similar with slight modifications, because of treatment differences in nature that probably have been attributed to the resemblance in antimicrobial activity. However, a slight difference in the

Figure 11. Antimicrobial activity of nanoparticles; activity against (a) E. coli, (b) S. aurues, (c) M. smegmatis, (d) P. aeruginosa, (e) MRSA, and (f) C. albicans. The symbols “B” and “M” denote C-NPs and M-NPs, respectively. 5983

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nanoparticles has been well-established in earlier studies.50,51 In addition, the size-dependent diffusion of nanoparticles in the agar medium might also be contributing to such a difference in antimicrobial activity. Furthermore, it was found that the antibacterial activity was higher than the anticandidal activity, since the silver does not enter into the fungal cell membrane at lower concentrations.52 Ultimately, this study has disclosed the advantage of different thermal treatments to manipulate desired attributes of the nanoparticles synthesized using biological sources.

ASSOCIATED CONTENT

S Supporting Information *

Supporting Information available for optimization of nanoparticle synthesis in various pH (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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4. CONCLUSION This study shows the distinct influence of thermal (physical) treatments (conventional thermal treatment (CTT) and microwave treatment (MWT)) on the biosynthesis of silver nanoparticles. Although the source of the synthesis is the same, CTT-assisted method synthesizes pure silver nanoparticles; in contrast, MWT-assisted method synthesizes silver oxide particles. This study also emphasizes the potential use of electrophoresis in the separation and characterization of nanoparticles synthesized using biological source under different thermal conditions, based on size and surface charge. Analysis of zeta potential shows that distinct surface modifications occurred during capping under MWT and CTT. The atomic force microscopy (AFM) image shows aggregated and nonsmooth M-NPs. Besides, the role of preheating the biological source (CFE) for synthesis has additionally been established. The synthesized C-NPs and M-NPs have also exhibited good antibacterial, including MRSA, and anticandidal activity.

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Article

AUTHOR INFORMATION

Corresponding Author

*Telefax: +91431 2407084. E-mails: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Ministry of Environment and Forests (MoEF), Government of India, for their financial support. The authors also acknowledge the service of Dr. Cheng Dong (Institute of Physics, Chinese Academy of Sciences, Beijing), for providing PowderX software and Mr. M. Kanagaraj (Research Scholar, Centre for High Pressure Research, School of Physics, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India), for his valuable suggestions regarding XRD data analysis. The service of Dr. K. Jeganathan, Assistant Professor and V. Rajiu, Research Scholar (both from Centre for Nanoscience and Nanotechnology, School of Physics, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India), for AFM analysis is also acknowledged by the authors. We thank Ms. R. Gayathri, M.Phil. project student, for assisting in antimicrobial assay. We thank Dr. V. Rajesh Kannan, Assistant Professor, Department of Microbiology, Bharathidasan Unviversity for providing MRSA strain. 5984

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