Pine cone-mediated green synthesis of silver nanoparticles and their ...

Appl Microbiol Biotechnol (2013) 97:361–368 DOI 10.1007/s00253-012-3892-8

ENVIRONMENTAL BIOTECHNOLOGY

Pine cone-mediated green synthesis of silver nanoparticles and their antibacterial activity against agricultural pathogens Palanivel Velmurugan & Sang-Myung Lee & Mahudunan Iydroose & Kui-Jae Lee & Byung-Taek Oh

Received: 2 December 2011 / Revised: 1 January 2012 / Accepted: 4 January 2012 / Published online: 31 January 2012 # Springer-Verlag 2012

Abstract The medicinal and physicochemical properties of nanoscale materials are strong functions of the particle size and the materials used in their synthesis. The nanoparticle shape also contributes significantly to their medicinal properties. Several shapes ranging from oval, spherical, rods, to teardrop structures may be obtained by chemical methods. Triangular and hexagonal nanoparticles have been synthesized by using a pine cone extract (PCE). Here, we report the discovery that PCE, when reacted with silver nitrate ions, yields a high percentage of thin, flat, single-crystalline nanohexagonal and nanotriangular silver nanoparticles. The nanohexagonal and nanotriangular nanoparticles appear to grow by a process involving rapid reduction with assembly at room temperature at a high pH. The nanoparticles were characterized by UV–Vis absorption spectroscopy, SEM-EDS, TEM, FTIR, and X-ray diffraction analyses. The anisotropy of the nanoparticle shape results in large near-infrared absorption by the particles. Highly anisotropic particles are applicable in

Palanivel Velmurugan and Sang-Myung Lee made equal contributions to this work. P. Velmurugan : S.-M. Lee : K.-J. Lee : B.-T. Oh (*) Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environment and Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk 570-752, South Korea e-mail: [email protected] P. Velmurugan Department of Environmental Science, Periyar University, Salem, Tamilnadu 636-011, India M. Iydroose Biodiversity and Aquatic Ecology Lab, Department of Environmental Sciences, Bharathiar University, Coimbatore, Tamilnadu 641-046, India

various fields, including agriculture and medicine. The obtained silver nanoparticles (Ag NPs) had significant antibacterial action on both Gram classes of bacteria associated with agriculture. Because the Ag NPs are encapsulated with functional group-rich PCE, they can be easily integrated in various applications. Keywords Antibacterial activity . Green synthesis . Silver nanoparticles . Pine cone extract

Introduction The optical, electrical, magnetic, and catalytic properties of metal nanoparticles have been intensively studied during the last two decades because of their unique properties (Bar et al. 2009). It is necessary to perform extensive research to control the size and shape of metal nanoparticles, which is crucial to tune and optimize their physical, chemical, and optical properties (Bruchez et al. 1998; Bar et al. 2009). Numerous chemical and physical methods have been employed to prepare metal nanoparticles, including chemical reduction (Petit et al. 1993; Vorobyova et al. 1999; Tan et al. 2002; Yu 2007), electrochemical reduction (Sandmann et al. 2000; Liu and Lin 2004;), photochemical reduction (Keki et al. 2000; Mallick et al. 2005), and heat evaporation (Bae et al. 2002; Smetana et al. 2005; Chandran et al. 2006). To avoid aggregation during nanoparticle production, surface passivation reagents are needed. Unfortunately, organic passivators are generally toxic and pollute the environment when large-scale nanoparticles are produced (Bar et al. 2009). Therefore, simple and greener procedures for the synthesis of silver nanoparticles (Ag NPs) which do not emit large quantities of toxic chemicals in solid, liquid, and gaseous forms in the environment are valuable (Parashar et al. 2009; Dubeya et al. 2010).

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Over the past several years, a number of biomimetic processes have been used for the synthesis of Ag NPs (Vigneshwaran et al. 2006; Abu Bakar et al. 2007; Thakkar et al. 2009; Dubeya et al. 2010). Certainly, in the past several years, nanoparticle research has focused on plants, algae, fungi, bacteria, and viruses for the low-cost, energy-efficient, and nontoxic production of metallic nanoparticles (Thakkar et al. 2009; Dubeya et al. 2010). However, to date, there has not been any report on the development of Ag NPs utilizing pine cone extract (PCE), which exhibits numerous medicinal properties (Harada et al. 1988; Bradleya et al. 2003; Bioflavonoids 2010). While microorganisms, such as bacteria, actinomycetes, and fungi continue to be investigated in metal nanoparticles synthesis, the use of parts of plants in similar nanoparticle synthesis methodologies is an exciting possibility, which is relatively unexplored and underexploited. Even though Ag NPs are considered bio-compatible, chemical synthesis methods may still lead to the presence of some toxic chemical species absorbed on their surface which may have adverse effects in industrial applications (Bar et al. 2009; Dubeya et al. 2010). The use of plant extract (pine cone) for the synthesis of nanoparticles is potentially advantageous over other environmentally benign biological processes by eliminating the elaborate requirement of maintaining cell cultures. Pine cone Pinus thunbergii (Black cone) occurs naturally in Eastern Asia from Ochotsk and southern Kamchatka to Korea and in the northern parts of Japan and China. P. thunbergii cones are widely used for household decorations and medicinal purpose. Traditionally, P. thunbergii cone extract has long been valued for its culinary, medicinal, cosmetic, and aromatherapy properties (Bioflavonoids 2010). However, there are no reports on the production of Ag NPs using PCE and their antibacterial activity against agricultural pathogens. In this study, a simple rapid green process to synthesize Ag NPs from PCE using water as an extracting solvent was developed. Silver nanoparticles can be prepared with lower amounts of PCE at room temperature and without any additional chemicals/and or physical steps. The effect of pH was evaluated to optimize the metal nanoparticle synthesis route, and the obtained Ag NPs were characterized using various techniques. In addition, the antibacterial activity of the Ag NPs against agricultural pathogens was evaluated.

Material and methods Silver nitrate (AgNO3) was acquired from Daejung Chemicals & Metals Co., Ltd., South Korea. All glassware was washed and cleaned in an ultrasonic sonicator bath, sterilized in an autoclave, and dried in an oven before use. Fresh mature pine cones were collected under the trees near the

Appl Microbiol Biotechnol (2013) 97:361–368

Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, South Korea. The collected pine cones were thoroughly washed and dried under shade for about 10 days. The dried pine cones were well ground to a fine powder using stone hand grinder made up of stone and transferred to a mixer grinder to obtain fine particles. The obtained powder was sieved using US Standard Sieve series 100 Mesh (149 μm). The obtained powder was stored in a polyethylene air tight container at room temperature before extraction. Next, 100 g of the fine powder was transferred to a 1,000-ml Erlenmeyer flask containing 400 ml deionized water. The mixture was boiled at 170 °C using hot plate with magnetic stirrer by covering the flask with aluminum foil to obtain 100 ml concentrated solution. The contents of the flask were then mixed thoroughly and filtered through Whatman no. 1 filter paper to obtain the extract. Finally, the extract was stored at 4 °C for further use. Syntheses of Ag Nanoparticles In 100-ml Erlenmeyer flasks, 45 ml Milli-Q Ultrapure water and 5 ml of PCE were added to synthesize the Ag NPs. One mM AgNO3 was added to 50 ml of the reaction mixture (PCE). The flasks were incubated at room temperature. Simultaneously, the reactions were also performed at different pH values (6, 7, 8, 9, and 10) and reaction times. Isolation of Ag Nanoparticles The Ag NPs synthesized after reaction with the PCE for a few minutes were separated from the mixtures. The reaction mixtures were filtered through 0.22 μm Steritop millipore filters and centrifuged at 12,000 rpm for 15 min to isolate the Ag NPs. The resulting pellets were resuspended in sterile Milli-Q Ultrapure water to eliminate any uncoordinated molecules. The process of centrifugation and redispersion in Milli-Q water was repeated three times to ensure better separation of free entities from the metal NPs. The obtained NPs were stored by freeze-drying to obtain a powder. Characterization of nanoparticles The synthesis of the Ag NPs was confirmed by monitoring the absorption maxima of the reaction mixtures between 200 nm and 800 nm on a UV-1800 UV–Vis spectrophotometer (Shimadzu, Japan). Scanning electron microscopy–energy dispersive spectroscopy (SEM–EDS) (JEOL-64000, Japan) analyses confirmed the formation of the Ag NPs. The morphologies, crystalline natures, and size distributions of the Ag NPs were analyzed using transmission electron


microscopy (TEM) (Hitachi, H-n650, Japan). X-ray diffraction (XRD) measurements of the Ag NPs on a drop-coated glass substrate were recorded by a Rigaku instrument operating at a voltage of 40 kV and a current of 30 mA using CuKα radiation with a λ of 1.5406 Å and a nickel monchromator filtering wave at a tube voltage of 40 kV and a tube current of 30 mA. The scanning was conducted in the 2θ range of 5–90° at 0.04°/min with a time constant of 2 s. Fourier transform infrared spectroscopy (FTIR) spectra of the Ag NPs were obtained using a Perkin– Elmer FTIR spectrophotometer (Norwalk, USA) in the diffuse reflectance mode at a resolution of 4 particles cm−1 in KBr pellets. Antibacterial assay The well diffusion method was used to study the antibacterial activity of the synthesized Ag NPs (Kora et al. 2010). All glassware, media, and reagents used were sterilized in an autoclave at 121 °C for 20 min. Bacillus megaterium (ATCC 35075), Pseudomonas syringae (KACC 10361), Burkholderia glumae (KACC 10138), Xanthomonas oryzae (KACC10331), and Bacillus thuringiensis (KACC 10168) acquired from Korean agricultural culture collection were used as model test strains for gram-positive and gram-negative bacteria, respectively. The bacterial suspensions were prepared by growing a single colony overnight in Luria–Bertani broth and by adjusting the turbidity to 0.5 McFarland standards (Mulvaney 1996; Kora et al. 2010). Luria–Bertani agar plates were inoculated with each bacterial suspension and 0.1 mg of the Ag NPs were dissolved in 1 ml deionized water. Approximately 50 μl of the resulting solution was added to the center well with a diameter of 8 mm. Control plates were made using wells containing PCE only. The plates were incubated at 37 °C for 24 h in a bacteriological incubator, and the zone of inhibition (ZOI) was measured by subtracting the well diameter from the total inhibition zone diameter. Three independent experiments were performed with each strain.

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Statistical analyses All statistical analyses were performed using SPSS (SPSS, version 10.0, Chicago, IL, USA), Sigma plot, and Microsoft Excel 2003 statistical software packages.

Results Syntheses and characterizations of Ag nanoparticles The aqueous silver nitrate solution turned brown within 2 min with the addition of PCE and the control AgNO3 solution (without PCE) showed no change of color, and there was no absorption peak in the UV–Vis spectrum (Fig. 1 and the inset). The intensity of the brown color increased in direct proportion to the incubation period. Kumar and Yadav (2009) detailed the synthesis of Ag NPs using various plant extracts. Similarly, in the present study, Ag NPs were synthesized using PCE. Interestingly, the Ag NPs were rapidly synthesized within 2 min of incubation at higher pH values of 8, 9, and 10 (Fig. 2 and the inset). This behavior may be due to the excitation of the surface plasmon resonance (SPR) effect and to the reduction of AgNO3 (Mulvaney 1996; Krishnaraj et al. 2010). Scanning electron microscopy–energy dispersive spectroscopy The energy dispersive spectroscopy (EDS) results revealed a strong signal in the silver region which confirms the formation of the Ag NPs (Fig. 3). Metallic silver nanocrystals generally show a typical optical absorption peak at approximately

Minimal inhibitory concentration The minimal inhibitory concentrations (MICs) of the Ag NPs were determined by applying the MTT assay by using a 96well microtitre plate (Krishnaraj et al. 2010). Live cells of B. megaterium, P. syringae, B. glumae, X. oryzae, and B. thuringiensis at concentrations of 105–106 CFU/ml were inoculated with different concentrations of Ag NPs, and their concentrations were recorded using a polar star optima micro plate reader (BMG LABTECH GmbH, Germany) after 24 h of incubation. The MIC was determined as the point where there was no increase in the OD595 when measuring the optical density at different concentrations.

Fig. 1 UV–Vis spectrum of a 10−3 M aqueous solution of AgNO3 with PCE. The inset shows the control and Ag NPs production

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Fig. 2 UV–Vis spectra of aqueous silver nitrate with PCE at different pH values. The inset shows the UV–Vis spectra of silver nitrate at different pH values

3 keV, due to the surface plasmon resonance (Bar et al. 2009; Magudapathy et al. 2001). There is also a strong signal for the coating agent (opium) in the EDS data, which results from the copper substrate.

Transmission electron microscopy Representative TEM micrographs of the Ag NPs obtained after 24 h of incubation are presented in Fig. 4. The smallest Ag NPs were subjected to different analytical techniques for characterization. The TEM micrographs revealed nanoparticles with variable shapes, and most of them had triangular and hexagonal shapes after reaction time of 3 min at pH 9 (Fig. 4a (100 nm), and b (20 nm)).

X-ray diffraction Figure 5 shows the XRD patterns of the vacuum-dried Ag NPs synthesized using PCE extracted by our simple technique. The XRD technique was used to determine and confirm the crystal structure of the Ag NPs. The XRD analysis revealed seven well-defined characteristic diffraction peaks at 38.6°, 44.2°, 46.2°, 65.2°, 68.1°, 78.2°, and 85.2°. The peak at 85.2° was unidentified and indexed to the 111, 200, 220, and 311 planes of the cubic face-centered silver. The lattice constant calculated from this pattern was a04.086 Å. The obtained data was matched to the database of the Joint Committee on Powder Diffraction Standards (JCPDS) file No. 040783. The average grain size of the Ag NPs formed in the bioreduction process as determined using Scherr’s formula, d0(0.9λx180) / βcosθπ, was estimated to be 35 nm (Fig. 5). This result is in agreement with a previous result, where Ag NPs were synthesized using leaf extract of Acalypha indica and their antibacterial activity against waterborne pathogens was investigated (Krishnaraj et al. 2010).

Fourier transform infrared spectroscopy

Fig. 3 EDS analysis of Ag NPs demonstrating characteristic peaks

The FTIR spectra of the PCE NPs were recorded in order to identify the functional groups of PCE involved in the reduction of the synthesized nanoparticles. Figure 6a, b show the FTIR spectra of the PCE before and after nanoparticle formation, respectively. The major absorbance bands present


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1,509 cm−1 could be assigned to the characteristic asymmetrical stretching of the carboxylate group. The symmetrical stretch of the carboxylate group can be attributed to bands present at 1,369 cm−1. The peak at 1,263 cm−1 indicates the acetyl group. The peaks at 1,160 and 1,057 cm−1 were due to the C–O stretching vibration of ether and alcohol groups, respectively. A shift in the absorbance peak from 3,449 to 3,406 cm−1 was observed along with increased band intensity, suggesting the binding of silver ions with hydroxyl groups. Based on these peaks, it can be inferred that both hydroxyl and carbonyl groups of the cone extract are involved in the synthesis of Ag NPs. These resultant peaks are very similar to the results of previous studies by Mohan et al. (2007) and Kora et al. (2010) in which silver nanoparticles were synthesized using Gum acacia and Gum kondagogu (Cochlospermum gossypium), respectively. Antibacterial activity

Fig. 4 TEM micrographs of a 100 nm and b 20 nm Ag NPs synthesized from 10−3 M AgNO3 solution and 5 ml of PCE at room temperature for 3 min at pH 9

in the spectrum of PCE were at 3,449, 2,922, 2,045, 1,718, 1,615, 1,509, 1,448, 1,369, 1,263, 1,160, 1,104, 1,057, and 1,029 cm−1. Meanwhile, the spectrum of the pine cone extract nanoparticles showed characteristic absorbance bands at 3,406, 2,914, 1,727, 1,630, 1,384, 1,254, 1,053, and 1,021 cm−1. The broad band observed at 3,449 cm−1 could be assigned to stretching vibrations of the O–H groups. The bands at 2,922 cm−1 correspond to asymmetric and symmetric C–H stretching. The peak at 1,718 cm−1 arises from carbonyl stretching vibrations. The stronger bands found at 1,615 and

To analyze the antibacterial activity of the PCE (5 ml)-mediated synthesized Ag NPs (1 mM AgNO3), NPs with an average size of 5–50 nm were used. The antibacterial activity of the Ag NPs against agricultural pathogens was observed after 24 h of incubation at 37 °C. Growth suppression was observed in plates loaded with 50 μl of Ag NPs. Meanwhile, the control plate with PCE only produced a minute zone of inhibition. The bacterial growth inhibition around the well is due to the release of diffusing inhibitory compounds from the Ag NPs (Kora et al. 2010). A ZOI of around 10 mm was observed for the gram-positive bacterial strain B. thuringiensis. The ZOIs for the gram-negative bacterial strains were 1.2, 8.9, 8.6, and 4.2 mm for B. megaterium, P. syringae, B. glumae, and X. oryzae, respectively. Minimal Inhibitory Concentration of Ag NPs The synthesized Ag NPs showed effective antibacterial activity against the test agricultural pathogens. The MIC was determined as the lowest concentration at which no visible growth of the test pathogens was observed. The MIC for the gram-positive B. thuringiensis was 9 μl/ml. The MICs for the gram-negative bacterial strains were 11, 6, 9, and 11 μl/ml for B. megaterium, P. syringae, B. glumae, and X. oryzae, respectively (Fig. 7).

Discussion

Fig. 5 XRD pattern of the Ag NPs synthesized from PCE

The present study reports the synthesis of Ag NPs from silver nitrate using PCE as a template. The adopted method is compatible with “Green chemistry” principles as the PCE serves as a matrix for both reduction and stabilization of the silver nanoparticles. Numerous approaches have been employed to

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Fig. 6 FTIR spectra of vacuum-dried powders of a PCE and b Ag NPs synthesized from 10−3 M AgNO3 and PCE

improve the synthesis of Ag NPs, including chemical and biological methods. However, recently, the synthesis of Ag NPs using plant extracts has become more popular (Mulvaney 1996; Magudapathy et al. 2001; Li et al. 2007; Song and Kim 2009; Krishnaraj et al. 2010). The characteristic absorption peak at 412 nm in the UV–Vis spectra at different pH values confirmed the formation of the Ag NPs. The SPR patterns and characteristics of metal nanoparticles strongly depend on the particle size, stabilizing molecules, surface-adsorbed particles, and the dielectric constant of the medium. These Ag NPs shapes have not previously been observed when using plant materials in the particle size ranged from 5 to 50 nm. The majority of the Ag NPs were aggregates with each other, only a few of them being individual NPs of varying sizes, as observed by TEM. B. megaterium is a rod-shaped, gram-positive, and endospore-forming species of bacteria used as a soil inoculant in agriculture and horticulture. Bacterium is arranged into the streptobacillus form. It is a plant pathogen which can infect

wheat, which shows a symptom of white blotch of wheat is characterized by severe white to very light tan blotchesand streaks on leaf blades, sheaths, and culus. P. syringae is a rodshaped and gram-negative bacterium with polar flagella. It is a plant pathogen which can infect a wide range of plant species. The environment in which it lives is known as the phyllosphere. Researchers speculate that most strains live primarily on the surface of leaf epidermis, thus distinguishing P. syringae as an epiphytic bacterium. B. glumae is a gram-negative, capsulated, motile, lophotrichous flagella, and pectolytic bacterium that causes bacterial panicle blight of rice, which is an increasingly important disease problem in global rice production. X. oryzae is a gram-negative rod, capsulated, and motile bacterium with a polar flagellum. The genus Xanthomonas, which mostly comprises phytopathogenic bacteria, is a member of the family Pseudomonadaceae. Among Xanthomonads, X. oryzae pv. oryzae causes bacterial blight of rice which is one of the most important diseases of rice in most of the rice-


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Fig. 7 Minimal inhibitory concentrations of PCE mediated synthesized Ag NPs against B. megaterium, P. syringae, B. glumae, X. oryzae, and B. thuringiensis

growing countries. B. thuringiensis is a gram-positive and soildwelling bacterium, commonly used as a biological pesticide; alternatively, the Cry toxin may be extracted and used as a pesticide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on the dark surface of plants. From the ZOI results, it can be concluded that the synthesized Ag NPs had significant antibacterial effects on both Gram classes of bacteria. The increased inhibition action on the gram-positive bacteria may be due to the differential sensitivity of gram-negative and gram-positive bacteria towards Ag NPs, which possibly depends on their cell surface characteristics and the interactions with the charged PCE-Ag NPs. The cell wall component and cell charge has a permeability to react with the charge holding Ag NPs. This observation is in excellent agreement with earlier studies (Sharma et al. 2009; Kora et al. 2010). Besides, the cell wall of gramnegative bacteria consists of an outer membrane composed of lipids, proteins, and lipopolysaccharides which acts as a barrier and provides effective protection against antibacterial agents whereas the cell wall of gram-positive bacteria does not consist of an outer membrane (Mohan et al. 2007; Maneerung et al. 2008; Kumar and Yadav 2009). The reduced MICs of the Ag NPs may be due to the smaller size of the nanoparticles, which leads to increased membrane permeability and cell destruction. However, the mechanism of the bactericidal actions of the Ag NPs is tentative and not well understood. However, a few previous reports hypothesized that the antimicrobial activity of Ag NPs is closely associated with the formation of “pits” in the cell wall of bacteria, leading to increased membrane permeability and resulting in cell death (Sondi and Salopek-Sondi 2004; Krishnaraj et al. 2010). Yamanaka et al. (2005), indicated that bactericidal actions of the silver ions are caused primarily by their interaction with

the cytoplasm in the interior of the cell (Sondi and SalopekSondi 2004). The silver ions appear to penetrate through ion channels without causing damage to the cell membranes as they denature the ribosome and suppress the expression of enzymes and proteins essential to ATP production, which renders the disruption of the cell (Sondi and Salopek-Sondi 2004; Krishnaraj et al. 2010). In this study, the bacterial cultures treated with Ag NPs demonstrated an increased conductance. This result may be attributed to the dissolution of the cellular contents in the culture broth, the disruption of the cell membrane structures with the loss of membrane permeability, or the inability to sustain ATP production, necessary for maintaining membrane dynamics (Krishnaraj et al. 2010). At a given PCE concentration, the efficiency of the nanoparticle synthesis increased with increased silver nitrate concentration and reaction time, a property attributable to the large reduction capacity of the PCE. Because the particle size of the nanoparticles can be controlled, this method can be implemented for the large-scale production of monodispersed and spherical nanoparticles 1–50 nm in size due to the availability of a low-cost plant-derived reducing agent. The abundance of hydroxyl and carboxylate groups facilitates the complexation of silver ions during reaction with the silver ions. Subsequently, these silver ions are reduced to elemental silver, possibly by in situ oxidation of hydroxyl groups and by the intrinsic carbonyl groups, as well as those produced by oxidation with air. The proposed mechanism was also substantiated by the FTIR data. The formed Ag NPs demonstrated significant antibacterial action on both gram-positive and gram-negative agricultural pathogenic bacteria. The surface reactivity facilitated by capping demonstrates that these functionalized nanoparticles are promising candidates for various agricultural, pharmaceutical, biomedical, and for future environmental applications.

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