This study investigates the phytosynthesis, characterization, and antibacterial efficacy of silver and gold nanoparticles (NPs) produced using the hot water extract of mixed woodchip powder. The woodchip extract (WCE) was successfully used as both a reducing and stabilizing agent for the phytosynthesis of both crystalline metal NPs. The effects of different physicochemical factors affecting the formation of the metal NPs including reaction pH, concentration of the precursor metal salts, amount of WCE, and external energy input were evaluated. The characterization of the metal NPs was performed by transmission electron microscopy, selected area electron diffraction (SAED), energy dispersive X-ray (EDX) spectroscopy, and X-ray diffraction (XRD) pattern analysis. In addition, the antibacterial efficacy of the phytosynthesized NPs was measured. The AgNPs showed clear antibacterial activity against four representative bacterial strains. However, the AuNPs did not exhibit bactericidal activity, probably due to their surface modifications and relatively large size. These results suggest that the phytosynthesis of the metal NPs using WCE is highly efficient, and its convenience makes it suitable for use in large-scale production.
Nanoparticles (NPs) are of great scientific interest due to their extremely small size and large surface-to-volume ratio, which contribute to both physical and chemical differences between their characteristics and those of larger particles of the bulk material [
Knowing the importance of emerging eco-friendly NP synthesis methods, many researchers have turned to using biological materials for NP synthesis [
Woodchips are one of the largest lignocellulosic plant biomasses on Earth, and they are produced all over the world as forestry residues and wood waste. Typically, they are used as pulp for paper, organic mulch, growing media for mushrooms, and renewable energy sources for biofuels. In this report, we demonstrated that woodchip extract (WCE) could be efficiently used to synthesize metal NPs when the process was properly optimized.
Barkless mixed woodchips were purchased from a local forestry company. Before experiments, the chopped (~7 cm) barkless woodchips were thoroughly washed with tap water three times and washed again three times with deionized (DI) water to remove extraneous matter. The washed woodchips were dried in an oven (Jeio Tech) at 70°C until a constant weight was achieved. The dried woodchips were ground to pass through a 420
The composition of the woodchip powder was analyzed by following a two-stage acid hydrolysis protocol developed by the National Renewable Energy Laboratory. Briefly, each sample was subjected to hydrolysis with 72% sulfuric acid at 30°C for 1 h and then hydrolysis with 3% sulfuric acid at 121°C for 1 h. The autoclaved hydrolysis solution was neutralized to a pH of 6.0 using calcium carbonate, and the solution was then vacuum filtered. The sugars released by acid hydrolysis were quantified on a high-performance liquid chromatography (HPLC) system (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) equipped with a refractive index detector (Agilent 1260) and a Bio-Rad Aminex HPX-87P column (Bio-Rad Laboratories, Hercules, CA, USA) was used with HPLC-grade water at a flow rate of 0.6 mL/min at 65°C. All of the samples were filtered through a 0.20 mm filter and diluted with an eluent before analysis on HPLC. Various concentrations of the pure monomeric sugar were used for standards. The water content was measured as the weight loss from 1 g of woodchip powder dried at 105°C to a constant weight. The acid-insoluble lignin (Klason lignin) content was defined as the weight of ash-free, oven-dried filter cake dried at 105°C to a constant weight. The elemental composition of the woodchip powder was determined using an inductively coupled plasma mass spectrometer (ICP-MS 7500, Agilent Technologies).
The woodchip powder (5%, w/v) was added to the appropriate volume of DI water and autoclaved at 121°C for 20 min. Then, the aqueous solution was separated by centrifugation at 4,000 rpm for 10 min. The supernatant was filtered through a GF/C grade glass fiber filter (GE Healthcare, Little Chalfont, UK) and then filtered again through a 0.45
The potential effects of the reaction parameters on the synthesis of NPs such as the pH, concentration of the precursor metal salts, amount of WCE, and external energy input were studied by varying one parameter at a time while keeping the others constant. Firstly, the effect of the AgNO3 concentrations was determined in the dark with the inherent pH condition while maintaining a constant reaction temperature (25°C) and WCE concentration (1%, w/v). Secondly, the pH of the WCE was varied while the concentrations of AgNO3 (10 mM) and WCE (1%, w/v) and the reaction temperature (25°C) were kept constant. The pH was adjusted using NaOH or HCl and the reaction was performed in the dark. Thirdly, the effect of the WCE concentration was studied in the dark while maintaining the other parameters as constant (10 mM AgNO3, pH 13, and 25°C). Fourthly, the effect of heat was evaluated in the dark with the other parameters being constant (10 mM AgNO3, pH 13, and 5% WCE). Lastly, to evaluate the effect of the light energy input, different strengths of light energy irradiated the mixtures using an equal ratio of warm and cool light-emitting diodes (LEDs), while all the other parameters were fixed (25°C, pH 13, and 10 mM AgNO3, and 5% WCE). The light intensity was measured using an LI-250A light meter (LI-COR Biosciences, Lincoln, NE, USA). The phytoreduction of the silver ions was confirmed by visually observing a color change in the reaction mixture. In addition, the optimum condition for each parameter was chosen by measuring changes in the surface plasmon resonance (SPR) band over time using a UV-Vis spectrophotometer (Biochrom, Cambridge, UK).
The effects of the reaction parameters on the phytosynthesis of AuNPs were determined similar to the method described in Section
Before characterization, reaction mixtures containing the metal NPs were purified at room temperature by dialysis against 3 L of DI water for 3 days to remove any remaining metal ions. The dialysis membrane (Spectra/Por 3 Dialysis Membrane, 3.5 kD MWCO) used for purification was purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). The purification process was completed by another dialysis step against DI water for 3 days and filtration through a 0.45
High-resolution transmission electron microscopy (HR-TEM) images, energy dispersive X-ray spectroscopy (EDX) line profiles, and selected area electron diffraction (SAED) patterns of the as-synthesized metal NPs were recorded using a FE-TEM Tecnai G2 F20 with an EDX detector (FEI, Tokyo, Japan) operated at 200 kV in the low-dose mode and a Gatan Digital Micrograph version 2.32 (Gatan Inc., Pleasanton, CA, USA). Specimens for TEM characterization were prepared directly from the reaction mixture by dropping 10
The atomic concentration of NPs in the reaction mixture was determined using the ICP-MS (Agilent Technologies) before the following measurement. The purified NP solution was digested using a Multiwave 3000 digestion unit (Anton Paar, Austria). For the digestion, 0.8 mL of NP solution was placed in 100 mL polytetrafluoroethylene-tetrafluoroethylene (PTFE-TFM) digestion vessels and 7.2 mL of nitric acid (60%) was added. The vessels were radiated for 20 min at 700 W and for 15 min at 1,100 W. Concentrations of the metal NPs were used to determine the conversion yield of the metal NPs based on
The average conversion yield of the as-synthesized metal NPs was determined by measuring the concentration of the NPs from three independently dialyzed samples.
To determine the exact crystallinity, high-resolution X-ray diffraction (XRD, PANalytical X’Pert Pro MRD, PANalytical, Holland) with Cu/K
The antibacterial activity of the synthesized metal NPs against two Gram-negative and two Gram-positive bacteria was measured by the disk diffusion method. The tested bacterial strains were grown in LB broth at 37°C with agitation (200 rpm) for 24 h. Exponentially grown cells (107 CFU/mL) were uniformly spread onto the surface of the LB agar medium. A sterile paper disc (8 mm) was placed on the inoculated medium and impregnated with 50
Reportedly, plant biomass or extracts have been successfully exploited for the biosynthesis of NPs [
Composition analysis of mixed woodchip powder.
Content (w/w, %) | SD (w/w, %) | |
---|---|---|
Glucose | 43.17 | 3.29 |
Mannose | 10.78 | 1.82 |
Xylose | 4.1 | 0.82 |
Galactose | 1.85 | 0.23 |
Arabinose | 0.74 | 0.15 |
Klason lignin | 27.55 | 3.65 |
Water | 10.12 | 2.12 |
SD; standard deviation.
Elemental analysis of mixed woodchip powder.
Element | Content (mg/kg) | SD (mg/kg) |
---|---|---|
Al | 16.51 | 3.92 |
As | ND | ND |
Ca | 446.37 | 41.51 |
Cd | ND | ND |
Cr | ND | ND |
Cu | 0.15 | 0.02 |
Fe | 90.58 | 11.88 |
K | 634.01 | 27.55 |
Mg | 338.54 | 46.47 |
Mn | 34.52 | 7.52 |
Mo | ND | ND |
Na | 25.3 | 4.6 |
Ni | 0.04 | 0.01 |
P | 103.08 | 15.75 |
Pb | 0.04 | 0.01 |
S | 101.13 | 15.22 |
Zn | 5.1 | 0.21 |
ND; not detected.
One of the most important factors for the economic and efficient synthesis of NPs is determining the optimum concentration of the precursor metal salt. Therefore, the effect of the precursor salt concentration was evaluated for both metal NPs as indicated by time-dependent SPR absorbance measurements. In the case of AgNPs, the absorbance of the SPR band increased with the increase in the salt concentration in a time-dependent manner (Figure
Effect of AgNO3 concentrations on the phytosynthesis of AgNPs after reaction for 24 h (a), 48 h (b), and 72 h (c). Reaction curve demonstrating the evolution of the SPR band at
In contrast to the phytosynthesis of AgNPs, the formation of AuNPs did not correlate with the increasing precursor salt (HAuCl4) concentration (Figure
Effect of HAuCl4 concentrations on the phytosynthesis of AuNPs after reaction for 24 h (a), 48 h (b), and 72 h (c). Reaction curve demonstrating the evolution of the SPR band at
Although the optimum concentration of metal salts could be varied by adjusting other reaction parameters, the above results indicate that the formation of the metal NPs using WCE is dependent on the species of metals used.
The pH of the reaction mixture affects the rate of NP synthesis, morphology, and stability. In the case of the phytosynthesis of AgNPs using WCE, the reaction pH was the most important factor (Figure
Effect of pH on the phytosynthesis of AgNPs after reaction for 24 hr (a), 48 hr (b), and 72 hr (c). Reaction curve demonstrating the evolution of the SPR band at
Effect of alkaline pH on the phytosynthesis of AgNP. UV-Vis spectrum after reaction for 24 h at various pH values (a), blue shift of the SPR band depicted by average
Effect of reaction pH on the phytosynthesis of AuNPs after reaction for 24 h (a), 48 h (b), and 72 h (c). Effect of pH values on the blue shift of the SPR band depicted by average
Earlier reports indicated that the pH of a reaction mixture greatly influenced the biosynthesis of NPs as well as their stability. According to Andreescu et al. [
The amount of plant extract is an important factor affecting the size, shape, and synthesis rate of NPs [
Effect of WCE concentration on the phytosynthesis of AgNP (a) and AuNP (b) after 24 h.
Any molecule that can reduce a metal ion to its zero valent state can be used to synthesize metal NPs. However, external energy may be required to stimulate the redox process by activating the molecules to act as a reducing agent in the reaction [
Effect of temperature on the phytosynthesis of AgNPs after reaction for 15 min (a), 30 min (b), 60 min (c), 120 min (d), and 240 min (e). Reaction curve demonstrating the evolution of the SPR band at
Effect of temperature on the phytosynthesis of AuNPs after reaction for 15 min (a), 30 min (b), 60 min (c), 120 min (d), and 240 min (e). Effect of temperature on the blue shift of the SPR band depicted by average
In the case of the visible-light-assisted phytosynthesis of AgNPs using WCE, the initial reaction occurred very rapidly, and the results demonstrated that light energy efficiently enhanced the yield of AgNPs (Figure
Effect of LED light irradiance on the phytosynthesis of AgNPs after reaction for 15 min (a), 30 min (b), and 60 min (c). Reaction curve demonstrating the evolution of the SPR band at
Effect of LED light irradiance on the phytogenic synthesis of AuNP after reaction for 15 min (a), 30 min (b), 60 min (c), 120 min (d), and 240 min (e). Reaction curve demonstrating the evolution of the SPR band at
The conversion yield from silver and gold metal ions to their NP forms using WCE was determined after evaluating the individual reaction parameters. The atomic concentration of the NP solution in Section
Yield of AgNPs and AuNPs synthesized by WCE.
The representative TEM image of the purified AgNPs in Figure
A representative TEM image of the AgNPs (a), a particle size distribution histogram of the AgNPs determined from TEM images (b), and EDX results of a single AgNP (c).
HR-TEM image of a single polycrystalline AgNP (a), SAED pattern with the diffraction rings indexed with reference to fcc silver (b), and XRD patterns of WCE (lower line) alone and the as-synthesized AgNPs (upper line) (c).
Microscopic (TEM) results indicate that the phytosynthesized AuNPs are well dispersed and irregular in shape (Figure
A representative HR-TEM image of the AuNPs (a), a particle size distribution histogram of the AuNPs determined from TEM images (b), and an EDX result of a single AuNP (c).
HR-TEM image of a single polycrystalline AuNP (a), SAED pattern with the diffraction rings indexed with reference to fcc gold, and XRD patterns of WCE alone (lower line) and the as-synthesized AuNPs (upper line) (c).
Two Gram-positive and two Gram-negative bacterial strains were used to evaluate the antibacterial efficacy of the phytosynthesized NPs. The results showed that the AgNPs possess antibacterial activity against all the tested strains, which demonstrates their potential as bactericidal agents in clinical and biomedical applications (Figure
Antibacterial efficacy of the as-synthesized AgNP against four representative Gram-positive and Gram-negative strains.
Recently, the biological synthesis of metal NPs has been extensively studied to produce biocompatible NPs using a cost-effective and eco-friendly method. Plants or their extracts can be used in large-scale production of NPs. However, to the best of our knowledge, all phytosynthetic methods have different reaction conditions for the efficient production of NPs. Therefore, the plant material must be selected before optimizing the reaction parameters. Woodchips are one of the most abundant bioresources in the world but their application in the production of high-value-added products has rarely been reported. In this study, AgNPs and AuNPs were phytosynthesized using WCE. The effects of reaction factors including the reaction pH, concentrations of the precursor salts and WCE, and external energy inputs (both heat and light) were investigated. In addition, their antibacterial efficacy against four representative Gram-negative and Gram-positive bacterial strains was evaluated. The formation of the AgNPs was greatly affected by the reaction pH, and the increase in the pH caused a blue shift in the SPR band. The increasing pH also sharpened the band, which indicates a narrow size distribution of the NPs. The WCE concentration was also important for increasing the productivity of the phytosynthesis of the AgNPs. In addition, the light energy input further enhanced the formation of the NPs. The effects of the reaction parameters for the phytosynthesis of AuNPs were somewhat different from those of AgNPs. The most important factors for the AuNP synthesis were reaction pH and the precursor concentration. Evident blue shifts occurred with the increasing reaction pH, temperature, and strength of visible light, indicating the complexity of the reaction. Compared with the phytosynthesis of AgNPs, the results for AuNPs indicate that more research is required to enhance the reaction rate for high-quantity production of the NPs. The AgNPs and AuNPs were spherical and irregular in shape, respectively. On average, the AuNPs (39 nm) were larger than the AgNPs (13 nm). The crystallinity of both NPs was determined by analyzing TEM images and XRD patterns. The phytosynthesized AgNPs showed antibacterial activity against the tested bacterial strains, thus indicating that the AgNPs could be used as a biocidal agent. However, the phytosynthesized AuNPs did not exhibit the bactericidal effect, which might have been caused by surface modification of the NPs by components from the WCE and their large size.
In this study, WCE was chosen for the phytosynthesis of AgNPs and AuNPs because it is a ubiquitous, inexpensive biomass. Although various plant extracts have been successfully used to synthesize NPs, our results demonstrate that the optimized phytosynthesis of both NPs using WCE could be one of the most effective methods considering the synthesis rate, conversion yield, cost effectiveness, and simplicity of the process.
The authors declare that they have no conflicts of interest.
This study was financially supported by the National Research Foundation of Korea (Grant no. NRF-2015M2A2A6A03045350).