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Eco-friendly synthesis and antibacterial activity of silver nanoparticles reduced by nano-wood materials Xiaobo Lin, Fengqi Wang, Shigenori Kuga, Takashi Endo, Min Wu, Dayong Wu & Yong Huang Cellulose ISSN 0969-0239 Volume 21 Number 4 Cellulose (2014) 21:2489-2496 DOI 10.1007/s10570-014-0251-1

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Author's personal copy Cellulose (2014) 21:2489–2496 DOI 10.1007/s10570-014-0251-1


Eco-friendly synthesis and antibacterial activity of silver nanoparticles reduced by nano-wood materials Xiaobo Lin • Fengqi Wang • Shigenori Kuga Takashi Endo • Min Wu • Dayong Wu • Yong Huang

Received: 8 January 2014 / Accepted: 27 March 2014 / Published online: 29 April 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Hydrothermal treatment of nano-structured wood, prepared by precision grinding, with cationic silver was found to give silver nanoparticles (Ag NPs) of 2–40-nm size range embedded in the wood tissue. The size and distribution of Ag NPs depended strongly on the starting silver ion concentration and reaction temperature. Higher temperature tended to give larger size and wider distribution. The obtained Ag NPs were characterized using various methods, including high-resolution transmission electron microscopy, UV–visible spectroscopy, and X-ray diffraction. The antibacterial effect of the product against Escherichia coli was evaluated by static and

Electronic supplementary material The online version of this article (doi:10.1007/s10570-014-0251-1) contains supplementary material, which is available to authorized users. X. Lin Shijiazhuang Tiedao University, 17 Beierhuan East Road, Shijiazhuang 050043, Hebei, China e-mail: [email protected] F. Wang  S. Kuga  M. Wu (&)  D. Wu (&)  Y. Huang (&) Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhongguancun East Road, Beijing 100190, China e-mail: [email protected]

dynamic culture experiments, revealing that the Ag NPs-loaded nano-wood materials have great promise as antimicrobial agents against E. coli. Keywords Silver nanoparticles  Wood  Antibacterial study  Synthesis

Introduction Silver nanoparticles (Ag NPs) have been extensively studied for their use in a large variety of applications including electron microscopy, chemical and biological sensors, and catalysis, etc. (Yan et al. 2006; Zhou et al. 2006; Saha et al. 2010; Tang et al. 2010; Dankovich and Gray 2007; Mhlanga et al. 2013). They S. Kuga e-mail: [email protected] S. Kuga Tokyo University, Tokyo, Japan T. Endo Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-32, Kagami-yama, Higashi-Hiroshima, Hiroshima 739-0046, Japan

D. Wu e-mail: [email protected] Y. Huang e-mail: [email protected]


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are also an excellent candidate for antibacterial materials owing to the Ag NP germicidal effect (Tankhiwale and Bajpai 2009; Sathishkumar et al. 2010). Ag NPs are usually prepared by chemical reduction of ionic silver by citrate (Faraday 1857; Ding and Fang 2007) or by the two-phase method (Brust et al. 1994; Wang and Chen 2008). Recent trends for environment-friendly processes, however, have led to the use of natural materials as reductants, such as leaf extracts, starch, bacterial cellulose, etc. (Vigneshwaran et al. 2006; Ifuku et al. 2009; Dubey et al. 2010a, b). For a long time, Ag has been used as an antibacterial agent in medical instruments and devices, fibers, water treatment, and food processing (Sathishkumar et al. 2010; Cheng and Su 2011; Mhlanga et al. 2013). The antibacterial effect of Ag NPs has been attributed to their small size and large surface area, which allows them to interact closely with microbes (Ruparelia et al. 2008). The contact causes leakage of intracellular substances leading to cell death. Wood is the most abundant renewable biomass, mainly consisting of lignin, cellulose, and hemicellulose (Quail 1979). The electron-rich features of phenolic, hydroxyl, and ether groups in these components allow them to have a reducing and stabilizing effect on metal NPs (Lin et al. 2011). In addition, wood tissues have three-dimensional porous structures with high mechanical toughness, which could be effective as a support material for metal particles. Also, wood can undergo natural degradation in soil and water, being environmentally friendly in this aspect. We here demonstrate a simple and green method for the controlled preparation of Ag NPs using nanowood materials as reducing and supporting agents. The obtained nanocomposites have been tested as antibacterial materials against E. coli. The antimicrobial effect was quantified by inhibition zone counting and dynamic antibacterial test. These nano-woodbased products have potential applications as biocompatible antibacterial materials.

Experimental Materials Nano-wood material was prepared as follows: Japanese Hinoki chips were cutter-milled to pass through a


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3-mm-size sieve using a cutter mill machine (MKCM3, 3-mm screen, Masuko Sangyo Co.). The obtained 3-mm wood flour was dispersed in water to make a 5 wt% solid suspension. The suspension was ground by a disk mill machine (MKZA6-2, Masuko Sangyo Co., Ltd.) through 15-cycle treatments to yield nanowood materials. The water suspension was treated with a tip sonicator (Ultrasound Cell Crusher, Scientz08, China) before use. Silver nitrate (Shenyang Jinke Reagent, China) was used as received. Preparation of Ag NPs This is a one-pot method for the green synthesis of Ag NPs by heating the mixture of silver nitrate and wood suspension directly. The metal precursor is silver nitrate 0.01 M. The concentration of the wood suspension is 0.1 wt%. In a typical synthesis experiment, a desired amount of 0.01 M AgNO3 solution (lL) was added to the 0.1 wt% nano-wood material suspension (10 or 2 ml) and stirred at 5–100 °C for 1 h. Characterization Specific surface analysis by nitrogen adsorption The suspension of nano-wood materials was subjected to solvent exchange to tert-butyl alcohol and freezedried. Nitrogen adsorption of the dried sample was performed by a Quantachrome NOVA 4000 (Yuasa Ionics, Tokyo). The specific surface area was determined by BET analysis provided by the accompanying software. UV–Vis absorption of silver nanoparticles UV–Vis absorption spectroscopy of Ag NPs was recorded at room temperature on a spectrophotometer (Cary 5000). Transmission electron microscopy The Ag NPs and their hybrids with nano-wood materials were observed on a Hitachi H-800 transmission electron microscope (TEM) operated at 100 keV or a JEOL FS-2200 high-resolution transmission electron microscope (HRTEM) operated at 200 keV. Specimens were prepared by depositing a drop of aqueous suspension onto carbon-coated Cu

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grids and drying in air. The number-averaged diameters of Ag NPs were estimated by counting the sizes of more than 100 particles in micrographs. Inductively coupled plasma atomic emission spectrometry After the reaction, the Ag NP-loaded nano-wood materials were isolated through centrifugation for three times against water. The supernatant was collected and analyzed by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP) (Varian 710 ES). X-ray diffraction XRD patterns of the composite films were obtained on a D8 Focus X-ray diffractomer (Bruker) using Cu Ka ˚ ) radiation with the X-ray generator oper(1.54059 A ating at 2.2 kW. The samples were prepared by packing about 5 mg of samples in a 9.0-mm cavity mount. Bactericidal experiments The bactericidal experiments were carried out with a gram-negative bacterium (Escherichia coli: E. coli). The bacterial suspension (3.7 9 106 cfu/ml) was applied uniformly on the surface of a nutrient agar plate before placing the sample on the plate (4 per plate). The plates were incubated at 35 °C for 24 h, after which the average diameter of the inhibition zone surrounding the sample was measured with a ruler with up to 1-mm resolution. In the dynamic antibacterial test, 10 mg blank sample and 10 mg antibacterial sample were added to 50 ml bacterial liquid. The initial bacterial concentration was 3.7 9 103 cfu/ml; then the average number of viable cells (cfu/ml) was record every 1 h.

Results and discussion After grinding, the nano-wood materials were suspended in water to form a large surface area with functional groups exposed and providing a site of deposition for the Ag NPs. The sample freeze-dried from t-butyl alcohol gave a BET surface area of 143 m2/g, which corresponds to a fiber width of


20 nm. Our previous work showed that hydroxyl groups of cellulose and hemicellulose as well as the phenolic groups in lignin can be effective reductants for Pt cations (Lin et al. 2011). Addition of Ag salts to the wood suspension caused yellow/brown coloration of the mixture because of Ag NPs formation. The reaction temperature and concentration of metal precursor are the two main factors in the seedmediated growth approach to metal NPs. In our protocol, the reaction temperature and concentration of precursor were investigated in detail. Figure 1 shows the TEM images of the Ag NPs obtained at reaction temperatures from 5 to 100 °C. Figure 2 shows the corresponding average size and size distribution of the particles. The average size of the four samples was 7.3 ± 1.2 nm, 7.8 ± 2.5 nm, 9.0 ± 4.0 nm, and 12.6 ± 8.7 nm, respectively. The TEM showed that the Ag NPs obtained at 5 °C are nearly monodispersed, while the high-temperature reaction broadened the size distribution. These results showed that Ag NPs with different sizes were successfully prepared and the nano-wood materials are effective supporting agents. The Ag NP formation at different reaction temperatures has been investigated using UV–visible spectroscopy in the 300–800-nm range (Fig. 3). All spectra show an absorption band in the range of 350–450 nm, a typical plasmon resonance band of Ag NPs. The absence of the absorption peaks above 300 nm in all of the samples shows the full reduction of the initial Ag ions. It can also be seen that the plasmon absorption band of the sample obtained at 5 °C is fairly sharp, indicating the narrow particle size distribution. At high temperature, the maximum absorbance of their surface plasmon band obviously increases as the temperature increases. At the same time, the absorbance of the plasmon band undergoes a blue shift from 363 to 427 nm. In contrast to low temperature, the plasmon band of Ag NPs obtained at high temperature showed some new features. The extinction peak became extremely broad, and a shoulder peak appeared at about 420 nm at reaction temperatures of 75 and 100 °C, which can be attributed to the formation of larger particles. All the UV–Vis spectral data are consistent with the TEM observation (Fig. 1). The influence of precursor concentration on the morphology of Ag NPs was investigated further. Figure 4 shows the TEM of the samples obtained by different concentrations of precursor under a fixed


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Fig. 1 TEM images of the 0.75-mM AgNO3 reaction with 10-ml wood suspension at a 5 °C, b 50 °C, c 75 °C, and d 100 °C

Fig. 2 Histogram of particle size distribution of the 0.75-mM AgNO3 reaction with 10-ml wood suspension at a 5 °C, b 50 °C, c 75 °C, and d 100 °C


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Fig. 3 The UV–Vis spectra of the 0.75-mM AgNO3 reaction with 10-ml wood suspension at a 5 °C, b 50 °C, c 75 °C, and d 100 °C

temperature of 100 °C. Figure S1 (supplementary information) shows the corresponding average size and size distribution of the obtained Ag NPs. Figure 4 shows that increasing the AgNO3 concentration from 0.1 to 2.0 mM increased the average size of the Ag NPs. Thus, the size of the Ag NPs could be controlled by the reaction temperature and the concentration of Ag ions. Additionally, since the Ag Ps loaded nanowood suspension is a mixture, it will separate if left to stand (Fig. S2, supplementary information). It can be observed that on standing the supernatant of the reaction mixture was transparent and colorless. Moreover, we could not find Ag NPs in supernatant by TEM observation. Therefore, all the Ag particles prepared in our protocol must be immobilized on the nano-wood materials eventually. The loading amount of Ag NPs can be calculated in this way: the added Ag? minus the remaining Ag in the supernatant. The remaining Ag in the supernatant, which can be isolated through centrifugation, was analyzed by ICP after the reaction (Table 1). It can be calculated that for all the reaction conditions, more than 94 % of the added Ag? was reduced to Ag NPs and loaded on the surface of the nano-wood materials. Figure 5 shows the UV–Vis absorption spectra of Ag NPs obtained for different concentrations of Ag precursor. The absorption bands of the reference sample are attributed to the absorption of Ag NPs. The absorbance of the blue plasmon band shifts from 379 to 440 nm as the concentration increases. The change


in the plasmon band is consistent with the particles size data in the TEM observation (Fig. 4). Apart from this, at an Ag ion concentration of 0.25 mM, a shoulder peak appeared at about 435 nm. The shoulder peaks increased, and the peaks became extremely broad as the concentration increased, representing the particle aggregates or clusters. This phenomenon may be due to the fast growth of the particles at high concentration. The formed nano-wood materials immobilized Ag NPs were further identified by powder X-ray diffraction (Fig. 6). Since hemicelluloses and lignin are amorphous, the peaks at 15.8°, 22.5°, and 34.4° in Fig. 6 are the reflections from the crystalline cellulose. The diffraction patterns are virtually unchanged by the metal-reducing reactions, indicating that the cellulose crystallinity has been retained. The peaks at 39.9°, 46.3°, 67.6°, and 81.7° are ascribed to the (111), (200), (220), and (311) planes of the face-centered cubic Ag crystal. These peaks grow sharper with the increase in particle size. Various functional composites of carbon, polymers, and inorganic particles, etc., have been prepared based on the antibacterial action of Ag NPs (Gong et al. 2007; Wang et al. 2009; Mhlanga et al. 2013). Compared to synthetic chemical materials, wood is absolutely nontoxic, a merit that may allow avoiding the problem of some toxic chemical species adsorbed on the surface of Ag NPs interacting with biological systems. Figure 7 shows the results of the zone inhibition test against E. coli as a model microorganism. In contrast to the blank sample, samples (a), (c), and (e) in Table 1 clearly show zones of inhibition indicating a bactericidal effect against E. coli. The width of the inhibition zone apparently increases with the increase in the size of the Ag particles and Ag NP loading. The observed findings may be attributed to more particles in the case of a high loading amount of Ag NPs. Figure 8 shows the dynamic antibacterial test against E. coli. Blank nano-wood materials without Ag NPs were used as control. After 1 h, the inhibition effect of antibacterial sample (e) in Table 1 was 99 %. After 2 h, the bacterial concentration decreased to less than 1 cfu/ml (Table S1), while the blank test showed no signs of a decrease in the concentration of E. coli. More data from the dynamic antibacterial study are shown in the supplementary information in Table S1. After 24 h, the E. coli concentration is 4.0 9 104 cfu/ml in


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Fig. 4 Photographs of a 0.1-mM, b 0.25-mM, c 0.5-mM, d 1.0-mM, and e 2.0-mM AgNO3 reactions with the 10-ml wood suspension. TEM images of a 0.1 mM, b 0.25 mM, c 0.5 mM, d 1.0 mM, and e 2.0 mM AgNO3 reaction with the 10-ml wood suspension

the blank test and less than 1 cfu/ml in the antibacterial test with sample (e). These results show that nano-wood material-supported Ag NPs have enduring


antimicrobial activity, potentially useful for medical devices, wound dressings, and water treatment equipment, etc.

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Table 1 Loading of Ag NPs to wood material* Conc. of Ag?/mM 0.1 0.25

Added Ag?/lg

Remaining Ag/lg











3.928 10.95 0.6009

Loading of Ag0/lg 21.32 53.94 104.0 204.8 431.0

* 2 mg nano-wood material dispersed in 2 ml of water

Fig. 7 Images of agar plates containing Ag NP-impregnated disks and the inhibition zone for E. coli. Sample a, c, and e: 0.1-, 0.5-, and 2.0-mM AgNO3 reaction with the 10-ml wood suspension

Fig. 5 The UV–Vis spectra of a 0.1-mM, b 0.25-mM, c 0.5mM, d 1.0-mM, and e 2.0-mM AgNO3 reactions with the 10-ml wood suspension

Fig. 8 The dynamic antibacterial study against E. coli: 10 mg blank sample and 10 mg antibacterial sample e were added to 50 ml bacterial liquid. The bacterial concentration is 3.7 9 103 cfu/ml


Fig. 6 XRD spectra of a 0.1-mM, b 0.25-mM, c 0.5-mM, d 1.0mM, and e 2.0-mM AgNO3 reactions with the 10-ml wood suspension

Silver nanoparticles with different sizes and size distributions were obtained using nano-wood materials as both the reducing and supporting agents. The size of the Ag NPs could be controlled by adjusting the reaction temperature and the concentration of Ag precursor. Moreover, since the main component of the herbaceous plant bodies is nearly the same, the raw materials in this method can be any tree species. Our work provides a way in which wood resources can be


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used to prepare Ag NPs without employing any other reductants, capping or dispersing agents. These obtained novel composites show enduring antimicrobial activity against the model bacteria E. coli. Therefore, the composites have potential applications in the development of biocompatible antibacterial materials. Acknowledgments This work was supported by the National Program on Key Basic Research Project (973 Program, no. 2011CB933700), the National Natural Science Foundation of China (51172247, 51043003, 50773086), and the Chinese Academy of Sciences Visiting Professorships.

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