Novel antimicrobial chitosan–cellulose composite films bioconjugated with silver nanoparticles

Industrial Crops and Products 70 (2015) 395–403

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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Novel antimicrobial chitosan–cellulose composite films bioconjugated with silver nanoparticles Shan Lin, Lihui Chen ∗ , Liulian Huang, Shilin Cao, Xiaolin Luo, Kai Liu College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, PR China

a r t i c l e

i n f o

Article history: Received 10 January 2015 Received in revised form 11 March 2015 Accepted 15 March 2015 Keywords: Chitosan Cellulose Silver nanoparticle Composite film Antibacterial activity

a b s t r a c t Cellulose-based membranes have emerged as an attractive alternative to non-biodegradable petrochemical materials. An important drawback, however, is that cellulose-based membranes are prone to biofouling. Silver nanoparticles (AgNPs) encapped with polyacrylic acid were conjugated with the chitosan/cellulose composite films to enhance the antimicrobial activities. Using the 1-ethyl-3-(3dimethylaminopropyl) carbodii-mide hydrochloride and N-hydroxysuccinimide as biocoupling agents, AgNPs with an average size of 9 nm were distributed evenly in the film without agglomeration. The presence of AgNPs in the chitosan/cellulose–AgNPs composite films was further confirmed by X-ray diffraction measurements. Fourier transform infrared spectroscopy analysis supported the presence of amide bonds between the primary amino groups of chitosan and the carboxylic residues of coordination to silver nanoparticles. The antimicrobial properties of the chitosan/cellulose and chitosan/cellulose–AgNPs composite films were determined using the disk diffusion tests with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). As compared to the chitosan/cellulose composite films, the chitosan/cellulose–AgNPs composite films showed significantly improved antimicrobial activities. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Membrane separation technology has attracted substantial interests and demonstrated great potential for waste water reclamation, drinking water purification, sea water desalination, and separation. However, most membranes have been prepared with non-biodegradable petrochemical materials. Accumulation of these non-degradable plastic materials used as disposable items is becoming a significant burden to the ecosystem and has negative environmental and health impacts (Lu et al., 2006). Cellulose can be used to prepare membranes for separation applications (Ruan et al., 2004; Jie et al., 2005; Madaeni and Heidary, 2011), and is attractive alternative to traditional petrochemical materials because it is biodegradable, biocompatible, reproducible, and inexpensive. However, the application of cellulose-based membranes has been limited by an important drawback, that biological matter can build up on the membrane surface and leads to biofouling (Worthley et al., 2011; Anitha et al., 2012). Hence, it is desirable to develop bio-membranes with antimicrobial activities (Liu et al., 2010; Zhu et al., 2010). Chitosan is non-toxic, has high

∗ Corresponding author. Tel.: +86 591 83715175; fax: +86 591 83715175. E-mail address: [email protected] (L. Chen). http://dx.doi.org/10.1016/j.indcrop.2015.03.040 0926-6690/© 2015 Elsevier B.V. All rights reserved.

antimicrobial activity, and has been widely used as wound dressing material (Hou et al., 2008; Alonso et al., 2009; Pandima Devi et al., 2012). Recently, chitosan/cellulose blend membranes were prepared to improve the antimicrobial properties of cellulose-based membranes (Shih et al., 2009; Morgado et al., 2011; Stefanescu et al., 2012). However, monocomponent antibacterial agents have been far from meeting requirements for some special conditions. Therefore, it is necessary to find composite antibacterial agents to solve this problem (Niu et al., 2009; Fu et al., 2011; Liu and Kim, 2012). In this work, we explore chemical modifications on chitosan to help overcome this major limitation and enhance the antimicrobial property. It has been recognized that silver based compounds have antimicrobial activities. For example, silver is an effective biocide either bound to a solid surface or in solution (Kusnetsov et al., 2001; Ibrahim et al., 2012). Silver nanoparticles (AgNPs) have even stronger antimicrobial activities compared to the bulk metal, likely due to much larger surface-to-volume ratios. It has been shown that even nanomolar concentrations of AgNPs can be effectively against microbes (Vimala et al., 2010; Zhou et al., 2012). However, if simply composed with other materials (such as cellulose and chitosan), AgNPs can be easily leached and the composite/membrane will gradually lose its antimicrobial activity (Pinto et al., 2012; Tripathi et al., 2011). Thus, it is necessary to incorporate chemical connec-

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tion among chitosan, AgNPs and cellulose to eliminate leaching and extend the functional life cycle of the composite membrane. In particular, free NH2 groups available in chitosan provide tethering sites for chemical modifications in synthesis of composite AgNPs/chitosan/cellulose materials with enhanced antimicrobial properties. In this work, we develop a new and green approach for synthesis of chitosan/cellulose–AgNPs composite films using water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a conjugating agent. EDC can be used for conjugating biomaterials through a biocompatible and non-toxic process (Cabana et al., 2011). It can be directly added to the reaction buffer without prior organic solvent dissolution (Wang et al., 2003). Therefore, the carbodiimide reaction provides an efficient means to form amide bonds between the NH2 groups and the carboxylic residues. We first encapped the silver nanoparticles with polyacrylic acid (PAA) through a polyol process (Hu et al., 2008). The chitosan/cellulose–AgNPs composite films were then prepared by using the EDC and N-hydroxysuccinimide (NHS) as biocoupling agents. The developed chitosan/cellulose–AgNPs composite films were then characterized using a range of analytical methods and evaluated for their antibacterial activities. 2. Materials and methods 2.1. Materials Chitosan (MW = 2 × 105 Da, degree of deacetylation = 90%) was obtained from Golden-Shell Biochemical Co., Ltd. (China). Bamboo cellulose was generously provided by Shaowu Bamboo Pulp Corporation (Fujian, China) and milled into powder (400 mesh screened) before used. The material was prepared by firstly kraft pulping and then ECF (elemental chlorine free, shorted as ECF) bleaching sequences. The mass average polymerization degree of the cellulose powder as prepared was determined to be 650. Then, both chitosan and cellulose were dried for 10 h at 60 ◦ C and then used without any further purification. Zinc chloride (ZnCl2 ) was purchased from Sinopharm Chemical Reagent Co., Ltd. (China) and used as received. Silver nitrate (AgNO3 , 99.9%) was purchased from Boda Chemical Industry (Shanghai, China). Short-chain polyacrylicacid PAA powder (MW = 1800, 99%), diethyleneglycol (DEG, 99%), EDC (99.9%) and NHS (99.9%) were purchased from Aladdin reagents Inc. All reagents were used without further purification. Ultrafiltration membranes (normal molecular weight limited (NMWL) = 14,000) were purchased from Jingke Hongda biotechnology (Beijing, China). 2.2. Preparation of chitosan/cellulose blend films The preparation of the chitosan/cellulose blend was carried out in ZnCl2 ·3H2 O solution according to a previously reported procedure (Lu and Shen, 2011). 7.2 g ZnCl2 was dissolved in 2.8 mL deionized water to obtain ZnCl2 ·3H2 O solution. Then, 0.4 g mixture of chitosan/cellulose (w/w = 1:6) was added to a 50 ml flask and further mixed with 10.0 g prepared ZnCl2 ·3H2 O solution at 80 ◦ C. During the process of dissolving, the mixture was heated and persistently stirred until a transparent homogeneous solution was obtained (Lin et al., 2012). The chitosan/cellulose composite films were prepared on a coater (GBC-A4, GIST, Korea). 2 g chitosan/cellulose blend in ZnCl2 ·3H2 O solution was firstly poured and then manually casted onto a glass slide. After the evaporation of water from blend, the gel sheet was formed. The gel sheet was then immediately immersed in water at ambient temperature for 15 min. The resulted fresh films were washed with running water and then deionized water to completely remove the solvent from the films. Finally, the films

were air-dried at room temperature. The regenerated chitosan and cellulose were prepared by the same method. 2.3. Synthesis of AgNPs AgNPs was synthesized by a modified polyol process (Hu et al., 2008). Briefly, AgNO3 (0.2 g) was dissolved in DEG (6 mL) at room temperature. It was then quickly injected into a boiling solution of DEG (30 mL) and PAA (0.060 M) with vigorous stirring under a protective nitrogen atmosphere. Samples were cooled to room temperature by high pressure air flow. The final nanoparticles were harvested by washing with excessive ethanol, and redispersed in water for further purification with ultrafiltration membranes. Carboxylate contents in the AgNPs solution were determined by the electric conductivity titration method (Saito et al., 2006). 2.4. Synthesis of chitosan/cellulose–AgNPs composite film EDC/NHS (10 mg/mL, the molar ratio of EDC to NHS = 1:1) was added to AgNPs solution with a carboxylate concentration of 0.01 mmol/mL, and the solution was incubated at 25 ◦ C for 4 h with shaking (150 rpm). 5 mg of the chitosan/cellulose film was then submerged into the solution for 24 h with shaking (150 rpm). In the procedure, as depicted in Fig. 1, the carboxyl groups of AgNPs were activated by EDC and NHS and subsequently reacted with the amino groups of chitosan in the chitosan/cellulose film. Finally, the modified film was taken out, washed with the deionized water. Following this process, the chitosan/cellulose–AgNPs composite film was obtained. 2.5. Characterization 2.5.1. Transmission electron microscopy (TEM) Morphology and size distribution of the AgNPs were characterized using a transmission electron microscope (Hitachi H7500) operating at 160 kV. For TEM measurements, samples were prepared by dropping 10–20 ␮L of hydrogel solution on a 400 mesh copper grid covered by an amorphous carbon supported film. The droplet was then allowed for full contact/spreading on the grid and dried at room temperature. The average diameter and size distribution were calculated from 50 pieces of AgNPs in the TEM image (Zhou et al., 2012). 2.5.2. Scanning electron microscopy (SEM) Scanning electron micrographs (SEM) were taken on a scanning electron microscope (JEOL JSM-7500F). Working distance of 10 mm was maintained and the acceleration voltage used was 5 kV with different magnification. Before observation, the films were mounted on metal grids by using double-sided adhesive tape and the surface was coated with a thin layer of gold under vacuum. 2.5.3. The X-ray diffraction (XRD) measurement The X-ray diffraction (XRD) measurement of the samples was conducted using a reflection method on a MiniFlex2 XRD diffractmeter (Japan Rigaku) using a monochromatized X-ray beam with a Cu K-radiation of 1.54 Å at 40 kV and 30 mA. Samples were gridded into powders so as to erase the effect of the crystalline orientation and freeze-dried. The patterns were collected in the region of 2Â from 5◦ to 85◦ at a scanning rate of 10◦ min. 2.5.4. Thermogravimetric analysis (TGA) The changes of material mass loss with temperature were carried out in a TG-DTA instrument (Netzsch STA 449 F3) at a heating rate of 10 ◦ C/min under nitrogen flow rate of 20 mL min−1 . Approx-

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Fig. 1. Schematic illustration of preparation of chitosan/cellulose–AgNPs composite film.

imately 2–3 mg of sample was weighed as a standard and heated from 25 ◦ C to 900 ◦ C. TG–DTA data were collected every second. 2.5.5. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectra were recorded on a Fourier transform infrared spectrometer (Thermo Nicolet 380). The films were gridded further into powder as small as possible and freeze-dried. Powder like samples were mixed with KBr and then pressed into disks (0.5 mm in thickness). 2.5.6. Antimicrobial test The antimicrobial activities of the chitosan/cellulose–AgNPs composite films were determined using the disc diffusion method.

The microorganism, Escherichia coli (E. coli) (ATCC25922) and Staphylococcus aureus (S. aureus) (ATCC25923), were chosen for this study, as E. coli and S. aureus are the most commonly found bacteria. E. coli and S. aureus were cultured in Tryptone Soya Broth (TSB) solution (30 g/L) (Zhu et al., 2010; Liu et al., 2010). Bacteria were first cultured in a flask with 30 mL of TSB. The incubation was performed at 37 ◦ C and oscillated at a frequency of 150 rpm for 24 h to obtain the overnight phase of the bacteria. Following that, about 1 mL amount of the E. coli and S. aureus were pipetted from the overnight phase into another flask with freshly prepared 30 mL of TSB, respectively. Bacteria were grown at 37 ◦ C in the incubator shaker for another 5 h to obtain the log phase of the bacteria with

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Fig. 2. (a) TEM images of the AgNPs solution; (b) the corresponding size distribution (c) TG curves of the AgNPs; SEM of (d) the chitosan/cellulose and (e) the chitosan/cellulose AgNPs composite films.

higher activity and more viable than in other growth phases (Liu et al., 2010). Antimicrobial tests were carried out for cellulose, chitosan/cellulose and the chitosan/cellulose–AgNPs composite films. Peptone (10.0 g), sodium chloride (10.0 g), and beef extract (6.0 g) were mixed in 2000 mL distilled water to make nutrient agar medium, and pH was adjusted to 7.0. Then, the agar (30.0 g) was added to the medium. The agar medium was sterilized in a conical flask at a pressure of 15 lbs for 30 min. This medium was transferred into sterilized Petri dishes in a laminar air flan (Rao et al., 2012). The stationary phase bacteria was diluted into a concentration at about 103 CFU/mL with NaCl solution (0.85%, w/v). Diluted suspensions (0.1 mL) of E. coli and S. aureus were transferred and

spread onto the solid surface of the media. In the inhibition experiment, the test samples were placed on the E. coli and S. aureus agar plates and incubated at 37 ◦ C for 24 h before the inhibition zone were measured. 3. Results and discussion 3.1. Size distribution of AgNPs The morphology and size distribution of the AgNPs were highly related to the formation process. According to the reported literature (Ge et al., 2007; Hu et al., 2008), the formation of Ag with nano-size can be simply described as follows: (1) a large number of

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ether linkages can be firstly formed between DEG molecular under certain condition; (2) Ag+ is then enfolded by these ether bond in DEG solution, producing a complex between Ag+ and ether bond; (3) subsequently, Ag+ in this complex is reduced to Ag0 by hydrogen atom from PAA molecular, which originated from the broken of PAA molecular if these mixture is mixed at high temperature (∼230 ◦ C) and with vigorous stirring (800 rpm); (4) finally, observed AgNPs would be explained by the accumulation of above reduced Ag0 as shown in TEM image in Fig. 2(a). From the TEM image in Fig. 2(a) and (b), it was found that the AgNPs have an average particle size of 9 nm and obvious aggregation between these AgNPs did not occur. It was mainly because of that the coordination of carboxylate on the silver surface can effectively prevent the aggregation and fusion between the nanoparticles (Hu et al., 2008). In addition, uncoordinated carboxylate groups extend into the aqueous solution, making these AgNPs with a high dispersibility in water (Ge et al., 2007), and therefore providing abundant anchoring points for further attachment of other biomacromolecules. To further confirm this, the organic content of AgNPs was determined by TGA in Fig. 2(c). Weight loss below 200 ◦ C can be attributed to the removal of bound water. An apparent degradation peak was observed between 313.1 ◦ C and 431.2 ◦ C and the weight loss rate was 36.96%, however the weight loss rate was only about 9.54% from 431.2 ◦ C to 900 ◦ C. Thus the organic content for AgNPs can be given as 46.50%. According to Xiao et al. (2012)’s report, the organic content was related with the particle size due to high specific surface of small size. The content of organic coating on the surface of AgNPs was relatively high for the strong coordination with carboxylate groups and the multiple anchor points for every single polymer chain. Furthermore, no more aggregation was observed after six months of storage, indicating the stability of this AgNPs solution and favoring for the industrial production and composition with other biomacromolecules. To confirm this hypothesis, some composite films of chitosan/cellulose and chitosan/cellulose–AgNPs were prepared with the procedures described in the sections of 2.2 and 2.4. The results were shown in Fig. 2(d) and (e). The images confirm that the AgNPs dispersed uniformly in the film without agglomeration for the coordination of carboxylate on the silver surface. Furthermore, the Schiff base reaction between the carboxyl groups on AgNPs and the amino groups of chitosan makes the AgNPs strongly attached to the chitosan/cellulose film by covalent bonding, which could be further confirmed by FI-TR results. The diameters of the AgNPs distributed in chitosan/cellulose films were observed as around 9 nm, which is consistent with the values observed by TEM, indicating that the Schiff base reaction did not change/affect the size of the AgNPs. The networks of chitosan/cellulose–AgNPs composite film look more porous than that of chitosan/cellulose film due to nanoparticles incorporation process (Vimala et al., 2010). The composite may be used as a wound dressing material. 3.2. Structural characteristics of the chitosan/cellulose–AgNPs films The results of XRD tests of chitosan, cellulose, regenerated chitosan, regenerated cellulose, chitosan/cellulose and chitosan/cellulose–AgNPs composite films were shown in Fig. 3. There were two prominent peaks at 2Â at 10.3◦ and 20.3◦ assigned to (0 2 0) and (1 0 0) reflection respectively in the diffraction pattern of chitosan (Tian et al., 2004). No significant change for diffraction pattern of regenerated chitosan was found but for the decreased intensity. During the process of dissolving the inter and intramolecular hydrogen bonds of chitosan might be destroyed and the crystal structure of regenerated chitosan was reconstituted, which resulted in a remarkable decrease in its crystallinity compared to that of chitosan. The peaks observed at 2Â = 16.4◦ , 22.9◦ , and 35.2◦ in

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cellulose regenerated cellulose

chitosan regenerated chitosan cellulose/chitosan (111) (200)

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40

cellulose/chitosan-AgNPs 60

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2θ (degree) Fig. 3. XRD patterns of the chitosan/cellulose and chitosan/cellulose–AgNPs composite films.

the XRD profile of cellulose were generally indexed as the cellulose crystalline plane (1 0 1), (0 0 2), and (0 4 0) (Lu et al., 2011). But there was only a peak at 2Â = 21.9◦ for the regenerated cellulose which indicated that the crystal type of cellulose was transferred from cellulose I to cellulose Ш. Moreover, the crystallinity of regenerated cellulose also decreased remarkably. These revealed that during the processes of dissolving and regeneration inter- and intra-molecular hydrogen bonds were disrupted as well as the crystal structure of cellulose. The diffraction peaks of chitosan/cellulose blend film were mainly at 11.8◦ and 21.1◦ and the crystallinity was between the corresponding values of chitosan and cellulose. It may be due to the reformation of hydrogen bonds between chitosan and cellulose during dissolution and regeneration processes according to the reports from Xu et al. (2005) and Luo et al. (2008). It was found that the crystallinities of chitosan and cellulose did not decrease during the Schiff base reaction. But the reflections around 34.0◦ and 41.0◦ observed in chitosan/cellulose–AgNPs composite films were assigned to (1 1 1) and (2 0 0) planes of the face centered cubic (fcc) of the AgNPs (Vimala et al., 2010). The results gave a clear evidence for the presence of AgNPs in the chitosan/cellulose–AgNPs composite film. 3.3. Thermal stability of the chitosan/cellulose–AgNPs films Thermal stability of chitosan/ cellulose and chitosan/ cellulose–AgNPs composite films were carried out on a TGA instrument and shown in Fig. 4. For these two composite films, the weight loss could be observed in two stages. The small initial drops occurring below 100 ◦ C in all cases were induced by the evaporation of retained water in materials. The next decrease stage (temperature ranged from 170 ◦ C to 360 ◦ C) could be caused by the thermal decomposition of the films. Only one mass degradation peak for two composites reflected the compatibility between cellulose, chitosan and AgNPs, also partly confirmed the uniform distribution in or physic-chemical connection with substrate materials (cellulose and chitosan). In decomposition stage, it was found that the chitosan/cellulose and chitosan/cellulose–AgNPs films started to degrade at the temperature of 314.2 ◦ C and 274.2 ◦ C, respectively. By derivation with TG curves, the maximum decomposition rate of two composites were determined at temperatures of 334.0 ◦ C and 294.3 ◦ C, illustrating the thermal stability of chitosan/cellulose–AgNPs films was inferior to that of chitosan/cellulose film without modification by AgNPs. This was possibly caused by looser networks as observed in the SEM images (Fig. 2(c) and (d)).

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Fig. 5. FT-IR spectra of the chitosan, cellulose, the chitosan/cellulose and chitosan/cellulose–AgNPs composite films.

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the primary amino groups of chitosan and the carboxylic residues of coordination to silver surface.

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Temperature(ºC) Fig. 4. TGA curves of the chitosan/cellulose and chitosan/cellulose–AgNPs composite films.

The residual qualities of the chitosan/cellulose–AgNPs composite film and the chitosan/cellulose film at 900 ◦ C were 19.3% and 12.3%, respectively. The 7.0 wt% weight loss difference between the chitosan/cellulose–AgNPs and chitosan/cellulose composite films represented the presence of silver nanoparticles in the chitosan/cellulose–AgNPs composite film (Vimala et al., 2010; Rao et al., 2012). Therefore, to improve the thermal stability of chitosan/cellulose–AgNPs, reforming its density via modifying mold process needs further studies. 3.4. Bonding mechanism of AgNPs with the chitosan/cellulose film The bonding mechanism of AgNPs with the chitosan/cellulose film was further studied by FT-IR spectra as shown in Fig. 5. It was well known that the characteristic absorption bands of chitosan appear at 1659.3 cm−1 (C O stretching), 1597.1 cm−1 ( NH2 bending) and 1380.4 cm−1 ( CH2 bending). And the absorption bands at 1154.5 cm−1 (anti-symmetric stretching of the C O C bridge) and 1087.9 cm−1 (skeletal vibrations involving the C O stretching) were characteristics of saccharide structure of chitosan (Saxena et al., 2010). The spectrum of the cellulose was similar to that of chitosan except for the absorption band at 1597.1 cm−1 . The carbonyl band of the chitosan/cellulose film was shifted to a lower frequency (1646.6 cm−1 ) and there was no NH2 bending observed. The band was shifted to a higher frequency overlapping with the carbonyl stretch in amides (Stefanescu et al., 2012). In the spectrum of the chitosan/cellulose–AgNPs composite film there was an absorption peak at 1573.5 cm−1 . It was ascribed to the amide bonds between

The antibacterial activities of the chitosan/cellulose and the chitosan/cellulose -AgNPs composite films were tested by E. coli and S. aureus based on a disc diffusion method (Fig. 6). The inhibitory activity was measured based on the diameter of the clear inhibition zone. If there was no clear zone surrounding, it was assumed that there was no inhibition zone. No inhibition zone against E. coli and S. aureus was observed around the cellulose films in Fig. 6(a) and (d), which indicated they did not show any inhibitory effect against the tested microorganisms. Substantial inhibition zones against E. coli (Fig. 6(b), ∼1.2 cm) and S. aureus (Fig. 6(e), ∼0.8 cm) were observed around the chitosan/cellulose based composite films. Recent studies in antibacterial activities of chitosan have revealed that chitosan show a broad range of activities against microorganisms (Alishahi and Aïder 2012; Niyas Ahamed et al., 2015). One of the reasons is the interactions between the positively charged chitosan amine groups and the negatively charged microbial cell membranes which lead to the leakage of proteinaceous and other intracellular constituents of the microorganisms (Zheng and Zhu, 2003; Alonso et al., 2009). Chitosan is also thought to have the innate characteristic of antimicrobial activity itself (Tripathi et al., 2011) and forms an impervious polymeric layer on the surface of the cell which prevents the transport of essential nutrients into the cell and thus results in the death of cell (Niu et al., 2009; Shi et al., 2006). Chitosan is more effective for gram-negative bacteria for the fact that hydrophilicity is significantly higher in gram-negative than in gram-positive bacteria, making them more sensitive to chitosan (Alishahi et al., 2012). Moreover, the outer membrane of Gram-negative bacteria such as E. coli consists of lipopolysaccharides (LPS) containing phosphate and pyrophosphate groups which make the cell surface negatively charged (Prescott et al., 2002). The cell wall can attach to chitosan by electrostatic interaction, more adsorbed chitosan would evidently result in greater changes in the structure and permeability of the cell membrane (Tripathi et al., 2011; Alishahi et al., 2012; Mansilla et al., 2013). The grampositive bacteria also have a thicker and more regid peptidoglycan cell wall (Shirvan et al., 2014). All these considerations agree with

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Fig. 6. The antibacterial activities of the films against E. coli (a) the cellulose films (b) chitosan/cellulose composite films (c) the chitosan/cellulose–AgNPs composite films and the antibacterial activities of the films against S. aureus (d) the cellulose films (e) chitosan/cellulose composite films (f) the chitosan/cellulose–AgNPs composite films.

evidence that the antimicrobial activity of the chitosan/cellulose based composite films against E. coli is greater than S. aureus. As shown in Fig. 6(c) and (f), substantial inhibition zones against E. coli (∼2.6 cm) and S. aureus (∼2.0 cm) were observed around the chitosan/cellulose–AgNPs films. AgNPs have been believed to function antimicrobially either as a release system for silver ions or as a contact-active material (Tripathi et al., 2011). They could bind to the sulphydryl groups of the metabolic enzymes and microbial DNA in bacterial electron transport chain. The former will inactivate them, and the latter will prevent the replication of bacteria (Zhou et al., 2012). Some of the AgNPs may penetrate the bacterial cells

(Feng et al., 2000) leading to leakage of proteins and other intracellular constituents, causing a significant increase in permeability and cell death (Lok et al., 2007). Other antimicrobial mechanism was reported that the surface of AgNPs were elucidated to generate free radicals, causing damage to the cellular membrane (Kim et al., 2007). Moreover, it has been reported that AgNPs with an average size of 12 nm had strong antimicrobial activities (Sondi and Salopek-Sondi, 2004). The nano particles increased the surface area and the contact probability of bacteria (Kim et al., 2007). In this study, AgNPs on the chitosan/cellulose–AgNPs composite films remained around 9 nm (Fig. 2(a) and (b)), and did not aggregate

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on the surface of the chitosan/cellulose composite film (Fig. 2(c) and (d)). This partially explains the observed enhanced antimicrobial activities of the chitosan/cellulose–AgNPs composite films. The results shown in Fig. 6 further suggest that the antibacterial properties of chitosan and AgNPs are not compromised by the chemical modifications. Instead, both two antimicrobial agents (chitosan and AgNPs) can work synergistically to provide a much better antimicrobial property. This is in accordance with other related composite antibacterial agents such as crosslinked chitosan coated Ag-loading nano-SiO2 composite (Niu et al., 2009), poly(vinyl alcohol)/chitosan blends containing silver nanoparticles(Hang et al., 2010) and so on. 4. Conclusions AgNPs encapped with PAA were successfully bioconjugated with the chitosan/cellulose composite films using the EDC and NHS as coupling agents. AgNPs were distributed evenly in the film with an average size of ∼9 nm and without agglomeration. XRD provided a clear evidence for the presence of AgNPs in the chitosan/celluloseAgNPs composite films with a mass fraction of about 7.0%. FT-IR spectrum confirmed the amide bonds between the primary amino groups of chitosan and the carboxylic residues of coordination to silver surface. Importantly, compared to the chitosan/cellulose composite films, the chitosan/cellulose-AgNPs composite films showed significantly improved antimicrobial activities and can thus be potentially used to prepare better biofouling-resistent degradable membranes. Acknowledgments The authors are grateful for the financial support from Nonprofit Industry Research and Special Funds of State Forestry Administration of China, and Fujian Development and Reform Commission of China (2014(482)), and National Natural Science Foundation of Fujian Province(2010J01271), China and Fujian Provincial Department of Education (JA12102), China. References Anitha, S., Brabu, B., John Thiruvadigal, D., Gopalakrishnan, C., Natarajan, T.S., 2012. Optical, bactericidal, and water repellent properties of electrospun nano-composite membranes of cellulose acetate and ZnO. Carbohydr. Polym. 87, 1065–1072. Alishahi, A., Aïder, M., 2012. Applications of chitosan in the seafood industry and aquaculture: a review. Food Bioprocess Technol. 5, 817–830. Alonso, D., Gimeno, M., Olayo, R., Vázquez-Torres, H., Sepúlveda-Sánchez, J.D., Shirai, K., 2009. Cross-linking chitosan into UV-irradiated cellulose fibers for the preparation of antimicrobial-finished textiles. Carbohydr. Polym. 77, 536–543. Cabana, H., Ahamed, A., Ledu, R., 2011. Conjugation of laccase from the white rot fungus Trametes versicolor to chitosan and its utilization for the elimination of triclosan. Bioresour. Technol. 102, 1656–1662. Fu, X.R., Shen, Y., Jiang, X., Huang, D., Yan, Y.Q., 2011. Chitosan derivatives with dual -antibacterial functional groups for antimicrobial finishing of cotton fabrics. Carbohydr. Polym. 85, 221–227. Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O., 2000. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. 52, 662–668. Ge, J.P., Hu, Y., Biasini, M., Dong, C., Guo, J., Beyermann, W.P., Yin, Y., 2007. One-Step synthesis of highly water-soluble magnetite colloidal nanocrystals. Chem. Eur. J. 13, 7153–7161. Hou, Q.X., Liu, W., Liu, Z.H., Duan, B., Bai, L.L., 2008. Characteristics of antimicrobial fibers prepared with wood periodate oxycellulose. Carbohydr. Polym. 74, 235–240. Hu, Y.X., Ge, J.P., Lim, D., Zhang, T.R., Yin, Y.D., 2008. Size-controlled synthesis of highly water-soluble silver nanocrystals. J. Solid State Chem. 181, 1524–1529. Hang, A.T., Tae, B., Park, J.S., 2010. Non-woven mats of poly(vinyl alcohol)/chitosan blends containing silver nanoparticles: fabrication and characterization. Carbohydr. Polym. 82, 472–479. Ibrahim, N.A., Eid, B.M., El-Batal, H., 2012. A novel approach for adding smart functionalities to cellulosic fabrics. Carbohydr. Polym. 87, 744–751. Jie, X.M., Cao, Y.M., Qin, J.J., Liu, J.H., Yuan, Q., 2005. Influence of drying method on morphology and properties of asymmetric cellulose hollow fiber membrane. J. Membr. Sci. 246, 157–165.

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