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ZnO nanocomposite with enhanced antimicrobial properties
International Journal of Biological Macromolecules 80 (2015) 121–129
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A versatile chitosan/ZnO nanocomposite with enhanced antimicrobial properties Madasamy Malini a , Munusamy Thirumavalavan b,∗∗,1 , Wen-Yi Yang b , Jiunn-Fwu Lee b , Gurusamy Annadurai a,∗,1 a Environmental Nanotechnology Division, Sri Paramakalyani Centre for Environmental Sciences, Manonmanium Sundaranar University, Alwarkurichi 627412, Tamilnadu, India b Graduate Institute of Environmental Engineering, National Central University, Chung-Li, Taoyuan County 320, Taiwan
a r t i c l e
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Article history: Received 17 March 2015 Received in revised form 27 May 2015 Accepted 18 June 2015 Available online 22 June 2015 Keywords: Chitosan membrane Nanocomposite Antibacterial activity
a b s t r a c t Porous chitosan membrane was fabricated by casting method using silica particles. Simultaneously nano ZnO was synthesized by green-synthesis method using tung ting oolong tea extract. Chitosan membrane was combined with nano ZnO in order to increase its antimicrobial activity. Through observations obtained from various techniques such as XRD, SEM, FT-IR, UV–visible and fluorescence emission analyses, chitosan was seen to be able to incorporate nano ZnO in the nanocomposite membrane. A blue shift (from 360 to 335 nm) was observed in the UV–visible spectrum of nanocomposite and fluorescence emission intensity of nanocomposite was considerably lower than that of nano ZnO. Gram negative organism Klebsiella planticola (MTCC2727) and Gram positive organism Bacillus substilis (MTCC3053) were used to test the antibacterial and antifouling activities of newly synthesized nanocomposite chitosan/ZnO membrane. The nanocomposite chitosan/ZnO membrane promisingly inhibited the bacterial growth when compared with as-synthesized chitosan. Gram negative K. planticola (MTCC2727) was comparatively more susceptible for inhibition than that of Gram positive Bacillus substilis (MTCC3053). In conclusion, nanocomposite obtained in this study showed enhanced antibacterial and antifouling activities. We believed that the enhanced physical properties of nanocomposite achieved by incorporating nano ZnO in the chitosan matrix could be beneficial in various applications. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In general fouling on bio-films is formed through colonization on ship hulls, pipelines, medical devices, wastewater, drinking water treatment, etc. Fouling is the unwanted growth and undesired deposition of microorganisms into communities on the surface [1]. Major and frequently formed fouling in water treatment in membrane technologies, resulting in a decrease in permeate water flux and membrane selectivity, an increase in energy consumption, and decrease in the extent of membrane life time. Any novel strategies are therefore required to decrease the extent of microorganism growth on membranes and better remedy for the biofouling would be the use of membrane with antimicrobial activity. Various
∗ Corresponding author. Tel.: +91 4634 283883; fax: +91 4634 283270. ∗∗ Co-corresponding author. Tel.: +886 34279455; fax: +886 34279455. E-mail addresses: [email protected] (M. Thirumavalavan), [email protected] (G. Annadurai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.06.036 0141-8130/© 2015 Elsevier B.V. All rights reserved.
numbers of polymers have been carrying out for the new product of membrane forming systems. Polymeric materials emerge with many fields rapidly producing new applications, especially in biotechnology, bioengineering and nanotechnology. Now-a-days, polymer is the most popular material for membrane manufacture. Commercially many polymers [2] such as polyacrylonitrile [3], glycidyl methacrylate and cellulose derivatives [4] are involved to make a synthetic membrane, Previous studies have also reported chitosan as an excellent matrix of membrane compared to other polymers due to its low cost, non-toxicity and easy biodegradability [5,6]. The reason for choosing chitosan was due to its linear structure composed of polymeric 2-amino-2-deoxy--d-glucose. It has been widely used to prepare polymer membranes because of it is good film forming and thermal properties, inexpensive, hydrophilic, biocompatible and good matrix for affinity ligand coupling that can be used in many areas such as food science, biochemistry, pharmaceuticals, medicine, agriculture and others [7–11]. But however, the so called chitosan membrane and films have poor mechanical strength. Recently, chitosan membranes with good mechanical properties have been successfully prepared
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using a novel method. Silica particles have some important benefits such as consistency and reproducibility of tight particle distribution of required pore size. Chitosan membranes are hydrophilic and highly reactive chemically (since they contain a large number of OH and NH2 groups). Due to these reasons, chitosan has been considered to be one of the important biomaterials [9–11]. But at neutral pH, chitosan does not show any antimicrobial activity and therefore to impart the antimicrobial activity of chitosan it is necessary to incorporate some antimicrobial agents [12]. Inorganic nanomaterials have attracted such particular interest for their antimicrobial activities. At the same time, inorganic nanomaterials alone will considerably reduce their applicability due to aggregation. Hence, one of the successful approaches used to prevent such aggregation and improve the dispersion of inorganic nanomaterials has been the fabrication of nanocomposite materials with enhanced properties by mixing both chitosan polymer membrane and nanomaterials. Nanotechnologies provide the tools required for preparation of such membranes, films or layers with embedded metal nanoparticles [13–16]. ZnO nanoparticles are biocompatible, nontoxic but noxious to microorganisms and these prospectives recommending their uses in food and agriculture fields [17,18]. The ZnO nanoparticles strongly inhibited the growth of microorganisms because it is disintegrating the cell membrane and thereby increasing the membrane permeability [19]. Many studies reported that metal oxide nanoparticles are stable under harsh processing, selective toxicity to microorganisms and exhibit minimal effect on mammalian cells [20]. Recently FDA (Food and Drug Administration, USA) listed ZnO is a safe material [21]. Compare to other metal oxides, ZnO nanoparticles have more advantages as white appearance, low production cost and UV-blocking [22]. Zinc oxide is a natural n-type semiconductor with wide band gap (3.37 eV) [23]. This has some noble properties such as high thermal conductivity and refractive index, photo oxidizing against biological species and chemicals, self-sterilization and antimicrobial action. So, it could be used in various fields such as cosmetics, concrete foods, pharmaceuticals, etc. [24]. Thus, ZnO has been considered to possess good antimicrobial properties. These ZnO nanoparticles can be synthesized by various methods such as chemical vapour deposition method [25], solvent thermal reaction method [26], hydro thermal method [27], wet chemical method [28], etc. These methods have unfavorable circumstance because they are involved various stages and also highly complicated. In contrast, synthetic procedure based on naturally occurring biomaterials so called “green-chemical” method provide an alternative, simple, environmental-friendly, inexpensive and non-toxic approach to obtain nanomaterials. The major content of these biomolecules are polyphenols which can act as both reducing and capping agents. Thus herein eco-friendly, green tea leaves mediated greensynthesis of nano ZnO using the extract of tung ting oolong green tea was reported. This tung ting oolong is one of the famous Taiwan green tea leaves. Thus, in this work, fabrication of chitosan/nano ZnO composite membrane using silica gel supported macroporous chitosan membrane and green tea mediated synthesized nano ZnO has been proposed. The significance of the present work is that the nanocomposite was prepared at room temperature by a simple dipping method without involving any high temperature thermal process. The dispersion of nano ZnO was believed to be an important factor affecting the functional properties of resultant chitosan/ZnO nanocomposite membranes. The obtained macroporous membrane, nano ZnO and bio-nanocomposite chitosan/ZnO membrane were characterized and confirmed by various techniques. Both antibacterial and antifouling activities against common pathogens Gram negative organism Klebsiella planticola (MTCC2727) and Gram positive organisms Bacillus substilis
(MTCC3053) were reported and discussed. A rapid, simple and environmentally benign technology was developed to evaluate the antimicrobial activities of resultant chitosan/ZnO nanocomposite membrane and the methodology reported in this work is very significant both technically and scientifically. Thus, the highlight of the present work is to explore the feasibility of combining the significant out comings of different feasible synthetic approaches in a single work. Hence, the purpose of the present study was mainly to prepare the nanocomposite chitosan/ZnO membrane with enhanced antimicrobial properties using a facile approach without any complication step. 2. Experimental 2.1. Chemicals Chitosan (<90% deacetylation degree, MW 100,000–300,000) was purchased from Marine chemicals, India. Silica particles (sizes in the range of 15–40 m) were purchased from Silicycle, Canada. Acetic acid was purchased from Thomas Baker, India. Zinc oxide (ZnO) was obtained from Sigma–Aldrich chemicals, USA and glycerol was purchased from RFCL limited, India. Gram negative organism K. planticola (MTCC2727) and Gram positive organism Bacillus substilis (MTCC3053) were purchased from MTCC, India. Fresh tung ting oolong tea leaves were collected from Lugu region of the central Taiwan. Deionized distilled water was used to prepare all solutions. 2.2. Preparation of macroporous chitosan membrane The chitosan membrane was prepared as follows: a solution of chitosan was first obtained by dissolving 1 g of chitosan in 100 mL of 1 vol% aqueous acetic acid solution. To this solution, silica particles were added, followed by vigorous stirring in order to disperse them uniformly. Then, the solution was poured onto a rimmed glass plate and the liquid was allowed to evaporate. The dried membrane was immersed in a 5 wt% aqueous NaOH solution and kept for 2 h at 80 ◦ C in order to dissolve the silica particles and to generate a porous membrane. Finally, the porous membrane was washed with distilled water to remove the remaining NaOH. In order to prevent its shrinkage during drying, the membrane was immersed in a 20 vol% aqueous glycerol solution (softening agent) for 30 min after the excess glycerol solution was removed, placed on a glass plate and allowed to dry. Thus, a strong and flexible chitosan membrane without shrinkage was obtained. 2.3. Preparation of tung ting oolong tea extract Fresh tung ting oolong tea leaves were collected from Lugu region of the central Taiwan. It was washed with distilled water and dried in shadow place for 1 week at room temperature. A 10 g of dried leaves was washed with double distilled water. Then the cleaned dry tea leaves were boiled in 100 mL of water for 10 min and filtered through 8 layers of muslin cloth. Filtered tea extract was stored at 4 ◦ C for further process. 2.4. Preparation of nano ZnO A 10 mL of tung ting oolong tea extract was boiled at 60 ◦ C. Then 90 mL of 1 M zinc nitrate aqueous solution was added to this and stirred at 60 ◦ C for 24 h to ensure thorough mixing. The reaction mixture was allowed to settle at room temperature. The color change of the tea extract from deep yellow to pale white vividly indicated the formation of nano ZnO. The nano ZnO particles were then separated by evaporating water from the solution,
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Fig. 1. Schematic representation of synthesis of (a) macroporous chitosan membrane; (b) Nano ZnO; (c) nanocomposite chitosan/ZnO.
dried and then collected. They were finally calcined at 100 ◦ C for 1 h and stored. The resultant nano ZnO was analyzed by UVspectrophotometer. 2.5. Preparation of nanocomposite chitosan/ZnO membrane Preparation of nanocomposite chitosan/ZnO membrane was performed as follows. Various circular formed discs were cut from the prepared chitosan membrane and were introduced in the suspension of nano ZnO in distilled water under magnetic stirring at 28 ◦ C for 24 h. The nanocomposite chitosan/ZnO discs were separated, rinsed with distilled water and then air dried. 2.6. Determination of water content and antimicrobial activities of as-synthesized and composite chitosan membranes The chitosan membranes were (as-synthesized and composite) soaked in distilled water for 24 h. The soaked membranes were mopped with blotting paper and weighed accurately. The dry weight of the membranes was measured after the wet membrane samples were placed in a dryer at 70 ◦ C for 48 h and cooled at room temperature in desiccators. The water content percentage was determined using the following formula: % water content =
wet sample weight−dry sample weight ×100 wet sample weight
Antibacterial activities of as-synthesized and nanocomposite chitosan/ZnO membranes were examined by disk diffusion analysis (membrane act as like a disk) against Gram negative organism
K. planticola (MTCC2727) and Gram positive organisms Bacillus substilis (MTCC3053). These microorganisms were cultured in Muller Hinton medium (MHM) and 12 h old culture of K. planticola and Bacillus substilis were swabbed on each plate. A small piece of as-synthesized chitosan membrane and nanocomposite chitosan/ZnO membrane was placed on the center of each inoculated plate. After 24 h of incubation at 37 ◦ C, the zone of inhibition against bacteria was observed. Antifouling activity was tested for both as-synthesized and nanocomposite chitosan/ZnO membrane by immersing them in the wastewater (collected nearby Sri Paramakalyani Centre for Environmental Sciences, Alwarkurichi, Tamil Nadu, India) at 37 ◦ C for 24 h. The membrane samples were removed from the wastewater by filtration. After that, the membranes were washed with distilled water and then immersed into 1% of glutaraldehyde at 4 ◦ C for 5 h to fix the microorganisms if any layer formed on the surface of the membranes. After 5 h, the membranes were taken and washed with ethanol [29]. The membrane samples were dried in a vacuum oven at 50 ◦ C overnight and then analyzed for antifouling activity. Thus, this study mainly focused on the synthesis of macroporous chitosan membrane, nano ZnO and nanocomposite chitosan/ZnO membrane using simple approaches as illustrated in Fig. 1. Various techniques such as XRD, SEM, FT-IR, UV–visible, fluorescence emission were employed to characterize the samples. SEM images of different samples are shown in Fig. 2 and XRD patterns are given in Fig. 3. As a core study, the antimicrobial activities were studied and compared in order to emphasize the superior behavior of nanocomposite chitosan/ZnO membrane over as-synthesized chtiosan as well as nano ZnO.
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Fig. 2. SEM images of (a) macroporous chitosan membrane; (b) nano ZnO; (c) nanocomposite chitosan/ZnO.
2.7. Characterization techniques X-ray diffraction (XRD) patterns were obtained using a Philips PW 1830 instrument. Scanning electron microscope (SEM) was carried out on Philip model CM 200, field emission scanning electron microscope. Attenuated total reflectance (ATR)-Fourier transform infrared (FT-IR) spectra were obtained on a Bruker vertex 80v instrument. UV–visible spectra were obtained using Perkin Elmer double beam UV spectrophotometer. Inverted fluorescence microscope (Nikon fluorescence microscope) was used to analysis the fluorescence emission.
3. Results and discussion 3.1. Macroporous chitosan membrane The chitosan membrane was prepared by casting method [30–32]. Fig. 1a clearly depicted the schematic representation of the formation of macroporous chitosan membrane. Chitosan was dissolved in an aqueous acetic acid solution containing silica particles (15–40 m) with vigorous stirring. The sol–gel transformation generated the pores because chitosan is soluble in acidic solutions and insoluble in basic solutions, whereas silica is insoluble in acidic solutions and soluble in basic media. These opposite properties were employed to form pores. For extracting the silica particles and to easily generate a porous membrane [31], the membrane was immersed in a 5% aqueous NaOH solution at 80 ◦ C for 2 h [32]. Chitosan was precipitated in NaOH solution, and the heat treatment was helpful for stabilizing the pore size and improving the mechanical as well as crystalline properties [33] of the chitosan. In this study, inorganic porogen (silica particles) was employed to generate the pore structure and the pore structure could be maintained only after vacuum drying. XRD peak around 2 = 20◦ corresponding to chitosan peak as shown in Fig. 3a, confirmed that the synthesized chitosan membrane showed only low crystalline character. This decrease in the crystallinity clearly demonstrated
that the conjugation of silica and chitosan during synthesis process suppressed the crystallization to some extent. It also suggested that silica and chitosan polymeric chain were mixed well at a molecular level. FT-IR spectra of macroporous chitosan membrane showed a peak around 3480 cm−1 due to (O H) and (NH2 ), a peak around (2800–2900 cm−1 ) due to alkyl (C H) and (N H), a peak around 1600 cm−1 due to (N H), a peak around 1725 cm−1 due to (C O), a peak around (1300–1500) cm−1 due to deformation of hydroxyl (OH) group, a peak around (1050–1100) cm−1 due to (C O) of chitosan and a peak around 800 cm−1 is due to aromatic (C H). The SEM image of macroporous chitosan membrane as shown in Fig. 2a indicated the opened pore surface on chitosan membrane. The porosity and the crystalline nature of the chitosan membrane would be expected to be altered upon the variation of the amount of silica added. The characteristics of the chitosan membrane are listed in Table 1. 3.2. Nano ZnO The synthetic process of nano ZnO is diagrammatically represented in Fig. 1b. The formation of nano ZnO was rapidly started with in a minute which was clearly identified by the color change of the reaction mixture from bright yellow to pale white. The pale white color after 24 h with no further color change [34] indicated the complete formation of nano ZnO. The color changes occurred in the reaction mixture during the formation of nano ZnO conveyed Table 1 Characteristics of as-synthesized and nanocomposite chitosan membranes. Characteristics
As-synthesized chitosan
Nanocomposite chitosan
Membrane material Configuration Thickness (mm) Membrane area (mm) Water content (%)
Chitosan + silica Flat membrane 0.30 60.04 84 .4
Chitosan + silica + nano ZnO Flat membrane 0.35 60.04 82.2
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that the fluorescence emission color of the nano ZnO nanoparticles could be tuned by varying the reaction time and reactant ratio [37]. Based on this, herein it is proved that tung ting oolong extract acts as a good stabilizing agent, which is responsible for the synthesis of nano ZnO in this study. In general, natural plant extract mostly resulted in the formation of reduced metal nanoparticles from the respective metal ions (metal salts), but interestingly in this case, ZnO nanoparticles were obtained from zinc salt. Thus, it is very clear that the tea extract used in this study acted as a stabilizing agent only and not as a reducing agent for Zn2+ ion. 3.3. Nanocomposite chitosa/Zno membrane
Fig. 3. XRD of (a) As-synthesized chitosan membrane and nano ZnO; (b) nanocomposite chitosan/ZnO.
the strong and quick light absorption behavior of nano ZnO in the visible region. This quick absorption is mainly due to the coherent oscillations of the free conduction electrons because of interacting with an electromagnetic field on the nanoparticle surface, which is called surface plasmon resonance [35]. XRD pattern of nano ZnO synthesized using green-method corresponded to close packed hexagonal Wurtzite structure as shown in Fig. 3a. The peaks around 2 = 31◦ , 33◦ , 36◦ , 48◦ , 56◦ , 62◦ , 67◦ , 68◦ , 69◦ and 77◦ are assigned to (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1) and (2 0 2) crystal planes respectively [36]. The surface morphology of biosynthesized nano ZnO as shown in Fig. 2b, indicated that these nanoparticles formed as agglomerated spherical nanosponges like shape. Also these shapes are not uniformly distributed, i.e. the particle distribution is undefined. The optical properties of synthesized samples were measured as shown in Fig. 4. The UV–visible absorption spectrum of nano ZnO, revealed a characteristic absorption peak located at 360 nm, which corresponds to the band gap of the zinc oxide nanoparticles. As the colloidal semiconductor ZnO nanocrystals are also known as novel inorganic fluorescence labeling agents, the fluorescence intensity of biosynthesized nano ZnO was measured and the fluorescence images are shown in Fig. 5. Fig. 5a clearly exhibited the bright fluorescence intensity of nano ZnO and this result supported to considering ZnO to be potentially used for bio-analytical and labeling application. It was proposed
The XRD pattern of nanocomposite chitosan/ZnO is illustrated in Fig. 3b. From Fig. 3b, two sets of diffraction peaks were observed corresponding to the chitosan and nano ZnO in the 2 range of 10–80◦ . The peaks in the 2 range 30–80◦ are corresponding to nano ZnO and the broad peak around 2 = 14◦ is corresponding to chitosan. This observation further indicated that the peak corresponding to chtiosan was shifted to lower 2 value (from 20◦ to 14◦ ) in the nanocomposite which could be due to possible interaction between nano ZnO and chitosan, but however the crystallinity of chitosan was greatly enhanced in the case of nanocomposite when compared with as-synthesized chitosan membrane. From Fig. 3b it was also observed that both nano ZnO and chitosan showed highest peak intensity and possessed good crystalline characters. The above results suggested the successful formation of nano ZnO dispersion on the chitosan matrix. There was no formation of a new peak in the nanocomposite compared with as-synthesized chitosan and nano ZnO, indicating that nanocomposite chitosan/ZnO membrane consisted of two phase structures, i.e. chitosan membrane and nano ZnO. These observations clearly witnessed the addition of nano ZnO caused an increase in the overall crystalline characters of chitosan membrane, which can be effective on the final properties of nanocomposite chitosan/ZnO membrane. From Table 1, it can be seen that the characteristics of chitosan membrane in the nanocomposite was slightly altered (or almost same). As the nano ZnO is doped, the thickness of the membrane somewhat increased, but the water content decreased slightly. However, the membrane area remained unaltered. This clearly suggested that incorporating nano ZnO into the chitosan matrix results in strong intermolecular interaction between the nano ZnO and chiotsan matrix [14] and consequently restricted the motion of the chitosan and thus promoted rigidity. Furthermore, increased crystallinity as observed by X-ray diffraction analysis could also be expected to enhance mechanical properties. The SEM image of nanocomposite chitosan/ZnO as shown in Fig. 2c, showed that the addition of nano ZnO caused surface morphology of membrane to be somewhat rough compared with as-synthesized chitosan membrane. The irregular distribution of uneven pore structures with some extent of pore blocking was observed on the surface of nanocomposite chitosan/ZnO membrane, suggesting that there exists good adhesion between nano ZnO and chitosan matrix. Such distribution of nano ZnO in the chitosan matrix would be expected to play a vital role in enhancing the mechanical properties of obtained nanocomposite chitosan/ZnO membrane. Also it could be concluded that stabilization of nano ZnO by chitosan membrane could help to increase the dispersion of nano ZnO in the chitosan matrix and prevents nano ZnO from more agglomeration. The FT-IR spectra of nanocomposite chitosan/ZnO revealed that the corresponding peak intensity of as-synthesized chitosan especially (O H), (N H) and (C O) was considerably diminished in the nanocomposite and the carboxyl characteristic band was almost disappeared. This observation obtained from the FT-IR spectra of chitosan before and after treating with nano ZnO confirmed the successful formation of nanocomposite chitosan/ZnO
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3.0
(a)
Nano ZnO as-synthesized chitosan membrane nanocomposite chitosan/ZnO membrane
Absorbance (a.u)
2.5
2.0
1.5
1.0
0.5
0.0 300
400
500
600
700
λ (nm) (b) 2.7
0.65
2.5
0.6
2.3
0.55
2.1
0.5
1.9
0.45
1.7
300-400 nm expanded region (nanocomposite)
0.4
300-40 0 nm expanded region (nano ZnO)
0.35
1.5 300
325
350
375
400
λ (nm)
0.3 300
325
350 λ (nm)
375
400
Fig. 4. UV–visible spectra of (a) Obtained samples; (b) 300–400 nm expanded region.
membrane. On comparing the optical properties of nanocomposite chitosan/ZnO with nano ZnO, the UV–visible absorption band of nano ZnO at 360 nm was blue shifted to 335 nm in the nanocomposite. Also the absorption peak of nanocomposite appeared as broad shoulder in shape with low intensity. However, the UV–visible absorption spectra of as-synthesized chitosan membrane did not show any obvious peak. In order to compare the fluorescence property of nanocomposite, the fluorescence image of nanocomposite
was obtained as shown in Fig. 5b. The brightness and intensity of the fluorescence emission of nanocomposite chitosan/ZnO was comparatively lower than that of nano ZnO. These shifts both in UV absorption and fluorescence emission are mainly due to the interaction between chitosan matrix and nano ZnO. Thus, it is believed that the nanocomposite chitsoan/ZnO may also protect against ultraviolet light and could be potentially used as UV shielding material.
Fig. 5. Fluorescence emission image of (a) Nano ZnO; (b) nanocomposite chitosan/ZnO.
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Fig. 6. Antibacterial activity of (a) As-synthesized chitosan membrane against Gram negative Klebsiella planticola; (b) As-synthesized chitosan membrane against Gram positive Bacillus substilis; (c) Nanocomposite chitosan/ZnO membrane against Gram negative Klebsiella planticola; (d) Nanocomposite chitosan/ZnO membrane against Gram positive Bacillus substilis.
3.4. Antibacterial and antifouling activity The disk diffusion method was carried out to examine the antibacterial activity of both as-synthesized and nanocomposite chitosan membranes as shown in Fig. 6, under in vitro conditions against common pathogens like a Gram negative organism K. planticola (MTCC2727) and Gram positive organism Bacillus substilis (MTCC3053). The formation zone was clearly observed around the disk containing nanocomposite indicating the antibacterial nature of nanocomposite. The results are shown in Fig. 6. The Bacillus substilis showed minimum range of inhibition 15.4 mm and K. planticola showed the maximum zone of inhibition 19.6 mm at 20 mm diameter disk. From Fig. 6, it is evident that assynthesized chitosan membrane and nanocomposite chitosan/ZnO membrane showed quite different activity from each other. The as-synthesized chitosan membrane could not inhibit significantly (very low antibacterial activity) the growth of both K. planticola and Bacillus substilis, where as in the case of nanocomposite chitosan/ZnO membrane, the antibacterial property was significantly enhanced due to the presence of nano ZnO. This result indicated that the nanocomposite chitosan/ZnO chitosan membrane showed better anti-bacterial properties against K. planticola and Bacillus substilis. Thus, it is very clear that the antibacterial activity was mainly occurred due to ZnO nanoparticles only. The presence of small amount of nano ZnO in the composite was enough to enhance the inactivation of bacteria as compared with as-synthesized chitosan membrane. The detailed mechanism for the antimicrobial effect of ZnO is still under debate. Many literature surveys [38–41] reported three mechanisms leading to the nano ZnO showing antimicrobial effect: (1) dissolution, (2) size and (3) ROS-Induction of reactive oxygen species. In dissolution method liberation of Zn ions from ZnO
nanoparticles has been shown to be the principal mechanism for their antimicrobial effect [38,39]. The formation active free radicals on the surface of the ZnO play a crucial role to harm microbial cells resulting in their decomposition or destruction. These mechanisms are related to each other and also combined together to involve the nano ZnO antimicrobial effect. Compared to that of Gram negative K. planticola, Gram positive Bacillus substilis was shown to be more sensitive to nano ZnO. Several factors play crucial role in determining the antimicrobial effect. The difference in antibacterial activity of the nanocomposite against these two types of bacteria could be related to structural and chemical compositional differences in the cell membrane, particularly difference in cell wall nature. The Gram negative pathogen’s outer membrane consists of thin peptidoglycon and the periplasmic region presents between outer and inner membrane acting as a barrier can easily be solubilized leading to enter the inhibitory substances. But the Gram positive pathogen’s outer membrane consists of thick peptidoglycon and lacks amount of periplasmic region and other components of lipoteichoic acids in cell wall substances and thus the entry the inhibitory substances is resisted [42,43]. The antifouling effect was performed to analyze the activity of membranes in the prevention of microorganism’s adhesion and reproduction on the membrane surface. The antifouling activity of as-synthesized as well as nanocomposite chitosan/ZnO membrane was tested with wastewater suspension after 24 h of immersion as shown in Fig. 7. The corresponding SEM results for the membrane samples as shown in Fig. 7 showed that the bacterial colonies on the surface of the as-synthesized chitosan membrane and nanocomposite chitosan/ZnO membrane were different from each other. From Fig. 7, it was observed that large amount of microorganisms was loaded on the as-synthesized chitosan membrane but nanocomposite chitosan/ZnO membrane had relatively clean
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Fig. 7. Observation of antifouling activity of (a) As-synthesized chitosan membrane (b) Nanocomposite chitosan/ZnO.
surface and free from growth of microorganisms. This result also indicated that microorganisms could not develop bio-film on the surface of the nanocomposite chitosan/ZnO membrane. Further investigations were also carried out in order to study the antifouling activities of the membranes. The microorganisms collected with the help of sterile buds by dipping and scratching on the surface of the membrane samples immersed in wastewater suspension were spread in petridish containing sterile nutrient agar as shown in Fig. 7. Then inoculated petridishes were incubated for 24 h at 37 ◦ C. The growth of microorganisms on petridishes was observed from the pictures taken as shown in Fig. 7. The results indicated that the extent of colonization of microorganisms on wastewater treated as-synthesized chitosan membrane is comparatively larger than that of wastewater treated nanocomposite chitosan/ZnO membrane, i.e., comparatively only smaller extent of microorganisms were colonized on nanocomposite chitosan/ZnO membrane. Thus, the nanocomposite chitaosan/ZnO membrane prevented the initiation (or) formation of the bio-films and showed good antifouling performance. 4. Conclusions In conclusion we have successfully synthesized nanocomposite chitosan/ZnO membrane using chitosan membrane as well as green-synthesized nano ZnO. The as-synthesized chitsoan membrane, as-synthesized nano ZnO and nanocomposite chitosan/ZnO membrane were characterized by different techniques. The XRD results confirmed the presence of two sets of diffraction peaks indicating the formation of chitosan/ZnO nanocomposite. The variation in SEM images also clearly witnessed the characteristics of synthesized samples. The difference in FT-IR spectra obtained for chitsoan before and after treating with nano ZnO also confirmed the successful formation of nanocomposite. The crystalline behavior of chitosan was greatly improved in the case of nanocomposite. Comparison of optical properties of nano ZnO and nanocomposite revealed that a shift in both UV absorption peak and fluorescence emission intensity was observed for nanocomposite and
thus confirming the favorable formation of nanocomposite. The antimicrobial activities of both as-synthesized and nanocomposite chitosan/ZnO membrane were studied against two different microorganisms, Gram negative K. planticola and Gram positive Bacillus substilis. In all cases, the Gram negative K. planticola was inhibited to larger extent comparatively than that of Gram positive Bacillus substilis. Among them, nanocomposite chitosan/Zno memembrane appeared to be very promising with regard to the aspects of strong antibacterial and antifouling activities. This is a facile room temperature synthetic approach without involving any high temperature hydrothermal reaction and calcination step to obtain a versatile nanocomposite chitosan membrane. This promising approach resulted into the development of novel kind of nanocomposite chitosan membranes and we also trust that these membranes would definitely have great promising applications in biological based nanotechnology. Acknowledgments We thank Department of Science and Technology (DST, Ref. no. /S/FST/ESI-101/2010), New Delhi, India and National Science Council (NSC, Grant No.: NSC100-2923-E-008-001-MY3), Taiwan, Republic of China (ROC) for financial support. References [1] H.F. Ridgway, H.-C. Flemming, Membrane bio fouling, in: P.E. J. Mallevialle, M.R. Odendall, M.R. Wiesner (Eds.), Water Treatment Membrane Processes, McGraw-Hill, New York, 1996 (Chapter 9). [2] L.D. Chambers, K.R. Stokes, F.C. Walsh, R.J.K. Wood, Surf. Coat. Technol. 201 (2006) 3642–3652. [3] G. von Sengbusch, S. Bowry, J. Viencken, Artif. Org. 17 (1993) 244–253. [4] N. Kubota, Y. Nakagawa, Y. Eguchi, J. Appl. Polym. Sci. 62 (1996) 1153–1160. [5] X.F. Zeng, E. Ruckenstein, J. Membr. Sci. 117 (1996) 271–278. [6] W. Paul, C.P. Sharma, STP Pharm. Sci 10 (2000) 5–22. [7] A. Domard, E. Piron, in: M.G. Peter, A. Domard, R.A.A. Muzzarelli (Eds.), Adv. Chitin Sci. 4 (2000) 295–301. [8] F. Sahidi, J.J.V. Arachchi, Y.J. Jeon, Trends Food Sci. Technol. 10 (1999) 37–51. [9] M.R. Gandhi, S. Meenakshi, Int. J. Biol. Macromol. 50 (2012) 650–657.
M. Malini et al. / International Journal of Biological Macromolecules 80 (2015) 121–129 [10] K. Lewandowska, A. Sionkowska, B. Kaczmarek, G. Furtos, Int. J. Biol. Macromol. 65 (2014) 534–541. [11] P.L. Kashyap, X. Xiang, P. Heiden, Int. J. Biol. Macromol. 77 (2015) 36–51. [12] S. Aoyagi, H. Onishi, Y. Machida, Int. J. Pharm. 330 (2007) 138–145. [13] V. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelhi, L. Sabbatini, T. Blevezacheo, M.D. Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255–5262. [14] P.M. Rahman, K. Muraleedaran, V.M.A. Mujeeb, Int. J. Biol. Macromol. 77 (2015) 266–272. [15] S. Saravanan, S. Nethala, S. Pattnaik, A. Tripathi, A. Moorthi, N. Selvamurugan, Int. J. Biol. Macromol. 49 (2011) 188–193. [16] M. Yadollahi, S. Farhoudian, H. Namazi, Int. J. Biol. Macromol. 79 (2015) 37–43. [17] A. Thill, O. Zeyons, O. Spalla, F. Chauvat, J. Rose, M. Auffan, A.M. Flank, Environ. Sci. Technol. 40 (2006) 6151–6156. [18] K.M. Reddy, K. Feris, J. Bell, D.G. Wingett, C. Hanley, A. Punnoose, Appl. Phys. Lett. 90 (2007) 2139021–2139023. [19] T. Jin, D. Sun, Y. Su, H. Zhang, H.J. Sue, J. Food Sci. 74 (2009) 46–52. [20] G. Fu, P.S. Vary, C.T. Lin, J. Phys. Chem. B 109 (2005) 8889–8898. [21] A. Emamifar, M. Kadivar, M. Shahedi, S.S. Zad, Food Sci. Emerg. Technol. 11 (2010) 742–748. [22] R. Dastjerdi, M. Montazer, Colloids Surf. B: Biointerfaces 79 (2010) 5–18. [23] M.G. Ma, Y.J. Zhu, G.F. Cheng, Y.H. Huang, Mater. Lett. 62 (2008) 507–510. [24] C. Klingshirn, Phys. Stat. Solidi 244 (2007) 3027–3073. [25] B.J.J. Wu, S.C. Liu, Adv. Mater. 14 (2002) 215–218. [26] P. Tonto, O. Mekasuwandumrong, S. Phatanasri, V. Pavarajarn, P. Praserthdam, Ceram. Int. 34 (2008) 57–62. [27] S. Baruah, J. Dutta, Sci. Technol. Adv. Mater. 10 (2009) 013001.
129
[28] A. Yadav, A.A. Virendra Prasad, Kathe, Sheela Raj, C. Deepti Yadav, N. Sundaramoorthy, Vigneshwaran, Bull. Mater. Sci. 29 (2006) 641–645. [29] L. Cen, K.G. Neoh, E.T. Kang, J. Biomed. Mater. Res. A 71 (2004) 70–80. [30] M. Zeng, Z. Fang, C. Xu, J. Appl. Polym. Sci. 91 (2004) 2840–2847. [31] M. Asaeda, M. Ishida, T. Waki, J. Chem. Eng. Jpn. 38 (2005) 336–343. [32] X.F. Zeng, E. Ruckenstein, Ind. Eng. Chem. Res. 35 (1996) 4169–4175. [33] M. Joworska, P. Sakurai Gaudon, E. Guibal, Polym. Int. 52 (2003) 198–205. [34] S. Rajeshkumar, C. Malarkodi, M. Vanaja, G. Gnanajobitha, K. Paulkumar, C. Kannan, G. Annadurai, Pharm. Chem. 5 (2013) 224–229. [35] M. Vanaja, S. Rajeshkumar, K. Paulkumar, G. Gnanajobitha, C. Malarkodi, G. Annadurai, Int. J. Mater. Biomater. Appl. 3 (2013) 1–4. [36] S. Mitra, P. Patra, S. Chandra, P. Pramanik, A. Goswami, Appl. Nanosci. 2 (2012) 231–238. [37] H.J. Zhang, H.M. Xiong, Q.G. Ren, Y.Y. Xia, J.L. Kong, J. Mater. Chem. 22 (2012) 13159–13165. [38] B. Wu, Y. Hang, Y.H. Lee, A. Horst, Z. Wang, D.R. Chen, R. Sureshkumar, Y.J. Tang, Environ. Sci. Technol. 44 (2010) 1484–1489. [39] S. George, S. Pokhrel, T. Xia, B. Gilbert, Z. Ji, M. Schowalter, A. Rosenauer, R. Damoiseaux, K.A. Bradly, L. Madler, A.E. Nel, ACS Nano 4 (2010) 15–29. [40] S. George, T. Xia, R. Rallo, Y. Zhao, Z. Ji, S. Lin, X. Wang, H. Zhang, B. France, D. Schoenfeld, R. Damoiseaux, R. Liu, S. Lin, K.A. Bradley, Y. Cohen, A.E. Nel, ACS Nano 5 (2011) 1805–1817. [41] G. Applerot, N. Perkas, G. Amirian, O. Girshevitz, A. Gedanken, Appl. Surf. Sci. 2565 (2009) 53–58. [42] N. Jones, B. Ray, R.T. Koodali, A.C. Manna, FEMS Microbiol. Lett. 279 (2008) 71–76. [43] Y. Xie, Y. He, P.L. Irwin, T. Jin, X. Shi, Appl. Environ. Microbiol. 77 (2011) 2325–2331.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A versatile chitosan/ZnO nanocomposite with enhanced antimicrobial properties Madasamy Malini a , Munusamy Thirumavalavan b,∗∗,1 , Wen-Yi Yang b , Jiunn-Fwu Lee b , Gurusamy Annadurai a,∗,1 a Environmental Nanotechnology Division, Sri Paramakalyani Centre for Environmental Sciences, Manonmanium Sundaranar University, Alwarkurichi 627412, Tamilnadu, India b Graduate Institute of Environmental Engineering, National Central University, Chung-Li, Taoyuan County 320, Taiwan
a r t i c l e
i n f o
Article history: Received 17 March 2015 Received in revised form 27 May 2015 Accepted 18 June 2015 Available online 22 June 2015 Keywords: Chitosan membrane Nanocomposite Antibacterial activity
a b s t r a c t Porous chitosan membrane was fabricated by casting method using silica particles. Simultaneously nano ZnO was synthesized by green-synthesis method using tung ting oolong tea extract. Chitosan membrane was combined with nano ZnO in order to increase its antimicrobial activity. Through observations obtained from various techniques such as XRD, SEM, FT-IR, UV–visible and fluorescence emission analyses, chitosan was seen to be able to incorporate nano ZnO in the nanocomposite membrane. A blue shift (from 360 to 335 nm) was observed in the UV–visible spectrum of nanocomposite and fluorescence emission intensity of nanocomposite was considerably lower than that of nano ZnO. Gram negative organism Klebsiella planticola (MTCC2727) and Gram positive organism Bacillus substilis (MTCC3053) were used to test the antibacterial and antifouling activities of newly synthesized nanocomposite chitosan/ZnO membrane. The nanocomposite chitosan/ZnO membrane promisingly inhibited the bacterial growth when compared with as-synthesized chitosan. Gram negative K. planticola (MTCC2727) was comparatively more susceptible for inhibition than that of Gram positive Bacillus substilis (MTCC3053). In conclusion, nanocomposite obtained in this study showed enhanced antibacterial and antifouling activities. We believed that the enhanced physical properties of nanocomposite achieved by incorporating nano ZnO in the chitosan matrix could be beneficial in various applications. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In general fouling on bio-films is formed through colonization on ship hulls, pipelines, medical devices, wastewater, drinking water treatment, etc. Fouling is the unwanted growth and undesired deposition of microorganisms into communities on the surface [1]. Major and frequently formed fouling in water treatment in membrane technologies, resulting in a decrease in permeate water flux and membrane selectivity, an increase in energy consumption, and decrease in the extent of membrane life time. Any novel strategies are therefore required to decrease the extent of microorganism growth on membranes and better remedy for the biofouling would be the use of membrane with antimicrobial activity. Various
∗ Corresponding author. Tel.: +91 4634 283883; fax: +91 4634 283270. ∗∗ Co-corresponding author. Tel.: +886 34279455; fax: +886 34279455. E-mail addresses: [email protected] (M. Thirumavalavan), [email protected] (G. Annadurai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.06.036 0141-8130/© 2015 Elsevier B.V. All rights reserved.
numbers of polymers have been carrying out for the new product of membrane forming systems. Polymeric materials emerge with many fields rapidly producing new applications, especially in biotechnology, bioengineering and nanotechnology. Now-a-days, polymer is the most popular material for membrane manufacture. Commercially many polymers [2] such as polyacrylonitrile [3], glycidyl methacrylate and cellulose derivatives [4] are involved to make a synthetic membrane, Previous studies have also reported chitosan as an excellent matrix of membrane compared to other polymers due to its low cost, non-toxicity and easy biodegradability [5,6]. The reason for choosing chitosan was due to its linear structure composed of polymeric 2-amino-2-deoxy--d-glucose. It has been widely used to prepare polymer membranes because of it is good film forming and thermal properties, inexpensive, hydrophilic, biocompatible and good matrix for affinity ligand coupling that can be used in many areas such as food science, biochemistry, pharmaceuticals, medicine, agriculture and others [7–11]. But however, the so called chitosan membrane and films have poor mechanical strength. Recently, chitosan membranes with good mechanical properties have been successfully prepared
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using a novel method. Silica particles have some important benefits such as consistency and reproducibility of tight particle distribution of required pore size. Chitosan membranes are hydrophilic and highly reactive chemically (since they contain a large number of OH and NH2 groups). Due to these reasons, chitosan has been considered to be one of the important biomaterials [9–11]. But at neutral pH, chitosan does not show any antimicrobial activity and therefore to impart the antimicrobial activity of chitosan it is necessary to incorporate some antimicrobial agents [12]. Inorganic nanomaterials have attracted such particular interest for their antimicrobial activities. At the same time, inorganic nanomaterials alone will considerably reduce their applicability due to aggregation. Hence, one of the successful approaches used to prevent such aggregation and improve the dispersion of inorganic nanomaterials has been the fabrication of nanocomposite materials with enhanced properties by mixing both chitosan polymer membrane and nanomaterials. Nanotechnologies provide the tools required for preparation of such membranes, films or layers with embedded metal nanoparticles [13–16]. ZnO nanoparticles are biocompatible, nontoxic but noxious to microorganisms and these prospectives recommending their uses in food and agriculture fields [17,18]. The ZnO nanoparticles strongly inhibited the growth of microorganisms because it is disintegrating the cell membrane and thereby increasing the membrane permeability [19]. Many studies reported that metal oxide nanoparticles are stable under harsh processing, selective toxicity to microorganisms and exhibit minimal effect on mammalian cells [20]. Recently FDA (Food and Drug Administration, USA) listed ZnO is a safe material [21]. Compare to other metal oxides, ZnO nanoparticles have more advantages as white appearance, low production cost and UV-blocking [22]. Zinc oxide is a natural n-type semiconductor with wide band gap (3.37 eV) [23]. This has some noble properties such as high thermal conductivity and refractive index, photo oxidizing against biological species and chemicals, self-sterilization and antimicrobial action. So, it could be used in various fields such as cosmetics, concrete foods, pharmaceuticals, etc. [24]. Thus, ZnO has been considered to possess good antimicrobial properties. These ZnO nanoparticles can be synthesized by various methods such as chemical vapour deposition method [25], solvent thermal reaction method [26], hydro thermal method [27], wet chemical method [28], etc. These methods have unfavorable circumstance because they are involved various stages and also highly complicated. In contrast, synthetic procedure based on naturally occurring biomaterials so called “green-chemical” method provide an alternative, simple, environmental-friendly, inexpensive and non-toxic approach to obtain nanomaterials. The major content of these biomolecules are polyphenols which can act as both reducing and capping agents. Thus herein eco-friendly, green tea leaves mediated greensynthesis of nano ZnO using the extract of tung ting oolong green tea was reported. This tung ting oolong is one of the famous Taiwan green tea leaves. Thus, in this work, fabrication of chitosan/nano ZnO composite membrane using silica gel supported macroporous chitosan membrane and green tea mediated synthesized nano ZnO has been proposed. The significance of the present work is that the nanocomposite was prepared at room temperature by a simple dipping method without involving any high temperature thermal process. The dispersion of nano ZnO was believed to be an important factor affecting the functional properties of resultant chitosan/ZnO nanocomposite membranes. The obtained macroporous membrane, nano ZnO and bio-nanocomposite chitosan/ZnO membrane were characterized and confirmed by various techniques. Both antibacterial and antifouling activities against common pathogens Gram negative organism Klebsiella planticola (MTCC2727) and Gram positive organisms Bacillus substilis
(MTCC3053) were reported and discussed. A rapid, simple and environmentally benign technology was developed to evaluate the antimicrobial activities of resultant chitosan/ZnO nanocomposite membrane and the methodology reported in this work is very significant both technically and scientifically. Thus, the highlight of the present work is to explore the feasibility of combining the significant out comings of different feasible synthetic approaches in a single work. Hence, the purpose of the present study was mainly to prepare the nanocomposite chitosan/ZnO membrane with enhanced antimicrobial properties using a facile approach without any complication step. 2. Experimental 2.1. Chemicals Chitosan (<90% deacetylation degree, MW 100,000–300,000) was purchased from Marine chemicals, India. Silica particles (sizes in the range of 15–40 m) were purchased from Silicycle, Canada. Acetic acid was purchased from Thomas Baker, India. Zinc oxide (ZnO) was obtained from Sigma–Aldrich chemicals, USA and glycerol was purchased from RFCL limited, India. Gram negative organism K. planticola (MTCC2727) and Gram positive organism Bacillus substilis (MTCC3053) were purchased from MTCC, India. Fresh tung ting oolong tea leaves were collected from Lugu region of the central Taiwan. Deionized distilled water was used to prepare all solutions. 2.2. Preparation of macroporous chitosan membrane The chitosan membrane was prepared as follows: a solution of chitosan was first obtained by dissolving 1 g of chitosan in 100 mL of 1 vol% aqueous acetic acid solution. To this solution, silica particles were added, followed by vigorous stirring in order to disperse them uniformly. Then, the solution was poured onto a rimmed glass plate and the liquid was allowed to evaporate. The dried membrane was immersed in a 5 wt% aqueous NaOH solution and kept for 2 h at 80 ◦ C in order to dissolve the silica particles and to generate a porous membrane. Finally, the porous membrane was washed with distilled water to remove the remaining NaOH. In order to prevent its shrinkage during drying, the membrane was immersed in a 20 vol% aqueous glycerol solution (softening agent) for 30 min after the excess glycerol solution was removed, placed on a glass plate and allowed to dry. Thus, a strong and flexible chitosan membrane without shrinkage was obtained. 2.3. Preparation of tung ting oolong tea extract Fresh tung ting oolong tea leaves were collected from Lugu region of the central Taiwan. It was washed with distilled water and dried in shadow place for 1 week at room temperature. A 10 g of dried leaves was washed with double distilled water. Then the cleaned dry tea leaves were boiled in 100 mL of water for 10 min and filtered through 8 layers of muslin cloth. Filtered tea extract was stored at 4 ◦ C for further process. 2.4. Preparation of nano ZnO A 10 mL of tung ting oolong tea extract was boiled at 60 ◦ C. Then 90 mL of 1 M zinc nitrate aqueous solution was added to this and stirred at 60 ◦ C for 24 h to ensure thorough mixing. The reaction mixture was allowed to settle at room temperature. The color change of the tea extract from deep yellow to pale white vividly indicated the formation of nano ZnO. The nano ZnO particles were then separated by evaporating water from the solution,
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Fig. 1. Schematic representation of synthesis of (a) macroporous chitosan membrane; (b) Nano ZnO; (c) nanocomposite chitosan/ZnO.
dried and then collected. They were finally calcined at 100 ◦ C for 1 h and stored. The resultant nano ZnO was analyzed by UVspectrophotometer. 2.5. Preparation of nanocomposite chitosan/ZnO membrane Preparation of nanocomposite chitosan/ZnO membrane was performed as follows. Various circular formed discs were cut from the prepared chitosan membrane and were introduced in the suspension of nano ZnO in distilled water under magnetic stirring at 28 ◦ C for 24 h. The nanocomposite chitosan/ZnO discs were separated, rinsed with distilled water and then air dried. 2.6. Determination of water content and antimicrobial activities of as-synthesized and composite chitosan membranes The chitosan membranes were (as-synthesized and composite) soaked in distilled water for 24 h. The soaked membranes were mopped with blotting paper and weighed accurately. The dry weight of the membranes was measured after the wet membrane samples were placed in a dryer at 70 ◦ C for 48 h and cooled at room temperature in desiccators. The water content percentage was determined using the following formula: % water content =
wet sample weight−dry sample weight ×100 wet sample weight
Antibacterial activities of as-synthesized and nanocomposite chitosan/ZnO membranes were examined by disk diffusion analysis (membrane act as like a disk) against Gram negative organism
K. planticola (MTCC2727) and Gram positive organisms Bacillus substilis (MTCC3053). These microorganisms were cultured in Muller Hinton medium (MHM) and 12 h old culture of K. planticola and Bacillus substilis were swabbed on each plate. A small piece of as-synthesized chitosan membrane and nanocomposite chitosan/ZnO membrane was placed on the center of each inoculated plate. After 24 h of incubation at 37 ◦ C, the zone of inhibition against bacteria was observed. Antifouling activity was tested for both as-synthesized and nanocomposite chitosan/ZnO membrane by immersing them in the wastewater (collected nearby Sri Paramakalyani Centre for Environmental Sciences, Alwarkurichi, Tamil Nadu, India) at 37 ◦ C for 24 h. The membrane samples were removed from the wastewater by filtration. After that, the membranes were washed with distilled water and then immersed into 1% of glutaraldehyde at 4 ◦ C for 5 h to fix the microorganisms if any layer formed on the surface of the membranes. After 5 h, the membranes were taken and washed with ethanol [29]. The membrane samples were dried in a vacuum oven at 50 ◦ C overnight and then analyzed for antifouling activity. Thus, this study mainly focused on the synthesis of macroporous chitosan membrane, nano ZnO and nanocomposite chitosan/ZnO membrane using simple approaches as illustrated in Fig. 1. Various techniques such as XRD, SEM, FT-IR, UV–visible, fluorescence emission were employed to characterize the samples. SEM images of different samples are shown in Fig. 2 and XRD patterns are given in Fig. 3. As a core study, the antimicrobial activities were studied and compared in order to emphasize the superior behavior of nanocomposite chitosan/ZnO membrane over as-synthesized chtiosan as well as nano ZnO.
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Fig. 2. SEM images of (a) macroporous chitosan membrane; (b) nano ZnO; (c) nanocomposite chitosan/ZnO.
2.7. Characterization techniques X-ray diffraction (XRD) patterns were obtained using a Philips PW 1830 instrument. Scanning electron microscope (SEM) was carried out on Philip model CM 200, field emission scanning electron microscope. Attenuated total reflectance (ATR)-Fourier transform infrared (FT-IR) spectra were obtained on a Bruker vertex 80v instrument. UV–visible spectra were obtained using Perkin Elmer double beam UV spectrophotometer. Inverted fluorescence microscope (Nikon fluorescence microscope) was used to analysis the fluorescence emission.
3. Results and discussion 3.1. Macroporous chitosan membrane The chitosan membrane was prepared by casting method [30–32]. Fig. 1a clearly depicted the schematic representation of the formation of macroporous chitosan membrane. Chitosan was dissolved in an aqueous acetic acid solution containing silica particles (15–40 m) with vigorous stirring. The sol–gel transformation generated the pores because chitosan is soluble in acidic solutions and insoluble in basic solutions, whereas silica is insoluble in acidic solutions and soluble in basic media. These opposite properties were employed to form pores. For extracting the silica particles and to easily generate a porous membrane [31], the membrane was immersed in a 5% aqueous NaOH solution at 80 ◦ C for 2 h [32]. Chitosan was precipitated in NaOH solution, and the heat treatment was helpful for stabilizing the pore size and improving the mechanical as well as crystalline properties [33] of the chitosan. In this study, inorganic porogen (silica particles) was employed to generate the pore structure and the pore structure could be maintained only after vacuum drying. XRD peak around 2 = 20◦ corresponding to chitosan peak as shown in Fig. 3a, confirmed that the synthesized chitosan membrane showed only low crystalline character. This decrease in the crystallinity clearly demonstrated
that the conjugation of silica and chitosan during synthesis process suppressed the crystallization to some extent. It also suggested that silica and chitosan polymeric chain were mixed well at a molecular level. FT-IR spectra of macroporous chitosan membrane showed a peak around 3480 cm−1 due to (O H) and (NH2 ), a peak around (2800–2900 cm−1 ) due to alkyl (C H) and (N H), a peak around 1600 cm−1 due to (N H), a peak around 1725 cm−1 due to (C O), a peak around (1300–1500) cm−1 due to deformation of hydroxyl (OH) group, a peak around (1050–1100) cm−1 due to (C O) of chitosan and a peak around 800 cm−1 is due to aromatic (C H). The SEM image of macroporous chitosan membrane as shown in Fig. 2a indicated the opened pore surface on chitosan membrane. The porosity and the crystalline nature of the chitosan membrane would be expected to be altered upon the variation of the amount of silica added. The characteristics of the chitosan membrane are listed in Table 1. 3.2. Nano ZnO The synthetic process of nano ZnO is diagrammatically represented in Fig. 1b. The formation of nano ZnO was rapidly started with in a minute which was clearly identified by the color change of the reaction mixture from bright yellow to pale white. The pale white color after 24 h with no further color change [34] indicated the complete formation of nano ZnO. The color changes occurred in the reaction mixture during the formation of nano ZnO conveyed Table 1 Characteristics of as-synthesized and nanocomposite chitosan membranes. Characteristics
As-synthesized chitosan
Nanocomposite chitosan
Membrane material Configuration Thickness (mm) Membrane area (mm) Water content (%)
Chitosan + silica Flat membrane 0.30 60.04 84 .4
Chitosan + silica + nano ZnO Flat membrane 0.35 60.04 82.2
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that the fluorescence emission color of the nano ZnO nanoparticles could be tuned by varying the reaction time and reactant ratio [37]. Based on this, herein it is proved that tung ting oolong extract acts as a good stabilizing agent, which is responsible for the synthesis of nano ZnO in this study. In general, natural plant extract mostly resulted in the formation of reduced metal nanoparticles from the respective metal ions (metal salts), but interestingly in this case, ZnO nanoparticles were obtained from zinc salt. Thus, it is very clear that the tea extract used in this study acted as a stabilizing agent only and not as a reducing agent for Zn2+ ion. 3.3. Nanocomposite chitosa/Zno membrane
Fig. 3. XRD of (a) As-synthesized chitosan membrane and nano ZnO; (b) nanocomposite chitosan/ZnO.
the strong and quick light absorption behavior of nano ZnO in the visible region. This quick absorption is mainly due to the coherent oscillations of the free conduction electrons because of interacting with an electromagnetic field on the nanoparticle surface, which is called surface plasmon resonance [35]. XRD pattern of nano ZnO synthesized using green-method corresponded to close packed hexagonal Wurtzite structure as shown in Fig. 3a. The peaks around 2 = 31◦ , 33◦ , 36◦ , 48◦ , 56◦ , 62◦ , 67◦ , 68◦ , 69◦ and 77◦ are assigned to (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1) and (2 0 2) crystal planes respectively [36]. The surface morphology of biosynthesized nano ZnO as shown in Fig. 2b, indicated that these nanoparticles formed as agglomerated spherical nanosponges like shape. Also these shapes are not uniformly distributed, i.e. the particle distribution is undefined. The optical properties of synthesized samples were measured as shown in Fig. 4. The UV–visible absorption spectrum of nano ZnO, revealed a characteristic absorption peak located at 360 nm, which corresponds to the band gap of the zinc oxide nanoparticles. As the colloidal semiconductor ZnO nanocrystals are also known as novel inorganic fluorescence labeling agents, the fluorescence intensity of biosynthesized nano ZnO was measured and the fluorescence images are shown in Fig. 5. Fig. 5a clearly exhibited the bright fluorescence intensity of nano ZnO and this result supported to considering ZnO to be potentially used for bio-analytical and labeling application. It was proposed
The XRD pattern of nanocomposite chitosan/ZnO is illustrated in Fig. 3b. From Fig. 3b, two sets of diffraction peaks were observed corresponding to the chitosan and nano ZnO in the 2 range of 10–80◦ . The peaks in the 2 range 30–80◦ are corresponding to nano ZnO and the broad peak around 2 = 14◦ is corresponding to chitosan. This observation further indicated that the peak corresponding to chtiosan was shifted to lower 2 value (from 20◦ to 14◦ ) in the nanocomposite which could be due to possible interaction between nano ZnO and chitosan, but however the crystallinity of chitosan was greatly enhanced in the case of nanocomposite when compared with as-synthesized chitosan membrane. From Fig. 3b it was also observed that both nano ZnO and chitosan showed highest peak intensity and possessed good crystalline characters. The above results suggested the successful formation of nano ZnO dispersion on the chitosan matrix. There was no formation of a new peak in the nanocomposite compared with as-synthesized chitosan and nano ZnO, indicating that nanocomposite chitosan/ZnO membrane consisted of two phase structures, i.e. chitosan membrane and nano ZnO. These observations clearly witnessed the addition of nano ZnO caused an increase in the overall crystalline characters of chitosan membrane, which can be effective on the final properties of nanocomposite chitosan/ZnO membrane. From Table 1, it can be seen that the characteristics of chitosan membrane in the nanocomposite was slightly altered (or almost same). As the nano ZnO is doped, the thickness of the membrane somewhat increased, but the water content decreased slightly. However, the membrane area remained unaltered. This clearly suggested that incorporating nano ZnO into the chitosan matrix results in strong intermolecular interaction between the nano ZnO and chiotsan matrix [14] and consequently restricted the motion of the chitosan and thus promoted rigidity. Furthermore, increased crystallinity as observed by X-ray diffraction analysis could also be expected to enhance mechanical properties. The SEM image of nanocomposite chitosan/ZnO as shown in Fig. 2c, showed that the addition of nano ZnO caused surface morphology of membrane to be somewhat rough compared with as-synthesized chitosan membrane. The irregular distribution of uneven pore structures with some extent of pore blocking was observed on the surface of nanocomposite chitosan/ZnO membrane, suggesting that there exists good adhesion between nano ZnO and chitosan matrix. Such distribution of nano ZnO in the chitosan matrix would be expected to play a vital role in enhancing the mechanical properties of obtained nanocomposite chitosan/ZnO membrane. Also it could be concluded that stabilization of nano ZnO by chitosan membrane could help to increase the dispersion of nano ZnO in the chitosan matrix and prevents nano ZnO from more agglomeration. The FT-IR spectra of nanocomposite chitosan/ZnO revealed that the corresponding peak intensity of as-synthesized chitosan especially (O H), (N H) and (C O) was considerably diminished in the nanocomposite and the carboxyl characteristic band was almost disappeared. This observation obtained from the FT-IR spectra of chitosan before and after treating with nano ZnO confirmed the successful formation of nanocomposite chitosan/ZnO
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3.0
(a)
Nano ZnO as-synthesized chitosan membrane nanocomposite chitosan/ZnO membrane
Absorbance (a.u)
2.5
2.0
1.5
1.0
0.5
0.0 300
400
500
600
700
λ (nm) (b) 2.7
0.65
2.5
0.6
2.3
0.55
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0.3 300
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350 λ (nm)
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Fig. 4. UV–visible spectra of (a) Obtained samples; (b) 300–400 nm expanded region.
membrane. On comparing the optical properties of nanocomposite chitosan/ZnO with nano ZnO, the UV–visible absorption band of nano ZnO at 360 nm was blue shifted to 335 nm in the nanocomposite. Also the absorption peak of nanocomposite appeared as broad shoulder in shape with low intensity. However, the UV–visible absorption spectra of as-synthesized chitosan membrane did not show any obvious peak. In order to compare the fluorescence property of nanocomposite, the fluorescence image of nanocomposite
was obtained as shown in Fig. 5b. The brightness and intensity of the fluorescence emission of nanocomposite chitosan/ZnO was comparatively lower than that of nano ZnO. These shifts both in UV absorption and fluorescence emission are mainly due to the interaction between chitosan matrix and nano ZnO. Thus, it is believed that the nanocomposite chitsoan/ZnO may also protect against ultraviolet light and could be potentially used as UV shielding material.
Fig. 5. Fluorescence emission image of (a) Nano ZnO; (b) nanocomposite chitosan/ZnO.
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Fig. 6. Antibacterial activity of (a) As-synthesized chitosan membrane against Gram negative Klebsiella planticola; (b) As-synthesized chitosan membrane against Gram positive Bacillus substilis; (c) Nanocomposite chitosan/ZnO membrane against Gram negative Klebsiella planticola; (d) Nanocomposite chitosan/ZnO membrane against Gram positive Bacillus substilis.
3.4. Antibacterial and antifouling activity The disk diffusion method was carried out to examine the antibacterial activity of both as-synthesized and nanocomposite chitosan membranes as shown in Fig. 6, under in vitro conditions against common pathogens like a Gram negative organism K. planticola (MTCC2727) and Gram positive organism Bacillus substilis (MTCC3053). The formation zone was clearly observed around the disk containing nanocomposite indicating the antibacterial nature of nanocomposite. The results are shown in Fig. 6. The Bacillus substilis showed minimum range of inhibition 15.4 mm and K. planticola showed the maximum zone of inhibition 19.6 mm at 20 mm diameter disk. From Fig. 6, it is evident that assynthesized chitosan membrane and nanocomposite chitosan/ZnO membrane showed quite different activity from each other. The as-synthesized chitosan membrane could not inhibit significantly (very low antibacterial activity) the growth of both K. planticola and Bacillus substilis, where as in the case of nanocomposite chitosan/ZnO membrane, the antibacterial property was significantly enhanced due to the presence of nano ZnO. This result indicated that the nanocomposite chitosan/ZnO chitosan membrane showed better anti-bacterial properties against K. planticola and Bacillus substilis. Thus, it is very clear that the antibacterial activity was mainly occurred due to ZnO nanoparticles only. The presence of small amount of nano ZnO in the composite was enough to enhance the inactivation of bacteria as compared with as-synthesized chitosan membrane. The detailed mechanism for the antimicrobial effect of ZnO is still under debate. Many literature surveys [38–41] reported three mechanisms leading to the nano ZnO showing antimicrobial effect: (1) dissolution, (2) size and (3) ROS-Induction of reactive oxygen species. In dissolution method liberation of Zn ions from ZnO
nanoparticles has been shown to be the principal mechanism for their antimicrobial effect [38,39]. The formation active free radicals on the surface of the ZnO play a crucial role to harm microbial cells resulting in their decomposition or destruction. These mechanisms are related to each other and also combined together to involve the nano ZnO antimicrobial effect. Compared to that of Gram negative K. planticola, Gram positive Bacillus substilis was shown to be more sensitive to nano ZnO. Several factors play crucial role in determining the antimicrobial effect. The difference in antibacterial activity of the nanocomposite against these two types of bacteria could be related to structural and chemical compositional differences in the cell membrane, particularly difference in cell wall nature. The Gram negative pathogen’s outer membrane consists of thin peptidoglycon and the periplasmic region presents between outer and inner membrane acting as a barrier can easily be solubilized leading to enter the inhibitory substances. But the Gram positive pathogen’s outer membrane consists of thick peptidoglycon and lacks amount of periplasmic region and other components of lipoteichoic acids in cell wall substances and thus the entry the inhibitory substances is resisted [42,43]. The antifouling effect was performed to analyze the activity of membranes in the prevention of microorganism’s adhesion and reproduction on the membrane surface. The antifouling activity of as-synthesized as well as nanocomposite chitosan/ZnO membrane was tested with wastewater suspension after 24 h of immersion as shown in Fig. 7. The corresponding SEM results for the membrane samples as shown in Fig. 7 showed that the bacterial colonies on the surface of the as-synthesized chitosan membrane and nanocomposite chitosan/ZnO membrane were different from each other. From Fig. 7, it was observed that large amount of microorganisms was loaded on the as-synthesized chitosan membrane but nanocomposite chitosan/ZnO membrane had relatively clean
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Fig. 7. Observation of antifouling activity of (a) As-synthesized chitosan membrane (b) Nanocomposite chitosan/ZnO.
surface and free from growth of microorganisms. This result also indicated that microorganisms could not develop bio-film on the surface of the nanocomposite chitosan/ZnO membrane. Further investigations were also carried out in order to study the antifouling activities of the membranes. The microorganisms collected with the help of sterile buds by dipping and scratching on the surface of the membrane samples immersed in wastewater suspension were spread in petridish containing sterile nutrient agar as shown in Fig. 7. Then inoculated petridishes were incubated for 24 h at 37 ◦ C. The growth of microorganisms on petridishes was observed from the pictures taken as shown in Fig. 7. The results indicated that the extent of colonization of microorganisms on wastewater treated as-synthesized chitosan membrane is comparatively larger than that of wastewater treated nanocomposite chitosan/ZnO membrane, i.e., comparatively only smaller extent of microorganisms were colonized on nanocomposite chitosan/ZnO membrane. Thus, the nanocomposite chitaosan/ZnO membrane prevented the initiation (or) formation of the bio-films and showed good antifouling performance. 4. Conclusions In conclusion we have successfully synthesized nanocomposite chitosan/ZnO membrane using chitosan membrane as well as green-synthesized nano ZnO. The as-synthesized chitsoan membrane, as-synthesized nano ZnO and nanocomposite chitosan/ZnO membrane were characterized by different techniques. The XRD results confirmed the presence of two sets of diffraction peaks indicating the formation of chitosan/ZnO nanocomposite. The variation in SEM images also clearly witnessed the characteristics of synthesized samples. The difference in FT-IR spectra obtained for chitsoan before and after treating with nano ZnO also confirmed the successful formation of nanocomposite. The crystalline behavior of chitosan was greatly improved in the case of nanocomposite. Comparison of optical properties of nano ZnO and nanocomposite revealed that a shift in both UV absorption peak and fluorescence emission intensity was observed for nanocomposite and
thus confirming the favorable formation of nanocomposite. The antimicrobial activities of both as-synthesized and nanocomposite chitosan/ZnO membrane were studied against two different microorganisms, Gram negative K. planticola and Gram positive Bacillus substilis. In all cases, the Gram negative K. planticola was inhibited to larger extent comparatively than that of Gram positive Bacillus substilis. Among them, nanocomposite chitosan/Zno memembrane appeared to be very promising with regard to the aspects of strong antibacterial and antifouling activities. This is a facile room temperature synthetic approach without involving any high temperature hydrothermal reaction and calcination step to obtain a versatile nanocomposite chitosan membrane. This promising approach resulted into the development of novel kind of nanocomposite chitosan membranes and we also trust that these membranes would definitely have great promising applications in biological based nanotechnology. Acknowledgments We thank Department of Science and Technology (DST, Ref. no. /S/FST/ESI-101/2010), New Delhi, India and National Science Council (NSC, Grant No.: NSC100-2923-E-008-001-MY3), Taiwan, Republic of China (ROC) for financial support. References [1] H.F. Ridgway, H.-C. Flemming, Membrane bio fouling, in: P.E. J. Mallevialle, M.R. Odendall, M.R. Wiesner (Eds.), Water Treatment Membrane Processes, McGraw-Hill, New York, 1996 (Chapter 9). [2] L.D. Chambers, K.R. Stokes, F.C. Walsh, R.J.K. Wood, Surf. Coat. Technol. 201 (2006) 3642–3652. [3] G. von Sengbusch, S. Bowry, J. Viencken, Artif. Org. 17 (1993) 244–253. [4] N. Kubota, Y. Nakagawa, Y. Eguchi, J. Appl. Polym. Sci. 62 (1996) 1153–1160. [5] X.F. Zeng, E. Ruckenstein, J. Membr. Sci. 117 (1996) 271–278. [6] W. Paul, C.P. Sharma, STP Pharm. Sci 10 (2000) 5–22. [7] A. Domard, E. Piron, in: M.G. Peter, A. Domard, R.A.A. Muzzarelli (Eds.), Adv. Chitin Sci. 4 (2000) 295–301. [8] F. Sahidi, J.J.V. Arachchi, Y.J. Jeon, Trends Food Sci. Technol. 10 (1999) 37–51. [9] M.R. Gandhi, S. Meenakshi, Int. J. Biol. Macromol. 50 (2012) 650–657.
M. Malini et al. / International Journal of Biological Macromolecules 80 (2015) 121–129 [10] K. Lewandowska, A. Sionkowska, B. Kaczmarek, G. Furtos, Int. J. Biol. Macromol. 65 (2014) 534–541. [11] P.L. Kashyap, X. Xiang, P. Heiden, Int. J. Biol. Macromol. 77 (2015) 36–51. [12] S. Aoyagi, H. Onishi, Y. Machida, Int. J. Pharm. 330 (2007) 138–145. [13] V. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelhi, L. Sabbatini, T. Blevezacheo, M.D. Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255–5262. [14] P.M. Rahman, K. Muraleedaran, V.M.A. Mujeeb, Int. J. Biol. Macromol. 77 (2015) 266–272. [15] S. Saravanan, S. Nethala, S. Pattnaik, A. Tripathi, A. Moorthi, N. Selvamurugan, Int. J. Biol. Macromol. 49 (2011) 188–193. [16] M. Yadollahi, S. Farhoudian, H. Namazi, Int. J. Biol. Macromol. 79 (2015) 37–43. [17] A. Thill, O. Zeyons, O. Spalla, F. Chauvat, J. Rose, M. Auffan, A.M. Flank, Environ. Sci. Technol. 40 (2006) 6151–6156. [18] K.M. Reddy, K. Feris, J. Bell, D.G. Wingett, C. Hanley, A. Punnoose, Appl. Phys. Lett. 90 (2007) 2139021–2139023. [19] T. Jin, D. Sun, Y. Su, H. Zhang, H.J. Sue, J. Food Sci. 74 (2009) 46–52. [20] G. Fu, P.S. Vary, C.T. Lin, J. Phys. Chem. B 109 (2005) 8889–8898. [21] A. Emamifar, M. Kadivar, M. Shahedi, S.S. Zad, Food Sci. Emerg. Technol. 11 (2010) 742–748. [22] R. Dastjerdi, M. Montazer, Colloids Surf. B: Biointerfaces 79 (2010) 5–18. [23] M.G. Ma, Y.J. Zhu, G.F. Cheng, Y.H. Huang, Mater. Lett. 62 (2008) 507–510. [24] C. Klingshirn, Phys. Stat. Solidi 244 (2007) 3027–3073. [25] B.J.J. Wu, S.C. Liu, Adv. Mater. 14 (2002) 215–218. [26] P. Tonto, O. Mekasuwandumrong, S. Phatanasri, V. Pavarajarn, P. Praserthdam, Ceram. Int. 34 (2008) 57–62. [27] S. Baruah, J. Dutta, Sci. Technol. Adv. Mater. 10 (2009) 013001.
129
[28] A. Yadav, A.A. Virendra Prasad, Kathe, Sheela Raj, C. Deepti Yadav, N. Sundaramoorthy, Vigneshwaran, Bull. Mater. Sci. 29 (2006) 641–645. [29] L. Cen, K.G. Neoh, E.T. Kang, J. Biomed. Mater. Res. A 71 (2004) 70–80. [30] M. Zeng, Z. Fang, C. Xu, J. Appl. Polym. Sci. 91 (2004) 2840–2847. [31] M. Asaeda, M. Ishida, T. Waki, J. Chem. Eng. Jpn. 38 (2005) 336–343. [32] X.F. Zeng, E. Ruckenstein, Ind. Eng. Chem. Res. 35 (1996) 4169–4175. [33] M. Joworska, P. Sakurai Gaudon, E. Guibal, Polym. Int. 52 (2003) 198–205. [34] S. Rajeshkumar, C. Malarkodi, M. Vanaja, G. Gnanajobitha, K. Paulkumar, C. Kannan, G. Annadurai, Pharm. Chem. 5 (2013) 224–229. [35] M. Vanaja, S. Rajeshkumar, K. Paulkumar, G. Gnanajobitha, C. Malarkodi, G. Annadurai, Int. J. Mater. Biomater. Appl. 3 (2013) 1–4. [36] S. Mitra, P. Patra, S. Chandra, P. Pramanik, A. Goswami, Appl. Nanosci. 2 (2012) 231–238. [37] H.J. Zhang, H.M. Xiong, Q.G. Ren, Y.Y. Xia, J.L. Kong, J. Mater. Chem. 22 (2012) 13159–13165. [38] B. Wu, Y. Hang, Y.H. Lee, A. Horst, Z. Wang, D.R. Chen, R. Sureshkumar, Y.J. Tang, Environ. Sci. Technol. 44 (2010) 1484–1489. [39] S. George, S. Pokhrel, T. Xia, B. Gilbert, Z. Ji, M. Schowalter, A. Rosenauer, R. Damoiseaux, K.A. Bradly, L. Madler, A.E. Nel, ACS Nano 4 (2010) 15–29. [40] S. George, T. Xia, R. Rallo, Y. Zhao, Z. Ji, S. Lin, X. Wang, H. Zhang, B. France, D. Schoenfeld, R. Damoiseaux, R. Liu, S. Lin, K.A. Bradley, Y. Cohen, A.E. Nel, ACS Nano 5 (2011) 1805–1817. [41] G. Applerot, N. Perkas, G. Amirian, O. Girshevitz, A. Gedanken, Appl. Surf. Sci. 2565 (2009) 53–58. [42] N. Jones, B. Ray, R.T. Koodali, A.C. Manna, FEMS Microbiol. Lett. 279 (2008) 71–76. [43] Y. Xie, Y. He, P.L. Irwin, T. Jin, X. Shi, Appl. Environ. Microbiol. 77 (2011) 2325–2331.