STRUCTURAL AND ANTIBACTERIAL STUDIES OF RICE STRAW BASED ZnONANOCOMPOSITE
AnjaliNambiar P K1,2 , DedhilaDevadathan1, Baiju V1, Raveendran R1
1 Nanoscience Research Laboratory, Department of Physics, SreeNarayana College, Kollam 691001, Kerala, India
2 Department of Physics,AmritaSchool of Arts and Sciences, Amritapuri, Amrita VishwaVidyapeetham University, Kollam 690525, Kerala, India
Email ID: [email protected]
Phone Number: 9744173070
Abstract: The present study is focused on the structural and antibacterial studies of rice straw based ZnO nanocomposite. For comparison, counter parts were also synthesized. Nanoparticles of ZnO were synthesized through combustion method and co-precipitation method. Rice straw and its nanocomposites were prepared through acid-alkali treatment method. Structural characterizations of all the synthesized samples were carried out using XRD. Antibacterial studies of the synthesized samples were carried out. The present study investigates the antimicrobial activity of the synthesized samples as antibacterial agents. The gram positive bacteria: Bacilus cereus (B. cereus) and the gram negative bacteria: Escherichia coli (E. coli) were the selected microbes.
Keywords: Nanocomposite, Combustion, Co-precipitation, Antibacterial agent, Biopolymer
Natural fiber has many advantages like biodegradability, flammability and non-toxicity 1, 2. The source of natural fiber are stalks, leaves, and seeds, for instance kenaf, sisal, flax, wheat straw and rice straw 3, 4. In most countries, straw is an abundant cellulosic by-product from the production of crops such as wheat, corn, soybean and rice. The utilization of cheap, renewable, accessible, and biodegradable farming residues like rice straw, rice husks and corn stover can be used to rationalize the cost effect. Rice straw is known to be a potential source of energy and also a value-added by-product 5. Due to the availability of natural/bio-fibers from the renewable resources, the use of bio-composites has been found to be expanding in the recent years. Nanocomposite materials formed by metal nanoparticles that have been appropriately incorporated into the polymer matrix were experimentally found to be very significant due to their diversity in electrical, catalytic and optical properties. These diversities have potential applications in the fields of electronic, photonic, catalysis and bioengineering 6. Synthesis of nanocomposites is an expanding trend of nanoscience. In order to exploit the full potential of the technological applications of the nanomaterials, the scientists were guided towards the conventional polymers as one component of the nanocomposite. In the present work we are focussed on the comparison of structural and antibacterial properties of nanocomposite of ZnO formed with rice straw
AR grade chemicals obtained from Merck were used for the preparation of rice straw based ZnO nanocomposite. ZnO nanoparticles were prepared by the co-precipitation method in presence of capping agent and also by combustion method.
Metal oxide ZnO was prepared as follows. Zinc nitrate dihydrate and sodium hydroxide were used as starting materials. Citric acid was used as stabilizer. Aqueous solutions of 25ml 0.1 M zinc nitrate, and 50ml 1 M of sodium hydroxide were slowly mixed drop wise into a beaker containing aqueous solution of 25 ml 0.02 M citric acid and stirred well using a magnetic stirrer for two hours. The stabilizer was used to prevent growth and agglomeration of the particles. The experimental parameters like concentration of the reactants, rate of mixing, pH, viscosity of the solutions etc governs the particle size. The stabilizers used for controlling the precipitation reaction should be easily and completely removable from the sample so as to avoid any possible contamination of the samples. After two hours of continuous stirring, the precipitate formed was in hydroxide form. To free from ions and other impurities the hydroxide precipitate formed was washed several times in distilled water. The wet precipitate obtained was dried at room temperature and thoroughly grounded using an agate motor to get the hydroxide precursor in the form of fine powder. The obtained powder was annealed at 6000C for 3 hours in a muffle furnace to get the respective metal oxide nanoparticles.
Nanoparticles of zinc oxide were prepared by combustion method using analytical grade chemicals. Zinc nitrate dehydrate and urea were used as starting materials. Urea was used as fuel. 5 g zinc nitrate was taken to which urea and glycol was added mixed to get a clear solution. pH of the solution was made neutral by controlling the amount of urea and glycol. The solution was then heated. The chemical reactions between the reactants produce exothermic energy and as a result flame is produced and fine powders of the nanoparticles were formed.
The obtained powder was annealed at 6000c for 3 hours in muffle furnace to obtain nanoparticles of zinc oxide.
After proper cleaning and drying process rice straw waste was used. For the treatment of rice straw waste, basic NaOH solution and methanol were used. Small pieces of 5g rice straw washed using distilled water. NaOH solution was prepared by measuring NaOH equal to the weight of 10% of rice straw (.5g) and making it upto 50ml by dissolving NaOH in distilled water. Liquor to fiber ratio was taken as 10:1, ie, 50g of methanol for 5g of rice straw. Liquor and NaOH solution were added to the rice straw and were cooked on a heater for 20 minutes until the solution reduced to some amount on open air. It cool for some time and the pulps were further applied for acid pulping, where nitric acid (69% concentration) diluted (2.5 ml of acid and making it upto 25 ml using distilled water) was used in the pulp for heating until it reduces. It was cooled for some time. Again 25 ml of concentrated acid was added. After reduction and cooling process another 25 ml of acid was added and heated. The pulp turned to particles. After cooling distilled water was added to it and stirred for 15 minutes on a magnetic stirrer. After some time the powder settled. Water was decanted. The process was repeated thrice and finally washing was done using 50 ml of methanol. The solution was filtered using filter paper and dried at room temperature. Dried rice straw powder was collected and used for analysis. In case of nanocomposite based on rice straw annealed ZnOprepared from combustion and co-precipitation methodwas added at the time of pulp formation, separately.
Table 1 Sample notations used in the present work
ZnO nanoparticles prepared from co-precipitation annealed at 6000C ZS
ZnO nanoparticles prepared from combustion method annealed at 6000C ZCS
Rice Straw RS
Rice Straw based Zinc Oxide nanocomposite RSZS
3 RESULTS AND DISCUSSIONS
Metal oxide prepared using co-precipitation method in the present study was obtained in the form of hydroxide. In the case of combustion method, black powder was obtained. Both annealed samples of zinc oxide are obtained as white powders. Rice straw and rice straw based nanocompositeswas obtained as pale yellow.
3.1 XRD Analysis
1. XRD analysis of ZS and ZCS
XRD studies were carried out using XPERT-PRO model powder diffractometer (PAN analytical, Netherlands) employing Cu- K? radiation (? = 1.54060Ao) operating at 40kV, 30mA.Figure 1 shows XRD of ZS and ZCS. The interplanar spacing (dhkl values), 2? values and relative intensity values of zinc oxide (ZS and ZCS prepared in the present work) corresponding to the observed diffraction peaks were compared with the standard values of zinc oxide as reported by JCPDS-International Centre for Diffraction Data which also matched with JCPDS-ICDD pattern number pattern number #79-0205 of zinc oxide. Both ZS and ZCS matched with the standard ZnO hexagonal system with primitive lattice of ZnO (JCPDS: #79-0205). For ZS the diffraction peaks at 2? values 31.71o, 34.39o, 36.27o, 47.49o, 56.61o, 62.68o, 66.29o, 67.99o, 69.1o, 72.75o and 77.12o corresponds to the crystal planes of (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) respectively with no characteristic peaks corresponding to the impurities, which further confirms the formation of pure stable ZnO phase. In case of ZCS, the diffraction peaks at 2? values 31.73o, 34.40o, 36.28o, 47.50o, 56.61o, 62.88o, 66.29o, 67.82o, 69.15o, 72.58o and 76.99o corresponds to the crystal planes of (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) respectively with no characteristic peaks corresponding to the impurities, which further confirms the formation of pure stable ZnO phase.
The average crystallite size of ZS and ZCS calculated from the line broadening of the XRD pattern, using FWHM values of three major peaks in the XRD spectrum making use of Scherrer formula are shown in Tables 2 and Table 3.The Scherrer equation is
t = K? /?hklcos ?hkl (1)
Here t is the average crystallite size normal to the reflecting planes, K is the shape factor and the values lies between 0.95 and 1.15 depending on the shape of the grains (K= 1 for spherical crystallites), l is the wavelength of X-rays used, ?hkl is the full width at half maximum of the diffraction line in radians and ?hkl is the Bragg angle corresponding to the diffraction line arising from reflections from the planes designated by Miller indices (h k l).
Table 2 Crystallite size calculation of ZCS using Scherrer equation
2? ? Cos? FWHM D
31.73 15.87 0.9619 0.3814 20.04
34.40 17.20 0.9553 0.3919 19.37
36.28 18.14 0.9503 0.4661 16.20
Average Crystallite size = 18.54 nm
Figure 1 XRD Spectrum of ZCS and ZS
Table 3 Crystallite size calculation of ZS using Scherrer equation
2? ? Cos? FWHM D
31.71 15.86 0.9619 0.3945 19.38
34.39 17.19 0.9553 0.3603 21.07
36.27 18.14 0.9504 0.4308 17.53
Average Crystallite size = 19.33 nm
2. XRD analysis of RS
The XRD pattern of RS was found to match well with the XRD patterns in literature 7. The XRD of RS showed the presence of a few crystalline peaks in the diffraction pattern having 2? values between 150 and 300 and broad peaks between 300 and 500. Figure 2 shows the XRD diffraction pattern of RS.
Figure 2 XRD Spectrum of RS
3. XRD analysis of RSZS and RSZCS
Figure 3 XRD Spectrum of RSZS and RSZCS
Figure 3 shows the XRD diffraction pattern of RSZS and RSZCS respectively. The variation of XRD of RSZS and RSZCS when compared with RS confirms the formation of nanocomposites. Since doping level of metal oxide was low no sharp peaks of metal oxide was obtained in case of nanocomposites.
There is a pressing demand in the present world to develop new antimicrobial agents and to find out novel strategies due to the emerging infectious diseases and the development of drug resistance in the pathogenic bacteria and fungi. The present study investigates the antimicrobial activity of the synthesized samples as antibacterial agents. The selected microbes for the present study are thegram positive bacteria: Bacilus cereus (B. cereus), the gram negative bacteria: Escherichia coli (E. coli). These test organisms were collected from Institute of Microbial Technology, Microbial Type Culture Collection Centre (IMTECH), Chandigarh. The strains were maintained on their respective medium in slants at 2-80C.
The antibacterial activities of the prepared samples in the laboratory had been calculated using the modified Kirby-Bauer disc diffusion method. The pure cultures of the given organisms were subcultured in the Müller-Hinton broth at a temperature range of 35°C ± 2°C on an orbital shaking incubator at 160 rpm. For the microbial growth, a large lawn of culture was prepared by spreading the 100 ?L freshly prepared culture having 106 colony-forming units (CFU)/mL of each testing organism on nutrient agar plates by the assistance of a sterile glass-rod spreader. Plates were left standing for 10 minutes to allow the culture to get absorbed into the plates. Thereafter, 8 mm wells were punched into the nutrient agar plates to test the nanomaterial antimicrobial activity. To avoid the leakage of nanomaterials from the bottom of the wells, the wells had been sealed with one drop of molten agar (0.8% agar). Using a micropipette, 100 ?L (50 ?g) of the prepared sample of nanoparticle suspension was poured onto each of the wells on all plates. After an overnight incubation at a temperature range of 35°C±2°C, the various levels of the zone of inhibition were measured around the disc in units of millimeter. The Solvent blank was used as negative control and Antibiotic tetracycline was used as a positive control.
The antibacterial activities of all the five samples were studied in detail. The antimicrobial activity of the nanoparticles with which is in contact with the microorganisms is generally known to be a function of the surface area. Since, the reactions take place at the surface of a chemical or material, smaller the size, higher is the surface to volume ratio i.e., in case of larger surface area, better interaction with the microbes is seen. Figure 4 shows the comparison of antibacterial activity of ZS, ZCS, RS, RSZS and RSZCS. Table 4 shows this comparison. Antimicrobial effects of ZnO nanoparticles can be attributed to several mechanisms: ROS (reactive oxygen species) generation results in induction of oxidative stress, due to accumulation of nanoparticles in the bacterial membrane, membrane disorganization occurs and also their cellular internalization, antimicrobial activity may also be due to the release of metal ions anf their binding to the membrane of microorganisms.
Figure 4 Comparison of Antibacterial activity of ZCS, ZS, RS, RSZS and RSZCS
The destruction of the outer membrane of bacteria by the generated superoxide anion radicals (•O2?),which acts as the reactive species, results in antibacterial activity. At the catalyst’s surface, the reactive species such as •OH and •O2? are generated. Hence the high surface area is very beneficial for degradation of bacteria. The activity was found to be highest in case of gram negative bacteria. All the five samples showed very low activity for B.cereus. The variation in the sensitivity or resistance to both gram positive and gram negative bacteria populations may be due to the differences in the cell structure, physiology, metabolism or degree of contact of organisms with nanoparticles.
Table 4 Zone of inhibition of ZS, ZCS, RS, RSZS and RSZS
Samples B.cereus E. coli
4 mm 8 mm
4 mm 9 mm
No zone 4 mm
No zone 10 mm
RSZCS No zone 12 mm
In the present work we are focussed on the comparison of structural and antibacterial properties of nanocomposite of ZnO formed with rice straw. ZnO nanoparticles were prepared using co-precipitation and combustion methods. Structural characterizations of all the five synthesized samples were carried out using XRD. Comparison of XRD’s of metal oxide with JCPDS confirmed that the formed metal oxide was ZnO. Crystallite sizes were calculated using Scherrer equation. Crystallite sizes calculated for ZCS was 18.54 nm and that of ZS 19.33 nm. The variation of XRD of RSZS and RSZCS when compared with XRD of RS confirms the formation of nanocomposites. Since doping level of metal oxide was low no sharp peaks of metal oxide was obtained in case of nanocomposites. The present study investigates the antimicrobial activity of the synthesized samples as antibacterial agents. The selected microbes for the present study are thegram positive bacteria: Bacilus cereus (B. cereus), the gram negative bacteria: Escherichia coli (E. coli). The activity was found to be highest in case of gram negative bacteria. All the five samples showed very low activity for B.cereus. The variation in the sensitivity or resistance to both gram positive and gram negative bacteria populations may be due to the differences in the cell structure, physiology, metabolism or degree of contact of organisms with nanoparticles.
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