Accepted Manuscript ZnO nanoflowers: Novel biogenic synthesis and enhanced photocatalytic activity R.M. Tripathi, Akhshay Singh Bhadwal, Rohit Kumar Gupta, Priti Singh, Archana Shrivastav, B.R. Shrivastav PII: DOI: Reference:
S1011-1344(14)00298-X http://dx.doi.org/10.1016/j.jphotobiol.2014.10.001 JPB 9850
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
25 May 2014 3 October 2014 4 October 2014
Please cite this article as: R.M. Tripathi, A.S. Bhadwal, R.K. Gupta, P. Singh, A. Shrivastav, B.R. Shrivastav, ZnO nanoflowers: Novel biogenic synthesis and enhanced photocatalytic activity, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.10.001
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ZnO nanoflowers: Novel biogenic synthesis and enhanced photocatalytic activity R.M.Tripathia, b*, Akhshay Singh Bhadwalb, Rohit Kumar Guptab, Priti Singhc, Archana Shrivastava,**, B.R.Shrivastavd a
Department of Microbiology, College of Life Sciences, Gwalior - 474 009 (M.P.), India.
b
Amity Institute of Nanotechnology, Amity University, Sector- 125, Noida- 201303(U.P.), India.
c
Department of Physics, Manav Rachna College of Engineering, Faridabad-121004 (Haryana), India d
Department of Surgical Oncology, Cancer Hospital & Research Institute, Gwalior - 474 009 (M.P.), India. *Corresponding author: [email protected], Phone No- +91-120-4392130, Fax No- +91-120-4392502 **Corresponding Author: [email protected], Phone No- +91-751-2336502, Fax No- +91-751– 2336506
R. M. Tripathi Asstt. Professor Amity Institute of Nanotechnology Amity University, Sector- 125 Noida- 201303, India. Phone No- +91-120-4392130 Fax No- +91-120-4392502 Email- [email protected], [email protected]
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Abstract: We demonstrate a novel, unprecedented and eco-friendly mode for the biosynthesis of zinc oxide (ZnO) nanoflowers at ambient room temperature using Bacillus licheniformis MTCC 9555 and assessed their photocatalytic activity. The photocatalytic degradation of methylene blue (MB) dye was analyzed under UV-irradiation. An enhanced photocatalytic activity of ZnO nanoflowers was obtained compared to the earlier reports on ZnO nanostructures and other photocatalytic materials. The mechanism behind the enhanced photocatalytic activity was illustrated with diagrammatic representation. It is assumed that due to larger content of oxygen vacancy ZnO nanoflowers shows enhanced photocatalytic activity. Photostability of ZnO nanoflowers was analyzed for consecutive 3 cycles. The size and morphology of ZnO nanoflowers have been characterized by SEM, TEM and found to be in the size range of 250 nm to 1 µm with flower like morphology. It was found that ZnO nanoflowers was formed by agglomeration of ZnO nanorods. Further the EDX established the presence of the elemental signal of the Zn and O. XRD spectrum of ZnO nanoflowers confirmed 2θ values analogous to the ZnO nanocrystal. FTIR analysis was carried to determine the probable biomolecules responsible for stabilization of ZnO nanoflowers. The plausible mechanism behind the synthesis of ZnO nanoflowers by Bacillus licheniformis MTCC 9555 was also discussed with diagram representation.
Keywords: Biosynthesis, ZnO nanoflowers, Enhanced Photocatalytic activity, Photostability.
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1. Introduction Nanotechnology is an interesting field of this era which deals with the formation of matter at nano range with more enhanced features comparative to their bulk counterparts [1]. It is an active area of research which mainly comprises the creation of nanomaterials of variable sizes, shapes and chemical composition [2]. Synthesis of various metal/semiconductor nanomaterials is an escalating area of this field due to its high applicability in varied fields of study like electronics [3], medical sciences [4], forensic science [5] and microbial science [6]. The nanoparticles found to have inimitable size dependent physiochemical properties in contrast to their bulk materials [7]. To meet the growing demands researchers have found many ways for the synthesis of nanoscale metal/semiconductor materials by chemical [8], physical methods [9] and biological methods concerning the use of plant extracts [10-11] and fungi[12-13]. But in recent time many researchers are using bacterial strains for synthesis of various nanoparticles, as an eco-friendly reducing and capping agent. Bacterial culture of Enterococcus sp. [14] was used for synthesis of cadmium sulfide nanoparticles. It has been reported that microorganisms are able to produce nanoparticles of varied size and shape [15]. In current years, synthesis of ZnO nanoparticles has attained huge interest due to their large band gap of 3.37 eV and large electron-hole binding energy of 60 meV [16]. ZnO nanoparticles are expected to increase the gas sensing and photon-to-electron conversion efficiency due to quantum confinement effect and large surface area [17]. These nanoparticles are used in biosensors [18], optical devices [19], solar cells [20] and photocatalysis [21]. Photocatalysis is an advance oxidation process (AOP) in which a semiconductor material absorbs light of suitable wavelength and help in generation of active species proficient of removing organic pollutants [22]. It is a promising technology for environmental remediation and solar energy transformation 3
[23]. A number of articles have been reported in literature using TiO2 nanoparticles as an efficient photocatalyst but ZnO is a more apposite material for photocatalytic activity owing its low cost, low toxicity and high activity [24-25]. Recent studies have accentuated on the usefulness of ZnO nanostructures for efficacious dye removal [26]. An earlier report by Xu et al. demonstrate that ZnO flower like morphology shows high photocatalytic activity compared to other ZnO structures [27]. Thus signifying importance of ZnO nanoflowers in photocatalytic process. Herein we have report the biosynthesis of ZnO nanoflowers using B. licheniformis, a thermophilic non-pathogenic bacteria capable of engendering protease [28]. B. licheniformis secretes certain proteins that serve as stabilization agent. The photocatalytic activity of biosynthesized ZnO nanoflowers was analysed against MB. Photostability of ZnO nanoflowers and mechanism behind enhanced photocatalytic activity has also been discussed. Further the plausible mechanism involved in biosynthesis of ZnO nanoflowers has also been given with diagrammatic representation. 2 Materials and methods 2.1. Materials The bacterial strains of B. licheniformis were procured from Department of Microbiology College of Life Sciences, Gwalior, India. Zinc acetate dihydrate (Zn(O2CCH3)2×2H2O and sodium bicarbonate (NaHCO3) were purchased from Qualigens Fine Chemicals, Mumbai, India. Nutrient media (NaCl 5 gm/litre, Peptone 5 gm/litre, Yeast and Beef extract 1.5 gm/litre each) were purchased from Hi-media, Mumbai, India.
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2.2. Bacterial culture and Biomass extraction B. licheniformis was inoculated into Erlenmeyer flasks containing essential sterile nutrient broth media. The culture was incubated in the shaker at 37±1°C with incessant shaking (200 rpm) for 36 h. After incubation, biomass was extracted from broth media by centrifugation at 8000 rpm for 10 mins and the supernatant was discarded. The collected bacterial biomass was washed with deionized water. The obtained biomass was used for the synthesis of ZnO nanoflowers.
2.3. Synthesis of ZnO nanoflowers (0.2 M) Zinc acetate dihydrate was dissolved in 50 ml of deionized water in 100 ml of Erlenmeyer flask and heated at 60°C for 15 mins followed by the addition of (0.6 M) sodium bicarbonate. Wet bacterial biomass (5 g) of B. licheniformis was then added into the mixture. The above solution was incubated under continuous stirring (200 rpm) for 48 h at 37±1°C. A control sample devoid of bacterial biomass was also maintained for comparison of the result. After 48 h of incubation white colour was observed in the bottom of the flask, indicating the formation of ZnO nanoflowers. The suspension was centrifuged to separate the bacterial biomass and used for characterization of ZnO nanoflowers.
2.4. Characterization of Nanoflowers A drop coated film of nanoflowers was prepared over the glass substrate to obtain the x-ray diffraction patterns. It was obtained using (X’Pert PRO PANanalytical X-ray Diffractometer) having CuKα radiation (λ = 1.5417Å). Morphology and size range of ZnO nanoflowers was determined by transmission electron microscopy (Philips CM-10) working at conventional 5
voltages 200.0 kV and having resolution of 20-450,000x and Scanning electron microscopy (Zeiss). The attendance of elements in the nanoflowers was analyzed by energy dispersive X-ray (Sigma). Finally, the sample was scanned by Fourier transform infrared spectroscopy (PerkinElmer) to determine the probable biomolecules. All the characterization was done according to standard operating procedure.
2.5. Photocatalytic activity of ZnO nanoflowers The photocatalytic performance of ZnO nanoflowers was observed in terms of degradation of MB dye under UV-irradiation. The UV-irradiation was provided by a 300 W high-pressure mercury lamp with main emission wavelength 365 nm (Shanghai Bilon Instruments Co. Ltd) and enclosed by a water jacket to minimize infrared radiation and cool the lamp. Photoreactor cell was filled with MB (100 µM l-1) solution and subsequently ZnO nanoflowers (0.25 g l-1) was then dispersed in the solution. The dispersion was kept in dark conditions for the establishing adsorption–desorption equilibrium. UV-Vis spectrophotometer (UV-1601 pc Shimadzu) was used to monitor the MB concentration after an irradiation for fixed time intervals. For comparison, of results the reaction was also performed in the absenteeism of ZnO nanoflowers (photocatalyst) both in dark and UV-irradiation. Above experiment was performed in triplicate. The degradation efficiency was estimated using the expression: Degradation= (1-C/C0) × 100% In, order to check the photostability, ZnO nanoflowers were collected by centrifugation and then reused for the degradation of MB for 3 cycles, experimental conditions were kept similar as that of the initial test. Mentioned experiments was performed in triplicate. The stated value
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correspond to mean value of three individual experiment. The error bars given in figures correspond to standard deviation calculated from three Individual analysis.
3 Results and discussion 3.1. X-ray diffraction X-ray diffraction analysis is performed by using a drop coated film of biosynthesized ZnO nanoflowers prepared over the glass substrate at ambient laboratory temperature. The spectra show diffraction peaks occurring at 2θ values = 31.730, 34.50, 36.220, 47.5040, 56.90 and 62.850 which correspond to (100), (002), (101), (102), (110) and (103) set of lattice planes respectively (Fig. 1). The diffraction peaks were in well conformity with JCPDS card No. 36-1451 which shows ZnO nanoflowers have hexagonal wurtzite structure and crystalline in nature. As, no other impurity peak were observed, so the synthesized nanoflowers were pure ZnO. The broadening of bragg’s peak indicate the formation on ZnO nanostructure in nanoregime.
3.2. Electron microscopy The size and morphology of ZnO nanostructures were analyzed by transmission electron microscopy. TEM reveals that the synthesized nanostructures have a flower like morphology (Fig. 2A). TEM micrograph clearly indicated that the nanoflowers are three dimensional in appearance with a range of 200 nm to 1 µm in size and have several nanopetals with about 40 nm in width and 400 nm in length. The average mean size of nanoflowers was found to be 620 nm (Fig. 2A). Figure 2B depicted a single nanoflower having the size 300 nm. The TEM micrograph of nanoflower was clearly portrayed that the nanopetals (nanorods) arise from the center of the flowers (Fig. 2C and D). The width of the nanopetals is about 40-50 nm and length is about 3207
400 nm (Fig. 2D and E). To know the basic structure of nanoflowers, the synthesized nanostructures were sonicated for 40 mins and subsequently micrograph was taken which shows several rods like structure. These rods are the basic unit of nanoflowers; means flowers are the resultant of agglomeration of rods (Fig. 2F). HRTEM shows ZnO nanostructures having dspacing of 0.18 nm (Fig. 3B). Fig. 3C shows selected area electron diffraction pattern of nanoflowers. Further the morphology of ZnO nanoflowers was also analysed by Lowmagnification transmission electron microscopy (Fig. 3A) and scanning electron microscopy (Fig. 4A and B).
3.3. Energy dispersive X-ray spectroscopy Elemental analysis was performed by energy dispersive x-ray; the biosynthesized ZnO nanoflowers were drop coated onto the carbon coated copper grid for EDX analysis. Graph was plotted between X-ray counts and energy in keV. EDX study validates the presence of elemental Zn (zinc) and O (oxygen) from the peak obtained by the emission of energy in the sample Thus confirming the formation of the ZnO nanoflowers (Fig. 5). Table 1 shows elemental composition of ZnO nanoparticles obtained through EDX analysis. There are additional peak occurring in the spectrum corresponding to the elemental copper as the sample was prepared on the copper grid.
3.4. Fourier transform infrared spectroscopy FTIR analysis was performed to determine the plausible biomolecules responsible for the stability of ZnO nanoflowers. FTIR spectra were recorded in the range of 150-4500 cm-1. In IR spectra of bacterial biomass (Fig. 6A) an intense peak at 3349.15.00 cm-1 corresponds to the stretching vibration of the -OH bond. Two peaks occurring at 3371.93 and 3400.97 cm-1 8
corresponds to N-H bond stretching. Two peaks were observed at 1640.53 cm-1 and 1530.31 cm-1 which corresponds to the carbonyl (-C-O-) stretching in typical amide I and amide II linkages of protein molecule respectively [29]. Peak was observed at 1333.21 cm-1 which corresponds to CN bond stretching. The IR peaks of the synthesized ZnO nanoparticles (Fig. 6B) showed distinct peaks at 3444.87 cm-1, 3415.93 cm-1, 3387.0 cm-1, 1643.36 cm-1, 1550.99 cm-1, 1340.22 cm-1 and 480.70 cm-1. The spectra shows changes in the peaks of –N-H and COO- as compare to the spectra of biomass. Moreover, there is an appearance of an extra peak at 480.70 cm-1 which is attributed to ZnO stretching vibration [30-31]. This provided evidence that ZnO nanoflowers were stabilized with protein secreted by bacterial. On the basis of FTIR studies, we can conclude that there are certain functional residues and proteins present in the sample that have the tendency to bind with the ZnO nanoflowers and provide them stability. This reveals that the biological molecules secreted by bacterial biomass could be responsible for the stabilization of ZnO nanoflowers. The obtained results were in consistency with previous research, which shows that microorganism provides protein for stabilization of nanoparticles [29].
3.5 Plausible mechanism of biosynthesis of ZnO nanoflowers. At initial phase zinc acetate was dissolved in water and heated to give zinc cation (Zn2+) in the solution. Sodium bicarbonate was then added to the solution which releases hydroxide ion (OH-) in the solution. The reactions are described below: NaHCO3
Na+ + HCO3
HCO3
OH + CO2
Zn(O2CCH3)2(H2O)2 Zn(O2CCH3)2 Zn2+ + 2OH
heating
Zn(O2CCH3)2
Zn2+ + 2[O2CCH3] Zn (OH)2 9
ZnO + H2O
The zinc cation and hydroxide anion combines to form Zn(OH)2 which in turn changes the pH towards the basic scale. Due to minimal heating conditions thermal decomposition of Zn(OH)2 leads to formation of ZnO nuclei in small quantity compared to [Zn(OH)4]2− growth units in the solution. The formation of ZnO nuclei leads to a stress condition for bacteria. Thus leading bacteria to adapt a stress responsive mechanism. Due to this, bacterial biomass secretes certain proteins to detoxify the stress condition. Meanwhile [Zn(OH)4]2− growth units present in the solution absorb on the surface of ZnO nuclei and enable their easy progression along the c-axis [0001] to minimize the energy of the hexagonal crystal structure. [32] This leads to the formation of nanorods which is being stabilized by protein secreted by bacterial biomass. These nanorods self-assemble leading to the formation of ZnO flower like nanostructure stabilized by protein secreted by bacteria. FTIR analysis also shows shifting of –N-H and –COO- peaks in synthesized ZnO nanoflowers (Fig. 6B) as compared to bacterial biomass (Fig.6A). This confirms that protein secreted by bacterial biomass is responsible for stabilization of ZnO nanoflowers. Fig. 7 represent the plausible mechanism behind synthesis of Nanoflowers.
3.6. Photocatalytic activity of ZnO nanoflowers The degrading ability of ZnO nanoflowers was analyzed by MB (C16H18N3SCl) considered as a model pollutant. In the absence of photocatalyst, there is barely any degradation of MB. Thus suggesting the fact that self-degradation of MB could be neglected which is revealed by control analysis. The time dependent absorption spectra of pollutant dye (MB) in the presence of photocatalyst (Fig. 8). The characteristic peak of MB (λ= 664 nm) was taken as an allusion for valuation of photocatalytic activity. A decline in the intensity of the absorbance peak was observed with an increase in the irradiation time. The reaction was done for 60 mins in presence 10
of ZnO nanoflowers and 83% degradation of MB was achieved shown in Figure 9A. Principally, the generation of electron-hole pairs by ZnO nanoflowers and its destination is the basic mechanism behind photodegradation in presence of ZnO nanoflowers. The photodegradation kinetics of MB was analyzed by Ln(C/C0) against time (t) graph (Fig. 9B), the reaction degradation constant of MB was found to be 4.4×10-3 s-1. The kinetics of degradation reaction followed first order kinetics at a low concentration of dye [33]. For comparison of results absorption spectra of pure MB (devoid of photocatalyst and UVirradiation) was also recorded (Fig.8), Also, degradation of MB under UV-irradiation and dark conditions was studied both in presence and absence of photocatalyst (Table 2). We have already studied the photocatalytic activity of CdS nanoparticles and found 37.15% degradation of MB in 60 mins13 whereas ZnO nanoflowers show 83% degradation in 60 mins. This shows that ZnO nanoflowers are effective catalyst than CdS nanoparticles. Barnes et.al showed 18% degradation of MB using ZnO nanoparticles in 60 mins [21]. This shows greater efficacy of ZnO nanoflowers for photodegradation of MB. 3.7. Photocatalytic stability The stability of photocatalyst is one of the crucial factors for its use in industrial applications. The photostability was examined by repetition of photocatalytic activity of the ZnO nanoflowers. The photodegradation efficiency after 3 consecutive (Fig. 10) recycles was found to be 74 %, indicating good photostability of ZnO nanoflowers. The rate of recovery was found to be about 90%, this shows great applicability of ZnO nanoflowers in industrial photocatalysis process. Further, the effect on morphology of ZnO nanoflowers after 3 recycles was also studied using
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scanning electron microscopy (Fig. 11). It was found that nanoflowers tends to agglomerate after three consecutive reuses. 3.8. Mechanism of Photocatalytic activity When ZnO nanoflowers were illuminated under UV light which have larger photon energy than the band gap of the ZnO nanoflowers it produces electron–hole pairs [34]. These electron–hole pairs act as powerful oxidizing and reducing agents [35]. The photo-generated electrons and holes can transfer to the surface of crystal, when particle size is small enough [34]. Thus, water (H2O) adsorbed on the ZnO nanoflowers and traps the hole (h+) which oxidized and leads to the formation of hydroxyl radical (•OH). Afterwards, the presence of oxygen (O2) hinders the recombination process of electron (e-) and hole (h+) pair, initiating to the formation of an anion radical (•O−2) by accepting electrons (e-) from the conduction band and further associate with proton to give •OOH. The aromatic ring of MB is degraded via opening it at the azo bond and the hydroxylated ring by ·OH and ·O2-. After the completion of reaction CO2, H2O, SO24-, NO3and NH4+ were formed [36]. The mechanism of the photocatalytic reaction (Fig. 12) is described as follows: ZnO + hν → ZnO (e− + h+)
(1)
H2O + ZnO (h+) → ·OH + H+ + ZnO
(2)
O2 + ZnO (e−) → ·O2- + ZnO
(3)
·O2-+ H+ → ·OOH
(4)
M.B +·OH/·O2- → Products
(5)
In, this study it is assumed that high photocatalytic activity of ZnO nanoflowers arises due to larger content of oxygen vacancy on the surface [37]. These defects act as an active center by 12
capturing photo-induced electrons, thus inhibiting photon-electron recombination [38] thus increasing the Photocatalytic activity [37]. 4. Conclusions In this we study reports a novel, simple and cost effective approach to prepare nanosized ZnO flowers at ambient laboratory temperature. ZnO nanoflowers has been developed by incubating bacterial biomass of B. licheniformis with Zinc precursors. The biosynthesized ZnO nanoflowers were characterized using x-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and fourier transform infrared spectroscopy (FTIR). It was found that the biosynthesized nanoflowers are three dimensional in appearance with a range of 250 nm-1 µm in size and have several nanopetals with about 40 nm in width and 400 nm in length. The average mean size of ZnO nanoflowers was found to be 620 nm. XRD shows a good crystallinity with hexagonal wurtzite structure. Based on FTIR analysis, bacterial biomass was found to be a prominent entrant for the biosynthesis of ZnO nanoflowers. The results of the study shows that ZnO nanoflowers can be easily synthesized at room temperature using biomass of B. licheniformis. The synthesized nanoflowers shows excellent photocatalytic activity against MB in the presence of UVillumination a degradation of 83% was observed in 60 mins. This remarkable increase in the photocatalytic activity was perceived mainly due to lager content of oxygen vacancy on the surface of ZnO nanoflowers. It was found that the biosynthesized ZnO nanoflowers possess decent photocatalytic stability even after 3 recurring cycles. Thus our research suggest that ZnO nanoflowers can be a potential candidate for its applications in photocatalytic degradation of organic contaminant.
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Acknowledgements The authors were grateful to Dr. Ashok K. Chauhan, Founder, President, Amity University (Noida, India) for his encouragement and providing excellent facilities for the above work. AS Bhadwal thanks to Mr. N. Kumar for his help rendered in carrying out the experiments. Authors are grateful to the management of college of life sciences, Gwalior, India for providing financial support for the above work.
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[36] D. P. Das, N. Biswal, S. Martha, K.M. Parida, Solar-light induced Photodegradation of organic pollutants over CdS-pillared zirconium-titanium phosphate (ZTP), J. Mol. Catal. A, 349 (2011) 36-41.
[37] Y. Wang, X. Li, N. Wang, X. Quan, Y. Chen, Controllable synthesis of ZnO nanoflowers and their morphology-dependent photocatalytic activities, Sep. Purif. Technol. 62 (2008) 727–732. [38] L. Q. Jing, Y. C. Qu, B. Q. Wang, S. D. Li, B.J. Jiang, L. B. Yang, W. Fu, H. G. Fu, J. Z. Sun, Review of photoluminescence performance of nano-sized semiconductor materials and its relationships with photocatalytic activity, Sol. Energy Mater. Sol. Cells 90 (2006) 1773–1787.
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Figure captions
Fig.1.
Fig.2.
XRD pattern of synthesized ZnO nanoflowers. TEM micrograph of ZnO nanoflowers synthesized by B. licheniformis (A) ZnO nanoflowers scanned at 500x magnification showing size range from 200 nm-1µm (B) ZnO nanoflowers scanned at 8000x magnification having size range of 300 nm (C) ZnO nanoflowers scanned at 8000x magnification (D and E) TEM micrograph showing nanopetals having length of 320 nm-400 nm (F) TEM micrograph showing rod like structure.
Fig. 3. (A) Low-Magnification TEM image of Single ZnO nanoflower (B) HR-TEM of ZnO nanoflower (C) SAED pattern of Nanoflower. Fig. 4. (A, B) FE-SEM analysis of ZnO nanoflowers. Fig.5. EDX mapping of the ZnO nanoflowers synthesized by B. licheniformis. Fig. 6. (A) FTIR Spectrum of B. licheniformis biomass. (B) FTIR of synthesized ZnO nanoflowers. Fig. 7. Mechanism behind Biosynthesis of ZnO nanoflowers. Fig.8 Absorbance spectra of MB in the presence of ZnO nanoflowers as a function of time. Fig.9. (A) Photodegradation of MB under UV-illumination as a function of time (B) Photodegradation kinetics of MB. Fig. 10. Cyclic runs showing photostability of ZnO nanoflowers. Fig. 11. SEM image of ZnO nanoflowers after 3 reuses. Fig.12. Schematic diagram showing photocatalytic mechanism of synthesized ZnO nanoflowers. 21
Table. 1 Elemental composition of ZnO nanoflowers obtained through TEM-EDX analysis. Table. 2 Percentage degradation of MB Dye.
22
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8
Fig. 9.
Fig. 10.
Fig. 11.
Fig. 12
Table 1.
37
Table. 2
38
39
Highlights 1. 2. 3. 4. 5.
Unprecedented one pot biosynthesis of ZnO nanoflowers at ambient conditions. Superior photocatalytic activity of ZnO nanoflowers. Photocatalytic mechanism with diagrammatic representation. Mechanism of biosynthesis of nanoflowers. Simple, cost effective & eco-friendly procedure for mass scale production.
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S1011-1344(14)00298-X http://dx.doi.org/10.1016/j.jphotobiol.2014.10.001 JPB 9850
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
25 May 2014 3 October 2014 4 October 2014
Please cite this article as: R.M. Tripathi, A.S. Bhadwal, R.K. Gupta, P. Singh, A. Shrivastav, B.R. Shrivastav, ZnO nanoflowers: Novel biogenic synthesis and enhanced photocatalytic activity, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.10.001
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ZnO nanoflowers: Novel biogenic synthesis and enhanced photocatalytic activity R.M.Tripathia, b*, Akhshay Singh Bhadwalb, Rohit Kumar Guptab, Priti Singhc, Archana Shrivastava,**, B.R.Shrivastavd a
Department of Microbiology, College of Life Sciences, Gwalior - 474 009 (M.P.), India.
b
Amity Institute of Nanotechnology, Amity University, Sector- 125, Noida- 201303(U.P.), India.
c
Department of Physics, Manav Rachna College of Engineering, Faridabad-121004 (Haryana), India d
Department of Surgical Oncology, Cancer Hospital & Research Institute, Gwalior - 474 009 (M.P.), India. *Corresponding author: [email protected], Phone No- +91-120-4392130, Fax No- +91-120-4392502 **Corresponding Author: [email protected], Phone No- +91-751-2336502, Fax No- +91-751– 2336506
R. M. Tripathi Asstt. Professor Amity Institute of Nanotechnology Amity University, Sector- 125 Noida- 201303, India. Phone No- +91-120-4392130 Fax No- +91-120-4392502 Email- [email protected], [email protected]
1
Abstract: We demonstrate a novel, unprecedented and eco-friendly mode for the biosynthesis of zinc oxide (ZnO) nanoflowers at ambient room temperature using Bacillus licheniformis MTCC 9555 and assessed their photocatalytic activity. The photocatalytic degradation of methylene blue (MB) dye was analyzed under UV-irradiation. An enhanced photocatalytic activity of ZnO nanoflowers was obtained compared to the earlier reports on ZnO nanostructures and other photocatalytic materials. The mechanism behind the enhanced photocatalytic activity was illustrated with diagrammatic representation. It is assumed that due to larger content of oxygen vacancy ZnO nanoflowers shows enhanced photocatalytic activity. Photostability of ZnO nanoflowers was analyzed for consecutive 3 cycles. The size and morphology of ZnO nanoflowers have been characterized by SEM, TEM and found to be in the size range of 250 nm to 1 µm with flower like morphology. It was found that ZnO nanoflowers was formed by agglomeration of ZnO nanorods. Further the EDX established the presence of the elemental signal of the Zn and O. XRD spectrum of ZnO nanoflowers confirmed 2θ values analogous to the ZnO nanocrystal. FTIR analysis was carried to determine the probable biomolecules responsible for stabilization of ZnO nanoflowers. The plausible mechanism behind the synthesis of ZnO nanoflowers by Bacillus licheniformis MTCC 9555 was also discussed with diagram representation.
Keywords: Biosynthesis, ZnO nanoflowers, Enhanced Photocatalytic activity, Photostability.
2
1. Introduction Nanotechnology is an interesting field of this era which deals with the formation of matter at nano range with more enhanced features comparative to their bulk counterparts [1]. It is an active area of research which mainly comprises the creation of nanomaterials of variable sizes, shapes and chemical composition [2]. Synthesis of various metal/semiconductor nanomaterials is an escalating area of this field due to its high applicability in varied fields of study like electronics [3], medical sciences [4], forensic science [5] and microbial science [6]. The nanoparticles found to have inimitable size dependent physiochemical properties in contrast to their bulk materials [7]. To meet the growing demands researchers have found many ways for the synthesis of nanoscale metal/semiconductor materials by chemical [8], physical methods [9] and biological methods concerning the use of plant extracts [10-11] and fungi[12-13]. But in recent time many researchers are using bacterial strains for synthesis of various nanoparticles, as an eco-friendly reducing and capping agent. Bacterial culture of Enterococcus sp. [14] was used for synthesis of cadmium sulfide nanoparticles. It has been reported that microorganisms are able to produce nanoparticles of varied size and shape [15]. In current years, synthesis of ZnO nanoparticles has attained huge interest due to their large band gap of 3.37 eV and large electron-hole binding energy of 60 meV [16]. ZnO nanoparticles are expected to increase the gas sensing and photon-to-electron conversion efficiency due to quantum confinement effect and large surface area [17]. These nanoparticles are used in biosensors [18], optical devices [19], solar cells [20] and photocatalysis [21]. Photocatalysis is an advance oxidation process (AOP) in which a semiconductor material absorbs light of suitable wavelength and help in generation of active species proficient of removing organic pollutants [22]. It is a promising technology for environmental remediation and solar energy transformation 3
[23]. A number of articles have been reported in literature using TiO2 nanoparticles as an efficient photocatalyst but ZnO is a more apposite material for photocatalytic activity owing its low cost, low toxicity and high activity [24-25]. Recent studies have accentuated on the usefulness of ZnO nanostructures for efficacious dye removal [26]. An earlier report by Xu et al. demonstrate that ZnO flower like morphology shows high photocatalytic activity compared to other ZnO structures [27]. Thus signifying importance of ZnO nanoflowers in photocatalytic process. Herein we have report the biosynthesis of ZnO nanoflowers using B. licheniformis, a thermophilic non-pathogenic bacteria capable of engendering protease [28]. B. licheniformis secretes certain proteins that serve as stabilization agent. The photocatalytic activity of biosynthesized ZnO nanoflowers was analysed against MB. Photostability of ZnO nanoflowers and mechanism behind enhanced photocatalytic activity has also been discussed. Further the plausible mechanism involved in biosynthesis of ZnO nanoflowers has also been given with diagrammatic representation. 2 Materials and methods 2.1. Materials The bacterial strains of B. licheniformis were procured from Department of Microbiology College of Life Sciences, Gwalior, India. Zinc acetate dihydrate (Zn(O2CCH3)2×2H2O and sodium bicarbonate (NaHCO3) were purchased from Qualigens Fine Chemicals, Mumbai, India. Nutrient media (NaCl 5 gm/litre, Peptone 5 gm/litre, Yeast and Beef extract 1.5 gm/litre each) were purchased from Hi-media, Mumbai, India.
4
2.2. Bacterial culture and Biomass extraction B. licheniformis was inoculated into Erlenmeyer flasks containing essential sterile nutrient broth media. The culture was incubated in the shaker at 37±1°C with incessant shaking (200 rpm) for 36 h. After incubation, biomass was extracted from broth media by centrifugation at 8000 rpm for 10 mins and the supernatant was discarded. The collected bacterial biomass was washed with deionized water. The obtained biomass was used for the synthesis of ZnO nanoflowers.
2.3. Synthesis of ZnO nanoflowers (0.2 M) Zinc acetate dihydrate was dissolved in 50 ml of deionized water in 100 ml of Erlenmeyer flask and heated at 60°C for 15 mins followed by the addition of (0.6 M) sodium bicarbonate. Wet bacterial biomass (5 g) of B. licheniformis was then added into the mixture. The above solution was incubated under continuous stirring (200 rpm) for 48 h at 37±1°C. A control sample devoid of bacterial biomass was also maintained for comparison of the result. After 48 h of incubation white colour was observed in the bottom of the flask, indicating the formation of ZnO nanoflowers. The suspension was centrifuged to separate the bacterial biomass and used for characterization of ZnO nanoflowers.
2.4. Characterization of Nanoflowers A drop coated film of nanoflowers was prepared over the glass substrate to obtain the x-ray diffraction patterns. It was obtained using (X’Pert PRO PANanalytical X-ray Diffractometer) having CuKα radiation (λ = 1.5417Å). Morphology and size range of ZnO nanoflowers was determined by transmission electron microscopy (Philips CM-10) working at conventional 5
voltages 200.0 kV and having resolution of 20-450,000x and Scanning electron microscopy (Zeiss). The attendance of elements in the nanoflowers was analyzed by energy dispersive X-ray (Sigma). Finally, the sample was scanned by Fourier transform infrared spectroscopy (PerkinElmer) to determine the probable biomolecules. All the characterization was done according to standard operating procedure.
2.5. Photocatalytic activity of ZnO nanoflowers The photocatalytic performance of ZnO nanoflowers was observed in terms of degradation of MB dye under UV-irradiation. The UV-irradiation was provided by a 300 W high-pressure mercury lamp with main emission wavelength 365 nm (Shanghai Bilon Instruments Co. Ltd) and enclosed by a water jacket to minimize infrared radiation and cool the lamp. Photoreactor cell was filled with MB (100 µM l-1) solution and subsequently ZnO nanoflowers (0.25 g l-1) was then dispersed in the solution. The dispersion was kept in dark conditions for the establishing adsorption–desorption equilibrium. UV-Vis spectrophotometer (UV-1601 pc Shimadzu) was used to monitor the MB concentration after an irradiation for fixed time intervals. For comparison, of results the reaction was also performed in the absenteeism of ZnO nanoflowers (photocatalyst) both in dark and UV-irradiation. Above experiment was performed in triplicate. The degradation efficiency was estimated using the expression: Degradation= (1-C/C0) × 100% In, order to check the photostability, ZnO nanoflowers were collected by centrifugation and then reused for the degradation of MB for 3 cycles, experimental conditions were kept similar as that of the initial test. Mentioned experiments was performed in triplicate. The stated value
6
correspond to mean value of three individual experiment. The error bars given in figures correspond to standard deviation calculated from three Individual analysis.
3 Results and discussion 3.1. X-ray diffraction X-ray diffraction analysis is performed by using a drop coated film of biosynthesized ZnO nanoflowers prepared over the glass substrate at ambient laboratory temperature. The spectra show diffraction peaks occurring at 2θ values = 31.730, 34.50, 36.220, 47.5040, 56.90 and 62.850 which correspond to (100), (002), (101), (102), (110) and (103) set of lattice planes respectively (Fig. 1). The diffraction peaks were in well conformity with JCPDS card No. 36-1451 which shows ZnO nanoflowers have hexagonal wurtzite structure and crystalline in nature. As, no other impurity peak were observed, so the synthesized nanoflowers were pure ZnO. The broadening of bragg’s peak indicate the formation on ZnO nanostructure in nanoregime.
3.2. Electron microscopy The size and morphology of ZnO nanostructures were analyzed by transmission electron microscopy. TEM reveals that the synthesized nanostructures have a flower like morphology (Fig. 2A). TEM micrograph clearly indicated that the nanoflowers are three dimensional in appearance with a range of 200 nm to 1 µm in size and have several nanopetals with about 40 nm in width and 400 nm in length. The average mean size of nanoflowers was found to be 620 nm (Fig. 2A). Figure 2B depicted a single nanoflower having the size 300 nm. The TEM micrograph of nanoflower was clearly portrayed that the nanopetals (nanorods) arise from the center of the flowers (Fig. 2C and D). The width of the nanopetals is about 40-50 nm and length is about 3207
400 nm (Fig. 2D and E). To know the basic structure of nanoflowers, the synthesized nanostructures were sonicated for 40 mins and subsequently micrograph was taken which shows several rods like structure. These rods are the basic unit of nanoflowers; means flowers are the resultant of agglomeration of rods (Fig. 2F). HRTEM shows ZnO nanostructures having dspacing of 0.18 nm (Fig. 3B). Fig. 3C shows selected area electron diffraction pattern of nanoflowers. Further the morphology of ZnO nanoflowers was also analysed by Lowmagnification transmission electron microscopy (Fig. 3A) and scanning electron microscopy (Fig. 4A and B).
3.3. Energy dispersive X-ray spectroscopy Elemental analysis was performed by energy dispersive x-ray; the biosynthesized ZnO nanoflowers were drop coated onto the carbon coated copper grid for EDX analysis. Graph was plotted between X-ray counts and energy in keV. EDX study validates the presence of elemental Zn (zinc) and O (oxygen) from the peak obtained by the emission of energy in the sample Thus confirming the formation of the ZnO nanoflowers (Fig. 5). Table 1 shows elemental composition of ZnO nanoparticles obtained through EDX analysis. There are additional peak occurring in the spectrum corresponding to the elemental copper as the sample was prepared on the copper grid.
3.4. Fourier transform infrared spectroscopy FTIR analysis was performed to determine the plausible biomolecules responsible for the stability of ZnO nanoflowers. FTIR spectra were recorded in the range of 150-4500 cm-1. In IR spectra of bacterial biomass (Fig. 6A) an intense peak at 3349.15.00 cm-1 corresponds to the stretching vibration of the -OH bond. Two peaks occurring at 3371.93 and 3400.97 cm-1 8
corresponds to N-H bond stretching. Two peaks were observed at 1640.53 cm-1 and 1530.31 cm-1 which corresponds to the carbonyl (-C-O-) stretching in typical amide I and amide II linkages of protein molecule respectively [29]. Peak was observed at 1333.21 cm-1 which corresponds to CN bond stretching. The IR peaks of the synthesized ZnO nanoparticles (Fig. 6B) showed distinct peaks at 3444.87 cm-1, 3415.93 cm-1, 3387.0 cm-1, 1643.36 cm-1, 1550.99 cm-1, 1340.22 cm-1 and 480.70 cm-1. The spectra shows changes in the peaks of –N-H and COO- as compare to the spectra of biomass. Moreover, there is an appearance of an extra peak at 480.70 cm-1 which is attributed to ZnO stretching vibration [30-31]. This provided evidence that ZnO nanoflowers were stabilized with protein secreted by bacterial. On the basis of FTIR studies, we can conclude that there are certain functional residues and proteins present in the sample that have the tendency to bind with the ZnO nanoflowers and provide them stability. This reveals that the biological molecules secreted by bacterial biomass could be responsible for the stabilization of ZnO nanoflowers. The obtained results were in consistency with previous research, which shows that microorganism provides protein for stabilization of nanoparticles [29].
3.5 Plausible mechanism of biosynthesis of ZnO nanoflowers. At initial phase zinc acetate was dissolved in water and heated to give zinc cation (Zn2+) in the solution. Sodium bicarbonate was then added to the solution which releases hydroxide ion (OH-) in the solution. The reactions are described below: NaHCO3
Na+ + HCO3
HCO3
OH + CO2
Zn(O2CCH3)2(H2O)2 Zn(O2CCH3)2 Zn2+ + 2OH
heating
Zn(O2CCH3)2
Zn2+ + 2[O2CCH3] Zn (OH)2 9
ZnO + H2O
The zinc cation and hydroxide anion combines to form Zn(OH)2 which in turn changes the pH towards the basic scale. Due to minimal heating conditions thermal decomposition of Zn(OH)2 leads to formation of ZnO nuclei in small quantity compared to [Zn(OH)4]2− growth units in the solution. The formation of ZnO nuclei leads to a stress condition for bacteria. Thus leading bacteria to adapt a stress responsive mechanism. Due to this, bacterial biomass secretes certain proteins to detoxify the stress condition. Meanwhile [Zn(OH)4]2− growth units present in the solution absorb on the surface of ZnO nuclei and enable their easy progression along the c-axis [0001] to minimize the energy of the hexagonal crystal structure. [32] This leads to the formation of nanorods which is being stabilized by protein secreted by bacterial biomass. These nanorods self-assemble leading to the formation of ZnO flower like nanostructure stabilized by protein secreted by bacteria. FTIR analysis also shows shifting of –N-H and –COO- peaks in synthesized ZnO nanoflowers (Fig. 6B) as compared to bacterial biomass (Fig.6A). This confirms that protein secreted by bacterial biomass is responsible for stabilization of ZnO nanoflowers. Fig. 7 represent the plausible mechanism behind synthesis of Nanoflowers.
3.6. Photocatalytic activity of ZnO nanoflowers The degrading ability of ZnO nanoflowers was analyzed by MB (C16H18N3SCl) considered as a model pollutant. In the absence of photocatalyst, there is barely any degradation of MB. Thus suggesting the fact that self-degradation of MB could be neglected which is revealed by control analysis. The time dependent absorption spectra of pollutant dye (MB) in the presence of photocatalyst (Fig. 8). The characteristic peak of MB (λ= 664 nm) was taken as an allusion for valuation of photocatalytic activity. A decline in the intensity of the absorbance peak was observed with an increase in the irradiation time. The reaction was done for 60 mins in presence 10
of ZnO nanoflowers and 83% degradation of MB was achieved shown in Figure 9A. Principally, the generation of electron-hole pairs by ZnO nanoflowers and its destination is the basic mechanism behind photodegradation in presence of ZnO nanoflowers. The photodegradation kinetics of MB was analyzed by Ln(C/C0) against time (t) graph (Fig. 9B), the reaction degradation constant of MB was found to be 4.4×10-3 s-1. The kinetics of degradation reaction followed first order kinetics at a low concentration of dye [33]. For comparison of results absorption spectra of pure MB (devoid of photocatalyst and UVirradiation) was also recorded (Fig.8), Also, degradation of MB under UV-irradiation and dark conditions was studied both in presence and absence of photocatalyst (Table 2). We have already studied the photocatalytic activity of CdS nanoparticles and found 37.15% degradation of MB in 60 mins13 whereas ZnO nanoflowers show 83% degradation in 60 mins. This shows that ZnO nanoflowers are effective catalyst than CdS nanoparticles. Barnes et.al showed 18% degradation of MB using ZnO nanoparticles in 60 mins [21]. This shows greater efficacy of ZnO nanoflowers for photodegradation of MB. 3.7. Photocatalytic stability The stability of photocatalyst is one of the crucial factors for its use in industrial applications. The photostability was examined by repetition of photocatalytic activity of the ZnO nanoflowers. The photodegradation efficiency after 3 consecutive (Fig. 10) recycles was found to be 74 %, indicating good photostability of ZnO nanoflowers. The rate of recovery was found to be about 90%, this shows great applicability of ZnO nanoflowers in industrial photocatalysis process. Further, the effect on morphology of ZnO nanoflowers after 3 recycles was also studied using
11
scanning electron microscopy (Fig. 11). It was found that nanoflowers tends to agglomerate after three consecutive reuses. 3.8. Mechanism of Photocatalytic activity When ZnO nanoflowers were illuminated under UV light which have larger photon energy than the band gap of the ZnO nanoflowers it produces electron–hole pairs [34]. These electron–hole pairs act as powerful oxidizing and reducing agents [35]. The photo-generated electrons and holes can transfer to the surface of crystal, when particle size is small enough [34]. Thus, water (H2O) adsorbed on the ZnO nanoflowers and traps the hole (h+) which oxidized and leads to the formation of hydroxyl radical (•OH). Afterwards, the presence of oxygen (O2) hinders the recombination process of electron (e-) and hole (h+) pair, initiating to the formation of an anion radical (•O−2) by accepting electrons (e-) from the conduction band and further associate with proton to give •OOH. The aromatic ring of MB is degraded via opening it at the azo bond and the hydroxylated ring by ·OH and ·O2-. After the completion of reaction CO2, H2O, SO24-, NO3and NH4+ were formed [36]. The mechanism of the photocatalytic reaction (Fig. 12) is described as follows: ZnO + hν → ZnO (e− + h+)
(1)
H2O + ZnO (h+) → ·OH + H+ + ZnO
(2)
O2 + ZnO (e−) → ·O2- + ZnO
(3)
·O2-+ H+ → ·OOH
(4)
M.B +·OH/·O2- → Products
(5)
In, this study it is assumed that high photocatalytic activity of ZnO nanoflowers arises due to larger content of oxygen vacancy on the surface [37]. These defects act as an active center by 12
capturing photo-induced electrons, thus inhibiting photon-electron recombination [38] thus increasing the Photocatalytic activity [37]. 4. Conclusions In this we study reports a novel, simple and cost effective approach to prepare nanosized ZnO flowers at ambient laboratory temperature. ZnO nanoflowers has been developed by incubating bacterial biomass of B. licheniformis with Zinc precursors. The biosynthesized ZnO nanoflowers were characterized using x-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and fourier transform infrared spectroscopy (FTIR). It was found that the biosynthesized nanoflowers are three dimensional in appearance with a range of 250 nm-1 µm in size and have several nanopetals with about 40 nm in width and 400 nm in length. The average mean size of ZnO nanoflowers was found to be 620 nm. XRD shows a good crystallinity with hexagonal wurtzite structure. Based on FTIR analysis, bacterial biomass was found to be a prominent entrant for the biosynthesis of ZnO nanoflowers. The results of the study shows that ZnO nanoflowers can be easily synthesized at room temperature using biomass of B. licheniformis. The synthesized nanoflowers shows excellent photocatalytic activity against MB in the presence of UVillumination a degradation of 83% was observed in 60 mins. This remarkable increase in the photocatalytic activity was perceived mainly due to lager content of oxygen vacancy on the surface of ZnO nanoflowers. It was found that the biosynthesized ZnO nanoflowers possess decent photocatalytic stability even after 3 recurring cycles. Thus our research suggest that ZnO nanoflowers can be a potential candidate for its applications in photocatalytic degradation of organic contaminant.
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Acknowledgements The authors were grateful to Dr. Ashok K. Chauhan, Founder, President, Amity University (Noida, India) for his encouragement and providing excellent facilities for the above work. AS Bhadwal thanks to Mr. N. Kumar for his help rendered in carrying out the experiments. Authors are grateful to the management of college of life sciences, Gwalior, India for providing financial support for the above work.
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Figure captions
Fig.1.
Fig.2.
XRD pattern of synthesized ZnO nanoflowers. TEM micrograph of ZnO nanoflowers synthesized by B. licheniformis (A) ZnO nanoflowers scanned at 500x magnification showing size range from 200 nm-1µm (B) ZnO nanoflowers scanned at 8000x magnification having size range of 300 nm (C) ZnO nanoflowers scanned at 8000x magnification (D and E) TEM micrograph showing nanopetals having length of 320 nm-400 nm (F) TEM micrograph showing rod like structure.
Fig. 3. (A) Low-Magnification TEM image of Single ZnO nanoflower (B) HR-TEM of ZnO nanoflower (C) SAED pattern of Nanoflower. Fig. 4. (A, B) FE-SEM analysis of ZnO nanoflowers. Fig.5. EDX mapping of the ZnO nanoflowers synthesized by B. licheniformis. Fig. 6. (A) FTIR Spectrum of B. licheniformis biomass. (B) FTIR of synthesized ZnO nanoflowers. Fig. 7. Mechanism behind Biosynthesis of ZnO nanoflowers. Fig.8 Absorbance spectra of MB in the presence of ZnO nanoflowers as a function of time. Fig.9. (A) Photodegradation of MB under UV-illumination as a function of time (B) Photodegradation kinetics of MB. Fig. 10. Cyclic runs showing photostability of ZnO nanoflowers. Fig. 11. SEM image of ZnO nanoflowers after 3 reuses. Fig.12. Schematic diagram showing photocatalytic mechanism of synthesized ZnO nanoflowers. 21
Table. 1 Elemental composition of ZnO nanoflowers obtained through TEM-EDX analysis. Table. 2 Percentage degradation of MB Dye.
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Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8
Fig. 9.
Fig. 10.
Fig. 11.
Fig. 12
Table 1.
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Table. 2
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Highlights 1. 2. 3. 4. 5.
Unprecedented one pot biosynthesis of ZnO nanoflowers at ambient conditions. Superior photocatalytic activity of ZnO nanoflowers. Photocatalytic mechanism with diagrammatic representation. Mechanism of biosynthesis of nanoflowers. Simple, cost effective & eco-friendly procedure for mass scale production.
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