Accepted Manuscript Leucas aspera mediated multifunctional CeO2 nanoparticles: Structural, Photoluminescent, Photocatalytic and Antibacterial properties J. Malleshappa, H. Nagabhushana, S.C. Sharma, Y.S. Vidya, K.S. Anantharaju, S.C. Prashantha, B. Daruka Prasad, H. Raja Naika, K. Lingaraju, B.S. Surendra PII: DOI: Reference:

S1386-1425(15)00545-4 http://dx.doi.org/10.1016/j.saa.2015.04.073 SAA 13624

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

9 November 2014 2 March 2015 22 April 2015

Please cite this article as: J. Malleshappa, H. Nagabhushana, S.C. Sharma, Y.S. Vidya, K.S. Anantharaju, S.C. Prashantha, B. Daruka Prasad, H. Raja Naika, K. Lingaraju, B.S. Surendra, Leucas aspera mediated multifunctional CeO2 nanoparticles: Structural, Photoluminescent, Photocatalytic and Antibacterial properties, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.04.073

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Leucas aspera mediated multifunctional CeO2 nanoparticles: Structural, Photoluminescent, Photocatalytic and Antibacterial properties J. Malleshappa a, H. Nagabhushana b*, S. C. Sharma c, Y.S. Vidya d, K.S. Anantharaju e, S. C. Prashanthae, B. Daruka Prasad f, H. Raja Naika g, K. Lingarajug, B. S. Surendrae a

Department of Physics, University College of Science, Tumkur University, Tumkur 572 103, India b Prof. C. N. R. Rao Centre for Advanced Materials Research, Tumkur University, Tumkur 572 103, India c Vice Chancellor, Chattisgarh Swami Vivekanand Technical University, Bhilai, Chattisgarh, India d Department of Physics, Lal Bahadur Shastry Government First Grade College, Bangalore 560 032, India e Research Center, Department of Science, East West Institute of Technology, Bangalore 560 091, India f B. S. Narayan Centre of Excellence for Advanced Materials, B. M. S. Institute of Technology, Yelahanka, Bangalore 560 064, India g Department of Studies and Research in Environmental Science, Tumkur University, Tumkur 572 103, India Abstract Spherical shaped cerium dioxide (CeO2) nanoparticles (NPs) were synthesized via bio mediated route using Leucas aspera (LA) leaf extract. The NPs were characterized by PXRD, SEM, UV– Visible techniques. Photoluminescence (PL), photocatalysis and antibacterial properties of NPs were studied. PXRD patterns and Rietveld analysis confirm cubic fluorite structure with space group Fm-3m. SEM results evident that morphology of the NPs was greatly influenced by the concentration of LA leaf extract in the reaction mixture. The band gap energy of the NPs was found to be in the range of 2.98-3.4 eV. The photocatalytic activity of NPs was evaluated by decolorization of Rhodamine-B (RhB) under UVA and Sun light irradiation. CeO2 NPs show intense blue emission with CIE coordinates (0.14, 0.22) and average color coordinated temperature value ~148953 K. Therefore the present NPs quite useful for cool LEDs. The superior photocatalytic activity was observed for CeO2 NPs with 20 ml LA under both UVA and Sunlight irradiation. The enhanced photocatalytic activity and photoluminescent properties were attributed to defect induced band gap engineered CeO2 NPs. Further, CeO2 with 20 ml LA exhibit significant antibacterial activity against Escherichia coli (EC) and Staphylococcus aureus (SA). These findings show great promise of CeO2 NPs as multifunctional material for various applications. Key words: CeO2 NPs; Leucas aspera; Photoluminescence; Photocatalysis; Antibacterial activity *Corresponding authors: E-mail: [email protected] (H. Nagabhushana), Tel.: +91 945954010 1

1. Introduction Currently nanoparticles (NPs) are attracting attention due to their exceptionally small size, large surface to volume ratio and many technological applications which cannot be achieved when compared to bulk of the same chemical composition. Design and fabrication of materials with novel applications can be achieved by controlling the shape and size at nano metre scale [1-5]. Due to their unique physical, chemical and optical properties nanostructured materials find wide range of applications namely catalysis, sensors, antibacterial, luminescent materials etc. [6-15]. In the field of nanoscience and nanotechnology, design and synthesis of NPs with tailor-made structural properties is a very challenging task. Consequently, appropriate synthetic routes are developed to ensure a reliable supply of such NPs in sufficient quantities [16, 17]. Green synthesis route is highly relevant as it is nontoxic, decreasing / completely eliminating the use and generation of substances hazardous to human health and the environment. The use of extracts obtained from plants, particularly the easily available parts such as leaves and stem bark are highly useful. Also, the collection of these plant parts does not damage the plant itself and storage of these plant materials is very easy [18-25]. The synthesis of nanostructured materials with controlled shape, size and structural properties can be achieved through many different approaches. Most of the wet chemical routes utilize high temperature and many of the reactants, starting materials; stabilizers and solvents used are toxic and potentially hazardous. Therefore, plant based bio mediated synthetic routes are almost hazard-free and at the same time economically viable and environment friendly. Leucas aspera (Willd) Linn (Family: Lamiaceae) normally known as 'Thumbai’ is distributed throughout India from the Himalayas down to Ceylon. Leucas aspera is an annual, branched herb erecting to a height of 15-60 cm with stout and hispid acutely quadrangular stem 2

and branches. The plant is used traditionally as an antipyretic and insecticide. Flowers are valued as stimulant, expectorant, aperient, diaphoretic, insecticide and emmenagogue. Leaves are considered to be useful in treating chronic rheumatism, psoriasis and other chronic skin eruptions.

Leucas aspera is used for the treatment of respiratory tract disorders, edema,

gastrointestinal disorders, pain and as an antidote to poison. In Indian traditional medicine, the leaf juice of Leucas aspera is used to treat psoriasis, chronic skin eruptions and chronic rheumatism. The ethanolic extract of roots of the plant is shown to contain anti-inflammatory, antinociceptive, antioxidant and cyto-toxic activities. Further, leaf extract of the plant has been shown to demonstrate antiplasmodial activity against chloroquine-sensitive strain of Plasmodium falciparum [26-29]. Ceria a crystalline material, is a major compound in the rare earth family and has received significant attention in recent years due to many distinctive characteristics such as unique ultraviolet radiation absorbing ability, high stability at higher temperature, high hardness index and its reactivity [27]. Extensive literature survey has been made in the synthesis of metal NPs using extracts obtained from bacteria, fungi and plant parts namely geranium leaves, lemongrass, neem leaves, aloe vera and others [28-32]. However, a few reports are available on the green synthesis of ZnO, CuO, SnO2, ZnAl2O3, Dy2O3 through E-tirucalli, Calotropis procera, Aloe vera gel extracts [32-36]. Owing to the multifunctional applications of CeO2 NPs, the present work addresses the various morphologies of CeO2 NPs synthesized via eco-friendly combustion route using different concentrations of LA leaf extract as fuel. The obtained products are characterized by PXRD, SEM, TEM, UV – Visible absorption techniques. Further, the CeO2 NPs are evaluated for photoluminescent, photocatalysis and antibacterial properties. The proposed methodology is 3

simple, limited usage of chemicals and doesn’t involve doping agents, such as metals, nonmetals, capping agents, etc., and has potential applications on commercial scale. 2. Materials and methods 2.1 Preparation of leaf extract and CeO2 NPs Mature and fresh leaves of Leucas aspera (LA) were collected from Tumkur university campus, Tumkur. Ceric nitrate (Ce (NO3)3 .6H2O) was procured from Sigma Aldrich (99.9%) and used without further purification. The detailed preparation of leaf extract and CeO2 NPs were given supplementary section and the flow chart for the preparation of CeO2 NPs using LA was shown in Fig. S1. The resultant products were calcined at 400° C for 3 h and labeled as S1, S2, S3 and S4. 2.2 Characterization The powder X-ray diffraction (PXRD) patterns were recorded on Shimadzu 7000 diffractometer using CuKα radiation (λ=1.54 nm). Morphology of the sample was analyzed by employing Hitachi Table Top Microscope Model TM 3000. Transmission Electron Microscopy (TEM) and the selected-area electron diffraction pattern (SAED) images were recorded in TECNAI F-30 TEM at an operating voltage of 200 kV. PL measurements were carried out using Horiba, USA made model Fluorolog-3 spectrofluorimeter at room temperature (RT). The absorption spectrum of the sample was measured on a Shimadzu UV-1800 UV-Vis spectrometer. Lattice constants were estimated using Fullprof suit programme and packing diagram was drawn using diamond software. 2.3 Photocatalytic studies The photocatalytic experiments were carried out at RT using a circular glass reactor whose surface area was 176.6 cm2 using 125 W medium pressure mercury vapor lamp as the UV light 4

source. The photon flux was found to be 8.1 mW / cm2 by ferrioxalate actinometric method [37]. The emission wavelength was in the region of 350 – 400 nm with maximum emission at 370 nm. No additional step was taken to eliminate the light of different wavelengths, since this process reduces the intensity of light. The irradiation was carried out by directly focusing light into the reaction mixture in the open air condition at a distance of 23 cm. All the experiments were carried out using double distilled water. Solar light experiments were performed under Sunlight directly between 11 am and 2 pm when the solar intensity fluctuations were minimal and Sunrays are not oblique. The experiments were conducted in the months of April - May at Bangalore, India. The latitude and longitude are 12.60 N and 77.31 E respectively. The average solar intensity was found to be 0.751 kW m-2 (using solar radiometer). The intensity of solar light was concentrated by using a convex lens and the reaction mixture was exposed to the concentrated Sunlight. The solar radiation as a function of wavelength was measured by photometer, which shows a maximum wavelength around 450 - 500 nm. To compare the photocatalytic activity of all the photocatalysts, the experiments were simultaneously conducted to avoid the error arising due to the fluctuations in solar intensity. A typical experiment contains 60 mg of photocatalyst dispersed in 250 ml of 20 ppm Rhodamine B (RhB) solution. The reaction mixture was stirred vigorously using magnetic stirrer for entire time span of the experiment. Prior to irradiation, the reaction mixture was stirred for 30 min to ensure the establishment of adsorption / desorption equilibrium. The extent of adsorption was found from the equation Q = (C0 – C) V / W, where ‘Q’; the extent of adsorption, C0 and C; the concentrations before and after adsorption, V; the volume of the reaction mixture and W; the amount of catalyst present in grams. The unit of Q was ppm ml mg-1. The 5 ml aliquots were collected at definite time intervals and immediately centrifuged and filtered through 0.45 µm 5

Millipore filter to remove the catalyst particles and subjected for spectrophotometric analysis and the residual concentration of RhB was determined. 2.4 Antibacterial activity Antibacterial activity was screened by Agar well diffusion method [38] against four bacterial strains namely Gram –ve bacteria Klebsiella aerogenes (NCIM-2098), Escherichia coli (NCIM5051), Pseudomonas desmolyticum (NCIM-2028) and Gram + ve bacteria Staphylococcus aureus (NCIM-5022). Nutrient agar plates were prepared and swabbed using Sterile Lshaped glass rod with 100 µl of 24 h mature broth culture of individual bacterial strains. The well is made by using sterile cork borer and 6 mm wells were created into the each Petriplate. Various concentrations of CeO2 NPs (500 µg and 1000 µg/well) were used to assess the antibacterial activity. The sample prepared in sterile water was added into the wells by using sterile micropipettes. Simultaneously the standard antibiotics (as positive control) were tested against the pathogens. Ciprofloxacin (Hi Media, Mumbai, India) was used as positive control and then the plates were incubated at 37 °C for 36 h. After the incubation period, the zone of inhibition of each well was measured and the values were noted. The antibacterial activity was assayed in triplicates with NPs and the average values were calculated for the antibacterial activity. 3. Results and discussion 3.1 Powder X-ray diffraction studies Fig. 1 shows the PXRD patterns of CeO2 NPs prepared by different concentrations (5-20 ml) of LA leaf extract. All the diffraction patterns (111), (200), (220) and (222) were well indexed to a pure cubic fluorite structure of CeO2 (JCPDS card No. 81-0792) [39]. No identifiable impurity peaks were observed which indicates the single phase of ceria NPs. According to the full width at half-maximum (FWHM) of the diffraction peaks, the average size of the particles (crystallites) 6

were estimated from the Scherrer’s equation. The variation of crystallite size, lattice constant, dislocation density, strain, stacking fault and texture co-efficient with different concentrations of LA leaf extract for the ceria NPs were estimated and shown in Table 1. The concentration of LA leaf extract strongly affects the structural parameters such as crystallite size and lattice constant (Table 1). The various structural parameters namely crystallite size, lattice constant, dislocation density, strain, stacking fault and texture coefficient with different concentrations of leaf extract in (111) direction are determined using the following relations [40]: nλ 2 sin θ kλ D= β cosθ 1 δ= 2 D β cosθ ε= 4   2π 2 SF =  β 1   45(3 tan θ ) 2 

………………… 1

d=

I (h k l )  1 TC = 0 i i i  I S (hi k i li )  N

I 0 (hi ki li )  ∑  i =1 I S (hi ki li ) 

………………… 2 ………………… 3 ………………… 4 ………………… 5 −1

………………… 6

Where, d; interplanar spacing, λ; wavelength of X-ray, θ; Braggs angle, D; the crystallite size, β; full width at half maximum (FWHM), δ; dislocation density, ε; the micro strain, SF; stacking fault, hkl; Miller indices and TC ; texture co-efficient. The peak shift and line broadening in PXRD profiles arise due to the presence of micro strain in NPs. Williamson and Hall (W-H) plots were used to estimate the micro strain in S1, S2, S3 and S4 NPs using the relation [41]:

7

β cos θ =

kλ + 4ε sin θ D

------------------------- 7

where ε; strain associated with the nanoparticle. Eqn. 7 represents a straight line between 4 sinθ (X-axis) and βcosθ (Y-axis). The slope of line gives the strain (ε) and intercept (kλ/D) of this line on Y-axis gives grain size (D). Fig. 2 shows the W-H plots of S1, S2, S3 and S4 NPs. In order to estimate the accurate lattice parameters (a, V), FullProf suit programme was used for the estimation of lattice parameters utilizing pseudo-Voigt function [42]. The experimental and fitted XRD profile of S4 is shown in Fig. 3. The estimated unit cell parameters for S1, S2, S3 and S4 NPs were listed in Table 2. The estimated lattice parameter (a = 5.427(5) Ǻ) was well matched to theoretically calculated values of cubic fluorite structure. The Goodness

of Fit (GOF) was found to be less than one which confirms good fitting between experimental and theoretical plots. Packing diagram of CeO2 NPs obtained using Diamond software after utilizing the obtained refined parameters is shown in inset of Fig. 3. The value of lattice constant was slightly higher compared to the bulk counter parts due to its increased oxygen vacancies [43]. 3.2 UV – Visible absorption studies

UV–Visible absorption studies are used to track the effect of morphological evaluation on the UV absorption ability of CeO2 NPs (Fig. 4). It was observed that a broad absorption peak located at around ~330 nm for all samples prepared under different concentrations of LA leaf extract. The 330 nm peak may originate from the charge transfer between the O 2p and Ce 4f states. Evidently, the UV absorption range is wider for the nanostructured materials. Since, it is generally believed that a good UV-shielding material should hold the ability to absorb the UV rays with a wavelength of less than 400 nm; therefore our prepared CeO2 NPs may act as an 8

efficient UV-shielding candidate. With this, we can find that the position of the peak will shift to higher wavelength with increase in concentration of LA extract. It indicates that the absorption positions depend on the morphology and particle size of CeO2 NPs [44]. The UV absorption ability of CeO2 NPs was related to band gap energy, so it is possible that CeO2 NPs with different shapes should have different band gap energies. The absorption data follow a power- law behavior of Wood -Tauc relation [45] given by αhν = A (hν − Eg )1/2 .Where A; energy independent constant, α; absorption co-efficient and Eg;

optical band gap. The absorption co-efficient α is defined using the relation:

α=

2.303 × 10 3 Aρ ………………………. 8 Lc

Where A; absorbance of the sample, ρ; density of CeO2 NPs (7.28 g cm-3), L; path length of the quartz cell (~1 cm) and c; concentration of the ceria suspensions. In order to determine the optical band gap, we have plotted (αhυ)2 as a function of photon energy. This plot gives a straight line, as shown in inset Fig. 4. The optical band gap is determined by extrapolating the linear portion of the plot to (αhυ) 2 = 0. The calculated values of the optical band gap from the plot were found to be 3.4, 3.3, 3.1and 2.98 eV for S1, S2, S3 and S4 NPs respectively. The red shift was mainly caused by quantum confinement energy, which is inversely proportional to the square of the radius. Thus, a decrease in the nanocrystals size should be accompanied by an increase of the effective width of the forbidden band Eg, the energy difference between the lower energy of conduction band and the upper energy of valence band, which affects the chargetransfer directly. As a result, the absorption band was shifted to the range of lower energy and red shift is observed [46]. Yin et al. reported the direct band gap values ranging from 3.03 - 3.68 eV for CeO2 NPs synthesized using sonochemical synthesis [47]. Ho et al. reported the direct 9

band gap values ranging from 3.36-3.62 eV for mesoporous CeO2 NPs prepared using a polyol method [48]. Most recently, Chen and Chang reported direct band gap values ranging from 3.56 3.71 eV for CeO2 NPs prepared using a precipitation method [49]. The decrease in band gap was attributed to oxygen vacancies and responsible for the unique properties for photocatalytic applications [50]. Since, band gap narrowing is directly proportional to the photocatalytic activity; CeO2 NPs are expected to show more activity in the visible region of the electromagnetic spectrum. Therefore, CeO2 NPs used for dye decolorization under Solar light irradiation. 3.3 Photoluminescence (PL) studies

PL is used primarily to determine the effectiveness of trapping, migration and transfer of charge carriers, as well as to understand the fate of the e-–h+ pairs in semiconductors [51]. In the present work PL is used to understand the optical properties, surface states, oxygen vacancy and defects of the CeO2 NPs. Inset Fig. 5 shows the excitation spectra of CeO2 NPs by monitoring the emission at 425 nm. The excitation spectra consist of a sharp peak at 388 nm corresponding to intra – configuration (f-f) transitions of Ce4+ ions. CeO2 is an important class of luminescent rare earth material. PL spectra of the CeO2 NPs measured using a Xenon laser of 388 nm as excitation source at RT (Fig. 5). The spectra of the CeO2 NPs S1, S2, S3 and S4 show emission bands at 425 (broad and strong), 454, 463 nm (week; blue) along with week blue green band at 478 nm and green bands at 512 and 529 nm. Yu et al. observed a strong blue emission with a peak at 425 nm (2.92 eV) in CeO2 NPs with average size of particles (1.8 nm) prepared by hydrothermal method [52]. They also observed violet / blue light emission peaks at 405, 389, 382, and 368 nm under different concentrations of CeO2 NPs diluted with double-distilled water. The weak blue and weak blue-green emissions of 10

CeO2 NPs were possibly due to surface defects in the CeO2 NPs, and the low intensity of the green emission may be due to low density of oxygen vacancies during the preparation of the CeO2 NPs [53]. The PL intensity was found to increase with increase in LA concentration as shown in inset Fig. 6 (S1 > S2 > S3 > S4). This was attributed to the presence of either oxygen vacancies and /or defects in CeO2 NPs, which leads to an increase in their optical properties. Oxygen vacancies and defects were reported to bind the photo-induced electrons easily to form excitons, so that the PL signal can occur easily. Therefore, the PL signature increased with the increasing content of oxygen vacancies or defects [54-56]. Further, highest PL intensity is observed in S4 sample when compared to S1, S2 and S3. This was attributed to the high concentration of oxygen vacancies and other structural defects which was in accordance with the UV-Visible absorbance spectra. Therefore, PL emission properties of these green synthesized CeO2 NPs can be controlled by their morphology indicating the capabilities of these NPs in photoluminescent, photocatalytic and antibacterial properties. The Commission International De I-Eclairage (CIE) chromaticity coordinates for CeO2 NPs (S1, S2, S3 and S4) were calculated and shown in inset Fig. 6a. It was observed that the CIE co-ordinates of CeO2 NPs was fall in blue region of chromaticity diagram and their corresponding location was marked with star in blue region (Fig. 6a). Coordinated color temperature (CCT) was estimated by Planckian locus, which was only a small portion of the (x, y) chromaticity diagram and there exist many operating points outside the Planckian locus. CCT was used to define the color temperature of a light source and it was found to be 148953 K for CeO2 NPs (Fig. 6b) and CCT coordinates were shown (inset Fig. 6b). However, lamps with a CCT rating below 3200 K are usually considered as “warm” light sources, while those with a 11

CCT above 4000 K are usually considered as “cool” in appearance. Therefore, the present phosphor is highly potential for the fabrication of blue component of white light emitting diodes with cool appearance. 3.4 Morphological studies

Fig. 7 (a-d) shows SEM micro graphs of CeO2 NPs (S1, S2, S3 and S4). From the micrographs it is clear that CeO2 microspheres were relatively uniform and arranged in the form of beads which can be attributed to the mild bio mediated synthesis process. The aggregation of NPs increases with increase in LA concentration from 5 to 20 ml. The main constituents of LA leaves were found to be Phytol (24.55 %), 9, 12, 15-Octadecatrienoic acid (22.97 %), n-Hexadecanoic acid (17.17%), Squalene (5.28%) and 1, 2-Benzenedicarboxylic acid, bis (2-methylpropyl) ester (4.44%) [57]. Structures of Phytol and 9, 12, 15-Octadecatrienoic acid were shown in Fig. S2. When cerium nitrate mixed with LA leaf extract, the Ce4+ ions distribute uniformly and form a three dimensional network structure with Phytol and 9, 12, 15-Octadecatrienoic acid. The resulting networks undergo slow decomposition when subjected to heat treatment. The mechanism of incorporation of CeO2 NPs into the Phytol and 9, 12, 15-Octadecatrienoic acid chains can be represented in Fig. 8 (a, b). Phytol and 9, 12, 15-Octadecatrienoic acid molecules that interact with tetravalent Ce4+ cations forming bridges between two hydroxyl / carbonyl groups from two different chains comes in close contact. The tetravalent ions keep the molecules together and form various structures. This mechanism forms the polymeric nature of bindings and their agglomeration further binds the CeO2 NPs more strongly. This polymeric binding is responsible for the conjugation of all these families of compounds present in the extract and exhibit a synergistic effect to get the complex structures.

12

TEM, high resolution TEM (HRTEM) images and selective area electron diffraction (SAED) patterns were used to study the crystalline characteristics of the CeO2 sample S4 (Fig. 9 (a-c)). TEM image of sample S4 consists of large number of agglomerated cubic flakes (Fig. 9a). The estimated crystallite size was found to be in good agreement with the crystallite sizes determined by Scherrer’s equation. The HRTEM image clearly reveals the lattice fringes with the interplanar spacing of 3.13 Å corresponding to the (111) plane of the cubic S4 NPs suggesting high quality cubic nanocrystals (Fig. 9b). The SAED pattern clearly indicate the high crystalline nature and the circular concentric circles well indexed to (111), (200), (220) and (222) planes of cubic CeO2 NPs (Fig. 9c). 3.5 Photocatalytic studies

RhB is a water soluble fluorescent xanthene dye widely used in textile industries for dyeing cotton, wool and silk. The dye is potentially toxic and carcinogenic to humans and animals and can cause irritation to the skin, eyes, gastrointestinal and respiratory tracts [58]. Fig. 10A and 10B shows the plot of C/C0 versus time for the decolorization of hazardous RhB as a model dye at RT under UVA (370 nm) and Sunlight irradiation for a duration of 90 min. The decolorization efficiency of CeO2 NPs in the dark was less than 5 %, which indicated the value of dye removal by adsorption was insignificant compared to photocatalysis. The decreasing decolorization capacity and rate constant of the catalysts were of the order under UVA irradiation: CeO2 NPs (S4 > S3 > S2 > S1) and under Sunlight irradiation: CeO2 NPs (S4 > S3 > S2 > S1) were tabulated inset (Fig. 10A and 10B) respectively. The photocatalytic efficiencies of the CeO2 NPs increased from 55 to highest activity of 73 % under UVA light irradiation and 61 % to highest activity of 80 % under Sunlight irradiation.

13

The crystallite size of CeO2 NPs plays an important role in the photocatalytic activity. Recently Lei et al. reported CeO2 nanostructure with smaller particle size showed enhanced photocatalytic activity for the decolorization of RhB [59]. The number of active surface sites increases as the particle size decreases hence enhanced photocatalytic activity. Among the CeO2 NPs, S4 had a smaller crystallite size distribution indicated by the XRD data, which showed higher photocatalytic activity for the decolorization of RhB compared to other CeO2 NPs under both UVA and Sunlight irradiation. This indicates that photocatalytic activity shows a proportional relationship to the crystallite size. Therefore, by varying the size of CeO2 NPs, it is possible to enhance the red-ox potential of the valence band holes and conduction band electrons and also the diffusion of photo induced electrons from bulk of CeO2 to NPs surface become fast, which may lead to enhancement of photocatalytic activity under both UVA and Sunlight irradiation. Besides crystallite size, significant by higher UVA and Sunlight light-induced photocatalytic activities of CeO2 NPs can be explained by their decreased energy band gap values. The decreased band gap values of CeO2 NPs with increase in the concentration of LA as indicated by UV−Visible spectroscopy. The enhanced photocatalytic activity of S4 sample compared to other photocatalysts was attributed to its decreased band gap. PL was employed for further investigation of the photocatalytic activities of CeO2 NPs. As the particle size of CeO2 NPs decreases, they demonstrated to have more oxygen vacancies [60]. The decrease in band gap has also been attributed to oxygen vacancies, which is responsible for the unique properties for photocatalytic applications [50]. All the samples S1, S2, S3 and S4 synthesized in the present study shows the presence of oxygen vacancies as indicated by the PL technique and it follows the trend: S4 > S3 > S2 > S1. The higher photocatalytic activity of S4 was attributed to lower 14

recombination between free electrons and energized holes with oxygen vacancies serving as electron traps [61, 62]. The reason can be interpreted as high concentration of oxygen vacancies can trap the electrons, resulting in the holes free to diffuse to the CeO2 surface where oxidation of organic species can occur. Therefore, effective content of oxygen vacancies will improve the photocatalytic process by separating the electron-hole pairs effectively. Further, the differences in photocatalytic activity were highly related to the concentration defects on the surface of the NPs [63-65]. The CeO2 sample S4 with small crystallite size has high level surface defects attributed to the increased concentration of both electron and hole traps as indicated by PL technique. As the particle size decreases, surface defects increases and hence the charge carrier recombination rate decreases which results in the increased photocatalytic activity. In addition to this, when the size of CeO2 nanoparticle decreases the amount of dispersion of particles per volume in the solution will increase, resulting in the enhancement of photon absorbance. Thus, the variation in the photocatalytic activity of CeO2 NPs was supported by PXRD, UV-Visible, PL, SEM and TEM analysis. These results clearly show that the UVA and Sunlight driven photocatalytic activity of CeO2 NPS can be improved greatly by narrowing the band gap and due to the formation of oxygen vacancies and defects. We have further studied the stability and reusability of the S4 photocatalyst by collecting and reusing the photocatalyst for the decolorization of RhB (20 ppm) under UVA and Sunlight irradiation (Fig. 11). Four consecutive recycles were performed, a negligible decrease in the decolorization efficiency occurred which was attributed to loss of photocatalyst during recycling process. It was demonstrated that S4 can be an efficient photocatalysts for the decolorization of RhB dye with high stability and reusability potential as a result it is highly useful for various practical applications. 15

3.6 Antibacterial activity

The antibacterial activity of the prepared S4 nanoparticle towards Gram–ve bacteria Escherichia coli and Gram + ve bacteria Staphylococcus aureus were investigated and the zone of inhibition observed for the concentration of 50 and 100 µL (Fig.12 (b, c)). It was evident from figure that a significant growth inhibition was observed in Escherichia coli and Staphylococcus aureus with diameter 4.67 and 3.33 mm. The formation of zones around the samples demonstrate that the presence of CeO2 NPs effectively prevent the growth of these bacteria. With increase in the CeO2 nanostructure sample concentration, an increase in zone of inhibition was observed in the bacterial growth. This implies the proficient antibacterial activity of CeO2 NPs against E.coli and S. aureus. This might be due to the direct interaction between CeO2 NPs and membrane surface of the bacterial cell causing disruption of the cell membrane. The size of inhibition zone varied depending upon the type of the bacteria, concentration of the antibacterial agent, surface area of the sample, shape and size of the NPs. Further, the difference in activity against E.coli and S.aureus could be attributed to the structural and configurational differences of the cell membrane [66]. The gram –ve bacteria (E.coli) showed the maximum zone of inhibition with 4.67 mm when compared to gram +ve bacteria (S. aureus). The cell wall of gram positive bacteria was mainly composed of a thick peptidoglycon layer consisting of linear polysaccharide chain cross linked by short peptides forming more rigid structure as a result; it was difficult to penetrate CeO2 NPs. However, in gram negative bacteria, the cell wall possesses thinner peptidoglycon layer. In general, gram –ve bacteria have a lipopolysaccharide which protests the cytoplasm membrane from external chemicals. The outer cell membrane of bacteria was damaged by reactive species namely O2-, OH and H2O2, when the tests are performed under UV light 16

irradiation [67]. In addition, the antibacterial activity of CeO2 NPs against Klebsiella aerogenes and Pseudomonas desmolyticum was also tested and the observed zone of inhibition was shown in Fig.12 (a, d). The zone of inhibition data for 50 and 100 µL concentrations were given in Table 3. 4.0 Conclusions

In summary, for the first time CeO2 NPs were successfully synthesized by an inexpensive, nontoxic and ecofriendly bio mediated combustion route using Leucas aspera leaf extract as fuel. The structure, morphology, photoluminescence and photocatalytic properties were sensitively dependent on the concentration of LA leaf extract. CeO2 NPs exhibit blue emission with CIE chromaticity coordinates (0.14, 0.22) and average CCT value was ~ 148953 K. Hence the present phosphor is highly potential for the fabrication of blue component of white light emitting diodes with cool appearance. The photocatalyst S4 showed superior photocatalytic activity for the decolorization of RhB compared with other photocatalysts under both UVA and Sunlight irradiation attributed to crystallite size, oxygen vacancies, surface defects, decreased band gap and capability for reducing the electron-hole pair recombination. The synthesized S4 photocatalyst remain stable even after four consecutive runs demonstrated its high reusability potential which is favorable for the potential applications. Further, CeO2 NPs S4 has significant activity against E. coli and S. aureus with the zone of inhibition diameter 4.67 and 3.33 mm respectively. Acknowledgement

One of the authors, H. Nagabhushana thanks DST Nanomission for sanctioning of the project.

17

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22

Figure captions Fig. 1 PXRD patterns of as formed CeO2 NPs using (a) 5 (b) 10 (c) 15 (d) 20 ml LA leaf extract. Fig. 2 W – H plots of as – formed CeO2 NPs with LA leaf extracts Fig. 3 Rietveld refinement for CeO2 NPs with 20 ml LA leaf extract (Inset: Packing diagram of CeO2 with 20ml LA leaf extract) Fig. 4 UV-Visible spectra of CeO2 NPs with different of LA leaf extract concentration (Inset: Energy band gap diagram of CeO2 NPs obtained with different LA leaf extract concentration). Fig. 5 PL emission spectra of CeO2 NPs obtained with different LA leaf extract concentration excited at 388nm (Inset: Variation of PL intensity of CeO2 NPs versus LA concentration in ml and excitation spectra of CeO2 sample S4 ) Fig. 6 (a) CIE chromaticity diagram of CeO2: LA nanophosphor (Inset: CIE chromaticity coordinates of CeO2 NPs with different LA leaf extract concentrations); (b) CCT diagram of CeO2 NPs with different concentration of LA leaf extract. Fig. 7 SEM images of the CeO2 NPs synthesized using different concentrations of LA leaf extract (a) 5 ml (b) 10 ml (c) 15 ml (d) 20 ml. Fig. 8 Mechanism incorporation of tetravalent Ce4+ ion into structures (a) Phytol (b) 9, 12, 15Octadecatrienoic acid Fig. 9 (a) TEM (b) HRTEM images and (c) SAED patterns of CeO2 NPs with 20 ml LA leaf extract Fig. 10 (A) Plot of C/C0 for the decolorization of RhB under UVA light illumination (Inset: rate constant and percentage decolorization for the decolorization of RhB); (B) Plot of C/C0 for the decolorization of RhB under Sunlight irradiation (Inset: rate constant and percentage decolorization of RhB) Fig. 11 Reusability of the CeO2 NPs (20 ml LA) photocatalyst for 4 consecutive recycles runs under A) UVA light irradiation B) Sunlight irradiation Fig.12 Photograph showing antibacterial activity against (a) Klebsiella aerogenes (b) Escherichia coli; (c) Staphylococcus aureus and (d) Pseudomonas desmolyticum

23

Table captions Table 1 Estimated crystallite size (Scherrer’s formula and W – H method), strain, dislocation density and surface factor of CeO2 NPs. Table 2 Rietveld refinement data of CeO2 NPs with 20 ml LA leaf extract Table 3 Antibacterial activity of CeO2 NPs (20 ml LA) on pathogenic bacterial strains.

24

C eO

2

Intensity (arb.units)

20 m l

15 m l

20

30

(222)

(220)

(200)

(111)

10 m l

5 m l 40

2 θ (d e g r e e )

Fig. 1

25

50

60

C eO

20 m l

2

β cosθ

15 m l

10 m l

5 m l

1 .0

1 .2

1 .4 4 sin θ

Fig. 2

26

1 .6

1 .8

CeO2(20 ml LA) •

Y obs Y calc Y obs – Y calc Bragg _ position

Fig. 3

27

CeO2

-1 2

20 ml

2

(α hν ) (eVcm )

1.2

336 nm 222 nm

Absorbance (arb.units)

1.6

15 ml 10 ml 5 ml

2

0.8

3

4

hν (eV)

5

6

20 ml 15 ml 10 ml

0.4

5 ml 0.0 200

300

400

500

Wavelength (nm) Fig. 4

28

600

700

λex = 388 nm

6

425 nm

6

4x10

6

6

3x10

6

2x10

6

1x10

6

6

1x10

5

10

15

20

LA concentration

0 280

300

320

340

360

380

400

Wavelength (nm)

15 ml

6

1x10

529 nm

512 nm

6

2x10

20 ml

463 nm

454 nm

PL Intensity (arb. units)

6

3x10

5x10

388 nm 2x10

4x10

λemi: 425nm

6

3x10

PL Intensity (arb. units)

5x10

CeO2

PL Intensity

6

10 ml 5 ml

0

420

440

460

480

500

Wavelength (nm) Fig. 5

29

520

540

0.9

CeO2 (5-20 ml LA)

a

X

Y

5 ml 0.14729 0.22367 10 ml 0.14732 0.22782 15 ml 0.1473

0.22811

20 ml 0.14703 0.22852

Y

0.6

0.3

0.0 0.0

0.2

0.4

0.6

0.8

X 0.6 0.5

CeO2 (5-20 ml LA)

b

V'

0.4 0.3

~ 148953 K

0.2 0.1

0.1

0.2

0.3

U'

Fig. 6 30

-5 ml

U' 0.1093

10 ml

0.1083

0.377

142165

15 ml

0.1083

0.3772

140332

20 ml

0.1079

0.3775

138090

0.4

V' 0.3735

0.5

CCT 175226

0.6

a

b

c

d

Fig. 7

31

CH3 H3C

CH3

CH3

CH3

CH3

H3C

CH3

CH3

CH3 CH3

Phytol 4+

+

CH3

H3C

H3C

HO

OH

Phytol

CH3

Ce

CH3

CH3

H3C

HO

OH

CH3 CH3

Phytol

Phytol

CH3

CH3

Phytol chain

O

Phytol chain

O 4+

CH3

CH3

Ce

Phytol chain

Ce

Phytol chain

O

O 4+

- Phytol complex

(a) O

O

H 3C

OH

HO

CH3 9,12,15 - Octadecatrienoic acid

9,12,15 - Octadecatrienoic acid O H 3C

+

Ce

4+

O HO

OH

9,12,15 - Octadecatrienoic acid

9,12,15 - Octadecatrienoic acid

O

O

9,12,15 - Octadecatrienoic acid chain

O Ce

4+

O

O

9,12,15 - Octadecatrienoic acid chain

O

O Ce

4+

9,12,15 - Octadecatrienoic acid chain

O

- 9,12,15 - Octadecatrienoic acid Complex

(b)

Fig. 8

32

9,12,15 - Octadecatrienoic acid chain

CH3

a)

b) d = 3.13Å

c) 4 3

1

2

Fig. 9

33

1 – (111) 2 – (200) 3 – (220) 4 – (222)

CeO2 under UVA

1.0

A Photocatalysts

%D

k x 10-3 (min -1)

S1

55

9.0

S2

62.5

11.1

S3

67.5

12.3

S4

73.0

14.3

C/C0

0.8

0.6

0.4

0.2

S1 S2 S3 S4

0

20

40

60

80

Time (min) CeO2 under Sunlight

1.0

B

C/C0

0.8

Photocatalysts

%D

k x 10-3 (min-1)

S1

61.0

10.6

S2

68.0

12.7

S3

73.5

14.7

S4

80.0

17.6

0.6

0.4 S1 S2 S3 S4

0.2 0

20

40 60 Time (min) Fig. 10 34

80

0.3

CeO2 (S4) under UVA

CeO2 (S4) under Sunlight

A

B

0.2

C/C0

C/C0

0.2

0.1

0.1

0.0

1

2 3 Recycle runs

0.0

4

Fig. 11

35

1

2 3 Recycle runs

4

a

b

c

d

Fig. 12

36

Table 1

CeO2 NPs

S1

S2

S3

S4

hkl 111 200 220 311 111 200 220 311 111 200 220 311 111 200 220 311

Crystallite size (nm) Scherer’s 11.7 12.5 12.0 12.8 6.5 7.2 6.3 6.0 4.7 4.8 4.0 4.9 4.6 4.5 4.3 4.4

W. H Plots

Dislocation density

Strain

Stacking fault

Texture coefficient

0.0073 0.0064 0.0068 0.0060 0.0239 0.0195 0.0251 0.0272 0.0452 0.0435 0.0635 0.1021 0.0476 0.0502 0.0544 0.0507

2.968 2.781 2.880 2.725 7.381 7.235 5.498 5.730 7.381 7.237 8.748 11.090 7.576 7.779 8.093 7.863

0.0061 0.0053 0.0048 0.0042 0.0101 0.0094 0.0091 0.0090 0.0153 0.0094 0.0146 0.0174 0.0157 0.0150 0.0135 0.0123

0.7998 1.1283 1.0097 1.0623 0.7556 1.2025 0.9913 1.0505 0.7046 1.2682 0.9736 1.0536 0.7454 1.2329 0.9585 1.0630

11.0

8.0

5.0

5.0

37

Table 2 Crystal system = Cubic, Space group = F m-3m (No. 225), Lattice parameters, a = 5.427(5) Å, Cell volume = 159.87(3) Å3 R factors (%), Rp = 1.19, Rwp = 1.50, RExp = 6.46, χ2 = 0.05, RBragg = 0.23, RF = 0.16.

Lattice parameters (a) Cell volume R- factors Rp Rwp RExp χ2 RBragg RF

5 ml 10 ml 15 ml 20 ml 5.405(5) 5.415(2) 5.419(6) 5.427(5) 157.86(3) 158.77(8) 158.18(2) 158.87(3) 0.99 1.35 6.03 0.05 0.24 0.21

1.76 2.19 6.24 0.13 0.94 0.65

1.05 1.38 6.21 0.04 0.34 0.25

1.19 1.50 6.46 0.05 0.23 0.16

Atoms Oxidation Wyckoff x y z Biso Occupancy State notations Ce +4 (4a) 0.0000 0.0000 0.0000 0.50 1 O -2 (8c) 0.2500 0.2500 0.2500 0.50 1

38

Table 3

Treatment Standard (5µg/50µL) CeO2 (500µg/50µL)

Klebsiella Aerogenes (B1) (Mean ± SE) 7.67 ± 0.33**

1.00 ± 0.00

Escherichia coli (B2) (Mean ± SE)

Staphyloccus aureus (B4) (Mean±SE)

Pseudomonas desmolyticum (B5) (Mean ± SE)

12.67 ± 0.33**

11.33 ± 0.33**

12.00 ± 0.00

2.67 ± 0.33**

1.67 ± 0.33**

1.00 ± 0.00

3.33 ± 0.67

1.67 ± 0.33**

2.33 ± CeO2 4.67 ± 0.33** 0.33** (1000µg/100µL) Values are the mean ± SE of inhibition zone in mm.

*Symbols represent statistical significance, *P < 0.05, **P < 0.01 as compared with the control group.

39

GRAPHICAL ABSTRACT

CeO2 under Sunlight

1.0

C/C0

0.8

0.6

0.4 S1 S2 S3 S4

0.2 0

20

40 60 Time (min)

80

0.9

CeO2 (5-20 ml LA)

X

Y

5 ml 0.14729 0.22367 10 ml 0.14732 0.22782 15 ml 0.1473 0.22811 20 ml 0.14703 0.22852

Y

0.6

0.3

0.0 0.0

0.2

0.4

X

Bio-mediated multifunctional CeO2 NPs

40

0.6

0.8

Research Highlights •

Bio-mediated route has been employed to prepare multifunctional CeO2 NPs.

•

CeO2 nanophosphor could be a promising candidate for warm blue LEDs.

•

CeO2 NPs showed superior photo catalytic activity under UVA and Sunlight.

•

NPs exhibit significant antibacterial activity against Escherichia coli and Staphylococcus aureus.

41