Studies on catalytic degradation of organic pollutants and anti-bacterial property using biosynthesized CuO nanostructures

Journal of Molecular Liquids 242 (2017) 690–700

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Studies on catalytic degradation of organic pollutants and anti-bacterial property using biosynthesized CuO nanostructures N. Sreeju, Alex Rufus, Daizy Philip ⁎ Department of Physics, Mar Ivanios College, Thiruvananthapuram 695015, India

a r t i c l e

i n f o

Article history: Received 12 April 2017 Accepted 19 July 2017 Available online 20 July 2017 Keywords: Copper oxide nanostructures UV–vis spectrum Psidium guajava FTIR spectrum Catalysis Anti-microbial activity

a b s t r a c t Metal oxide nanostructures have become an inevitable class of materials explored for the environmental and biomedical applications. Thus there is a budding demand to develop a method that purge the need of toxic chemicals. It is a report on a novel and eco-friendly method for the rapid synthesis of copper oxide (CuO) nanostructures using Psidium guajava leaf extract. The synthesis route follows green protocols and exploits the phytochemicals present in the leaves. These nanostructures have been characterized by XRD, UV–vis spectroscopy, FTIR spectroscopy and TEM. BET specific surface area analysis reveals the information of availability of active surface sites in CuO nanostructures. Particle size obtained from XRD, TEM and BET studies are found to be ~17– 20 nm. The XRD pattern, UV absorption at 270 nm and the characteristic IR bands in the region from 400 to 600 cm−1 confirms the formation of nanostructured CuO. The catalytic activity of the synthesized CuO nanostructures has been evaluated by monitoring the degradation of the dyes methylene blue, methyl orange, methyl red and eosin yellow. The synthesized CuO nanostructures could also very effectively catalyze the reduction of the pollutants 2-NP, 3-NP and 4-NP to the corresponding amino compounds. The degradations are observed to be complete within 4–12 min. Anti-microbial activity of CuO nanocrystals against E. coli and S. aureus has also been assessed by agar well diffusion method. This study provides a promising route for the facile and cost-effective synthesis of bio-active CuO nanocatalysts which could find applications in biomedical field and waste water management. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Cupric oxide (Tenorite-CuO), being a heterogeneous catalyst and ptype semiconductor with a narrow band gap of 1.2 eV [1] plays an inconceivable role and has paramount importance in many technological and scientific areas because of their industrial applications. Its potential applications in various fields include electronics, ceramics, sensors, fuel cells, pigments, cosmetics and catalysis [2–3]. There are many reports on the synthesis of nanostructured CuO including nanoribbons, nanobundles, nanorods, nanowires, nanoplates and several shape tunable nanostructures [4–8]. Owing to their outstanding structural flexibility, CuO nanostructures possess unique properties associated with their highly anisotropic geometry and size confinement mechanism [9–10]. From the industrial point of view, the combination of new and conventional properties makes the research of CuO nanostructures with a peculiar morphology and desired functionality very relevant [11–16]. Urchin-like CuO microspheres have been successfully synthesized by a mixed-solvothermal route [17]. Ibrahim et al. [18] have synthesized CuO nanofilms through a spin coating method. Rose-like CuO ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (D. Philip).

http://dx.doi.org/10.1016/j.molliq.2017.07.077 0167-7322/© 2017 Elsevier B.V. All rights reserved.

nanostructures by a mild solution phase route without any templates have also been reported [19]. Flower-like and leaf-like Cuo nanostructures have been synthesized using some ionic liquids through microwave assisted approach [20]. Nanosheets, peachstone-like architecture, nanowires and rectangular shaped nanobot-like CuO nanostructures have also been reported [21–24]. Nanostructures fabricated by chemical methods involve toxic reagents as well as hazardous by-products. This can be avoided by a cost-effective, eco-friendly method termed green approach. The green synthesis includes use of plant parts, biological agents such as bacteria, fungi, antinomycetes and yeast. Sarkar et al. [25] have synthesized CuO nanoparticles using Carica papaya leaves extract. Honari et al. [26] have reported the green synthesis of CuO nanoparticles stabilized using Pencillium aurantiogriseum and Pencillium citrinum. Using gum karaya as a bio template Padil and Černik [27] have fabricated CuO nanoparticles. Through solution combustion synthesis Naika et al. [28] have reported the preparation of CuO nanoparticles using Gloriosa superba leaves extract as a fuel. However, the green approaches towards the synthesis of CuO nanostructures are not thoroughly exploited for dye degradations and anti-microbial activity. The catalytic performance of materials is strongly influenced by their crystallographic nature and morphology. CuO has already been

N. Sreeju et al. / Journal of Molecular Liquids 242 (2017) 690–700

exploited as an upcoming category of heterogeneous catalyst for the complete conversion of hydrocarbons into carbon dioxide and water, nitrous oxide degradation and in improving selective catalytic reduction of nitric oxide with ammonia [29–34]. A large fraction of hazardous environmental pollutants including non-degradable carcinogenic and chemically stable dye stuffs discharged into the water bodies by the textile and paper industries has become an alarming concern among the environmentalists. Technologies such as UV irradiation, coagulation, membrane separation, adsorption techniques and hydrogen peroxide oxidation are not effective for colour degradation. The photocatalytic method of effective decolourization of dyes has also been carried out [25,35]. During such reaction, the semiconducting materials absorb photons, creating charge carriers which further give rise to potential oxidizers of organic dyes. Adsorption has been registered as an effective dye degradation process from waste water effluents [36]. The potential catalytic activity of CuO nanoparticles in the degradation of methylene blue has been reported recently [37]. Besides, CuO nanoparticles synthesized using renewable materials and eco-friendly solvents viable to be applied in the degradation of dye effluents has inspired our current work. Plant mediated synthesis is economic when compared with chemical and physical methods utilized so far. Psidium guajava is a tropical phytotherapic plant known for its potential pharmacologic applications such as anti-oxidant, antiplasmodial, anti-inflammatory and anti-microbial activities. Its leaves contain essential oils enriched in pinene, limonene, menthol, isopropyl alcohol, tannins, flavanoids and terpenic acids, being main carriers behind their bioactivities. The present article focuses on the synthesis of CuO nanostructures using P. guajava leaf extract. Chemocatalytic efficiency and antimicrobial activity of the synthesized CuO nanostructures have been incorporated. The decolourization of the dyes methylene blue (MB), methyl orange (MO), methyl red (MR), eosin yellow (EY) and isomers of nitrophenol (NP)(2-NP, 3-NP and 4-NP) in presence of sodium borohydride (NaBH4) has been chosen as the probe reactions to demonstrate the catalytic capability of the green synthesized CuO nanostructures. The investigation of plant systems as efficant nanofactories has attracted attention in the biological applications of nanomaterials. Moreover, high sensitivity of CuO nanoparticles towards prokaryotes and eukaryotes compared to metal nanoparticles has motivated us to investigate its antimicrobial activities. Copper based products for human use has been approved by the US Environmental protection agency [38]. Hence, the synthesized CuO nanostructures have been tested against pathogenic Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). This cost-effective plant mediated synthesis can be extended as a commercial substitute for the large-scale production of CuO nanoparticles.

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colour of the supernatant solution after precipitation. The precipitate was then centrifuged at 10,000 rpm for 15 min and the product was washed thoroughly using deionised water several times followed by dilute acetone and dried under ambient conditions. The dried precipitate was annealed at 300 °C for 2 h to obtain CuO nanoparticles. 2.2. Catalytic studies The catalytic efficiency of synthesized CuO nanostructures was determined by monitoring the colour deteriorization of the organic pollutants MB, MO, MR, EY, isomers of NP (2-NP, 3-NP and 4-NP). To about 1 mL of 1 mM constantly stirred dye solution 1 mL of 10 mM aqueous solution of NaBH4 was added. The reaction mixture was further made up to 10 mL followed by vigorous stirring for 15 min. About 1 mg of CuO nanopowder in 10 mL of de-ionised water was dispersed using a magnetic stirrer and was then ultrasonicated for 20 min until a transparent homogeneous solution obtained keeping sonication temperature at about 25 °C. Aliquots of this solution were transferred drop wise to all the dye solutions keeping stirring for two more minutes. For a given time interval, 2 mL of the solution mixture was then taken in a quartz cuvette (path length-10 mm), and its absorption spectra was observed using a UV–vis spectrophotometer to establish an equilibrium between CuO nanocrystals and dyes. The degradation reactions were monitored through the declined absorbance at wavelength corresponding to the maximum absorption for each toxic pollutant in the UV–vis region, measured as a function of time according to the Beer's law. The same procedure was repeated with 1 mL of 10 mM isomers of NP and 1 mL of 0.25 M of NaBH4 in 25 mL aqueous solution. 2.3. Antimicrobial studies Antimicrobial activities of the CuO nanostructures were examined against Gram negative E. coli bacterium and Gram positive S. aureus bacterium through agar well diffusion method. Suitably grown microbials were allowed to interact with 20 mL of Muller-Hintor Agar medium taken on to 100 petriplates. The turbidities of the suspensions of microorganisms were adjusted to a 0.5 McFarland standard. 10 mm sized wells were punctured using a well cutter and different volumes (25, 50 and 100 μL) of CuO nanocrystals (0.001% concentrated) were incorporated; followed by incubation of the plates at 37 °C for 24 h. By measuring the diameter of the inhibition zone, a confluent margin lawn of growth, the bactericidal efficiency of as-prepared CuO nanocrystals was ascertained. 10 mg/mL concentrated streptomycin was taken as standard antimicrobial agent. 2.4. Characterization

2. Materials and methods Fresh leaves of P. guajava were collected from the botanical garden of Mar Ivanios College. Copper (II) sulphate pentahydrate (CuSO4·5H2O (N99%)) from Sigma-Aldrich was used as the precursor. All organic pollutants were of analytical grade and were used without further purification. All glass wares were cleaned using aqua-regia and deionised water was used throughout the synthesis. 2.1. Synthesis of CuO nanostructures 10 g fresh leaves of P. guajava were boiled in 100 mL of deionised water for about 2 min. The clear solution obtained after filtration was used for further experiments. The precursor solution was prepared by mixing 0.05 M CuSO4·5H2O crystals in 100 mL deionised water and stirred well for 5 min using a magnetic stirrer. The solution is then allowed to boil and 15 mL P. guajava leaf extract was added drop wise under constant stirring. The blue colour of the solution gradually appeared pale green followed by the formation of a turbid brown precipitate. The volume of the extract has been optimized by observing the

X-ray diffraction (XRD) pattern of CuO nanostructures were recorded on a Bruker AXS D8 Advanced Focus X-ray diffractometer using Cu Kα radiation (λ = 0.1542 nm) operated at 40 kV and 40 mA. UV–vis absorption spectrum was obtained using Varian Cary 5000 UV–vis-NIR spectrophotometer. Absorption studies of catalysis were carried out on Perkin-Elmer Lambda-35 UV–vis spectrophotometer. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) observations were performed on a JEOL JEM 2100 transmission electron microscope at an acceleration voltage of 200 kV. Thermo Nicolet Avatar 370 spectrophotometer was used to obtain the FTIR spectra of the extract and nanoparticles. 3. Results and discussion 3.1. Structure and morphology of CuO nanostructures Crystallinity and crystallite size of CuO nanostructures have been probed by X-ray diffraction technique. Fig. 1(a) shows the XRD pattern of the synthesized product just before annealing. The complete phase

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Fig. 1. X-ray diffractogram of as-prepared material (a) before annealing and after annealing at (b) 300 °C (c) 600 °C and (d) 900 °C and (e) the Williamson-Hall plot of CuO nanostructures.

formation of CuO has not been achieved as evident from the peaks. The XRD patterns of CuO nanopowder obtained after annealing at three different temperatures (300 °C, 600 °C and 900 °C) are shown in Fig. 1(b–d). All the diffraction peaks can be indexed to the monoclinic phase of CuO (space group C2/c (15)), lattice parameters a ¼ 4:618 A, b ¼ 3:431 A and c ¼ 5:119 A which are in good agreement with JCPDS card No. 89-2529. Characteristic diffraction peaks are observed at 2θ values of 32.6°, 35.6°, 38.8°, 48.9°, 53.0°, 58.2°, 61.6°, 66.3°, 67.9°, 72.1° and 74.9° corresponding to the crystal planes (110Þ; ð1 11Þ; ð200Þ; ð202Þ; ð020Þ; ð202Þ; ð113Þ; ð310Þ; ð220Þ; ð311Þ and (044) of tenorite respectively [39]. No other peaks are observed indicating the high purity of as-prepared CuO nanostructures. The relatively broader diffraction peaks suggest the smaller crystallite size of the CuO structures. The particle size increases as annealing temperature rises to 600 °C, then to 900 °C indicating the increase in the crystallinity of the product. By the use of Scherrer formula [40], D =(0.891 λ)/(βhkl cos θ), where λ is the X-ray wavelength and θ and β are the diffraction angle and fullwidth at half maximum (FWHM) of the reflections respectively, the average crystallite size (D) calculated is found to be 17.91 ± 1.36 nm.

Larger particle size of the nanostructures reduces the surface activity; hence the further studies have only been focused on the CuO nanostructures annealed at 300 °C. Broadening of the diffraction peaks can mainly be attributed due to the deviations from perfect crystallinity extended infinitely in all directions. In addition to the crystallite size, lattice strain including grain boundary, triple junction, stacking faults can also be extracted from the peak width analysis. Lattice strain increases the peak width and intensity shifting the 2θ peak positions accordingly and varies as tan θ from the peak width. Williamson-Hall analysis is an integral-breadth method, according to the equation,

βhkl cosθ ¼

  kλ þ 4ε sinθ D

ð1Þ

where strain (ε) and particle size are calculated from the slope and y-intercept of the fitted line (Fig. 2(e)), which are found to be 2.9 × 10−3 and 20.09 nm, respectively.

Fig. 2. TEM micrographs (a–b) of CuO nanospheres at different magnifications. (c) SAED pattern.

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Further morphological characterization of the CuO nanostructures have been probed by TEM. Fig. 2 shows TEM images of the as-prepared CuO nanostructures under two magnifications along with SAED pattern. The particles are found to be rather elongated spheroids. The average particle size is found to be 19.19± 1.61 nm, which is in agreement with XRD studies. Crystallinity of the nanoparticles is evident from the SAED pattern. 3.2. Optical characterization of CuO nanostructures To investigate the energy structures and optical properties of semiconductor nanocrystals, absorption measurements in the UV–vis region has been explored extensively [41]. The UV–vis absorption spectrum of the synthesized CuO nanostructures is shown in Fig. 3(a). There is absorption band centred at 270 nm with a shoulder at around 388 nm. An obvious blue-shift has been observed in comparison with the urchin-like CuO nanospheres having absorption band at 360 nm reported by Hong et al. [17]. Such a blue-shift has been reported for CuO quantum dots in literature [42], due to the quantum confinement effects. The exciton Bohr radius, the characteristic size below which a fundamental shift in electronic and optical properties has been observed is reported in the range of 6.6–28.7 nm for tenorite [42]. The nanostructures synthesized through this route are found to be within this range and therefore within a strong confinement regime. Further, a classical Tauc approach has been employed to elucidate the bandgap ( Eg ) of CuO nanostructures according to the equation αEphoton ¼ B Ephoton −Eg


m2

ð2Þ

where B is a constant, α is the absorption coefficient of the nanoparticles. The exponent m depends on the nature of the transition of electron between valance and conduction band, m= 1 , 4 ,3 , 6 for allowed direct, allowed indirect, forbidden direct and forbidden indirect transitions respectively. Fig. 3(b) shows the Tauc plot of CuO nanostructures. The energy intercept of the plot of (αEphoton)2 versus Ephoton yields Eg for the direct transition. The absorption value α ≥ 104 cm−1 is a consequence of direct band transitions [43–45]. The optical energy gap has been estimated as 2.45 eV by extrapolating the linear portion of this plot at (αEphoton)2 = 0. Compared with that of the bulk (1.2 eV), the value has been blue-shifted by 1.25 eV due to quantum size effects. This result promises the suitability of these phytosynthesized CuO nanocrystals in optoelectronic and photovoltaic applications. The occurrence of surface-related defects significantly influences the absorption phenomena. The role of electronic defects such as oxygen vacancies and Cu1+ has been reported by Ovchinnikov et al. [46]. It is observed that the CuO nanostructures are formed by aggregating a number of CuO

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nanoparticles, and hence the structure effectively affects in quantum confinement mechanism [47]. Detailed studies are necessary to substantiate such explanations. 3.3. FTIR analysis of CuO nanostructures FTIR studies have been carried out to characterize the surface of the CuO nanostructures as well as to determine the presence of organic species on them. Metal oxides generally give absorption bands below 1000 cm−1 arising due to interatomic vibrations. Fig. 4 shows the comparative FTIR spectra of the synthesized CuO nanostructrures and P. guajava leaf extract. A few bands in the spectrum of extract exhibit varied intensity and appreciable shifts when compared with the spectrum of CuO nanostructures. The sharp band centred at 1626 cm−1 in the CuO nanocrystals and at 1641 cm−1 is due to the bending mode of hydrated water and carbonyl stretching vibrations. The two strong bands at 1548 and 1411 cm−1 in the extract which arise from amide I and amide II vibrations [48–50] appear as very weak bands in CuO indicating the presence of organic phase on the surface of the synthesized nanomaterial. This is supported by the presence of medium intense band at 1025 cm− 1 due to C\\N stretch in the IR spectrum of the extract which appears as very weak and broad in the spectrum of CuO. This spectral analysis supports the involvement of phytochemicals such as proteins and flavanoids in the formation of CuO nanostructures. CuO crystallizes in the space group C2/c with four molecules in the unit cell [6]. The irreducible representations excluding acoustic modes are distributed among the different symmetry species as follows: Γ Cuo ¼ Ag þ 2Bg þ 3Au þ 3Bu

ð3Þ

Among these six zone centre modes are IR active (3Au + 3Bu). The bands observed around 581, 536, 485 and 410 cm−1 are due to Cu\\O stretching vibrations [51]. The size induced lattice variations and crystal defects like edge dislocations can be pointed out as the reason behind the shifting of IR bands. No bands due to the Cu(II)-O vibrational mode in the range of 605 cm− 1 to 660 cm−1 has been observed which totally rules out the existence of Cu2O phase and the result supports the purity of CuO nanocrystals [52]. The role of P. guajava leaf extract as an exceptional stabilizer on the surface of copper nanoparticles has been reported recently [53]. The water soluble phytocomponents such as isopropyl alcohol, polyphenols, avicularin, pinene, limonene, carboxylic acids and ketones releases hydrophilic hydroxyl radicals into the precursor solution forming Cu(OH)2. The pale green colour of the solution indicates the beginning of the formation of hydroxide and heating process accelerates the same. The volume of leaf extract has to be optimized in such a manner

Fig. 3. (a) UV–vis absorption spectrum of CuO nanostructures. (b) Tauc plot for the direct allowed transition of CuO nanostructures.

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However, it is important to control the morphology of CuO nanostructures since morphology plays an important role in catalysis. 3.5. Catalytic degradation of dyes Large surface area and more active sites in CuO nanostructures lead to potential catalytic capabilities [54]. In the present work, certain toxic organic pollutants such as MB, MO, MR, EY, and isomers of NP in aqueous solution have been chosen as representative dye stuffs to evaluate the chemocatalytic performance of CuO nanostructures. Throughout the process NaBH4 has been used as reducing agent. 1 mL aqueous solution of CuO (10−2%) is taken as the optimum amount whose catalytic proficiency has been examined.

Fig. 4. FTIR spectra of CuO nanostructures and P. guajava leaf extract.

that all the Cu ions in the solution get precipitated. On annealing, the hydroxide ultimately transforms into oxide with well defined crystallinity. 3.4. Surface area determination of CuO nanostructures Active surface sites and surface area are important factors that determine the catalytic performance of nanocrystals. Using BrunauerEmmett-Teller (BET) method by Micromeritics Gemini 2360 surface area analyzer, the surface area of CuO nanostructures has been estimated. The nanocrystals were prepared using a Gemini Vac Prep degasser. The measurement of the surface of the crystal is taken from the surface where nitrogen gas molecules are allowed to adsorb. Dry and degassed samples were analysed using multipoint adsorption technique. Mean particle size (D) of the nanocrystals can be estimated as, D = 6/(δ × AS), where δ is the density of CuO (6310 kg/m3) and AS is the specific surface area of the nanocrystals. Using BET method, assuming the particles are spherical in shape and separated apart, the mean surface area obtained is 48.453 m2/g. The average particle diameter is found to be ~ 19.58 nm, which is in good agreement with TEM and XRD studies. BET results showing large surface area of CuO nanostructures, has further encouraged us to investigate its catalytic activity.

3.5.1. Catalytic degradation of MB Being a promising candidate in the textile industry, MB causes several environmental issues due to its toxicity. This cationic dye has two characteristic absorption bands, one prominent band at around 665 nm and another shoulder band at around 612 nm. Fig. 5(a) displays the typical time-dependent UV–vis absorption spectrum of MB solution. During the course of degradation, the colour of MB goes on fading and the intensity of absorption decreases gradually implying a strong reduction of MB. Finally, the band became very broad and weak suggesting nearly complete degradation of MB into its protonated form, leucomethylene blue. The reaction was almost complete within 12 min. The rate of disintegration decreases rapidly with increase in contact time from 1 to 9 min and reaches an optimum level when the degradation equilibrium achieves. The rate of removal of N75% has been observed in the first 5 min (Fig. 5(b)). 3.5.2. Catalytic degradation of MO MO, representing an anionic azo dye is a well-known carcinogenic substance and a major effluent from cosmetic and analytic industries. Its removal from water bodies is a major challenge to environmental scientists. A reductive degradation of MO has been included to probe out the catalytic efficiency of as-prepared CuO nanocrystals. The aqueous solution of MO shows a characteristic wide band centred at around 460 nm due to π− π∗ electronic transitions arises from\\N_N\\group [55].

Fig. 5. (a) UV–vis spectrum showing degradation of MB supported by CuO nanocrystals. (b) Time profiles of lnðAA0t Þ and percentage of dye degradation. (c) Schematic representation of degradation of MB into leuco-methylene blue.

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In the absence of any catalyst, the reduction of MO attains a low pace in the presence of NaBH4 even though the reaction is thermodynamically favourable. The reduction is found to proceed faster after the incorporation of CuO nanocrystals, as it is evident from the suppression of band at 460 nm shown in the Fig. 6(a). The dye ultimately decolourizes completely forming hydrazine derivatives within 4 min. About 85% of the dye has been found to be degraded within the first 2 min, after that the degradation attains a steady state Fig. 6(b). The results show that the dye removal process is supposed to be independent of the concentration of NaBH4 in comparison to the dye concentration. 3.5.3. Catalytic degradation of MR The catalytic studies have also been extended to the degradation of yet another azo dye, MR which shows a prominent absorption band at around 430 nm. This band is responsible for the π − π∗ electronic transitions of the yellow anion form of O-MR. The intensity of MR could be taken as an indication of its amount left in the aqueous solution. On the addition of NaBH4, the intensity of absorption band decreases very slowly due to the large kinetic barrier and low encounter probability [56–60]. The CuO nanocatalysts accelerate the reduction mechanism and the results are shown in the Fig. 7(a). The significant decline in the absorption is due to the cleavage of azo bonds into colourless amino compounds [57], which results in the concomitant appearance of a weak band at around 300 nm. A rapid decolourization has been initiated between 1 and 2 min. After 3 min the reduction attains equilibrium and completes within 4 min (Fig. 7(b)). 3.5.4. Catalytic degradation of EY As a fluorescent anionic dye, EY which displays greenish yellow fluorescence is used in printing, dyeing and leather industries [58]. EY is characterized by an absorption band at around 514 nm, and decolourizes from orange to pale yellow during to its reduction from ES2 − to ESH2. Fig. 8(a) shows the absorption spectrum corresponding to the degradation of EY supported by CuO nanocrystals which is complete within 4 min having N 85% of dye degradation during first 2 min.

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3.5.5. Catalytic degradation of nitrophenols Nitrophenols are hazardous anthropogenic pollutants substantially used in the field of drug delivery, whose severe effects from the environment can be eradicated through the insertion of nanostructures. The present article describes the catalytic degradation of three isomers of nitrophenols such as 2-NP, 3-NP and 4-NP in the presence of excess NaBH 4 . Nitrocompounds are characterized by an intense absorption at around 400 nm corresponding to nitrophenolate ion that impart greenish yellow colour. In parallel a new band at around 290 nm is found to appear corresponding to their respective aminophenols as reaction proceeds [59]. In the absence of any catalyst, the completion of reaction takes several days. The greenish yellow colour diminishes completely in support of CuO nanocrystals within 4 min, 7 min and 6 min for 2-NP, 3-NP and 4-NP respectively as shown in the Fig. 9(a–c). The results show almost similar time dependence for dye degradation. For all the three dyes, within the first 3 min the decoulorization has almost completed N 50%. In comparison to 2-NP and 4-NP, the degradation of 3-NP attains a steady state after 3 min until completion (Fig. 10(b)).

3.5.6. Catalytic efficiency of CuO nanocrystals and kinetic studies During the degradation of dyes, the large redox potential barrier between donor (BH 4 − Þ and acceptor (dyes) kinetically hinders its rapid deteriorization. In addition to the intermediate redox potential with respect to the aforementioned donor and acceptors, the availability of large surface active sites as evident from BET measurements, unambiguously favours the catalytic reaction of as-prepared CuO nanocrystals. It initiates the double electron transfer (electron relay effect) adsorbing the dye stuffs owing to an electrostatic force of attraction and terminates the desirable reactions within few minutes [60]. The presence of excess NaBH4 in all the reactions confirms the reactions to follow a pseudo first order kinetics [61] since there is a linear correspondence  between ln ðAA0t ) and time as per the equation, lnðAt A Þ ¼ −kt; 0

where k is the pseudo first order rate constant and At, A0 are the corresponding absorbance values of dye stuffs at the tth and 0th minutes

Fig. 6. (a) UV–vis spectrum showing degradation of MO supported by CuO nanocrystals. (b) Time profiles of lnðAA0t Þ and percentage of dye degradation. (c) Schematic representation of degradation of MO into hydrazine derivatives.

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Fig. 7. (a) UV–vis spectrum showing degradation of MR supported by CuO nanocrystals. (b) Time profiles of lnðAA0t Þ and percentage of dye degradation. (c) Schematic representation of degradation of MR into hydrazine derivatives.

respectively. The percentage of decolourization at time t has been calculated using the equation,  Decolourization % ¼

At 1− A0

  100

ð4Þ

The percentage of dye degradation (% D), rate constants (k) and the time for degradation for different dyes are given in Table 1.

3.6. In-vitro antimicrobial activity of CuO nanostructures From the industrial point of view, the organic compounds used for the purpose of disinfection are limited due to its toxicity towards healthy cells and instability at high temperatures and pressures. Thus we are at the cutting edge stage to find the alternative effective agent to deactivate microorganisms. Here comes the significance of metal oxide nanoparticles fabricated through green method.

Fig. 8. (a) UV–vis spectrum showing degradation of EY supported by CuO nanocrystals. (b) Time profiles of lnðAA0t Þ and percentage of dye degradation. (c) Schematic representation of degradation of EY.

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Fig. 9. UV–vis spectra showing degradation of (a) 2-NP, (b) 3-NP and (c) 4-NP supported by CuO nanocrystals.

Antimicrobial efficiency of CuO nanostructures has been evaluated against E. coli and S. aureus with different volumes (20, 50 and 100 μL) by agar well diffusion method. Preliminary research on the antimicrobial capabilities of nanoparticles has shown that CuO nanoparticles release Cu++ ions that cause local pH and conductivity changes. The liberation of these metal ions will gain the capability to deactivate microbials. Cu++ ions are little enough required to disrupt bacterial cell membranes and enzymatic functionalities. Due to the complex cell wall structure, both E. coli and S. aureus show resistance to mostly available drugs. High surface area and reduced

particle size of CuO nanostructures promise higher chemical and biological reactivity towards microorganisms in the form of inhibition zones as shown in the Fig. 11(a) and (b). The size of the inhibition zone and thus antimicrobial potency is found to be linearly dependent with CuO dosage (Fig. 11(c)). The results reveal that CuO nanocrystals exhibit superior bactericidal susceptibility against E. coli. The difference in the activity against these two bacteria could be attributed to the structural and compositional variations of the cell membranes [62]. The inhibition zones for both the bacterials are much clear without any white patches, which

Fig. 10. Time profiles of (a) lnðAA0t Þ and (b) percentage of dye degradation.

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Table 1 The percentage of dye degradation (% D), rate constants (k) and the time for degradation for different dyes. Dye MB MR MO EY 2-NP 3-NP 4-NP

Rate constant k (min−1)

Time for degradation (min)

Percentage of degradation

0.419 0.789 0.734 1.601 0.820 0.885 0.261

12 4 4 4 4 7 6

91 89 80 97 95 86 99

demonstrate the anti-bactericidal efficiency of CuO nanostructures. The lowest concentration of CuO nanocrystals causing anti-bactericidal effect, the minimum bactericidal concentration (MBC) determined from the batch culture studies along with zone of inhibition (ZOI) for both bacteria are tabulated in Table 2. Based on the results, it can be concluded that the green synthesized CuO nanostructures has significant antibacterial action against both of the Gram classes of bacteria. The greater abundance of amines and carboxyl groups on their cell surface enhances the affinity of Cu ions towards these groups [63], which results in high antimicrobial proficiency of CuO nanostructures. The differential sensitivity of CuO nanocrystals against selected bacteria particularly depends upon its particle size and morphology. Antimicrobial studies can also be extended in search of some morphological alterations by varying experimental conditions such as concentration of extract and precursor solutions, temperature, stirring time and pH rather elongated spherical nanostructures.

Table 2 The zone of inhibition (ZOI), minimum bactericidal concentration (MBC) for CuO nanostructures and the positive control chosen against E. coli and S. aureus. Bacteria

Sample/control

Zone of inhibition (mm)

MBC (μL)

E. coli

CuO Streptomycin CuO Streptomycin

16 39 12 39

b75 b20 b50 b20

S. aureus

4. Conclusions We demonstrate a single-step, fast, reliable and scalable route towards the successful synthesis of CuO nanocrystals using P. guajava leaf extract followed by thermal treatment at 300 °C for 2 h. This method is found to be novel and environmental-friendly and the synthesis parameters have been optimized in order to avoid agglomeration. The formation of CuO nanostructures substantially exploits the water-soluble phytocomponents present in the leaf extract. The crystallite size calculated by XRD and TEM studies are 17.91 nm and 19.19 nm, respectively. Additionally, UV–vis spectroscopic studies disclose the information regarding morphology dependence of absorption and band gap (2.45 eV) of CuO nanospheres. The involvement of phytochemicals during synthesis has been demonstrated through FTIR responses. Moreover, the exceptional catalytic efficiency of the synthesized CuO nanocrystals has been evaluated by monitoring the degradation of a series of dyes such as MB, MO, MR, EY and nitrophenols (2-NP, 3-NP and 4-NP) in presence of NaBH4, which are found to follow pseudo first order kinetics. BET surface studies have also been encouraged to investigate the bio potency of synthesized CuO nanocrystals besides chemocatalytic activities. This includes the anti-microbial activity

Fig. 11. Microbial growth inhibitions by CuO nanocrystals against (a) E. coli and (b) S. aureus. (c) Dose dependency on zone of inhibitions.

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against E. coli and S. aureus through agar-well diffusion method. It is expected that this promising green approach of synthesis can be extended in industrial scale for potential applications including waste water treatment and disinfection purposes.

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