Evidence of oxygen defects mediated enhanced photocatalytic and antibacterial performance of ZnO nanorods

Colloids and Surfaces B: Biointerfaces 184 (2019) 110541

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Evidence of oxygen defects mediated enhanced photocatalytic and antibacterial performance of ZnO nanorods

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Jaspal Singha, , Subhavna Junejab, Shatrudhan Palsaniyac, Ashis. K. Mannad,e, R.K. Sonia, Jaydeep Bhattacharyab a

Department of Physics, Indian Institute of Technology Delhi, New Delhi, 110016, India School of Biotechnology, Jawaharlal Nehru University, New Delhi, India c Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, India d Institute of Physics, Sachivalaya Marg, Bhubaneswar, Odisha 751005, India e Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400085, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Oxygen defect ZnO Nanorod Photocatalytic activity Antibacterial activity

Defect engineered one-dimensional (1D) ZnO nanostructures have found great interest in diverse fields, including water detoxification and environmental remediation. In this article, we report a facile, low-temperature hydrothermal synthesis of defect enriched ZnO nanorods at different pH conditions. The dimension of all the synthesized ZnO nanostructures was restricted to 1D with changes only in their aspect ratios, unlike previous reports where change in morphology accompanies the effect of pH. With an increment in the pH value of the reaction mixture, oxygen defect concentration was controlled and confirmed using XPS and Raman spectroscopy. Considerable increase in optical light absorption and reduction in the bandgap, as inferred from the UV–vis study, corroborating the pH-dependent enrichment of defect states in 1D ZnO. Superior photosensitivity of oxygen defect rich ZnO nanorods was utilized to study their sunlight-induced photocatalytic and bactericidal activity towards its application in wastewater treatment. Within 4 h and 30 min of sunlight exposure (900 W/ cm2), a 100% bacterial population (S.aureus, 106 cells/m) killing and complete degradation of methylene blue dye (10μM) were achieved. Enhanced reactive oxidative species (ROS) formation due to the presence of additional oxygen defect states is ascribed to be the prime factor facilitating improved degradation efficiency. Additionally, during the optimization study, ZnO nanorods were found to be active against bacterial cells even in the absence of light opening avenues in antimicrobial food packaging and protective surface coatings.

1. Introduction Characterized as functional and versatile, one-dimensional (1D) metal-oxide semiconductor nanostructures find extensive use in different applications such as gas sensors [1], photovoltaics [2], photodetectors [3], energy storage [4] and environmental remediation [5]. Their efficient and fast electron flow along with a large surface area to volume ratio, light sensitivity and high chemical activity makes them an outstanding material for fabricating light-harvesting and optoelectronic devices [6]. Semiconductor metal oxide assisted photocatalysis, as a green energy harvesting alternate for energy production, toxin degradation and antimicrobial killing has been garnering enough attention universally [1,5,6]. One dimensional metal oxide nanostructures with high mobility and improved charge separation are known to enhance the photocatalytic

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activity significantly [7,8]. Marked by a direct wide bandgap (3.3 eV), large binding energy (60 meV), environmental benignity, low cost, high quantum yield and high photostability, ZnO metal-oxide semiconductor fulfils all the basic requirements of an ideal photocatalyst justifying its abundant use in photocatalytic reactions [9]. ZnO reciprocates to UV light absorption by generating charge photocarriers (electron-hole pair) in their conduction and valance band, respectively. The photo-charge carriers upon interaction with water to yield ROS, which attacks the organic pollutants leading to their degradation. Adverse to its advantages, ZnO nanostructures are also identified with a high photoinduced charge carrier recombination rate and selective sensitivity to UV light, limiting its photocatalytic efficiency. Numerous strategies aimed at improving light responsiveness and retardation of recombination rates have been adopted for achieving higher degradation efficiencies [10–16]. Metal doping [17,18], nonmetal doping [19–21],

Corresponding Author at: Department of Physics, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. E-mail address: [email protected] (J. Singh).

https://doi.org/10.1016/j.colsurfb.2019.110541 Received 5 June 2019; Received in revised form 26 September 2019; Accepted 29 September 2019 Available online 03 October 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. (a–d) FESEM images of ZnO nanorods sample ZP, ZP1, ZP2 and ZP3 respectively, (e–h) High-resolution FESEM images of samples ZP, ZP1, ZP2 and ZP3 respectively.

complexities or complex mechanism [26]. In addition to altering the wettability and hydrophobic properties in ZnO, oxygen defects have proven to modulate charge separation characteristics, subsequently leading to a modified photocatalytic and antibacterial efficiency [27–29]. Several research efforts have been directed towards preparation of self-doped ZnO [30–32] with markedly improved photocatalytic activity owing to a reduced bandgap sequential to the generation of defect states below the conduction band. Different research groups [33–38] have synthesized vacancy rich ZnO for improved treatment of

decoration with noble metal nanoparticle [22,23] and semiconductor heterojunction formation [24,25] are a few of them. Although beneficial, these methods involve complex synthesis processes and high costs making it difficult to scale up for industrial production and application, inviting of simpler cost-effective routes. Introducing additional defect states in the form of oxygen vacancies has been proposed recently as a facile method to improve the photocatalytic efficiency of ZnO. Introduction of defect states can be achieved by simple tuning of the chemical reaction parameters without the use of any synthetic 2

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Fig. 2. (a) X-ray diffraction patterns of ZnO nanorods sample ZP, ZP1, ZP2 and ZP3, respectively, (b) Raman spectra of ZnO nanorods sample ZP, ZP1, ZP2 and ZP3.

Fig. 3. (a) UV–vis spectra of sample ZP, ZP1, ZP2 and ZP3, (b) Tauc plots for the sample ZP, ZP1, ZP2 and ZP3, (c) Photo-luminescence spectra of sample ZP, ZP1, ZP2 and ZP3.

concentration induced into ZnO nanorods sample by variation of reaction pH. In addition to its influence on photocatalytic and antibacterial efficiency, defect associated changes in optical and structural properties were also investigated. Variation in defect states in ZnO nanorods synthesized at different pH was precisely studied through XPS and collaborated to its photolytic and antibacterial activity. A linear relationship between activity and defect state concentration was established where increasing defect state presence was associated with higher ROS species generation and thus higher killing and degradation efficiency. Gram-positive, S.aureus and organic textile dye, Methylene Blue were used as model contaminants for the study. We have highlighted the importance of the antibacterial aspect of ZnO nanorods photocatalyst which is rarely reported.

organic, inorganic and biological contaminations in wastewater. Sabahi et al. [39] established a linear relationship between oxygen defect states and the photocatalytic efficiency of ZnO. They showed that ZnO nanorods with the highest density of surface defects exhibited four times better photocatalytic degradation for 10 ppm solution of phenol under visible light compared to pristine ZnO. Orou et al. [40] reported the preparation of ZnO nanodisc and nanorod like structures and compared their antimicrobial activity under UV light. They achieved complete inactivation of Bacillus subtilis and S. aureus using each type of ZnO at 128 and 256 μg/ml respectively. In another study, Dutta et al. [41] studied the effect of different capping agents on the ZnO nanoparticles to tune their oxygen defect concentration. The synthesized ZnO nanoparticles were later employed to study their antibacterial activity against gram-negative E. Coli bacteria strain. Bacterial inactivation was found to be strongly influenced by defect state concentration, with maximum killing obtained with ZnO with highest defects concentration. In this report, we studied a systematic variation of defect state 3

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Fig. 4. (a–d) Gaussian fitted XPS spectra of O1s peak for sample ZP, ZP1, ZP2 and ZP3, respectively.

2. Experimental detail

the optical absorption was performed on a PerkinElmer UV WinLab. Photoluminescence spectra of ZnO nanostructures were recorded through Perkin Elmer lambda with Xenon lamp source (excitation −325 nm). The surface area of the as-synthesized ZnO nanostructures were estimated through the BET (Brunauer-Emmett-Teller) method. For each BET measurement, the conventional degassing-adsorption cycle was followed. Following high temperature (150 °C, 5 h) degassing under vacuum conditions, sample chamber was inserted into measurement zone where liquid nitrogen maintained at a flow rate of 0.7 kg/cm2 is in flux. Changes in effective pressure within the chamber with Nitrogen gas adsorption on ZnO nanostructures were measured and tabulated for assessing the surface area of the sample. The BET system used for measurement was Micromeritics Instrument, TriStar II 3020 Version 3.02.

2.1. Materials Zinc nitrate hexahydrate (Zn(NO3)2.6H2O), Hexa Methyl Tetra Amine (HMTA) (C6H12N4) and sodium hydroxide (NaOH) employed for the preparation of ZnO nanostructures. Zinc nitrate hexahydrate, HMTA, NaOH and methylene blue (MB) were purchased from SRL, India. Luria Bertani (LB) Agar and Luria Bertani Broth were purchased from HiMedia, India. All chemicals were used as purchased. 2.2. Synthesis of ZnO nanorods For the synthesis of ZnO nanostructures, a 100 ml aqueous solution of zinc nitrate (1 M) was thoroughly mixed with 100 ml aqueous solution of HMTA (1 M). The reaction mixture was maintained under constant stirring and heating at 70 °C for the next 5 h. After the requisite time, heating was switched off, and the sample was allowed to cool down to room temperature gradually. Following sample precipitation, through washing and centrifugation was performed to remove any unreacted reactants. The centrifuged sample was transferred to a crucible and allowed to air-dry overnight in a hot air oven maintained at 80 °C. The pH of the reaction mixture was varied from 5.0 to 6.5 by using 2 M NaOH solution. The ZnO samples synthesized at pH value 5.0, 5.5, 6.0 and 6.5 named as ZP, ZP1, ZP2 and ZP3, respectively.

2.4. Photocatalysis test The photo-degradation capability of ZnO nanostructures was assessed spectrophotometricallyby studying the degradation behaviour of methylene blue dye under solar light. For each test, an ultrasonically well-dispersed ZnO solution (1 mg/ml) was mixed with 10μM MB dye solution. The reaction solution was initially maintained in the dark for 30 min to attain adsorption-desorption equilibrium followed by its subsequent exposure to sunlight (900 W/cm2). Within the stipulated time duration for the reaction, an equal amount of reaction mixture was aliquoted at regular intervals (5, 10, 20 and 30 min) for acquiring the absorption data. Change in absorption intensity at characteristic 664 nm absorption peak for MB dye was used to collaborate effective dye concentration in the reaction mixture. Notably, before UV–vis absorption measurement, ZnO sample from the aliquot was removed by centrifugation at 3000 rpm.

2.3. Characterization techniques Morphological characteristics of as-synthesized ZnO nanostructures were studied using FESEM (Gemini 300) and TEM (JEOL-200), while the crystallographic and structural information was analyzed using powder XRD (X'pert Pro diffractometer). Raman spectrum of each sample was acquired using LabRam HR800, Jobin Yvon with 488 nm excitation source. Optical nature of the nanostructures was studied using UV–vis technique and photoluminescence spectroscopy, where

2.5. Bacterial cell growth Gram-positive S.aureus was used as a representative bacterial strain 4

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Fig. 5. (a–d) Representative optical absorption spectra of MB dye treated with different photocatalyst material (ZP, ZP1, ZP2 and ZP3) as a function of time, (e) Comparative degradation kinetic curve for samples ZP, ZP1, ZP2 and ZP3, (f) Degradation bar graph presenting photocatalytic efficiency of different synthesized ZnO nanorods against MB dye using sunlight as the activator. Each plotted value is the mean of triplicate reading while the error bar represents standard deviation (n = 3).

without nanorods, 1 ml of bacterial culture was added, which was equivalent to approximately 106 CFU. All these culture tubes were then properly sealed and maintained in the shaker incubator at 37 °C and 200 rpm overnight. Before incubation, 500 μl of culture mix was aliquoted for 0 h plating. Following 20 h of overnight incubation, spread plating was done to quantify the killing efficiency of the nanorods. After each plating routine (0 and 20 h), the plates were maintained in the incubator for 8–10 h. After incubation, the plates were used to record the colony forming units for each sample and each concentration. All the experiments were performed in triplicates for statistical significance.

to study the bactericidal efficiency of synthesized ZnO nanorods. S.aureus mother culture was obtained by inoculating a single bacterial colony into 5 ml of freshly autoclaved LB broth. This culture was allowed to grow aerobically for 12 h in a shaker incubator maintained at 37 °C and 200 rpm. Following incubation, 1 ml of the mother culture was again inoculated into 15 ml of fresh LB broth to attain exponential growth phase for the bacterial cells. Regular OD measurements were made to monitor cell growth. The killing assay experiments were performed with cell population at about 108 CFU/ml (OD at 600 nm corresponding to ∼ 0.5). 2.6. Antibacterial killing assay (spread plate method)

2.7. Sunlight mediated killing assay Stock particle suspensions of all the different nanorod samples with concentration 2 mg/ml were made. The nanomaterial stock suspensions were diluted in autoclaved LB media to obtain effective load corresponding to 0.03, 0.06, 0.09 and 0.125 mg/ml. Culture tube carrying no nanomaterial served as control. To all the culture tubes, with and

For studying the photolytic killing of S.aureus cells in the presence of nanorods, the cells were grown and maintained as described earlier. Following the addition of cells and nanomaterials into the culture tubes, these tubes were exposed to sunlight for 4 h. Both prior and post5

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Fig. 6. Schematic representation of the proposed mechanism of decomposition of MB dye molecules using oxygen defect enrich ZnO nanostructures as photocatalyst under sunlight.

exposure, the culture mix from each tube was aliquoted for spread plating. The culture tube carrying no nanorods was the control sample. To minimize any influence of temperature/heat, regular supply of cold water was used to maintain the temperature at 37 °C.

(2)

Zn+2 + 2OH− → ZnO + H2 O

(3)

Firstly, at pH∼5.0 (acidic medium), the effect of the HMTA is mostly neutralized due to the high concentration of H+ ions, consequently resulting in the formation of irregular one-dimensional nanostructures were obtained. Hence in sample ZP, a mixture of nonuniform structures one-dimensional nanostructures as observed for sample ZP. At reaction pH 5.5, maintained by addition of NaOH, rate of generation of OH− ions surpasses neutralizing effect of HMTA, allowing the growth of perfect nanorod structures compared to that in ZP. Moreover, as Zn+2 nucleation rate is higher than OH− ions in acidic pH conditions, competitive nucleation leads to the formation of hexagonal surfaces at the nanorod terminals. On the other hand, the further increment in the pH (∼ 6.0) of the reaction suspension enhances the concentration of OH− ions which were responsible for the transformation of hexagonal ZnO nanorods to the smaller ZnO nanorods [43]. Moreover, at pH ∼ 6.5, the reaction suspension contains a high concentration of OH− ions which further helps to HMTA for the generation of rod-like nanostructures. In order to analyze the crystal structures and crystallinity of the sample, XRD measurements were carried out at room temperature. Fig. 2(a) reveals the XRD diffraction pattern and crystalline nature of the sample ZP, ZP1, ZP2 and ZP3. All four samples ZP, ZP1, ZP2 and ZP3 exhibit three major peaks and two minor peaks in the XRD patterns. The observed five peaks (100), (002), (101), (102) and (110) confirmed the existence of ZnO in hexagonal wurtzite structure (JCPDS Card no 891397). The measured average crystallite size for the sample ZP, ZP1, ZP2 and ZP3 were 54, 62, 56 and 58 nm respectively. In addition to affirming wurtzite crystal structure presence, Raman spectroscopy was used to gather initial information on ZnO defect states. Fig. 2(b) illustrates the Raman spectra of sample ZP, ZP1, ZP2 and ZP3. Raman spectrum of sample ZP shows four peaks at 327.5, 376.3, 437.2 and 581.8 cm−1. The major peak at 437.2 originates due to the vibration of oxygen atoms and is ascribed to E2(high) mode of ZnO [45]. The other three minor peaks at 327.5, 376.3 and 581.8 correspond to the E2(high) - E2(low), E1(TO) and 1E1(LO) respectively [46]. In ZnO, the E2(low) mode corresponds to the vibration of Zn sublattice while E2(high) mode is associated with the oxygen atoms [47]. Existence of E2(high) mode confirms the ZnO wurtzite structure which was found consistent with the XRD results [47]. E1(TO) and E1(LO) modes of ZnO which are associated with the lattice bond strength and concentration of defects states (Oxygen and Zn interstitial) respectively were

3. Result and discussion Surface characteristics of samples ZP, ZP1, ZP2 and ZP3 were studied through FESEM. As acquired microscopic images in low and high magnification are presented as Fig. 1 (a–d) and Fig. 1 (e–f) respectively. For ZP, nanostructure synthesized at pH 5.0, FESEM images (Fig. 1(a) and (e)) reveals the formation of one dimensional ZnO nanorod aggregates with rough edges and slightly tapered ends resembling a pencil tip. The average length of the one-dimensional aggregate of ZnO nanostructures was estimated to be ca. 187 nm. For sample ZP1, where the reaction pH was 5.5, although the basic morphology was observed to be restricted to being one-dimensional however unlike ZP its ends were observed to be hexagonal in appearance (Fig. 1(b) and (f)), and their average diameter and length were found to be 300 and 465 nm respectively. Further increment in reaction pH was accompanied by a small variation in nanorod length but an almost halved nanorod diameter. At pH 6.0, for ZP2, the nanorod dimensions, average length and diameter, were tabulated to be 329 nm and 107 nm respectively. The corresponding FESEM images are presented as (Fig. 1(c) and (g)). On approaching near-neutral pH, nanorod sample was seen to undergo a further reduction in their average length while the diameter was calculated to be closely similar (Fig. 1(c) and (g)). For ZP3, average length and diameter as estimated were ca.187 nm and 106 nm respectively. Notably, we observe that restricting pH below basic conditions, allows restricting the ZnO nanostructure growth in one dimension; however, it modulates its aspect ratio. For samples ZP-ZP3, we observed an aspect ratio variation within the range of 1.5–2.7. Morphological transition within various ZnO nanorods sample can be understood in terms of an ionic interplay sequestered in response to the changing reaction mixture pH. Zinc nitrate reacts with HMTA, which helps to form crystalline and stable ZnO nanorods by controlling the rate of OH− ions during the reaction [42]. At acidic pH conditions, the reaction suspension helps the growth of rod-like nanostructures by promoting growth along the preferred c-axis [43]. The chemical reaction between Zinc nitrate and HMTA is chemically expressed as [42,44].

C6 H12 N4 + 6H2 O → 6HCHO + 4NH3

NH3 + H2 O → NH4+ + OH−

(1) 6

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Fig. 7. (a–e) Digital images of bactericidal test performed on S.aureus cells in absence of light, with various as synthesized ZnO nanorod samples at an effective concentration of 0.125 mg/ml, (f) Representative bar graph depicting antibacterial activity of different ZnO nanorod samples. Histogram readings present mean ± standard deviation (n = 3).

spectra of sample ZP1, ZP2 and ZP3 were found to show higher sensitivity in the visible region indicated in the wider absorption region compared to sample ZP. Using the Tauc plot method, the optical band gap of ZnO samples were estimated which are presented in Fig. 3(b). The computed bandgap of sample ZP, ZP1, ZP2 and ZP3 were found to be 3.03, 2.84, 2.58 and 2.42 eV respectively. Tauc plot results clearly indicate bandgap narrowing in the ZnO nanorod samples, supporting our postulation of defect state presence in the ZnO nanorods synthesized at different pH values Previously, Ansari et al. [50] have demonstrated positive correlation between band gap narrowing and improved visible light responsiveness of ZnO to the presence of oxygen defects in ZnO thin films.

examined thoroughly to deduce information about the defect content in ZnO as a function of pH values [48]. The 1E1(LO) mode was found to increase continuously from sample ZP to ZP3 indicating increment of defect states with pH values [48,49]. Although the increment in the E2(high) mode indicates improvement in the crystalline quality of the ZnO sample the significant enhancement and shift towards lower wavenumber of the 1E1(LO) mode from sample ZP to ZP3 implies increment in the defects state concentration. The optical properties of ZnO samples were investigated using UV–vis absorption spectroscopy. Fig. 3(a) depicts the optical absorption spectra of the synthesized samples. The characteristic ZnO absorption peak at 379 nm was observed for all the samples. The optical absorption 7

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Fig. 8. (a–b) Digital images of S.aureus culture on Agar plates; control and treated with ZP3 sample at 0.125 mg/ml under solar light, (c–d) AFM images of control and treated S.aureus cells with ZP3 sample under sunlight.

respectively. XPS spectrum of sample ZP shows three peaks at 530, 531.4 and 532.5 eV which are assigned to the lattice oxygen, oxygen defect and chemisorbed oxygen species respectively [54–56]. Interestingly, it can be seen that the intensity of the peak associated with the oxygen defects was continuously increased from the sample ZP to ZP3, which inferred that oxygen defects states continuously increased in the ZnO nanorods sample with the increment in the pH values. XPS studies highlighted the increment in the defect states from the sample ZP to ZP3 which presumably controls the photodegradation behaviour of the synthesized ZnO samples predominantly. Photodegradation behaviour of ZnO samples was explored by analyzing the decomposition rate of methylene blue dye solution under sunlight illumination using sample ZP, ZP1, ZP2 and ZP3. Fig. 5(a–d) shows the optical absorption spectra of methylene blue dye solution using sample ZP, ZP1, ZP2 and ZP3 respectively as photocatalysts. The photodegradation studies reveal that sample ZP3 was found to be the most efficient photocatalyst as compared to other photocatalyst samples ZP, ZP1 and ZP2. Fig. 5(e) highlights the rate kinetics of the ZnO nanostructure samples and reveals that the reaction follows first-order kinetics. Rate kinetics curves also confirmed the highest photocatalytic behaviour of sample ZP3 among other samples. The measured rate constant for sample ZP, ZP1, ZP2 and ZP3 are 0.04218, 0.04301, 0.06043 and 0.07151/ min respectively. Fig. 5(f) illustrates the photodegradation efficiency of sample ZP, ZP1, ZP2 and ZP3 under sunlight exposure. Sample ZP3 degrades to 86% of 10μM methylene blue dye while sample ZP, ZP1 and ZP2 decompose only 65%, 67% and 79% of MB dye solution respectively. The photocatalytic behaviour of ZnO nanorods and its underlying

To explore the defect states and optical nature of ZnO nanorods samples, photoluminescence spectroscopy was carried out at room temperature. Fig. 3(c) illustrates the PL spectra of ZnO samples ZP, ZP1, ZP2 and ZP3. PL spectrum of sample ZP shows five discrete peaks at 391.0, 441.7, 467.0, 493.0 and 562.0 nm. The observed peak at 391.0 nm (3.17 eV) can be assigned to the band edge emission of ZnO while the peak at 438 nm (2.83 eV) ascribed to the shallow traps of Zn interstitial [48]. A major peak at 467 nm (2.65 eV) originates due to the deep level emission [48], while two other peaks at 493 nm (2.51 eV) and 562 nm (2.20 eV) correspond to the oxygen vacancies exists in ZnO nanostructures [50]. Interestingly, the band edge peaks for samples ZP1, ZP2 and ZP3 are found to be systematically red-shifted and marked at 393.6 nm, 394.4 nm and 396.7 nm respectively (ESI, Table 1). This redshift in the PL spectrum is ascribed to variation in nanostructure shape and size [51]. Along with a shift in band edge wavelength a distinct difference in signal intensity for samples ZP-ZP3 is also observed which are often associated with differential defect state density in the samples [52,53]. It was observed that the PL intensity of peaks corresponding to defects states were continuously increasing, suggesting increment in the defects concentration as we move from sample ZP to ZP3. The PL spectra also used to tabulate the bandgap in the ZnO nanostructures, and erewere found to be 3.17 eV, 3.15 eV, 3.14 eV and 3.12 eV for sample ZP, ZP1, ZP2 and ZP3 respectively. The bandgap calculations were well in accordance with the optical absorption spectroscopy results. For an in-depth insight into the oxygen chemical state in ZnO nanorods samples, XPS measurements were performed. Fig. 4(a–d) reveals the Gaussian fitted O1s spectra of sample ZP, ZP1, ZP2 and ZP3, 8

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against S.aureus cells. ZP and ZP1 which considerably lesser defect states show no significant killing of bacterial cells under treatment in the dark (Fig. 7 (b–c)). As the oxygen vacancy content increases within the samples, namely ZP2 and ZP3, significant improvement towards cellular killing is observed (Fig. 7(d)). Sample ZP3 with highest oxygen defect concentration, exhibiting the superlative antibacterial activity could all the inactivate the S.aureus bacterial cells within 20 h without any light exposure (Fig. 7(e)). The comparison of the antibacterial activity for the inactivation of S.aureus through sample ZP, ZP1, ZP2 and ZP3 are depicted in Fig. 7(f). The bar graph for the antibacterial activity of sample ZP, ZP1, ZP2 and ZP3 elucidated that sample ZP3 exhibits superior antibacterial activity as compared to sample ZP, ZP1 and ZP2. Since ZP3 was found to be the most effective photocatalytic and antibacterial agent, we chose to study its antimicrobial activity as a function of light as well. The cell and sample load concentration erewere used as optimized from experiments performed in the dark. To study the contributive effect of sunlight towards antibacterial activity, ZP3 sample with optimized concentration bacteria, cell load of∼ 106 CFU/ml was exposed to sunlight. An additional culture tube carrying only the cells in the absence of any ZnO samples was also placed in the sunlight, which served as the control to minimize any assay estimation error due to heat inactivation in bacteria. Within 4 h of sunlight assisted inactivation study, ZP3 sample at an effective concentration of 0.125 mg/ml could achieve a 100% killing efficiency (Fig. 8(a–b)). The observed exceptionally high bactericidal efficiency of ZP3 is ascribed to pronounced ROS generation, on account of increased oxygen vacancies presence in ZP3 compared to rest [63–65]. Compared to dark, ROS generation under light activation is rapid and pronounced leading to an accelerated cidal rate in bacterial cells. The control and treated bacterial samples were viewed after that under an atomic force microscope (AFM) to observe any differences in cellular appearances. AFM images of control and treated cells are depicted in Fig. 8(c–d). High surface perturbations such as ruptures and pits were seen on the cell walls of ZP3 treated S.aureus while in control cells, the cell walls were intact, smooth and unperturbed, features indicative of healthy cells. Sample ZP3 with maximum oxygen defect concentration under sunlight exposure generate the hydroxyl radicals more efficiently consequently inactivation of S.aureus bacteria process occurs more efficiently as compared to the killing activity in the dark. Like photocatalytic degradation of MB dye solution, the photolytic killing of bacterial cells is postulated to depend on water splitting efficiency of ZP3, which leads to the generation of ROS subsequently leading to cell death by creating a stressful environment. Further comparison of our study with recent reported studies is represented in ESI (Table 3) [60,66–70].

principle has been presented diagrammatically as Fig. 6 and be understood as follows (Fig. 6). ZnO as a wide bandgap semiconductor generates electron-hole pair in its conduction and valance band respectively under sunlight exposure. The electrons in the conduction band of ZnO, convert the oxygen molecules into superoxide radicals (%O2−) while holes in the valance band decompose the water molecule into the hydroxyl radicals (%OH−). These two unsaturated radicals interact with the organic molecules and decompose them [57,58]. We have demonstrated that hydroxyl radicals (%OH−) radicals are majorly responsible for the degradation of MB dye molecule (supporting information, Fig. 2). Recently, Zhang et al. [59] reported that increment in defect concentration in ZnO nanostructures could significantly improve their photocatalytic activity. They demonstrated that by using 250 mg of ZnO nanorods, 5 mg/l methylene blue dye solution was decomposed in 160 min under UV light exposure (400 W, mercury lamp). Flores et al. [60] demonstrated the improvement in photocatalytic decomposition of MB dye under visible light due to the increment in the defect concentration of ZnO nanostructures. In their photocatalytic experiment, 10 ppm MB dye was degraded in 300 min by using 100 mg ZnO nanostructures under UV light. Recently, Chen et al. [61] demonstrated significant enhancement in the photodegradation ability of ZnO due to the incorporation of oxygen defects through ball milling method. They performed their photocatalytic degradation studies under UV light (100 W) and decomposed 50 ml MB dye solution of 20μM by using 25 mg ZnO in 40 min. They have also shown that holes and hydroxyl radicals (%OH−) radicals majorly influence the photo-degradation behaviour of ZnO. In our case, 10μM of MB dye was degraded within 30 min of sunlight exposure using 5 mg of oxygen defect enriched ZnO nanorods. Parallel to previous reports, increase in surface defect states leading to bandgap narrowing is ascribed to be the driving force responsible for improved photocatalytic behaviour of the nanorods, especially ZP3 which has been found to possess maximum defect state concentration of all the samples. To investigate any contribution from the ZnO surface area to MB dye photodegradation, the specific surface area of each sample was measured using the Brunauer–Emmett–Teller (BET) desorption adsorption method. For samples, ZP-ZP3 the surface area values were estimated to be ∼4.603 m2/g, 5.6 m2/g and 2.3 m2/g respectively. Clearly, no linear relationship between surface area and activity could be established, confirming photodegradation activity is independent of surface area. As mentioned briefly during the introduction section ZnO nanostructures are also an ideal candidate for inactivation of biological contaminants such as bacteria. As proved with our photocatalysis results, we are aware that the defect rich ZnO nanostructures are well capable of ROS generation in light, however owing to some recent reports postulating ROS generation even in the dark [62], we tested the antimicrobial activity of our ZnO samples both in dark and sunlight. In our study, the relative antibacterial activity of different ZnO nanorods towards S.aureus was studied by spread plate method. The results for the antibacterial assay in the dark for all the four different ZnO samples are shown in Fig. 7. A clear demarcation in the differential ability of the ZnO nanorods in killing the bacterial cells was observed. ZP3 sample was found to be most efficient, followed by ZP2 while ZP and ZP1 were unable to cause any considerable cell death within the stipulated time of study and tested concentrations. The killing activity of ZnO samples was found to be concentration-dependent with no cidal activity below 0.09 mg/ml. The results are tabulated in Table 2 (ES1). The superior antibacterial activities of sample ZP3 in the dark can be ascribed to improved ROS generation ability of oxygen vacancies enriched nanostructures. Presence of oxygen vacancies facilitates the formation of superoxide radicals by water splitting, which eventually is converted into reactive hydroxyl ions. [62]. These highly active radicals then target bacterial cells leading to their killing consequential to the generation of state of oxidative stress. In our study, ZnO samples with different oxygen vacancy content show differential cidal activity

4. Conclusion Defect enriched ZnO nanorods with different aspect ratios were successfully fabricated through a low temperature hydrothermal method. pH mediated evolution on their optical, structural and morphological characteristics along with their photodegradation and antibacterial efficiencies were studied elaborately. With a change in growth conditions, an ordered variation in defect state presence was observed which proved to be an important parameter in deciding the chemical and biological contaminant degradation efficiency. Nanostructures and their properties were well studied using FESEM, Raman, UV–vis, PL and XPS. Sample ZP3 with maximum defect state content was found to be the most efficient photocatalytic and antibacterial agent on account of bandgap narrowing and improved ROS generation capabilities when compared to other samples ZP, ZP1 and ZP2. In summary, defect state tuning forms an integral synthetic parameter defining nanomaterial performance for any designed application. These results inferred that by tuning the oxygen defect concentration of ZnO nanorods, significant enhancement could be achieved in their antibacterial and photocatalytic behaviour. This study may provide a new insight into the field of the advanced photocatalyst. 9

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Acknowledgements

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