Green Synthesis of Copper Oxide Nanostructures using Cynodon dactylon and Cyperus rotundus Grass Extracts for Antibacterial Applications

Journal Pre-proof Green Synthesis of Copper Oxide Nanostructures using Cynodon dactylon and Cyperus rotundus Grass Extracts for Antibacterial Applications S. Suresh, R. Ilakiya, G. Kalaiyan, S. Thambidurai, P. Kannan, K.M. Prabu, N. Suresh, R. Jothilakshmi, S. Karthick Kumar, M. Kandasamy PII:

S0272-8842(20)30326-6

DOI:

https://doi.org/10.1016/j.ceramint.2020.02.015

Reference:

CERI 24238

To appear in:

Ceramics International

Received Date: 27 November 2019 Revised Date:

27 January 2020

Accepted Date: 2 February 2020

Please cite this article as: S. Suresh, R. Ilakiya, G. Kalaiyan, S. Thambidurai, P. Kannan, K.M. Prabu, N. Suresh, R. Jothilakshmi, S. Karthick Kumar, M. Kandasamy, Green Synthesis of Copper Oxide Nanostructures using Cynodon dactylon and Cyperus rotundus Grass Extracts for Antibacterial Applications, Ceramics International (2020), doi: https://doi.org/10.1016/j.ceramint.2020.02.015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Green Synthesis of Copper Oxide Nanostructures using Cynodon dactylon and Cyperus rotundus Grass Extracts for Antibacterial Applications S. Suresha*, R. Ilakiyaa, G. Kalaiyana, S. Thambiduraia, P. Kannanb, K.M. Prabua, N. Suresha, R. Jothilakshmic, S. Karthick Kumard, M. Kandasamye a

PG & Research Department of Physics, Sri Vidya Mandir Arts & Science College, Uthangarai – 636 902, Krishnagiri, Tamil Nadu, India b Department of Physics, Sri Moogambigai Arts and Science College (Women), Palacode – 636 805, Dharmapuri, Tamil Nadu, India c Department of Physics, Vel Tech Rangarajan Dr. Sagunthala R & D Institute of Science and Technology, Avadi – 600 062, Chennai, Tamil Nadu, India d Department of Physics, Sethu Institute of Technology, Kariapatti – 626 115, Virudhunagar, Tamil Nadu, India e Department of Inorganic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai – 625 021, Tamil Nadu, India

Abstract Copper oxide (CuO) nanostructures were synthesized using Cynodon dactylon and Cyperus rotundus grass extracts. XRD analysis revealed formation of end-centered monoclinic structured CuO with high crystallinity. SEM images of CuO nanostructure prepared by C. dactylon grass extract disclosed rice spikelet-like morphology, while CuO nanostructure synthesized by C. rotundus grass extract exhibited composite morphology with nanoparticles, nanorods and nanoprisms. EDAX spectra clearly revealed presence of Cu and O elements that confirms purity of CuO nanostructures. Strong absorption peaks observed in FTIR spectra of monoclinic CuO nanostructures revealed high purity of CuO nanostructures synthesized by grass extracts. Potential antibacterial activity exhibited by CuO nanostructures against Gram negative Klebsiella pneumoniae bacterial species with zone of inhibition of 28 mm can be ascribed to diverse factors, such as mechanical damage, oxidative injury and gene toxicity. Thus, C.

1

dactylon and C. rotundus grass extracts can be regarded as sustainable and abundant natural resources towards green synthesis of CuO nanostructures for potential antibacterial applications. Keywords: Green Synthesis; Grass extracts; CuO nanostructures; Antibacterial activity; Reactive oxygen species *Corresponding author: E-mail address: [email protected] (S. Suresh) 1. Introduction Nanotechnology is an emerging field that connects interdisciplinary subject areas of physics, chemistry, biology, material science and medicine. It is primarily concentrated on synthesis of nanosized metal and metal oxides of various shapes, sizes, chemical compositions and dispersity. Research efforts towards preparation and characterization of metal and metal oxide nanomaterials have gained immense attention owing to their potential applications in biomedicine, agriculture, food, cosmetics, paints, catalysis and textiles [1]. Nanoscale metal oxides are regarded as key constituents in micro/nanoscale devices due to their specific size and size oriented physico-chemical properties. Among different nanostructured metal oxide semiconductors, cupric oxide (CuO) with a narrow band gap energy of 1.2 eV has provoked proficient attention because of its large surface area, remarkable magnetic and superhydrophobic properties, high electrical and thermal conductivity, superior catalytic activity and selectivity [2,3].

Copper in its elemental form has been found as toxic to human beings, animals, aquatic life and environment when it surpasses its permissible limit. On the other hand, copper and its complexes have been employed as water purifier, antibacterial agents and pesticides over many years [4]. It is highly appropriate to mention here that the US Environmental Protection Agency

2

(USEPA) has recognized application of materials based on copper for human use [5]. Therefore, CuO nanostructures have been employed in diverse applications including gas sensors, catalysis, solar energy conversion, batteries and heat transfer [6–8]. Besides, they have also been effectively used as antimicrobial, anti-fungal and anti-biotic agents in textiles, coatings, plastics, etc. [9]. CuO nanostructures can be used for neither treating infection nor identifying pathogens and these features improve smart drug delivery systems by tailoring antimicrobial compound surfaces using CuO nanostructures [10]. CuO nanostructures have emerged as notable antimicrobial candidates for widespread pathogens that are usually resilient to commercial antimicrobial agents [11]. Antibacterial activity of CuO nanostructures presumably depends on size and surface area to volume ratio of nanoparticles with unusual crystal morphology that facilitates close interaction of CuO nanostructures with cell membrane of bacteria and kills bacteria by releasing metal ions inside the cell [12].

CuO nanostructures are prepared by a variety of physical and chemical synthesis methods, viz. electrochemical reduction, sonochemical methods, chemical precipitation, sol-gel, hydrothermal, gamma-irradiation, microwave irradiation, pyrolysis, solid state reaction and thermal decomposition [1]. Nevertheless, there are lot of significant limitations associated with these synthesis routes, namely usage of harmful and hazardous chemicals, liberation of significant quantity of waste stuffs into environment, high cost, need of sophisticated equipment, consumption of high energy, high temperature treatment, difficulty in cleansing of nanomaterials, etc., which create serious problems to the environment [13]. Furthermore, toxic chemicals adsorption on the surface of nanostructure prepared by chemical method is considered as inappropriate for its biological and medical applications [14]. Hence, there is a prevailing

3

interest to prepare CuO nanostructures through a most controllable, accelerated, energy efficient, eco-friendly and non-toxic method [15]. In this context, alternative and eco-friendly synthesis of nanomaterials has enumerated incredible attention. For the past one decade, reliable, low-cost and eco-friendly approaches have been established to synthesis metal oxide nanostructures in view of their diverse biological applications.

Biosynthesis procedures encompasses utilization of organisms originated from natural origin, viz. extract from various plant parts of terrestrial [16,17] and aquatic [18,19] origin, bacteria [20,21], yeast and fungi, as appropriate, facile, eco-friendly and non-toxic chemicals [22]. Among natural sources, plants are regarded as ideal candidates to synthesis of metal oxide nanomaterials. This can be attributed to facile sampling, good stability, low-cost, efficient, safe to use, presence of a large number of biomolecules, faster reaction rate in the absence of high energy, pressure and toxic chemicals [23]. Most desirable green synthesis approach to prepare nanostructured semiconductor metal oxides is bio-reduction that involves reaction between bioactive products separated from plants with reduced state of metal oxides. Phyto-chemicals present in plant extract play both as reducing and stabilizing agents during synthesis of metal oxide nanostructures [24]. Presence of rich metabolite constituents, namely flavonoids, tannins, terpenoids and proteins in plant extract crucially act as reducing and stabilizing agents during green synthesis [25].

In the past, green synthesis of CuO nanoparticles using diverse nanomaterials organized from biological origin, such as green tea, brown alga, coffee, yeast, bacteria and fungi has been demonstrated [26]. Nevertheless, synthesis of CuO nanostructures with an aid of green grasses has not been reported elsewhere to the best of our knowledge. In this respect, this work is

4

performed to synthesis CuO nanostructures using green reducing and stabilizing agents from Cynodon dactylon (C. dactylon) and Cyperus rotundus (C. rotundus) grass extracts. Photographs of C. dactylon and C. rotundus grass species are shown in Figure 1. The reason behind the selection of these two elegant perennial grass species extracts to synthesis CuO nanostructures is that C. dactylon is a major tropical grass found in all tropical and subtropical areas and highly tolerant to drought and heavy grazing, while C. rotundus grass grows everywhere throughout India. Hence, they can be considered as sustainable and abundant natural resources towards green synthesis of CuO nanostructures. The prepared CuO nanostructures have been systematically characterized by X-ray diffraction, scanning electron microscopy, energydispersive X-ray spectroscopy and Fourier transform infrared spectrometer. In terms of novelty, this is a first work, which is devoted to green synthesis of diverse CuO nanostructures using grass extracts, since numerous works in the past have been demonstrated green synthesis of semiconductor metal oxide nanoparticles rather than different nanostructures. In view of the prepared CuO nanostructures towards biological application, they have been employed as antimicrobial agents against Gram positive bacterial species, viz. Bacillus cereus (MTCC 430) and Staphylococcus aureus (MTCC 3160) and Gram negative bacterial species, such as Escherichia coli (MTCC 1698) and Klebsiella pneumoniae (MTCC10309), by adopting a standard well diffusion method and moreover mechanism for antibacterial activity of CuO nanostructures are discussed.

5

2. Materials and Methods 2.1. Chemical and reagents High pure copper nitrate trihydrate (Cu(NO3)2 ·3H2O) was purchased from Merck. All other chemicals employed in this work were of analytical grade and used as such. Unless otherwise specified, deionized double distilled water was used to prepare aqueous solutions and washings.

(a)

(b)

Figure 1. Photographs of (a) C. dactylon and (b) C. rotundus grass species.

6

2.2. Synthesis of copper oxide (CuO) nanostructures Fresh grass species of Cynodon dactylon (C. dactylon) and Cyperus rotundus (C. rotundus) were collected from the campus of Sri Vidya Mandir Arts and Science College, Katteri, Uthangarai, Krishnagiri District Tamil Nadu, India. Collected grasses were thoroughly cleaned using tap water until all surface adsorbed impurities were washed out. Subsequently, grasses were washed by double distilled water few times and dried under shadow at room temperature in the absence of any light illumination to remove all moisture present on the surface of grasses. Then, five gram of each fresh grass species was separately weighed in a physical balance which were cut into small pieces using a scissor. Small pieces of grass species were grounded using a mortar and pestle for 10 min and extract of grass species were transferred into beakers containing 150 mL of distilled water. The beakers were placed on a magnetic stirrer and stirred the content at 600 rpm for 1 h under heating at 80 °C. Beakers containing grass extracts were filtered using Whatman Grade No. 1 filter paper. Grass extracts (30 mL) were taken in separate beakers, in which one gram of Cu(NO3)2·3H2O was added under stirring a 80 °C. During stirring process, colour of solutions was changed from bluish to dark green that revealed formation of CuO. The stirring was continued until solutions were turned into green colour paste. The pastes were washed there times by distilled water to remove impurity ions and excess biomolecules. Finally, the obtained precipitates were transferred to silica crucibles, which were calcined at 200 °C for one hour in a muffle furnace. During calcination process, green colour precipitates were changed into dark coloured powders that disclosed formation of CuO nanostructures. Steps involved in synthesis of CuO nanostructure using C. dactylon grass extract are illustrated in Figure 2.

7

Collected Grass Species

Grounded Grass

Chopped Grass

Stirring & Heating at 80 °C

Filtered Grass Extract

Grass Extract + Cu(NO3)2·3H2O

Stirring & Heating at 80 °C

Green Colour CuO Paste

Calcined at 200 °C

CuO Nanopowder

Figure 2. Steps involved in synthesis of CuO nanostructure using C. dactylon grass extract.

8

2.3. Characterization studies Prepared CuO nanostructures were characterized by X-ray diffraction (RD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDAX) and Fourier transform infrared (FTIR) spectrometer. XRD analysis of CuO samples was carried out using PANalytical X'pert3 powder X-ray Diffractometer with Cu Kα radiation (λ = 1.5418 Å). SEM micrographs were recorded in VEGA 3 TESCAN scanning electron microscopy. EDAX analysis of samples was subsequently carried out during SEM analysis by Bruker Energy-dispersive Xray spectrometer. FTIR spectra of CuO samples and grass extracts were recorded in the region between 4000 and 400 cm−1 using PerkinElmer FTIR spectrometer (Model: Spectrum Two).

2.4. Antimicrobial activity evaluation of CuO nanostructures Antimicrobial activity of synthesized CuO nanostructures was examined by standard well diffusion method. Liquid Mueller Hinton agar media and petri dishes were sterilized using autoclave at a temperature of 121 °C and pressure of 15 lbs for 30 min. In the presence of aseptic conditions in laminar airflow chamber, 20 mL of agar medium was dispensed into each petri dish so as to obtain a uniform depth of 4 mm. After solidification of media, 18 h culture of Gram positive bacterial species, viz. Bacillus cereus (MTCC 430) and Staphylococcus aureus (MTCC 3160) and Gram negative bacterial species, such as Escherichia coli (MTCC 1698) and Klebsiella pneumoniae (MTCC10309), procured from Institute of Microbial Technology (IMTECH), Chandigarh, India were swabbed on the surface of agar plates. Well was prepared using cork borer followed by loading 50 and 100 µL concentrations of CuO nanostructured samples; sterile distilled water as negative control and tetracycline with a concentration of 30

9

mcg/disc as positive control in distinct wells. The plates were then incubated at 37 °C for 24 h to observe zone of inhibition [27,28].

3. Results and Discussion 3.1. X-ray diffraction analysis X-ray diffraction (XRD) patterns of CuO nanostructures prepared using C. dactylon and C. rotundus grass extracts were recorded and the resultant XRD patterns are showed in Figures 3. CuO nanostructure prepared using C. dactylon grass extract exhibited diffraction peaks at 2θ values of 32.27, 35.24, 38.49, 48.70, 53.31, 57.96, 61.38, 66.18, 67.70, 72.09 and 74.94°, which can be ascribed to (1 1 0), (0 0 2), (1 1 1), (2 0 2), (0 2 0), (2 0 2), (1 1 3), (3 1 1), (1 1 3), (2 2 1) and (0 0 4) diffraction planes, respectively produced by end-centered monoclinic CuO (JCPDS Card No. 80-1916). Major diffraction peaks with high intensities imply high crystalline nature of CuO samples prepared through the present green synthesis method (Figure 3(a)). CuO nanostructure prepared using C. rotundus grass extract showed diffraction peaks at 2θ values of 33.25, 35.12, 38.38, 48.44, 53.10, 58.02, 61.27, 65.98, 67.70, 72.19 and 74.95° that can be ascertained to (1 1 0), (0 0 2), (1 1 1), (2 0 2), (0 2 0), (2 0 2), (1 1 3), (3 1 1), (1 1 3), (2 2 1) and (0 0 4) diffraction planes, respectively generated by end-centered monoclinic CuO. Absence of impurity peaks correspond to Cu(OH)2 and Cu2O were not observed in the XRD patterns, which indicates phase pure formation of CuO [29]. However, intensities of major diffraction peaks are relatively high over intensities of CuO nanostructure synthesized by C. dactylon grass extract that disclosed relatively high crystallinity of CuO samples prepared by C. rotundus grass extract (Figure 3(b)). The X-ray diffraction patterns observed in this study are similar to previous reports based on green synthesis of CuO nanoparticles using different natural sources [30,31].

10

Crystalline size (D) of CuO nanostructures for major diffraction peaks was theoretically calculated using the following equation of Scherrer [32]. XRD parameters, such as diffraction angle, h k l plane value, FWHM, crystalline size and lattice strain calculated for CuO nanostructures are given in Table 1. Average crystalline size of CuO nanostructure prepared using C. rotundus grass extract was found out as 16.63 nm, which was lower than crystalline size (22.81) of CuO nanostructure synthesized using C. dactylon grass extract.

= λ/

ℎ

where, D is average crystalline size, k is shape factor, λ is X-ray wavelength and β is full-width at half maximum (FWHM) corresponding to respective major diffraction planes.

11

Figure 3. XRD patterns of CuO nanostructures synthesized using (a) C. dactylon and (b) C. rotundus grass extracts.

Table 1. XRD parameter of CuO nanostructures synthesized using (a) C. dactylon and (b) C. rotundus grass extracts.

Source for CuO Nanostructures C. dactylon Grass Extract C. rotundus Grass Extract

2Theta

hkl

FWHM

Crystalline size

Lattice strain

35.18 38.42

002 111

0.3570 0.4140

24.39 21.23

0.0049 0.0052

35.27

002

0.4720

18.45

0.0065

38.47

111

0.5935

14.80

0.0074

12

3.2. Scanning electron microscopy examination Scanning electron microscopic (SEM) images were recorded to examine surface morphology of CuO nanostructures prepared by C. dactylon and C. rotundus grass extracts. Higher and lower magnified SEM images of CuO nanostructure synthesized using C. dactylon grass extract are shown in Figure 4. It is highly apparent from SEM images that C. dactylon grass extract have played a crucial role both as reducing and capping agent towards formation of ensuing CuO nanostructure. Lower magnified (20 and 10 µm) SEM micrographs exhibited undefined, aggregated and clusters of sponge-like CuO morphology that are uniformly distributed with appreciable voids. Obviously, convinced size of CuO morphology could not be measured from the lower magnified SEM micrographs. However, higher magnified SEM micrographs (5 and 2 µm) disclosed that CuO nanostructure is composed of rice spikelet-like morphology that are uniformly distributed by covering whole surface of SEM image without any appreciable voids. Average length of rice spikelet-like structure ranges from 1 µm to 1.5 µm with an average width of ~500 nm. Rice spikelet-like CuO nanostructure is transparent, uniformly oriented and densely distributed without any voids. In terms of antimicrobial activity, this kind of CuO nanostructure morphology is highly beneficial as they offer large surface area towards surface interaction of bacterial species over three dimensional spherical CuO nanoparticles.

13

Figure 4. SEM images of CuO nanostructure synthesized using C. dactylon grass extract.

Higher and lower magnified SEM images of CuO sample synthesized using C. rotundus grass extract are illustrated in Figure 5. Lower magnified (20 µm) SEM micrographs of CuO sample prepared by C. rotundus grass extract disclosed highly transparent and uniformly distributed CuO nanostructure, whose average size is confined below 2 µm. CuO nanostructure

14

morphology contains relatively meager voids when compared to CuO nanostructure prepared using C. dactylon grass extract. Careful examination of higher magnified SEM micrographs (5 and 10 µm) obviously revealed that CuO nanostructure is composed of composite morphology, such as nanoparticles, nanorods and nanoprisms. These composite nanostructures are highly transparent and randomly aligned. Average diameter of CuO nanoparticles ranges between 0.5 and 1 µm, length of CuO nanorods varied from 0.5 µm to 1.5 µm and their width differs between 0.5 and 1 µm and size of CuO nanoprisms varied from 3 µm to 5 µm. This CuO composite nanostructure covered whole surface with minimum proportion of voids. Since this composite nanostructure is composed of dissimilar dimensional CuO nanostructures, viz. one dimensional nanorods, two dimensional nanoprisms and three dimensional nanoparticles, it is worthwhile to mention here that a favorable and new kind of performance can be expected during their application as antibacterial agent against selected bacterial species.

15

Figure 5. SEM images of CuO nanostructure synthesized using C. rotundus grass extract.

3.3. Energy dispersive X-ray spectroscopy investigation Energy-dispersive X-ray (EDAX) spectra of CuO nanostructure synthesized by grass extracts were concurrently recorded during SEM analysis in view of examining proportion of elements present in the prepared samples. EDAX spectrum of CuO nanostructure synthesized by C. dactylon grass extract clearly indicates peaks corresponding to Cu and O elements that

16

confirm formation of monocrystalline CuO nanostructure through this green synthesize route and moreover sample is pristine in nature without any foreign elements (Figure 6). Atomic weight percentage between Cu and O for the CuO nanostructure is found out as ~ 1:1, which discloses that CuO nanostructure encompasses only CuO, which is in accordance with corresponding XRD result (Table 2).

Figure 6. EDAX spectrum of CuO nanostructure synthesized using C. dactylon grass extract.

Table 2. Weight percentage of Cu and O atoms present in CuO nanostructure synthesized using C. dactylon grass extract Element Cu

Anomic Number 29

O

8 Total

Series K-Series

Unn. [wt.%] 69.69

C Norm. [wt.%] 80.71

C Atom. [wt.%] 51.30

(1 Sigma) [wt.%] 2.04

K-Series

16.65

19.29

48.70

2.87

86.34

100.00

100.00

17

EDAX spectrum of CuO nanostructure prepared by C. rotundus grass extract also obviously discloses presence of peaks analogous to Cu and O elements that confirms formation of monocrystalline CuO nanostructure through this green synthesize route and furthermore sample is pristine without any foreign impurities (Figure 7). Atomic weight percentage between Cu and O for CuO nanostructure is found out as ~ 1:4, which unveils that CuO nanostructure contains only CuO, which is association with corresponding XRD result (Table 3).

Figure 7. EDAX spectrum of CuO nanostructure synthesized using C. rotundus grass extract.

Table 3. Weight percentage of Cu and O atoms present in CuO nanostructure synthesized using C. rotundus grass extract Element Cu

Anomic Number 29

O

8 Total

Series K-Series

Unn. [wt.%] 47.24

C Norm. [wt.%] 50.58

C Atom. [wt.%] 20.49

(1 Sigma) [wt.%] 1.47

K-Series

46.15

49.42

79.51

9.22

93.39

100.00

100.00

18

3.4. Fourier-transform infrared spectroscopy analysis Dual role played by grass extracts as reducing and capping agent by means of few functional groups is ascertained by FTIR analysis of CuO nanostructures. FTIR spectra of CuO nanostructures synthesized using C. dactylon and C. rotundus grass extract are shown in Figure 8. The strong absorption peaks observed at 438.14, 543.02, 589.33, 528.04 and 589.33 cm−1 are caused by asymmetrical stretching, which corresponds to deformation vibration of Cu–O along (2 0 2) direction that confirms presence of monoclinic CuO [33,34]. Weak absorption peaks noticed at 823.61 and 1022.47 cm−1 are attributed to M–O stretching vibrations of CuO (M = Cu) [35]. Several small absorption bands positioned 1100 and 2000 cm−1 might be due to chemisorbed and/or physisorbed H2O and CO2 molecules present on the surface of CuO nanostructures [36]. Sharp intense peaks originated in mid FTIR region of the spectra at 2355.96 and 2358.68 cm−1 could be ascribed to existence of atmospheric CO2 [37]. The band located at 2884.45 cm−1 is due to C–H stretching vibrations of alkane. The CuO nanostructures with large surface to volume ratio absorb some moisture from environment and as a consequence they exhibited meager absorption peaks at 3028.83, 3005.68, 3346.20, 3444.27 and 3694.82 cm−1 [38]. The virtually flat FTIR spectra apart from strong absorption peaks obtained for monoclinic CuO revealed high purity of CuO nanostructures synthesized using both C. dactylon and (b) C. rotundus grass extracts.

19

Figure 8. FTIR spectra of CuO nanostructures synthesized using (a) C. dactylon and (b) C. rotundus grass extracts.

20

3.5. Formation mechanism Green synthesis of nanomaterials using plant extracts as bio-reductants can have advantages over other biological processes because it eliminates the process of maintaining cell culture and can be appropriately scaled up for large-scale synthesis. In the present case, the formation of CuO nanostructure was confirmed primarily on the basis of change in colour of the reaction mixture. As CuNO3.3H2O solution was added to the grass extract, color of the solution changed from light blue to dark green after stirring with heat and subsequent calcination process lead to formation of CuO nanostructure (Figure 2). The biomolecules, such as terpenoids, proteins, flavonoids and alkanoids present in the C. dactylon and C. rotundus grass extracts might be responsible for the reduction of Cu2+ ions from CuNO3.3H2O and subsequent transformation into CuO nanostructure [39,40]. The probable mechanism involved during the synthesis of CuO nanostructure is given in the following equations. Cu2+ + Grass Extract [Cu/ Grass Extract] 2+

[Cu/ Grass Extract] 2+

---------------- (1)

Stirring (80 °C) [Cu(OH)2/ Grass Extract]

---------------- (2)

Calcined (200 °C) [Cu(OH)2/ Grass Extract]

CuO Nanostructure

---------------- (3)

3.6. Antibacterial activity evaluation Antibacterial activity potential of CuO nanostructures synthesized by C. dactylon and C. rotundus grass extracts was examined by adopting standard well diffusion method using Muller Hinton agar media, in view of their exploitation as possible antibacterial agent owing to inferior toxicity and thermal resistance of the CuO nanostructures. Gram positive bacterial species (Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus)) and Gram negative bacterial

21

species (Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) were employed as model bacterial species to investigate antibacterial activity of CuO nanostructures. Evaluation of antibacterial activity using sterile distilled water revealed absence of any antibacterial activity (nil zone of inhibition (ZOI)) against all four bacterial species. Antibacterial activity results of CuO nanostructure synthesized by C. dactylon grass species, positive and negative controls against bacterial species are shown in Figure 9 and relevant data values are given in Table 4.

Among the bacterial species, CuO nanostructure showed highest antibacterial performance against K. pneumoniae species with ZOI values of 26 and 28 mm for CuO nanostructure dosage concentration of 50 and 100 µL, respectively. It is highly peculiar to mention here that positive antibacterial control agent (tetracycline with a concentration of 30 mcg/disc) exhibited relatively lower ZOI of 24 mm) against same bacterial species. Next better antibacterial activity was noticed against S. aureus species with ZOI values of 24 and 26 mm for 50 and 100 µL dosage concentration of CuO nanostructure, respectively and positive control showed a ZOI value of 22 mm. Third better antibacterial activity was spotted against B. cereus species with 20 and 24 mm ZOI respectively for 50 and 100 µL CuO nanostructure dosage concentration and 24 mm ZOI for positive control. Least antibacterial activity was noticed against E. coli with ZOI values of 16 and 20 mm, respectively for 50 and 100 µL CuO nanostructure dosage. A ZOI of 22 mm was detected for positive control.

22

(a)

(b)

(c)

(d)

Figure 9. Antibacterial activity of CuO nanostructure synthesized by C. dactylon grass extract, positive and negative controls against bacterial species (a) B. cereus, (b) S. aureus, (c) E. coli and (d) K. pneumoniae.

23

Table 4. Antibacterial activity results of CuO nanostructure synthesized by C. dactylon grass extract, positive and negative controls against different bacterial species

Zone of Inhibition (mm) Sl. No.

Microorganism

Control (Sterile Distilled Water)

CuO Nanostructure

Standard Antibiotic (Tetracycline)

Dosage Level 100 µL

50 µL

100 µL

30 µg/disc

1

B. cereus

Nil

20

24

24

2

S. aureus

Nil

24

26

22

3

E. coli

Nil

16

20

22

4

K. pneumoniae

Nil

26

28

24

Antibacterial performance results of CuO nanostructure synthesized by C. rotundus grass species, positive and negative controls against diverse bacterial species are shown in Figure 10 and associated data values are furnished in Table 5. In this case also superior antibacterial activity was disclosed against K. pneumoniae species with ZOI values of 22 and 26 mm for CuO nanostructure dosage concentration of 50 and 100 µL, respectively. Second best antibacterial performance was observed against E. coli species with ZOI values of 20 and 26 mm for 50 and 100 µL dosage concentration of CuO nanostructure, respectively. Third better antibacterial performance was detected against S. aureus species with 18 and 24 mm ZOI values for 50 and 100 µL CuO nanoparticles dosage concentration. Low antibacterial activity was observed against B. cereus species with ZOI values of 14 and 24 mm, respectively for 50 and 100 µL CuO nanostructure dosage. Positive antibacterial control agent, tetracycline with a dosage concentration of 30 mcg/disc disclosed a ZOI value of 24 mm against all bacterial species. 24

(a)

(b)

(c)

(d)

Figure 10. Antibacterial activity of CuO nanostructure synthesized by C. rotundus grass extract, positive and negative controls against bacterial species (a) B. cereus, (b) S. aureus, (c) E. coli and (d) K. pneumoniae.

25

Table 5. Antibacterial activity results of CuO nanostructure synthesized by C. rotundus grass extract, positive and negative controls against different bacterial species

Zone of Inhibition (mm) Sl. No.

Microorganism

Control (Sterile Distilled Water)

CuO Nanostructure

Standard Antibiotic (Tetracycline)

Dosage Level 100 µL

50 µL

100 µL

30 µg/disc

1

B. cereus

Nil

12

24

24

2

S. aureus

Nil

18

24

24

3

E. coli

Nil

20

26

24

4

K. pneumoniae

Nil

22

26

24

Antibacterial performance analysis revealed that the CuO nanostructures are highly effective in killing / inhibiting growth of selected bacterial species. One of the probable causes for this superior antibacterial activity performance could be direct interaction of CuO nanostructures with external cell membrane of the bacteria. The presence of CuO generates reactive oxygen species and their interaction with bacterial cell membrane facilitates penetration of individual CuO nanostructures into the cell. The inhibition in growth bacterial species is possibly due to disturbances of cell membrane by CuO nanostructures that results in malfunction of cell enzyme [41]. The size of bacterial cells is usually in the micrometer range and pores of their cellular membranes have nanometer size range. The CuO nanostructures with size less than the pore size of the bacteria easily crossed the cell membrane without any interruption [42]. CuO nanostructures could destruct bacterial membranes by means of reactive oxygen species (superoxide and hydroxyl) radicals or direct cell damage since superoxide and hydroxyl radicals

26

are two chief reactive oxygen species produced by metal oxides [43–46]. Another probable reason is profusion of amines and carboxyl groups present on cell surface of bacterial species improves affinity of Cu2+ ions towards these groups, which results in superior antimicrobial performance of CuO nanostructures [47]. The variations in sensitivity of CuO nanostructures against selected bacteria chiefly rely on their size and morphology. Based on these results, it can be concluded that the green synthesized CuO nanostructures using C. dactylon and C. rotundus grass extracts have substantial antibacterial activity against both Gram positive and Gram negative bacterial species. Hence, the potential antibacterial mechanism can be related to mechanical damage, oxidative injury and gene toxicity. Our results also suggested that CuO nanostructures exert excellent antibacterial activity against K. pneumoniae species due to different mechanisms in combination with physical and chemical damage and gene expression inhibition (Figure 11). Comparison of present work with previous literatures pertaining to green synthesis of diverse CuO nanostructures are given in Table 6.

Table 6. Comparison of green synthesis of CuO nanostructures using diverse plant sources

Sl. No.

Plant Source

Shape of CuO

1.

Thymus vulgaris L

Spherical Nanoparticles

[48]

2.

Carica papaya L

Spherical Nanoparticles

[49]

3.

Bauhinia tomentosa

Spherical Nanoparticles

[13]

4.

Madhuca longifolia

Spherical Nanoparticles

[38]

5.

Sida acuta

Nanorods

[1]

6.

Calotropis procera

Cylindrical

[50]

7.

Ruellia tuberosa

Nanorods

[12]

8.

Seidlitzia rosmarinus

Cauliflower-like Nanoparticles

[51]

27

Reference

O2

CB

O 2¯ VB

ROS Generation

CuO Nanostructures

Capsule

Cell membrane

Cytoplasm

Pilus Flagellum

DNA

Mitochondria

Figure 11. Antibacterial mechanism of CuO nanostructures against bacterial species.

4. Conclusion Copper oxide (CuO) nanostructures were synthesized using Cynodon dactylon (C. dactylon) and Cyperus rotundus (C. rotundus) grass species extracts. Prepared CuO nanostructures showed diffraction peaks at 32.27, 35.24, 38.49, 48.70, 53.31, 57.96, 61.38, 66.18, 67.70, 72.09 and 74.94°, corresponding to (1 1 0), (0 0 2), (1 1 1), (2 0 2), (0 2 0), (2 0 2),

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(1 1 3), (3 1 1), (1 1 3), (2 2 1) and (0 0 4) diffraction planes of end-centered monoclinic CuO. Major diffraction peaks with high intensities imply high crystalline nature of CuO samples. SEM images of CuO nanostructure prepared by C. dactylon grass species extract exhibited rice spikelet-like morphology, whose average length ranges from 1 µm to 1.5 µm and their average width is ~500 nm. SEM micrographs of CuO nanostructure prepared by C. rotundus grass species extract showed composite morphology, such as nanoparticles, nanorods and nanoprisms. EDAX spectra of CuO nanostructures obviously indicate that peaks corresponding to Cu and O elements only are noticed that confirms pristine nature CuO nanostructures prepared through this green synthesize route without any foreign elements. FTIR spectra also indicated presence of monoclinic CuO. Better antibacterial activity noticed can be credited to formation of reactive oxygen species on surface of CuO nanostructures that facilitates death of bacterial species.

Acknowledgment The authors acknowledge the DST-FIST, Government of India, New Delhi for generously providing the Research Instruments Facility (Grant No. SR/FST/College-2017/140 (C), dt. 14.08.2018).

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Declaration of Interest Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper entitled “Green Synthesis of Copper Oxide Nanostructures using Cynodon dactylon and Cyperus rotundus Grass Extracts for Antibacterial Applications” to the journal Ceramics International. Yours sincerely, S/d. (Dr. S. Suresh) (On behalf of all the Authors)