Inorganic Chemistry Communications 134 (2021) 108984

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Inorganic Chemistry Communications

journal homepage: www.elsevier.com/locate/inoche

A novel bio-fabrication of ZnO nanoparticles using cow urine and study of

their photocatalytic, antibacterial and antioxidant activities
Harshal Dabhane a, b, Manohar Zate c, Ramdas Bharsat b, Ghanshyam Jadhav a,
Vijay Medhane a, d, *
a
Department of Chemistry, K.R.T. Arts, B.H. Commerce, and A.M. Science College, Nashik 422002, Savitribai Phule Pune University, Maharashtra, India
b
Department of Chemistry, G.M.D Arts, B.W Commerce and Science College, Sinnar, 422 103, Savitribai Phule Pune University, Maharashtra, India
c
Department of Physics, G.M.D Arts, B.W Commerce and Science College, Sinnar, 422 103, Savitribai Phule Pune University, Maharashtra, India
d
Department of Chemstry, S.V.K.T. Arts, Commerce and Science College, Nashik 422401, Savitribai Phule Pune University, Maharashtra, India

A R T I C L E I N F O A B S T R A C T

Keywords: In recent years, nanomaterials have piqued the interest of the research community due to intriguing claims in a
Biogenic synthesis variety of areas, the primary focus being the mode and methods of synthesis of applicative materials. Numerous
ZnO NPs methods are now used in the production of various nanomaterials, and these processes have an impact on their
Cow urine
properties. Considering that we describe a novel biogenic approach for the synthesis of Zinc Oxide nanoparticles
Photocatalytic degradation
Antibacterial
(ZnO NPs) using cow urine as a reducing and stabilizing agent, the confirmation and characterization of the
Antioxidant Study synthesized material were accomplished using spectroscopic and microscopic techniques. The primary confir­
mation of bio-fabrication of ZnO NPs was done with UV-DRS and the bandgap was found to be 2.96 keV. The
Wurtzite structure with 3.99 nm size was confirmed by the XRD pattern, whereas the morphology and purity
were confirmed via SEM and EDX techniques. Furthermore, the disc diffusion method’s antibacterial activity of
characterized ZnO NPs was tested against different bacterial strains. The radical scavenging activity was studied
using the OH and DPPH assays.

1. Introduction route for the synthesis of ZnO NPs which comprises the use of plant
extracts and microorganisms as reducing, stabilizing, and capping
Nanotechnology has been a great protagonist in the enlargement and agents. The extracts of countless medicinal plants are full of phyto­
evolution of research fields and other areas. The step-down and bottom- chemicals like polyphenols, alkaloids, terpenoids, alcohols, amino acids,
up approaches provide fascinating opportunities for the synthesis of carbohydrates, etc. which actively do bio-reduction of zinc metal salt at
wonder materials in the nano range. The nano size and higher surface basic conditions [12]. Attempts have been made to synthesize them
area of nanoparticles make them more special and have chemical, using microorganisms like bacteria, fungus, algae, and yeast [13]. There
physical, and biological applicability [1]. Researchers have reported are plenty of reports on the phytogenic synthesis of ZnO NPs which
numerous metal oxide nanoparticles. Among them, ZnO NPs have a explain the requirement for basic conditions via external addition of
unique identity because of their non-toxic nature and multifunctional bases like NaOH, and ammonia [14].
behavior [2]. Several applications of ZnO NPs in the biomedical field, Cow urine has been used for drinking purposes since the ancient era.
agriculture, water treatment, solar cells and devices, sunscreen, cos­ Their prominence was well explained in ancient literature like Shivambu
metics, and food packaging make them special [3–9], along with that (Human Urine), and in medicinal applications in Ayurveda [15–16]. The
ZnO NPs were act as photocatalyst and multifunctional material due to clinical analysis of cow urine shows the presence of iron, copper, ni­
their high band gap (3.37 eV) and free-exciton transition luminescence trogen, sulfur, manganese, carbolic acid, silicon, chlorine, magnesium,
in the UV range [10]. ZnO NPs were broadly synthesized using chemical citric, calcium salts, enzymes, mineral salts, vitamins like A, B, C, D, E,
and physical methods which involve toxic chemicals, are time- creatinine, uric acid, hormones, gold acids, hippuric acid, and aureum
consuming and do not follow the principles of green chemistry [11]. hydroxide [17]. At present, silver, cadmium, copper, palladium, and
Since the last decade, researchers have espoused a novel eco-friendly silver oxide nanoparticles have been reported using cow urine as fuel,

* Corresponding author.
E-mail address: [email protected] (V. Medhane).

https://doi.org/10.1016/j.inoche.2021.108984
Received 2 August 2021; Received in revised form 30 September 2021; Accepted 30 September 2021
Available online 5 October 2021
1387-7003/© 2021 Elsevier B.V. All rights reserved.
H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Fig. 1. Active biomolecules present in cow urine.

Fig. 2. Possible mechanism of cow urine mediated biogenic synthesis of ZnO NPs.

and their antibacterial, antifungal, and antioxidant activities have been India and cow urine were filtered via whatmann filter paper was used as
tested against pathogenic gram-positive and negative strains source substances for the synthesis of ZnO NPs.
[18,19,20,21].
Herein, we first report the biogenic synthesis of ZnO NPs via cow 2.2. Preparation of ZnO NPs.
urine and zinc sulfate as a precursor. The present study concentrates on
the biogenic synthesis of ZnO NPs by a one-step process which is The cow urine of the Holstein breed was selected for the biogenic
economical, quick, simple, and ecofriendly in nature. The synthesized synthesis of ZnO NPs. The fresh urine of a healthy cow was collected in a
ZnO NPs were confirmed and characterized by spectroscopic and clean and dry 1 L plastic bottle. 100 ml of cow urine was taken in a 250
microscopic techniques and were further used for the evaluation of their ml beaker and carefully filtered through Whatman filter paper no. 1 to
antimicrobial and antioxidant activities. The benefits of biogenic syn­ remove unwanted impurities and further used for the synthesis of
thesis of ZnO with cow urine can be well explained by comparing their nanoparticles. 1.61 g of zinc sulfate (0.01 M) was added to 100 ml of
biological activities with the reported work in the results and discussion filtered cow urine to get a final concentration of 0.01 M with constant
section. stirring for 1 hr. The change in color of cow urine indicates the
completion of the reaction. The precipitate was filtered by a Buckner
2. Experimental details funnel followed by washing through distilled water to remove the
organic content in cow urine. The resultant precipitate was dried in a hot
2.1. Materials. air oven at 100 ◦ C for 2 hrs, and after that, calcinated in a muffle furnace
at 400 ◦ C for 3 hrs to obtain ZnO NPs.
Analytical grade Zinc sulfate (ZnSO4) of 99% purity of sigma Aldrich

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

2.3. Characterization.

The preparation of ZnO NPs was confirmed by a UV–Visible spec­

trophotometer (JASCO V-770). The bandgap was obtained by plotting
(αhν)2 against hν according to the Tauc relation. The metal-oxygen bond
was confirmed through FT-IR spectroscopy (FT/IR-4600 type A). The
photoluminescence efficiency of ZnO NPs was also studied by evaluating
the photoluminescence spectrum (FP-8200). The surface area and
porosity were measured with the help of the Brunauer–Emmett–Teller
(BET) method, whereas a Powder x-ray diffactometer was used to study
the structure. The crystal plane and morphology and size were deter­
mined by examining scanning electron microscopy (VEGA3 TESCAN),
HR-TEM and elemental detection was done by EDX graph.

2.4. Photocatalytic Activity.

The photocatalytic degradation of Methylene Orange (MO) was

carried out in an aqueous solution using cow-mediated ZnO NPs under
UV irradiation (λ = 254 nm). In the present experiment, 10 ml of MO
was taken in a dry and clean 50 ml beaker with different amounts of
Fig. 3. UV–Visible spectra of synthesized ZnO NPs. synthesized ZnO NPs (5, 10, 15 mg) and stirred for a moment before
irradiation with UV light. The progress of the reaction was monitored by
a UV–Visible spectrometer by recording the absorbance of the reaction
mixture after centrifugation at a regular interval of 10 mins (λ = 460
nm).

2.5. Antimicrobial activities

2.5.1. Disk diffusion assay

The antibacterial potential of synthesized ZnO NPs was evaluated by
the disk diffusion assay method. In brief, each dried paper disk (What­
man filter paper No.1) contained ZnO NPs 50 µL (Conc.1 mg/ mL). Each
disk was then placed on the surface of the sterile solidified Mueller-
Hinton agar/ Potato Dextrose agar medium which was spread with 24
hr old inoculums of bacterial and fungal cultures respectively. Penicillin
(for S. typhi, B. subtilis, B. megaterium, E. coli,) Fluconazole (for R. oryzae,
P. chrysogenum, A. niger, C. albicans) were used as standards (at con­
centrations of 1 mg/mL). The plates were kept in the refrigerator for
diffusion for 3 hrs and then kept in an incubator at 37 ◦ C & 30 ◦ C
respectively for 24 hrs. After incubation, the zones around the discs were
measured by the zone scale.

2.5.2. Resazurin microtiter assay (REMA)

Fig. 4. FT-IR spectra of synthesized ZnO NPs.
The MIC was calculated using the REMA plate assay method. Briefly,
100 µL of Mueller-Hinton / Potato Dextrose broth medium was
dispensed into each well of a sterile flat-bottom 96-well plate, and serial
two-fold dilutions of standard/ZnO NPs were prepared directly on the
plate. 100 µl of inoculum were added to each well. Sterile cold water
was added to all perimeter walls to avoid evaporation during the incu­
bation. The plates were covered, sealed in a plastic bag, and incubated at
37 ◦ C for 24 hrs. After incubation, 30 µL of resazurin solution (0.01% in
sterile D/W) was added to each well, and the plate was pre-incubated
overnight. A change in color from blue to pink indicated the growth of
bacteria, and the MIC was defined as the lowest concentration of drug
that prevented this color change. The concentration ranges used were
0.97–500 µg/ml for ZnO NPs and standards. Minimum inhibitory con­
centrations (MIC) were studied using Penicillin and Fluconazole as
positive controls against bacterial and fungal strains respectively.

2.6. Antioxidant activity

The antioxidant activity of cow urine mediated ZnO NPs was scanned
by two different methods, namely the Hydroxyl and DPPH radical assay.

2.6.1. Hydroxyl radical assay (OH)

Fig. 5. Photoluminescence spectra of synthesized ZnO NPs.
The OH radical scavenging activity was demonstrated with the

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Fig. 6. (a) Adoption-desorption Study using BET, (b) Pore size of ZnONPs via adsorption plot.

that the final volume was made up to 3 ml, incubating the samples for
20 min, and reading the absorbance at 517 nm using (Shimadzu).
Ascorbic acid (1 mM) was used as a standard, the percent inhibition was
calculated using the following formula (1).

3. Result and discursion

3.1. The mechanism for the synthesis of ZnO NPs

The chemical analysis of cow urine confirms the presence of several

biomolecules, but some of them are more reactive and act as reducing,
stabilizing, and capping agents (Fig. 1). The biomolecules like allantoin,
creatinine and uric acid have polar sides which interact with positive
metal ions. The result is a metal–biomolecule complex, after calcination,
the complex results in ZnO NPs as shown in Fig. 2 [22,23]. At the
beginning of reaction, the zinc sulphate precursor was reacted with cow
urine to form zinc hydroxide precipitate in basic condition provided by
cow urine, further the zinc hydroxide precipitate was dry in oven and
calcinated in muffle furness at 400 ◦ C. This process involve the con­
version of zinc hydroxide in to zinc oxide by the removal of water
Fig. 7. XRD pattern of the synthesized ZnO NPs. molecules, during this process agglomerated zinc oxide particles start to
adopt specific morphology and size [24].

Table 1
XRD peaks and corresponding parameter of synthesized ZnO NPs. 3.2. Characterization of ZnO NPs

Peak Position (2θ) FWHM Crystalline size D (nm) D nm (Average)

3.2.1. Study of optical properties
31.89678 1.72737 4.783172487 The optical properties of nanoparticles are contingent on their size,
34.42571 1.75031 4.751604611
shape, and appearance, so cow urine mediated ZnO NPs were analyzed
36.2642 1.44603 5.780934195 4.399301113
51.48167 29.39775 0.300004748 with UV-DRS techniques which displayed a single absorption peak at
56.67332 1.41453 6.380789524 300 nm in Fig. 3 and this result matches with the previous reports [25].
This single peak is owing to surface plasmon resonance sensations and
the physical state of ZnO NPs, which evidences the spherical shape of
Fenton reaction. The typical reaction mixture contained 60 µL of FeCl2 ZnO NPs also approved by SEM image.
(1 mM), 90 µL of 1–10 phenanthroline (1 mM), and 2.4 ml of phosphate The optical band-gap of bio-fabricated ZnO NPs was intended using
buffer (0.2 M, pH 7.8), 150 µL of H2O2 (0.17 M), and 1.5 ml of ZnO NPs Tauc’s relation (2).
(1 mg/ml). The reaction was started by adding H2O2. After 5 min of ( )n
incubation at room temperature, the absorbance was read at 560 nm. αhν = hν − Eg (2)
Ascorbic acid (1 mM) was used as a reference. The radical scavenging
activity was calculated using Eq. (1). where α is absorption coefficient, Eg is optical band-gap, and hν is
photon energy [26]. The optical band-gap of bio-fabricated ZnO NPs was
% radical scavenging activity = 1 − T/C × 100 (1) found to be (Eg = 2.96 eV) shown in the inset of Fig. 3 which shows a
redshift when compared with a previous report (Eg = 3.175) [27].
2.6.2. 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH)
The electron donation ability of the crude extracts was measured by 3.2.2. FT-IR analysis
the bleaching of the purple-colored ethyl acetate solution of DPPH. The FT-IR spectrum was performed to study the interaction and role
DPPH is a stable reagent used in this spectrophotometric assay. The of bio-molecules present in cow urine in the synthesis of ZnO NPs. Fig. 4
assay was performed by mixing equal DPPH solution in methanol, so shows the FT-IR spectrum of cow urine mediated synthesis of ZnO NPs.

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Fig. 8. (a) Larger view of SEM image. (b) Closer view of SEM image. (c) EDS of synthesized ZnO NPs.

The analysis of the obtained FT-IR spectrum was done by referring to the size and pore volume of ZnO NPs were 33.073 nm and 0.3199 cm3/g
previous report on green synthesis of ZnO NPs using insulin plant extract respectively (Fig. 6b). The reported specific surface area of commercial
[28]. At first glance, the sharp peak below 500 cm− 1 of the Zn–O bond ZnO NPs is 3.23 m2/g. In comparison, synthesized ZnO NPs have ten
confirms the formation of ZnO NPs. The appearance of a broad peak times greater specific surface area [30]. In addition, the specific surface
centered at 3320 cm− 1 along with the carbonyl group peak at 1602 cm− 1 area of synthesized ZnO NPs is greater than plant-mediated synthesized
confirms the presence of the carboxylic acid functional group in uric ZnO NPs [31].
acid. The double absorption less intense peaks at 2879 cm− 1 and 2825 The adsorption–desorption isotherm of bio-fabricated ZnO NPs cor­
cm− 1 indicate the C–H stretching vibration in the aldehyde group. The responds to the type IV isotherm shown in Fig. 6a. The BET parameter
moderate peak around 1513 cm− 1 shows the aromatic bending vibra­ suggests that synthesized ZnO NPs might act as a good catalyst in
tions. The peak at 1126 cm− 1 shows the presence of C–O bond organic transformation and photocatalytic dye degradation.
stretching vibrations which lead to the existence of the phenolic group.
Finally, the FT-IR spectrum indorses the reducing, stabilizing, and 3.2.5. X-ray diffraction analysis
capping abilities of biomolecules present in cow urine. The crystal structure and size of bio-fabricated ZnO NPs also
confirmed by the XRD spectrum shown in Fig. 7. The diffraction peaks in
3.2.3. Photoluminescence study the XRD pattern of nanoparticles agree with the single-phase crystalline
The photoluminescence properties and defects present in cow urine nature, corresponding to the wurtzite hexagonal crystal structure
mediated synthesized ZnO NPs were studied with photoluminescence confirmed by data from JCDPS card No. 36-1451. The crystalline size of
spectra at room temperature. The first emission band appears in the UV synthesized ZnO NPs was calculated using the Debye-Scherrer formula
region, which might be due to band-band transition, and the second (3).
emission band is located at 600 nm, which is attributed to the presence
D = Kλ /βcosθ (3)
of a single ionized oxygen atom in bio-fabricated ZnO NPs (Fig. 5). The
obtained photoluminescence spectra were compared and confirmed where K is constant (0.9), λ is the wavelength of X-ray (1.54056 Ao),
with a previous report [29]. β is the corrected full width of half maximum (FWHM), and θ is Bragg’s
angle. The corresponding XRD peaks are listed in the Table 1, the
3.2.4. Gas adsorption studies calculated average crystalline size of synthesized ZnO NPs using the
The adsorption–desorption studies of bio-fabricated ZnO NPs were Debye-Scherrer equation was found to be 4.399 nm [32].
carried out with the BET method in a nitrogen atmosphere. The par­
ticulate properties of synthesized ZnO NPs such as specific surface area, 3.2.6. SEM and EDX analysis
pore size, and volume distribution are shown in Fig. 6. The BET surface The conformation of nano-size ZnO NPs was done with SEM micro­
area of synthesized ZnO NPs was determined by the pretreated sample at graph. Fig. 8a is a larger view of synthesized ZnO NPs showing the
250 ◦ C and found to be 38.691 m2/g (Fig. 6a). The surface area of NPs agglomeration, whereas the closer view in Fig. 8b shows the mesoporous
depends on the calcinated temperature. It is found that calcination at size in the range of 42–60 nm. The agglomeration might be due to the
low-temperature results in a high surface area of NPs. The average pore unequal calcination temperature. The purity and elemental composition

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Fig. 9. (a) HR-TEM image of low resolution, (b) HR-TEM image of high resolution, (c) d-spacing in crystal plane, (d) SAED pattern.

Fig.10. XPS graph of bi-fabricated ZnO NPs.

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Fig 11. Suggested mechanism of photodegradation of methyl orange using bio-fabricated ZnO NPs in sunlight.

Fig. 12. Photocatalytic activity of ZnO NPs (a) plot of absorbance vs. wavelength for the decolonization of methyl orange under sunlight, (b) Removal efficiency (%)
of methyl orange dye by ZnO NPs, and (c) its first order kinetics, (d) Reproducibility with error bar for 10 mg of photocatalyst.

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Table 2
Comparative study of photocatalytic activity of ZnO NPs with previous reports.
Entry Metal Reducing/ Stabilizing/ Capping Morphology & Size Organic Pollutant Light Time Degradation Ref.
source agents Source (Minute) efficiency in %

1 Zinc Pithecellobium dulce peel Spherical 30 nm Methylene Blue UV–Visible 120 63 [1]
Acetate
2 Zinc Nitrate Phyllanthus niruri plant leaves Spherical 25.61 nm Methylene Blue UV 30 100 [2]
Extract
3 Zinc Sechium edule leaf extract Spherical 36.2 nm Reactive Blue 160 UV 120 94.47 [3]
Acetate
4 Zinc Serratia nematodiphila strain Spherical 24.79 nm Methyl Orange UV 270 88 [4]
Sulphate ZTB15
5 Zinc Nitrate Leaf extract of Plectranthus Rod Shape 88 nm Methyl Red UV 180 92.45 [5]
amboinicus
6 Zinc Azadirachta Indica (Neem) Spherical 33.25 nm Methylene Blue UV 200 80.2 [6]
Acetate
7 Zinc Nitrate Rambutan (Nephelium Spherical 25.67 nm Methyl Orange UV 120 83.99 [7]
lappaceumL.) peel extract
8 Zinc Broccoli extract Spherical 14 nm Methylene Blue and UV 180 74 and 71 [8]
Chloride Phenol Red
9 Zinc Nitrate Extracts of Justicia spicigera Semi-spherical Methylene Blue UV 120 90 [9]
10–100 nm
10 Zinc Nitrate Thymus vulgaris leaf extract Irregular Shape Methylene Blue UV 30 96 [10]
50–60 nm
11 Zinc Cow Urine Spherical 4.399 Methyl Orange Sunlight 60 98 Present
Sulphate nm Work

oxygen interstitials (Oi) and oxygen vacancies (VO) point defects in ZnO
Table 3 and O 1 s state always contain three binding energies components
Antibacterial activities and MIC study of ZnO NPs.
contains three binding energies components corresponding respectively
Antibacterial Activity to low binding energy peak (LP) at 530.15 eV, middle binding energy
Entry Bacterial Strain ZnO NPs Activity Penicillin (Standard) peak (MP) at 531.25 eV and high binding energy peak (HP) at 532.40 eV
[34].
1 B. subtilis +++ +++
2 B. megaterium +++ +++ In the present study, the oxygen-deficient nature of bio-fabricated
3 E.coli +++ +++ ZnO NPs already suggested by EDAX analysis, the XPS survey spectra,
4 S. typhi +++ +++ and core spectrum of O1s and Zn 2p levels are shown in Fig. 10. The x-
Minimum Inhibitory Concentration (MIC)
ray photoelectron and Auger emission from zinc and oxygen in ZnO NPs
Bacterial Strain ZnO NPs (µg/ml) Penicillin (Standard) (µg/ml)
5 B. subtilis 31.26 1.95
observed in Fig. 10a, it exactly matches with the EDAX spectra of ZnO
6 B. megaterium 31.26 3.9 NPs confirm the purity and presence of zinc and oxygen ions in syn­
7 E.coli 7.8 3.9 thesized ZnO NPs. The oxygen deficiency suggested by EDAX techniques
8 S. typhi 7.8 1.95 can prove by the O 1 s core level (Fig. 10c), also the three binding energy
+ = <5 mm, ++ = >5 & <10 mm, +++ = >10 & < 18 mm, NZ = No zone. component peaks corresponding respectively low energy peak (LP) at
Results are the average mean of three parallel experiments. 530.47 eV, middle binding energy peak (MP) at 531.59 eV, and high
binding energy peak (HP) at 532.73 eV which are confirmed by previous
of ZnO NPs have been tested via the EDS spectrum Fig. 8c. The EDS report [35,36]. Similarly, the peak of Z 2p3/2 and Z 2p1/2 was found at
graph shows the three characteristic peaks of Zn at 1.2 keV, 8.6 keV, and 1022.25 eV and 1045.55 eV respectively, where the energy difference
10 keV respectively, and a single peak at 6 keV in the EDS spectrum between the two levels was found to be 23.3 eV shown in Fig. 10a.
which confirms the formation and purity of cow urine mediated ZnO
NPs [33]. 3.2.9. Adsorption study of methyl orange (MO) dye
The adsorption study was carried out by considering methyl orange
3.2.7. HR-TEM analysis as an organic pollutant and synthesized ZnO NPs as adsorbents. Fig. 12a
Fig. 9 shows the HR- TEM micrograph and SAED pattern of bio- represents the UV–Visible spectra of the adsorption kinetics of MO at a
fabricated ZnO NPs. Fig. 9a suggests the quasi-spherical shape of with regular time interval at wavelength of 460 nm, whereas Fig. 9b reports
different size, also the sharp edge of ZnO NPs structures was shown in the kinetic studies of MO dye plotted at a regular time interval on the x-
Fig. 9b. The crystalline size of the analyzed single crystal of ZnO NPs was axis and removal efficiency (%) on the y-axis. The removal efficiency is
found to be 7.04 nm which matches the average size calculate using the calculated by the following equation.
Debye-Scherrer formula using XRD data and the d-spacing distance was Efficiency (%) = C0 − Ct /C0 *100
found to be 0.28 nm matches (1 0 0) plane standard value 0.2814 nm
shown in the inset of Fig. 9c. The typically selected area electron where C0 is the initial absorbance at t = 0, Ct is the absorbance value at a
diffraction (SAED) pattern showing bright rings corresponding to (1 1 0), particular time interval. Several reports have been published on the
(1 0 2), (1 0 1), and (1 0 0) planes suggest the crystalline nature of bio- degradation of MO in the presence of UV light. This study mainly focuses
fabricated ZnO NPs (Fig. 9d). on the degradation of MO in the presence of sunlight using ZnO NPs as
adsorbents.
3.2.8. XPS analysis
XPS technique used to investigate the intrinsic electronic structure of 3.2.10. Photocatalytic study
synthesized solid material, metal oxide mainly deal with some defects One of the famous environmental-based applications of ZnO NPs is
due to the deficiency or access of positive and negative ions in the the photocatalytic degradation of organic dyes. This was studied
crystalline structure. XPS study of metal oxide confirm the presence of considering methyl orange as an organic pollutant. The photocatalytic

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Fig. 13. Antibacterial study of synthesized ZnO NPs indicated by Z1 (a) B. subtilis (b) B. megaterium (c) E. Coli (d) S. typhi.

Table 2 shows that bio-fabricated ZnO NPs synthesized for the first time
Table 4
using cow urine had excellent photocatalytic activity while exposed to
Antioxidant activities of ZnO NPs.
sunlight.
Entry Material Antioxidant Activity (%) The gas adsorption study of ZnO NPs confirms the high surface area,
OH DPPH pore size, and volume distribution in Fig. 6 and band gap were inves­
1 ZnO NPs 52.8 60.2
tigated using UV–Visible spectra and was found to be 2.96 keV. When
2 Ascorbic Acid (Standard) 82.3 86.4 such ZnO NPs were used as an adsorbent in the photodegradation of MO,
their removal efficiency was well explained by their absorption maxima
at wavelength of 460 nm. As per the literature survey, there are two
efficiency of bio-fabricated ZnO NPs was determined under sunlight. In different mechanisms was used to explain the photocatalytic mecha­
the present study, the aqueous solution of methyl orange at 10 ppm was nism, conventional type-II and z-scheme heterojuctions (Fig. 11) [37].
treated with different concentrations of catalyst (5, 10, 15 mg) under According to the conventional type-II mechanism ZnO NPs were
sunlight and their absorbance was measured at regular time intervals photoexcited in presence of solar radiation to generate e- h+ pairs, those
using a UV–Visible spectrophotometer at λ = 460 nm. Fig. 12a explores holes which are situated at CB will combine with water molecules to
the decrease in absorbance of methyl orange with regular intervals of form an OH radical. Conversely, electrons shifted in VB are absorbed by
time and different amounts of catalyst. The higher rate of degradation oxygen molecules to produce peroxide radical and that is responsible for
was observed with 15 mg ZnO NPs, which confirms the effect of catalyst photodegradation of methyl orange and finally result photodegradation
dose on the degradation efficiency of dye. However, the significant color product [38,39,40].
change was not seen with concentrations of 5 and 10 mg catalyst.
Fig. 12b explains the percent removal efficiency of ZnO NPs in methyl 3.2.11. Antibacterial activity
orange, where the maximum degradation was observed after 60 min in Bruna Lallo da Silva et al in his review explores the relationship
the presence of sunlight and Fig. 12c show the mechanism proceed via between structural properties of ZnO NPs and antibacterial efficiency by
first order kinetics. Reproducibility of results were tested by repeating considering four different types of mechanisms based on the production
the photocatalytic activity for triplicate (10 mg photocatalyst) with of reactive oxygen species (ROS), loss of cellular integrity following
error bar shown in Fig. 12d, minimum and maximum error was deter­ contact between ZnO NPs and the cell wall, the release of Zn2+ ions, and
mined 0.001 and 0.004 respectively. Many photocatalyst for the ZnO NPs internalization [41]. The ZnO NPs show reactivity towards
degradation of various organic pollutants have been described; howev­ bacteria due to the presence of both Zn2+ and superoxide ions, as ZnO
er, in this paper, we compare the technique of synthesis of ZnO NPs, NPs activate under UV and sunlight radiation or even in the dark and
their shape, and photocatalytic performance against methyl orange. produce superoxide ions which combine with water molecules to form

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H. Dabhane et al. Inorganic Chemistry Communications 134 (2021) 108984

Fig. 14. Antioxidant activity of synthesized ZnO NPs.

H2O2 molecules which leads to the death of the bacteria [42]. Another ZnO NPs was confirmed by XRD pattern which shows the wurtzite
mechanism is based on the release of Zn2+ ions [43]. Under acidic structure with a hexagonal arrangement. The size was determined from
conditions (pH = 4.5), ZnO NPs dissolve and produce Zn2+ which binds the Debye-Scherrer equation and authorized from SEM image was found
with biomolecules present inside the bacterial cell and inhibits its to be 4.399 and 47.37 nm respectively. The high surface area and pore
growth [44]. The morphology and structure of ZnO NPs also have a great size were investigated by the BET technique, which supports good
effect on the antibacterial efficiency. The higher surface area of ZnO NPs photocatalytic activity against methyl orange. It also shows excellent
is able to produce a large amount of superoxide ions efficiently, leading antibacterial activity against four different bacteria. The minimum in­
to the death of bacterial cell lines [45,46]. hibition study shows that only 7.8 µg/ml and 31.26 µg/ml concentra­
In the present work, cow urine mediated synthesized ZnO NPs were tions are enough to inhibit the growth of bacteria. The antioxidant
investigated against gram-positive and negative bacterial strains and activity was evaluated by DPPH and OH assays and showed 60.2 % and
showed excellent efficacy against B. megaterium, B. megaterium, E. Coli, 58.6 %. Finally, the ZnO NPs synthesized by this novel route expertly
and S. typhi when compared with the positive control Penicillin as show all activities and might be useful in the future as an antibacterial
standard (Table 3, entry 1, 2, 3, and 4) confirmed by the inhabitation agent.
zone shown in Fig. 13. The admirable antibacterial activity of synthe­
sized ZnO NPs might be due to the high surface area (38.691 m2/g), pore
size (33.073 nm) and pore volume (0.3199 cm3/g) confirmed by the BET Declaration of Competing Interest
analysis. The minimum inhibitory concentration (MIC) study also re­
veals high antibacterial potential, especially against E. Coli, and S. typhi. The authors declare that they have no known competing financial
The MIC study shows that 7.8 µg/ml dose of bio-fabricated ZnO NPs is interests or personal relationships that could have appeared to influence
enough to inhibit the bacterial colonies by the well diffusion method the work reported in this paper.
(Table 3, entry 7, and 8), whereas a 31.26 µg/ml dose is sufficient to
inhibit the growth of B. megaterium, B. megaterium (Table 3, entry 5, and Acknowledgement
6).
Authors are thankful to DST-SAIF Cochin, India for providing char­
3.2.12. Antioxidant activity acterization facility.
The radical scavenging activity of synthesized ZnO NPs was evalu­
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