Introduction

In modern materials science, the most active research area is nanotechnology [1]. The science of nanotechnology is concerned with the creation, processing, application, and range of materials in the nanoscale range, or nanomaterials [2]. The word "nano" is derived from the Greek word "dwarf," which describes a particle that is typically between one and one hundred nanometers in size. Richard Feynman gave the first lecture on the theoretical idea of nanotechnology in 1959 [3, 4].  It is the application of science to the molecular control of matter. Almost every aspect of civilization, including communications, computing, textiles, cosmetics, sports, therapy, automobiles, environmental monitoring, fuel cells and energy devices, water purification, the food and beverage industry, etc., is expected to be impacted by the truly multidisciplinary field of nanoscience and technology [3]. In addition, it is the study and application of small objects that has applications in material science, engineering, chemistry, biology, and physics. Surface-enhanced Raman Scattering (SERS), nanobiotechnology, bio nanotechnology, quantum dots, and applied microbiology are just a few of the new basic and applied frontiers in materials science and engineering that have been made possible by the tremendous expansion of nanotechnology [5]. The field of nanoscience and nanotechnologies is transforming our comprehension of matter and is anticipated to have significant effects on various industries, such as food and agriculture, energy generation and efficiency, automobiles, cosmetics, pharmaceuticals, home appliances, computers, and weapons. In an economical and ecologically responsible way, nanotechnology can help us avoid, identify, and eliminate environmental pollutants from the air, water, and soil [6]. Materials having at least one exterior dimension in the range of around 1-100 nm are referred to as nanomaterials (NMs). On the other hand, solid particles with all three exterior dimensions at the nanoscale, known as nanoparticles (NPs), have the ability to significantly alter physico-chemical properties in contrast to bulk material [7]. Regardless of their size, bulk materials have comparatively constant physical properties; however, this is frequently not the case at the nanoscale. The distinct optical, biological, and physico-chemical characteristics of the nanoparticles can be appropriately tailored for the intended uses [8]. Nanoparticles' small sizes are the cause of almost all of their characteristics. Owing to their incredibly small size and enormous surface area, nanoparticles have a variety of intriguing characteristics. They thus find novel applications in a wide range of fields, including communication technology, aircraft technology, heavy industry, agriculture, medicine and drug delivery, optoelectronics, magnetic, information storage, recording media, sensing devices, catalysis, chemistry, environment, energy, and consumer goods [9]. The process of obtaining materials from environmentally favorable or green sources through the use of a good reducing agent, a solvent, and a harmless stabilizing ingredient is known as "green synthesis"[10]. This synthesis technique yields more stable molecules and is also simple, economical, reliable, sustainable, and somewhat repeatable [11]. Numerous physical and chemical synthesis methods call for extremely hazardous reductants, stabilizing agents, and high radiation levels, all of which have negative impacts on marine life and humans. On the other hand, the environmentally friendly one-pot or one-step technique of green synthesis of metallic nanoparticles uses very little energy to start the reaction. This reduction technique is economical as well [12, 13]. In recent years, bacteria have been employed to produce a range of nanoparticles, such as copper oxide nanoparticles. Through intracellular or extracellular means, bacteria have been used to manufacture a variety of materials with intriguing forms and nanoscale dimensions. The ability to produce nanoparticles from bacteria is very high. They have quick generation times, are simple to grow, provide great stability, benign experimental settings, extracellular nanoparticle formation, and are easily genetically modified [14]. CuO, an inorganic metal oxide nanoparticle, exhibits notable antibacterial properties and selective toxicity, suggesting possible uses for these materials in medical devices, diagnostics, therapies, and nanomedicine to combat human diseases. With a range of potentially useful physical properties, copper oxide is the most basic copper chemical in the family. Because of its unique properties, such as stability, conductivity, catalytic activity, and antibacterial and anticancer effects, copper oxide (CuO) has attracted more attention than other nanomaterials. In humans, copper is involved in several processes, such as producing neuropeptides, regulating cell signaling pathways, protecting against free radicals, and supporting immune cells [15]. The antimicrobial, antibacterial, antifungal, magnetic phase change, gas sensing, biocidal, superconductive, catalytic, and optical properties of copper oxide, on the other hand, are quite different [16].

II. Material and methods

A.     Selection of bacteria species

The Siddhachalam Laboratory's (CG) collection of E. Coli bacteria were cleared out. Using this specific species of E. Coli was used as a template, and CuO nanoparticle synthesis was done.

B.     Preparation of culture media for bacteria

250 ml - 7 g of high-Nutrient Agar Media, 250 ml of distilled water.

C.     Isolation and observation of bacteria

High Media Nutrient Agar Media [NAM] and Agar were added in distilled water and mixed homogenize it, and then, after autoclaving, and under laminar air flow, the media will be poured and inoculated by bacteria from the inoculating loop in sterile petri plate. Poured   plates were incubated at 37 an incubator for 24 hours, and bacterial species [E. Coli] was observed.

D.     Stock Preparation:

Sterilized stock solution (1000mg/l) of selected metals Copper sulphate, of concentration (100 ppm for bacteria was repaired in distilled water which was further diluted as per the requirement.

E.     Synthesis of CuO nanoparticles

About 100 ml of distilled water was taken in a conical flask and 2.4969 gm of CuSO₄ was added to it. The solution was stirred on the magnetic stirrer for 1 hours and the bacterial suspension and ethylene glycol solution were added drop wise at an interval of 5 minutes. After that, the complete solution was placed in the sonicator for 20 to 25 minutes for complete dissolving. After that, the solution is kept an autoclave for 2 hours (15 psi). The solution was reduced in a hot air oven for 3 hours (150). The solution was washed by water for 4 to 5 times in an interval of 20 minutes and was placed a centrifuge to obtain the precipitate and filtrate. After that, the precipitate was washed with ethanol, transferred to a beaker, and placed a hot plate at 50 for drying. The dried precipitate was placed a hot air oven under 110 for about 5 hours. After that the precipitate was transferred to a Muffle Furnace for 2 hours at 500 and then it was cooled in a desiccator and collected.

UV-Vis spectra analysis

CuO particles were characterized by UV-Vis Spectroscopy 1900i Shimadzu. Before putting the sample in UV-Vis Spectroscopy, the CuO sample is diluted with water or ethylene glycol because CuO Nanoparticles is soluble in ethylene glycol. The sample was directly placed in Spectroscopy, due to which the absorption of CuO nanoparticles was recorded in the transmittance mode in the region of 200-800 by UV-Vis light, and the spectrum of the sample is visible on the screen, which is displayed as a graph.

FT-IR

The chemical composition of the synthesized copper oxide nanoparticles was studied by using a FT-IR spectrometer (Perkin elemer LS-65 Luminescence spectrometer). The solution was dried at 75 and the dried powder were characterized in the range 4000-400 cm^-1 using the KBr pellet method.

X-ray diffraction (XRD) analysis:

A common method for figuring out crystallographic shape and structure is X-ray diffraction. The amount of element causes the intensity to change either way. This method is used to determine whether a particle is metallic, provides information on the unit cell's size, shape, and translational symmetry based on peak positions, and determines the electron density—that is, the location of the atoms within the cell—based on peak intensities [17].

Methylene Blue Degradation Analysis

For MBD analysis, the CuO sample solution with D/W was used because CuO shows solubility with water. Vis spectroscopy was used, and different absorption graphs were obtained in every cycle showing the degradation of Methylene Blue by metal oxide over a time interval.

Antimicrobial Test

The well diffusion method for the antimicrobial testing of CuO NPs. Well diffusion method is widely used for antimicrobial testing of antibiotics and other metal compounds. This method is also used to obtain the minimal inhibitory concentration of a compound. In this method, the microorganisms to be tested are spread and small wells formed through the cork borer and are filled with the solution of metal oxide nanoparticles and left for diffusion of the nanomaterials. The nanoparticles show the effect of inhibiting the cultured organisms.

Antibacterial test

NAM media should be prepared for antibacterial testing of CuO Nanoparticles

Peptone 5.0g, HM Peptone B 1.50g, Yeast Extract / beef Extract 1.50g, Sodium Chloride 5.0g, Agar 15g, D/W 1000ml, and Final Ph 7.4 (± 0.2)

Media is prepared, poured into petri plates and well wear formed, pure culture of a bacterial colony is inoculated by spreading and well formed, Little amount of CuO NPs solution is added to the well and incubated at 37 for overnight and observed.

Antifungal Test

PDA media should be prepared for antifungal testing of CuO NPs Infusion from potatoes 200g, Dextrose (Glucose) 20g , Agar -15g - D/W 1000ml Final pH 7.4(±0.2) Media is prepared, poured into petri plates and well were formed, pure culture of the fungal colony is inoculated by spreading and well formed. A small amount of CuO NPs solution is added to the well, incubated at 28 ℃ for 48 hours, and observed.

III. Result and Discussion

A.     UV -Vis spectra analysis

The UV-Vis spectrum of the generated NPs is measured by the UV-Vis spectrophotometer [18].

Figure 1: UV-Vis spectra of CuO NPs synthesized from (E. Coli) bacteria

Based on the computation of UV-Vis spectra, the band gap of the synthesized CuO NPs was found to be 4.6. At one point, we were able to determine the produced CuO NPs' absorbance. Based on UV absorbance spectra, we inferred that CuO undergoes photocatalysis to break strong covalent bonds when exposed to UV light.

B.     Band gap

We apply the Touc Relation, which is provided by the equation, for band gap;

αhv = A (hv – Eg)n

Where,

α = Absorption Coefficient

hv = Incident Wavelength

Figure 2:  Band gap of CuO NPs synthesized from (E. Coli) bacteria

From the UV-Vis spectrophotometer measures the UV-Vis spectrum of produced NPs [19]. The band gap of the synthesized CuO NPs was computed from UV-Vis spectra, and it was determined to be 4.6.

Band gap calculation from the graph:= 4.6

C.     FT-IR analysis

FT- IR spectrum is used to analyze the functional group of CuO NPs. The absorption spectrum in the IR region was acquired by using FT-IR spectrophotometer. The peak at 3571, and 3484cm^-1in the spectra are due to stretching and bending vibration of the –OH group. A small peak at 1071 cm^-3 shows stretching vibration of CuO [20].

Figure 3: FTIR spectrum of CuO NPs synthesized from (E. Coli) bacteria

D.     X-ray diffraction (XRD) analysis:

Structural and Crystallographic Analyses Fig. 4 shows the XRD profile at 36.55º, 38.76º, 48.78º,53.54º, 58.32º, 61.58º, and 68.1º, corresponding to (002), (111), (-202),(020), (202), (-113), and (220) crystal planes, respectively. The XRD profile revealed that CuO NP has a monoclinic structure, which was affirmed by the JCPDS card no. 01-080-1268. The mean diameter size of the CuO NP was calculated using Debye-Scherrer’s equation. which was around 5±1 nm [21].

Figure 4: X-ray diffraction spectrum of CuO NP

E.     Methylene Blue Degradation Analysis

Methylene blue is a heterocyclic aromatic chemical compound with the molecular formula C₁₆H₁₈C₁N₃S. The extent of degradation of Methylene blue using CuO NPs as catalysts was monitored by UV-Vis spectroscopy [22].  The absorption peaks for Methylene blue dye in water was found to be centered at 664 nm in the visible region.

Figure 5: Showing Methylene blue dye degradation

Photocatalytic activity of synthesized CuO NPs was studied by Methylene Blue Dye degradation with a regular time interval. 10^-1 Methylene blue dye was used for the test, and a small amount of CuO NPs sample solution was added to the MB solution. The change in color of MB from deep to light blue shows the reduction in dye and photo catalytic effect, and the sample data is recorded by a UV-Vis spectrophotometer for about 40 cycles in at regular interval of time and the graph is plotted. The decrease in absorbance at a different wavelength with time, and intervals shows the photo catalytic activity of CuO NPs.

Figure 6: CuO Photo Catalytic Absorbance Graph

F.      Antimicrobial Test

For the antimicrobial test solid agar medium is used, and the well diffusion method is used. In the bacterial culture plate, a clear zone around the well containing CuO NPs was observed, showing the antibacterial properties of synthesized CuO NPs, whereas in the fungal culture plate, a zone or effect is observed, it shows that antifungal activity is observed in synthesized CuO NPs [23].

(A)                                     (B)

Figure 7 (A): CuO Antibacterial Test Staphylobacillus Figure (B): CuO Antifungal Test, Alternaria

CuO NPs have been focused on the loss of membrane integrity caused by oxidation of phospholipids, which led to an increase in membrane fluidity, leakage of cellular content, and eventually cell lysis [24].(Correa et al., 2020) (Dicastillo et al, 2020) 

Table 1: Zone of inhibition for Antimicrobial study

IV. CONCLUSION

At last, we conclude that, the synthesis of NPs was successfully done by using a green synthesis method. The UV–Vis spectroscopy (264nm) data gives confirmation about the absorbance, and the result shows that the NPs were synthesized properly, and the FT-IR data confirms the vibration of CuO NPs. The absorbance is observed at the wavelength of 320 nm, by which we can calculate the band gap of about 4.9 eV which resembles the absorbance of the CuO NPs. The photo catalytic effect is confirmed by the Methylene Blue reduction test and the dye reduction of Methylene blue for about 40 cycles for 3 hours shows by the lightening of the blue color, and the removal of 1% is obtained at about 70%, Antibacterial activity shows the antimicrobial effects of NPs by inhibiting the bacteria Staphylobacillus.  After 24 hours of incubation, it was observed that the clear zones around the well contained. CuO NPs. And we can say that the CuO NPs are easily used as an antimicrobial agent and as an antibacterial and also they as it can also be used as a for prevention method for water pollution.