Synthesis and Characterization of CuO Nanoparticles and CuO/PVA-PVP Nanocomposites: Adsorption Activity for Malachite Green (MG) Dye and Antibacterial Property in Wastewater Treatment

Contaminated waterways, particularly effluent from the dye industry, are one of the major issues today. Among various methods adsorption being the most straight forward, low-cost, effective, and successful to treat such effluents. For this purpose, Copper oxide nanoparticles (CuO) and Copper oxide nanoparticles embedded within Polyvinyl alcohol (PVA) and Polyvinyl pyrrolidone (PVP) mixed polymer (CuO/PVA-PVP) were synthesized using precipitation methods and different analytical techniques such as dynamic light scattering (DLS), powdered X-ray diffraction (XRD), fourier-transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) were used to characterize morphology and size. Synthesized nanomaterials were used for the adsorption of malachite green (MG) dye in aqueous solution by the batch adsorption method. Various experimental conditions e.g., contact time, concentrations, pH, were optimized to obtain best adsorption outcome. Adsorption kinetics and isotherm suggested that it follows Pseudo-second-order-kinetic and Freundlich isotherm. The invitro antibacterial activity was investigated using disk diffusion and minimum inhibitory concentration (MIC) test against Staphylococcus aureus (S.aureus), a gram positive and Escherichia coli (E.coli), a gram negative bacteria, where both the synthesized nanoparticles showed a decent antibacterial activity against both the bacterial strain.


Introduction
Nanotechnology is one of the most important and useful innovations of contemporary science, and has gained an enormous attention during the last three decades because of vast range of applications [1]. Nanotechnology is a branch of study which deals with extremely small particles. In the realm of material science, where tiny size plays a key role in defining fundamental characteristics, amazing evolutions have occurred during recent time. As the subject of study expands, commercial exploration evolves day by day [2]. The nanoparticle having smaller surface area and greater activity in interacting with metallic species outperforms the larger particles in the adsorption phenomenon.
Pollution of potable water resources has become one of the main headaches for the scientific society. Researchers are trying to develop applications to reduce pollution, which is a tragic problem in the recent situation and especially in the case of water. Water pollution has become one of the most severe issues nowadays due to being polluted by various toxic materials, such as agricultural practices, household waste, especially industrial pollution. Among which textile dye is one of the most polluting aspects of the global fashion industry. Though the progress of the textile and dye industries is a profitable venture, the drawback is the creation of dangerous organic wastes, which eventually pollute the water and disturb the ecosystem. Dyes are mainly contained with complex mixture of dye bases, salts, acid, compounds, organ chlorinated, auxiliary chemicals, and heavy metals which are water soluble so it is very difficult to remove them with the conventional methods. Coloring dye is the first contaminant detected in water, and it must be removed from wastewater before it may be discharged into bodies of water. Which can obstruct light penetration, slows photosynthetic activity and biota growth, and has a proclivity for chelating metal ions, resulting in micro-toxicity in fish and other species and reduction in dissolved oxygen level [3][4][5][6][7].Among many harmful dyes, malachite green (MG) is one of them, which is cationic in nature and used as a coloring agent in silk, leather, paper industries also used as a tropical antiprotozoal agent, fungicide, and antiseptic in the aquaculture industry to control disease and fish parasites. However, MG is highly cytotoxic and also can be a liver tumor enhancing agent. The toxicity of MG dye increases with exposure time, temperature, and concentration. There are many conventional procedures to remove dyes from water, such as electro coagulation [8], potentiometric [9], degradation using visible light [8,10], adsorption [11] and bio-gradation [12,13].
Among the different dye removal techniques adsorption process is an extensively used process with various advantages. Because of its great efficiency in removing harmful contaminants, ease of operation and low cost, liquid phase adsorption offers a significant advantage over other approaches [14].Due to small size and high adsorption effectiveness, nanoscale metal oxides possibly provide a more cost-effective water treatment and remediation technique [15]. Many metal oxides (Zn, Sn, Fe, Co, Ni, Cu, etc.) are used to remove toxic dyes from waste water [16,17].
Among them, Copper oxide (CuO) nanoparticles have been discovered to be a good adsorbent for heavy metals and dyes [18][19][20]. CuO nanoparticles also act as a photo catalyst under sunlight irradiation. It has gained a tremendous role in science and technology due to its unique features such as high specific surface area, chemical stability, electrochemical activity, high electron communication features, fungicide, optical switch, metallurgy reagent, gas sensor, pigment, field emission (FE) emitter, magnetic storage media and so on. In addition, also one of the valuable antimicrobial agents due to being cheap and easily mixed with water and polymers [21,22].The goal of this study is to make CuO nanoparticles that are coated with a PVA and PVP blend matrix in a simple and straightforward way. Both amorphous polymers are highly water soluble, synthetic, and biocompatible. When two polymers are mixed their aqueous blending form inter chain hydrogen bonds with in PVA and PVP develop potential applications that are more similar to those of the homopolymer. The stability and performance of nanoparticle in their application field become better with coated with these blend matrices [23][24][25][26]. Our present work is to determine the effect of the morphology of the prepared nanoparticles and compare the MG dye adsorption efficiency as well as antibacterial activity between CuO nanoparticles (NPs) and CuO/PVA-PVP nanocomposites (NCs).

Synthesis of Copper oxide (CuO) nanoparticles
CuO nanoparticles were synthesized by aqueous precipitation method [27]. 200 mL of 7% CH3COOH solution was prepared and 4gm of copper (II) acetate mixed with it, then the mixture was stirred for a few minutes until the solution turned green. A 0.9 (M) sodium hydroxide solution was mixed with it in a dropwise manner, maintaining its pH~12, which is followed by heating at 100°C. The mixture was cooled at room temperature and a black precipitate was obtained and separated by centrifugal force (4000 rpm) and washed several times with distilled water and ethanol. The resulting product was dried at 80°C in a hot air oven for 8 h to obtain the dry powder of CuO nanoparticles.

Synthesis of CuO/PVA-PVP blend nanocomposite
Equal amount (1 gm of each polymer mixed with double distilled water) of PVA and PVP solution was dissolved in 120 ml water under magnetic stirring at 100°C [23]. After the dissolution of polymer composites, CuO nanoparticles (pre prepared in section 2.2) were mixed with the polymer solution and sonicated for 1 h until homogenous solution was obtained. The mixture was stirred for next 1 h, and further ultrasonication was done for complete dispersal of CuO in PVA-PVP solution. It was then spread out on a clean, level glass plate and dried in a hot air oven for 24 hours at 60 ̊ C. The dried films were taken from the glass plate and cut into little pieces for later usage.

Characterizations
The hydrodynamical size distribution of particles were measured by Litesizer 500 (Anton paar, Austria), at a scattering angle of 90° at 28°C. For the optical characteristics of the samples UV/Vis spectroscopy had been applied by using a UV/Vis spectrophotometer (Thermo Scientific For the centrifugation force a centrifuge (Remi R-4C) was used. And the pH of the solutions was adjusted using a pH meter (Fisher Scientific, Accumet)

Adsorption studies
The adsorption experiment of CuO NPs and CuO/PVA-PVP NCs were performed by batch method at room temperature. Known amount of malachite green and nanoparticles were taken in conical flask, and then the mixture was shaken thoroughly for 150 min. The dye absorbances were measured at the maximum absorption wavelength, and the samples were collected at predetermined intervals i.e., 618 nm for MG using a 1 cm path length cuvette in UV-vis spectrophotometer Various parameters like contact time (30-150 min), dye concentration in solution (1.0-5.0 mg/L) and nanomaterial concentration (2-10 mg), pH (2-10) were varied during the experiments, pH was adjusted using dilute NaOH and dilute HCl.
The acquired results were verified using adsorption isotherm models such as Langmuir and Freundlich, as well as pseudo-first-order and pseudo-second-order equations to simulate the kinetics of the experiments.
The amount of adsorption percentage (%) and adsorption capacity was evaluated by two equations (1) and (2), respectively.

………….. (2)
Co and Ce designate the colorant's initial and final concentrations (mg/L), qe the amount of colorant removed by the adsorbents (CuO and CuO/PVA-PVP) at the final phase of the reaction (mg/g), V is the volume (L), and M is the weight of adsorbents (CuO and CuO/PVA-PVP) (gm).

Bacteria Culture Preparation
A gram negative bacteria E.coli (ATCC 25922) and a gram positive bacteria S.aureus (ATCC 29213) were cultured in Luria Broth (LB) for 24h at 37 °C and standardized to 0.5 McFarland standard.

Disk Diffusion Test
The antibacterial effect of CuO NPs and CuO/PVA-PVP NCs were performed against E.coli and S.aureus by disk diffusion method. 100 µL of each bacteria cell suspension (0.5 McFarland Standard) was spread over the LB-Agar plate and allowed to dry for 5 minutes. Then sterile paper disk (5 mm) was placed on the plate and 10 µL of solution (concentration of 1 mg/mL) of respective compounds were poured on each disk. The plates were then sealed with parafilm and incubated at 37°C for 24 hours, after which the diameter of each inhibitory zone was measured.
Water was taken as negative control [28].

Minimum inhibitory concentration (MIC) determination
The minimum inhibitory concentration of the CuO NPs and CuO/PVA-PVP NCs were determined by broth dilution method. 50 µL LB was dispensed in each well of column 1-10.50 µL of then 50 µL serially diluted solution of tested compound was poured in column 1-10, concentration sequence from 4 mg/mL to 0.0078 mg/mL followed by addition of standardized bacteria inoculum (1×10 5 cfu/ml). Column 11 was for positive control (only bacteria) and column 12 contain 100 µL LB (for sterility). And the micro plates were incubated at 37 0 C for 24 h. And the lowest concentration of compound with no visible growth of bacteria was determined as MIC [29,30].

DLS experiment
CuO NPs and CuO/PVA-PVP NCs were characterized by dynamic light scattering (DLS) measurements from which the hydrodynamical size distribution was investigated. Both CuO NPs and CuO/PVA-PVP NCs showed single peak having hydrodynamic diameter of 421 nm and 512 nm and polydispersity index 28.5% and 11.3% respectively. These results reveals that both samples were monodispersed i.e., all particles were in the similar size range (Fig.S1).

UV-Vis analysis
The UV-Vis spectra of both nanoparticles CuO NPs and CuO/PVA-PVP NCs are shown in the Fig. 1 which range was in between 300-400 nm with confirm their good formation and appeared due to surface plasmon resonance(SPR) of metal oxide [31].This UV-Vis characterization mainly observes for the confirmation of nanoparticle by knowing their absorption peak.

Powder XRD analysis
The CuO nanoparticle XRD pattern in Fig.3(a)   and similarly the XRD pattern of CuO/PVA-PVP NCs in Fig.3

TEM and EDS analysis
Transmission electron microscopy (TEM) reveals the size and morphology of CuO NPs and CuO/PVA-PVP NCs shown in Fig.4 with a regular and rod-shaped narrow size distribution  To determine the adsorption isotherm, which basically established the equilibrium relationship of adsorption between dye and nanoparticles, two equilibrium models, the Langmuir and the Freundlich were interpreted and correlation coefficients were calculated to identify the best fit.
Adsorption isotherms represent the adsorbate's interaction with the adsorbent and provide information on the material's adsorption capability. The correlation coefficients (R 2 ) and statistical characteristics were examined using the linear form of Langmuir and Freundlich models.

……… (3)
Where Q e is the amount of adsorbed dye on the adsorbent at equilibrium (mg/g),C e is the equilibrium concentration of dye solution (mg/L),K L is the Langmuir constant (L/mg) and the maximum adsorption capacity is Q m .A slope of 1/Q m is obtained from the plot Another dimensionless constant which called equilibrium parameter is RL to describe the essential characteristics of Langmuir isotherm using equation (4) below:

………. (4)
Where C 0 (mg/g) is the initial adsorbate concentration, K L is the Langmuir constant. The value of RL is classified as 0<RL<1 the adsorption process is satisfactory and for RL≥1 for unsatisfactory.
The Freundlich isotherm describes the heterogeneous surface adsorption. The linear form of this equation is given in equation (5).

Dye adsorption kinetic study for MG dye
One of the key factors in determining adsorption efficiency is kinetic adsorption. The kinetic adsorption experiences were done by taking a fixed amount of CuO NPs and CuO/PVA-PVP NCs (10 mg) in 10 mL MG dye (2 mg/L) solution at room temperature and natural pH for 150 minutes and samples were collected at every 25 minutes interval and the absorbance was measured. The adsorption process, which included mass transfer and chemical reaction, were investigated using kinetics models and kinetic study was investigated using two models: Pseudo-first-order and pseudo-second-order models. The linear form equations of these models are given below: Pseudo-first-order kinetic model:log(Q e − Q t ) = log(Q e ) − K 1 t

….. (7)
Where Q e and Q t are the adsorption capacity of dye at equilibrium time and time t (mg/g).K 1 (1/min) and K 2 (g/mg min) are the equilibrium rate constant of Pseudo-first-order and Pseudo-second-order respectively All kinetic data for both models are represented in table 2.   are listed. The correlation factor (R 2 ) value is used to determine the best fitted kinetic model (Fig.   7). According to Table 2, Pseudo-second-order-kinetic model with higher correlation co-efficient value (R 2 ) is higher than Pseudo-first-order-kinetic model for both nanoparticles, these results suggest that the Pseudo-second-order kinetics will be used in the adsorption of MG dye onto CuO NPs and CuO/PVA-PVP NCs. This shows chemical adsorption and lack of internal diffusion resistance. The adsorbate and adsorbent exchange or share electrons, resulting in valency forces.

Effect of pH
The most important variable impacting dye adsorption onto adsorbent has been discovered as the pH of the solution. The effect of pH on the removal efficiency of the MG dye was investigated by changing the pH from 2 to 10 keeping at room temperature, dye concentration (2 mg/L, 10 mL) and adsorbent amount (10 mg) fixed. Solution pH is one of important parameter during the adsorption process. In Fig. 8 it was found that the nanoparticle removal efficiency increases with increasing the pH value; the highest removal efficiency of the dye was at pH 10 for both nanoparticles. The percentage of removal efficiency is comparatively less in the acidic range of pH [39]. Increasing pH leads to formation of negative charge on the adsorbent surface whereas at low pH the presence of excess H + ions, the surface of these nanoparticles was positively charged.
The negative charge enables high removal efficiency due to the electrostatic attraction between the adsorbent and positive charged adsorbate (MG dye), but other hand at low pH due to the strong repulsive force of the two positive charged dye and adsorbents, the decolorization process decreases [40].

Antibacterial activity of CuO NPs and CuO/PVA-PVP NCs
The antibacterial activity of CuO and CuO/PVA-PVP against both E.coli and S.aureus in terms of ZOI is shown in Fig. S4   The CuO/PVA-PVP NCs showed a slightly better ZOI value than that of CuO NPs against both the bacteria (Fig. S4). Negative control did not show any zone of inhibition. The MIC value against E.coli and S.aureus was found to be 0.5 mg/mL for CuO, whereas CuO/PVA-PVP exhibited a value of 0.25 mg/mL and 0.5 mg/mL (Table 3). By which it is confirmed that both the synthesized nanoparticles has a reasonable and indistinguishable antibacterial property against E.coli and S.aureus.

Conclusions:
Copper oxide nanoparticle (CuO) and copper oxide nanoparticle embedded within PVA and PVP mixed polymer (CuO/PVA-PVP) had been prepared successfully by the chemical precipitation method. These two nanomaterials were examined for their ability to remove malachite green (MG) color. The results indicated that both nanoparticles fit well with the Freundlich isotherm and follow the pseudo-second-order kinetic model. The maximum adsorption capacity of CuO NPs and CuO/PVA-PVP NCs. were found 3.63 and 7.57 mg/g respectively which indicating that CuO/PVA-PVP NCs. had a higher adsorption capacity than CuO NPs. Besides, in case of antibacterial test against E.coli and S.aureus CuO/PVA-PVP NCs. showed a better result than CuO NPs. This research found that CuO/PVA-PVP NCs might be worth more than CuO NPs and it would be an environmentally acceptable and efficient option for treating organic dyes and decent antibacterial agent in the sectors of water environment and resource management.

Declaration of Competence Interest
The authors declare no competing financial interest.