Antibacterial and photo dye degradative ability of copper oxide nanoparticles from Pleurotus cystidiosus

The present study deals with in vitro experiments for to develop synthesized copper oxide nanoparticles using Pleurotus cystidiosus. Mushroom-based nanoparticles, such as copper nanoparticles have become more significant because of potential biocatalytic properties, including antibacterial activity. The blue-to-brown color shift suggests CuONPs, as confirmed by absorbance maxima at 246 nm in ultraviolet spectroscopy. FTIR spectra reveal the existence of carboxyl, alcohol, and aldehyde carbon; the amine group represents the myco-synthesized copper nanoparticle. Scanning electron microscopy (SEM) nanoparticles show spherical to oval nano aggregates of 80–110 nm in size. The EDX spectrum confirms the presence of Cu at 8.00 keV in myco-synthesized copper oxide nanoparticles. The antimicrobial efficacy of produced copper oxide nanoparticles on human pathogens showed a maximum zone of inhibition against coliform bacteria such as Enterococcus faecalis (32 ± 2), Enterobacter aerogenes (30 ± 1), E. coli (28 ± 1) and least zone of inhibition about 25 ± 1 mm recorded in Streptococcus mutans. The photocatalytic activity of Congo red dye, Malachite green dye, and Remazol blue dye by copper oxide nanoparticles from Pleurotus cystidiosus was investigated, and the results showed a maximum degradation efficiency of 99.2% in Congo red within 90 min. However, moderate degradation was noted in malachite green, and Remazol blue dye has a low degradation efficiency. Thus, myco-synthesized copper oxide nanoparticles from Pleurotus cystidiosus have potent antimicrobial and photocatalytic dye degradation efficacy.


Introduction
In recent years, nanotechnology is great innovative research sources, to develop different biomaterials to treat various human diseases in worldwide.Pleurotus cystidiosus, also known as maple oyster mushrooms, is a member of the Pleurotus genus and is edible and nutritious.They have useful properties in areas such as nutrition, biotechnology, medicine, and hygiene [1].Numerous reports have detailed the many useful characteristics of the Pleurotus genus, confirming the appeal of this inexpensive machinery equipment in easing the load of environmental concerns [2,3].Nanotechnology has been widely studied in areas of materials technology research, with nanoparticles (NPs) being minor particles that measures from 1-100 nm in one dimension [4].Myco nanoparticle synthesis has drawn lots of attention recently due to its therapeutic potential, low-cost, sustainable, non-toxic, and eco-friendly nature of the mushrooms.Previously many researchers are reported synthesis of different plants, bacteria, fungi and different metal nanoparticles such as iron, gold, silver, platinum, selenium, iron oxide, manganese, copper, zinc, graphene, etc Additionally, compared to these (silver, gold, copper) nanoparticles have been various biological activity and also very cheaper, easily available and ecofriendly.Copper's high selectivity and inexpensiveness make it a useful material in many fields, including medicine and environmental cleanup [5,6].Unique chemical and physical properties make copper (Cu) nanoparticles preferable and highly toxic to organisms but not animal cells, and this metal killed bacteria [7].
Copper and copper oxide nanoparticles stand out among other nanoparticles because of their remarkable electric, thermal, and catalytic effects and their versatile claims, which include fungicidal, dye degradation, sensor, and nematicidal treatments [8][9][10].Studies show that CuNPs is a highly effective agent with minimal side effects, making it ideal for use in biomedicine [11].Numerous studies show that the antioxidant and antimicrobial nanoparticles made of copper oxide and copper show promising properties during the conflict against bacterial resistance to antibiotics [1,12].Owing to their huge surface area to volume ratio when compared to bulk, Nps (nanoparticles) are now attracting a lot of interest in the detoxification of dangerous dyes, with a particular emphasis on the photocatalytic degradation of dyes utilising metallic nanoparticle [13].Complex dyes, of which heaps are produced and released annually, have a negative impact on aquatic life.Researchers face a difficult task in devising efficient dye degradation methods.When it comes to successfully degrading dyes, however, photo induced degradation using a photocatalyst of semiconductor nature is an economical and environmentally friendly option [10].Malachite green (MG), Remzol Brilliant Blue R (RBBR), and Congo Red (CR) are widely used dyes in manufacturing industries.MG is a cationic tint employed in dyeing cotton, paper, silk and has several side effects, such as mutagenic and teratogenic effects in human [14].RBBR is an anthraquinone dye extensively used in the textile dying industry.It has toxic effects on aquatic life and vegetation and is recalcitrant [15].The textile and paper industries have adopted Congo red, an anionic di azo dye, for dyeing purposes [5].Dye effluents should be reconditioned prior to discharge into the water.The large surface area of nanoparticles has attracted a lot of interest recently as a potential method for degrading toxic dyes [16,17].Recently, there has been a lot of research focused on the photocatalytic degradation of dyes using metallic nanoparticles [10,15,16].The present study investigates, CuONPs biosynthesis by the water extract of Pleurotus cystidiosus and its biological activities such as antimicrobial and photocatalytic dye degradation activity.

Materials and methods
Collection and identification of mushroom samples Pleurotus cystidiosus was collected from the tree trunk of Spathodea campanulata on the premises of Shrimathi Devkunvar Nalal Bhatt Vaishnav College, located at Chromepet, Chennai-600044, Tamil Nadu, India.

Preparation of mushroom extracts
Pleurotus cystidiosus was dried under shade condition for a week.The dried mushrooms were ground into a powder in a blender.10 mg of powdered mushrooms were taken, mixed with 100 ml of deionized water, and kept in a boiling water bath for ten minutes.When cooled to room temperature, the extract was filtered using filter paper (Whatmann No. 1) and refrigerated at 4 C until further investigation.

Synthesis of copper oxide nanoparticles (CuONPs)
The ratio of mushroom extract to 5 mM aqueous CuSO 4 was 1:2 ratio and allowed to stand until further color changes occurred.The reaction blend was heated at 60 °C to accelerate the formation of CuONPs and incubated for 10 to 20 min based on the formation of nanoparticles.The color shift was compared to an indicator for nanoparticle synthesis with a blank (water and 5 mM CuSO 4 ) solution.Visual color changes in the solution were examined and further confirmed using characterization studies.For further studies the nanoparticles was concentrated by centrifugation at 10000Xg and the pellet was dried into powder.

Characterization of myco-synthesized CuONPs
A UV-visible spectrophotometer (GENESYS™ 140/150 Thermo Scientific India) was used for the initial characterization of synthesized CuONPs within the range of 200-800 nm [18].Fourier Transmission Infra-Red Spectroscopy (FTIR) (Thermoscientific Nicolet Summit X, India) was executed to find the underlying functional categories in CuONPs.The FTIR analysis was performed on the samples from 4000 to 500 cm −1 .Field Emission Scanning Electron Microscope (FESEM) with Energy Dispersive x-ray spectroscopy (EDX) attachment (FEI Quanta 200 ESEM) was used to examine CuONPs and determine their surface morphology.For examination, a carbon-coated grid was used to examine a thin film of the sample under the FESEM.Elemental analysis on myco-synthesized CuONPs was performed using Energy dispersive x-ray microanalysis attachment.

Inhibition of bacterial growth
The disc diffusion method was used to study the antibacterial properties of green-synthesised CuONPs [19].LB broth was used to grow bacterial inoculums for 24 h at 28 °C and 200 rpm and re-suspended in LB medium.One hundred microliters of each bacterial suspension were spread on nutrient agar using the spread plate method.A disc containing a colloidal solution of copper oxide nanoparticles was prepared and utilised with a set of concentrations (25, 50 μl).The two distinct concentrations of the CuONPs as well as positive control ampicillin in respective discs were positioned over a bacterially-cultivated lawn.Incubation of the agar plates lasted for 24 h at 37˚C.Plates were examined for Zone of Inhibition (ZOI) and measured in mm.

Photocatalytic activity (degradation of dye)
The phenomenon of photocatalysis of Pleurotus cystidiosus mushroom-synthesised synthesized CuONPs was examined for the photo degradation of the dyes Congo red, malachite green, and Remzol brilliant blue.Under ultraviolet light, photocatalytic reactions took place.Typically, dye stock solution of Congo red, fast green, and brilliant blue were prepared by mixing 10 mg dye with 1L of double-distilled water.About 20 mg of biosynthesized copper oxide nanoparticles were mixed with each dye solution (Congo red, Remzol Brilliant Blue, and Malachite Green) containing 100 ml.In addition, a condition devoid of copper oxide nanoparticles served as a control.The suspension was kept under UV light to initiate the photocatalytic degradation.5 ml of reaction mixture was taken out at regular intervals (0, 30, 60, 75, and 90 min interval), centrifuged, and analyzed using UV-vis spectroscopy.Decreases in the strength of a particular absorption peak of the Congo red dye (483 nm), Malachite green dye (633 nm), and Remazol brilliant blue dye (615 nm) were measured in percent.

Results and discussion
Visual observation of myco-synthesized CuONPs from P. cystidiosus The mycosynthesis of CuONPs was observed in a blue-to-brown colour transition, as shown in figure 1.Initially, the colour was light blue, but that changed into a blue to brown in 10 min at 60 °C (figure 1).Similar change in color was noted in the previous studies [20,21].The mechanism of colour change might be due to the conversion of copper (II) sulphate to CuO nanoparticles with biomolecules and proteins present in the extract [18].The protein content of the aqueous mushroom extract is high, and so are the levels of other amino acids and essential amino acids like tryptophan, glutamic acid, and lysine.Riboflavin and other essential minerals can be found in high concentrations in mushroom extracts [22].Previous studies show the flavoproteins in mushroom concentrates cause the aggregation of copper atoms into nanoparticles [13].

Characterization of myco-synthesized CuONPs using P. cystidiosus UV-vis spectroscopy
To examine the presence of CuONPs in the biosynthesized solution, the absorption spectra were examined using UV-visible spectroscopy.Strong absorbance is seen in the UV-vis spectra between 200 and 300 nm, signifying copper nanoparticle synthesis (figure 2).The resonance of copper oxide nanoparticles in water is due to the surface plasmons synthesized.Pleurotus cystidiosus exhibited a peak at 246 nm, the absorbance max falls under UV range.The outcome is in concordance with the previous studies by Saravanakumar et al where he synthesized mycogenic copper oxide nanoparticles using indigenous fungi and showed an absorbance max at 285-295 nm with average particle size of about 110 nm [23].In another study by Asemani et al the Copper oxide nanoparticles were synthesized from Juglans regia leaf extract, which also showed its surface plasmon resonance at 226 nm with an average particle size of 80 nm [24].In the present study the formation of brown color was in contrast with SPR at 246 nm, this might be due to combination of the color of extract and biosynthesis of the copper oxide nanoparticles.Similar results were noted in previously bio synthesized copper/ copper oxide nanoparticles [20,21,25].These results clearly illustrate the synthesis of CuONPs.

FTIR analysis
FTIR was used to look at the CuONPs from 400 cm −1 to 4000 cm −1 array for the existence of functional molecules in their environs.The FT-IR spectra of myco-synthesized CuONPs were revealed in figure 3 and represent different functional groups that act as nanoparticle reducers and/or capping agents.The peaks at 3680, 3250, 2349 cm −1 indicate OH stretching, NH2 vibrations, and O=C=O vibrations [26] which represents flavonoids and proteins present in the extract responsible for CuONPs synthesis [19].Similarly, bands present at 2038, 1512, 1398, and 1233 cm −1 represent isothiocyanate N, C, S-stretching; polyphenol skeletal; phenol and amide groups present phenolic compounds, alkaloids, and tannins present in the fruiting body of mushrooms [27].Mycogenic nanoparticle synthesis protocols typically call for the presence of these groups as a capping agent [18].There are many biomolecules in mycelial extract, and their activity is what converts metal ions to their reduced state [28].

Field emission scanning electron microscopy
The FESEM picture of the Myco-synthesized CuONPs represented in figure 4(a) shows the development of polydisperse CuONPs with a spherical to elliptical nature and particle size measuring about the average size of 80-110 nm.The results were in agreement with the mycogenic copper oxide nanoparticles of T. asperellum [18,29].EDX analysis of myco-synthesized CuONPs is shown in figure 4(b).The energy band spectrum was performed using EDX in the range between 0 KeV and 9 keV.CuONPs exhibited a peak at1 kev and 8 keV, representing the CuNPs.The composition analysis of the CuONPs represented in table 1 showed the occurrence of C, O, and Cu by 56.92% 37.03%, and 3.02% of weight, respectively.The above results coincide with similar EDX spectral studies carried out by Gaba et al in T. asperellum [18] and by Amirtham et al in Lentinus squarrosulus [13].

Antimicrobial activity of CuONPs
CuONPs' antimicrobial effect is well known, and they are widely used in the pharmaceutical industry for antiinfection applications [30].The antibacterial activity of different concentrations of CuONPs on two grampositive pathogenic bacteria, Streptococcus mutans and Entercoccus faecalis, and two gram-negative organisms, Enterobacter aerogenes and Escherichia coli, respectively.The standard ampicillin drug in two different concentrations was used as a positive control.The zones of inhibition measured (in mm) were shown in figure 5 and table 2. The results show that the antibacterial activity of myco-synthesised CuONPs showed a higher degree of antibacterial activity against coliform bacteria such as Entercoccus faecalis (34 mm) and Enterobacter aerogenes (30 mm).Moderate activity was observed against Escherichia coli (28 mm) and Streptococcus mutans (25 mm).Many researchers also showed CuONPs are good at antibacterial activity against coliform bacteria [31,32] and they have different accepted mechanisms for antibacterial activity [33].CuONPs dramatically enhance the cellular ROS level, followed by combined disruption and penetration [34].Moreover, it also enhances killing by irreparable damage to proteins, RNA, and DNA molecules, followed by mutilation of the cell membrane by lipid peroxidation activities [32].Biologically synthesized nanoparticles shows better antibacterial activity when compared to chemically synthesized nanoparticles [35].This difference in activity might be due to biologically  active component present in the biologically synthesized nanoparticles [36] which may induce oxidative stress in the target cells [37].
Photo catalytic dye degradation using CuONPs from P. cystidiosus Photocatalytic activities of CuONPs have been analysed through a degradation process of Congo red dye, malachite green, and Remazol brilliant blue under UV light irradiation.The Pleurotus cystidiosus mushroom synthesized copper oxide nanoparticles showed a higher photo catalytic degradation efficiency of about 99% in Congo red dye, followed by 71.3% malachite green dye, which has a moderate degradation efficiency, and Remazol blue dye, which has a low degradation efficiency of about 49.2% upon treatment with UV(figure 6).However, the dyes without UV treatment showed a very low percentage of degradation even after 90 min.The highest degradation of 3% was noted in Congo red dye, even after 90 min (figure 6).The research studies by Aroob et al showed CuO nanoparticles are efficient in photocatalytic degradation of textile dyes under UV light [10].Another study showed good derivative potential against various dyes using porus nanosheets, where ultra- Table 2. Antimicrobial effect of CuONPs for disc diffusion method.

Zone of inhibition
Copper oxide nanoparticle (50 μg/disc) Ampicillin (50 μg/disc) Enterobacter aerogenes Entercoccus faecalis 32 ± 2 1 8 ± 1 6 fast photocatalytic activity was achieved to a degradation efficiency of about 96.99%.In this study, CuONPs have a higher degradation potential, similar to the previous literature by Nazim et al and Aruna devi et al [38,39] .In this study, a better degradation rate was observed in malachite green by CuONPs, similar to the study by Abir et al where he showed the monometallic CuONPs photocatalyst showed better photo degradation potential compared to bimetallic Cu-Co Nps [40].Moreover, the lowest photocatalytic degradation rate was recorded in remazol blue due to the complexity of the dye, wavelength of UV light, and pH of the solution [41].

Conclusions
This study effectively described an extraordinary environment-friendly edible mushroom, Pleurotus cystidiosus, which was used in a mycosynthesis technique to create CuONPs.The biologically active components in the fruiting bodies act as stabilizing and reducing cofactor for CuONPs, which have potent antimicrobial and photocatalytic dye degradation properties.These promising results of new materials for costeffective and environmentally friendly photocatalysts for effective removal of harmful dyes from textile effluents.

Table 1 .
Elemental composition of CuONPs determined by EDX.