Green Synthesis of Copper Nanoparticles Using a Bioflocculant from Proteus mirabilis AB 932526.1 for Wastewater Treatment and Antimicrobial Applications

Abstract

Nanotechnology offers effective solutions for removing contaminants and harmful bacteria from polluted water. This study synthesized copper nanoparticles using a carbohydrate-based bioflocculant derived from Proteus mirabilis AB 932526.1. The bioflocculant is a natural polymer that facilitates the aggregation of particles, enhancing the efficiency of the nanoparticle synthesis process. Characterization of the bioflocculant and copper nanoparticles was conducted using Fourier Transform Infrared Spectroscopy, Scanning Electron Microscopy, Energy-Dispersive X-ray Spectroscopy, Ultraviolet-Visible Spectroscopy, X-ray Diffraction, and Transmission Electron Microscopy techniques to assess their properties, flocculation efficiency, and antibacterial characteristics. The optimal flocculation efficiency of 80% was achieved at a copper nanoparticle concentration of 0.4 mg/mL, while a concentration of 1 mg/mL resulted in a lower efficiency of 60%. The effects of biosynthesized copper nanoparticles on human-derived embryonic renal cell cultures were also investigated, demonstrating that they are safe at lower concentrations. The copper nanoparticles effectively removed staining dyes such as safranin (90%), carbol fuchsine (88%), methylene blue (91%), methyl orange (93%), and Congo red (94%), compared to a blank showing only 39% removal. Furthermore, when compared to both chemical flocculants and bioflocculants, the biosynthesized copper nanoparticles exhibited significant nutrient removal efficiencies for nitrogen, sulfur, phosphate, and total nitrates in coal mine and Vulindlela domestic wastewater. Notably, these biosynthesized copper nanoparticles demonstrated exceptional antibacterial activity against both Gram-positive and Gram-negative bacteria.

1. Introduction

Water is a critical resource for all living things, playing important roles in various chemical, biochemical, and biological processes [1]. However, the increasing global population, rising costs, and environmental concerns have led to a significant shortage of clean water, and the World Health Organization estimates that over 1.1 billion people do not have access to appropriate drinking water [2]. Traditional wastewater treatment procedures have been developed, to address this pressing issue; however, such procedures often fall short of successfully eliminating pollutants, resulting in insufficient water quality [3].

The limitations of conventional wastewater purification methods highlight the urgent need for innovative, environmentally acceptable, and cost-effective solutions for organic pollutant removal. In addition to water shortage, the growing problem of microbial resistance to antibiotics and disinfectants poses a substantial threat to public health [4]. As a result, there is increasing emphasis on creating novel antimicrobial agents to combat harmful bacteria and pathogens.

In this regard, bioflocculants have emerged as a promising alternative for wastewater purification [5]. Bioflocculants, which are classified as secondary metabolites, are produced by microbes such as bacteria, algae, and fungi during their development process [6]. These big molecules, such as proteins, carbohydrates, glycoproteins, and nucleic acids, are created by microbial metabolism and cell breakdown [7]. Notably, bioflocculants are environmentally harmless and biodegradable and do not contribute to secondary, contamination, making them an attractive option in wastewater treatment [8]. However, problems such as costly production and less efficiency compared to traditional flocculants impede their economic viability [9].

The advancement of nanoscience and nanotechnology opens up opportunities for studying the bactericidal impact of groundbreaking nanomaterials comprising metals with high bioactivity, such as silver, copper, and zinc, to name a few [10]. Nanotechnology is considered the next industrial transformation due to its status as the preeminent fast-progressing field capable of creating a foundation for the convergence of technology and biology [11]. This technique has been used in a variety of academic fields and organizations, including medicine, physics, chemistry, and biology [12]. In light of their better biological and physicochemical properties as compared to bulk substances, nanoparticles have enormous potential in a diversity of scientific fields. Nanoparticles are compounds that vary in chemical and physical characteristics and range in size from 1 to 100 nm. They demonstrate optical, electrical, and thermal conductivity, as well as catalytic, antioxidant, antibacterial, and anticancer properties [13].

Copper nanoparticles (CuNPs) have received a lot of interest in the treatment of wastewater due to their catalytic, optical, antifungal, and antibacterial capabilities and their high electrical conductivity [14]. Copper (Cu), a minor metal essential for existence, play a role in several functions and has been utilized by people for over ten millennia [15]. Cu has recently gained scientific attention for its antibacterial capabilities and claimed low toxicity in humans [15]. Cu materials exhibit activity toward both positive-staining and negative-staining microbes [16]. Their antibacterial effect varies according to size. To optimize antibacterial effectiveness, CuNPs should be synthesized to a size that enhances their contact with bacterial surfaces, resulting in a more potent antimicrobial action compared to their standard size, thereby targeting a wide range of bacteria, including multidrug-resistant strains [15].

Physical and chemical methods have been reported for nanoparticle synthesis [17]. Even though such approaches create large amounts of nanoparticles, they are not preferred as they create toxic substances, are cost-ineffective, and require more time [18]. Recently, researchers have considered the production of nanoparticles using biological approaches [19]. This method is simple, direct, non-toxic, and user-friendly [20]. It produces nanoparticles using biological organisms, such as bacteria, algae, plants, actinomycetes, and fungi [21]. This approach monitors the optimal size and form of nanoparticles, which might have environmental advantages [22]. However, there is limited information about the nanoparticles generated using biological approaches, and there is even less on synthesis using microbial bioflocculant [23,24,25,26].

In this work, a green method for synthesizing Cu nanoparticles utilizing a bioflocculant derived from Proteus mirabilis AB 932526.1 is described. An evaluation of their antimicrobial efficacy against Gram-positive and Gram-negative bacteria is also presented. Further applications of the biosynthesized Cu nanoparticles in the treatment of wastewater and dye removal are discussed.

2. Materials and Methods

2.1. Source and Production Medium of Bioflocculant

The bioflocculant used in this study was produced from P. mirabilis, isolated from activated sludge wastewater. This strain was identified as P. mirabilis AB 932526.1, using the 16S rRNA technique [23]. To create the bioflocculant production medium, the method outlined by Zheng, Ye [24] was followed. The medium consisted of 1 L of sludge-filtered water, supplemented with 20 g of glucose, 0.2 g of MgSO₄·7H₂O, 0.2 g of (NH₄)₂SO₄, 5 g of K₂HPO₄, 0.5 g of urea, 0.5 g of yeast extract, and 2 g of KH₂PO₄, adjusted to a pH of 6. This mixture was autoclaved at 121 °C for 15 min to ensure sterility. After cooling, it was inoculated with a fresh culture of P. mirabilis AB 932526.1 that had been revived overnight and incubated for 60 h at 35 °C with agitation at 120 rpm.

2.2. Bioflocculant Extraction and Purification

The process for extracting and purifying the bioflocculant from Proteus mirabilis AB 932526.1 as published by Karthiga Devi and Natarajan [25] was used with minor modifications. A fermentation mixture was made by mixing 10 g of sucrose, 0.1 g of (NH4)₂SO₄, 1.0 g of KH₂PO₄, 2.5 g of K₂HPO₄, 0.05 g of NaCl, and 0.1 g of MgSO₄ with 500 mL of filtered sludge water. The medium was adjusted to a pH of 6 before sterilizing in an autoclave at 121 °C for 15 min and inoculating with 3% (v/v) culture broth. The fermentation mixture filled with the culture was stored in an incubator at 35 °C, 120 rpm for 3 days. Once 96 h of fermentation was completed, the microbial flocculant from the cultivation medium was collected by centrifugation at 5000× g for 15 min at 4 °C. One volume of distilled water was added to the supernatant phase to isolate insoluble materials, and we centrifuged for 15 min at 5000× g and 4 °C. The culture supernatant was mixed with two (2) liters of ice-cold ethanol, stirred, and kept at 4 °C for 12 h. The residue was subjected to vacuum drying to produce the raw microbial flocculant. The raw microbial flocculant was redissolved in 100 mL of purified water, and one liter of a chloroform-n-butyl alcohol combination (5:2 v/v) was added. The mixture was incorporated properly and kept at room temperature for 12 h. To produce a pure bioflocculant, the supernatant was centrifuged at 5000× g for 15 min at 4 °C before being vacuum dried. The weight of the dry bioflocculant was given in grams per liter of culture.

2.3. Biosynthesis of Copper Nanoparticles

A procedure utilizing green approaches was followed to fabricate copper nanoparticles from copper sulfate (CuSO₄) as a metal precursor. A 200 mL solution of 3 mM copper sulfate was prepared using distilled water. Thereafter, 0.5 g of pure microbial flocculant was added in a prepared copper sulfate solution, and the mixture was agitated at 200 rpm for 10 min at room temperature. Following that, the combination was placed in the dark at an ambient temperature for 24 h, while the solid residue was gathered through centrifugation at 8000 revolutions per minute for 15 min at 4 °C. Visual inspection of a color shift from colorless to blue indicated CuNP production. Analysis of the newly synthesized substance employing multiple analytical methods confirmed the production of CuNPs. A copper sulfate solution without bioflocculant was used as a control. The vacuum-dried nanoparticles were stored safely for subsequent study [26].

2.4. Characterization of the Bioflocculant–Copper Nanoparticles

The characterization of the bioflocculant and biosynthesized copper nanoparticles was conducted using several advanced techniques. Scanning Electron Microscopy (SEM) micrographs were obtained to analyze the morphology of both samples, with specific regions further characterized using Energy-Dispersive X-ray Spectroscopy (EDX) to determine the elemental composition. The structural analysis was complemented by Fourier Transform Infrared Spectroscopy (FT-IR) using a Bruker Tensor 27 spectrometer (Bruker, Gauteng, South Africa), which recorded spectra of desiccated and finely powdered solid substances in the range of 4000 to 400 cm⁻¹ at a resolution of 4 cm⁻¹. X-ray Diffraction (XRD) analysis was performed with a Bruker AXS D8 Advance diffractometer (Johannesburg, South Africa) to assess the crystalline nature of the synthesized materials. Additionally, absorption measurements were taken using a UV-Vis spectrophotometer (Orion AquaMate AQ8100, Gauteng, South Africa), where samples were diluted and analyzed over a wavelength range of 200 to 700 nm at room temperature. Further examination of the biosynthesized CuNPs was conducted using Transmission Electron Microscopy (TEM) with a JEOL 1010 microscope (JEOL USA, Inc., Peabody, MA, USA), providing high-resolution images. Additional SEM analysis was performed with a Sipma VP-03-67 microscope (Johannesburg, South Africa) at a resolution of 1000 keV/wavelength for enhanced imaging of both the bioflocculant and CuNPs [27].

2.5. Determination of the Concentration of the Biosynthesized Copper Nanoparticles

The formed Cu powder was mixed with purified water to form preparations containing 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL [28]. Every mixture was produced independently by thinning the CuNPs to the appropriate levels. Kaolin powder was added, which acts as a test substance. A solution was formed by mixing one volume of purified water and 4 g of kaolin powder. In a 250 mL conical flask, 100 mL of the kaolin powder was combined with 3 mL of 1% (w/v) CaCl₂ and 2 mL of Cu nanoparticles. Following intense stirring for a minute, the solution was moved to a graduated cylinder (100 mL) and set aside uninterrupted for 5 min at 25 °C, for sedimentation. The same process was followed for the control, except instead of 2 mL of Cu nanoparticles, 2 mL of deionized water was utilized. To test flocculation performance, a pipette was used to collect the translucent top layer of the supernatant and deposit it in a cuvette. The aggregation speed of CuNPs and the control sample was assessed by analyzing the light absorbance (OD550 nm) of the solution utilizing a UV-light spectrophotometer (PerkinElmer, Gauteng, South Africa), which was placed 1 cm below the fluid surface. The spectrophotometer readings served as the basis for the measurements [29]. The flocculation activity (FA) was calculated using the following equation:

Flocculating Activity (%) = [(C − D)/C] × 100

(1)

where C represents the optical density (OD) at 550 nm of the untreated control, and D represents the OD of the solution treated with Cu nanoparticles.

2.6. Evaluation of Antibacterial Performance Test of the Bioflocculant–Copper Nanoparticles

Revival of Microbial Strains

Dissimilar Gram-positive and Gram-negative microbes were initially revived by introducing them into sterile growth media and cultured at 37 °C for 18–24 h. We added 1 mL of Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia, and Klebsiella pneumoniae, respectively, to separate test tubes containing 9 mL of sterile nutrient. The cultures were then placed on Mueller–Hinton agar and incubated overnight at 37 °C. Every test tube’s absorbance was measured at 600 nm utilizing an ultraviolet-visible spectrophotometer. The turbidity was adjusted to a range of 0.1–0.5, which meets McFarland’s acknowledged standard. This was carried out following Akapo [30] procedure.

2.7. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

The biosynthesized CuNPs were tested for antibacterial activity using 96-well plates, following Eloff’s 1998 approach. Firstly, the nutritional broth was prepared by weighing 23 g in 1 volume of purified water, and the prepared medium was autoclaved at 121 °C for 15 min. Ciprofloxacin (40 μL) served as a positive control. A 96-well plate was used to generate a set of dilution factors. We poured 50 μL of sterilized nutritional broth, 50 µL of the isolated bacteria, and 50 μL of the biosynthesized CuNPs into each well in descending order onto a microwell plate. Purified water served as a −ve control, and ciprofloxacin was adopted as a +ve control. The plates were grown at 37 °C overnight, and then 40 μL of 0.2 mg/mL of p-iodonitrotetrazolium (INT) was employed as an indicator in each well for the examination of color changes in the wells. The plates were examined for the minimum bactericidal concentration. Each strain culture was streaked onto Mueller–Hinton nutrient agar with a culture-filled loop. Thereafter, they were placed at 37 °C for 12 h. The MBC was defined as the smallest quantity of biosynthesized CuNPs required to eliminate the test microbes [30].

2.8. Evaluation of the Biosafety of the Bioflocculant–Copper Nanoparticles

The following method was adapted from a published protocol to assess the substances in the particles (bioflocculant and CuNPs) harmful for the developing kidney of a human (HEK 293) cell line [31]. A 96-well microplate was prepared with cell suspensions at a concentration of 1 × 10⁵ cells/mL. Cells were treated with biosynthesized Cu nanoparticles at varied doses (25–100 µg/µL) utilizing a tenfold dilution series technique. After a 48 h incubation phase, biosynthesized CuNPs were supplied in a medium containing 1% fetal bovine serum (FBS), and the plates were incubated for an additional 48 h. Following this, 15 µL of MTT solution (5 mg/mL) in phosphate-buffered saline (PBS) was added to each well and incubated for 4 h at 37 °C. The MTT medium and the formed formazan crystals were extracted from the wells and dissolved in 100 mL of dimethyl sulfoxide. Optical densities were measured at a wavelength using a microplate reader, and the percentage of cell inhibition was determined based on the given equation:

% Cell viability (%) = (Ao − A)/Ao × 100

(2)

where Ao and A indicate the optical density (OD) values of untreated and treated samples at 540 nm. The inhibitory concentration of 50% (IC50) was determined using linear regression analysis with GraphPad Prism (V6.1).

2.9. Application of Bioflocculant–Copper Nanoparticles

Removal of Pollutants from Wastewater

The removal of contaminants from wastewater using bioflocculant and biosynthesized Cu nanoparticles utilizing bioflocculant P. mirabilis AB 932526.1 required fresh samples to be collected from two locations, namely, the Vulindlela domestic sewage water in the KwaDlangezwa area of KwaZulu-Natal, RSA, and Tendele coal mine in the KwaSomkhele district of Mtubatuba, KwaZulu-Natal. The pH was adjusted using 1 N NaOH and 1 N HCl to meet the pH requirements for each sample (pure bioflocculant and as-synthesized Cu nanoparticles). Parameters including biological oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (N), sulfate (SO₄²⁻), phosphate (PO₄³⁻), and nitrate (NO³⁻) were evaluated using test kits, according to the manufacturers’ specifications, before and after treatment [32]. Briefly, in 250 mL conical flasks, 100 mL of wastewater adjusted to pH 6, along with 3 mL of a 1% (w/v) metal ion solution, and 2 mL of the produced sample solution, were added. Conventional flocculants were used as standards. The mixtures in separate flasks were stirred at 200 rpm for 3 min, followed by a reduction in speed to 45 rpm for 5 min at room temperature. Subsequently, the solution in the flasks was poured into graduated 100 mL cylinders and left for 5 min at room temperature to allow flocculation. The topmost solution was collected and used to assess COD, BOD, N, SO₄²⁻, PO₄³⁻, and NO³⁻ concentrations, as well as their flocculating activity. Spectrophotometric measurements were taken at 680 nm with a spectroquant (Pharo 300, Merck KGAa, Darmstadt, Germany) and test kits [32]. The subsequent equation was employed to determine the elimination efficiencies (EEs):

EE (%) = [Pb − Pa]/Pb × 100

(3)

where Pb and Pa represent the starting and final levels prior to and post-treatment with flocculating agents and produced CuNPs, correspondingly.

2.10. Assessment of Flocculants in Color Elimination from Different-Colored Solutions

The color elimination effectiveness of produced NPs was assessed in comparison with a blank (control) using the technique detailed by Shende and MITRA [33]. The study utilized several color solutions (4 g/L), including Congo red, methylene blue, safranin, carbol fuchsin, and methyl orange. To eliminate the color from these solutions, 100 mL of each color solution was combined with 3 mL of 1% (w/v) metal ions preferred by each produced Cu nanoparticle and 2 mL of an effective Cu nanoparticle dosage concentration. The blank (control) was used for comparison. The solution was vigorously mixed for 60 s at a speed of 200 rpm. After shaking, the mixture was poured into a 100 mL graduated measuring cylinder and allowed to settle at ambient temperature for 10 min for sedimentation. Following sedimentation, the upper clear solution was collected and subjected to examination at different wavelengths (nm) using a UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) for both untreated and treatment solutions. The decolorization removal efficiency was estimated using the original dye concentration and the final dye concentration following treatment, as well as the residual dye concentration in the samples. The estimated dye elimination capability of the samples was determined using the equation below:

Removal efficiency (%) = D_i − D_f/D_i × 100

(4)

where D_i represents the first value prior to treatment, and D_f the last value after being treated with samples.

2.11. Statistical Analyses

The studies were conducted in triplicate, and the qualitative results were provided as mean values with standard deviation for errors. To distinguish between statistical groups, one-way analysis of variance (ANOVA) was utilized. Statistical significance was considered present, utilizing GraphPad Prism Version 6, with a confidence level of 0.05 (p < 0.05). Percentage flocculating activities marked with different letters in the figures are significantly different from each other, while values sharing the same letter are not significantly different from each other. Error bars indicate standard deviation.

3. Results and Discussion

3.1. Biosynthesis of Copper Nanoparticles (CuNPs) Through Visual Means

The biosynthesis of CuNPs was initially identified through a distinct color change in the reaction mixture, where the pale blue copper sulfate solution transitioned to a deeper blue upon the addition of a bioflocculant produced by P. mirabilis AB932526.1. This color variation is attributed to surface plasmon resonance, characteristic of metallic nanoparticles [34]. Importantly, the bioflocculant plays a crucial dual role in this process as it acts as both a reducing agent and a capping agent [35]. The functional groups present in the bioflocculant, such as hydroxyl (-OH) and carboxyl (-COOH), facilitate the reduction of Cu²⁺ ions to elemental copper (Cu⁰) by donating electrons [36]. Simultaneously, the bioflocculant stabilizes the formed CuNPs by adsorbing onto their surfaces, preventing agglomeration and ensuring a uniform size distribution. This stabilization is vital for maintaining the nanoparticles’ properties and potential applications. The evidence from studies by Dlamini, Basso [37] and Tsilo, Basson [38] further supports our findings, demonstrating that microbial bioflocculants effectively reduce metal ions while stabilizing the resulting nanoparticles. Thus, understanding the multifaceted role of bioflocculants enhances our insights into environmentally friendly methods for nanoparticle production.

3.2. Characterization of Bioflocculant–Copper Nanoparticles

3.2.1. Fourier Transform Infrared Spectroscopy (FT-IR) Examination of the Bioflocculant and the Biosynthesized Copper Nanoparticles

The FT-IR analysis conducted in this study offers valuable insights into the chemical composition and functional groups present in both the biosynthesized copper nanoparticles and the bioflocculant. Microorganisms produce bioflocculants with a diverse chemical composition, which directly influences their flocculating action [39]. The functional groups within this produced bioflocculant act as binding sites for various colloids in solution, playing a crucial role in the flocculation process [40]. This analysis aims not only to elucidate these functional groups but also to explore their roles in stabilizing and capping the Cu nanoparticles [41].

The FT-IR spectra illustrating the functional groups present in both the bioflocculant and the biosynthesized copper nanoparticles are presented in Figure 1. The bioflocculant exhibits prominent peaks at 1706 cm⁻¹, 1241 cm⁻¹, 1067 cm⁻¹, and 925 cm⁻¹. The peak at 1706 cm⁻¹ corresponds to hydroxyl (-OH) stretching vibrations, which play a vital role in binding metal ions, thereby enhancing the bioflocculant’s capacity to aggregate colloidal particles in solution [42]. This interaction is crucial for effective flocculation, as it facilitates the formation of larger aggregates that can be easily removed from wastewater.

The peak at 1241 cm⁻¹ suggests the presence of carbonyl (C=O) groups, contributing to the chemical diversity of the bioflocculant. These groups can participate in various interactions with metal ions, further enhancing flocculation efficiency [43]. Additionally, the bands at 1067 cm⁻¹ and 925 cm⁻¹ are likely to correspond to bending vibrations associated with aromatic compounds and other functional groups, emphasizing the complex nature of the bioflocculant [44].

The FT-IR spectrum of the biosynthesized CuNPs reveals peaks at 3364 cm⁻¹, 2223 cm⁻¹, 1709 cm⁻¹, 1311 cm⁻¹, 1067 cm⁻¹, 869 cm⁻¹, and 566 cm⁻¹. The peak at 3364 cm⁻¹ indicates the presence of hydroxyl (-OH) groups, which can enhance the stability of the nanoparticles through hydrogen bonding. The peak at 2223 cm⁻¹ may be related to alkyne or nitrile functional groups, suggesting possible stabilization mechanisms.

The observed disappearance of bands at 1706 cm⁻¹ and 1064 cm⁻¹ from the bioflocculant spectrum can be attributed to chemical interactions during nanoparticle formation. Specifically, as copper ions are reduced to form nanoparticles, functional groups from the bioflocculant may bind to the metal surface or undergo structural changes that diminish their vibrational intensity [45]. This transformation is evidenced by new peaks at 1709 cm⁻¹ and 1067 cm⁻¹ in the biosynthesized CuNP spectrum, indicating that these bands may represent newly formed interactions between copper and functional groups from the bioflocculant.

The curve at 1709 cm⁻¹ might be due to carbonyl (C=O) stretching, signifying the presence of carbonyl functionalities, such as ketones or aldehydes, which may facilitate complexation with metal ions. Additionally, peaks at 1311 cm⁻¹ and 1067 cm⁻¹ suggest the occurrence of C-O stretching vibrations, likely from ethers or alcohols, which may help stabilize the Cu nanoparticles and prevent agglomeration. The curve at 869 cm⁻¹ could be indicative of C-H bending vibrations; these are possibly related to aromatic compounds, which contribute to the structural integrity of the biosynthesized CuNPs. The band at 566 cm⁻¹ is often attributed to Cu–O vibrations, indicating the formation of copper oxide phases within the nanoparticles. This formation can enhance their catalytic and antimicrobial properties [46].

Nasrollahzadeh, Momeni [47] examined FT-IR spectra of CuNPs synthesized using Plantago asiatical leaf extract and revealed strong absorption bands at 3480, 1725, 1590, and 1300 cm⁻¹, which might be attributed to the binding of C=O, OH, C-O, and C=C vibration, respectively.

Nzilu, Madivoli [48] utilized an aqueous extract of Parthenium hysterophorus to synthesize copper oxide nanoparticles, serving as a reducing, stabilizing, and capping agents in the process. The Fourier Transform Infrared Spectroscopy analysis indicated the presence of secondary metabolites on the surface of the CuO NPs, with a notable Cu–O stretching band observed at 522 cm⁻¹.

A study focused on the green synthesis of CuNPs using Piper retrofractum extracts highlighted the role of FT-IR in confirming the presence of metal–oxygen (Cu–O) bonds within the range of 550–570 cm⁻¹. Additionally, bending absorption related to Cu–O–H bonds was observed around 870–880 cm⁻¹, indicating the successful formation of CuNPs and their stabilization through bioactive compounds present in the extract [49].

These findings emphasize the role of FT-IR spectroscopy in elucidating the chemical environment and interactions that contribute to the properties and effectiveness of the biosynthesized copper nanoparticles in various applications.

3.2.2. Scanning Electron Microscopy with Energy Dispersive X-Ray (SEM-EDX) Analysis of the Bioflocculant and Biosynthesized Copper Nanoparticles

SEM-EDX is crucial for revealing the elemental composition of the produced materials [50]. Figure 2 shows the SEM-EDX of the bioflocculant and the biosynthesized CuNPs. The bioflocculant and biosynthesized CuNPs contain several elements, as the SEM-EDX data demonstrated. SEM-EDX analysis of the bioflocculant revealed the following elemental composition: carbon (5.90 wt%), oxygen (25.00 wt%), magnesium (2.47 wt%), phosphorus (43.35 wt%), calcium (14.35 wt%), sodium (3.18 wt%), aluminum (2.18 wt%), sulfur (1.00 wt%), chloride (2.00 wt%), and potassium (0.86 wt%). These elements play a crucial role in the bioflocculant’s effectiveness in wastewater treatment, making it an effective agent for removing suspended particles and improving water quality. The presence of elements such as oxygen and carbon suggest that the bioflocculant is predominately carbohydrate in nature, and the absence of nitrogen confirms that it cannot be a glycoprotein. Similar results were obtained by Okaiyeto, Nwodo [51] in a bioflocculant MBF-UFH, where elements such as C, O, Na, Mg, P, S, Cl, K, and Ca were found.

The SEM-EDX spectrum of the produced CuNPs confirms the Cu at distinct peaks at 8 keV and 9 keV, indicating that the synthesis of copper nanoparticles was successful. The presence of copper in the biosynthesized CuNP samples results from copper binding onto the surface of the bioflocculant, as evidenced by the SEM-EDX results, while the bioflocculant shows no copper in the sample. However, the quantitative analysis revealed that the biosynthesized CuNPs contained only 3.44 wt% copper, alongside significant amounts of oxygen (54.05 wt%), phosphorus (18.90 wt%), magnesium (14.11 wt%), and carbon (8.04 wt%). The presence of these elements is crucial, as they not only contribute to the structural integrity of the Cu nanoparticles but also influence their reactivity and stability, which are essential for various applications, including environmental remediation and medical treatments [52]. While the study achieved a successful synthesis of CuNPs, it produced a notably lower copper content compared to previous research. For instance, Dlamini, Basso [37] reported 35.8 wt% of Cu in CuNPs synthesized from a bioflocculant derived from Alcaligenis faecalis, while Tsilo, Basson [38] found 6.38 wt% of Cu in the biosynthesized CuNPs produced using a bioflocculant from Pichia kudriavzevii. Oli, Sharma [53] reported a significantly higher copper content of 67.8 wt% in the biosynthesized Cu nanoparticles from Zingiber officinate extract. Gondwal and Joshi Nee Pant [54] SEM-EDX analysis of biosynthesized copper nanoparticles using Cassia occidentalis revealed a significant presence of metallic copper, at 19.09 wt%, alongside a high oxygen content of 74.32 wt%.

The lower copper percentage observed in this study may be attributed to the lack of effective copper-binding proteins or mechanisms necessary for accumulating copper ions in the bioflocculant P. mirabilis AB932526.1, which was used for the synthesis of CuNPs. Other factors, such as exposure to ambient conditions during synthesis or storage, could lead to oxidation, forming compounds that do not contribute to elemental copper detection. Environmental factors like pH, temperature, and other stabilizing agents may also influence the yield and composition of Cu. The minor peaks for calcium (1.040 wt%) and potassium (0.06 wt%) suggest trace elements that may originate from either the bioflocculant or the synthesis process itself.

Furthermore, the high level of oxygen detected in the SEM-EDX analysis of the biosynthesized CuNPs could result from the bioflocculant used, which facilitates the development of functional groups on the surface of the CuNPs, enhancing their interaction with contaminants, and makes them more effective in wastewater treatment applications. These comparative results highlight the unique elemental profile of CuNPs synthesized using P. mirabilis AB932526.1, demonstrating its effectiveness as both a capping and reducing agent in the synthesis process.

3.2.3. Ultraviolet (UV) Spectra of the (a) Bioflocculant and (b) Biosynthesized Copper Nanoparticles

UV-Vis spectra of a bioflocculant and produced copper nanoparticles are shown in Figure 3. The UV-visible absorbance peak is caused by the collective oscillation of the surface electrons [55]. The bioflocculant exhibited a prominent absorption peak at 300 nm (Figure 3a), indicating its composition of proteins, polysaccharides, and various functional groups that are crucial for flocculation processes. This is consistent with previous reports, such as that by Lu, Zhang [56], which observed an absorption peak at 280 nm for a bioflocculant produced by Enterobacter aerogenes. When the bioflocculant reacted with copper ions, a noticeable color change occurred, transitioning from pale blue to deep blue, signifying the successful reduction of Cu²⁺ ions to Cu⁰ nanoparticles. This color change is indicative of the formation of CuNPs and is attributed to the bioactive compounds present in the bioflocculant facilitating this reduction process [57]. The surface plasmon resonance (SPR) peak appeared at 583 nm (Figure 3b), confirming the formation of CuNPs [58]. The absence of an absorption band at 280 nm in the CuNP spectrum suggests a lack of aromatic amino acids or organic compounds typically associated with proteins, further distinguishing these nanoparticles from the bioflocculant [59].

This finding aligns with previous studies that report SPR for CuNPs generally falling within the range of 500 to 600 nm, indicating effective synthesis [60,61,62]. For instance, Khani, Roostaei [63] reported the absorption peak at 586 nm in the biosynthesized CuNPs utilizing Ziziphus spina-christi (L.). Tiwari, Jain [64] synthesized CuNPs using Bacillus cereus and observed a similar peak value at 570–620 nm. Another study by Karimi and Mohsenzadeh [65] stated that Aloe vera-mediated CuNPs were characterized by an intense absorbance peak at about 578 nm. However, a study by Noman, Ahmed [66] showed that CuNPs synthesized using a native Escherichia sp. exhibited an absorption peak at 325.89 nm, which differs from the results of this study. Earlier research has shown that variations in particle size can influence spectral characteristics; specifically, larger nanoparticles exhibit broader peaks that shift to longer wavelengths [67]. These findings suggest that the bioflocculant plays a significant role in both reducing copper ions and stabilizing the resulting nanoparticles during synthesis.

3.2.4. X-Ray Diffraction (XRD) of the Bioflocculant and Biosynthesized Copper Nanoparticles

XRD was used to assess the crystallinity and structural properties of the produced CuNPs and bioflocculant. Figure 4 shows various diffraction patterns of the produced materials. The XRD patterns for both samples indicate that the samples have a crystalline nature with a characteristic structure. In the main diffraction peaks of CuNPs, the peaks at 2ɵ values of 30°, 35^°, 42.4°, 51°, and 78° correspond to 110, 111, 200, 220, and 311 planes, respectively. For the bioflocculant, we found peak values at 2ɵ of 11°, 14.8°, 15.6°, 22°, 25°, and 30°, corresponding to 001, 111, 112, 200, 211, and 212, respectively. These peaks confirm the face-centred cubic (fcc) structure of CuNPs and bioflocculant with the standard JCPDS (No. 040836) data [68]. The 35° 2ɵ (degree) peak indicates the formation of CuO (111). There were no impurity peaks in either sample. The appearance of the sharp peaks indicates a highly crystalline material, whereas the widening of peaks indicates nanocrystalline particles. The peak locations are consistent with metallic copper values reported in the literature [69]. The widening of the peaks suggests that the average crystallite size was determined to be around 20 nm, as predicted by peak (111) (Figure 4) using the Scherrer equation, D = Kλ/βcosɵ. Here, K = 0.94, D = average crystallite size, β = full width at half maximum, λ = wavelength of Cu Kα radiation (λ = 0.1546 nm), and ɵ = half diffraction angle [70]. The XRD patterns reported by Suárez-Cerda, Espinoza-Gómez [71] in the synthesis of Cu nanoparticles using native cyclodextrins as stabilizing agents were reported to be 43.6, 50.8, and 74.4, corresponding to 111, 200, and 220 lattice planes of Cu.

3.2.5. Scanning Electron Microscopy (SEM) Characterization of the Bioflocculant and Biosynthesized Copper Nanoparticles

The surface morphology of a bioflocculant and biosynthesized CuNPs was assessed using SEM (Figure 5). The SEM image of the bioflocculant, which facilitated the synthesis of CuNPs, revealed a cumulus-like morphological structure (Figure 5a). Chen, Zhao [72] reported a porous structure in bioflocculants produced by Pseudomonas sp. XD-3, suggesting that such structures may enhance the interaction between the bioflocculant and Cu ions during nanoparticle synthesis. The SEM image of the biosynthesized CuNPs indicates that the particles are not only small but also exhibit a uniform shape (Figure 5b). The uniformity in size and shape is important, as it directly influences the nanoparticles’ properties, such as their surface area and reactivity, and plays a crucial role in pollutant removal during wastewater treatment [73]. Moreover, while SEM analysis shows a tendency for agglomeration among CuNPs, likely due to electrostatic attractions, it is important to note that this agglomeration can impact their functional properties [74].

For instance, Al-Khafaji, Al-Refai’a [75] reported spherical CuNPs with an average size of 38.5 nm, which exhibited effective pollutant removal capabilities. In contrast, Parvathalu, Rajitha [76] observed irregularly shaped copper nanoparticles with dimensions ranging from 0.5 to 1.5 µm when synthesized using Tinospora cordifolia extract; these larger and less uniform particles may demonstrate different reactivity profiles. Agglomeration with different sizes and shapes in the biosynthesized CuNPs using a P. granatum seed extract as a reducing and capping agent was reported by Nazar, Bibi [77]. Therefore, the presence of these morphologies serves as a vital tool for ensuring the effectiveness of bioflocculants and produced CuNPs in environmental applications.

3.2.6. Transmission Electron Microscopy (TEM) Analysis of the Bioflocculant and Biosynthesized Copper Nanoparticles

TEM has proven to be an invaluable tool for characterizing biosynthesized CuNPs, revealing important details about their size, shape, and agglomeration behavior [78]. Figure 6 shows the TEM images of the bioflocculant and biosynthesized CuNPs at a 100 nm scale. The analysis reveals that the CuNPs are predominantly spherical and exhibit noticeable agglomeration. This spherical morphology is particularly advantageous for various applications, including catalysis and drug delivery, as it enhances the surface area and reactivity [79]. The average particle size of the synthesized CuNPs was reported to be around 20 nm, consistent with findings from previous studies that noted sizes ranging from 20 to 30 nm [80,81], indicating that the biological synthesis method is reliable and effective. However, the agglomeration observed in the TEM images suggests that while biosynthesis can efficiently produce nanoparticles, it may also lead to clustering, which can significantly impact their physical properties [82]. This agglomeration could affect the solubility and reactivity of CuNPs in biological systems or industrial processes, making it essential to understand these interactions in order to optimize their practical applications [83]. The study by Shantkriti and Rani [84] explored the biological synthesis of copper nanoparticles (CuNPs) using Pseudomonas fluorescens. They found that the average particle size was 49 nm, with the nanoparticles exhibiting both spherical and hexagonal shapes.

3.3. Effect of Dosage Concentration on the Flocculating Activity of the Biosynthesized Copper Nanoparticles

The study by Okaiyeto, Nwodo [51] emphasizes the importance of appropriate dosage sizes in reducing costs and enhancing flocculating efficiencies in industrial processes. Dosage concentrations ranging from 0.2 to 1.0 mg/mL were investigated on the produced copper nanoparticles, and the results are shown in Figure 7. A comparison between the efficiencies of the formed copper nanoparticles and bioflocculant was also presented.

The biosynthesized copper nanoparticle solution at 0.2 mg/mL had a flocculating activity of 78%, 0.4 mg/mL had 80%, 0.6 mg/mL had 93%, 0.8 mg/mL had 68%, and 1.0 mg/mL had 60%, respectively. There was no significant difference in flocculating activity between 0.4 mg/mL and 0.6 mg/mL; thus, additional experimentations were conducted employing 0.4 mg/mL as the preferred dose size, because a minimal dose is preferred, given that it is advantageous in large-scale uses for lowering costs. The microbial bioflocculant revealed the greatest flocculating performance of 82% at 0.4 dosages in this study (published) [85], indicating that this will be effective for copper NPs synthesized using bioflocculant derived from Alcalegenis faecalis. Sadia, Cherutoi [58] reported a concentration of 0.0125 mg/mL for the synthesized copper nanoparticles using Senna didymobotrya extract.

3.4. List of Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) Values Obtained from Tests of the Bioflocculant–Copper Nanoparticles

To effectively combat the spread of waterborne diseases, it is crucial to eliminate microorganisms during water treatment and purification processes [86]. While flocculation can remove some microorganisms, it is not universally effective. Therefore, the combined use of flocculants and disinfectants is common in water treatment to ensure comprehensive purification. However, these agents can sometimes interact negatively, potentially diminishing the overall effectiveness of the purification process. To streamline water treatment and reduce costs, utilizing a flocculant that possesses both flocculation and inhibitory properties is recommended.

Recent studies have highlighted the antimicrobial potential of metallic nanoparticles, including copper, gold, silver, zinc, and titanium. Notably, copper nanoparticles have demonstrated antibacterial, antifungal, anti-parasitic, and even anticancer properties [87]. Their applications extend to food packaging and wound dressings. In this study, we assessed the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of biosynthesized CuNPs against various bacterial strains such as Gram-positive (Streptococcus pneumoniae and Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria. The results are summarized in

Table 1. List of minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for the produced copper nanoparticles.

Bacterial Strain	Cu Nanoparticles		Ciprofloxacin		Bioflocculant
	MIC (mg/mL)	MBC (mg/mL)	MIC (mg/mL)	MBC (mg/mL)	MIC (mg/mL)	MBC (mg/mL)
S. pneumonia	25.0	-	6.25	12.5	-	-
S. aureus	3.125	6.25	1.56	3.125	-	-
K. pneumonia	12.5	25.0	3.125	6.25	-	-
P. aeruginosa	6.25	12.5	6.25	6.25	-	-

.

In the findings, the biosynthesized CuNPs exhibited significant antimicrobial activity against both types of bacteria. The lowest MIC and MBC values were recorded for S. aureus at 3.125 mg/mL and 6.25 mg/mL, respectively. When compared to the commercial antibiotic ciprofloxacin used as a control drug, the Cu nanoparticles showed comparable effectiveness against all tested organisms. Ciprofloxacin was administered at a concentration of 20 µL and successfully inhibited all bacterial strains tested. While CuNPs demonstrated significant antimicrobial properties, the bioflocculant used for their synthesis did not exhibit any antimicrobial effects on the tested organisms. This lack of activity may be attributed to the bioflocculant’s different mode of action, primarily flocculation or coagulation, which was not evaluated in terms of its antimicrobial potential [88]. Lower MIC and MBC values signify that smaller amounts of an antimicrobial agent are required to inhibit microbial growth, suggesting enhanced efficacy [32]. The implications of this study suggest that the synthesized Cu nanoparticles could serve as effective antibacterial agents across various sectors, particularly in wastewater treatment and food production. These findings align with previous research by Ebrahimi, Shiravand [89], which reported potent antimicrobial effects of CuNPs synthesized from Capparis spinosa fruit extract against both Gram-negative and Gram-positive bacteria. Sadia, Cherutoi [58] found that the copper nanoparticles produced through biological synthesis showed strong antimicrobial effects against Escherichia coli and Staphylococcus aureus. The zones of inhibition were measured at 26.00  ±  0.58 mm for E. coli and 30.00  ±  0.58 mm for S. aureus. In comparison, the standard antibiotic amoxicillin-clavulanate resulted in smaller inhibition zones of 20  ±  0.58 mm for E. coli and 28.00  ±  0.58 mm for S. aureus.

3.5. Cytotoxicity Assay of Bioflocculant–Copper Nanoparticles on HEK 293 Cells

The toxicity of nanoparticles is a crucial consideration due to their increasing use and environmental introduction. To investigate the effects of a bioflocculant and copper nanoparticles (CuNPs) on human embryonic kidney 293 (HEK 293) cells, an MTT assay was conducted. The assay measures the ability of viable cells to reduce MTT into blue formazan products, evaluating mitochondrial function [90]. The results showed that the bioflocculant had significant effects on cell viability, while increasing concentrations demonstrated a minimally significant cytotoxic effect, showing that the microbial flocculant extracted from P. mirabilis AB932526.1 was safe for environmental or industrial application. Previous studies have demonstrated the survival of purified bioflocculant cells in the environment, with cell survival ranging from 95.1 to 84.2% [91].

The fabricated CuNPs induced a cytotoxic effect at higher concentrations, revealing dose-dependent cytotoxicity (Figure 8). The study findings can be attributed to copper’s essential role in metabolic pathways, but that significant levels of copper can disrupt cell functions, leading to cell growth suppression [92]. Similar results have been reported for CuNPs on human breast cancer cells and non-tumorigenic human breast epithelial cells [92]. Additionally, CuNPs synthesized from Mentha pulegium leaf extract showed a dose-dependent cytotoxic effect on normal human lymphocytes [93]. These findings highlight the importance of evaluating the toxicity of nanoparticles, particularly CuNPs, to ensure their safe application.

3.6. Application of As-Produced Bioflocculant–Copper Nanoparticles in Wastewater

3.6.1. List of Contaminants Removed from Tendele Coal Mine Using Bioflocculant–Copper Nanoparticles

The wastewater generated by the coal chemical industry comprises a wide range of organic and inorganic contaminants that impair the environment, mostly through the presence of poisonous and refractory chemicals [94]. So far, several approaches have been utilized to remediate coal chemical effluent; nevertheless, the methods involve toxic substances [95]. To address these issues, an affordable and eco-friendly approach is needed. In this study, synthesized Cu nanoparticles from a bioflocculant P. mirabilis AB932526.1 were introduced as an environmentally favorable method for wastewater purification.

Table 2. List of contaminants eliminated from Tendele coal-mine wastewater by biosynthesized copper nanoparticles, compared to microbial bioflocculant and FeCl₃.

Flocculants	Water Quality	BOD (mg/L)	COD (mg/L)	N (mg/L)	SO42− (mg/L)	PO42− (mg/L)	NO3− (mg/L)	Flocculating Activity (%)
Microbial	Before	168.3 ± 0.0	154 ± 0.1	9.0 ± 0.0	35 ± 0.0	4.0 ± 0.0	9.0 ± 0.0	2.781
	After	27.3 ± 0.0	35.1 ± 0.3	3.2 ± 1.0	10 ± 1.0	0.58 ± 0.0	2.5 ± 0.0	0.264
	Removal rate (%)	84	83	64	71	86	72	90
CuNPs	Before	168.3 ± 3.0	154 ± 0.0	9.0 ± 0.0	35 ± 0.0	4.0 ± 1.0	9.0 ± 0.0	2.781
	After	17.4 ± 0.0	27.2 ± 0.0	3.3 ± 0.0	13 ± 2.0	0.50 ± 0.0	1.8 ± 0.0	0.123
	Removal rate (%)	90	87	63	63	88	80	97
FeCl3	Before	168.2 ± 2.0	154 ± 2.0	9.0 ± 0.0	35 ± 2.0	4.0 ± 0.0	9.0 ± 1.0	2.781
	After	37.2 ± 0.3	41.3 ± 0.3	2.8 ± 0.0	9.9 ± 3.0	0.58 ± 1.0	2.0 ± 0.1	0.296
	Removal rate (%)	78	82	69	64	86	78	89

indicates the effectiveness of the biosynthesized CuNPs in comparison to a bioflocculant and commonly used commercial flocculant (iron (III) chloride).

Table 2 summarizes the findings, demonstrating that the biosynthesized copper nanoparticles show considerable promise for treating wastewater from the Tendele coal mine. These nanoparticles effectively reduced various contaminants, achieving removal rates of BOD (90%), COD (87%), total nitrogen (64%), sulfates (63%), phosphates (88%), and nitrates (80%), in addition to exhibiting a high flocculation efficiency of 97%. However, their performance in removing total nitrogen and sulfates was notably less effective compared to the bioflocculant and FeCl₃. This may be due to the lower binding affinity of CuNPs for these specific compounds. Additionally, the presence of competing ions in complex wastewater can inhibit the adsorption capabilities of CuNPs, further reducing their efficiency in removing total nitrogen and sulfates [96].

The microbial bioflocculant demonstrated impressive removal rates for BOD (84%), COD (81%), total nitrogen (64%), sulfates (71%), phosphates (86%), and nitrates (72%), with a flocculation efficiency of 90%. However, its performance in nitrogen and nitrate removal was relatively lower compared to the effectiveness of commercially used Fe (III) chloride, potentially due to the complex nature of nitrogen compounds and the specific metabolic pathways required for their degradation [97].

The removal rates for FeCl₃ were observed to be 78% for BOD, 82% for COD, 69% for nitrogen, 64% for sulfates, 86% for phosphates, and 78% for nitrates, with a flocculating activity of 89%. However, this performance was less effective than that of microbial bioflocculants and biosynthesized CuNPs.

According to Dhandayuthapani, Sarumathi [98], the removal of COD and BOD from wastewater is crucial to protecting aquatic life and preventing eutrophication. High levels of these contaminants can deplete dissolved oxygen, suffocating aquatic organisms and promoting excessive algal growth, which further reduces oxygen levels as the algae decompose [99]. High amounts of nitrate and phosphate lead to eutrophication, causing an imbalance in the aquatic ecosystem [100], while sulfur and hydrogen may combine to produce an unpleasant H₂S that harms marine life [101]. The results of this research indicate that copper nanoparticles have considerable potential for a range of industrial uses, especially in cleaning wastewater, where they can effectively eliminate the biochemical oxygen demand (BOD), chemical oxygen demand (COD), phosphates, and nitrates. Bhagat, Anand [102] employed copper nanoparticles (CuNPs) to extract pollutants like phosphorus and sulfur from effluent samples collected from wastewater treatment facilities.

Batool, Shahid [103] demonstrated substantial reductions in various pollutants in textile wastewater using copper nanoparticles synthesized from the Bacillus flexus strain. The study reported a reduction of 39.659% in chemical oxygen demand (COD), 43.157% for sulfates, and 49.493% for phosphates. Patel and Bhatt [104] achieved remarkable removal rates, with COD reduction quantified at 55,263.3 ± 3298.5 mg/m³·min and BOD reduction at 30,560.3 ± 1987.5 mg/m³·min, utilizing copper nanoparticles produced from S. polyrhiza.

3.6.2. List of Pollutants Removed from the Vulindlela Wastewater Treatment Plant Using Biosynthesized Copper Nanoparticles, in Comparison to the Microbial Bioflocculant and FeCl₃

A water sample collected from the Vulindlela Wastewater Treatment Plant was analyzed to determine the removal efficiency of synthesized copper nanoparticles. This study focused on the removal of elements such as nitrogen, sulfates, phosphates, and nitrates using the biosynthesized CuNPs. Additionally, the removal rates of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) in the wastewater sample were evaluated. The results of these analyses are presented in

Table 3. List of pollutant removal efficiencies achieved using bioflocculant–copper nanoparticles from the Vulindlela Wastewater Treatment Plant.

Flocculants	Water Quality	BOD (mg/L)	COD (mg/L)	N (mg/L)	SO42− (mg/L)	PO42− (mg/L)	NO3− (mg/L)	Flocculating Activity (%)
Microbial	Before	187.3 ± 0.0	437 ± 0.0	1.76 ± 1.0	5.0 ± 0.0	3.0 ± 0.0	4.0 ± 0.0	2.781
	After	47.3 ± 0.0	90.1 ± 0.0	0.12 ± 0.0	1.0 ± 0.0	0.58 ± 0.2	1.0 ± 0.2	0.227
	Removal rate (%)	75	79	93	80	81	75	91
CuNPs	Before	187.1 ± 1.0	437 ± 0.4	9.0 ± 0.0	4 ± 0.0	3.0 ± 0.0	4.0 ± 0.0	2.781
	After	17.4 ± 0.3	27.2 ± 2.0	0.2 ± 1.0	0.3 ± 0.0	0.50 ± 1.0	0.7 ± 0.0	0.123
	Removal rate (%)	91	94	97	93	83	82	95
FeCl3	Before	183.2 ± 0.0	407 ± 0.0	3.0 ± 0.0	4 ± 0.0	3.0 ± 0.0	4.0 ± 0.0	2.781
	After	37.2 ± 1.0	44.3 ± 0.0	1.1 ± 0.0	1.2 ± 0.0	0.58 ± 0.0	1.2 ± 0.0	0. 296
	Removal rate (%)	78	87	63	70	81	70	89

.

In Table 3 above, the biosynthesized Cu nanoparticles show the ability to remove the BOD (91%), COD (94%), nitrogen (97%), sulfates (93%), phosphates (83%), and nitrates (82%), with a flocculation activity of 95%. The removal capabilities of these pollutants by the biosynthesized CuNPs may be attributed to their functional groups, chemical constituents, and surface structure [105]. The microbial bioflocculant exhibited varying removal efficiencies in removing BOD (75%), BOD (79%), nitrogen (93%), sulfates (80%), phosphates (81%), and nitrates (75%), with a flocculating activity of 91%. Notably, the removal rates of microbial bioflocculant for BOD and COD were significantly lower than those achieved with the commercially used flocculant FeCl₃. This suggests that the concentration of the microbial bioflocculant may have been insufficient during treatment, highlighting the need for dosage optimization to improve outcomes. Furthermore, while FeCl₃ exhibited a flocculating activity of only 89%, the results emphasize the potential of biological methods like CuNPs and microbial bioflocculants as effective alternatives for wastewater treatment. Since the biosynthesized copper nanoparticles possess a high potential removal efficiency, they can be employed in industrial applications. Furthermore, the effectiveness of CuNPs leads us to suggest that they also have the potential to reduce the adverse effects of chemical flocculants being used. The study conducted by Dlamini, Basson [68] found that biosynthesized CuNPs were able to remove 80% of phosphate, 89% of total nitrogen, 63% of nitrate, 62% of aluminum, 64% of sulfate, 72% of COD, and 96% of BOD in domestic wastewater. Almisbah, Mohammed [106] stated that CuONPs synthesized using Hibiscus sabdariffa L. extract proved promising in eliminating biochemical oxygen demand (BOD) and chemical oxygen demand (COD) by 56% in wastewater.

3.7. Staining Dye Removal Using Biosynthesized Copper Nanoparticles

The dyes present in water effluents are extremely harmful and have the potential to cause cancer in both human populations and mammals [107]. Therefore, it is crucial to eliminate them from water effluents before they are discharged into water bodies. Dyes have the property of being resistant to light and cannot be broken down by biological means. They also exhibit resilience against aerobic digestion, making them one of the most challenging groups to eliminate from industrial wastewater [108].

The biosynthesized CuNPs in this study were evaluated for dye removal, and the results are shown in Figure 9. The tested dyes were safranin (90%), carbol fuschin (88%), methylene blue (91%), methyl orange (93%), and Congo red (94%). The blank (control) was used as a comparison and resulted in a 39% flocculation efficiency. It can be seen that all the dyes tested had a strong affinity for the biosynthesized copper nanoparticles, and their removal effectiveness was above 85%. The highest removal efficiency was observed on Congo red, with a removal efficiency of 94%, and the lowest flocculation rate was carbol fuschin, with an 88% removal efficiency. There was no significant difference among the dyes tested. That means any of these dyes can be effectively removed using CuNPs synthesized using the bioflocculant P. mirabilis AB 932526.1. Meanwhile, Rafique, Shaikh [109] reported a dye removal efficiency of 93.75% for methylene blue.

Fathima, Pugazhendhi [110] reported on copper nanoparticles synthesized with L-ascorbic acid, aimed at treating textile wastewater. They found that these biosynthesized Cu nanoparticles effectively removed 91.53% of basic blue 9, 84.89% of Congo red, and 73.89% of acid red 2. Soomro and Nafady [111] indicated that copper nanoparticles exhibited remarkable catalytic activity in breaking down dyes, achieving a removal efficiency of 91.53% for methylene blue and 73.89% for methyl red, while the effectiveness for phenol red was not detailed. The study’s outcomes suggest that CuNPs synthesized through biological methods could serve as powerful agents for the treatment of industrial wastewater, especially in reducing dye pollution from a range of color solutions.

4. Conclusions

This study effectively utilized microbial bioflocculant to biosynthesize copper nanoparticles (CuNPs), showcasing their significant potential for environmental applications, particularly in wastewater treatment. A comprehensive range of characterization techniques, including Fourier Transform Infrared Spectroscopy (FT-IR), Ultraviolet-Visible Spectroscopy (UV-Vis), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray Spectroscopy (EDX), Transmission Electron Microscopy (TEM), and X-ray Diffraction (XRD), provided critical insights into the properties and efficacy of these nanoparticles. FT-IR analysis revealed the presence of key functional groups associated with the bioflocculant and CuNPs, indicating interactions that may stabilize the nanoparticles. The UV-Vis spectra provided clear evidence of nanoparticle formation, with the bioflocculant showing a prominent absorption peak at 300 nm, indicative of its protein and polysaccharide composition essential for flocculation. A notable color change from pale blue to deep blue upon reaction with copper ions confirmed the successful reduction of Cu²⁺ ions to Cu⁰ nanoparticles. The surface plasmon resonance (SPR) peak observed at 583 nm further validated the formation of CuNPs, while the absence of an absorption band at 280 nm in the CuNP spectrum indicated a lack of aromatic amino acids or organic compounds typically found in proteins. SEM images showed the surface morphology of the CuNPs, confirming their predominantly spherical shape and providing visual evidence of some degree of agglomeration. EDX analysis further validated the elemental composition of the nanoparticles, predominantly consisting of copper and oxygen, with no significant impurities detected. TEM analysis offered detailed insights into the size and shape of the CuNPs, reaffirming their spherical morphology, with an average size of approximately 20 nm. XRD results confirmed the crystalline nature of the CuNPs, displaying distinct peaks that correspond to a face-centered cubic (fcc) structure, indicating high crystallinity and the absence of impurities. The biosynthesized CuNPs achieved an impressive flocculating efficacy of 80% at an optimal concentration of 0.4 mg/mL against kaolin solutions, significantly outperforming traditional chemical flocculants like iron (III) chloride in removing contaminants from Tendele coal mine and Vulindlela domestic wastewater. The biosynthesized CuNPs were effective in eliminating all the tested dyes, with an optimal eliminating effectiveness of above 85%. Additionally, these nanoparticles exhibited substantial antibacterial activity against both Gram-negative and Gram-positive pathogens, highlighting their potential for antimicrobial applications. The cytotoxic effects on HEK 293 cell lines varied with the dosage, indicating a need for careful consideration in therapeutic contexts. However, the observed agglomeration behavior suggests that while biosynthesis can efficiently produce nanoparticles, it may also lead to clustering that could impact their physical properties and effectiveness. Therefore, future studies will focus on further characterization of CuNPs through Thermogravimetric Analysis (TGA), optimization of synthesis parameters, and exploring applications in treating other types of wastewater, to enhance their performance across a broader range of environments.