Impact of metal nanoparticles biosynthesized using camel milk on bacterial growth and copper removal from wastewater

2.1 Microorganism

The microorganisms used in the present study were Bacillus subtilis ATCC 6633, MRSA ATCC, Staphylococcus aureus ATCC 29213 as the Gram-positive bacteria, the Gram-negative bacteria were Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25966, and Candida tropicalis ATCC 66019 as the yeast isolate, all were provided from the microbiology department of the College of Science, King Saud University.

2.2 Chemicals and reagents

Pure materials and solvents from Sigma Aldrich (Hamburg, Germany) were used in this study and were not further purified: Zinc sulfate monohydrate (≥99.0%), nickel nitrate hexahydrate (99.9%), hydrated copper sulfate (CuSO₄.5H₂O, 98.0%), sodium hydroxide (99.0%), hydrochloric acid (37.5%), and ethanol (≥99.8%).

2.3 Instruments

Ulrospec-7500 UV-vis spectrophotometer (Ottawa, Canada), Fourier transform infrared (FTIR, PerkinElmer, Llantrisant, United Kingdom), X-ray diffractometer (Thermo Electron, Ecublens, Switzerland), transmission electron microscope (TEM, JEOL-1400, Tokyo, Japan), JEOL scanning electron microscope in combination with energy dispersive X-rays (SEM, JEOL, Tokyo, Japan) and Zetasizer Advance Range analyzer (Malvern, United Kingdom).

2.4 Nanoparticles preparation from camel milk sample

The camel milk samples were collected from healthy camels from a Saudi farm in Riyadh, Saudi Arabia. The milk was filled into sterile bottles and stored on ice until transportation to the laboratory.

2.5 Camel milk-mediated synthesis of NPs

The synthesis of ZnO NPs or NiO NPs was carried out by mixing 200 mL of a 2.0 mM aqueous zinc sulfate solution or nickel nitrate hexahydrate separately with 50 mL of camel milk and heating the mixed solution to 80°C. Then, a few drops of 0.2 M sodium hydroxide were added for 30 min, with constant stirring to reach a pH of 8. The resulting precipitate was centrifuged at 4,500 rpm for 15 min and decanted, and the formed ZnO NPs or NiO NPs precipitate were rinsed with deionized water (DI) and then with ethanol to remove organic or excess sodium hydroxide [26].

The resulting NPs were collected and subjected to calcination at 100°C for 12 h before being stored in an airtight container for further studies (Scheme 1).

Scheme 1

Biosynthesis illustration of metal oxide NPs using camel milk.

2.6 Characterization

The formation of ZnO NPs and NiO NPs was confirmed by various spectroscopic and microscopic analyzes. These methods include UV-Vis spectroscopy for behavioral investigation, FTIR spectroscopy for functional groups, dynamic light scattering and zeta potential (ZP) for particle size distribution and surface charge, X-ray diffraction (XRD) analysis for grain size, EDX and mapping analysis, and surface morphology, size and aggregation of NPs studied by J-Image software in TEM, and 3D images of SEM/atomic force microscopy (AFM).

2.7 In vitro antimicrobial assay

The standard well-diffusion agar technique was performed to determine the antibacterial potential of ZnO NPs and NiO NPs synthesized in camel milk media. Pure cultures of all microbial isolates, cultured overnight, were smeared evenly onto Muller-Hinton (MH) plates (Oxoid, USA) using sterile cotton swabs. 6 mm wells were made on the surface of the agar plates using a sterile gel puncture. 100 µL of each camel milk NPs was added to the corresponding well and then incubated at 37°C for 18–24 h. The assay was repeated three times and the average of inhibition zones was measured and expressed in mm. Standard tetracycline (30 µg) was used as a positive control. The camel milk metal NP showing the highest antimicrobial activity was selected for further experiments.

2.8 MIC determination

The MIC of the metal NPs was determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines. In brief, two-fold serial dilutions of the NPs in the concentration range of 1.0–0.01 mg·L⁻¹ were prepared in 2.0 mL MH broth tubes. 5 µL of the microbial strains were inoculated into each tube. After incubation at 37℃ for 18–24 h, 100 µL of the observed MICs were spread on the surface of MH agar plates for plate count.

2.9 Time killing assay

Susceptible strains were inoculated in sterile tubes containing 1.0 mL of the metal NPs and incubated at 0, 2, 4, 18, and 24 h time intervals. Following incubation, 100 µL of each time interval were loaded and spread on MH agar plates for direct bacterial plate count.

2.10 Elimination of Cu(ii) ions from wastewater

3.1 Mechanism of formation of NPs

The biosynthesis of ZnO NPs and NiO NPs using camel milk involves a series of biochemical processes that leverage the natural components of the milk, including proteins, lipids, and lactose, which act as reducing and stabilizing agents. The process begins with the collection of fresh camel milk, which contains a rich array of bioactive compounds, including enzymes and metabolites. To initiate nanoparticle synthesis, the milk is often subjected to mild heating or pH adjustment to create an optimal environment for nanoparticle formation. Zinc and nickel salts are used. Upon the introduction of these metal salts, the bioactive compounds in the milk facilitate the reduction of metal ions to their respective metal oxides. For ZnO NPs, the carboxyl and hydroxyl groups present in the milk proteins interact with the zinc ions, leading to nucleation and subsequent growth of NPs. Similarly, for NiO NPs, the proteins and other organic molecules in camel milk help in reducing nickel ions, promoting the formation of NiO through controlled aggregation. Following synthesis, the NPs are usually purified by centrifugation, and the residual milk components can be removed to yield a cleaner product. Characterization techniques are employed to analyze the size, shape, and crystalline structure of the NPs, confirming their formation and properties (Scheme 2).

Scheme 2

Mechanism of biosynthesis of ZnO NPs and NiO NPs using camel milk constituents as reducing, stabilizing, and capping agents.

3.2 Characterization of the prepared nanosorbents

UV-Vis spectroscopy was used to evaluate the optical properties of the individual synthesized metal oxide NPs. The UV-Vis absorption spectra of zinc sulfate together with the camel milk and the metal NPs were shown (Supporting information S1a). Three peaks at 258, 277, and 374 nm were observed for zinc sulfate, camel milk, and ZnO NPs, respectively. These results are consistent with those reported in the current literature [27]. The absorption spectra (Supporting information S1b) again revealed three distinct peaks at 250, 277, and 350 nm, which are assigned to Ni(NO₃)₂·6H₂O, camel milk, and NiO NPs, respectively. The results obtained are in agreement with those reported in the literature [28].

The optical band gaps (Figure 1a–d) of the synthesized ZnO NPs and NiO NPs can be determined by the Tauc relationship (3):

where A is the constant of transition possibility, hυ is the energy of the incident photon, and q is the optical absorption index, respectively. The calculated band gaps of ZnO NPs and NiO NPs were found to be 3.32 and 3.54 eV, respectively.

Figure 1

Optical spectra and Tauc plot of (a) and (b) ZnO NPs and (c) and (d) NiO NPs synthesized using camel milk, respectively.

FTIR analysis is utilized to investigate the active functional groups of present compounds in pre-synthesized metal oxide NPs using camel milk. The FTIR spectra were indicated spanning between 4,000 and 500 cm⁻¹. The camel milk FTIR spectrum (Figure 2a) revealed one wide peak positioned at 4,343 cm⁻¹ (sharp O–H stretching of free alcohol), 3,402 cm⁻¹ (broad O–H stretching intermolecular bonded of alcohol), 2,924 and 2,854 cm⁻¹ (weak C–H and –CH₂ stretching of fats), 2,368 cm⁻¹ (strong stretching O═C═O of carbon dioxide), 1,743 cm⁻¹ (strong C═O stretching of aldehyde), 1,658 and 1,550 cm⁻¹ (strong stretching C═O of amide I and amide II in the protein), 1,458 cm⁻¹ (medium –CH₃ bending), 1,141 cm⁻¹ (strong stretching of C–OH), and 609–331 cm⁻¹ (strong stretching C-halide compounds) [29].

Figure 2

FTIR spectra of (a) camel milk, (b) ZnO NPs, and (c) NiO NPs measured at 4,000–500 cm⁻¹.

The synthesized ZnO NPs FTIR analysis indicated different functional groups (Figure 2b) with a broad peak at 3,741 cm⁻¹ (sharp O–H stretching of free alcohol), 3,425 cm⁻¹ (broad O–H stretching of the intermolecular bond of alcohol), 2,924 and 2,854 cm⁻¹ (weak C–H and –CH₂ stretching of fats), 2,368 cm⁻¹ (strong O═C═O stretching of carbon dioxide), 1,743 cm⁻¹ (strong C═O stretching of aldhydes), 1,651 cm⁻¹ (strong C═O stretching of amide I in the proton), 1,381 cm⁻¹ (average −CH₃ bending), 1,118 cm⁻¹ (strong C–OH stretching), and 609 cm⁻¹ (strong C-halide stretching), the 432 cm⁻¹ band indicated Zn–O bonding [30].

Similarly, NiO NPs FTIR spectrum (Figure 2c) exhibited various absorption bands positioned at 3,749 cm⁻¹ (sharp O–H stretching of free alcohol), 3,441 cm⁻¹ (broad O–H stretching of the intermolecular bond of alcohol), 2,924 cm⁻¹ (weak C–H and –CH₂ stretching of fats), 2,376 cm⁻¹ (strong O═C═O stretching of carbon dioxide), 1,743 cm⁻¹ (strong C═O stretching of aldehydes), 1,651 cm⁻¹ (strong C═O stretching of amide I in the proton), 1,465 cm⁻¹ (average –CH₃ bending), 1,111 cm⁻¹ (strong C–OH stretching), and 624–393 cm⁻¹ attributed to Ni–O bonding [31].

Both metal nanoparticles’ sizes were determined using a zetasizer. In the DI water, the synthesized ZnO and NiO NPs’ particle size distribution revealed hydrodynamic average diameter sizes of 60.25 and 35.5 nm, with polydispersity index values of 0.368 and 0.298, respectively (Supporting information, S2). On the other hand, the ZPs of ZnO and NiO NPs suspended in a DI water stock solution were found to be −10.0 and −10.8 mV, respectively, indicating greater hydrodynamic sizes than their actual sizes in aqueous media. In addition, the particle exhibited an aggregating tendency, caused by a rise in ionic concentration, and resulted in weakened repulsion forces between the NPs (Supporting information, S3).

Through XRD profiles in the 2θ range of 20–80° with a scan speed of 0.03°/min, the crystallinity and crystalline phase of synthesized samples were ascertained. The XRD peak was used to calculate the crystallite size of the NPs. Scherrer’s Eq. 4 was used to calculate the average grain size of the NPs.

where the CuKα radiation wavelength is represented by λ (0.15406 Å), the full-width at half-maximum (FWHM) is indicated by β, the shape factor is represented by k (k = 0.9), and the Bragg’s angle of the peak is indicated by θ. The XRD pattern of ZnO NPs (Figure 3a) shows various diffraction peaks at 2θ values of 31.64°, 34.43°, 36.29°, 47.75°, 56.51°, 62.90°, 67.98°, and 69.23° related to (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (1 1 2), and (2 0 1), respectively. The results revealed the formation of hexagonal ZnO NPs with an average crystallite size of 18.39 ± 1.05 nm. It was found that the ZnO-NPs diffractogram matched the standard ZnO (JCPDS card No-36-1451) [32].

Figure 3

XRD profiles of (a) ZnO NPs and (b) NiO NPs synthesized using camel milk.

Through the XRD pattern of NiO NPs (Figure 3b), the crystallinity and crystalline phase of the synthesized sample showed NiO with spherical NPs with a face-centered cubic structure at 2θ values of 37.46°, 43.51°, 63.16°, 75.65°, and 79.71°, which corresponded to (1 1 1), (2 0 0), (2 0 2), (3 1 1), and (2 2 2), respectively. This diagram indicated that no more peaks were observed for the presence of pure NPs. The collected data (JCPDS # 96-101-0096) matched the standard pattern [33]. The average crystallite size of NiO NPs was found to be 19.92 ± 1.6 nm.

ZnO NPs and NiO NPs synthesized with camel milk were further detected using TEM analysis revealed the size, shape, and agglomeration state of the synthesized NPs (Figure 4a–d). The dislocation density of the synthesized ZnO NPs and NiO NPs was calculated as follows:

where δ is the dislocation density and D is the crystallite size of the sysnthesized nanoparticles. The diclocation densities at 100°C for ZnO NPs and NiO NPs were found to be 0.0029 and 0.0025 for the abovementioned NPs, respectively. The micro strain of the synthesized ZnO NPs and NiO NPs was calculated as follows:

where ε, ß, θ is the micro strain of NPs, FWHM of the peak, and Bragg angle, respectively. The micro strain is able to give information about the defects present in the lattice [34]. The calculated micro strains at 100°C for ZnO NPs and NiO NPs were found to be 5.04 × 10⁻³ and 4.17 × 10⁻³ for the abovementioned NPs.

Figure 4

TEM images of (a) and (b) ZnO NPs and (c) and (d) NiO NPs measured at different magnifications 100,000–200,000×.

Both ZnO and NiO-NP samples were analyzed for surface morphology and particle size using Image-J software. The NPs were observed as polydisperse hexagonal and spherical particles, with a particle size distribution of 62.63 ± 8 and 29.15 ± 5 nm for the aforementioned nanomaterials, respectively (Figure 5a–d).

Figure 5

TEM and particles size using image-J software of (a) and (b) ZnO NPs and (c) and (d) NiO NPs.

The field emission scanning electron microscope (FESEM) is the study of material surface phenomena, where numerous qualitative details of a material, including composition, morphology, and crystallography can be obtained. Put differently, FESEM offers details about the sample’s surface characteristics and texture, as well as the size, form, and particles distribution [35]. The primary elements that have a significant impact on an adsorbent’s capacity to absorb biomolecules are its surface area, shape, charge, and aggregation state.

The FESEM, in the present study, was utilized to verify the size and the 3D profile as well as to assess the surface characteristics of the synthesized nanomaterials. Results revealed well-defined hexagonal and spherical shapes with a solid, dense structure, and an average size of 80 and 45 nm for ZnO NPs and NiO NPs, respectively (Figure 6a and b).

Figure 6

(a) and (b) FESEM images and (c) and (d) AFM 3D images of ZnO NPs and NiO NPs synthesized using camel milk.

The elemental configuration of ZnO NPs and NiO NPs synthesized using camel milk and biogenically synthesized using SEM/EDX spectrometer were confirmed (Figure 7a and b). In the ZnO NPs sample, the weight percentages of Zn and O were 73.31% and 26.31%, respectively, while the atomic percentages were 40.67% and 59.33%. NiO NPs showed different weight percentages, 54.88% for Ni and 45.12% for O, with atomic percentages of 24.89% and 75.11%, respectively (Figure 7c and d).

Figure 7

(a) and (b) SEM images and (c) and (d) EDX analysis of ZnO NPs and NiO NPs synthesized using camel milk.

Moreover, the X-rays are restricted to the surface area and excited by the tiny electron beam, enabling the detection of particles or specific area spectra. As a result, by scanning the beam, spectral data for the full-view field can be acquired, producing an element map. The ZnO NPs and NiO NPs EDX mapping is displayed in (Supporting information, S4).

3.3 Surface area and pore diameter

ZnO and NiO NPs’ Barret–Joyner–Halenda (BJH) pore size distribution diagrams and N₂ adsorption/desorption isotherms were examined. At a relative pressure (P/P _o) in the range of 0.1–1.0, both samples exhibited a typical IV pattern of the IUPAC isotherm type with a prominent hysteresis loop in the low-pressure region of the open structured mesopores. ZnO NPs and NiO NPs, respectively, had surface areas of 134.2 ± 0.1 and 187 ± 0.4 m²·g⁻¹ and pore volumes of 0.47 and 0.51 cm³·g⁻¹, according to surface area analysis using the Brunauer–Emmett–Teller (BET) method (Figure 8a and b).

Figure 8

Pore diameter and surface area using BET/BJH for (a) ZnO NPs and (b) NiO NPs.

3.4 Thermal stability of the synthesized nano sorbents

The thermogravimetric stability (TGA) of the formed ZnO NPs and NiO NPs was examined (Figure 9) with 25–600°C as the temperature range of thermal stability. The ZnO NPs sample’s TGA curve demonstrated the highest stability. A 10% weight reduction was noted at 380°C. On the other hand, the NiO NP sample’s curve displayed various weight loss regions. Thermal degradation caused by moisture and solvent was observed at temperatures between 160°C and 250°C, resulting in weight losses of approximately 2.5% and 7%, respectively. At 380°C, a 25% decrease in weight was observed as a result of the camel milk polysaccharides’ carbon chains used in the synthesis breaking down. The last areas of weight loss were discovered to be 50%, 68%, and 69% of body weight, respectively, at a temperature range of 420–600°C.

Figure 9

TGA analysis of ZnO NPs and NiO NPs synthesized using camel milk.

3.5 Greenness of the synthesis process using complex green analytical procedure index (GAPI) and analytical greenness metric (AGREE) score software

The greenness profile was used to evaluate the analytical environmentally friendly method used relative to other documented methods. Four requirements were evaluated: corrosiveness, toxicity, hazardousness, or bioaccumulative property (with a pH range between 2 and 12), and the waste following consumption should weigh less than 50 g/sample [36].

Ultrapure water and camel milk are known to be non-corrosive and non-hazardous, and for this, they were used in this analysis. Furthermore, not a lot of waste was generated. Consequently, the suggested method conforms fairly well to the five greenness profile requirements (Figure 10a) and is considered more environmentally friendly in terms of time and reagents with a green score greater than 0.6 (Figure 10b) when compared to other chemical methods previously used in the synthesis of nanomaterials [37]. The new co-precipitation approach for the preparation of ZnO and NiO NPs using camel milk revealed a final AGREE score of 0.87 for its greenness features. The parameter 2 with orange color of sample amount means that the sample treatment was not performed online. The parameter 8 with yellow color indicates partial compliance with the greenness criteria.

Figure 10

(a) GAPI pictogram and (b) AGREE score for assessment of the greenness of synthesis method of metal oxide nanoparticle.

Greenness index using the spider diagram metric: The spider diagram greenness index was used to evaluate the reagents used in the proposed synthesis procedure. The Safety Data Sheets (SDS), which detail the numerous solvent properties and their environmental, health, and safety impacts at each stage of the process, provide the data on which this tool is based. By combining five categories of assessment criteria (health effects, general properties, odor, fire safety, and stability), a hierarchical spider diagram was created that provides a visual representation of the overall level of sustainability of the chemicals used. Following a predetermined methodology, each cluster is assigned a score from −5 (least green) to +5 (greenest). For each of the five key criteria, the results are presented visually in the form of a primary spider diagram, which provides an overview of the environmental friendliness of the solvents. The spider diagrams allow a more detailed analysis of the specific characteristics of each criterion [38]. The SDS sections of several solvents are not complete. Each attribute with missing data is scored zero. The “greenness index table” contains a list of all criteria and the percentage of available data used to generate the greenness index results. The percentage indicates the level of confidence in the assessment. It is noteworthy that this study is one of the first to use the greenness index in the analytical domain. The primary spider diagram shows that the overall greenness index of DI water and ethanol is on the safe side (Figure 11). The supporting data for the other values are included in Supporting information, S5. In addition, the eco-friendliness index provides the average values and the percentage of valuable data currently available for the solvents used are summarized in Table 1.

Figure 11

Primary spider diagram for DI water and ethanol in the green synthesis process of nanomaterials.

3.6 Antimicrobial activity assay

Nanobodies in camels were investigated and found to be efficient for the treatment of microbial infections as well as targeting cancer cells, adding as such more unique features to camel milk [39]. The current work established the potential activity of ZnO NPs and NiO NPs, both synthesized from camel milk against some pathogenic bacteria (Figure 12). Higher antibacterial activity was mainly noted with ZnO NPs against Gram-negative bacteria (P. aeruginosa and E. coli) (16 and 11 mm) and the yeast isolate C. tropicalis (13 mm) than against Gram-positive bacteria (S. aureus, B. subtilis, and MRSA) (Table 2). Similar observation was noted with previous report [40], where S. aureus showed no significant effect to ZnO NPs activity.

Figure 12

Well diffusion method showing the inhibition zone of both metal oxide NPs against the selected strains.

While the Gram-positive bacteria were more resistant to the nano extracts, the Gram-negative bacteria and the yeast isolate showed greater sensitivity to ZnO NPs when compared to nickel NPs. These findings led to the selection of ZnO NPs for additional examination.

The MIC for the two selected microorganism strains: P. aeruginosa and C. tropicalis were observed at MIC 50 of (0.5 mg·mL⁻¹) with a lower bacterial count (200 and 180 colonies/plate) compared to MICs 25 and 12 with a bacterial count of >300 (Figure 13a and b).

Figure 13

(a) MIC in vitro assay of P. aeruginosa and C. tropicalis showed similar MIC 50 of 0.5 mg·mL⁻¹ concentration against ZnO NPs indicated by (b) a lower count on MH agar plates.

The best time for killing the bacterial strains was observed after 2 and 24 h of incubation, as shown in Table 3. No microbial growth was observed on MH agar plates after 2 and 24 h of incubation, while only a few bacterial colonies were observed after 4 h of incubation.

This could be due to the thicker cell wall structure, which makes it more difficult for ZnO NPs to enter the cell [41]. The exact mechanism by which ZnO-NPs influence bacterial growth is not yet fully understood. In general, it appears that these NPs release metal ions that act directly on the bacterial cell wall and disrupt its permeability [8], thereby damaging the first line of defense [42]. Once the metal NPs penetrated the bacterial cells, they impair the biochemical processes of the bacterium and cause DNA damage and protein denaturation, which leads to cell death, as the bacteria can no longer multiply at this point [43] as indicated in Scheme 3.

Scheme 3

Possible mechanism of metal NPs (Zinc oxide as an example) synthesized from camel milk against bacterial cells. NPs attach to the bacterial cell, they start to release metal ions that act directly on the bacterial cell wall and disrupt its permeability. Once inside the bacterial cell, they impair the biochemical processes of the bacterium by oxidative stress (reactive oxygen species, ROS), cause DNA damage, and protein denaturation leading to cell death.

Several studies investigating the membrane interactions of metal NPs with the bacterial cell have shown that this binding based mainly on the ionic charges of both the NPs and the bacterial cell membrane. The cationic properties of ZnO NPs allow them to attach to the negatively charged bacterial cell surface through electrostatic interaction, which leads to their accumulation and thus to cell damage. This in turn is based on the different cell wall structures of Gram-positive and Gram-negative bacteria, which represent a natural barrier for the uptake of many antimicrobial agents [42]. Another earlier study too stated that binding of titanium oxide NPs to Gram-negative E. coli depolarized the membrane potential, which increased cell permeability and led to leakage of intracellular proteins, DNA, and ions, which in turn resulted in cell death, but this was not the case with Gram-positive Staphylococcus aureus [44]. This is consistent with the available data, in which Gram-negative bacteria showed a higher sensitivity to the zinc oxide NPs than Gram-positive bacteria. Another antibacterial activity of metallic NPs could be attributed to oxidative stress (ROS), which having high reactivity and oxidizing potential, is considered hazardous to microorganisms [45]. NPs similarly, once are in contact with the bacterial surface, trigger a free radicals accumulation inducing as such oxidative stress similar to the effect of high energy electron production when NPs are exposed to UV light induction [46].

In addition, both the shape and size of the metallic oxide NPs play an important role in determining their efficiency as antimicrobial agents. Another study [47] stated that hexagonal ZnO NPs caused lethal damage to various bacteria. Moreover, a single-pot biogenic synthesis of NiO NPs with spherical morphology for antimicrobial activities was developed [48]. Thus, the strong antimicrobial activity shown in this study is also due to the hexagonal and spherical shape of the metal oxide NPs, which were investigated as ZnO and NiO NPs, respectively. Hence, due to their surface charge, size, and shape, or by combining all three, NPs might exhibit their antibacterial activity [49]. The buildup and toxicity of metal NPs at high concentrations in human tissues restricts their broad application, even though bactericidal activity is sought [50].

3.7 Adsorption removal of Cu(ii) from wastewater

3.7.1 Factors influencing adsorption potential

3.7.1.1 Effect of sorbent dose

The influence of biogenic synthesized ZnO NPs using camel milk on the efficient removal of Cu(ii) ions was investigated with adsorbent dosages of 0.1–1.0 g·L⁻¹ using 50 mL of 200 mg·L⁻¹ Cu(ii) ions at 25°C, agitation speed of 150 rpm, 90 min contact time, and  pH of 6.2. The % removal of Cu(ii) ions dramatically increased with adsorbent doses from 0.1 to 0.5 g, referred to an increase in the active sites number [51]. It was noticed that at adsorbent doses higher than 0.5 g, adsorption was uniform. As a result, for all upcoming experiments, an adsorbent dose of 0.5 g was determined to be the ideal dose for a generally acceptable Cu(ii) ions removal efficiency (Figure 14a).

Figure 14

Optimal experimental conditions (a) ZnO NPs dosage (g·L⁻¹), (b) contact time (min), (c) initial Cu(ii) concentration (mg·L⁻¹), and (d) pH of test solution from 2 to 8.

3.7.1.2 Effect of contact time

The contact time greatly affected the sorbate kinetics at a specific initial concentration [52]. The effect of contact time on the removal efficiency of 200 mg·L⁻¹ was tested by changing the time period from 0 to 180 min, and using an equilibrium dose of ZnO NPs (0.5 g·L⁻¹) under agitation speed of 150 rpm, pH = 6.2 at room temperature (25°C). The quickest adsorption of Cu ions was observed at 30 min (Figure 14b). Then, the process slowed down and reached equilibrium at 60 min. Increased contact time up to 90 min showed unsignificant increase in the adsorption (less than 1%), suggesting that adsorption might be completed in the contact time less than 90 min. As a result, further studies were performed during contact time 90 min.

3.7.1.3 Effect of initial Cu(ii) concentration

Under the abovementioned conditions, the removal % and uptake values of ZnO NPs were investigated by altering the initial Cu(ii) concentration from 100 to 500 mg·L⁻¹. The ZnO NPs were separately dispersed for 90 min to ascertain the impact of initial Cu(ii) concentration on adsorption quantity. Figure 14c presents the results which state that when Cu(ii) concentration rises, the adsorption rate slows down after initially increasing. The adsorption process slows down as Cu(ii) concentration increases due to competition for active adsorption sites [53]. According to the data, the removal percentage using ZnO NPs sorbent decreased slightly from 99.9% at 100 ppm to 99.7% at 200 mg·L⁻¹, and it significantly decreased to 50.47% at 500 mg·L⁻¹. The initial Cu(ii) concentration in subsequent experiments was 200 mg·L⁻¹.

3.7.1.4 Effect of initial pH values and sorption mechanism

An essential component of the adsorption process is the pH of the solution which influences the adsorbent’s surface charge, the ionization level, and the speciation of the adsorbent. In this study, the effect of pH on the copper adsorption under the above determined parameters, the adsorption of heavy metals Cu(ii) ions onto 0.5 g·L⁻¹ of adsorbent at room temperature, an initial concentration of 200 mg·L⁻¹ Cu(ii) ions, 150 rpm stirring speed, and 90 min contact time, was investigated. The pH of the solution was adjusted to 2–8 with either a 0.1 M HCl or NaOH solution. It was found that with increased pH values from 2 to 6, the removal efficiency increased to reach 96.8%. The surface charge of the adsorbent can be used to explain the fluctuations in the removal of the heavy metals Cu(ii) in function of the pH value. At a strong acidic solution (pH 2), no copper ions are held in solution. This can be explained by the high concentration of H⁺ cations in the solution, causing competition between the H⁺ cations and the Cu²⁺ cations for the negatively charged space. Thus, pH 6.2 was selected for further studies (Figure 14d).

Eq. 7 can be used to calculate the distribution coefficient K_d to assess the adsorbent surface’s affinity.

(7) K d = C s C w

where C _s and C _w stand for the Cu(ii) concentration in the adsorbent (mg·g⁻¹) and the concentration of Cu(ii) that remains after removal (mg·L⁻¹), respectively. The obtained K _d values as a function of pH are displayed in Figure 15a. It is important to keep in mind that the target pollutant’s binding capability increases with its K _d value [54].

Figure 15

(a) Initial pH of 50 mg·L⁻¹ Cu(ii) solution vs log K _d and (b) initial pH of Cu(ii) solution (50 mg·L⁻¹) vs final pH of Cu(ii) solution using ZnO NPs.

The findings demonstrated the efficacy of ZnO NPs in removing Cu(ii) ions from water samples. The zero-point charge of the adsorbent accounted for the variation in absorbance with respect to the starting pH of the sample. Figure 15b shows the plot of the pH of the Cu(ii) working solution at the beginning vs the end to determine the zero point of ZnO NPs. The findings showed that the zero-point charges of ZnO NPs at pH = 8.4. Moreover, the maximum sorption capacities of the adsorbent at 25°C was found to be 3.5 mg·g⁻¹.

3.7.1.5 Effect of competing ions

It was investigated how co-ions in contaminated water, including Ca²⁺, Mg²⁺, Mn²⁺, Ag⁺, Na⁺, and K⁺, affect the ability to remove Cu(ii) ions. At room temperature, the Cu(ii) content was stable at 200 mg·L⁻¹, while the ion concentration was 500 mg·L⁻¹. The study was done under the following conditions: a Cu(ii) concentration of 200 mg·L⁻¹, a contact time of 90 min, an adsorbent dose of 0.5 g·L⁻¹, a pH of 6.2 and a temperature of 25°C. In the presence of Ca²⁺, Mg²⁺, Mn²⁺, Ag⁺, Na⁺, and K⁺ ions, the adsorption capacity is drastically reduced (Figure 16a). This indicated that the competition of these ions with the active sites on the adsorbent surface delayed Cu(ii) removal [55].

Figure 16

(a) Removal % of Cu(ii) ions in the presence of various cations (b) The effect of temperature in the range from 25°C to 45°C on the removal % of Cu(ii) ions.

3.7.1.6 Effect of different temperatures

There are two ways that temperature affects the adsorption process. As the viscosity of the solution reduces, it is known that a rise in temperature speeds up the dispersion of the adsorbed molecules between the inner pores and the outer layer of the ZnO NPs. Furthermore, for a given adsorbate, the temperature alters the adsorbent’s equilibrium capacity [56]. 0.5 g of the researched sorbent was used to remove 200 mg·L⁻¹ Cu(ii) ions from aqueous solutions. The temperature effect was evaluated at various temperatures between 25°C and 45°C, with a pH of 6.2, a stirring speed of 150 rpm, and a contact period of 90 min.

The data showed that the percentage of dissolved Cu(ii) ions decreased slightly with increasing temperature (Figure 16b). The Cu(ii) adsorption capacity dropped from 99.9% to 98.75% when the temperature was increased from 25°C to 45°C. This trend indicated that the sorption of Cu(ii) from aqueous solutions is an exothermic process. The Le-Chatelier principle [57] states that an increase in the mobility of metal ions can facilitate the reverse desorption process, which in turn can explain the decrease in copper(ii) absorption at high temperatures.

3.8 Adsorption kinetics

To understand the correlation between adsorption time, concentration, and equilibrium adsorption capacity, an understanding of adsorption kinetic models is required. The control steps of the adsorption rate of ZnO NPs can be understood using the pseudo first-order Eq. 8.

The kinetics of Cu(ii) ions adsorption on the surface of ZnO NPs was also investigated utilizing Eq. 9 of pseudo-second order.

where the values q _e (mg·g⁻¹) and q _t (mg·g⁻¹) represent the adsorption capacity of ZnO NPs on Cu(ii) ions at adsorption equilibrium and at a specific time point. However, the K ₁ (h⁻¹) and K ₂ (g·mg⁻¹·h⁻¹) correspond to the first- and second-pseudo-order rate constants [58]. Figure 17 shows the linear plots of pseudo-first-order ln ( q e − q t ) and pseudo-second-order t q t vs time.

Figure 17

(a) Pseudo-first-order and (b) pseudo-second-order Cu(ii) ions removal using ZnO NPs.

The intercepts and slopes were utilized to estimate the kinetic values of the Cu(ii) ion adsorption on the surface of ZnO NPs. The kinetics of Cu(ii) ion adsorption onto nanomaterials were investigated using two models: pseudo-first order and pseudo-second order. Table 4 provides an overview of the calculated parameters.

The most appropriate model was selected based on the correlation coefficient (R ²) and the numerical matching between the tested and estimated qe values. The pseudo-second-order kinetic model for the above sorbent provides a better explanation for the experimental results. The adsorption capacity was 77.39 mg·g⁻¹ for ZnO NPs which was confirmed by the correlation coefficient R ² which has a value of 0.9958.

3.9 Adsorption isotherms

To evaluate and understand the adsorption system between both adsorbent and adsorbate, adsorption isotherms were studied [59].

Langmuir adsorption isotherm model (10):

Freundlich adsorption isotherm model (11):

where C _e is the equilibrium concentration of Cu(ii) ions (mg·L⁻¹), q _e and Q _m are the adsorption capacity and saturation adsorption capacity (mg·g⁻¹), respectively, and K _L is the Langmuir constant (L·mg⁻¹). A Freundlich constant (K _F, L·mg⁻¹) is a constant that might represent the saturated adsorption quantity and 1/n is the adsorption intensity.

The adsorption mechanism was studied in detail during the sorption process using the adsorption isotherm model. The adsorption effect was significantly influenced by the temperature of the adsorption environment. Compared to a diagram generated using the Freundlich adsorption isotherm model, a diagram generated using the Langmuir model is more similar to the experimental diagram.

The recorded results are summarized in Table 5. The slope and intercept of the linear graphs were calculated (Figure 18a). The values for R _L (0.0002 L·mg⁻¹), the constant K _L (169.7 L·mg⁻¹), and Q _max (100.0 mg·g⁻¹) were determined. The regression coefficient for the correlation of the adsorbent was 0.9943. The Langmuir isotherm model described better removal of Cu(ii) ions when using ZnO NPs, with a higher Langmuir correlation coefficient (r ²). For the Freundlich adsorption isotherm model, the K _F values (1.403), 1/n (1.1897), and correlation coefficient (0.9905) were estimated (Figure 18b).

Figure 18

(a) Langmuir and (b) Freundlich adsorption isotherms for copper removal using ZnO NPs.

3.10 Thermodynamic studies

Standard enthalpy (∆H°, kJ·mol⁻¹), standard entropy (∆S°, kJ·mol⁻¹), and change in Gibbs free energy (∆G°, kJ·mol⁻¹) are the thermodynamic parameters that are taken into account to ascertain the spontaneity and other aspects of a given adsorption process. Five different temperatures 298, 308, 318, 328, and 333 K were used in this study. The adsorption thermodynamics were estimated using the following formulas:

where R is the molar gas constant (8.314 JK·mol⁻¹), T is the temperature (K), and K _c is the thermodynamic constant.

Figure 19 shows the thermodynamic plot of Cu(ii) ions adsorption on the surface of ZnO NPs. The slope and intercept of the given graph were used to calculate the values of ∆H° and ∆S°. The chemisorption method or physical adsorption was examined using the parameter ∆G°. When the ∆G° value is negative, adsorption happens on its own. The positive ∆H° readings indicate endothermic process. During adsorption, an increase in disorder at the solid–liquid interface is shown by positive ∆S° values [60]. The obtained results were found to be ∆H° value (−4,127.4 J·mol⁻¹), ∆S° (−88.5774 kJ·mol⁻¹), and ∆G° values (−9,910.29, −11,523.2, −12,928.4, −14,998.5, and −16,556 J·mol⁻¹), for the abovementioned temperature degrees, respectively. The negative ∆H° value indicated the exothermic adsorption process. The affinity of adsorbent materials for retaining Cu(ii) ions on their surfaces consistently with limited mobilities of copper ions was revealed by a negative entropy of adsorption. The negative ∆G° values indicated that the elimination process of Cu(ii) ions on the sorbent is a physical process, spontaneous, with electrostatic interaction, and does not require a time of induction (Table 6).

Figure 19

Thermodynamic graph of copper removal using ZnO NPs as sorbent.

3.11 Elimination of copper from wastewater samples and reusability of adsorbents

In this study, five wastewater samples containing a Cu(ii) ion concentration of 200 mg·L⁻¹ were subjected to treatment with ZnO NPs (0.5 g·L⁻¹). The main objective was to evaluate the effectiveness of ZnO NPs in removing Cu(ii) ions from wastewater. After treatment, the results showed a significant reduction in Cu(ii) concentration, indicating that the ZnO NPs are very effective in adsorbing and precipitating these ions. Various factors may have contributed to this effectiveness, including the high surface area of the NPs, which enhances interaction with the Cu(ii) ions, and their potential catalytic properties that could facilitate the removal process.

ZnO NPs show significant antibacterial efficacy in wastewater treatment due to several key properties as stated earlier [42–48]. Their antimicrobial mechanism primarily involves the generation of ROS which can damage bacterial cell membranes, DNA, and proteins, ultimately leading to cell death. The high surface-to-volume ratio of ZnO NPs improves their interaction with bacteria, increases adsorption, and facilitates more effective antimicrobial action [42]. Factors such as the size and shape of the NPs play a crucial role, with smaller particles often exhibiting greater efficacy due to their increased surface area and ability to penetrate bacterial cells more effectively [42–48]. Additionally, the concentration of ZnO NPs in wastewater is critical; higher concentrations generally yield greater bacterial inhibition, although there exists a threshold beyond which further increases may not enhance effectiveness and could potentially be toxic to beneficial microorganisms. However, the emergence of bacterial resistance is a concern, as excessive or continuous use of ZnO NPs may lead to the development of resistant strains, necessitating careful management.

Additionally, the regeneration of the synthesized ZnO NPs was also studied. The regeneration study was conducted by sonicating the filtrated sorbent with 3 mL of ethanol followed by 3 mL of DI water, filtered, and then oven dried at 100°C for 1 h. The starting elimination effectiveness was assumed to be 100%, and the consequent performance was approximated. The average efficiency of ZnO NPs was 96.75% with relative standard deviation values of 0.52%. However, the lowest efficiency was 76.32% for the above sorbent. These results indicated that the penetration of copper ions into the internal sorbent sites was easy and hindered the recovery (Figure 20).

Figure 20

Regeneration % of sorbent after five removal cycles.

3.12 Potential limitations for industrial applications

This study has some limitations that should be considered for future applications. First, the scalability of the biogenic synthesis process for industrial use is a crucial factor. While the current method effectively produces ZnO and NiO NPs at laboratory scale, scaling up to larger quantities suitable for commercial applications poses challenges related to cost, production time, and consistent quality. The efficiency of camel milk as a stabilizing agent on a larger scale needs to be evaluated to ensure that the synthesis process remains economically viable. Second, the long-term stability of the synthesized NPs in real wastewater environments is another important aspect. The effectiveness of ZnO and NiO NPs can be influenced by factors such as pH fluctuations, temperature changes, and the presence of organic matter or competing ions in actual wastewater. Research is necessary to determine how these variables impact the structural integrity, reactivity, and overall performance of the NPs over time. Moreover, assessing potential leaching of the NPs into the environment is vital, as this could pose environmental risks. Evaluating the performance of these NPs in diverse wastewater conditions will provide insight into their practical applicability and durability, ensuring that they can deliver sustained results in real-world scenarios.