Batch adsorption treatment of poultry slaughter-house wastewater using modified Tunisian smectite for organic pollutant removal

Abstract

Industrial wastewater discharge into aquatic environments poses a significant threat to both human health and aquatic life. Among the available water treatments, adsorption is a particularly efficient approach for removing pollutants. In this context, chemically-modified Tunisian smectite clay (named 1CEC and 2CEC) was used as an adsorbent material for removing proteins from poultry slaughterhouse wastewater. The structural modification of smectite clay, detected using X-ray diffraction, Fourier-transform infrared spectroscopy and scanning electron microscopy, confirmed the intercalation of the surfactant used (CTAB) into smectite layers. According to X-ray results, the basal spacing (d₀₀₁) of the newly produced composite material (2CEC) was determined to be 13.63Å. The appearance of N-H stretching vibrations near to 3000 cm⁻¹ along with the C-H stretching bands from CH₂ groups, in the modified samples provides clear evidence of CTAB interaction. Batch adsorption experiments were carried out as a function of sorbent dose, initial protein concentration, pH solution and contact time. Turbidity and chemical oxygen demand (COD) were also assessed. The results revealed that modified clay 2CEC showed the best adsorption capacity of protein (3251±104 mg/g), with optimum conditions achieved with sorbent dose of 10 mg, a contact time of 4 h, pH = 9 and an initial protein concentration of 10 mg/l. However, raw and 1CEC-modifed smectite showed adsorption capacities of 2746±103 mg/g and 1578±100 mg/g, respectively. Turbidity measurements showed a value of 8.175 NTU for 2CEC, compared to 53 NTU for 1CEC and 57 NTU for raw smectite. The adsorption isotherm analysis revealed that the equilibrium data were best described by the Freundlich model (R² = 0.99) for all samples, suggesting a multi-layer protein sorption onto smectite. The pseudo-second order (R² = 0.99) fitted well the three used clays, suggesting a chemisorption process. Interestingly, while they remain prone to water sorption and associated to a swelling behavior, the studied modified smectite clay could be an effective and environmentally adsorbent for proteins’ removal from poultry wastewater. This study opens the perspectives for exploring other types of modifications to further enhance the performance of clay as a natural adsorbent for wastewater treatment.

Introduction

Water pollution has immerged as a major global environmental challenge, due to the rapid growth of industrialization and urbanization. In particular, poultry slaughterhouses generate large volumes of wastewater containing organic matter, proteins, nitrogen, suspended solids, blood, fat feathers, and cleaning chemicals (Mohan et al., 2020).

To satisfy the demands of a rapidly expanding population and their elevated consumption patterns, numerous industrial sectors continuously manufacture a diverse array of industrial and engineering products. During production and processing, these activities generate considerable quantities of effluents that are daily discharged, and frequently released into the environment without adequate treatment of filtration. This is primarily attributable to insufficient regulatory oversight, the absence of stringent environmental policies, and the escalating costs associated with effluent management.

The composition of this wastewater can diverge depending on factors such as the size of the slaughterhouse, the specific processes used, and the extent of wastewater treatment implemented. The accumulation of these organic wastes provides an excellent medium for microbial growth that will lead to oxygen depletion, disruptions in ecological balance and decrease water quality (Rachna et al., 2022). Globally, the poultry industry production is in a continuous increase (Terán Hilares et al., 2021). According to the Food and Agriculture Organization (FAO) data from 2020, global poultry meat production reached nearly 128 million metric tons in 2019, representing a 3% increase compared to the previous year's production levels. Consequently, managing poultry slaughterhouse wastewater is increasingly critical to reduce environmental impacts, comply with regulations, and protect human and ecological health. Hence, it is essential to treat this wastewater by developing simple and cost-effective treatment that minimize secondary pollution, and thereby reducing environmental contamination and preserving ecosystem heath (Rahman et al., 2023a).

Several methods, such as anaerobic digestion (Wang et al., 2024), electrocoagulation (Sandoval et al., 2024), advanced oxidation process (Kanafin et al., 2022), anaerobic membrane bioreactor (Ramadan et al., 2023), tubular microfiltration membrane (Goswami and Pugazhenthi, 2020), precipitation (Terán Hilares et al., 2021), ion exchange, electro-dialysis, photo-electro catalysis, coagulation-flocculation, reverse osmosis and adsorption process (Khan et al., 2020; Amiri et al., 2020; Zhang et al., 2021; Rashid et al., 2021; Nure and Nkambule, 2023; Sheikh et al., 2023; Goswami et al., 2020), have been implemented for the remediation of industrial effluents continuously discharged into the environment. Several of these techniques can generate secondary toxic waste, further limiting their sustainability (Choudhury et al., 2022).

Adsorption has emerged as a promising alternative for industrial wastewater treatment due to its high efficiency and cost effectiveness. It is particularly favored over conventional methods, which often suffer from low removal efficiency, large spatial requirements and high operational costs associated with expensive equipment. Adsorption technology has attracted the attention of several researchers. The adsorption method is significantly more effective, highly appealing, and cost-efficient, capable of successfully removing toxins from industrial wastewater solutions (Rahman et al., 2023a). Conventional fossil-based synthetic adsorbents are widely used for wastewater treatment; however, they suffer from several drawbacks, such as high production costs, energy-intensive synthesis processes, poor biodegradability, and the generation of secondary pollutants. Numerous researchers have developed various types of biomaterials, such as those based on cellulose and chitosan fibers and their nanocrystals (Dewan et al., 2025; Hossain et al., 2024; Rahman et al., 2024d; Rahman et al., 2024e; Rabby et al., 2025; Sheikh et al., 2023). In addition, chitosan-modified clay bio-nanocomposites have been developed and applied for the purification of wastewater (Rahman, 2024a).

Several clay-based materials, including, montmorillonite, kaolinite, laponite, smectite and hydrotalcite, have been described for removing various contaminants from wastewater. In fact, Majid et al. (2023) had include alkali-activated kaolinite for the adsorption of antibiotic rifampicin from aqueous solutions, Amrhar and Yacoubi (2024) developed a kaolinite for the adsorption of anionic dyes, Shi et al. (2024) used a nano-clay montmorillonite for removing tetracycline from water, and Dai et al. (2022) developed a modified-iron laponite and diatomite composite photo-fenton for methyl orange dyes degradation. Jamil (2024) revealed the effectiveness of modified ball clay/MnO₂ on removing chemical oxygen demand and color from textile wastewater, while Rani et al. (2025) reported the efficiency of clay ceramic membrane in treating wastewater from local dairy and palm oil industries.

Clays are abundant, environmentally friendly, and possess unique features such as high surface area, cation exchange capacity (CEC), swelling behavior, and tunable surface charge, all of which enhance adsorption performance (García-Guzmán et al., 2023). Based on these characteristics, the adsorption capacity of clays can be further improved by intercalation or pillaring, which expands interlayer spacing, increases active sites and enhances surface charge interactions (Baloyi et al., 2018). This increased surface area provides more active sites for adsorption, catalysis, and other surface-mediated processes. The surface charge of clay minerals plays a pivotal role in several key processes. It influences the ability of clay minerals to adsorb various ions and molecules, with positively or negatively charged surfaces attracting and holding onto oppositely charged particles, thereby enhancing adsorption capacity. Surface charge also determines electrostatic interactions between clay particles and substances in the surrounding environment, which are crucial for processes like flocculation, where particles aggregate to form larger clusters (Worasith and Goodman, 2023).

Clay minerals, particularly smectite, possess a high cation exchange capacity (CEC), which allow them to exchange cations between their surface and the surrounding solution. This property is especially important for wastewater treatment, as it enables smectite to interact and bind protein molecules present in poultry slaughterhouse wastewater. Proteins, being charged, can be adsorbed onto the clay surface through electrostatic interactions, ion exchange, and surface complexation (Mnasri et al., 2017), which reduce their concentration in wastewater. The surface charge of clay enhanced these interactions by attracting oppositely charged molecules, promoting their stabilization on the clay surface and reducing their mobility and bioavailability in the environment.

Nevertheless, the application of clays in poultry slaughterhouse wastewater treatment has not yet been reported. This study aims to evaluate the effectiveness and the performance of the cetyltrimethylammonium bromide (CTAB)-modified smectite clay for the removal of proteins from poultry wastewater, compared to the raw material. The performance of the adsorption process is systematically investigated by examining the influence of key operational parameters, including contact time, pH solution, initial protein concentration, sorbent dose, turbidity and chemical oxygen demand, and by comparing these findings to the literature. Kinetic and isotherm adsorption models were done to understand the synergistic action (chemisorption, mono and multilayer physisorption, and interparticular diffusion) and to elucidate the role of smectite surface modification in enhancing protein removal from poultry wastewater.

Materials and methods

Preparation of organoclay (smectite) samples

Preparation of purified organoclay

The natural Haidoudi clay (Sm), used as the raw material in this study, was collected from the Gabes region in southeastern Tunisia. The samples were obtained from the upper layers of the Aleg Formation (Maider II) corresponding to Coniacian–Santonian deposits exposed at Jebel Haidoudi. The steps involved in clay preparation, from extraction to the production of the modified composite material, are summarized in a flow diagram presented in Supplementary material 1.

Purification of the collected clay (smectite) was performed using a conventional procedure: the raw smectite was initially oven-dried at 80 °C and subsequantly treated with a 0.5 M hydrochloric acid solution (HCl, solvent pure, purity: 36 % - 38 %, liquid) to remove carbonates impurities. The treated clay powder was then subjected to six successive cation exchanges steps with a 1 N sodium chloride solution (NaCl, Chem Center, purity 99%, solid). Excess chloride ions were removed by repeated washing with distilled water until neutrality was achieved. The resulting suspension was centrifuged (at 6000 rpm, 30 min (GT4R Expert, Fisherbrand) and dried at 60 °C following the method described by Churchman et al. (2006). The purified clay was finally stored for further experimental use.

Preparation of modified organoclay

Two organoclay samples were prepared from the previously purified smectite following the procedure described by Yahya et al. (2021). For each preparation, 8 g of purified smectite was mixed with 500 mL of cetyltrimethylammonium bromide (C₁₉H₄₂BrN, 99%, Biochemia, solid) solution at concentrations equivalent to one and two times with respect to the clay cation exchange capacity (CEC). The CEC of the smectite was determined to be 66.66 meq/100 g. The resulting materials were designed as Sm (purified smectite), 1CEC (purified smectite modified with CTAB at one CEC equivalent) and 2CEC (purified smectite modified with CTAB at two CEC equivalents). CTAB, with an average molecular weight of 384.44 g·mol⁻¹ (Carvalho et al. (2022), was used as the organic modifier to induce interlayer modification (pillaring). The CTAB - smectite mixtures were mechanically agitated with a mechanical rotatory agitator (SLAB-9) for a specific period at 70 rpm for 24 hours at room temperature (25 ± 1 °C). Then, the materials were centrifuged and washed with bidistilled water to remove excess and unbound surfactant. The resulting modified smectites were then dried at 80 °C for 24 hours before being subjected to physicochemical characterization.

Wastewater characterization

The wastewater samples were collected from a poultry slaughterhouse operating at an industrial scale, and the effluent originated mainly from slaughtering, washing, and cleaning process lines. Grab sampling was performed at the outlet of the wastewater stream prior to any on-site treatment. Samples were collected on multiple sampling days to ensure representativeness. After collection, the wastewater was stored at 4°C and analyzed within 24 h to minimize biological and chemical changes. The preliminary physicochemical characteristics of an untreated wastewater sample collected from one representative facility is summarized in Table 1.

Table 1.

Characterization of poultry wastewater.

Characteristics	Unit	Value
pH	-	6.5
Turbidity	NTU	980
COD	mg.L−1	2850

Characterization methods

X-ray diffraction

To confirm the successful intercalation of cetyltrimethylammonium bromide (CTAB) into the smectite structure, both X-ray diffraction and Fourier-transform infrared spectroscopy analyses were performed on the clay samples before and after modification. XRD was utilized to investigate variations in the interlayer spacing, specifically focusing on the d(₀₀₁) reflection of the smectite structure. The initial smectite sample was analyzed using a PANalytical X’Pert diffractometer operating with Cu-Kα radiation (λ = 0.154056 nm) over a 2θ range of 2°–30°, at a scanning rate of 2°•min⁻¹. The resulting diffractograms were processed using High Score Plus software to evaluate peak shifts and structural changes associated with surfactant incorporation.

Fourier-transform infrared spectroscopy

Infrared spectroscopy was performed using a PerkinElmer FTIR Model 783 spectrometer (USA). The spectra were recorded over the wavenumber range of 400–4000 cm⁻¹, employing 64 scans per sample at a spectral resolution of 4 cm⁻¹ and a mirror velocity of 0.6329 cm.s⁻¹ Potassium bromide (KBr) pellets were prepared by homogenizing 1 wt% of the clay sample with 99 wt% KBr, followed by mechanical pressing to form translucent discs suitable for FTIR analysis.

Scanning electron microscopy (SEM)

The microstructural features of both purified and CTAB-modified smectite samples were investigated using a Thermo Scientific Quattro Environmental Scanning Electron Microscope (ESEM). It is a versatile imaging technique widely applied in materials science, biology, and related disciplines, offering the ability to examine specimens under controlled environmental conditions, including variable pressure settings and tailored gas atmospheres. This enables high-resolution imaging of hydrated or delicate samples without the need for extensive pre-treatment, making it particularly suitable for characterizing clay minerals and assessing surface morphology and textural changes induced by chemical modification.

Zeta potential

Smectite samples (0.5 g) was added to 40 mL distilled water in falcon tubes, then the dispersion solution was centrifuged at 6000 rpm for 10 min. About 20 mL of supernatant liquid from each dispersion solution was subjected to pH adjustment over a wide range (pH = 3-9) using either 0.1 M HCl or 0.1 M NaOH. Then, the sample solution was introduced into the electrophoresis cell for zeta potential measurements, using a Zetasizer Nano ZS (Malvern) instrument. All measurements were executed under 100 mV, 52.3 ms/cm specific conductivity.

Effluent turbidity test

Turbidity is a key parameter in water quality assessment, particularly within the domains of environmental monitoring and wastewater treatment, as it reflects the concentration of suspended particulate matter present in aqueous systems. It serves as a standard metric for evaluating the clarity or cloudiness of water samples. In this study, turbidity measurements were conducted using a calibrated turbidimeter (2100N laboratory turbidimeter, EPA, 115 VAC) to ensure accuracy and reliability (Matos et al., 2024). Water turbidity can be influenced by both pH variations and contact time, highlighting their combined role in water treatment efficiency. In this study, the effect of pH (3, 5, 7and 9) on protein removal was evaluated over different contact time (0 h, 12 h, 24 h, 36 h, 48 h) using both raw and CTAB-modified smectite organoclay The target pH levels were stabilized through the controlled addition of diluted HCl or NaOH at regular intervals. The turbidity was measured using a Nephelometric Turbidity Units (NTU).

Chemical oxygen demand test

The reactor digestion method is a widely adopted technique in environmental and wastewater analysis for evaluating water quality and quantifying pollution levels. In order to assess the protein removal efficiency from poultry slaughterhouse wastewater, it is essential to determine the chemical oxygen demand (COD) measurements were conducted by subjecting the wastewater sample to oxidation using a strong oxidizing agent, potassium dichromate (K₂Cr₂O₇, 99 % purity, solid state, NeutroPure), in the presence of concentrated sulfuric acid (H₂SO₄, 99 % purity, liquid state, Finar), as described by Gutiérrez González et al. (2025). The reaction mixture was heated within a controlled digestion apparatus to promote oxidative degradation. During this process, organic constituents within the sample are oxidized by dichromate ions, resulting in the formation of carbon dioxide and water. The quantity of dichromate consumed during digestion is directly proportional to the concentration of organic matter present. The COD value was determined by measuring the reduction in dichromate concentration pre- and post-digestion. The COD removal efficiency was evaluated as a function of pH solution and contact time using purified smectite and two CTAB-modified variants (1CEC and 2CEC). The experimental procedure involved the dispersion of 0.01 g of each clay material (Sm, 1CEC, and 2CEC) into 50 mL of poultry wastewater with an initial COD concentration of approximately 2850 mg/L. The suspension was mechanically agitated at 250 rpm for 48 hours at ambient temperature under different pH conditions (3, 5, 7, and 9). Following treatment, COD removal efficiencies were calculated at each pH value to evaluate the adsorption performance of the materials under varying acidity and alkalinity conditions. The chemical oxygen demand was calculated using the following formula (1).

$$\%\mathbf{CO}{\mathbf{D}}_{\mathbf{removal}}=\frac{\mathit{CO}{\mathit{D}}_{\mathit{i}}-\mathit{CO}{\mathit{D}}_{\mathit{f}}}{\mathit{CO}{\mathit{D}}_{\mathit{I}}}\times 100$$ (1)

COD_i and COD_f represent the first and last concentrations, respectively.

Protein concentration assay

Protein concentration was quantified using the Lowry method (LOWRY et al., 1951) a well-established analytical technique recognized for its accuracy and sensitivity in protein estimation. Protein concentration was determined as a function of contact time (1, 2, 3, and 4 h) using different clay samples. The procedure involves the interaction of proteins with copper ions in an alkaline medium (0.1 N NaOH, 99% purity, liquid state), leading to the formation of a chromogenic complex. The intensity of the resulting coloration is directly proportional to the protein concentration in the sample. Spectrophotometric analysis (Thermo, Helios OMEGA) was conducted at an absorption wavelength of approximately 750 nm to determine protein levels. A standard calibration curve was constructed using bovine serum albumin as the reference protein, prepared in the same alkaline medium (0.1 N NaOH). Nine concentration levels were prepared in triplicate: 0, to 1000 µg/mL. The resulting calibration equation was:

$$A_{750}=0.00182\times \left[C\right];R^2=0.9987$$

A reagent blank (0.1 N NaOH + Lowry reagent, no protein) was included in each analytical batch to correct for background absorbance. A positive quality control (QC) standard at 500 µg/mL and BSA was run at the beginning, middle, and end of each batch; QC acceptance criterion was set at ±5% of the nominal value. All QC results were within specification throughout the study.

Batch experiments for protein adsorption capacity using raw and modified clay

Batch experiments were conducted by introducing raw and modified clay samples (powder form) into specific flasks containing various concentrations of the poultry wastewater (C1, C2, C3 and C4; with C1 = 6782 mg/L, C2 = 5087 mg/L, C3 = 3391 mg/L, and C4 = 678 mg/L). A mechanical rotatory agitator (SLAB-9) for a specific period at 70 rpm was used to shake the suspension at room temperature (25 ± 1°C). After the centrifugation of the solutions (6000 rpm, 30 min), the residual concentration of protein was determined. The effects of contact time (1 hour – 6 hours), initial protein concentration (C1, C2, C3 and C4) and biosorbent dose (0.01 g – 0.04 g) and pH solution (3, 5, 7, and 9) were assessed. Experiences were detailed in Supplementary material 2. Optimum conditions were maintained in subsequent experiments. The removed protein percentage (R %) in solution as well as the protein adsorption capacity (qe mg/g) was calculated using the Formula 2 and 3, respectively (Rahman et al., 2024f):

$$R(\%)=\left(C0--Cf\right)/Ci)x100$$ (2)

$$qe\left(mg/g\right)=\left(C0-Ce\right)/m)xV$$ (3)

C0 and Cf are the initial and final concentration of wastewater solution. Ce is the equilibrium protein concentration (mg/l), m is the mass of used dried adsorbent (g) and V (l) is the volume of the reaction mixture. C0 = is the initial protein concentration in the wastewater (mg/l), expressed as the mass of protein per unit volume of protein. Ce (mg/l) represents the residual protein concentration remaining in the wastewater after adsorption process and is calculated following formula (4):

$$\begin{array}{c}\hfill \mathit{Ce}=C0-\mathit{Cads}\end{array}$$ (4)

Where Cads (mg/l) is the amount of protein adsorbed during the adsorption process determined directly after adsorption

Determination of protein sorption capacity over time

The effect of contact time on protein adsorption capacity is a critical factor in the optimization of adsorption-based treatment processes. A thorough understanding of adsorption kinetics and appropriate contact time selection is essential for enhancing the efficiency of systems designed for wastewater treatment, pollutant mitigation, and purification applications. In the present study, contact time was varied from 0 to 360 minutes under alkaline conditions (pH = 9) and at protein concentration C1, C2, C3 and C4 to evaluate its influence on adsorption dynamics.

Determination of protein sorption capacity as a function of sorbent dose

The effect of clay concentration on sorption capacity pertains to the ability of clay minerals to adsorb contaminants from aqueous solutions. Smectite, in particular, is characterized by its expansive surface area, substantial cation exchange capacity, and inherent porosity, making it an effective adsorbent for a broad range of pollutants. The effect of sorbent dose on protein adsorption capacity was investigated using 0.01 g, 0.02 g, 0.03 g, and 0.04 g of raw and modified material under optimal conditions, pH = 9, initial protein concentration C1 - C4 and a contact time of 240 minutes.

Adsorption kinetics and isotherms

Adsorption kinetics models

The use of theoretical adsorption models is well-established across disciplines such as environmental science, chemical engineering, and materials research, serving as essential tools for interpreting and optimizing adsorption processes. Kinetic models provide insights into the rate and mechanism of solute uptake, while isotherm models capture the equilibrium distribution of adsorbates between the solid surface and aqueous phase under constant temperature. A sound grasp of these modeling frameworks is key to understanding the nature of adsorption, whether governed by physisorption or chemisorption, as well as estimating adsorption capacity and evaluating process efficiency.

In this study, protein adsorption experiments were conducted by introducing 0.01 g of each adsorbent sample into poultry wastewater solutions. Reaction times ranged from 0 to 360 minutes, under a fixed protein concentration (C = 6782 mg/L) and controlled pH = 9, enabling detailed kinetic and equilibrium analyses.

To elucidate the nature of the protein adsorption mechanism, three widely recognized kinetic models, pseudo-first-order, pseudo-second-order and intraparticle diffusion, were applied to the experimental data. These models are instrumental in discerning whether the sorption process predominantly follows a physical or chemical pathway. The linearized forms of the pseudo-first-order and pseudo-second-order equations are represented by Eqs. (5) and (6), respectively:

$$Ln\left(q_e-q_t\right)=ln{q}_{e1}-\left(k_1/2.303\right)xt$$ (5)

Where q_e and q_t are the amounts adsorbed protein at equilibrium and at time (mg. g⁻¹), respectively, and k₁ (min⁻¹) is the rate constant of pseudo first-order adsorption (Ben Jmaa et al., 2026).

$$t/q_t=\left(1/\left(K_{2}x{q_e}^2\right)\right)+\left(\left(1/{q_e}^2\right)xt\right)$$ (6)

Where q_e and q_t are the amount of adsorbed protein at equilibrium (mg.g⁻¹) and k₂ is the equilibrium rate constant of pseudo-second-order sorption (Ben Jmaa et al., 2026).

Intraparticle diffusion often governs adsorption kinetics, as the overall rate is controlled by the transport of adsorbate molecules through the porous structure of the biosorbent. The adsorption process involves successive mass transfert steps including diffusion in the bulk solution, film diffusion at the liquid-solide interface, intraparticle diffusion and adsorption onto internal active sites (Ben Jmaa et al., 2026). This model was determined using the following Weber and Morris equation (Weber and Morris, 1963):

$$qt=Kx{t}^{0.5}+C$$ (7)

Where C is the thickness of the boundry layer and K denotes the intraparticle diffusion rate constant (mg/g/min^0.5) (Ben Jmaa et al., 2026).

Adsorption isotherm models

Adsorption isotherms characterize the interaction between solutes and adsorbents, serving as essential tools for optimizing sorbent dosage and understanding equilibrium dynamics. Quantifying contact time and sorbent dose is crucial for identifying the adsorption capacity of materials under defined experimental conditions. Once the system reaches equilibrium, the adsorbents retain the maximum achievable amount of solute, primarily governed by the saturation of active surface sites.

Accordingly, isotherm models are employed to describe the relationship between the quantity of solute adsorbed and its residual concentration in solution at equilibrium. In the present study, adsorption experiments were conducted at a fixed pH of 9, a contact time of 280 minutes, and a sorbent dose of 0.01 g for each material. The initial protein concentrations were C1 = 6782 mg/L, C2 = 5087 mg/L, C3 = 3391 mg/L, and C4 = 678 mg/L. To analyze sorption behavior and extract equilibrium parameters, the linearized forms of the Langmuir and Freundlich isotherm models were applied to the experimental data (Dada, 2012).

The Langmuir model assumes a reversible, dynamic equilibrium between adsorbed and non-adsorbed molecules. It is particularly applicable to systems where adsorption occurs on homogeneous surfaces, characterized by uniform adsorption energies and monolayer coverage (Jędras et al., 2022). This model provides valuable insight into sorbent saturation behavior and maximum adsorption capacity.

The linearized form of the Langmuir isotherm can be expressed as following formula (8):

$$Ce/qe=\left(1/KLxqmax\right)+\left(\left(1/qmax\right)xCe\right)$$ (8)

where qe is the protein adsorption capacity at equilibrium (mg·g⁻¹), Ce is the equilibrium concentration of protein remaining in the liquid phase (mg·L⁻¹). KL is the Langmuir constant reflects the adsorption intensity or affinity between the adsorbate and the sorbent, and qm is the maximum monolayer adsorption capacity (mg·g⁻¹) (Ben Jmaa et al., 2025).

The Freundlich equation is a widely utilized empirical model for characterizing the adsorption of solutes from liquid phases onto solid surfaces. It presumes adsorption onto a heterogeneous surface with a non-uniform distribution of adsorption sites and energies Jędras et al. (2022), making it particularly suitable for describing systems exhibiting multilayer adsorption and variable affinity. The linearized form of the Freundlich isotherm is expressed by the following formula (9).

$$Lnqe=\left(lnKf\right)+\left(1/nxlnCe\right)$$ (9)

Where Kf and n are empirical constants specific to each adsorbent–adsorbate system. The parameter Kf is indicative of adsorption capacity, while the exponent n represents the heterogeneity factor and provides insight into adsorption intensity across a range of surface sites. To determine these constants, the linear form of the Freundlich equation can be employed by plotting ln qe against ln Ce. The slope of this plot corresponds to 1/n, and the intercept yields ln Kf, thereby allowing for straightforward calculation of the model parameters and evaluation of sorption behavior (Ben Jmaa et al., 2025).

Reusability study

After completing the initial adsorption under the optimized conditions, the protein-loaded sorbent was treated with 0.1 M HCl solution to remove residual protein located in the smectite sites (Abdelrahmed et al., 2025). The sorbent was then rinsed with distilled water and dried. The obtained material was then employed in a successive adsorption cycles. This procedure comprising adsorption measurement, washing and drying was repeated for eight cycles, with the protein adsorption capacity evaluated after each cycle.

Statistical analysis

Analytical results were expressed as mean values accompanied by standard deviation (SD) to reflect variability within the dataset. Statistical analyses were performed using SPSS software, version 18.0 (IBM Corp., USA). A p-value less than or equal to 0.05 was considered indicative of statistically significant differences among treatments. All experiments were done in triplicate.

Results and discussion

Structural analysis of the biosorbent

X-ray diffraction

Fig. 1 presents the X-ray diffraction profiles of purified smectite, and CTAB-modified variants 1CEC and 2CEC. The diffractogram of smectite confirms the presence of smectite and kaolinite phases, with characteristic basal spacings of 12.70 Å and 7.12 Å, respectively. Upon incorporation of the cationic surfactant cetyltrimethylammonium bromide (CTAB), a discernible shift in the d₀₀₁ basal reflection was observed. Specifically, the original spacing of 12.62 Å for untreated smectite increased to 12.80 Å in 1CEC and further to 13.63 Å in 2CEC. This expansion is attributed to the intercalation of CTAB’s organic cations into the interlayer galleries via ion exchange mechanisms, as reported by Zhu et al. (2023). These results were described by Sayah et al. (2025)

Fig. 1.

X-Ray diffraction patterns of smectite samples after CTAB-treatment, compared to the raw clay.

The increase in basal spacing is closely correlated with the hydrocarbon chain length of the surfactant, which modulates the extent of interlayer expansion. Additionally, a persistent peak at 7.4 Å indicates the presence of kaolinite admixture; this mineral is known to resist intercalation by surfactants (Matusik et al., 2022). The effective incorporation of CTAB led to structural reorganization within the smectite layers, yielding variable arrangements in the basal spacing. These molecular configurations are governed by factors such as the surfactant's size, geometry, charge distribution, and its interaction with the clay surface (Yahya et al., 2021).

Moreover, the XRD diffractograms suggest the formation of bilayer assemblies of CTAB within the interlayer regions of both 1CEC and 2CEC. This structural modification enhances the physicochemical properties of the clay, promoting improved interaction with protein molecules. Compared to unmodified smectite, the CTAB-treated materials exhibit superior adsorption potential, attributable to their optimized layer charge distribution and elevated cation exchange capacity.

Whereas the XRD diffractograms did not expose any peaks referred to the excess of CTAB molecules which could have crystallized on the mineral surfaces indicating that CTAB molecules were successfully incorporated into the interlayer spaces of smectite rather than remaining free on the surface. This finding reinforces the effectiveness of CTAB modification in enhancing the functional properties of smectite for applications in wastewater treatment.

In a related study, Matusik et al. (2022) modified smectite group minerals using four cationic surfactants for the removal of styrene. Their findings proved that after modification of smectite with cationic surfactants (Alkyl-trimethylammonium surfactants, Dodecyl trimethylammonium-chloride (C12), Tetradecyltrimethylammonium chloride (C14) and Hexadecyltrimethylammonium chloride (C16)), resulted in an increase in the basal spacing. This increase was correlated with several factors including, the surfactant’ s hydrocarbon chain length, the layer charge of clay mineral, CEC, SBET and type of interlayer cations.

Fourier-transform infrared spectroscopy

Fourier-transform infrared spectroscopy was employed to identify vibrational bands corresponding to specific functional groups characteristic of clay minerals, and to evaluate the structural effects of CTAB intercalation into the smectite matrix. Fig. 2 and Table 2 display the spectra of purified smectite and its organo-modified forms (1CEC and 2CEC), and their corresponding major peaks, respectively.

Fig. 2.

FTIR spectra of smectite samples after CTAB-treatment, compared to the raw clay.

Table 2.

The predominant functional groups present in the smectite clay according to their specific adsorption peaks.

Particular peak (cm−1)	Intensity	Responsible functional group
3698-3624	Broad and Str	Free -OH
3000-2921	New appearence	N-H of amine
2853	Symmetric bending	C-H
1650-1637	Sharp and Str	H-O-H
-947-873	Sharp and Str	Si-O
524	Sharp and Str	Si-O-Al
468	Sharp and Str	Si-O-Si

The spectra showed similar baseline bands but with marked differences in bandwidth, intensity, and the emergence of new absorptions. Notably, the N–H stretching vibration at ∼3000 cm⁻¹ appeared exclusively in the modified samples, indicating successful CTAB intercalation. Additional peaks corresponding to C–H stretching bands from CH₂ groups and aliphatic chains further validate surfactant incorporation. In the spectrum of unmodified smectite, dominant bands were observed at 468 cm⁻¹ and 524 cm⁻¹, attributed to Si–O–Si and Si–O–Al bending vibrations, respectively. A pronounced peak at 947 cm⁻¹ was associated with the Si–O stretching, while the band at 894 cm⁻¹ suggests the presence of amorphous silica.

The H–O–H deformation band at 1637 cm⁻¹ reflects molecular water within the clay structure, and broad absorptions between 3622–3697 cm⁻¹ correspond to –OH stretching of structural hydroxyls (Abbas Bhatti et al., 2024). Two weaker bands at 2851 cm⁻¹ and 2921 cm⁻¹—assigned to symmetric and asymmetric C–H stretching vibrations—intensify in the 2CEC spectrum (Kenawy et al., 2018). The strong peak at 2921 cm⁻¹ is also linked to conformational shifts in trapped amine chains, from trans to gauche configuration, as surfactant loading increases. This transition implies altered interlayer organization and enhanced hydrophobicity within the organo-clay system.

The shift of the 1601 cm⁻¹ band (smectite) to 1637 cm⁻¹ (composite) indicates increased hydrophobic character, supported by greater water retention due to swelling. Post-adsorption, similar spectral shifts were observed across samples, with consistent bandwidth and intensity. Specifically, the 1605 cm⁻¹ band migrated to 1650 cm⁻¹ in the protein-adsorbed composite, confirming elevated hydrophobicity and water uptake. These FTIR results strongly align with XRD observations, substantiating the structural intercalation of CTAB and its functional role in enhancing protein adsorption performance. These findings were described by Sayah et al. (2025). Hussain and Ali (2021) reported that bentonite showed Si-O-Al bending and Si-O stretching vibrations in the tetrahedral sheets comparable to those observed in smectite. They also showed that the O-H stretching vibration of bentonite are similar to those of smectite. However, differences were observed in the hydration related bands: the hydration band appeared at 2513 cm⁻¹ for bentonite, whereas it was detected at 1637 cm⁻¹ for smectite. In addition, bentonite displayed C-H and C=C vibration bands associated with organic components in clay soil at 1795 cm⁻¹ and 1435 cm⁻¹, respectively, while smectite showed the appearance of N-H amine groups, with characteristic band at 2928 cm⁻¹.

Scanning electron microscopy

SEM imaging was performed to assess the morphological transformation of purified smectite following intercalation with the cationic surfactant CTAB. Fig. 3 shows the SEM micrographs of the purified and modified clays. The untreated smectite exhibited a dense, agglomerated texture composed of compact particle clusters—typical of unmodified clay minerals. Upon modification with CTAB at 1CEC, the microstructure displayed a more condensed and cohesive appearance, likely due to intraparticle binding facilitated by surfactant alkyl chains interacting at the surface.

Fig. 3.

SEM images of raw and modified smectite samples: (a) purified smectite (200 µm), (b) organo-smectite 1CEC (200 µm), (c) organo-smectite 2CEC (200 µm) and (d) organo-smectite 2CEC (500 µm).

At the higher modification ratio (2CEC), SEM micrographs revealed a shift toward stratified and sheet-like particle arrangements. This transition reflects intensified electrostatic attraction between the negatively charged smectite inner surfaces and the trimethylammonium head groups of CTAB (Msadok et al., 2020), promoting organized layering and interfacial expansion. The replacement of hydrated exchangeable cations with surfactant molecules induces surface reorientation and enhances hydrophobic character linked to interactions between the long hydrocarbon chains and the mineral framework.

Zeta potential of the samples

The zeta potential of smectite clay is determined to evaluate its surface charge behavior and electrostatic stability in aquoues media. The zeta potential as a function of pH is presented in Fig. 4. The results show that CTAB-modified smectite exhibited a positive zeta potential over the entire investigated pH range, in contrast to the raw smectite, which remains negatively charged. This behavior indicates that CTAB modification induces a surface charge reversal, whereby the inherently negative smectite surface becomes positively charged due to the anchoring of CTA⁺ head groups on the external surface and whithin the interlayer spaces of the clay. The shift of zeta potential from negative values for raw smectite to positive values after modification clearly confirms the successful intercalation of CTAB. The resulting positively charged surface enhances electrostatic attraction between the CTA⁺ groups of the modified smectite and negatively charged functional groups of proteins (-COO-), which suggested a high protein adsorption capacity for CTAB-modified smectite.

Fig. 4.

Zeta potential of smectite samples after CTAB-treatment, compared to the raw clay.

Effect of experimental conditions

pH effect on turbidity of wastewater treatment over time

pH is a crucial parameter in wastewater treatment, as it strongly influences the adsorption behavior of clay minerals. These minerals possess various surface functional groups that can undergo protonation or deprotonation depending on the pH solution. Thus, changes in pH modify their surface charge and, in turn, affect the adsorption of ions and molecules (Shoaib et al., 2022). The selection of an appropriate pH is a key factor in optimizing water treatment processes. Fig. 5 shows the effect of initial pH values (3 – 9) on the turbidity of poultry slaughterhouse wastewater after treatment with purified and CTAB-modified smectite. The obtained results showed that, after 48 hours of agitation, all smectite variants (Sm, 1CEC, and 2CEC) achieved significant turbidity reduction at pH = 9, indicating enhanced sorption efficiency under alkaline conditions. The measured turbidity values were 57 NTU for Sm, 53 NTU for 1CEC, and 8.175 NTU for 2CEC. The results further indicate that water turbidity was significantly lower when treated with 2CEC compared to Sm and 1CEC. This improvement can be attributed to surfactant-induced modification with cetyltrimethylammonium bromide at ratios corresponding to one and two times the cation exchange capacity. Such modification promotes the development of a positive surface charge on the clay particles, counteracting the intrinsic negative charge arising from isomorphic substitution and surface hydroxyl groups. The resulting positively charged layers promote stronger electrostatic interactions with negatively charged protein molecules, thereby enhancing colloid destabilization and turbidity removal (Moslemizadeh et al., 2016). When the solution pH exceeds the protein isoelectric point (pI), proteins acquire a net negative charge due to the deprotonation of functional groups such as amine (–NH₂) and carboxyl (–COO⁻). Under these alkaline conditions, electrostatic repulsion between protein molecules increases, while their electrostatic attraction toward positively charged smectite surfaces, particularly those modified with cationic surfactants is strengthened. This complementary charge interaction facilitates efficient protein adsorption onto the engineered clay surfaces (Yu et al., 2013; Balila and Vahdati, 2024).

Fig. 5.

Assessment of poultry wastewater turbidity using (a) purified smectite, (b) 1CEC modified smectite and (c) 2CEC modified smectite. a,b,c,d Different letters for the same pH value and different contact times indicate significant differences.

Jędras et al. (2022) reported that, under the studied experimental conditions, the precipitation of Cr (III) hydroxide begins to occur at pH values above 4.5. Mg/Fe–Na-montmorillonite materials exhibited the highest adsorption efficiencies at an equilibrium pH (pHₑ_q) of 6, indicating favorable sorption behavior under mildly acidic to neutral conditions. In contrast, Ni/Al–Na-montmorillonite showed negligible adsorption of Cr (III) at pH_ₑq = 4.1, likely due to limited hydroxide availability and insufficient surface deprotonation. Overall, materials for which equilibrium pH values approaching the Cr (III) precipitation threshold of 4.5 displayed a marked decrease in adsorption capacity, highlighting the strong influence of pH-dependent metal speciation and surface charge on Cr (III) retention.

The adsorption behavior observed in the present study is consistent with findings reported by Kenawy et al. (2018) who reported a pronounced decrease in rhodamine B adsorption capacity from 125.3 mg/g to 9.3 mg/g as the pH increased from 5 to 10. This trend highlights the strong sensitivity of organic molecule adsorption to pH variations, primarily due to changes in adsorbent surface charge and solute ionization. The agreement between both studies further confirms the dominant role of electrostatic interactions in controlling sorption efficiency, particularly under alkaline conditions where negatively charged functional groups prevail.

Contact time is a crucial parameter in determining wastewater turbidity, as it directly affects the efficiency of processes responsible for removing suspended solids. Fig. 1 illustrates the effect of contact time in wastewater turbidity. The results indicate that wastewater clarity improved with increasing contact time (over 48 h) for all clay samples. Among them, 2CEC (8.175 NTU) modified clay achieved the highest enhancement of wastewater clarity after 48 h compared to Sm (57 NTU) and 1CEC (53 NTU) at pH = 9. Hence, stirring time effect ensured uniform dispersion of particles and promoting reaction kinetics (Getahun et al., 2024).

Chemical oxygen demand

Fig. 6 illustrates the chemical oxygen demand (COD) of proteins in poultry wastewater as a function of pH (3, 5, 7 and 9) over t = 48 h. The data presented in Fig. 2 a clear relationship between solution pH and the efficiency of organic matter removal, as indicated by chemical oxygen demand. The obtained results indicated that, under strongly acidic conditions (pH = 3), all smectite samples exhibited a gradual and limited reduction in COD, suggesting minimal removal of organic constituents from the poultry wastewater. At pH = 5, COD levels unexpectedly increased, indicating that such pH values may inhibit organic matter removal, potentially due to destabilization of adsorption sites or unfavorable charge interactions.

Fig. 6.

COD removal efficiency of (a) purified smectite, (b) 1CEC modified smectite and (c) 2CEC modified smectite. a,b,c,d Different letters for the same pH value and different contact times indicate significant differences.

At pH= 7, a steady decline in COD was observed, indicating consistent adsorption performance of smectite materials toward organic pollutants. The most pronounced reduction occurred under alkaline conditions (pH = 9), where all three smectite variants Sm, 1CEC, and 2CEC exhibited a marked decrease in COD after approximately ten hours of treatment. This pronounced decline suggests that alkaline conditions enhance organic matter removal, likely due to improved electrostatic interactions and increased solubility and reactivity of organic compounds at elevated pH levels(Jeon et al., 2023).

From these results, the 2CEC- modified smectite consistently revealed superior COD removal efficiency over 48 h compared to the purified Sm and 1CEC variants. These findings underscore that increasing the cation exchange capacity through targeted modification significantly improves the adsorptive performance of smectite in wastewater treatment. The higher CTAB loading likely enhances surface reactivity, increases the number of positively charged adsorption sites, and strengthens electrostatic interactions with organic pollutants, thereby facilitating more efficient contaminant removal.

Evaluation of protein adsorption efficiency over time

Protein adsorption onto clay surfaces may occur via multiple physicochemical mechanisms, including electrostatic interactions, hydrogen bonding, hydrophobic forces, and site-specific binding at active functional groups on the mineral surface. These interactions are affected by various factors such as pH, ionic strength, surface charge distribution, and the structural characteristics of both the protein and the clay matrix. In this context, it is essential to evaluate the protein adsorption capacity of purified and CTAB-modified smectite variants (Sm, 1CEC, and 2CEC). Such a comparative analysis provides insight into the underlying sorption mechanisms and highlights the role of surface modification in enhancing protein retention from complex wastewater systems.

Fig. 7 illustrates the protein removal efficiency of purified and CTAB modified smectite clays as a function of contact time. The results demonstrated a progressive reduction in protein concentration over time for the three-smectite types. All samples exhibited a decrease in protein concentration, with the lowest protein adsorption efficiency observed for purified smectite compared to 1CEC and 2CEC at pH = 9. The maximum protein adsorption concentration were reached after 4 hours of treatment, with values of 3625 mg/L for Sm, 1289 mg/L for 1CEC and 280 mg/L for 2CEC, indicating that the highest adsorption efficiency is achieved with the 2 CEC- modified smectite. The reduction in protein concentration can be attributed to the electrostatic interactions between the negatively charged surfaces of unmodified or modified smectite and the positively charged amino acids residues on the protein molecules (Yu et al., 2013) . Contact time is also a crucial parameter in protein removal efficiency, as it allows sufficient interaction between the protein present in the poultry wastewater and the smectite surface, enabling the system to reach maximum adsorption efficiency (time = 4 h). Previous studies have also reported that protein – clay interactions are strongly influenced by pH solution and the protein’s isoelectric point (pI), which govern the balance of attractive and repulsive forces between oppositely charged surfaces. In this context, the protein adsorption capacity from poultry wastewater onto purified and CTAB-modified smectite (1CEC; 2CEC) increased as the pH solution rose from 3 to 9. At pH values above the pI, protein carry a net negative charge due to the deprotonation of functional groups such as -NH₂ and -COO⁻ groups, which leads to increased repulsive forces between protein molecules (Yu et al., 2013) . These findings highlight the critical role of pH in modulating interactions between proteins and clay surfaces and provide insights into the complex adsorption behavior of these molecules.

Fig. 7.

Determination of protein adsorption concentration of purified and modified smectite samples at pH = 9 over 240 min using 0.01 g of sorbent weight.

Among the tested materials, 2CEC emerged as particularly effective for treating poultry wastewater, outperforming conventional treatment approaches. Its high cation exchange capacity, enables efficient adsorption and removal of heavy metals, organic pollutants, and other contaminants (Wang et al., 2013). While electrocoagulation is often considered efficient and cost-effective, it may not remove certain pollutants as effectively as 2CEC- modified smectite (Ngobeni et al., 2021). Membrane technologies, such as microfiltration, ultrafiltration, nanofiltration, and reverse osmosis, can be costly and may not efficiently target all pollutant types (Fatima et al., 2021). Biological treatment methods, which rely on microbial degradation of organic matter, are often slow and may not fully eliminate all pollutants (Wang et al., 2013). Overall, 2CEC-modified smectite offers a more comprehensive solution for poultry wastewater treatment, combining high efficiency in pollutant removal with potential for sustainable application.

In a related study, Wang et al. (2013) developed a simplified foam separation column incorporating internal baffles to enhance protein adsorption from soy whey wastewater. The baffles improved bubble stability and increased the available surface area for adsorption, optimizing protein recovery. Their experiments demonstrated the potential of such systems for industrial-scale application, particularly the recovery of valuable protein fractions from agro-industrial effluents. By enhancing interfacial adsorption and mass transfer, this innovative approach provides a promising avenue for improving protein recovery processes while contributing to the sustainability and efficiency of food processing operations.

Determination of experiments conditions

Initial protein concentration and contact time effect

Protein adsorption kinetics were evaluated under controlled conditions (25 °C, pH = 9) over contact times ranging from 0 to 360 minutes to evaluate the effect of initial protein concentration as well as the contact time on adsorption performance. Four concentrations obtained from poultry wastewater were selected: C1 = 6782 mg/L, C2 = 5087 mg/L, C3 = 3391 mg/L and C4 = 678 mg/L. These concentrations were used to investigate the adsorption behavior of purified smectite (Sm) and CTAB-modified variants (1CEC and 2CEC).

Fig. 8 shows the effect of initial protein concentration as well as the contact time on protein adsorption capacity. As shown, the protein adsorption occurred rapidly during the first 60 minutes, followed by a more gradual increase until equilibrium was reached at approximately 240 minutes. At equilibrium, the adsorption capacities (mg/g) for Sm were: 1578 (C1), 1284 (C2), 889 (C3), and 157 (C4); for 1CEC exhibited enhanced values of 2746 (C1), 2260 (C2), 1573 (C3), and 284 (C4); while for 2CEC achieved the highest capacities of 3451 (C1), 2738 (C2), 1925 (C3), and 325 (C3). The protein adsorption capacity increased with the increase of initial protein concentration until equilibrium was reached. The obtained results indicate that all tested smectite exhibited high adsorption capacities at an initial protein concentration of C1 = 6782 mg/L. This suggests that the adsorption sites on the clay surfaces became saturated at this concentration (Ben Jmaa et al., 2025), highlighting the efficiency of the clays in capturing proteins from poultry wastewater under the tested conditions. Rahman et al. (2024c) reported that chitosan coated bentonite clay nanosorbents effectively removed Ni2+ and Eosin Y from industrial wastewater, exhibiting adsorption capacities of 186.42 mg/g and 238.37 mg/g, respectively at low concentration (10–30 ppm), highlighting the strong affinity of the composite toward divalent metal ions, whereas Rahman et al. (2023b), demonstrated that a chitosan-modified coal nanocomposite achieved enhanced adsorption of pb²⁺ and crystal violet(CV) at low pollutant concentrations (20 ppm-40 ppm). Yakkerimath et al. (2024), who found that Cr⁶⁺ removal from effluent using iron-rich clay minerals was strongly dependent on initial protein concentration.

Fig. 8.

Effect of contact time on the protein adsorption capacity using 0.01 g of sorbent; (a) Sm, (b) 1CEC and (c) 2CEC at pH=9.

The superior performance of 2CEC highlights the effectiveness of CTAB modification at a 2:1 CEC ratio, likely due to increased hydrophobicity and expanded interlayer spacing as confirmed by FTIR and XRD analysis, respectively. The observed relationship between initial solute concentration and adsorption capacity reflects enhanced mass transfer and greater probability of interaction between protein molecules and active sites on the clay surface (Yahya et al., 2021). Similar trends have been reported in previous studies. Amor et al. (2023) reported that Pb(II) and Cu(II) removal efficiencies by geopolymers increased over time, reaching 52 mg/g and 79 mg/g, respectively, at 120 minutes. Similarly, Gulen and Demircivi, (2020) demonstrated time-dependent adsorption of ciprofloxacin, achieving a maximum of 13.64 mg/g within one hour.

Sorbent dose effect

The effect of sorbent dosage on protein removal efficiency was systematically investigated by varying the sorbent mass from 0.01 g to 0.04 g in 20 mL of poultry wastewater over a 4-hour contact period. Fig. 9 shows the protein sorption capacity per unit mass of Sm, 1CEC and 2CEC. The obtained results revealed a pronounced decrease of protein sorption capacity with increasing sorbent dose for all samples. In fact, as the sorbent weight increased from 0.01 g to 0.04 g, the protein sorption capacity declined from 994 mg/g to 137 mg/g for Sm, from 1171 mg/g to 640 mg/g for 1 CEC, and from 1222 mg/g to 800 mg/g for 2CEC. This inverse relationship between sorbent dosage and adsorption capacity can be attributed to the progressive saturation of available active sites. In addition, the protein removal efficiency increased linearly with increasing sorbent dose from 0.01 g to 0.04 g for all smectite variants. Increasing the sorbent weight from 0.01g to 0.04 g resulted in protein removal efficiencies rising from 13% to 53% for Sm, from 22% to 85% for 1CEC and from 26% to 97% for 2CEC.

Fig. 9.

Effect of sorbent dose on (a) protein removal efficiency and (b) protein adsorption capacity of smectite samples at pH = 9, time = 4 h.

This increase in protein removal efficiency could be attributed to the availability of unsaturated sites available in the smectite surfaces. At higher sorbent loadings, the number of available binding sites exceeds the quantity of protein molecules present in solution, leading to incomplete surface coverage and reduced protein uptake per unit mass of sorbent. Such behavior is commonly observed in adsorption systems and reflects diminished surface loading due to sorbent excess.

Kinetic studies

In general, adsorption refers to the transfer of surfactant molecules from the bulk phase to the solid–liquid interface, a process that becomes particularly complex in porous materials such as smectite due to their layered structure, heterogeneous charge distribution, and diverse surface reactive sites (Moslemizadeh et al., 2016). A comprehensive understanding of the adsorption behavior of smectite-based materials requires the application of kinetic and isotherm models to define the governing mechanisms.

In this study, adsorption kinetics were analyzed using pseudo-first-order, pseudo-second-order and intraparticle diffusion models as summarized in Table 3, with the linear plots in Supplementary material 3. The results showed that the pseudo-second-order model provided the best fit to the experimental for all investigated materials (Sm, 1CEC, and 2CEC), yielding an excellent correlation coefficient (R² = 0.999), compared to that of pseudo-first-order, proving that the clay smectite removed the protein from poultry wastewater with chemical bonds by iions exchange called chemisorption. The 2CEC- modified smectite exhibited enhanced adsorption performance, reflecting its greater surface reactivity and improved structural accessibility.

These findings highlight the importance of smectite swelling behavior, which promotes molecular diffusion and facilitates protein entrapment both within the interlayer spaces and on external surfaces. The obtained results are consistent with previous studies. For instance, Jean Baptiste et al. (2020) reported efficient adsorption of pigments and free fatty acid using acid-activated Cameroonian smectite, while Muslim et al. (2024) observed similar pseudo-second- order kinetic behavior (R² = 0.9996) during the adsorption of Cs-137 radioactive ions onto bentonite clays.

To further investigate the mass transport mechanism, the intraparticle diffucion kinectic model was employed to evaluate the transport behavior of protein within the porous structure of the adsorbents. The model parameters, namely the intraparticle diffusion rate constant (K) and the intercept (C), were determined from linear plots (Fig. 10b) and are reported in Table 3 for 1CEC, 2CEC and Sm. The results revealed a decrease in the diffusion rate constant (K) accompanied by an increase in the intercept (C) values for all initial protein concentration C1, C2, C3 and C4. The reduction in K values indicates a weakened contribution of intraparticle diffusion to the overall adsorption process, whereas the increase in C indicates a stronger influence of surface adsorption and boundary layer effects. Higher intercept values are typically associated with greater resistance to mass transfer at the external surface of the adsorbent.

Fig. 10.

Reusability study of smectite samples to remove proteins from poultry wastewater after eight cycles of adsorption at optimal conditions (pH = 9, t = 4 h, sorbent weight = 0.01 g, initial protein concentration = 6782 mg/l).

Table 3.

Kinetic rate parameters of the adsorption of protein onto Sm, 1CEC and 2CEC.

	Initial protein concentration (mg/L)
1CEC		C1	C2	C3	C4
Pseudo-first-order	Qe1’ (mg.g−1) R2 K1’	1382 0.9963 0.9946	1133 0.7388 0.9712	735 0.6179 0.953	136 0.9228 0.5215
Pseudo- second-order	Qe2’ K2’ R2’	3333 0.001 0.9999	2500 0.0008 0.999	1666 0.0012 0.9982	303 0.0077 0.9967
Intraparticle diffusion	K	450.4	484.31	348.12	67.55
	C	1798.4	1053.9	705.64	138.38
	R2	0.8639	0.8277	0.8241	0.8448
2CEC		C1	C2	C3	C4
Pseudo-first-order	Qe1 (mg.g−1) R2 K1	2442 0.9185 1.1965	895 0.9276 0.388	712 0.9153 0.3628	85 0.9056 0.2365
Pseudo- second-order	Qe2 K2’ R2	3333 0.0128 0.9965	2500 0.016 0.9966	1666 0.0018 0.9983	322 0.024 0.9956
Intraparticle diffusion	K	565.68	437.39	379.79	42.797
	C	2092.1	1539.1	890.47	234.4
	R2	0.9492	0.9483	0.9115	0.9661

Sm		C1	C2	C3	C4
Pseudo-first-order	Qe1’ (mg.g−1) R2 K1’	711 0.7753 1.0098	434 0.7738 0.8784	304 0.7594 0.7959	65 0.7164 0.4083
Pseudo- second-order	Qe2’ K2’ R2’	1666 0.0018 0.9912	1250 0.002 0.9944	909 0.003 0.9912	172 0.018 0.9956
Intraparticle diffusion	K	350.4	244.29	179.39	37.391
	C	872.09	691.76	431.18	87.702
	R2	0.6257	0.645	0.644	0.7748

Consequently, these findings imply that the diffusion of protein from the external surface of the Tunisian clay into the internal pore structure occurs at a relatively slow rate, with surface adsorption playing a dominant role in the adsorption mechanism.

Isotherm study

To evaluate the protein adsorption behavior onto purified and CTAB-modified smectite surfaces, the experimental equilibrium data were analyzed using the Langmuir and Freundlich adsorption isotherm models. All calculated parameters are summarized in Table 4, Table 5. As presented, the Langmuir separation factor values were all less than 1, indicating the favorable nature of protein adsorption over the investigated concentration range. Comparative isotherm analysis revealed that the Freundlich model provided a best fit to the experimental data, with correlation coefficients of 0.9996, 0.9984, and 0.9957 for Sm, 1CEC, and 2CEC, respectively, compared to that of Langmuir isotherm model. These high R² values suggest that protein adsorption process occurs on a heterogeneous surface, involving sites with varying affinities and multi-layer sorption behavior. The Freundlich intensity parameter (n) fell within the favorable range (0 < n <10), further confirming the high affinity and effective retention of protein on both unmodified and CTAB-modified smectite surfaces.

Table 4.

Adsorption isotherms constants for protein onto Sm, 1CEC and 2CEC.

		Sm	1CEC	2CEC
Langmuir	Qmax (mg. g−1)	5000	1666	2500
	R2	0.9745	0.9079	0.9756
	KL’	0,00012	0.0015	0.0076
Freundlich	n	1.129	1.064	1.116
	Kf	1.145	3.471	26.335
	R2	0.9996	0.9984	0.9957

Table 5.

Equilibrium parameter (R_L) values.

	C1	C2	C3	C4
Sm	0.35	0.42	0.44	0.48
1CEC	0.07	0.15	0.20	0.38
2CEC	0.018	0.04	0.06	0.20

Consistent observations have been reported in the literature. Yuan and Lu (2024) demonstrated that magnetic chitosan-modified clay exhibited efficient Cr (VI) adsorption, with freundlich isotherm model, indicating heterogonous adsorption. In contrast, Rasilingwani et al. (2024) demonstrated that the Congo red dye removal using metal oxide-clay nanocomposites was better represented by the Langmuir model (R² = 0.96) than by Freundlich isotherm model (R² = 0.92), suggesting predominant monolayer adsorption under their experimental conditions.. Mundkur et al. (2022) also reported that alginate modified clay exhibited effective removal of methylene blue, with the adsorption behavior following monolayer adsorption mechanism, consistent with the Langmuir isotherm model.

Together, these findings highlight that adsorption mechanisms are strongly dependent on the nature of the adsorbent-adsorbate system and operating conditions, with heterogeneous multilayer adsorption dominating in protein smectite interactions, while monolayer adsorption may prevail in other pollutant systems.

Structural characterization of smectite after adsorption

After the use of the three-smectite variants, FTIR and SEM characterization (Supplementary materials 4 and 5, respectively) revealed differences between the material before and after adsorption. In fcat, based on the FTIR spectra, a noticeable decrease and shrinking in the intensity of all characteristic clay absorption bands. This behavior indicates that electrostatic interactions between the clay surface and the adsorbate species were progressively weakened, likely due to the partial saturation, depletion, or alteration of active adsorption sites after eight successive reuse cycles. In addition, after adsorption, the SEM micrographs of the 2CEC sample showed marked modifications in the adsorption sites on the smectite surface, but it remained porous and able to proceed for a new adsorption cycle.

Mechanism

In general, the primary mechanisms governing protein adsorption onto smectite surfaces include electrostatic interactions with surface hydroxyl groups and ion exchange processes at sites of constant charge. In aqueous medium, protein molecules exist as polyanions due to the deprotonation of carboxylate group (-COO⁻). The CTAB-modified smectite provides a positively charged interlayer environment through quaternary ammonium head groups, with bromide ions acting as a mobile counter-ions. Upon contact with protein-containing wastewater, the negatively charged protein molecules diffuse toward the organoclay surface and progressively replace bromide ions via an anion-exchange mechanism. Yahya et al. (2021) demonstrated that anion removal in similar systems predominantly occurs via anion exchange.. These bromide ions, embedded within the interlayer space through hydrophobic interactions with CTAB molecules, serve as exchangeable counter-ions. This substitution is favored due to the higher charge density, multivalent nature, and stronger electrostatic stabilization of protein anions compared to monovalent Br⁻ ions. The expanded interlayer spacing induced by CTAB facilities protein penetration, while electrostatic attraction constitutes the dominant driving force of adsorption. As illustrated in Supplementary material 6, the proposed mechanism confirmed the results obtained by kinetic and isotherm studies, which leads to a multilayer adsorption mechanism via ion exchange with chemical bonds.

Reuse study

Reusability is a key criterion in assessing the performance of metal-ion sorption processes. The capacity to regenerate and reuse biosorbents provides substantial economic advantages and reduces energy consumption, thereby enhancing the overall sustainability of adsorption. Adsorption emerges as a more cost-effective and environmentally friendly alternative to conventional wastewater treatment technologies. Fig. 10 presents the results of the reusability test for 2CEC, 1CEC and Sm samples, evaluated over eight consecutive cycles for the removal of proteins from poultry slaughterhouse wastewater. The data revealed a remarquable reduction in adsorption capacity as the number of cycles increased for the Sm clay sample. This decrease was observed over three successive cycles, during which the sorption capacity declined from 1548 mg/g to 925 mg/g. For 2CEC and 1CEC clay samples, there is a slight reduction in the adsorption capacity over seven cycles of reuse, indicating strong reusability potential. This slight decline (3251 mg/g to 3249 mg/g and 2260 mg/g to 2230 mg/g, for 2CEC and 1CEC, respectively indicates that the modified smectite clay samples retained good stability and adsorption efficiency, enabling their effective reuse for up to eight adsorption-desorption cycles before a significant loss in performance was observed. This slight decline in sorption performance could be attributed to the slight material loss of the clay samples or structural changes during the regeneration process (Abdelrahman et al., 2025). Overall, the findings demonstrated that the CTAB- modified smectite samples maintained satisfactory performance and could be effectively reused for up to eight consecutive cycles in the removal of proteins from poultry wastewater. Santhana et al. (2012) reported that the modified cellulose-clay composite could be reused up to ten cycles of adsorption-desorption to remove chromium from industrial wastewater. After seven adsorption-desorption cycles, the repeated use of the clay samples led to a significant decline in adsorption performance. This reduction could be attributed to weakened interaction between the adsorbent and the adsorbate, resulting from the progressive non- availability of active adsorption sites in proteins. Basha et al. (2025) reported that the MC (600) nanocomposites could be reused for only up to five consecutives adsorption cycles to remove the brilliant green dye from wastewater.

Comparison to other sorbents

In this study, chemical treatment using CTAB was investigated as an innovative and an environmentally friendly to modify clays and enhance their capacity for adsorption of proteins from poultry wastewater. The obtained results were compared to those reported for various chemically treated natural clays. A summary of different treatment methods and their corresponding adsorption capacities is presented in Table 6. The results showed that modified smectite clay powder produced a highly and pronounced adsorbed materials, with notable removal capacities of 3451 mg/g for 2CEC and 2260 mg/g for 1CEC, compared to raw smcetite (Sm) with 1548 mg/g for most of the investigated adsorbate. These findings highlight the significant role of the CTAB in enhancing the structural properties of the smectite clays and consequently, their effectiveness in capturing pollutants from wastewater.

Table 6.

Comparison of CTAB-modified smectite clay to other sorbents in the literature.

Adsorbent	Adsorbate	Removal Capacity (mg/g)	Mode of adsorption	Experimental Conditions	References
Thermally modified smectite clay	Reactive Red 198	3.32	Chemisorption	Sorbent dose = 50 g [initial concentration] = 1-1000 mg/g	Pajak et al. 2021
H2SO4-modified Smectite		10.32
NaOH-modified Smectite		5.06
Raw Smectite		2.92
Graphene oxide–cellulose nanocrystal (GO–CNC) nano-filter composite membrane	Cr³⁺, Co²⁺, Ni²⁺, Pb²⁺, Cd²⁺, Methylene blue	∼99% removal	Electrostatic interactions (implied)	-	Sheikh et al. 2023
Tetraethylenepentamine-based nanocomposites	Heavy metals (HMs)	High removal	Chemisorption monolayer	-	El Messaoudi et al. 2024
Crystalline nanocellulose-activated char (CNC–AC) nanocomposite	Pb2+	538.91 mg/g	Physisorption Chemisorption	-	Rahman et al. 2024b
	CR dye	455.70 mg/g
Nanocrystals Based on Cellulose (CNC/Eg-A)	Brilliant Crystel Blue	98 mg/g	Chemisorption / monolayer	pH = 8 [initial conc] = 20 mg/L;	Chabane and Bouras 2025
	Titanium Yellow	44 mg/g		pH = 2 [initial conc] = 20 mg/L;
Pb2(CrO4)O/MgO/MgCrO4/C nanocomposites (MP600)	Malachite green dye	425.53 mg/g	Chemisorption; Langmuir	pH = 10, t = 50 min, [malachite dye] = 50 mg/l	Abdelrahman et al. 2025
Pb2(CrO4)O/MgO/MgCrO4/C nanocomposites (MP800)		362.32 mg/g		pH = 10, t = 70 min, [malachite dye] = 50 mg/l
MgO based chromate nanocomposite at 600°C (MC600)	Brilliant green dye	246.91 mg/g	Physisorption Monolayer	pH = 10, t = 50 min, [green dye] = 50 mg/l	Basha et al. 2025
MgO based chromate nanocomposite at 800°C (MC800)		229.89 mg/g		pH = 10, t = 70 min, [green dye] = 50 mg/l
Chitosan-modified coal nanocomposites	Crystal Violet	238.51 mg/g	Chemisorption / Monolayer	[initial concentration] = 20 ppm -40 ppm Sorbent dose = 1 g and 2 g	Rahman et al. 2023b
	Pb2+	243.08 mg/g
CTAB-modified smectite	Proteins	3251 mg/g	Chemisorption / Multilayer	pH = 9 sorbent dose= 10 mg [initial concentration] = 6782 mg/l t = 240 min	This study

When compared with other sorbents reported in the literature, the CTAB- modified smectite powder showed the highest adsorption performance for clay-protein removal, with a capacity of 3251 mg/g. This value markedly exceeds those reported for other materials, including modified clay MC600 (246 mg/g) and MC 800 (229.89 mg/g) used for removing brilliant green dye removal (Abdelrahmen et al., 2025),Cellulose nanocrystals (CNC), which showed adsorption capacities of 98 mg/g for brilliant blue and 44 mg/g for titanium yellow (Chaabane and Bouras, 2025), and chitosan modified coal nanocomposites, with capacities of 238.51 mg/g for crystal blue and 243.08 mg/g for Pb²⁺ (Rahman et al., 2023b). In addition, modified smectite clay-based adsorbents, including thermally-treated (3.32 mg/g), H₂SO₄-treated (10.32 mg/g) and NaOH-treated (5.06 mg/g) clays, showed considerably lower adsorption capacities of reactive red 198 a (Pajak, 2021).

Regardless of the type of pollutants or adsorbents reported in the literature, CTAB modification consistently proved to be the most effective method for enhancing sorbent performance.

Conclusions

In conclusion, smectite exhibits significant potential as an effective protein sorbent for the treatment of poultry slaughterhouse wastewater. Successful intercalation of the cationic surfactant CTAB into smectite structure was confirmed through comprehensive physicochemical analyses. Among the investigated materials, 2CEC demonstrated the highest performance, achieving an optimal protein removal concentration of approximately 6782 mg/L and a maximum protein adsorption capacity of about 3251 mg/g after 4 hours at pH = 9.0. FTIR analysis revealed enhanced hydrophobicity and strong interactions with surface hydroxyl (-OH) groups for 2CEC compared to 1CEC, which indicated to its superior protein sorption capacity. Adsorption kinetic and isotherm modelling indicated that protein uptake on 2CEC proceeds via a combined chemisorption and multilayer adsorption mechanism, consistent with heterogeneous surface interactions. The modified smectite clay could be reused for up to eight cycles, compared to the purified clay, owing to its enhanced structural stability These findings highlight the effectiveness of CTAB-modified smectite, particularly at a 2:1 CEC ratio, as a promising and sustainable adsorbent for protein wastewater treatment. Owing to the natural mineral origin and low toxicity of smectite, controlled landfill disposal may be considered acceptable, provided that leaching tests confirm compliance with environmental regulations. The strong adsorption affinity abserved for 2CEC further supports its suitability for disposal without significant risk of contaminant release. Thermal treatment of protein –loaded smectite may be applied to decompose organic matter, after which the residual mineral phase could be potentially reused as a filler or safely disposed of. Future investigations should focus on dynamic adsorption conditions to further elucidate the operational performance and scalability of modified smectite systems for protein recovery and wastewater remediation.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Funding

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2601).

Declaration of generative AI and AI-assisted technologies in the writing process

No new scientific content was created using AI. OpenAI’s ChatGPT was used solely to improve the clarity of the manuscript. All content was thoroughly reviewed by the authors, who take full responsibility for the final version of the study.

CRediT authorship contribution statement

Nermine Sayah: Writing – original draft, Methodology, Conceptualization. Senda Ben Jmaa: Investigation, Data curation. Kawthar Yahya: Investigation, Data curation. Hamdi Bendif: Writing – review & editing, Validation, Supervision. Walid Elfalleh: Writing – review & editing, Validation, Supervision. Noureddine Hamdi: Writing – review & editing, Validation, Supervision. Mourad Jridi: Writing – review & editing, Validation, Supervision.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.