HAp@Cell bio-films engineered from local resources involving molecular mechanisms of dye adsorption and antibacterial activity

Abstract

This work describes the eco-friendly synthesis and multifunctional performance of hydroxyapatite/cellulose (HAp@Cell) bio-films prepared from Moroccan natural phosphate and cotton-derived cellulose through a solvent-free, low-temperature (< 100 °C) route. Structural (XRD) and morphological (SEM) analyses confirmed nanocrystalline HAp homogeneously dispersed in a semi-crystalline cellulose matrix, creating a rough and porous network favorable for molecular interactions. The optimized film (PC5) exhibited outstanding adsorption toward methylene blue (qₑ,max ≈ 85 mg g⁻¹) and natural indigo (~ 95% removal). Kinetic data fitted the pseudo-second-order model, while the Freundlich isotherm best described multilayer adsorption, indicating a heterogeneous surface with high affinity for cationic dyes. At the molecular scale, non-covalent interactions such as hydrogen bonding, electrostatic attraction, and π–π coupling between dye molecules and surface hydroxyl/phosphate groups dominate the adsorption process. The same surface chemistry governs the antibacterial mechanism, where the controlled release of Ca²⁺ and PO₄³⁻ ions combined with electrostatic contact destabilizes bacterial membranes, producing inhibition zones of 25 mm for S. aureus and 20 mm for E. coli. The films maintain over 85% adsorption efficiency after five reuse cycles, demonstrating high stability and regeneration potential. These results position the HAp@Cell composite as a sustainable, dual-function material for wastewater decolorization and antimicrobial protection within Morocco’s eco-circular strategy.

Introduction

The development of advanced biomaterials that are simultaneously biocompatible, antibacterial, mechanically robust, and environmentally sustainable remains a pressing challenge in both biomedical engineering and environmental science¹. Conventional polymer-based composites and synthetic bio-films have found widespread applications in implants, wound dressings, and wastewater treatment systems². However, these materials are frequently limited by their poor biocompatibility, insufficient antibacterial properties, and ecological concerns arising from non-biodegradability and toxic by-products³. Addressing these shortcomings requires the design of multifunctional, eco-friendly composites derived from renewable natural resources⁴.

Hydroxyapatite (HAp), a calcium phosphate mineral structurally analogous to the inorganic matrix of human bone, has long been recognized as one of the most promising biomaterials for medical applications⁵. Its intrinsic osteoconductivity promotes bone cell adhesion and proliferation, while its excellent biocompatibility minimizes adverse immune responses⁶. Moreover, HAp can exert an antibacterial effect through the controlled release of calcium and phosphate ions, which disrupt microbial cell membranes and metabolic pathways, thereby reducing the risk of post-implant infections⁷. These features have led to its widespread use in bone graft substitutes, coatings for orthopedic and dental implants, and as a scaffold material in tissue engineering⁸. However, despite these advantages, bulk or pure HAp is inherently brittle, with low tensile and flexural strength, which severely limits its applicability in load-bearing or flexible biomedical devices⁹. Its poor toughness and susceptibility to microcracking under stress also hinder its processing into thin, stable films or adaptable coatings¹⁰. This mechanical limitation necessitates its combination with polymeric or biopolymeric matrices to enhance flexibility, durability, and practical usability in advanced biomedical and environmental systems¹¹.

Cellulose, the most abundant renewable biopolymer on Earth, is widely valued for its versatility and eco-friendliness¹². Its molecular structure, composed of linear chains of β(1→4)-linked D-glucose units, imparts remarkable mechanical flexibility and enables the formation of stable hydrogen-bonded networks¹³. This makes cellulose an ideal candidate for producing biodegradable materials with low environmental impact. In addition to its mechanical adaptability, cellulose exhibits high chemical stability, thermal resistance, and the capacity to form porous matrices with large surface areas, which are advantageous for adsorption processes and as support structures in composite systems¹⁴. These features explain its extensive use in packaging, filtration membranes, and biomedical scaffolds¹⁵. However, cellulose lacks intrinsic antibacterial activity and, when used alone, often struggles to generate stable, homogeneous bio-films capable of maintaining their integrity under physiological or industrial conditions¹⁶. Its hydrophilicity may also lead to rapid degradation in aqueous environments, limiting its long-term performance¹⁷. Consequently, combining cellulose with bioactive inorganic fillers such as hydroxyapatite (HAp) emerges as a promising strategy: while cellulose provides flexibility, matrix stability, and eco-sustainability, HAp introduces antibacterial, osteoconductive, and mechanical reinforcement properties¹⁸. This synergy enables the design of multifunctional composites that overcome the shortcomings of each material when used individually¹⁹.

Recent studies have attempted to combine calcium phosphate minerals with natural or synthetic polymers; however, existing approaches often suffer from limitations such as rapid degradation, weak antibacterial effects, and reliance on environmentally unfriendly synthesis pathways²⁰. There remains a critical need for biofilm materials that are not only effective in biomedical contexts but also versatile for environmental remediation especially in water purification, where organic dyes, pharmaceutical residues, and heavy metals present growing global threats^21,22.

The central objective of this research is to develop and validate a new class of multifunctional bio-film composites that simultaneously meet biomedical and environmental demands. Specifically, our goal is to engineer hydroxyapatite/cellulose (HAp/Cell) films that combine the osteoconductivity, antibacterial ion release, and biocompatibility of HAp with the flexibility, chemical stability, and porous adsorption capacity of cellulose, thereby overcoming the limitations of each material when used alone. To achieve this, we designed a scalable and eco-friendly synthesis route based on natural Moroccan phosphate and cotton-derived cellulose, enabling precise control over composite ratios to tailor material properties. The study seeks to demonstrate that these bio-films not only exhibit enhanced antibacterial activity against clinically relevant pathogens (E. coli and S. aureus), but also achieve high-efficiency removal of organic dyes from contaminated water, with proven durability across multiple regeneration cycles. Ultimately, this work aims to position HAp/Cell bio-films as sustainable, dual-purpose materials capable of addressing two pressing global challenges healthcare-associated infections and water pollution while promoting the use of renewable resources and environmentally responsible design.

Materials and methods

Materials

Natural phosphate ore was obtained from Moroccan deposits (Office Chérifien des Phosphates, Morocco) and used as the precursor for hydroxyapatite (HAp) synthesis. Analytical-grade nitric acid (HNO₃, 65%), sodium hydroxide (NaOH, ≥ 98%), and urea (≥ 99%) were obtained from Sigma-Aldrich (Merck, Germany), while aqueous ammonia solution (< 30%) was supplied by Fisher Scientific (Waltham, MA, USA). Sodium hypochlorite (NaClO, 12% solution) was purchased from Carlo Erba Reagents (Milan, Italy), and hydrogen peroxide (H₂O₂, 30% w/v) from VWR Chemicals (Radnor, PA, USA). Methylene blue (analytical grade, Sigma-Aldrich) was used as a model dye for adsorption experiments.

Methods

Preparation of hydroxyapatite (HAp)

Hydroxyapatite was synthesized from Moroccan natural phosphate through an acid–base precipitation method designed to mimic conventional wet-chemical synthesis while valorizing a local mineral resource. In a typical procedure, raw phosphate ore was first finely ground and gradually dissolved in concentrated nitric acid (HNO₃, 65%) under continuous stirring, maintaining the pH 2 to ensure efficient release of calcium and phosphate ions into solution. The suspension was subjected to vacuum filtration to eliminate insoluble residues such as silicates and trace metal oxides. The clear filtrate containing dissolved calcium and phosphate species was then slowly neutralized using aqueous ammonia solution (< 30% NH₃) under vigorous stirring, which induced the controlled precipitation of hydroxyapatite crystals. Precipitation was allowed to proceed until complete pH stabilization, ensuring maximum yield. The resulting white precipitate was recovered by a second vacuum filtration, thoroughly washed with deionized water to remove residual nitrates and ammonium ions, and dried overnight at 100 °C to obtain a stable crystalline powder. Finally, the dried HAp was finely ground in a ceramic mortar to produce a homogeneous, micron-sized powder suitable for subsequent composite film preparation.

Extraction and purification of cellulose

Raw cotton fibers were purified through a two-step chemical treatment to obtain cellulose suitable for composite film preparation. In the first step, the fibers were subjected to alkaline boiling in sodium hydroxide (NaOH, 5 wt%) at 90 °C for 2 h under constant stirring. This treatment is known to effectively remove surface impurities such as waxes, oils, pectins, and hemicelluloses, yielding a cellulose-rich fraction. The treated fibers were then thoroughly washed with deionized water until neutral pH was reached to eliminate residual alkali.

In the second step, bleaching was performed using hydrogen peroxide (H₂O₂, 30%) at 90 °C for 60 min in order to further reduce residual lignin and colored impurities. Hydrogen peroxide bleaching selectively oxidizes lignin chromophoric structures and aromatic groups, thereby enhancing fiber whiteness and apparent cellulose purity, as widely reported in the literature. It should be noted that this bleaching step was intended to reduce residual lignin rather than ensure complete lignin removal, and no direct quantitative lignin determination was performed in this study.

After bleaching, the fibers were rinsed repeatedly with deionized water to remove residual oxidizing agents and salts, followed by drying at 70 °C. The resulting purified cellulose exhibited high whiteness and chemical stability and was suitable for subsequent dissolution in the NaOH/urea system and composite film preparation.

Preparation of HAp/Cellulose bio-films

Purified cellulose was dissolved in a NaOH/urea aqueous solution (typically 7 wt% NaOH and 12 wt% urea) that had been pre-cooled to − 12 °C in order to enhance cellulose solubilization. At this low temperature, the solvent system disrupted the extensive hydrogen bonding network within cellulose chains, thereby facilitating dissolution and minimizing re-aggregation of polymer chains. Hydroxyapatite (HAp) powder, previously prepared, was added directly into the cellulose solution at defined molar ratios to produce uniform dispersions. Three compositions were investigated, corresponding to HAp-to-cellulose ratios of 1:2, 1:1, and 2:1 (designated PC1:2, PC1:1, and PC2:1, respectively). The PC1:1 formulation, corresponding to an equimolar HAp-to-cellulose ratio, is hereafter also referred to as PC5, as it was identified as the optimized composition based on its structural and functional performance.

For comparison, control films of pure cellulose (C0) and pure HAp (P0) were also prepared following the same procedure (Fig. 1). The resulting viscous gels were stirred until complete homogenization, then poured into Petri dishes and cast into thin films. After drying at 40 °C under controlled conditions, rigid and homogeneous bio-films were obtained, suitable for subsequent characterization and performance testing.

Fig. 1

Schematic representation of the preparation process of cellulose/hydroxyapatite (HAp) bio-films.

Characterization methods

Chemical composition analysis

The chemical composition of the fibers before and after alkaline treatment was determined using standardized wet chemical analysis procedures commonly applied for lignocellulosic biomass characterization. Prior to analysis, extractives such as waxes and oils were removed by solvent extraction. Hemicellulose and pectin were subsequently solubilized under alkaline conditions, while the remaining insoluble fraction was considered as cellulose-rich material.

The contents of cellulose, hemicellulose, pectin, and waxes/oils were quantified by gravimetric determination based on mass balance calculations before and after each selective extraction step. All measurements were carried out at least in triplicate, and the results are reported as mean values ± standard deviation. This analysis was used to quantitatively evaluate the effectiveness of the alkaline treatment in removing non-cellulosic components from the fibers.

X-ray diffraction (XRD)

The crystalline structure of hydroxyapatite, cellulose, and their composites was analyzed using an X-ray diffractometer (Bruker D8 Advance, Germany) equipped with Cu Kα radiation (λ = 1.5406 Å), operating at 40 kV and 30 mA. Data were collected over a 2θ range of 10–70° with a step size of 0.02° and a scanning speed of 2°/min. The obtained diffraction patterns were compared with reference data from the Joint Committee on Powder Diffraction Standards (JCPDS) to confirm phase purity and crystallinity.

In addition to phase identification, quantitative microstructural parameters were extracted from the XRD patterns to further characterize the structural evolution of pure cellulose (C0), pure hydroxyapatite (P0), and HAp–cellulose composite films. The average crystallite size (D) of hydroxyapatite domains was calculated using the Scherrer equation applied to the most intense diffraction peak:

$$\mathbf{D}=\frac{\varvec{K}\varvec{\lambda}}{\varvec{\beta}\varvec{c}\varvec{o}\varvec{s}\varvec{\theta}}$$

Where K is the shape factor (0.9), λ is the Cu Kα wavelength (1.5406 Å), β is the full width at half maximum (FWHM) in radians, and θ is the Bragg angle.

The lattice microstrain (ε), associated with crystal lattice distortions, was estimated using²³:

$$\varvec{\upepsilon}=\frac{\varvec{\beta}}{4\varvec{t}\varvec{a}\varvec{n}\varvec{\theta}}$$

The dislocation density (δ), which reflects the density of crystallographic defects, was calculated according to:

$$\varvec{\updelta}=\frac{1}{\varvec{D}\check{\rm S}}$$

The crystallinity index (CrI) of cellulose-containing samples was determined using the Segal method:

$$\mathbf{C}\mathbf{r}\mathbf{I}\left(\mathbf{\%}\right)=\frac{{\varvec{I}}_{200}-{\varvec{I}}_{\varvec{a}\varvec{m}}}{{\varvec{I}}_{200}}\times100$$

where I₍₂₀₀₎ corresponds to the intensity of the crystalline cellulose peak at 2θ ≈ 22°, and I₍am₎ represents the amorphous background intensity at 2θ ≈ 18°. For hydroxyapatite-rich samples, relative crystallinity was assessed by comparing the integrated area of crystalline peaks to the total diffracted area.

Thermogravimetric analysis (TGA)

Thermal stability and decomposition profiles were determined using a thermogravimetric analyzer (PerkinElmer STA 6000, USA). Approximately 10 mg of each dried sample was heated from room temperature to 800 °C at a rate of 10 °C/min under a nitrogen atmosphere. Weight loss curves were recorded to evaluate moisture content, organic decomposition, and the influence of HAp content on composite stability.

Antibacterial assay

Antibacterial activity was assessed by the agar disk diffusion method against Escherichia coli (ATCC 25922, Gram-negative) and Staphylococcus aureus (ATCC 25923, Gram-positive). Sterile circular films (10 mm diameter) were placed on inoculated agar plates and incubated at 37 °C for 24 h. Zones of inhibition (mm) were measured using a Vernier caliper. All tests were performed in triplicate, and mean values were reported.

Adsorption experiments

The adsorption capacity of the bio-films was evaluated using methylene blue (MB) dye as a model organic pollutant. All experiments were performed in batch mode at room temperature (25 ± 2 °C) under constant stirring (150 rpm). Circular bio-film samples (average dry mass 50 ± 5 mg, diameter ~ 1 cm) were immersed in 50 mL MB solutions with initial concentrations ranging from 10 to 50 mg/L. At predetermined time intervals (0, 5, 10, 20, 30, 60, 120 min), 3 mL aliquots were collected, filtered, and analyzed by UV–Vis spectrophotometry (Shimadzu UV-1800, Japan) at λ_max = 664 nm. The adsorption capacity at time t (qₜ, mg/g) and the removal efficiency (%) were calculated as:

The adsorption capacity of composites was calculated using Eqs. (1) and (2)²⁴:

$$\text{\%}\text{Removal}=\frac{{C}_{0}-{C}_{e}}{{C}_{0}}\cdot 100$$

(1)

$${Q}_{e}=\frac{{C}_{0}-{C}_{e}}{W}V$$

(2)

where C₀ denotes the beginning metal ion and C_e denotes the equilibrium methylene blue concentration in ppm. Q_e is the equilibrium adsorption capacity in ppm, W is the absorbent foam weight in mg, and V is the solution volume in L.

Isotherm of the adsorption process

The Langmuir and Freundlich isotherm models were followed in this study to conclude the adsorption behavior of methylene blue (MB) dye on bio film. The Langmuir isotherm model is presented in Eqs. (3) and (4)⁸:

$$\frac{{C}_{e}}{{Q}_{e}}=\frac{1}{{q}_{max}}{C}_{e}+\frac{1}{{q}_{max}{K}_{L}}$$

(3)

where C_e is the methylene blue (MB) dye concentration in ppm, Q_e is the amount of metal ion removed per unit mass of bio film at equilibrium (mg/g), q_max is the foam’s greatest single layer adsorption capacity (mg/g), and K_L (L/mg) is the Langmuir constant.

The Langmuir isotherm model can be used to predict whether adsorption will be favorable or unfavorable by utilizing the dimensionless constant separation factor given in Eq. (4)⁸.

$${\text{R}}_{\text{L}}=\frac{1}{1+{K}_{L}{C}_{e}}$$

(4)

where C₀ represents the initial (MB) and K_L represents the Langmuir constants. If the R_L value exceeds one, the adsorption is considered unfavorable; otherwise, it is considered favorable or linear if it is between one and one.

The heterogeneous surface energy of non-ideal adsorption process is represented by the Freundlich isotherm model indicated in Eqs. 5 and 6⁸.

$$\hbox{In}(q_e)=\hbox{In} k_f + \frac{1}{n}C_e$$

(5)

$$Qe=KFCe1/n$$

(6)

where 1/n represents the adsorption intensity and K_F denotes the relative adsorption capacity¹². Adsorption is advantageous if 1/n is between 0.1 and 0.5; it is unfavorable if 1/n.

Kinetics of the adsorption process

The pseudo first-order and second-order kinetic models, described below, were used to study the rates of MB adsorption on the surfaces of bio film. Equation 7 to 10 were employed to compute the linearized versions of the rate Eq.⁸.

$$\text{ln}({q}_{e}-{q}_{t})=\text{ln}{q}_{e}-{K}_{1}\text{t}$$

(7)

$$\frac{\text{t}}{{\text{q}}_{\text{t}}}=\frac{1}{{\text{K}}_{2}{q}_{e}^{2}}+\frac{\text{t}}{{\text{q}}_{\text{e}}}$$

(8)

$$Qt=Kidt1/2+Z$$

(9)

$$\text{ln}\frac{K (T_2)}{K(T_1)} = \frac{Ea}{R}\cdot \left(\frac{1}{T_1}- \frac{1}{T_2}\right)$$

(10)

where Q_t denotes temperature-dependent adsorption capacity and q_e denotes equilibrium adsorption capacity (mg/g).

K₁ represents the pseudo-first-order rate constant (min), and K₂ represents the pseudo-second-order rate constant (g/ mg. min). Z (mg/g) can be used to calculate the boundary layer thickness, where Kid is the diffusion rate constant measured in mg/g.min^1/2.

Reusability tests

To assess reusability, adsorption–desorption cycles were performed for five consecutive runs. After each adsorption test, films were desorbed in an ethanol-HCl solution (1:1 v/v), rinsed with deionized water, and dried at 50 °C before reuse. The efficiency of each cycle was calculated relative to the first adsorption run.

Computational modeling and analysis of adsorption mechanisms

Computational modeling techniques, particularly those using the Forcite module and semi-empirical methods, are powerful tools for elucidating adsorption mechanisms by providing molecular-level insight into interactions among bacteria, pollutants, and adsorbent materials such as activated carbon and hybrid composites. In this work, a sequential computational workflow combining geometry optimization and post-processing interaction analysis was adopted. The Forcite module of Materials Studio 6.0 was used to optimize the geometries of the HAp/Cell composite, bacterial systems (Staphylococcus aureus and Escherichia coli), and the model dyes (methylene blue and natural indigo), with ultra-fine settings to achieve precise energy minimization and identify the most stable adsorption configurations. In addition, DFT calculations were performed on methylene blue and natural indigo using the DMol³ module (DNP basis set, GGA–PBE functional) to analyze electronic properties, HOMO–LUMO distributions, global quantum chemical descriptors, and molecular electrostatic potential (ESP) maps. These results provided complementary information on charge distribution and non-covalent interaction sites, supporting and validating the adsorption mechanism on the HAp@Cellulose surface. Following geometry optimization, the optimized structures were exported in compatible coordinate formats and subsequently employed for post-processing analysis using the Multiwfn program. To complement these optimizations, the Reduced Density Gradient (RDG) and Non-Covalent Interaction (NCI) isosurfaces were calculated based on electron density analysis in Multiwfn, and visualized using Gnuplot and VMD visualization tools^25,26. These analyses enabled the identification and detailed characterization of weak intermolecular forces such as hydrogen bonding, van der Waals interactions, and steric effects that govern the adsorption behavior and interfacial stability of the investigated systems. This sequential workflow geometry optimization in Materials Studio followed by electron-density-based interaction analysis in Multiwfn has been widely adopted for the investigation of adsorption and interfacial phenomena in complex molecular systems. Overall, this integrated computational framework provides a comprehensive molecular-level perspective on the interaction mechanisms, adsorption dynamics, and structural stability of the HAp/Cell–bacteria/dye complexes.

Protein structure validation

We obtained the three-dimensional structures of the target proteins from the RCSB Protein Data Bank, using PDB IDs 1JIJ for Escherichia coli and 1KZN for Staphylococcus aureus. Before running molecular docking, we checked the stereochemical quality of the selected protein models by using PROCHECK and reviewing their Ramachandran plots. This validation step was carried out to confirm that the protein conformations used in the docking simulations were structurally reliable.

The stereochemical validation of the protein structures was performed using PROCHECK implemented in the SAVES v6.0 server (UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, USA)²⁷. Ramachandran plots were generated to evaluate backbone dihedral angles (φ and ψ) and to determine the percentage of residues located in favored, allowed, and disallowed regions²⁸. This validation step ensured the structural quality and suitability of the proteins for subsequent molecular docking studies.

Molecular docking

Prior to the docking simulations, the hydroxyapatite/cellulose complex were initially drawn and visualized using ChemDraw 16.0²⁹. The generated structures were subsequently subjected to geometry optimization using the MM2 force field in order to obtain energetically stable conformations^30,31.

Molecular docking studies were performed using the Molecular Operating Environment (MOE) software package³². The three-dimensional crystal structures of the target proteins were retrieved from the RCSB Protein Data Bank (RCSB-PDB)³³. Protein preparation involved removal of crystallographic water molecules, correction of missing side chains, when necessary, addition of hydrogen atoms, and protonation according to physiological conditions. We added hydrogen atoms to the ligands and then gave them their partial atomic charges. We first used the MMFF94x force field to minimize the energy, which helped us make sure things were stable before we started docking. We then saved the best-performing ligands as MDB files, ready for the next step of docking^34,35.

To get the receptors ready, we put in hydrogen atoms and then used the MOE Site Finder to locate the active site. This tool basically creates some spheres to give us a good idea of where the binding pocket is. We ran docking simulations by putting the ligands right inside the active site. We used the Triangle Matcher placement method to do this. We made a bunch of different shapes for each ligand and then picked the best-fitting ones, looking at which had the lowest binding energies³⁶. First, the London dG scoring function was used to get the initial ranking. After that, the best poses were made even better by minimizing their energy. We looked at how the ligand and protein stuck together, checking things like how much energy it took to bind, any hydrogen bonds, watery bits that touched, and electrical pushes and pulls. We made some maps and pictures of the surface to really see how it all fits in the catalytic pocket^37,38.

In this study, particular attention was given to the hydroxyapatite/cellulose complex exhibiting the highest antibacterial activity, with the objective of elucidating its potential inhibitory mechanism against the selected bacterial targets.

Results and discussion

Chemical composition analysis

The chemical composition analysis provides direct quantitative evidence of the effectiveness of the alkaline treatment in removing non-cellulosic components from the fibers. As shown in Fig. 2; Table 1, a significant increase in cellulose content is observed after treatment, rising from 62.4 ± 1.2% in untreated fibers to 82.7 ± 1.0% in alkali-treated fibers. This increase reflects the enrichment of the cellulose phase resulting from the selective removal of amorphous constituents.

Fig. 2

Chemical composition of fibers before and after alkaline treatment.

Table 1 Chemical composition of fibers before and after alkaline treatment.

In contrast, substantial reductions are observed for hemicellulose, pectin, and waxes/oils following alkaline treatment. The hemicellulose content decreases from 18.6 ± 0.8% to 6.2 ± 0.5%, while the pectin content is reduced from 7.4 ± 0.4% to 2.1 ± 0.3%, indicating effective solubilization of these polysaccharides under alkaline conditions. Similarly, the waxes and oils content decreases markedly from 4.8 ± 0.3% to 0.9 ± 0.2%, demonstrating efficient removal of surface extractives.

The relatively small error bars associated with all components confirm the good reproducibility of the chemical composition analysis and the consistency of the alkaline treatment process. These quantitative results are in good agreement with the FTIR analysis, which shows attenuation or disappearance of absorption bands related to ester, acetyl, and carboxyl functional groups characteristic of hemicellulose and pectin.

The removal of non-cellulosic components exposes hydroxyl-rich cellulose domains and increases surface accessibility, which favors stronger interfacial interactions with hydroxyapatite in the composite films. This structural purification contributes directly to the enhanced adsorption capacity and antibacterial activity observed for the HAp–cellulose composites, establishing a clear relationship between chemical composition, structure, and functional performance.

FTIR analysis

Figure 3 presents the FTIR spectra of pure hydroxyapatite (P0), pure cellulose (C0), and the HAp/cellulose composite bio-film (PC5), allowing a direct comparison of the chemical structures and interactions between the components.

Fig. 3

FTIR spectra of pure hydroxyapatite (P0), pure cellulose (C0), and the HAp/cellulose composite bio-film (PC5).

The FTIR spectrum of pure cellulose (C0) is characterized by a broad absorption band in the region 3200–3500 cm⁻¹, attributed to the stretching vibrations of hydroxyl (–OH) groups associated with extensive intra- and intermolecular hydrogen bonding in cellulose chains. The band around ~ 2890 cm⁻¹ corresponds to C–H stretching vibrations of aliphatic groups, while the intense band in the 1050–1150 cm⁻¹ region is assigned to C–O–C and C–O stretching vibrations of the glycosidic backbone. These features are typical of native cellulose and confirm the polymeric nature of the C0 film.

The spectrum of pure hydroxyapatite (P0) shows characteristic phosphate (PO₄³⁻) vibrational modes. The strong bands observed at approximately ~ 1090 and ~ 960 cm⁻¹ are attributed to asymmetric and symmetric stretching vibrations of PO₄³⁻ groups, respectively, while the bands near ~ 602 and ~ 560 cm⁻¹ correspond to phosphate bending modes. A weak band around ~ 3570 cm⁻¹ is associated with structural hydroxyl groups of hydroxyapatite. These bands confirm the formation of crystalline hydroxyapatite with preserved phosphate structure.

For the HAp/cellulose composite film (PC5), the FTIR spectrum combines the main features of both cellulose and hydroxyapatite, confirming successful composite formation. Notably, the broad –OH band becomes attenuated and slightly shifted compared to pure cellulose, indicating strong hydrogen bonding interactions between cellulose hydroxyl groups and surface hydroxyl/phosphate groups of hydroxyapatite¹⁸. In addition, the phosphate bands of hydroxyapatite remain clearly visible in the composite, with modified intensities, suggesting good dispersion of HAp within the cellulose matrix rather than simple physical mixing. These spectral changes provide clear evidence of interfacial interactions between cellulose chains and hydroxyapatite particles, which are essential for the improved structural stability and adsorption performance observed for the composite bio-films²¹.

X-ray diffraction

The XRD diffractogram shows distinct patterns for pure hydroxyapatite (P0), cellulose (C0), and their composites (PC(2:1) and PC5), (Fig. 4).

Fig. 4

X-ray diffraction (XRD) patterns of pure cellulose (C0), hydroxyapatite (P0), and HAp–cellulose composite films with different ratios.

The P₀ pattern displays characteristic HAp reflections at 2θ ≈ 25.9° (002), 31.7° (211), 32.9° (300), and 34° (202), confirming the formation of a single hydroxyapatite phase with nanocrystalline structure. The broadness of these peaks indicates small crystallite domains and partial lattice disorder, which are typical of HAp derived from natural sources. In contrast, the C₀ sample exhibits a broad diffraction halo centered near 22°, characteristic of semi-crystalline cellulose, confirming the absence of mineral phases.

For the composite films PC(2:1) and PC5, both patterns display the main HAp peaks, demonstrating the preservation of its crystalline structure within the cellulose matrix. In PC(2:1), HAp peaks are sharp and intense, suggesting good crystallinity and efficient dispersion of HAp in the biopolymer framework. In PC5, however, the peaks become broader and less intense, and the amorphous background between 15–25° increases, reflecting smaller crystallite sizes and a higher degree of HAp dispersion or confinement within the cellulose network³⁹. This structural evolution highlights the synergistic integration between the inorganic and organic components, where cellulose limits crystal growth and enhances film uniformity.

These structural features have a direct influence on both adsorption and antibacterial activity. The reduced crystallite size and greater amorphous character in PC5 increase the specific surface area and expose more reactive hydroxyl and phosphate groups, improving the adsorption of pollutants and metal ions. Simultaneously, the intimate HAp–cellulose interface favors the gradual release of Ca²⁺ and PO₄³⁻ ions, which, as noted in the patent text, destabilize bacterial membranes and inhibit microbial metabolism²⁴. Thus, the XRD evidence of nanocrystalline, highly dispersed HAp within a porous cellulose matrix explains the composite’s dual performance enhanced adsorption efficiency due to larger active surface exposure, and potent antibacterial action driven by ionic exchange and surface reactivity⁴⁰.

• Microstructural parameters and crystallinity analysis.

The microstructural parameters derived from XRD analysis are summarized in Table 2. Clear differences in crystallite size, lattice strain, defect density, and crystallinity were observed between pure components and composite films.

Table 2 Microstructural parameters derived from XRD analysis.

Pure hydroxyapatite (P0) exhibits the largest crystallite size (≈ 42 nm) and the lowest microstrain, reflecting well-developed crystalline domains. In contrast, regenerated cellulose (C0) shows significantly smaller apparent crystallite size and higher microstrain, consistent with its semi-crystalline nature and the presence of a substantial amorphous fraction. For the composite films, a progressive refinement of crystallite size is observed as cellulose content increases. The PC(2:1) composite displays intermediate crystallite dimensions, while PC5 shows the smallest HAp crystallite size (≈ 24 nm), indicating that the cellulose matrix effectively restricts crystal growth and promotes nanoconfinement of hydroxyapatite particles. This confinement induces lattice distortion, as evidenced by the increased microstrain and dislocation density values in PC5.

The crystallinity index follows a similar trend. While pure hydroxyapatite exhibits the highest crystallinity, the incorporation of cellulose leads to a gradual decrease in crystallinity, particularly in PC5. This reduction is attributed to strong interfacial interactions between hydroxyapatite nanocrystals and cellulose chains, which disrupt long-range ordering and increase the amorphous fraction.

These structural modifications play a critical role in the functional behavior of the composite films. Reduced crystallite size, higher defect density, and partial amorphization increase surface reactivity, enhance ion exchange capacity, and expose more active sites. This explains the superior adsorption performance and antibacterial activity observed for PC5, establishing a clear structure property relationship.

• Hydroxyapatite/cellulose crystal structure.

Figure 5 illustrates the interfacial structure of the hydroxyapatite/cellulose composite, represented as a planar view of the crystallographic (001) face of hydroxyapatite. This model highlights the atomic organization and potential interactions between the organic matrix (cellulose) and the mineral phase (hydroxyapatite), which are essential to the stability and functional properties of the composite.

Cellulose forms a polymer chain rich in hydroxyl groups (–OH), which can form hydrogen bonds with oxygen ions and phosphate groups (PO₄³⁻) present on the surface of hydroxyapatite. These interfacial interactions promote strong adhesion between the two phases and contribute to better organo-mineral compatibility. The presence of calcium ions (Ca²⁺), coordinated by the oxygen atoms of the phosphate groups, also plays a key role in anchoring cellulose to the crystalline surface of hydroxyapatite.

The polyhedral representation of PO₄ units highlights the structural connectivity of the apatite network and the availability of active sites on the surface, facilitating interaction with cellulose. This organization suggests a preferential orientation of cellulose along the (001) surface, leading to a structured and energetically stable interface.

These structural characteristics give hydroxyapatite/cellulose composites properties that are particularly interesting for biomedical and environmental applications. The synergy between the rigidity of the mineral phase and the flexibility of the organic matrix not only mimics the structure and behavior of natural bone tissue, but also provides a surface rich in active sites (hydroxyl and phosphate groups) that are conducive to the adsorption of heavy metals. This organo-mineral architecture facilitates the interaction and fixation of toxic metal ions present in aqueous environments, making these composites promising materials for pollution control and water treatment applications.

Fig. 5

Hydroxyapatite/ cellulose structure planar view of (001) face.

Scanning electronic microscopy (SEM)

The surface morphology and elemental composition of the bio-composites were examined using scanning electron microscopy (SEM). (Fig. 6)

Fig. 6

SEM images of (a) C0, (b) P0, (c) PC(1:2), (d) PC(2:1) and (e) PC5.

The SEM image of pure cellulose (C0) reveals a smooth, compact, and homogeneous surface with few visible defects or textural irregularities. Such morphology is typical of regenerated cellulose films, where the dense hydrogen-bonding network between polymer chains leads to limited surface roughness. The absence of granular features or cracks confirms the good cohesion of the polymeric matrix; however, this dense structure is also associated with limited accessibility of surface functional groups, which is consistent with the relatively low adsorption capacity and antibacterial activity observed for this sample⁴¹.

In contrast, the pure hydroxyapatite (P0) micrograph exhibits agglomerated clusters of irregularly shaped particles with rough surfaces and interparticle voids. These aggregates are commonly attributed to the high surface energy of hydroxyapatite particles synthesized by wet precipitation methods. The granular texture observed at the microscale is consistent with the polycrystalline nature identified by XRD analysis. Nevertheless, the absence of a continuous polymeric matrix results in poor mechanical cohesion and limited structural integrity, which likely contributes to the moderate adsorption and antibacterial performances recorded for P0.

The morphology of the composite films (PC(1:2), PC(2:1), and PC5) shows a progressive evolution toward more uniform and interconnected structures. In PC(1:2), hydroxyapatite is incorporated within the cellulose matrix but remains partially distinguishable as dispersed bright domains, suggesting good but incomplete interfacial integration⁴². PC(2:1) exhibits a denser and more continuous microstructure, with a more homogeneous distribution of the inorganic phase and reduced surface discontinuities. Among the investigated composites, PC5 presents the most cohesive and texturally homogeneous surface, characterized by enhanced surface roughness and reduced particle agglomeration at the microscale⁴³.

These morphological features correlate well with the adsorption and antibacterial performances reported in this study. The improved textural homogeneity and interfacial integration in the composite films, particularly for PC5, favor dye–surface interactions through accessible hydroxyl and phosphate groups, in agreement with the high removal efficiencies and pseudo-second-order kinetic behavior. At the same time, the intimate contact between hydroxyapatite and the cellulose matrix contributes to antibacterial activity, likely through surface-mediated interactions and localized ion release, as reported in similar composite systems²¹. The SEM analysis supports the presence of a synergistic microstructural organization that underlies the enhanced functional performance of the optimized HAp–cellulose composite films.

Thermogravimetric analysis (TGA)

Figure 7, illustrates weight loss (%) as a function of temperature (°C), showing the decomposition of cellulose, the stabilizing effect of HAp, and the improved thermal resistance of the composites compared to cellulose alone.

Fig. 7

Thermogravimetric analysis (TGA) curves of pure cellulose (C0), pure hydroxyapatite (P0), and HAp/Cellulose composite bio-films with different ratios (PC1:2, PC2:1, and PC5).

The thermogravimetric analysis provides insight into the thermal stability and compositional integrity of the purified cellulose and HAp–cellulose composite films, which are relevant to their structural robustness and functional performance. As shown in Fig. 5, pure cellulose (C0) exhibits a major weight loss in the temperature range typically associated with the thermal degradation of the polysaccharide backbone. This behavior is characteristic of regenerated cellulose materials and reflects the decomposition of glycosidic linkages and volatile products formed during heating²⁴. The relatively low residual mass at high temperature confirms the predominantly organic nature of the C0 sample.

In contrast, pure hydroxyapatite (P0) displays minimal mass loss over the entire temperature range, demonstrating the high thermal stability of the inorganic phase. This behavior is consistent with previous reports on calcium phosphate materials and confirms that hydroxyapatite remains structurally stable under the applied thermal conditions⁴⁴. The composite films show intermediate degradation profiles between those of pure cellulose and pure hydroxyapatite, with the residual mass at high temperatures increasing progressively with increasing HAp content, confirming the effective incorporation of the inorganic phase into the cellulose matrix.

Notably, the composite films exhibit a slight shift of the main degradation region toward higher temperatures compared to pure cellulose, particularly for PC(2:1) and PC5. This enhanced thermal stability can be attributed to interfacial interactions between cellulose chains and hydroxyapatite particles, which restrict polymer chain mobility and delay thermal decomposition. Similar stabilization effects have been reported for inorganic–polymer composite systems, where strong interfacial interactions contribute to improved thermal resistance⁴⁵.

Although thermogravimetric analysis does not directly measure adsorption capacity or antibacterial activity, the observed thermal stabilization is relevant to the functional behavior of the composite films. Improved thermal and structural stability reflects a more cohesive composite architecture, which supports the preservation and accessibility of surface hydroxyl and phosphate functional groups involved in dye adsorption. In addition, the stable incorporation of hydroxyapatite within the cellulose matrix contributes to sustained surface-mediated antibacterial activity by maintaining close contact between the inorganic phase and bacterial cells during exposure.

Among the investigated samples, PC5 exhibits a balanced degradation profile characterized by enhanced thermal stability and a significant inorganic residue, which is consistent with its superior adsorption efficiency and antibacterial performance reported in this study.

Antibacterial assay

The antibacterial activity of the prepared bio-films was evaluated using the agar diffusion method against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). As shown in Fig. 8; Table 3, clear differences in inhibition zones were observed depending on the sample composition.

Fig. 8

Antibacterial activity of pure cellulose (C0), pure hydroxyapatite (P0), and composite bio-films with different HAp/Cellulose ratios (PC1:2 and PC2:1) against Staphylococcus aureus and Escherichia coli.

Table 3 Inhibition zone diameters (mm) of cellulose (C0), hydroxyapatite (P0), and composite bio-films (PC1:2 and PC2:1) against Staphylococcus aureus and Escherichia coli.

Pure cellulose (C0) did not exhibit any inhibition zones against either bacterial strain, confirming the absence of intrinsic antibacterial properties in cellulose. In contrast, pure hydroxyapatite (P0) produced moderate inhibition zones, measuring approximately 14 mm for S. aureus and 12 mm for E. coli. This is consistent with the well-documented antibacterial action of hydroxyapatite, which is attributed to the release of calcium and phosphate ions that can interfere with bacterial membrane integrity and metabolic processes⁴⁶.

The composite films displayed significantly improved antibacterial effects compared to pure cellulose, with the degree of inhibition strongly dependent on the HAp-to-cellulose ratio. The cellulose-rich composite (PC1:2) generated inhibition zones of about 18 mm against S. aureus and 17 mm against E. coli, demonstrating that even a lower concentration of HAp within the cellulose matrix can enhance antimicrobial activity. However, the most pronounced results were obtained for the HAp-rich composite (PC2:1), which produced inhibition zones of 25 mm against S. aureus and 20 mm against E. coli.

These results indicate a dose-dependent effect of hydroxyapatite content on antibacterial activity within the range of composite formulations investigated in this study. The presence of cellulose alone does not contribute to antimicrobial effects, but it provides a stable and homogeneous support for HAp dispersion, facilitating ion release and enhancing surface contact with bacterial cells. The strong inhibition observed for PC(2:1) highlights the potential of HAp-rich bio-films for biomedical applications such as implant coatings and wound dressings, where resistance to bacterial colonization is essential⁴⁷.

Comparative antibacterial study

The Table 4 compares the antibacterial activity of the present HAp/Cellulose PC(2:1) bio-film with previously reported hydroxyapatite-based composites against S. aureus and E. coli. The present material exhibits the highest inhibition zones, reaching approximately 25 mm for S. aureus and 20 mm for E. coli, indicating superior antibacterial performance. In comparison, the HApM/CNC composite shows moderate activity, with inhibition zones of about 17 mm and 13.5 mm, respectively, which may be attributed to the reinforcing effect of cellulose nanocrystals. Meanwhile, Al-HAp-AgNPs display lower antibacterial efficiency, suggesting that metal doping alone is not sufficient to achieve optimal performance.

Table 4 Comparative antibacterial activity of hydroxyapatite (HAp)–polymer films against S. aureus and E. coli.

The enhanced activity of the present bio-film can be mainly attributed to the homogeneous dispersion of nano-HAp within the cellulose matrix, improved surface reactivity, and synergistic interactions between both components, which promote stronger bacterial membrane disruption and ion release.

Adsorption experiments

Effect of adsorbent mass

The effect of adsorbent dosage on the removal efficiency of methylene blue (MB) is presented in Fig. 9.

Fig. 9

Effect of adsorbent mass on methylene blue (MB) removal efficiency for pure cellulose (C0), pure hydroxyapatite (P0), and HAp/Cellulose composites PC(1:2), PC(2:1), and PC5 (pH 7, initial MB concentration (C₀) = 20 mg/L, and contact time = 120 min).

For all samples, the percentage removal increased steadily with rising adsorbent mass, reflecting the greater number of available active sites and larger surface area that favor adsorption. Beyond 0.4–0.5 g, the removal efficiency tended to plateau, indicating site saturation and equilibrium between MB molecules and available binding sites.

Comparative analysis among the different formulations reveals significant differences in adsorption performance. The composite PC5 exhibited the best performance, reaching 95% removal at 0.7 g, confirming its highly optimized balance between cellulose’s porous network and hydroxyapatite’s active ion-exchange sites⁵⁰. The PC (2:1) composite achieved approximately 80% removal, outperforming PC (1:2) (75%) and highlighting the advantage of higher HAp content in creating abundant active sites for MB adsorption. Conversely, the PC (1:2) composite, though cellulose-rich, provided favorable diffusion pathways but fewer HAp binding sites, resulting in slightly lower efficiency. The individual components performed the least efficiently. Pure cellulose (C0) reached only 69% removal, relying mainly on physical sorption and hydrogen bonding interactions. Pure hydroxyapatite (P0) exhibited the lowest efficiency (60%), due to limited accessible surface area when used alone, despite its intrinsic ionic exchange capability⁵¹.

Overall, these results confirm the synergistic effect of combining cellulose and HAp: while each component alone shows modest performance, their integration significantly enhances adsorption efficiency. PC5, in particular, demonstrates that carefully tuned ratios maximize both adsorption kinetics and equilibrium capacity, making it the most promising candidate for wastewater treatment applications⁵².

Effect of contact time

The effect of contact time on the adsorption of methylene blue (MB) is illustrated in Fig. 10. All samples exhibited a rapid increase in removal efficiency during the initial stage (0–30 min), which corresponds to the availability of abundant active sites and the fast diffusion of dye molecules onto the adsorbent surface. As time progressed, the adsorption rate slowed down and approached equilibrium between 60 and 120 min, after which no significant increase was observed up to 180 min.

Fig. 10

Effect of contact time on MB removal efficiency for pure cellulose (C0), pure hydroxyapatite (P0), and composites PC(1:2), PC(2:1), and PC5 (pH = 7, initial MB concentration (C₀) = 20 mg/L, adsorbent mass = 0.2 g).

Among the different materials, PC5 achieved the highest removal efficiency (95%) within 30 min, confirming its superior adsorption kinetics and maximum capacity. The composites PC (2:1) and PC(1:2) reached equilibrium at 80% and 75%, respectively, showing good performance but slightly lower than PC5 due to their unbalanced cellulose/HAp ratios. In contrast, the individual components performed less efficiently: pure cellulose (C0) stabilized at 69% removal and pure hydroxyapatite (P0) at 60%. These results highlight the synergistic effect of combining cellulose and HAp, which not only improves equilibrium capacity but also accelerates the adsorption rate⁵³.

The kinetic behavior, with an initial rapid uptake followed by a slower approach to equilibrium, is consistent with pseudo-second-order kinetics, suggesting that chemisorption involving electrostatic interactions and surface complexation governs the process⁵⁴.

Kinetic models

The nonlinear kinetic fitting results presented in Fig. 11, together with the kinetic parameters summarized in Table 5, provide clear insight into the adsorption behavior of methylene blue on pure cellulose (C0), pure hydroxyapatite (P0), and the HAp–cellulose composite films. A marked difference in fitting quality is observed between the pseudo-first-order (PFO) and pseudo-second-order (PSO) models, indicating distinct mechanistic implications.

Fig. 11

(a) Pseudo-first-order and (b) Pseudo-second-order for methylene blue adsorption onto bio-films.

Table 5 Nonlinear Kinetic Parameters for MB Adsorption on HAp/Cellulose Composites.

In the case of the pseudo-first-order model (Fig. 11a), noticeable deviations between the experimental data and the fitted curves are observed throughout the adsorption process, particularly at the initial stage and near equilibrium. This behavior is quantitatively reflected in Table 5, where the calculated equilibrium adsorption capacities (q_ₑ,calc) are systematically lower than the experimental values (q_ₑ,exp) for all samples. While the rate constant k₁ shows a slight increase from the pure components to the composite films, the relatively low correlation coefficients (R² = 0.349–0.444) indicate that the PFO model does not adequately describe the adsorption kinetics. These results suggest that the adsorption process cannot be explained solely by physisorption or diffusion-controlled mechanisms.

In contrast, the pseudo-second-order model (Fig. 11b) exhibits an excellent agreement between the experimental data and the nonlinear fitted curves over the entire contact time range. The calculated equilibrium adsorption capacities closely match the experimental values for all samples, with differences remaining within experimental uncertainty. This strong agreement is supported by the very high correlation coefficients obtained for the PSO model (R² = 0.995–0.999), confirming its superior fitting performance compared to the PFO model⁵⁵.

The PSO rate constant k₂ increases progressively from pure cellulose and hydroxyapatite to the composite films, reaching its maximum value for PC5. This trend indicates faster adsorption kinetics and enhanced surface reactivity resulting from the synergistic interaction between hydroxyapatite and the cellulose matrix. The porous structure of cellulose facilitates rapid mass transfer, while the presence of hydroxyapatite introduces additional active sites, such as phosphate and hydroxyl groups, that promote strong interactions with dye molecules.

The kinetic analysis demonstrates that methylene blue adsorption on the HAp–cellulose bio-films is predominantly governed by chemisorption mechanisms involving surface functional groups and ion-exchange interactions. The superior kinetic performance of PC5, characterized by the highest adsorption capacity and fastest uptake rate, is consistent with its optimized structural features identified by XRD and FTIR analyses, further establishing a clear structure–property relationship in the developed composite materials⁵⁶.

Initial concentration effect.

The effect of the initial methylene blue concentration on the adsorption capacity of the prepared bio-films is presented in Fig. 12.

Fig. 12

Effect of initial MB concentration on removal efficiency for pure cellulose (C0), pure hydroxyapatite (P0), and composites PC(1:2), PC(2:1), and PC5 (pH = 7,, t = 3 h, adsorbent mass = 0.2 g).

As expected, the amount of dye adsorbed per unit mass of adsorbent (qe) increased progressively with increasing C₀ for all samples. This trend can be attributed to the stronger driving force at higher solute concentrations, which enhances the diffusion of dye molecules towards the surface of the adsorbents. At low initial concentrations (10–20 mg/L), the qe values were relatively modest and close between samples, since most of the available active sites were sufficient to capture the dye molecules.

At higher concentrations (60–100 mg/L), the differences between the materials became more pronounced. The PC5 composite consistently exhibited the highest qe, reflecting its larger number of active binding sites and better surface accessibility. The binary composites PC (2:1) and PC(1:2) also demonstrated superior adsorption capacities compared to the pure components, confirming the synergistic effect of combining hydroxyapatite with cellulose. In contrast, the pure cellulose (C0) and pure hydroxyapatite (P0) showed lower qe values, which highlights their limitations when used individually⁵⁷.

Overall, the increasing qe with C₀ and the superior performance of composite films suggest that the adsorption process is not limited by initial dye concentration, but rather by the availability of active sites and surface affinity. The optimized PC5 composite, in particular, provides significantly higher adsorption efficiency, underscoring its potential for wastewater treatment applications where high pollutant concentrations are encountered.

Isotherm models

Fig. 13

Linearized (a) Langmuir and (b) Freundlich adsorption isotherms for methylene blue onto C0 (Cellulose), P0 (HAp), PC(1:2), PC(2:1), and PC5 bio-composites.

The linearized Langmuir plots (Ce/qe vs. Ce) exhibit only moderate linearity across the series (R² = 0.721–0.799) (Fig. 13), indicating that MB uptake on these films does not follow an ideal monolayer process on a homogeneous surface. Nevertheless, the fitted capacity parameter increases systematically with HAp content: q_{e, max} rises from 40 mg·g⁻¹ (C0) and 35 mg·g⁻¹ (P0) to 55 mg·g⁻¹ [PC(1:2)], 65 mg·g⁻¹ [PC(2:1)], and peaks at 85 mg·g⁻¹ (PC5). The affinity term β (×10⁻³ L·mg⁻¹) follows the same order (40 → 80) (Table 6), implying progressively stronger dye, surface interactions as the composite becomes richer in HAp and as the cellulose matrix organizes accessible sites. Still, the sub-optimal R² values reveal noticeable site heterogeneity and/or multilayer contributions that the Langmuir model cannot capture⁵⁸.

Table 6 Langmuir and Freundlich adsorption isotherm parameters for methylene blue uptake onto HAp and HAp–cellulose composites.

By contrast, the Freundlich linear plots (ln qe vs. ln Ce) deliver excellent fits (R² = 0.975–0.991), consistent with heterogeneous surface adsorption and possible multilayer accumulation of MB. The capacity index K_F grows markedly from 2.50 (C0) and 1.90 (P0) to 4.40 [PC(1:2)], 6.40 [PC(2:1)], and 11.20 (PC5), mirroring the rise in q_{e, max} and confirming that the composites offer a denser population of effective binding sites (Table 6). The intensity factor nnn remains > 1 for all materials (1.55–2.00), demonstrating favorable adsorption throughout the concentration range; higher nnn for PC5 (≈ 2.00) indicates stronger affinity and less sensitivity to concentration changes at equilibrium.

Taken together, these results show that while the composites possess high theoretical capacities (Langmuir q_{e, max}), the Freundlich model better explains the experimental behavior, evidencing surface heterogeneity introduced by combining HAp crystallites with the cellulose network. The performance ranking is consistent across all metrics PC5 > PC(2:1) > PC(1:2) > C0 > P0 with PC5 delivering the highest capacity (85 mg·g⁻¹) and strongest affinity (K_F = 11.20; n = 2.00). Practically, this means PC5 will sustain high removal efficiencies over a broad range of MB concentrations, making it the most promising candidate for dye-laden wastewater treatment among the tested films.

Adsorption mechanism

The adsorption of dye molecules on hydroxyapatite/cellulose (HAp/Cell) bio-films proceeds through a multistep heterogeneous chemisorption mechanism involving both physical and chemical interactions. Initially, dye molecules diffuse from the bulk solution toward the external surface of the composite film. This process is facilitated by the porous and hydrophilic nature of the cellulose matrix, which allows rapid penetration of water and solute molecules. The cellulose network, composed of β(1→4)-linked D-glucose units rich in hydroxyl (–OH) groups, forms an open and hydrated structure that enables efficient dye diffusion toward the embedded hydroxyapatite particles⁴⁵.

Upon reaching the surface, methylene blue (MB⁺) or indigo molecules undergo surface binding through weak van der Waals forces, π–π stacking between aromatic dye rings and the glucopyranose units of cellulose, and hydrogen bonding with surface hydroxyl groups. These initial interactions represent a fast physisorption stage that accounts for the sharp increase in dye uptake observed during the first 30 min of contact. The dye molecules then migrate deeper into the internal pores of the composite through intraparticle diffusion, encountering additional active sites located on both cellulose and hydroxyapatite surfaces⁵⁹.

Once the dye reaches the HAp domains, chemisorption becomes the dominant step. The hydroxyapatite surface, consisting of phosphate (PO₄³⁻) and hydroxyl (OH⁻) groups coordinated to calcium ions (Ca²⁺), carries a net negative charge at neutral pH, promoting electrostatic attraction with the cationic MB⁺ molecules. These positively charged dye ions are drawn toward negatively charged phosphate sites through Coulombic forces, forming surface complexes such as ≡ PO₄⁻···MB⁺. Additionally, ion-exchange reactions may occur between MB⁺ and surface Ca²⁺ ions according to the equilibrium⁶⁰:

$$\equiv Ca^{2+} + 2\hbox{MB}^{+}_{aq} \leftrightarrow \equiv (\hbox{MB})_2 + \hbox{Ca}^{2+}_{(aq)}$$

This exchange mechanism enhances dye fixation and contributes to the strong chemisorptive nature of the process. Concurrently, hydrogen bonds form between –OH groups of both cellulose and HAp and heteroatoms (N, O) within the dye molecules, stabilizing the adsorbed layer.

The kinetic modeling reveals that the process follows a pseudo-second-order kinetic law (R² ≈ 0.999), indicating that chemisorption rather than simple diffusion controls the rate of adsorption. This involves electron sharing or exchange between dye molecules and surface functional groups. The synergy between cellulose and HAp is critical: cellulose provides a large accessible surface and diffusion pathways, while HAp contributes reactive ionic sites and enhances electrostatic and chemical affinity⁶¹.

Ultimately, the adsorption mechanism is a synergistic interplay of (i) electrostatic attraction and ion exchange on HAp, (ii) hydrogen bonding and π–π stacking on cellulose, and (iii) multilayer accumulation governed by surface heterogeneity⁶². This combination explains the high removal efficiencies (up to 95% for MB and 65% for indigo) and the excellent recyclability of the HAp/Cell bio-films, confirming their potential as eco-friendly and robust adsorbents for wastewater treatment.

Adsorption of indigo dye from dar dbagh de Fès

To broaden the scope of this study beyond model pollutants such as methylene blue, adsorption experiments were carried out using a natural indigo dye solution collected from the traditional leather tanning site (Dar Dbagh, Fez, Morocco). Indigo is one of the oldest dyes used in Moroccan artisanal processes and represents a real pollutant in tannery effluents. The aim was to evaluate whether the prepared HAp/Cellulose bio-films are capable of treating complex natural dyes under realistic conditions.

The raw indigo solution was filtered (0.45 μm), diluted to 50 mg·L⁻¹, and characterized by UV–Vis spectroscopy (λmax ≈ 615 nm). Batch adsorption tests were performed using 50 mL of dye solution and 0.2 g of each bio-film (C0, P0, PC1:2, PC2:1, and PC5) at pH 7, 25 ± 2 °C, and 150 rpm for 30 min. The discoloration was followed visually and confirmed by UV–Vis absorbance monitoring.

Fig. 14

Photographic illustration of the adsorption process of indigo dye onto the biofilm (PC5) over time.

The solution’s intense blue coloration progressively faded upon immersion of the bio-films, with marked differences between the samples (Fig. 14). Pure cellulose (C0) and hydroxyapatite (P0) showed limited adsorption performance, whereas composite films exhibited significantly higher removal efficiencies.

Fig. 15

Percentage removal of indigo dye by HAp/Cellulose bio-films under batch adsorption conditions (C₀ = 50 mg/L, V = 50 mL, m = 0.2 g, pH 7, 25 ± 2 °C, 150 rpm, 30 min).

Table 7 Removal efficiency and adsorption capacity of bio-films against indigo dye.

The adsorption performance of the HAp/Cellulose bio-films against indigo dye shows a clear dependence on the composite formulation (Fig. 15; Table 7). Pure cellulose (C0) achieved a moderate removal efficiency of 43 ± 2% (qe = 28.4 mg/g), confirming its ability to act as a porous support, although limited by its lack of intrinsic affinity for dyes. Hydroxyapatite alone (P0) exhibited a slightly lower removal of 35 ± 2% (qe = 23.3 mg/g), which may be attributed to its limited surface area accessibility and reduced interaction with the indigo chromophore. When cellulose and HAp were combined, a significant improvement in removal was observed. The PC (1:2) composite achieved 61 ± 3% removal (qe = 41.0 mg/g), while PC (2:1) further improved the adsorption to 73 ± 3% (qe = 49.8 mg/g). These results highlight a clear synergistic effect, where cellulose provides a flexible, porous matrix that enhances dispersion and stabilization of HAp particles, while HAp contributes additional active sites and ionic interactions⁶³. The most remarkable performance was obtained with PC5, which achieved 95 ± 3% removal (qe = 65.2 mg/g). This outstanding efficiency confirms that optimizing the HAp-to-cellulose ratio maximizes both dye surface interactions and accessibility of adsorption sites⁶⁴. The near-complete discoloration of indigo by PC5 demonstrates the strong potential of this composite as a sustainable adsorbent, outperforming both individual components and lower-ratio blends.

Beyond the laboratory context, these results hold direct industrial relevance. Indigo is one of the key colorants released by traditional tanneries (Dar Dbagh, Fès) and textile effluents, which are known to cause severe water pollution due to their intense coloration and persistence. The ability of HAp/Cellulose bio-films particularly PC5 to achieve nearly complete removal highlights their suitability as a low-cost, eco-friendly solution for industrial wastewater treatment and elimination of colorants from leather and textile industries.

Regeneration and reusability studies

The regeneration and reusability of the HAp/Cellulose-based biofilms were evaluated to assess their potential for repeated applications in wastewater treatment. After the initial adsorption of Methylene Blue (MB), the spent adsorbents were desorbed using a 0.1 M ethanol–HCl (1:1 v/v) solution under mild stirring for 1 h. The regenerated films were then washed with distilled water until neutral pH and dried at 50 °C before being reused in successive adsorption cycles under the same experimental conditions (Fig. 16).

Fig. 16

Regeneration of Biofilm HAp/Cellulose (PC5).

Fig. 17

Regeneration and reusability of HAp/Cellulose biofilms over five adsorption–desorption cycles for methylene blue removal.

The regeneration and reusability tests of the HAp/Cellulose biofilms were conducted over five consecutive adsorption–desorption cycles using methylene blue as a model dye. As shown in Fig. 17, all biofilms maintained relatively high performance across repeated uses, indicating good mechanical integrity and surface stability⁶⁵.

During the first cycle, the PC5 composite achieved a 95% removal efficiency, confirming its outstanding initial adsorption capacity. However, a gradual decline in removal efficiency was observed with successive cycles due to the partial occupation of active sites and limited desorption of dye molecules strongly bonded to the surface. After five cycles, the PC5 film retained about 85–88% of its initial adsorption capacity, whereas the PC (2:1) and PC(1:2) films retained 80–83% and 77–79%, respectively. In contrast, pure cellulose (C0) and hydroxyapatite (P0) films showed a more pronounced performance loss, with only ~ 55–60% of their initial capacity remaining after five cycles.

This behavior confirms the robust recyclability and chemical stability of the hybrid HAp/Cellulose composites, which can undergo multiple regeneration steps without significant degradation. Their sustained efficiency suggests strong potential for industrial wastewater treatment, especially in the repeated removal of colorants such as methylene blue or natural indigo dyes from tanneries (e.g., Dar Dbagh of Fes).

Electronic structure and charge-transfer characteristics governing methylene blue and natural indigo adsorption

DFT calculations within the generalized gradient approximation (GGA) were conducted to elucidate the electronic properties governing the adsorption of methylene blue and natural indigo on the HAp@Cellulose surface. The optimized molecular geometries, frontier molecular orbitals (HOMO and LUMO), and molecular electrostatic potential (ESP) maps are presented in Fig. 17. HOMO electron density is primarily localized on heteroatom-rich regions (N, O, and S), designating these as the main electron-donating sites, whereas LUMO distributions are largely delocalized over the conjugated aromatic frameworks, highlighting regions favorable for electron acceptance and charge-transfer interactions. The relatively high HOMO energies and narrow HOMO–LUMO gaps (Fig. 18) indicate enhanced reactivity and strong affinity toward the adsorbent. The maximum electron transfer parameter (ΔNₘₐₓ) values of 6.182 for methylene blue and 6.222 for natural indigo further confirm a strong propensity for electron donation to the HAp@Cellulose surface, supporting a charge-transfer-driven adsorption mechanism. ESP maps reinforce these findings, revealing electron-rich regions around heteroatoms and electron-deficient zones over protonated or aromatic moieties, which facilitate electrostatic attraction and hydrogen-bond formation with surface hydroxyl and phosphate groups. Together, the HOMO–LUMO analysis, ΔNₘₐₓ values, and ESP mapping provide clear molecular-level evidence that the adsorption of both dyes is dominated by non-covalent interactions, particularly electrostatic forces and charge-transfer processes.

Fig. 18

Optimized Structures, HOMO–LUMO Distributions, and ESP Maps of Natural Indigo and Methylene Blue.

Molecular analysis of non-covalent interactions governing bacterial and dye adsorption on the HAp@Cell composite surface

To gain deeper insight into the experimental observations and elucidate the interfacial interactions between bacteria (Staphylococcus aureus and Escherichia coli), model dyes (methylene blue and natural indigo), and the composite surface, computational analyses were performed to complement the experimental findings. NCI and RDG methods were employed to characterize the nature of weak intermolecular forces at the HAp$/$Cell interface. The simulations were conducted using the Forcite module of Materials Studio, whereas RDG and NCI isosurfaces were computed with Multiwfn and visualized via Gnuplot and VMD. These analyses provide a molecular-level understanding of weak interactions governing the adsorption process on the HAp$/$Cell film, including steric repulsion, van der Waals forces, and hydrogen bonding⁶⁶. The RDG isosurfaces and corresponding scatter plots display variations in the $\text{s}\text{i}\text{g}\text{n}({\uplambda}₂){\uprho}$ function through color mapping, effectively distinguishing the type and strength of non-covalent interactions^67,68. The electron density (${\uprho}$) reflects the interaction magnitude, while the sign of the second eigenvalue (${\uplambda}₂$) of the electron-density Hessian matrix differentiates attractive (${\uplambda}₂<0$) from repulsive (${\uplambda}₂>0$) regions. Specifically, blue regions denote hydrogen bonding with negative $\text{s}\text{i}\text{g}\text{n}({\uplambda}₂){\uprho}$ values, green areas correspond to van der Waals interactions near $\text{s}\text{i}\text{g}\text{n}({\uplambda}₂){\uprho}\approx0$, and red zones indicate steric repulsion where $\text{s}\text{i}\text{g}\text{n}({\uplambda}₂){\uprho}$ is positive^69,70. As shown in Fig. 19, the intense blue regions confirm the formation of strong hydrogen bonds between bacterial or dye molecules and the Hap$/$Cell surface. The coexistence of green and red areas further reveals that adsorption proceeds through a mixed mechanism involving hydrogen bonding, van der Waals attraction, and localized steric effects. These computational findings are in excellent agreement with the experimental results, confirming that both bacterial adhesion and dye adsorption are primarily governed by synergistic non-covalent interactions at the biofilm interface.

Fig. 19

Top view of the NCI and RDG isosurfaces illustrating the structural stability and non-covalent interaction network within the HAp/Cell–bacteria/dye complexes.

Ramachandran plot analysis

The results of the Ramachandran plot of the 1JIJ and 1KZN proteins generated using PROCHECK are shown in the Fig. 20.

Fig. 20

Ramachandran plot of the 1JIJ and 1KZN protein generated using PROCHECK, showing the distribution of residues in favored and allowed regions.

Ramachandran plot assessment of the 1JIJ structure showed that 76% of residues fall within the favored regions, supporting acceptable backbone geometry for the model. Only 3% of residues were found in the most favored regions (21)0.5% in other permitted regions, and 2%. A 2% level was observed in regions where it was permitted, and no residues were detected in regions where it was not permitted. Because no residues appear in disallowed conformational regions, the structure has acceptable stereochemical quality for use in molecular docking.

For 1KZN, the reported value is 89. In the structural assessment, 9% of residues were located in the most favored regions (8). The rate was 8% in the regions where it was permitted and 0% in all other areas. The proportion was 6% in regions where allowances were more generous, and it was 0% in all other regions. 6% of the sample originated from regions classified as disallowed. Because over 99% of residues fall within allowed conformational regions, the structure of 1KZN appears reliable.

The Ramachandran analysis indicates that both protein structures have acceptable stereochemical quality, which supports their use in the next stage of docking studies.

Molecular docking

The aim of this study was to investigate the molecular interactions and dynamic behavior of the target system after treatment with the proposed inhibitors. Molecular docking simulations were carried out using the MOE software package. The hydroxyapatite/cellulose complex was docked into the active sites of the antibacterial protein crystal structures 1JIJ and 1KZN obtained from the Protein Data Bank (PDB). The docking outcomes are illustrated in the two figures below.

Fig. 21

Best docking interaction of hydroxyapatite/cellulose with 1JIJ receptor.

The interaction between the hydroxyapatite/cellulose complex and the 1JIJ receptor (E. coli activity) was evaluated through molecular docking analysis. The best-ranked binding pose exhibited a docking score (S value) of − 8.4567 kcal/mol, indicating a favorable binding affinity toward the target enzyme (E. coli activity). Such a negative binding energy suggests the formation of stable hydroxyapatite/cellulose − 1JIJ interactions within the active site of 1JIJ protein.

As illustrated in Fig. 21, the hydroxyapatite/cellulose complex demonstrating the highest predicted E. coli activity establishes four key interactions within the binding pocket. Specifically, hydrogen bond interactions are observed with the amino acid residues Asp195, Asp 84, Cys 37, Gly 193, Ala 39, Lys 84, Asp 80 and Asp 40. These interactions contribute to the stabilization of the ligand within the catalytic site and may explain its predicted antibacterial (E. coli activity) potential.

Fig. 22

Best docking interaction of hydroxyapatite/cellulose with 1KZN receptor.

The molecular docking analysis of the hydroxyapatite/cellulose complex, with 1KZN receptor (Staphylococcus aureus receptors) (PDB ID: 1KZN). The calculated docking scores (S values) reached up to − 6.5945 kcal/mol, indicating a favorable binding affinity toward the active site of the staphylococcus aureus receptors. These negative binding energies suggest stable hydroxyapatite/cellulose − 1KZN complex formation within the catalytic pocket.

As shown in Fig. 22, the hydroxyapatite/cellulose complex exhibiting the highest predicted anti-S. aureus activity adopts a well-oriented conformation inside the binding cavity. The ligand establishes five key interactions with amino acid residues Arg 136, Arg 76, Glu 50, Asp 49, and Ile 90. These interactions include hydrogen bonding and stabilizing contacts that contribute to the overall binding stability of the complex.

The strength and multiplicity of these interactions support the potential inhibitory effect of the hydroxyapatite/cellulose complex against antibacterial activity (E. coli activity and Staphylococcus aureus). Consequently, the anchoring results observed underline the potential of these hydroxyapatite/cellulose molecules as promising antibacterial candidates.

Comparative study

This comparison shows that your HAp/Cellulose PC5 film achieves a high qe, max (~ 85 mg·g⁻¹) among film architectures, outperforming typical HAp/biopolymer films such as HAp/HPMC (52 mg·g⁻¹), HAp/Starch (45.5 mg·g⁻¹), and a CMC/AC/HAp film-like composite (43.9 mg·g⁻¹). The advantage is consistent with your SEM/XRD evidence of nano-HAp well dispersed in a porous cellulose network, which increases accessible active sites and promotes rapid PSO kinetics (Table 8).

Across the literature, films often exhibit lower capacities than porous beads/aerogels because their accessible surface area and pore volume are constrained by the flat architecture. Even within that constraint, PC5 performs at the upper end of the film range, indicating that your formulation (balanced HAp loading + cellulose diffusion pathways) effectively mitigates the usual film limitations.

Table 8 Comparative adsorption performances of hydroxyapatite (HAp)–polymer films toward methylene blue (MB).

Finally, the PSO kinetic preference reported for several entries (including PC5) supports a surface process dominated by chemisorption/electrostatic interactions on heterogeneous sites, in line with your functional groups (–OH/PO₄) and the observed synergy between HAp and cellulose. In practice, this means PC5 combines meaningful capacity with fast uptake and recyclability, making it a strong candidate for dye removal in thin-film modules.

Sustainability and industrial relevance

The development of the HAp/Cellulose bio-films aligns strongly with Morocco’s national vision for eco-circular innovation and sustainable material production. Both natural phosphate extracted from abundant Moroccan deposits and cotton-derived cellulose sourced from local agricultural waste are renewable and locally available resources. Their valorization not only reduces dependence on imported raw materials but also adds value to regional by-products, contributing to a circular and environmentally responsible economy.

From an environmental standpoint, the proposed synthesis route is particularly green and low-impact. The entire process is conducted without the use of organic solvents and at low temperatures (< 100 °C), which drastically minimizes energy consumption and eliminates toxic emissions typically associated with conventional polymer–ceramic fabrication methods. The mild processing conditions ensure worker safety, low carbon footprint, and ease of scalability, making the methodology suitable for both laboratory and pre-industrial settings. In addition to its environmental advantages, the composite exhibits strong industrial potential within the Moroccan context.

On one hand, its high adsorption capacity toward methylene blue and natural indigo dyes makes it an ideal candidate for treatment of textile effluents, particularly from traditional dyeing and leather tanning sectors such as Dar Dbagh in Fez. On the other hand, the proven antibacterial performance of the HAp/Cellulose film positions it as a promising material for antimicrobial bone coatings and wound dressings, offering a dual biomedical–environmental application pathway. This dual-purpose functionality underscores Morocco’s capability to transform local mineral and agricultural resources into advanced, sustainable materials of high added value.

Relationship between adsorption capacity and antibacterial activity: toward dual-function applications

Although dye adsorption and antibacterial activity are often evaluated separately, both functions originate from the same surface chemistry and microstructural features of the HAp–cellulose composite films. In the present system, the enhanced adsorption performance and antibacterial efficacy are intrinsically linked through shared physicochemical mechanisms governed by surface charge, functional groups, porosity, and defect density.

From an adsorption perspective, the high removal efficiency of cationic dyes such as methylene blue and indigo arises from the abundance of negatively charged phosphate (PO₄³⁻) groups, surface hydroxyl (–OH) groups, and defect-rich nanocrystalline domains. These features promote electrostatic attraction, hydrogen bonding, and surface complexation, particularly in the optimized PC5 composite. The reduced crystallite size, increased microstrain, and higher dislocation density identified by XRD further enhance surface reactivity by increasing the density of accessible active sites.

The antibacterial activity is governed by closely related surface interactions. The same negatively charged phosphate groups that attract cationic dye molecules also promote electrostatic interactions with positively charged regions of bacterial cell membranes. In addition, the nanocrystalline hydroxyapatite domains embedded within the cellulose matrix enable a sustained release of Ca²⁺ and PO₄³⁻ ions, which destabilize bacterial membranes, disrupt ionic homeostasis, and interfere with essential metabolic processes. The increased defect density and partial amorphization observed in PC5 facilitate ion exchange and surface contact, thereby amplifying antibacterial efficacy.

Importantly, the porous cellulose network acts as a common structural platform for both functions. It enhances mass transfer and diffusion of dye molecules toward adsorption sites while simultaneously promoting close contact between the composite surface and bacterial cells. This dual role explains why the composite exhibiting the highest adsorption capacity (PC5) also demonstrates the strongest antibacterial activity, establishing a direct structure–function correlation.

• Bridging Biomedical Engineering and Environmental Science.

The coexistence of adsorption and antibacterial functionalities positions the HAp–cellulose bio-films at the interface of biomedical engineering and environmental science, enabling applications where both contaminant removal and microbial control are required. In environmental contexts, such as textile and tannery wastewater treatment, the material can simultaneously remove toxic dyes and suppress bacterial proliferation, reducing biofouling and secondary contamination. In biomedical and healthcare-related environments, the same surface chemistry enables adsorption of biological fluids, toxins, or metabolites while preventing bacterial colonization.

Common application domains bridging these two fields include antimicrobial filtration membranes, wound dressings for contaminated environments, antibacterial adsorbent coatings, water purification systems with biofouling resistance, and implant or device surfaces exposed to biological fluids and pollutants. In all these cases, the ability to combine adsorption-driven contaminant capture with contact-active antibacterial behavior is critical for performance and durability.

Therefore, the HAp–cellulose composite films developed in this work represent a class of multifunctional materials where adsorption and antibacterial activity are not independent properties, but rather synergistic outcomes of a unified surface chemistry and hierarchical structure. This dual-functionality underscores their relevance for integrated biomedical–environmental solutions.

Conclusion

The cellulose/hydroxyapatite (HAp) bio-films developed in this study demonstrate a successful integration of renewable Moroccan resources natural phosphate and cotton cellulose through a low-temperature, solvent-free process, aligning with eco-circular and sustainable material strategies. Structural analysis by XRD confirmed the presence of well-crystallized HAp nanophases uniformly distributed in the cellulose matrix, while SEM micrographs revealed a progressive evolution of morphology from dense to highly porous networks, particularly in PC5, where the homogeneous dispersion of nanosized HAp grains generated micro- and nano-scale porosity.

This optimized texture directly enhances both adsorption and antibacterial performances. For dye removal, the PC5 film achieved the highest removal efficiencies 95% for indigo and ~ 85 mg.g⁻¹ for methylene blue confirming the synergistic role of cellulose (porous diffusion network) and HAp (active ion-exchange sites). The kinetic data followed a pseudo-second-order model, suggesting that chemisorption dominated the process via electrostatic attraction and hydrogen bonding between dye molecules and the surface hydroxyl/phosphate groups. The Freundlich isotherm provided the best fit, indicating a heterogeneous surface with multilayer adsorption behavior.

Antibacterial assays revealed a dose-dependent inhibitory effect on Staphylococcus aureus and Escherichia coli, with inhibition zones increasing from ~ 14 mm (pure HAp) to 25 mm for S. aureus and 20 mm for E. coli for the HAp-rich composite. The strong antibacterial activity of PC5 arises from the intimate HAp dispersion enabling sustained Ca²⁺/PO₄³⁻ ion release, which destabilizes bacterial membranes and disrupts vital cell functions. The synergistic correlation between microstructural refinement (observed by SEM) and dual functional response (adsorption + antibacterial) confirms that the composite’s hierarchical porosity and surface chemistry govern both pollutant capture and microbial inhibition.

Overall, these eco-designed films exhibit a balanced combination of mechanical stability, environmental efficiency, and biological activity. Their fabrication from abundant Moroccan biogenic resources, mild synthesis conditions (< 100 °C), and proven reusability make them promising candidates for industrial wastewater decolorization (textile and tannery effluents) and antimicrobial biomedical coatings.