Photocatalytic Remediation of Harmful Alexandrium minutum Bloom Using Hybrid Chitosan-Modified TiO₂ Films in Seawater: A Lab-Based Study

Abstract

The uncontrolled growth of harmful algal blooms (HABs) can negatively impact the environment and pose threats to human health and aquatic ecosystems. Titanium dioxide (TiO₂) is known to be effective in killing harmful algae through flocculation and sedimentation. However, TiO₂ in a dispersed form can harm other non-target marine organisms, which has raised concerns by environmentalists and scientists. This research seeks to explore the utility of immobilized titanium oxide as a photocatalyst for mitigation of HABs, where the Alexandrium minutum bloom was used as a model system herein. Chitosan was modified with 0.2 wt.% TiO₂ (Chi/TiO₂ (x mL; x = 1, 3 and 5 mL) and the corresponding films were prepared via solvent casting method. Scanning electron microscope (SEM) images of the films reveal a highly uneven surface. X-ray diffraction (XRD) analysis indicates the reduction in chitosan crystallinity, where the presence of TiO₂ was negligible, in accordance with its dispersion within the chitosan matrix. The photocatalytic mitigation of A.minutum was carried out via a physical approach in a laboratory-scale setting. The negative surface charge of the films was observed to repel the negatively charged A.minutum causing fluctuation in the removal efficiency (RE). The highest RE (76.1 ± 13.8%) was obtained when Chi/TiO₂ (1 mL) was used at 72 h, where the hydroxyl radicals generated were inferred to contribute to the deactivation of the algae cells by causing oxidative stress. An outcome of this study indicates that such hybrid films have the potential to replace the non-immobilized (dispersed) TiO₂ for HAB mitigation. However, further investigation is required to deploy these films for field applications at a larger scale.

1. Introduction

Harmful algal blooms (HABs) are a phenomenon caused by the uncontrolled and rapid growth of toxic and harmful algae. HABs can be generated from a wide range of organisms such as toxic cyanobacteria, phytoplankton, microalgae and benthic algae [1]. The frequent occurrence of HABs is attributed to global warming and the high input of nutrient species that contain nitrogen and phosphorus from industrial sewage, agricultural and aquaculture runoff and discharge into water bodies [2,3]. In addition, natural environmental factors such as temperature, pH, light irradiance and water currents also contribute to the occurrence of HABs [3]. These harmful algal blooms (HABs) generally form in warm and still water, where HABs appear foamy or scummy on the surface of water bodies, which may occur in both fresh and marine water bodies [4]. A single outbreak of HABs can cause millions of dollars in losses to the fisheries industry, mostly fish farms and the tourism industry. Ingestion of contaminated seafood products by humans through skin contact with toxin-contaminated water, or the inhalation of aerosolized toxins, or noxious compounds can be lethal [5].

Mitigation of HABs is the term used to describe the actions taken to deal with an existing or ongoing bloom by taking necessary precautions to reduce its negative impact [6]. Various biological, chemical and physical approaches have been reported to be successful in HAB mitigation. Although effective, most techniques to control HABs have several limitations such as high maintenance cost, short-term effectiveness and the need for repeated treatments [7]. Control technologies for the mitigation of HABs should be more sustainable at a low maintenance cost with long-term effects that should not cause any direct or indirect impact on human life and aquatic ecosystems.

In wastewater remediation, photocatalysis is one of the widely used advanced oxidation processes (AOPs) that utilize photogenerated electron/hole pairs generated by semiconductors when irradiated with light. In turn, the photogenerated electron/hole pairs can react with moisture and dissolved oxygen to generate reactive oxygen species (ROS), which can cause damage to the structure of the algae and degrade its toxins [8]. Various semiconductors such as TiO₂, ZnO, SnO₂, CuO, CdS and Fe₂O₃ have been used as photocatalyst materials to alter the structure and function of algae cells. Baniamerian et al. reported that Fe₂O₃–TiO₂ nanoparticles show a high removal rate of Chlorella vulgaris (99.8%) within 24 h under visible light irradiation [9]. The g-C₃N₄/Bi-TiO₂ floating photocatalyst reported by Song et al., 2021 led to the removal of Microcystis aeruginosa (99.2%) within a 3 h exposure [10]. On the other hand, Fan et al., 2022 have reported that the ZnFe₂O₄/Ag₃PO₄/g-C₃N₄ (ZFO/AP/CN) photocatalyst had a removal efficacy for M. aeruginosa (94.31%) and MC-LR (76.92%) under visible light conditions [11].

Chitosan is a biopolymer with antimicrobial activity that contains glucosamine units with a variable level of N-acetylation at C2, where primary and secondary hydroxyl groups at C6 and C3 that can be used for synthetic modification [12,13]. The chitosan/TiO₂-based nanocomposite films have been extensively studied for wastewater treatment. Such types of chitosan (CS) films reveal remarkable adsorption and photocatalysis of organic and inorganic pollutants. Razzaz et al. found that chitosan functionalized with TiO₂ nanoparticles prepared by the entrapment method exhibited better adsorption of Cu(II) and Pb(II), as compared with nanocomposite particles prepared by the coating method [14]. In 2018, Saravanan et al. reported that chitosan-TiO₂ composites showed great degradation efficiency toward methyl orange dye [15]. Zhao et al. reported that Ag₂O/TiO₂-modified CS-based film was able to completely degrade ampicillin and methyl orange within 180 min and 30 min, respectively [16] Abdullah Al Balushi et al. reported that a chitosan-TiO₂ film with 12 layers of coating was able to completely degrade 0.60 ppm of methylene blue (MB) within 120 min of contact [17].

To the best of our knowledge, no reports can be found on the application of chitosan-TiO₂ film as a strategy for the mitigation of HAB. In this study, chitosan-TiO₂ films were developed via a solvent casting method for the mitigation of Alexandrium minutum (A. minutum), which is a common algae species frequently found in the northeastern coast of peninsular Malaysia [18]. The lab-scale mitigation study reported herein represents a first example that combines a physical and chemical approach involving adsorption and photocatalysis. This study will demonstrate the effectiveness of this photocatalytic approach of HAB mitigation with its feasibility for scalability and sustainability.

2. Results

As outlined in the introduction, a series of chitosan films were prepared that contain an incremental volumetric dosage of 0.2% w/v of anatase (TiO₂) during the film preparation. In the sections below, the structure of the films were characterized by various spectral (IR, XRD, SEM-EDX and digital microscopy) and physicochemical characterization (TGA, pH dependence of the surface charge, solvent swelling, and water contact angle) methods. In turn, the composite films were evaluated for their photocatalytic efficacy of the neutralization of algal cells (A. minutum) in seawater to evaluate the potential of such materials for remediation of harmful algal blooms (HABs) in marine environments, as part of future studies in photocatalytic remediation to control HABs.

2.1. Characterization of the Fresh Hybrid Chitosan-Modified TiO₂ Films

2.1.1. Attenuated Total Reflectance Fourier Transformed Infrared Spectroscopy (ATR-FTIR) Analysis

The ATR-FTIR spectra of the films are shown in Figure 1a. Based on the FTIR spectrum of the chitosan film (Chi), the band at 1024 cm⁻¹ corresponds to the symmetric stretching of the C–O–C bond. The band at 1072 cm⁻¹ relates to the skeletal vibration of C–O and is usually assigned as the fingerprint band for the structure of chitosan [19]. The deformation vibration of –NH₂ is indicated by the appearance of an IR signature at 1554 cm⁻¹. The bands at 1384 cm⁻¹ and 1317 cm⁻¹ correspond to the asymmetric C–O–C stretching vibrations and C–O stretching vibration of CH–OH [20]. The band at 1645 cm⁻¹ represents the C=O stretching of the amide group [21,22]. The small shoulder at 1151 cm⁻¹ is due to the C–H vibration from C–O–C of chitosan.

The higher intensity of the IR band in the region of 3200–3500 cm⁻¹ of chitosan/TiO₂ (Chi/TiO₂) films compared to Chi is due to the interaction between Ti with –OH and –NH functional groups of chitosan via hydrogen bonding [23]. The band formation at 2870 cm⁻¹ was attributed to C–H asymmetric and symmetric vibrations of the Ti–OH functional group [23]. The chemical reaction between the chitosan and TiO₂ is confirmed by the presence of an IR band at 1019 cm⁻¹, where this signature corresponds to the Ti–O–C bond [24,25]. The IR band associated with Ti–OH and Ti–O bonds can be seen at 1387 cm⁻¹ [26], whereas the IR band related to the angular deformation of N–H bond of chitosan was observed at 1590 cm⁻¹ [27]. The absorption at the lower frequency (650 cm⁻¹) is due to the symmetric stretching vibration of Ti–O–Ti and O–Ti–O flexion vibration of the anatase phase [28]. These bands support the presence of TiO₂ in the chitosan matrix.

In acidic media, the concentration of surface hydroxyl groups on the TiO₂ surface will be higher. These hydroxyl groups can react with those of chitosan via a condensation reaction to form Ti–O–C bonds. The cross-linking between the TiO₂ and the chitosan backbone is expected to reinforce the biopolymer and form a net-like structure that can capture and immobilize the algal cells. The presence of cross-linking is supported by the presence of an IR band at 1019 cm⁻¹ for the Chi/TiO₂ films. The reaction scheme indicates an illustration of the cross-linking between the two groups, as shown in Scheme 1.

2.1.2. X-ray Diffraction (XRD) Analysis

The XRD profiles of Chi and the Chi/TiO₂ films are shown in Figure 1b. From the XRD diffractogram of Chi, three diffraction peaks can be observed at 2θ = 10.1°, 19.8° and 22° that correspond to the respective crystallographic planes of chitosan: (002), (101) and (220) [28,29].

The XRD peaks of chitosan became attenuated and broadened upon the incorporation of the TiO₂, which indicates a reduction in the crystallinity of chitosan and a decrease in the intermolecular hydrogen bonding of the chitosan matrix [28]. The characteristic peaks of anatase TiO₂ which occur at 2θ (°) values also correspond to the crystallographic planes denoted in parentheses: 25.42° (101), 38.08° (004), 38.93° (112), 48.35° (200), 63.02° (204), 69.17° (116), 70.62° (220), 75.43° (215) and 83.14° (224). The XRD lines of TiO₂ were not detected due to the low concentration of TiO₂ or uneven dispersion in the film matrix [30,31].

2.1.3. Scanning Electron Microscopy (SEM) Analysis

Figure 2 shows the SEM images for the surface morphology of the films (left side) and cross-sectioned images (right side). The formation of crater-like structures occurs due to the evaporation of water vapor and organic solvent during the film drying process. Meanwhile, the cross-section of Chi is observed to be dense and coarse but with no visible boundaries between chitosan and TiO₂ (Figure 2A,a). The surface of the Chi/TiO₂ films was observed to be different from Chi (Figure 2B–D). The surface of Chi/TiO₂ films was uneven and possibly due to the protrusion of irregularly shaped TiO₂ nanoparticles or due to the agglomeration of TiO₂ particles during the drying process. The cross-section images of the Chi/TiO₂ films (Figure 2b–d) appear to be less coarse compared to Chi. The increase in the dosage of TiO₂ reduces the compact appearance of the films. Calero et al. have reported that a more compact structure is essential to enhance the film’s capabilities for various applications [32].

2.1.4. Point of Zero Charge (pH_pzc)

The point of zero charge (pH_pzc) is the pH value at which the surface of the solid is considered to have no net electrical charge [33]. The ability of any surface to adsorb ionic species of target pollutants is determined by the pH_pzc. The net surface charge on the particle is affected by the pH of the liquid media in which the solid is dispersed. The pH_pzc determination of Chi and Chi/TiO₂ films is shown in Figure 3. The pH_pzc of Chi/TiO₂ was lower compared to the pH_pzc of a Chi film. The decrease indicates that TiO₂ altered the electronic structure and energy states of chitosan [34,35]. At a solution pH above the pH_pzc, the surface of the films is negatively charged, whereas the surface becomes more positively charged at pH values below pH_pzc.

2.1.5. Wettability and Swelling Index Analysis

The hydrophilic or hydrophobic surface characteristics of the prepared films were established through a study of the variation in the water contact angle (θ) variation for films of variable composition. The θ-values for each film are presented in

Table 1. The measured contact angle (θ) and swelling index of chitosan films.

Film Sample 1	Swelling Index (%)	Contact Angle (θ; °)
Chi	63.7 ± 1.05	98.2 ± 0.84
Chi/TiO2 (1 mL)	143.8 ± 2.67	93.0 ± 0.54
Chi/TiO2 (3 mL)	150.7 ± 1.15	75.6 ± 0.03
Chi/TiO2 (5 mL)	144.2 ± 1.09	92.8 ± 0.05

The values were expressed in mean ± standard deviation with a significant difference (p < 0.05); ¹ The volume quantities in parentheses refer to the dosage of 0.2% w/v TiO₂.

, where the highest θ-value is noted for the Chi film (θ = 98.2°) due to the hydrophobic domains of the biopolymer [36]. The contact angle for the Chi film was observed to be similar to the reported values of chitosan films in the literature [37,38]. The θ-values of Chi/TiO₂ (1 mL and 5 mL) are slightly lower than Chi, whereas the Chi/TiO₂ (3 mL) shows the lowest contact angle (75.6°), which indicates a notable increase in the film hydrophilicity. The apparent increase for θ is due to the higher concentration of hydroxyl groups available to interact with water in the Chi/TiO₂ films, compared to a Chi film without TiO₂. The FTIR analysis indicates a greater level of hydroxyl groups in the Chi/TiO₂ films and none in the Chi film (Section 2.1.1). The lowest contact angle of Chi/TiO₂ (3 mL) may relate to the greater uniform distribution of TiO₂ nanoparticles compared to the other Chi/TiO₂ films. The even distribution of the TiO₂ increases the amount of hydrogen bond sites that may interact with water. The role of cross-linking may cause the microstructural surface heterogeneity or roughness of the film, which contribute to variable contact angle effects. From the SEM images (Section 2.1.3), it can be observed that Chi/TiO₂ (1 mL and 5 mL) appear uneven when compared to Chi/TiO₂ (3 mL). Greater surface heterogeneity or roughness can contribute to hydrophobic effects, which may inhibit the diffusion transport of water molecules through the film [39]. Hence, the contact angle of Chi/TiO₂ (3 mL) film is lower than Chi/TiO₂ (1 mL and 5 mL) systems.

The swelling index of the Chi/TiO₂ films was greater when compared to Chi films (without TiO₂), which is expected since both TiO₂ and Chi have hydrophilic properties. The TiO₂ is more likely to interact with the functional groups of chitosan such as –OH and –NH₂ which can likewise form hydrogen bonds with water molecules [40]. The functional groups increase the swelling of the film and also influence the hydrophilic character of the film [41,42]. The highest swelling index (%) of Chi/TiO₂ (3 mL) is due to its greater hydrophilic nature.

2.2. Photocatalytic Mitigation Studies

The removal efficiency (RE; %) values of the A. minutum sp. for Chi and Chi/TiO₂ films are shown in Figure 4. Based on the pH_pzc of the films, the films will be negatively charged in seawater (pH = 6.8). Hence, the negatively charged algal cells will be repelled from the film surface. The slow swelling process of the film may also facilitate the trapping of algae cells in the net-like structure of the composite film to enable release and exchange with the external aqueous media. This is evident from the fluctuating RE values (%). The Chi, Chi/TiO₂ (3 mL) and Chi/TiO₂ (5 mL) films achieve ca. 20% removal of A. minutum at 72 h. Even though the swelling indices of the Chi/TiO₂ films were similar, Chi/TiO₂ (1 mL) has the lowest fluctuation and the highest RE (76.1 ± 13.8%). The lower RE value for Chi/TiO₂ (3 mL) may relate to its excessive swelling, which facilitates the release of the trapped algae to the aqueous media. The Chi/TiO₂ (3 mL) had the highest swelling index value. Whereas the lower RE value for Chi/TiO₂ (5 mL) was related to the agglomeration of TiO₂ nanoparticles, especially as the TiO₂ concentration was increased up to the highest level of film incorporation. Agglomeration and aggregation of the metal oxides such as TiO₂ were reported to affect the absorption of photons. In turn, a decrease in the ability to generate reactive oxygen species (ROS) such as the hydroxyl radical (^•OH), which is responsible for the photodegradation of organic pollutants [43,44]. Since the agglomeration of TiO₂ in CH/TiO₂ (1 mL) is lower, more ROS can be generated to induce oxidative stress toward the algal cells. In turn, a greater production of ROS can cause organelle dysfunction, cell structure alteration and mutagenesis [45].

The mitigating ability of Chi/TiO₂ (1 mL) towards algal removal was compared to other semiconductor-based mitigating agents reported in the literature. Based on the results in

Table 2. Removal efficiency of various HAB species using semiconductor-based mitigation agents, compared with Chi/TiO₂ (1 mL).

Mitigation Agent	HAB Species	Mitigation Approach	Removal Efficiency (%)	Lamp Intensity	Ref.
Fe2O3–TiO2 NPs	Chlorella vulgaris	Chemical	High removal rate of algal cells (99.8%) within 24 h was achieved under visible light irradiation.	55 W/m2	[9]
Ag/AgCl@ZIF-8 floating	Chlorophyll a M. aeruginosa Other algae	Physical	After 6 h of exposure to sunlight, the chlorophyll a degraded by 99.9%, Microcystis aeruginosa (92.6%) and biomass of the other algae decreased by about 80%.	Sunlight	[46]
Z-scheme g-C3N4-MoO3 (Mo-CN) composite photocatalysts	M. aeruginosa	Chemical	15Mo-CN achieved a removal efficiency of 97% for the algal cells after 3 h of visible light irradiation.	48.1 W/m2	[47]
γFe2O3/TiO2 nanoparticle	M. aeruginosa A. circinalis	Physical	Within 1 h, M. aerugonisa (99.99%) and A.cricinalis (95.49) was removed.	32 W/m2	[4]
Ag/AgCl@LaFeO3 (ALFO) photocatalyst	Phytoplankton	Chemical	ALFO-20% had a higher photocatalytic activity with a near 100% removal rate of chlorophyll a within 150 min.	10,000 W/m2	[48]
g-C3N4/Bi-TiO2 floating photocatalyst	M. aeruginosa	Physical	Within 6 h of visible light illumination, 75.9% of M. aeruginosa was removed.	NA	[10]
Z-scheme Ag3PO4@PANI core–shell photocatalyst	Microcystis aeruginosa	Chemical	99.2% was Microcystis aeruginosa was removed within 3 h.	NA	[11]
SNP-TiO2	Karenia mikimotoi	Chemical	Under visible light irradiation, 81.8% was removed within 96 h.	NA	[49]
ZnFe2O4/Ag3PO4/g-C3N4 (ZFO/AP/CN) photocatalyst	M. aeruginosa Microcystin-LR (MC-LR)	Physical	The photocatalytic removal of M. aeruginosa and MC-LR was 94.3% and 76.9%, respectively, under visible light.	NA	[11]
Floating BiOCl0.6I0.4/ZnO photocatalyst	Microcystis aeruginosa	Physical	The removal rate of chlorophyll a was 89.3% after 6 h of photocatalytic reaction under visible light.	42 W/m2	[50]
Chi/TiO2 (1 mL)	Alexandrium minutum	Physical	The removal of Alexandrium minutum was 76.1 ± 13.8% within 72 h.	70 μmol photons m2s−1	This study

, the RE value of Chi/TiO₂ (1 mL) is lower and relates to the high swelling rate of the film in seawater. In turn, the greater swelling contributes to the release of entrapped algae cells back to the surrounding aqueous environment. The intensity of the light source plays an important role in the generation of photogenerated electron/hole pairs. The intensity of the light used in this research was far lower (70 μmol photons m²s⁻¹), based on the 16:8 h light: dark cycle, compared with the cited studies. The light intensity in this research might not be enough to generate higher levels of photogenerated electron/hole pairs. Hence, this effect may account for the lower overall RE values reported herein. Regardless, the current study showed that Chi/TiO₂ (1 mL) displayed better RE values under solar light irradiation. Future studies are planned where higher levels of solar radiation will be used to study algal removal to better establish the deployment of this method for field-based applications.

2.3. Proposed Reaction Mechanism

One of the drawbacks of utilizing chitosan in seawater is the weakening of the netting and the bridging properties of the chitosan film, according to the high alkalinity and ionic strength of seawater [51,52]. Cross-linking of chitosan with TiO₂ will serve to strengthen its net-like structure for better adsorption of the algae cells. However, the adsorption of algae cells onto the films was limited due to repulsion forces between the negatively charged algae and the negatively charged film surface. Hence, it can be inferred that the mitigation process took place via a photocatalytic mechanism. The relationship between the conduction band (CB) and valence band (VB) potential of the TiO₂ was determined using Equation (1) to identify the ROS responsible for photocatalytic process.

$${\mathrm{E}}_{\mathrm{CB}}=\mathrm{X}-{\mathrm{E}}^{\mathrm{e}}-0.5{\mathrm{E}}_{\mathrm{g}}$$

(1)

E_CB is the CB energy, X is the geometric mean of the electronegativity of the constituent atoms (5.82), E^e is the energy of a free electron on the hydrogen scale (approximately 4.5 eV) and E_g is the bandgap energy of the semiconductor (TiO₂ = 3.03 eV). The VB potential was determined through the following Equation (2).

$${\mathrm{E}}_{\mathrm{VB}}={\mathrm{E}}_{\mathrm{CB}}+{\mathrm{E}}_{\mathrm{g}}$$

(2)

The CB potential of the TiO₂ (−0.2 V) was determined to be less negative than E° (O₂/O₂^•−) = −0.33 V vs. NHE, indicating that the electrons produced in the reaction will not react with the adsorbed O₂ to produce O₂^•−, which became channeled towards chitosan. The transfer of the photogenerated electrons to chitosan will prevent the electrons from combining with the holes. The VB potential of TiO₂ (2.38 V) was calculated to be higher than E° (^•OH/OH⁻) = +1.99 V vs. NHE indicating that the photogenerated h⁺ can oxidize OH⁻ into ^•OH. The generation of ^•OH is simplified by the illustration in Figure 5.

2.4. SEM and Digital Microscopy Analyses of the Films after Mitigation

The films after mitigation were subjected to SEM analysis to observe any structural changes of the films and the algae. As depicted in Figure 6a, the surface of the used Chi film became uneven with the appearance of distinctive hook-shaped particles which represent the apical pore complex of A. minutum. The surface of the Chi/TiO₂ films was constructed with irregularly shaped particles after the mitigation process, which may relate to the attachment of the algae cells (Figure 6b–d). This trend is in agreement with the SEM images before (Figure 2) and after (Figure 6) the photochemical treatment of the algae.

The used films were further analyzed using a digital microscope, where the corresponding images revealed that the algae cells attached to the Chi film were ruptured (shown in red arrow) (Figure 6e). The presence of positively charged amine groups of chitosan promote electrostatic interaction with the negatively charged algal cells, which contribute to cell rupture [53,54]. The algae cells in Figure 6f–h show that the cells were preserved after the mitigation process. The seawater systems have elevated ionic strength that hinders the unique surface characteristics of amorphous TiO_2, which prevents rupture of the algae cells [55]. The absence of rupturing relates to charge screening effects due to the presence of sufficient ionic strength in the aqueous media.

2.5. Physical Appearance of Used Film Studies and Weight Change after Mitigation

The films were observed to undergo rupture and have a gel-like texture from absorbing the seawater during the mitigation studies. The weight of the dried use films decreased after mitigation suggesting that some of the films may have disintegrated during the process. The physical appearance of fresh and used films and their weight change after mitigation are given in Figure S1 and Table S1 (cf. Supplementary Materials).

3. Materials and Methods

3.1. Materials

Sodium hydroxide (NaOH) pellets (Qrec, 99%, Rawang, Malaysia), glacial acetic acid (Qrec, 100%, Rawang, Malaysia) and chitosan powder (Sigma-Aldrich, medium molecular weight, product id: 448877, St. Louis, MO, USA) were used without further purification. The anatase TiO₂ nanoparticle solution (0.2% w/v) was obtained from Prof. Ir. Dr. Srimala Sreekantan from the School of Materials & Mineral Resources, Engineering Campus, Universiti Sains Malaysia, Penang. The TiO₂ nanoparticles were prepared according to the method reported by N.H. Ahmad Barudin et al. [53], along with characterization of the physicochemical properties of the anatase TiO₂. The filtered seawater was supplied by Fisheries Research Institute (FRI), Batu Maung, Penang, Malaysia. The seawater pH before and after film immersion was recorded using a pH meter (Model Hanna edge^pH), where the seawater remained constant at pH~6.8.

3.2. Preparation of Hybrid Chitosan-Modified TiO₂ Film

A chitosan solution (2% w/v) was prepared by dissolving chitosan powder (2 g) in 100 mL of acetic acid solution (1% v/v). The solution was stirred for 4 h at 50 °C followed by centrifugation at 4000 rpm for 15 min. The solution was then filtered to remove any undissolved chitosan powder. Different dosages (1 mL, 3 mL and 5 mL) of 0.2% w/v of anatase TiO₂ were added into the chitosan solution and stirred for another 2 h to minimize the formation of bubbles and to form a homogeneous solution. The film-forming solution was then poured into a square-shaped Teflon mold, followed by oven-drying for 21 h at 50 °C. A NaOH solution (2% w/v) was prepared by dissolving 10.0 g of NaOH pellets in 500 mL distilled water. The dried film was soaked in 2% w/v NaOH aqueous solution for a minute and then rinsed with distilled water to neutralize it. The film was then dried at room temperature for 24 h and kept in a desiccator for further use. The films were labelled as Chi (chitosan film) and Chi/TiO₂ (x mL; x = 1, 3 and 5 mL)

3.3. Characterization

3.3.1. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy

The ATR-FTIR spectrophotometer (Perkin Elmer FT-NIR spectrometer with Universal ATR sampling accessory, Waltham, MA, USA) was used to identify the film’s functional groups. The spectra of the films were analyzed over the spectral range of 4000 to 600 cm⁻¹ with 64 scans.

3.3.2. Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDX)

The surface topology, cross-section and elemental composition of the films were analyzed using SEM-EDX analysis (SEM Leica Cambridge S360-EDX Falcon System, Cambridge, UK). The films were cut into 1 cm × 1 cm prior to being fixed onto a stub containing carbon adhesive and sputtered with gold for 10–15 min in an airtight sputter coater (Armetech, Ozolnieki, Latvia) to sharpen the SEM images.

3.3.3. Thermogravimetric Analysis (TGA)

The changes in the mass of the films in relation to the changes in temperature were determined using a TGA analyzer (Perkin Elmer). Approximately 6 mg of the films was heated from 30 to 600 °C at 10 °C min⁻¹ under a nitrogen flow (50 cm³/min).

3.3.4. X-ray Diffraction (XRD)

A Bruker-D8 Advance Powder X-ray diffraction (XRD) (Billerica, MA, USA) was used to determine the crystalline phases and the degree of crystallinity of the films. The diffractogram was obtained for each film at 2θ angle of 5° to 50°. The diffractometer was equipped with CuKα radiation, λ = 0.1541 nm, voltage = 40 kV and current = 30 mA.

3.3.5. Swelling Index (SI)

The swelling index (SI) of the films was estimated by submerging the pre-weighed dry films into 100 mL of seawater for 24 h at room temperature. After 24 h, the swollen films were removed, wiped with filter paper to remove residual excess water and then weighed. The SI was calculated using Equation (3) as reported by Sabzevari et al. [54]. Three films were tested at each film composition to obtain the average swelling index.

$$\mathrm{SI}\left(\%\right)=\frac{\left({\mathrm{W}}_{\mathrm{f}}-{\mathrm{W}}_{\mathrm{i}}\right)}{{\mathrm{W}}_{\mathrm{i}}}\times 100$$

(3)

W_f is the weight of the swollen film after 24 h and W_i is the weight of dry film before being submerged in the aqueous media.

3.3.6. Wettability Test (WS)

The film’s wettability or water contact angle (CA) was tested via the static CA using a goniometer (Ramé-Hart Instrument Co., Succasunna, NJ, USA) based on the Sessile drop method. Deionized water (4 µL) was dropped using a microsyringe onto the smooth surface of the film at room temperature. Then, a microscope was used to capture the micrograph images. This step was repeated for five different spots of the membrane sample to calculate the average CA.

3.3.7. Point of Zero Charge (pzc)

The surface charge of the films was performed using the reported method by Shah et al. with some modification [33].Firstly, 100 mL of distilled water was poured into six 200 mL beakers. The initial pH of the distilled water (pH_i) was adjusted from pH 2 to 12 using 0.1 M HCl and/or 0.1 M NaOH solution. Then, the film was submerged into each beaker and shaken for 24 h at a shaking rate of 250 rpm. The final pH of the distilled water (pH_f) was recorded. The difference in pH values (∆ pH) was calculated using Equation (4):

$$\Delta \mathrm{pH}={\mathrm{pH}}_{\mathrm{f}}-{\mathrm{pH}}_{\mathrm{i}}$$

(4)

Finally, the ∆ pH value was plotted against the pH_i to determine the intersection point at ∆ pH zero to estimate the pzc. This pH indicates the surface charge density of the film.

3.3.8. Mitigation of Alexandrium Minutum

A. minutum cells were grown in ES-Dk medium at 25 °C under a light intensity of 70 μmol photons m²s⁻¹ using a 16:8 h (light:dark) photocycle [55]. Filtered natural seawater diluted to 15 ppt was used as solution medium for A. minutum culture. The cell removal experiments were performed when the culture reached the exponential growth phase. Cultures of A. minutum with a cell concentration of 2 × 10⁴ cell/mL were used for the experiments.

The mitigation of A.minutum was carried out via physical method in a 250 mL beaker that contained 100 mL of the cell culture that was placed on a table in a static condition. The film was hung so that ¾ of its dimension was submerged into the reaction medium. Approximately 1 mL of the sample from 2 cm below the solution surface was collected and preserved with one drop of Lugol’s solution at each specific time interval. The preserved cells were counted using the Sedgewick-rafter counter under a light microscope (Leica CME, Wetzlar, Germany) at 10× magnification. The change in the structure of the cells on the surface of the films was observed under the digital microscope (Keyence VHX E-100, Osaka, Japan). The removal efficiency (RE) was calculated using Equation (5) [56]. All the RE data were expressed as the mean ± standard deviation (S.D.).

$$\mathrm{RE}\left(\%\right)=1-\left[\frac{\mathrm{Final}\mathrm{cell}\mathrm{concentration}\mathrm{in}\mathrm{sample}}{\mathrm{Final}\mathrm{cell}\mathrm{concentration}\mathrm{in}\mathrm{control}}\right]\times 100$$

(5)

Upon completion of the mitigation process, the films were separated for further characterization using SEM and a digital microscope.

3.3.9. Statistical Analysis

The statistical analysis was performed using GraphPad Prism 5 version 5.01. The obtained data were expressed as the mean ± standard deviation of the triplicate measurements. The distinction between the experimental groups was evaluated using a one-way variance (ANOVA) analysis. A p-value less or equal to 0.05 was estimated to be statistically significant.

4. Conclusions

Hybrid chitosan-modified TiO₂ thin films (CH/TiO₂ (x mL; x = 1, 3 and 5 mL)) were successfully synthesized via solvent casting method for the mitigation of A.minutum in a lab-scale experimental setup. The negatively charged surface of the films repelled the negatively charged algae cells and caused the RE values (%) to fluctuate. The CH/TiO₂ (1 mL) was able to remove 76.1 ± 13.8% of algae cells within 72 h due to its ability to generate hydroxyl radicals which can cause oxidative stress to the adsorbed and free algae. The results obtained from this study showed that the hybrid chitosan-modified TiO₂ film can be used to mitigate harmful algal blooms. In particular, future studies are planned to study the effect of higher levels of solar radiation on the efficacy of algal removal in order to establish this method for field-based applications. Further studies are also required to determine the suitability of this mitigation method in marine-based environments to determine the effect of such materials towards other marine biota.