This study was conducted at the Membrane Materials and Water Treatment Laboratory, School of Environmental Science and Engineering, Suzhou University of Science and Technology (SUST), located in Suzhou, Jiangsu Province, China. The laboratory provides advanced facilities for membrane fabrication, surface modification, and performance evaluation under controlled conditions. Suzhou lies in the Yangtze River Delta region (30˚47' - 32˚02'N, 119˚55' - 121˚20'E), an area characterized by extensive water networks and proximity to Taihu Lake. The region experiences a subtropical monsoon climate, with average temperatures of 15˚C - 17˚C and annual rainfall of 1000 - 1200 mm. These warm and humid conditions promote microbial growth, making membrane fouling a significant challenge in water treatment systems. The study area is highly relevant due to water quality issues such as organic pollution, oil-water emulsions, natural organic matter, and microbial contamination associated with rapid industrialization and urbanization. Although experiments were conducted in the laboratory, they simulated real-world conditions using common foulants such as BSA, humic acid, oil emulsions, and bacteria.

All chemicals used in this study were of analytical grade and used as received without further purification. Pullulan was purchased from Macklin Biochemical Co., Ltd. (China), while hydrophobic polyvinylidene fluoride (PVDF) membranes (0.45 µm pore size, 47 mm diameter), sodium periodate (NaIO₄), sodium hydroxide (NaOH), calcium chloride (CaCl₂), and ethylene glycol were obtained from Tansoole. Branched polyethyleneimine (PEI) with an average molecular weight of approximately 25,000 Da was supplied by Sigma-Aldrich (USA). Deionized water was used throughout the study for solution preparation, oxidation reactions, membrane coating, and washing procedures.

The experimental work was conducted at the Membrane Materials and Water Treatment Laboratory, School of Environmental Science and Engineering, Suzhou University of Science and Technology (SUST), Suzhou, Jiangsu Province, China. The study focused on the synthesis of oxidized pullulan (O-Pul), fabrication of O-Pul/PEI-coated PVDF membranes, and evaluation of the influence of PEI concentration on membrane surface modification.

Membrane characterization was performed using several analytical techniques. Fourier Transform Infrared Spectroscopy (FTIR) was used to identify functional groups and confirm chemical modifications on the membrane surface, while X-ray Photoelectron Spectroscopy (XPS) was employed to determine surface elemental composition and chemical states. Scanning Electron Microscopy (SEM) was used to examine membrane surface morphology and coating uniformity, and a contact angle goniometer was utilized to evaluate membrane hydrophilicity by measuring water contact angles. Standard laboratory glassware, including beakers, was used for solution preparation, oxidation, and dip-coating processes, whereas Petri dishes were employed for antibacterial evaluation through zone-of-inhibition tests.

The scope of the investigation was limited to laboratory-scale membrane fabrication and physicochemical characterization. Performance evaluations focused on membrane surface properties, including wettability, coating formation, antifouling behaviour, and antibacterial activity, rather than pilot-scale or full-scale water treatment applications. This controlled laboratory approach enabled systematic investigation of the effects of PEI concentration on the structure and surface performance of O-Pul/PEI-coated PVDF membranes.

The aldehyde content and degree of oxidation (DO) of oxidized pullulan (O-Pul) were determined using the hydroxylamine hydrochloride titration method, a widely accepted technique for quantifying aldehyde groups in dialdehyde polysaccharides ([7]). In this method, aldehyde groups in O-Pul react quantitatively with hydroxylamine hydrochloride to form oximes, releasing an equimolar amount of hydrochloric acid (HCl), according to the following reaction:

R − CHO + NH₂OH∙HCl → R − CH = NOH + HCl + H₂O

The liberated HCl was titrated with standardized sodium hydroxide (NaOH), and the volume of NaOH consumed was used to determine the aldehyde content of the oxidized polymer.

The aldehyde content (mmol∙g⁻¹) was calculated using:

Aldehyde Content= (V_s − V_b) × CNaOH/m

where (V_s) is the volume of NaOH consumed by the sample (L), (V_b) is the volume consumed by the blank (L), (CNaOH = nNaOH/VNaOH) is the NaOH concentration (mol∙L⁻¹), and (m) is the mass of O-Pul (g). The results were expressed as millimoles of aldehyde per gram of O-Pul (mmol∙g⁻¹).

The degree of oxidation (DO) was calculated by relating the number of aldehyde groups formed to the number of pullulan anhydroglucose units (AGU; molecular weight = 162 g∙mol⁻¹), assuming a theoretical maximum of two aldehyde groups per oxidized AGU:

DO= VNaOH × NNaOH/2 × (m/162)

The degree of oxidation was expressed either as mol aldehyde per mol AGU or as percentage oxidation:

DO (%) = DO × 100

The filtration and antifouling performance of the pristine and O-Pul/PEI-modified PVDF membranes were evaluated using a laboratory-scale dead-end filtration system with an effective membrane area of 33.18 cm² (0.003318 m²). Prior to testing, each membrane was compacted with deionized water at 1.5 bar for 30 min to stabilize the membrane structure and permeate flux. The operating pressure was subsequently reduced to 1.0 bar, and the pure water permeate flux was determined using:

J= ΔV/A × Δt

where J is the permeate flux (L∙m⁻²∙h⁻¹), ΔV is the permeate volume (L), A is the effective membrane area (0.003318 m²), and Δt is the filtration time (h).

The antifouling properties of the membranes were assessed using 1.0 g∙L⁻¹ bovine serum albumin (BSA) as a model protein foulant in a cross-flow filtration system operated at 0.15 MPa. The initial pure water flux (J_w1) was measured after membrane compaction, followed by filtration of the BSA solution to obtain the fouling flux (J_p). Flux values were recorded at 5-minute intervals until a steady state was achieved. After fouling, the membranes were rinsed thoroughly with distilled water for 30 min, and the recovered pure water flux (J_w2) was measured to evaluate membrane cleaning efficiency.

Membrane fouling behaviour was assessed using the normalized flux, flux recovery ratio (FRR), total fouling ratio (R_t), reversible fouling ratio (R_r), and irreversible fouling ratio (R_ir), calculated using the following equations:

Normalized Flux= J_p/J_w1

F_rr = J_w2/J_w1 × 100%

R_t = (J_w1 − J_f)/J_w1 × 100%

R_r = (J_w2 − J_f)/J_w1 × 100%

R_ir = (J_w1 − J_w2)/J_w1 × 100%

where J_w1 is the initial pure water flux before fouling, J_p is the flux during BSA filtration, and J_w2 is the pure water flux after membrane cleaning.

The O-Pul/PEI-modified PVDF membranes were expected to exhibit enhanced filtration performance compared with pristine PVDF due to the formation of a hydrophilic coating that promotes water transport, minimizes protein adsorption, and suppresses irreversible fouling. Consequently, the modified membranes are anticipated to demonstrate higher permeate flux, greater flux recovery, and lower irreversible fouling, indicating improved long-term operational stability for water treatment applications.

The study focused on the fabrication and surface modification of polyvinylidene fluoride (PVDF) membranes using oxidized pullulan (O-Pul)/polyethyleneimine (PEI) coatings to improve membrane hydrophilicity, antifouling behaviour, antibacterial properties, and surface stability for potential water treatment and wastewater reuse applications. All experimental work was conducted under laboratory conditions at the Membrane Materials and Water Treatment Laboratory, Suzhou University of Science and Technology (SUST), China.

2.5.1. Membrane Pretreatment and Fabrication

Pristine hydrophobic PVDF membranes (0.45 µm pore size, 47 mm diameter) were first immersed in ethanol for 30 min to remove residual contaminants, thoroughly rinsed with deionized water, and designated as M0 (Figure 1). Surface modification was then carried out using a simple two-step dip-coating method. The pretreated membranes were immersed in a 2.5 wt.% O-Pul solution for 1 min at room temperature and immediately transferred into PEI solutions with concentrations of 0.2, 0.3, 0.4, and 0.5 wt.% for an additional 1 min. The coated membranes were dried at 40˚C for 15 min to stabilize the crosslinked coating layer and subsequently immersed in deionized water for more than 24 h, with the washing solution replaced every 12 h to remove unreacted chemicals. The resulting membranes were designated M1, M2, M3, and M4, corresponding to increasing PEI concentrations.

Figure 1. Membrane fabrication and characterization.

2.5.2. Synthesis of Oxidized Pullulan (O-Pul)

Oxidized pullulan was synthesized by sodium periodate oxidation. Briefly, 4 g of pullulan was dissolved in 70 mL of deionized water at 40˚C, and the solution pH was adjusted to 4.0. A sodium periodate solution (1.2 g in 30 mL deionized water) was then added, and the reaction mixture was stirred in the dark at 40˚C for 4 h. Oxidation was terminated by adding ethylene glycol, after which anhydrous CaCl₂ was introduced to precipitate calcium iodate. The precipitate was removed by filtration, and the product was purified by dialysis followed by freeze-drying to obtain O-Pul. Successful oxidation and aldehyde formation were confirmed using FTIR, XPS, and aldehyde titration (Figure 2).

Figure 2. The FTIR spectrum of synthesized Pul, O-Pul.

2.5.3. Crosslinking and Membrane Characterization

The O-Pul coating was crosslinked with branched PEI (Mw ≈ 25,000 Da) through Schiff-base (imine) formation and Michael-type reactions between aldehyde and amine groups, producing a stable hydrophilic coating on the PVDF membrane surface (Figure 3). The influence of PEI concentration on coating formation and membrane performance was investigated.

The modified membranes were characterized using Scanning Electron Microscopy (SEM) to examine surface and cross-sectional morphology, Fourier Transform Infrared Spectroscopy (FTIR) to identify functional groups and confirm chemical bonding, and X-ray Photoelectron Spectroscopy (XPS) to determine surface elemental composition and verify successful coating formation. Membrane hydrophilicity was evaluated using a water contact angle (WCA) goniometer, while coating stability was assessed under sonication, varying pH, saline conditions, and thermal stress. These characterization techniques provided comprehensive information on coating morphology, chemical composition, wettability, and stability, enabling evaluation of the effects of PEI concentration on the physicochemical properties and surface performance of the modified PVDF membranes.

Figure 3. Dip coating of pristine PVDF into O-Pul/PEI solution.

FTIR confirmed successful pullulan oxidation (new 1733 cm⁻¹ band) and formation of the crosslinked network (broad 3400 - 2800 cm⁻¹ band from O-H/N-H overlap; new bands at ≈1472 and 1560 cm⁻¹ linked to N-H and C-N vibrations), consistent with Schiff-base formation ([33]; [13]). XPS showed the emergence of O1s and N1s peaks on coated surfaces and increasing C=O/C-O contributions with coating ratio, while high-resolution N1s spectra revealed −NH/−NH₂, C-N, and C=N species (evidence of covalent crosslinking). SEM images displayed a thin, uniform coating layer for intermediate PEI concentrations (e.g., M3, 0.4 wt.%), whereas excessive PEI led to poorer adhesion and the reappearance of pores likely due to homogeneous crosslinking in solution rather than on the surface, indicating an optimal PEI window for in-situ coating formation ([48]; [65]) Collectively, chemical and morphological analyses demonstrate that the controlled oxidation of pullulan yields reactive aldehyde groups and that PEI crosslinking produces a stable network on substrates ([43]; [2]). The spectroscopic signatures (C=N and C-N formation) and XPS elemental changes indicate successful surface chemistry modification, rather than mere physisorption, which explains the improved durability in subsequent stability tests ([36]; [60]).

The surface morphology of the pristine PVDF membrane and those coated with various modifications was examined using SEM, focusing on both the top surface and cross-sectional views. As shown in Figures 4(a1)-(a3), the pristine PVDF membrane displays a porous structure with a smooth framework. However, its top surface is irregular, which negatively affects its anti-fouling capabilities. In contrast, after the deposition of O-Pul and cross-linking with PEI on the membrane surface, noticeable differences among the coated membranes are apparent in Figures 4(b1)-(e3) as the PEI concentration varies. For M1, the low PEI concentration used as the cross-linking agent causes minimal changes in the membrane’s morphology, which is also reflected in the subsequent water flux measurements. As the O-Pul concentration remains steady and the PEI concentration increases, noticeable changes in the surface pore structure of M3 can be seen. The modifier successfully adheres to the original PVDF membrane’s skeleton without altering its basic structure at a PEI concentration of 0.4 wt.%. Figure 4(d2) shows that the surface of the modified membrane M3 no longer has visible pores. Additionally, Figure 2(d3) presents the SEM cross-sectional image of a thin, smooth, and uniform modified layer firmly bonded to the PVDF membrane after co-deposition of O-Pul and PEI. This confirms that the PVDF membrane surface has been effectively coated with O-Pul/PEI ([59]; [8]).

The likely reason for this is the formation of covalent bonds between the aldehyde groups of O-Pul and the amine groups of PEI through Michael addition and Schiff base reactions under the applied conditions. This strong interaction ensures the stable attachment of the O-Pul/PEI coating to the PVDF membrane, enhancing its structural integrity and anti-fouling properties. ([37]). However, in Figures 4(e1)-(e3), distinct pore structures reappear on the modified membrane. This phenomenon may be attributed to the high concentration of PEI. When the membrane, which had already undergone the first deposition of O-Pul, is immersed in the PEI solution, the rapid reaction rate between O-Pul and PEI causes the cross-linked material to form in the aqueous solution rather than firmly adhering to the membrane surface. As a result, the cross-linked material fails to stabilize on the membrane, leading to the reappearance of pores on the modified membrane surface. This suggests that an excessively high PEI concentration can hinder the effective formation of a stable coating, emphasizing the importance of optimizing the PEI concentration for successful membrane modification ([10]; [34]; [23]).

Selection of 0.4 wt.% PEI (M3) as the optimum coating concentration was based on an overall qualitative assessment of membrane surface characteristics, including coating continuity, uniformity, adhesion, and structural integrity, rather than surface morphology alone. M1 (0.2 wt.% PEI) exhibited limited surface modification because the PEI concentration was insufficient to form a continuous crosslinked O-Pul/PEI layer. M2 (0.3 wt.% PEI) showed improved coating coverage; however, noticeable surface openings remained, indicating incomplete membrane coverage. In contrast, M3 (0.4 wt.% PEI) produced the most uniform, continuous, and well-adhered coating while preserving the underlying porous PVDF support structure, suggesting an optimal balance between crosslinking and surface deposition. At the highest concentration, M4 (0.5 wt.% PEI) exhibited partial pore reappearance and reduced coating stability, likely because excessive PEI accelerated bulk-solution crosslinking instead of controlled interfacial film formation. Consequently, 0.4 wt.% PEI (M3) was selected as the optimum formulation, as it provided the best balance between coating coverage, surface uniformity, membrane structural preservation, and expected filtration and antifouling performance.

Figure 4. SEM images of the various modified and unmodified membranes.

In addition, the successful preparation of the coated membranes was further confirmed through surface physicochemical characterization using FTIR and XPS. The FTIR spectra of Pul and O-Pul are shown in Figure 5. Compared to Pul, O-Pul exhibits a characteristic peak at 1733 cm⁻¹, corresponding to the aldehyde (C=O) group. This peak confirms the successful aldehyde modification of Pul, indicating the introduction of aldehyde functional groups into the O-Pul structure. This modification is crucial for the subsequent cross-linking reactions with PEI, as the aldehyde groups enable the formation of covalent bonds with the amine groups of PEI through Schiff base reactions. The presence of this peak in the FTIR spectrum provides strong evidence for the chemical alteration of Pul and supports the effectiveness of the coating process ([33]; [54]; [45]). The test results confirm the successful introduction of aldehyde groups into pullulan. The FTIR spectra of the pristine PVDF membrane and the O-Pul cross-linked PEI-modified membrane (M3) are shown in Figure 4. Compared to the spectrum of the original PVDF membrane, the spectrum of M3 exhibits a broad peak in the range of 3400 - 2800 cm⁻¹, which is likely due to the overlapping stretching vibrations of hydroxyl (O-H) and amine (N-H) groups. Additionally, a new absorption peak at 1472 cm⁻¹ corresponds to the N-H vibration, while a new peak at 1560 cm⁻¹ is attributed to the C-N bond. These observations indicate that the amine groups in PEI and the aldehyde groups in O-Pul have formed chemical cross-links through Schiff base reactions, successfully coating the PVDF membrane ([25]; [1]; [21]).

Figure 5. The FTIR spectrum of Pul, O-Pul, and M3.

As shown in

Figure 6

, compared to the XPS spectrum of the pristine PVDF membrane, which only shows C and F signal peaks, the XPS spectrum of the coated membrane reveals two new peaks corresponding to O 1s and N 1s. With increasing coating ratios, the high-resolution C1s spectrum shows a significant decrease in the area of the C-F bond while the areas of the C=O and C-O bonds steadily increase. Furthermore, the N1s signal peak of the coated membrane M3 was fitted using the Gaussian-Lorentzian algorithm, and the high-resolution XPS spectrum is presented in

Figure 6

. It was observed that the surface of the O-Pul and PEI-coated membrane primarily contains −NH/−NH

2

, C-N, and C=N bonds (

Zhang et al., 2017

). The formation of the C=N bond is likely due to the Schiff base reaction between the aldehyde groups of O-Pul and the amine groups of PEI. These findings align with the FTIR results, confirming that O-Pul and PEI were effectively coated on the membrane surface (

Xie et al., 2023

;

Bandara et al., 2019

;

Teng, 2023

). These observations demonstrate that the dip-coating method is effective, rapid, and compatible with mild conditions. Performance, including hydrophilicity, flux, and fouling resistance, correlates strongly with coating morphology. A continuous film (M3) exhibits the most pronounced hydrophilic effect and superior antifouling performance with coatings that are too thin (M1) or poorly adhered (M4) underperform (

Xie et al., 2023

;

Teng, 2023

).

Figure 6. XPS spectrum comparing pristine PVDF with the O-Pul/PEI-coated membrane (M3).

The water contact angle (WCA) and water flux bar charts are consistent (Figure 7). Generally, enhanced hydrophilicity ultimately improves the anti-fouling efficiency of the membrane ([31]). This is because a more hydrophilic surface tends to form a hydration layer, which repels foulants and reduces their adhesion to the membrane surface ([31]). As a result, membranes with lower water contact angles (higher hydrophilicity) typically exhibit higher water flux and better anti-fouling performance, as demonstrated by the data in the bar charts. This correlation highlights the importance of surface hydrophilicity in designing membranes with superior anti-fouling properties ([29]). The wettability of the membranes was investigated by measuring their water contact angles (WCA). The figure shows the different contact angles of the various membranes. The pristine PVDF membrane maintains a water contact angle of approximately 97.5˚, indicating its strong hydrophobic nature. However, as the concentration of the cross-linking agent PEI increases, water droplets on the coated membranes rapidly spread into the membrane, causing the water contact angle to decrease quickly and reach zero within ten seconds. This phenomenon is likely due to the synergistic effect of the hydrophilic layers containing hydroxyl and amine groups from O-Pul and PEI ([32]). Additionally, these results suggest that increasing the PEI concentration promotes stable Schiff base cross-linking of the copolymer on the membrane surface, enabling effective coating on the PVDF membrane. This demonstrates the successful enhancement of membrane hydrophilicity through the O-Pul/PEI modification process.

Figure 7. A Graph showing the Water contact angle (˚) result of the modified and Unmodified membranes and water flux test (L∙m⁻²∙h⁻¹).

The antifouling performance of membranes is a critical factor in evaluating their practical applications. As shown in Figure 8, three different pollutants-bovine serum albumin (BSA), humic acid (HA), and sodium alginate (SA) were used as simulated wastewater to assess the antifouling performance of the membranes. Compared to the pristine PVDF membrane, all coated membranes exhibited significantly higher flux recovery ratios (FRR). This improvement is likely due to the introduction of a hydrophilic coating and the enhancement of surface smoothness. Generally, surface roughness and hydrophilicity influence the deposition of pollutants and, consequently, the antifouling performance of membranes ([56]; [3]). The coated membrane surfaces are covered with a hydrophilic cross-linked layer of O-Pul and PEI, which readily binds with water to form a hydration layer. This hydration layer can repel BSA molecules. As observed in previous SEM images, the surface of the coated membranes becomes smoother with changes in the coating ratio, which also contributes to the repulsion of pollutants.

In the case of BSA as a simulated pollutant, the flux of all membranes decreased at the beginning of filtration. This is likely because BSA was separated by the membrane and deposited on the surface, thereby blocking the membrane pores ([56]). As BSA filtration continued, a balance was gradually reached between the deposition of BSA on the membrane surface and its detachment from the deposited layer ([56]), leading to a stabilization of the permeate flux. Furthermore, the flux decline was more pronounced during HA filtration, possibly because HA molecules are smaller than BSA and, thus, more prone to blocking membrane pores. However, this had little impact on the subsequent cleaning process. During SA filtration, SA tends to aggregate and precipitate, forming a gel layer that is difficult to remove through ordinary water rinsing. As a result, SA’s flux recovery ratio was relatively lower than the other two pollutants.

Figure 8. Graphical simulation of wastewater to assess the antifouling performance of the membranes using Bovine serum albumin (BSA), humic acid (HA), and sodium alginate (SA).