Development of a novel polyethersulfone ultrafiltration membrane with antibacterial activity and high flux containing halloysite nanotubes loaded with lysozyme

Abstract

In this study, halloysite nanotubes (HNTs) were used to immobilize lysozyme via a covalent binding reaction. Immobilized lysozyme (HNTs–Ly) was then added to a polyethersulfone (PES) polymer solution to prepare hybrid antibacterial ultrafiltration membranes via classic phase inversion. The results showed that the surface hydrophilicity and the water flux of the hybrid membranes were significantly improved after adding HNTs–Ly. When the content of HNTs–Ly was 3.0 wt%, the water flux of the resultant membranes could achieve values as high as 400 L m⁻² h⁻¹ and maintain higher rejections for PEG 20 000 (69%) and PVA 30 000–70 000 (99.6%). The tensile strength and the elongation at the break of the hybrid membranes were increased after adding HNTs–Ly, which revealed that the mechanical strength of the membranes was also enhanced. Moreover, the hybrid membrane showed a good antibacterial activity against Gram-negative bacteria (E. coli) with a high bacteriostasis rate of 63%.

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Introduction

Ultrafiltration membranes, one of the new developments in chemical engineering, have been playing a momentous role in the removal of low concentration pollutants in wastewater treatment, preparing ultra pure water, concentrating, separating and purifying medicine, and food processing because of their low energy consumption and selective separation.^1–6 Polyethersulfone (PES) is always used to prepare ultrafiltration membranes on account of its excellent chemical and thermal stability, oxidation resistance and mechanical strength.^7,8 Nevertheless, the inherent hydrophobic character of pure PES ultrafiltration membranes often leads to fouling during the separation process, which reduces productivity, shortens membrane life, and changes membrane selectivity.⁹ Plenty of studies have been devoted to improving the ability of membranes to resist protein-fouling and biofouling. For protein-fouling, the commonly used approach is to increase the hydrophilicity and reduce the roughness of the membrane surface.¹⁰ Moreover, associated efforts to decrease biofouling always include the introduction of antibacterial groups to the membrane matrix or utilization of antibacterial polymers into the membrane material.¹¹ To date, various types of methods have been adopted to fabricate antibacterial membranes such as coating,¹² surface grafting polymerization¹³ and blending modification.¹⁴ Several antibacterial agents have been used to fabricate antibacterial hybrid membranes via blending modification because of easy operation, mild condition and neglectful influence for the surface and the cross-section structure of the membranes.^2,15–18 However, the resulting high-cost and slow response meant that the use of inorganic antibacterial agents was somewhat limited their applications. Organic antibacterial agents (PHMG and PVP) were selected as alternative candidates owing to their high antibacterial activity; however, disadvantages associated with these compounds include poor chemical and thermal stability as well as the development of drug-resistant bacteria.^7,19 Compared with inorganic and organic antibacterial agents, natural antibacterial agents, like chitosan, chitin and many enzymes, are better for the environment and for human health.^20–22

Enzymes are biocatalysts, which are highly active, selective and specific. Herein, lysozyme belongs to the hydrolase family, a group of enzymes which destroy the cell walls of microbes, making it a good candidate in antibacterial applications. Nevertheless, the reaction condition of enzymes is so rigorous that many ways to improve enzyme features during the chemical processes are needed, such as the mildest experimental and environmental conditions. If the conditions are reasonably designed, it will improve almost all enzyme properties.²³ Moreover, the direct addition of enzymes to a membrane matrix is less efficient because of the loss of enzyme,²⁴ whereas immobilization of enzymes could outstandingly enhance their stability.²⁵ Saeki et al.²² prepared a polyamide reverse osmosis (RO) membrane through the covalent immobilization of enzymes, which decreased the water flux but maintained the salt rejection ratio, and retained sufficient antibacterial activity against the Gram-positive bacteria, Micrococcus lysodeikticus and Bacillus subtilis. Wang et al.²⁶ reported the successful immobilization of lipase enzyme in fibers via the electrospinning of an aqueous mixture of lipase and polyvinyl alcohol (PVA). It was confirmed that the catalytic activity of the immobilized enzyme was the same as that of the crude enzyme.

Halloysite nanotubes (HNTs), a type of aluminosilicate clay, possess a unique hollow and lathy tubular structure consisting of a silicon–oxygen tetrahedron on the external surface and an aluminum–oxygen octahedron on the internal surface. The superior physical and chemical properties of HNTs comprising a high length–diameter ratio and a large specific surface area have attracted increasing attention in a wide field.^27,28 Chen et al.²⁹ synthesized halloysite nanotubes–chitosan–Ag nanoparticles (HNTs–CS@Ag) and then blended them with PES to prepare an antibacterial PES ultrafiltration hybrid membrane, which showed good antibacterial activity against Escherichia coli and Staphylococcus aureus. HNTs also exhibited environmentally safe properties. Fakhrullina et al.³⁰ have studied the toxicity of halloysite clay nanotubes in vivo by employing Caenorhabditis elegans nematode and found that HNTs were not capable of severely damaging the nematode organism.

Herein, HNTs were functionalized with carboxylic groups (HNTs–COOH) and then lysozyme was covalently immobilized onto the surface of modified HNTs. Subsequently, a novel antibacterial PES ultrafiltration hybrid membrane was prepared via blending with HNTs–Ly through a phase inversion method. These hybrid membranes were expected to display good antibacterial properties in water treatment.

Experimental

Materials

Polyethersulfone (PES, WM = 58 kDa) was supplied by BASF Company, Germany. Halloysite nanotubes (HNTs) were refined from clay minerals in Henan province, China. 1,6-Hexamethylene diisocyanate (HMDI), dibutyltin dilaurate, lysozyme and fluorescein isothiocyanate were purchased from J&K. Butanedioic anhydride was purchased from Shanghai Chemical Reagent Research Institute. All the other chemicals (analytical grade) were obtained from Tianjin Kermel Chemical Reagent Co., Ltd., China and were used without further purification. The test strains, E. coli (8099), used for this study were provided by the College of Public Health of Zhengzhou University. The water used is deionized water.

Synthesis of HNTs–Ly

The grafting of isocyano on HNTs (HNTs–NCO). Powders of HNTs were obtained by milling, sieving and then drying in a muffle furnace at 573 K for 12 h. HNTs powders (2 g) were dispersed into acetone (60 g) and treated with ultrasound for 1 h to form a suspension of HNTs. Thereafter, HMDI (1 g) was added to the abovementioned suspension and then dibutyltin dilaurate (0.1 g) was added and stirred for 4 h at 70 °C under reflux conditions under a nitrogen atmosphere. After cooling to room temperature, the obtained pink powder was collected by filtering, washing with acetone for several times and drying at 50 °C under vacuum overnight.

The grafting of carboxyl on HNTs–NCO (HNTs–COOH). As-prepared HNTs–NCO (2 g) was dispersed in acetone (60 g) and treated with ultrasound for 1 h, and then butanedioic anhydride (3 g) and dibutyltin dilaurate (0.1 g) were added under a nitrogen atmosphere to react at 70 °C for 3 h under constant stirring and reflux conditions. At last, the resulting precipitate was collected by filtration, washed with ethyl alcohol 3 times, and dried in a vacuum oven at 50 °C for 24 h.

The immobilization of lysozyme on HNTs–COOH. EDC (5 mg) and NHS (6 g) were added to 100 mL of phosphate buffer (0.2 M, pH = 6.2), then HNTs–COOH (0.3 mg) was dispersed into the aforementioned solution with the assistance of ultrasound at room temperature for 30 minutes. Lysozyme (120 mg) was added to a HNTs–COOH suspension in phosphate buffer and shaken continuously at 200 rpm for 24 h at 4 °C. The precipitate was acquired by centrifugation, then washed with phosphate buffer several times and freeze-dried under vacuum. The reaction principle of immobilizing lysozyme on HNTs is shown in Fig. 1.

Fig. 1 Reaction principle of immobilizing lysozyme on HNTs.

Fabrication of PES/HNTs–Ly antibacterial hybrid membrane

The PES membrane and the PES/HNTs–Ly antibacterial hybrid membrane were prepared by a phase inversion method using casting solutions, including PES (18 wt%), polyvinylpyrrolidone (PVP) (at 8 wt% concentration) as a pore former, small volumes of acetone and different concentrations of HNTs–Ly powder (1 wt%, 2 wt%, and 3 wt% by weight of PES) in N,N-dimethylacetamide (DMAc) (73.2 wt%) as the solvent. To prepare the homogeneous casting solution, firstly, a certain amount of HNTs–Ly was dispersed into DMAc with ultrasonic treatment; subsequently, PES, acetone and PVP were added to the HNTs–Ly suspension under mechanical stirring at room temperature for 15 h to fabricate a transparent and homogeneous casting solution, which was filtered and degassed under vacuum at room temperature for at least 4 h without stirring. Finally, the abovementioned solution was cast onto a clean glass plate using a self-made casting knife with a thickness of 0.1 mm and then immersed in a coagulation bath (deionized water, 40 °C) for precipitation. After complete precipitation, the membranes were kept in deionized water at ambient temperature for at least 24 h to complete phase separation and remove the remaining solvent prior to further characterization.

Characterization of HNTs

Fourier transform infrared spectroscopy (FTIR). The chemical compositions of raw HNTs and modified HNTs (HNTS–NCO, HNTs–COOH, and HNTs–Ly) were all determined using an FT-IR Thermo Nicolet IR 200 spectroscope (Thermo Nicolet Corporation, USA). Typically, 64 scans were signal-averaged to reduce spectral noise. The spectra were recorded in the 400–4000 cm⁻¹ range using KBr pellets.

Thermo-gravimetric analysis (TGA). TGA measurements were performed on a TG-DTA, DT-40 system (Shimadzu, Japan). For this purpose, ca. 3 mg samples were placed in an alumina crucible and heated from room temperature to 800 °C at 10 °C per min under flowing nitrogen.

Transmission electron microscopy (TEM). The morphologies of HNTs and modified HNTs were investigated using a FEI model TECNAI G² transmission electron microscope (200 kV acceleration voltages). The samples for analysis were dispersed in ethanol with the aid of ultrasound and then the suspended particles were transferred to and allowed to dry on a copper grid (400 meshes) coated with a strong carbon film.

Fluorescent microscopic analysis. Lysozyme was labeled with fluorescein isothiocyanate (FITC–Ly) and then the labeled samples were used for the immobilization process. The immobilization and distribution of lysozyme on the surface of HNTs were examined by a BM-21AY fluorescence microscope (Shanghai BM optical instrument manufacturing Co., Ltd, China) with a fluorescence excitation wavelength of 450–490 nm.

Characterization of membranes

Water contact angle. The water contact angle of the membranes was measured on a contact angle goniometer (OCA20, Dataphysics Instruments, Germany) at 25 °C and 50% relative humidity. 1 μL of deionized water was carefully dropped on the top surface and the contact angle between water and membrane was measured until no further change was observed. To minimize the experimental error, the contact angle was measured at five random locations for each sample and then the average was reported.

Scanning electron microscopy (SEM). A scanning electron microscope (JSM-6700F, JEOL, Japan) was used for the morphology observations of the membrane cross-section and plane. Samples of the membranes were frozen in liquid nitrogen and then fractured. The membranes were sputtered with gold then viewed with the microscope at 10 kV.

Mechanical properties. Tensile strength and percentage elongation were measured on testing strips using a model UTM2203 electronic universal testing machine (Jinan Huike Test Instrument Co., Ltd., China) mounted with a 100 N load cell at room temperature at a constant crosshead speed of 5 mm min⁻¹ with an aluminum sample holder. The membranes were cut into 40 mm × 10 mm pieces and the thickness of the membranes was obtained from the SEM results for the membranes. Particular attention was given to the macroscopic homogeneity of membranes and only apparently homogeneous membranes were used for the mechanical tests.

Separation performance of membranes. The separation performances of the prepared membranes were measured by a cross-flow filtration system. The effective area of each flat sheet membrane piece is 22.2 cm². Each membrane was compacted at 0.2 MPa for 1 h prior to performing the ultrafiltration experiments. Then, the pressure was lowered to 0.1 MPa and all the ultrafiltration experiments were carried out at this pressure. After compaction, the pure water flux was recorded at ambient temperature. The PEG 20 000 solution (0.5 g L⁻¹) and the PVA 30 000–70 000 solution (0.5 g L⁻¹) were forced to permeate through the membrane and the permeate solutions were collected. The permeation flux (J) and rejection (R) were calculated using the following equation:where V is the volume of permeate pure water (L), A is the effective area of the membrane (m²), and T is the permeation time (h).where C_p is the permeate concentration and C_f is the feed concentration (mg L⁻¹). The concentrations of PEG 20 000 and PVA 30 000–70 000 were obtained by a UV-vis spectrophotometer (Shimadzu, Japan). The detailed procedure for PEG or PVP determination was given elsewhere.⁹

Antibacterial activity tests. Minimal inhibitory concentration (MIC) tests were performed to study the quantitative antibacterial properties of HNTs–Ly. A quantity of E. coli cells suspended in a solution (10⁶ CFU per mL) was prepared to reserve. Antibacterial agents (ca. 0.6826 mg) were dissolved in 1 mL of Luria–Bertani (LB) liquid nutrient medium with a concentration of 512 μg mL⁻¹. Subsequently, the suspension of E. coli cells (1 mL) was added to solutions containing different concentrations of antibacterial agents (512, 256, 128, 64, 32 μg mL⁻¹). Samples containing only 2 mL of the bacterium solution and only 2 mL of LB were regarded as control groups. Then, the seven samples were shaken for 2 h at 37 °C. After that, the bacterium solution was diluted with sterile water until its concentration was reduced to 10⁻⁴ of that of the original culture, 0.2 mL of the diluted solution was uniformly coated on plates and these were incubated at 37 °C for 12 h. The numbers of colonies on the plates were determined by the plate count method. The bacteriostasis rate (B_R) was calculated using the following equation.where A is the number of bacterial colonies on the plates from the control group and B is the number of bacterial colonies on the plates from the experiment group.

Moreover, the bacteriostasis rate was also used in order to quantitatively analyze the antibacterial activity of the membranes. A quantity of E. coli cells suspended in solution (10⁶ CFU per mL) was prepared to reserve. All the membrane samples (ca. 0.06 g) were cut and sterilized by autoclaving for 20 min. To test the antibacterial activity, the membranes were added to a 5 mL solution containing about 10⁶ cells per mL from the E. coli bacterium solution, and then incubated at 37 °C for 4 h with shaking. Furthermore, membranes were retrieved from cultures and washed with normal saline. The washed solutions were collected and diluted with deionized water until their concentrations were reduced to 10⁻⁵ of their original values. 0.1 mL of the dilution solution was spread onto an LB culture medium and all plates were incubated at 37 °C for 24 h. The numbers of colonies on the plates were determined by the plate count method and the bacteriostasis rate (B_R) was defined by the abovementioned eqn (3).

Long time water flux performance of membranes. Long time (48 h, per 30 min) water flux performance of the membrane was also measured by a cross-flow filtration system with pure water. The effective area of each flat sheet membrane piece is 22.2 cm². The pressure was set at 0.1 MPa and all the tests were carried out at this pressure. After compaction, the pure water flux was recorded at ambient temperature every 30 minutes. Flux drop (ΔJ) was calculated using the following equation:where J₁ is the water flux for 30 min and J_x is the water flux for 30x min (x = 1, 2, ⋯ 96).

Results and discussion

Characterization of HNTs–Ly

The chemical structures of the unmodified and modified HNTs were characterized by FTIR. Fig. 2 shows the FTIR spectra of raw HNTs, HNTs–NCO, HNTs–COOH and HNTs–Ly. Compared with Fig. 2(a) (the raw HNTs), the peaks around 2933 cm⁻¹ and 2852 cm⁻¹ are ascribed to the C–H stretching vibration in CH₂ and CH₃. The peaks around 2269 cm⁻¹ are assigned to the –NCO asymmetric stretching vibration and the absorption peak at 1579 cm⁻¹ is the stretch vibration of the –NHCO– bonds in the HNTs–NCO. In Fig. 2(c), the C=O stretching vibrations in the carboxyl group of N–COOH are obviously visible around 1750 cm⁻¹, whereas the peaks around 2269 cm⁻¹ disappear, which indicates that –NCO was turned into –COOH. In Fig. 2(d), the CH₂ absorption peaks at 2933 cm⁻¹ and 2852 cm⁻¹ are stronger, which is because the densities of the methyl and methylene groups are increased after the immobilization of lysozyme. The above results confirmed that lysozyme was immobilized on the HNTs.

Fig. 2 FT-IR spectra of (a) raw HNTs, (b) HNTs–NCO, (c) HNTs–COOH and (d) HNTs–Ly.

In order to further confirm the modification of HNTs, Fig. 3 displays the TGA curves of (a) raw HNTs, (b) HNTs–NCO and (c) HNTs–COOH. For the curve of raw HNTs, an obvious mass loss occurred in the range of 450–550 °C. This mass loss was assigned to the dehydroxylation of structural Al–OH groups of the HNTs.³¹ The first mass loss in the range 50–150 °C was due to physically adsorbed water.³² Curves (b) and (c) both showed distinct mass losses, which can be attributed to decomposition of the HMDI and SAA of the modified HNTs, which also demonstrated that –COOH groups were grafted onto the HNTs successfully. From the TGA analysis, the –NCO and –COOH contents could be obtained and are about 0.3 g (–NCO) per g (HNTs) and 0.13 g (–COOH) per g (HNTs–NCO).

Fig. 3 TGA curves of (a) raw HNTs, (b) HNTs–NCO, and (c) HNTs–COOH.

Fig. 4 displays TEM images of HNTs (a) and HNTs–Ly (b). As shown in Fig. 4(a), the morphology of raw HNTs apparently exhibits hollow tubular and open-ended structures. From Fig. 4(b), the surface of the HNTs is covered with irregular viscous deposits, which have large particle sizes. Therefore, the results demonstrated that lysozyme was successfully immobilized on the surface of HNTs–COOH.

Fig. 4 TEM images of (a) raw HNTs and (b) HNTs–Ly.

A fluorescence microscopic image of HNTs–Ly is shown in Fig. 5. FITC–Ly was immobilized on the HNTs–COOH by covalent bonding. Immobilized enzymes showed bright yellow-green fluorescence under a fluorescence microscope with a 490 nm excitation wavelength. This result demonstrated that lysozyme was successfully immobilized on the surface of HNTs–COOH.

Fig. 5 Fluorescence microscopic image of HNTs–Ly.

The antibacterial effect is shown in Fig. 6. Compared with the control (see Fig. 6(a)), Fig. 6(b) shows that HNTs–Ly particles exhibit significant antibacterial activity with less E. coli colonies. Therefore, the results indicated that HNTs–Ly had antibacterial efficacy for E. coli. The MIC and the bacteriostasis rate of HNTs–Ly were 512 μg mL⁻¹ and 94.9%, respectively.

Fig. 6 Antibacterial effect of (a) control and (b) HNTs–Ly against E. coli.

Characterization of PES/HNTs–Ly hybrid membranes

Morphology of membranes. Fig. 7 displays the SEM images of (a and b) pure PES membranes and (c and d) hybrid membranes with an HNTs–Ly loading amount of 3 wt%. As can be seen in the SEM images (a) and (c), the surface of the PES/HNTs–Ly hybrid membrane was much smoother compared with the pure PES membrane. It is generally recognized that membranes with smooth surfaces exhibit higher hydrophilicity and superior antifouling behavior compared to those with rough surfaces.³³ Fig. 7(b) and (d) show the cross-section morphologies of the tested membranes. Both the PES membrane and the hybrid membrane have a dense skin layer and a finger-like structural support layer. However, it can be observed that there is a significant increase in the pore size of the macrovoids in the support layer, like the scale of Fig. 7(b) and (d), which would enhance the permeation flux of the membrane. The enlarged pore size of macrovoids may result from the increase in thermodynamic instability of the cast film, which promotes a rapid phase separation.³⁴

Fig. 7 SEM images of the surface morphology of (a) PES membrane, (c) PES/HNTs–Ly hybrid membrane and the cross-sections of (b) PES membrane, (d) PES/HNTs–Ly hybrid membrane.

Hydrophilicity of membranes. Surface hydrophilicity is one of the most important factors for ultrafiltration membranes in determining their antifouling properties. However, PES is an intrinsically hydrophobic polymer; thereby, hydrophilic modification is indispensable for PES ultrafiltration membranes. In this study, water contact angle measurement was selected to evaluate the relative hydrophilicity or hydrophobicity of the membrane surface and provide information on the interaction energy between the surface and liquid.⁷ It is generally accepted that a lower contact angle represents a greater tendency for water to wet the membrane, a higher surface energy and a higher hydrophilicity.⁹ From Fig. 8 it can be seen that the pure PES membrane presents the highest contact angle of 82° and this value decreased as the HNTs–Ly content was increased, which indicated that the hybrid membrane exhibited superior hydrophilicity on account of the introduction of HNTs–Ly. During the formation process of the hybrid membranes, HNTs–Ly in the casting solution tended to migrate spontaneously to the surface of the hybrid membranes to reduce the interface energy,³⁴ which lead to an increase in membrane hydrophilicity. As shown in Fig. 8, the lowest contact angle was 67° when the HNTs–Ly content reached 3 wt%. These results proved that the accretion of HNTs–Ly could increase the hydrophilicity of PES membranes.

Fig. 8 Effect of HNTs–Ly content on water contact angle of PES/HNTs–Ly hybrid membranes.

Permeation property of membranes. The pure water flux and the rejections for PEG 20 000 and PVA 30 000–70 000 for hybrid membranes with different HNTs–Ly contents (1 wt%, 2 wt%, 3 wt%) are illustrated in Fig. 9. The pure water flux increased quickly after adding HNTs–Ly. When the content of HNTs–Ly was 3 wt%, the pure water flux reached a maximum of 400 L m⁻² h⁻¹ compared with 198.5 L m⁻² h⁻¹ for pure PES membranes. This phenomenon is consistent with the results of contact angle measurements. Moreover, the rejection for PVA 30 000–70 000 remained above 99.6%. These results might be explained as follows: physical sieving by pores is believed to be the main driving factor in the ultrafiltration membrane. The diameter of a water molecule is smaller than the pore size on the skin layer of the PES membrane, so it could be permitted through the membrane under the pressure difference. However, the water flux of the pure PES membrane was lower than that of the PES/HNTs–Ly membrane because of the inherent hydrophobic character of PES, and the enhanced pure water flux might also be attributed to the addition of hydrophilic HNTs–Ly and the hollow structure of HNTs, which may facilitate water transmission. The principle of increasing water flux for the hybrid membranes is illustrated in Fig. 10. These performances are consistent with the latest research.^9,35 Moreover, the large particle size of HNTs–Ly may result in more interface voids around HNTs–Ly particles, thereby producing additional pathways for water molecules. However, a slight increase in PVA 30 000–70 000 rejection may result from the thickened skin layer (as observed in Fig. 7), which provides more steric resistance for PVA molecules. Such an increased rejection for PVA 30 000–70 000 was consistent with our previous study.³⁶ The rejection of the membranes against PEG 20 000 decreased only slightly as a result of the addition of HNTs–Ly due to its smaller molar mass compared with PVA 30 000–70 000.

Fig. 9 Effect of HNTs–Ly content on the separation performance of PES/HNTs–Ly hybrid membranes.

Fig. 10 Principle of increasing water flux for PES/HNTs–Ly hybrid membranes.

Mechanical properties of membranes. Fig. 11 shows the stress–strain curves of the test membranes obtained from mechanical measurements. As shown in Fig. 11, tensile strength increased from 1.13 MPa to 3.30 MPa with increasing HNTs–Ly content from 0 wt% to 3 wt%. This phenomenon is consistent with the widespread theory that organic–inorganic hybrid membranes combine the flexibility of an organic membrane with the stiffness of an inorganic membrane. However, elongation at break changed erratically, which might be caused by the agglomerations and voids of immobilized lysozyme in the membranes and the decrease in homogeneity of the hybrid membranes. When the content of HNTs–Ly is increased to 3 wt%, both achieve a maximum value. Briefly, the mechanical properties of membranes are mainly affected by the cross-linking of additive molecules and the homogeneity of membranes. Thus, the above results indicated that the incorporation of HNTs–Ly could observably improve the mechanical stabilities of PES membranes, making them suitable for use under high pressure conditions.

Fig. 11 Stress–strain curves of (a) PES membrane, (b–d) PES/HNTs–Ly membranes with different HNTs–Ly content (1 wt%, 2 wt%, 3 wt%, respectively).

Antibacterial performance of membranes. The antibacterial effect is reflected in Fig. 12 and the bacteriostasis rate is presented in Table 1. Compared with the pure PES membrane, it is clearly seen that the number of colonies on the plates treated with hybrid membranes containing 3 wt% HNTs–Ly decreased significantly. The bacteriostasis rates of the hybrid membranes against E. coli were 63%, which indicated the excellent inhibiting effect of immobilized lysozyme for E. coli, because it is generally deemed that a higher bacteriostasis rate demonstrates a better antibacterial ability. In addition, it was also certified that immobilized lysozyme was intercalated into the PES membranes. Simultaneously, Fig. 13 clearly exhibits the antibacterial mechanism of the membranes with HNTs–Ly. The abovementioned results suggested that PES/HNTs–Ly membranes had a preferable antibacterial ability, which was mainly attributed to the introduction of HNTs–Ly.

Fig. 12 Antibacterial effect of (a) PES membrane and (b) PES/HNTs–Ly hybrid membranes with 3 wt% of HNTs–Ly against E. coli.

Table 1 Antibacterial rates of the PES/HNTs–Ly hybrid membranes against E. coli

	E. coli
	Bacterial colonies (CFU)	Antibacterial rate (%)
Pure PES membrane	314	—
Hybrid membranes with 3 wt% HNTs–Ly	116	63

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Fig. 13 Antibacterial mechanism of the PES/HNTs–Ly hybrid membranes.

Long time water flux performance of membranes. A long time (48 h) water flux test of the PES membrane and the PES/HNTs–Ly membrane with 3 wt% HNTs–Ly is shown in Fig. 14(a). It can be seen that the water flux of the membranes is decreasing with respect to time, which may be caused by the blocking of the pores of the membranes. Fig. 14(b) illustrates the flux drop of membranes. Interestingly, the flux drop of the PES/HNTs–Ly membrane is smaller than for the PES membrane. This indicates that the addition of hydrophilic HNTs–Ly has efficaciously reduced the pollutant adhered to the membrane surface and improved the antifouling properties of PES membranes.

Fig. 14 (a) Water flux and (b) flux drop of PES and PES/HNTs–Ly hybrid membranes in long time run.

Conclusions

In summary, a novel antibacterial particle, HNTs–Ly was successfully prepared via covalently immobilized lysozyme on the surface of modified HNTs. Subsequently, PES/HNTs–Ly hybrid ultrafiltration membranes were fabricated by a classical phase inversion method. The hydrophilicity and mechanical properties of the hybrid membranes increased significantly compared with the pure PES membranes. In addition, the antibacterial properties of the hybrid membranes were also improved. Therefore, these membranes exhibit the potential to reduce fouling in water treatment.

Acknowledgements

This study was financially sponsored by the National Natural Science Foundation of China (nos 21376225 and 21106137) and the China Postdoctoral Science Foundation (nos 2014T70686 and 2013M531684). We sincerely acknowledge the financial assistance of the visiting research program in the University of New South Wales by the China Scholarship Council (no. 201208410135).

Notes and references

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