Achieving Highly Effective Non-biofouling Performance for

The TM:SA ratios examined in the monomer solutions were 1:0, 2:1, 1:1, 1:2, and 0:1, respectively. ... brushes on the membrane surface was calculated ...
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J. Phys. Chem. B 2010, 114, 2422–2429

Achieving Highly Effective Non-biofouling Performance for Polypropylene Membranes Modified by UV-Induced Surface Graft Polymerization of Two Oppositely Charged Monomers Yong-Hong Zhao, Xiao-Ying Zhu, Kin-Ho Wee, and Renbi Bai* DiVision of EnVironmental Science and Engineering, Faculty of Engineering, National UniVersity of Singapore, 9 Engineering DriVe 1, Singapore 117576, Singapore ReceiVed: August 25, 2009; ReVised Manuscript ReceiVed: January 4, 2010

A major problem in membrane technology for applications such as wastewater treatment or desalination is often the loss of membrane permeability due to biofouling initiated from protein adsorption and biofilm formation on the membrane surface. In this study, we developed a relatively simple and yet versatile approach to prepare polypropylene (PP) membrane with highly effective non-biofouling performance. Copolymer brushes were grafted to the surface of PP membrane through UV-induced polymerization of two oppositely charged monomers, i.e., [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TM) and 3-sulfopropyl methacrylate potassium salt (SA), with varying TM:SA molar ratios. Surface analysis with scanning electron microscope (SEM) clearly showed the grafted copolymer brushes on the membrane surfaces and that with X-ray photoelectron spectroscope (XPS) revealed a similar TM:SA ratio of the grafted copolymer brushes to that of the monomer solution used for the polymerization. Water contact angle measurements indicated that the hydrophilicity of the membrane surfaces was remarkably improved by the grafting of the TM/SA copolymer brushes, with the lowest water contact angle of 27° being achieved at the TM:SA ratio of around 1:1. Experiments for antiprotein adsorption with bovine serum album (BSA) and lysozyme (LYZ) and antibiofilm formation with Escherichia coli (E. coli) demonstrated a great dependence of the membrane performance on the TM:SA ratios of the grafted copolymer brushes. It was found that the characteristics of the surface charges of the membrane surfaces played a very important role in the non-biofouling performance, and the membrane surface with balanced positive and negative charges showed the best non-biofouling performance for the proteins and bacteria tested in this study. Introduction The decay of membrane performance in many separation applications, such as water reclamation and desalination, due to biofouling arising from protein adsorption and biofilm formation is of great concern because of the significant reduction in system productivity and remarkable increase in system operational cost.1,2 Many conventional polymeric membranes available in the market are known to be hydrophobic and often prone to severe biofouling. For example, when a protein molecule approaches and is in contact with the surface of those hydrophobic polymeric membranes, the water molecules between the protein and the membrane surface will be displaced. This causes the protein molecule to lose its bound water and thus induce conformational changes in its structure, which results in an irreversible adsorption of the protein on the membrane surface.3 Similarly, even though the interaction between a bacterium and the separation membrane surfaces may be much more complex, bacteria adhesion to the membrane surface can be simply initiated by the nonspecific adsorption (e.g., through hydrophobic interaction). Once attached, the bacteria can grow on the membrane surface and synthesize insoluble exopolysaccharides (EPS) that encase the adhered bacteria in a threedimensional matrix. With the accumulation of EPS and the reproduction of bacteria, a mature biofilm that cannot be easily removed will be developed on the membrane surface.4 * Corresponding author. E-mail: [email protected]. Phone: +65 6516 4532. Fax: +65 6774 4202.

One of the widely adopted methods to increase the nonbiofouling property of commercially available filtration membranes is surface modification, particularly through surface graft polymerization of various functional monomers, such as poly(ethylene glycol) (PEG) derivatives, amines, basic and zwitterionic monomers, hydroxyl-containing monomers, strong and weak acids, etc.5 It has often been assumed that a surface resistant to nonspecific protein adsorption would also resist biofilm formation. However, the work by Whitesides and coworkers showed that the ability to reduce protein adsorption at a surface did not necessarily correlate to the ability to reduce bacterial adhesion that initiates biofilm formation.6 This finding was further supported by a number of later studies. For example, Kingshott et al.7 reported that stainless steel modified with PEG resists protein adsorption, but not bacterial adhesion. Roosjen et al.8 found that PEG-coated materials can effectively resist nonspecific protein adsorption and short-term bacterial adhesion, but have limited success in preventing long-term biofilm formation. Zwitterionic polymers, including poly(phosphorylcholine) (PPC), poly(sulfobetaine methacrylate) (PSBMA) and poly(carboxybetaine methacrylate) (PCBMA), etc., are a kind of biomimetic materials, in which cation and anion groups are located on the same monomer residue and maintain an overall charge neutrality.9 Because of the strong capacity to form a hydration layer via electrostatic interaction between zwitterions and water molecules, zwitterionic polymers are recognized as unique type of materials that have excellent nonfouling

10.1021/jp908194g  2010 American Chemical Society Published on Web 02/01/2010

Non-biofouling Performance for PP Membranes

Figure 1. Monomers used to create mixed charge copolymers via UVinduced graft polymerization.

properties.10-14 The strategy of using zwitterionic polymers to increase the nonfouling performance of various separation membranes has drawn great attention recently.15-17 However, most of the studies have only focused on the antiprotein adsorption property of the zwitterionic polymer modified membranes, and very few attempts have been made to examine the antibiofilm formation performance of these membranes as well. A key factor that determines the nonfouling property of zwitterionic polymers has been attributed to the balanced charge of the zwitterions.18 It has also been demonstrated that mixed positively and negatively charged self-assembled monolayers (SAMs) of balanced overall charges were highly resistant to protein adsorption.19-21 Based on these studies and other related work,22 it has been hypothesized that any materials composed of a homogeneous blend of charged components with balanced charges would prevent protein adsorption. This hypothesis was supported by the results from Bernards et al.,23 who prepared polymer brush coatings via surface-initiated atom transfer radical polymerization (ATRP) from two oppositely charged monomers. Through the measurement of nonspecific adsorption for fibrinogen, lysozyme (LYZ), and bovine serum album (BSA), they found that when the coating was formed as copolymer brushes with balanced charges, nonfouling properties for all the three probe proteins were observed. Since cationic monomers and anionic monomers are more readily available than zwitterionic monomers, it is of great interest for research and practical applications to develop nonbiofouling membrane surfaces using oppositely charged monomers. In this work, we report our new development in the modification of polypropylene (PP) membranes through a simple UV-induced surface graft polymerization from a mixture of two typical but oppositely charged monomers, i.e., positively charged [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TM) and negatively charged 3-sulfopropyl methacrylate potassium salt (SA), as shown in Figure 1. The two oppositely charged monomers were polymerized from a mixture containing them in varying molar ratios. The non-biofouling properties of the modified PP membranes were examined in a series of experiments in terms of BSA and LYZ adsorption as well as E. coli adhesion and growth on the membrane surfaces. One of the unique features of the present work is that it provides a more complete evaluation of the non-biofouling performance of the membrane surfaces grafted with different ratios of positive and negative charges (i.e., not just balanced). Experimental Section Materials. PP porous membrane, in the configuration of woven PP fibers and with an average pore size of 0.6 µm, was purchased from Whatman. TM, SA, BSA, LYZ, and benzophenone (BP) were supplied by Sigma-Aldrich and used as received. Acetone, sodium chloride, and sodium hydrate were in analytical

J. Phys. Chem. B, Vol. 114, No. 7, 2010 2423 grade and were used without further purification. Deionized (DI) water (18 MΩ) purified with a Milli-Q system from Millipore was used in the study to prepare solutions as needed. The phosphate-buffered saline (PBS, pH ) 7.4) was prepared by the addition of the prepackaged buffer salts (Aldrich) into DI water and was used to dissolve BSA and LYZ. UV-Induced Surface Graft Polymerization of TM/SA Monomers. Before UV-induced polymerization, the PP membrane sample was washed with acetone to remove any possible impurities on the surface and then dried in a vacuum oven at 40 °C to a constant weight (W0). BP (0.033 M), TM, and SA (the total mole concentration of TM and SA was fixed at 0.9 M) were dissolved in an acetone/water (1:1) mixture solvent together to prepare the monomer solutions. The TM:SA ratios examined in the monomer solutions were 1:0, 2:1, 1:1, 1:2, and 0:1, respectively. A piece of the dried PP membrane samples was then immersed in 10 mL of a freshly made monomer solution in a Petri dish and subsequently exposed to UV irradiation for 30 min in an ultraviolet system (UVP, B-100AP, USA) equipped with a 100 W high-pressure mercury lamp in a nitrogen gas environment. After the surface graft polymerization, the modified membrane was washed in acetone for 4 h with vibration, and then in DI water for 20 h. Finally, the membrane was dried in the vacuum oven at 40 °C again to a constant weight (W1). The grafting degree (Dg) of copolymer brushes on the membrane surface was calculated by Dg ) [(W1 W0)/W0] × 100%. Characterization of Membranes. Measurement of Water Contact Angle (CA). CA was measured using an optical contact angle analyzer (Model 250, Rame´-Hart Instrument Co., USA) at room temperature with the static captive bubble method. The membrane sample was immersed into DI water in the measuring cell and fixed at a horizontal position with the active layer surface facing down. An air bubble (5-10 µL) was injected from a microsyringe with a stainless steel needle onto the active layer surface in water. Then, the instrument was started to automatically measure and record the CA values. Ten measurements at different locations on the membrane surface were made, and the average was used to represent the CA value for the membrane sample examined. ObserWation of Surface Morphology. The surface morphologies of the PP and modified PP membranes were observed using a field emission scanning electron microscope (FESEM, JEOL JEM-6700) under the standard high-vacuum conditions. Each sample was dried under vacuum and coated with gold powder by a vacuum electric sputter coater (JEOL JFC-1300) before being mounted onto the sample stud for the SEM observation. Analysis of Surface Chemical Compositions. The surface chemical compositions of the membranes were characterized by X-ray photoelectron spectroscopy (XPS). XPS analyses were carried out on an AXIS HIS spectrometer (Kratos Analytical Ltd., UK) with an Al KR X-ray source (1486.71 eV of photons). The X-ray source was run at 250 W with an electron take-off angle of 45° relative to the sample surface. The pressure in the analysis chamber was maintained at about 5 × 10-7 Pa during the analysis. Determination of Zeta Potentials. The zeta potentials of the membrane surface were measured using an electrokinetic analyzer (Anton Paar GmbH, Austria) equipped with a clamping cell. The streaming potential was measured between two Ag/ AgCl electrodes on the opposite ends of the clamping cell. The analysis was conducted following the standard procedures and the analyzer automatically converted the measured streaming potential to zeta potential as the output.24 The reported values

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are the average of the zeta potentials determined in two opposite flow directions and with at least three samples for each type of membranes. Measurement of Membrane Flux Change Due to Protein Fouling. The pure water flux as well as the filtration flux of protein solutions through the membranes under dynamic fouling was investigated using a dead-end filtration system, consisting of a nitrogen gas cylinder, a pressure controller (Alicat Scientific PCD, USA), a clear reservoir, and an Advantec stirred cell (effective filtration area 11.9 cm2, Advantec UHP-43, Japan) coupled with a magnetic stirrer. The transmembrane pressure was precisely controlled via the pressure controller with an accuracy of (0.5%. Permeate weight was measured by an electronic balance (Precisa XT-220-A, Switzerland) that was serially linked to a computer for automated data collection at desired time intervals. In the present study, the transmembrane pressure was set at 0.1 MPa and the data collection was made at every 10 min. Although there may be a preference to use fibrinogen in the nonfouling test due to its possibly greater hydrophobic “stickness”,23 BSA and LYZ were used as the probe proteins in this study because they are more commonly encountered in applications and, especially, much cheaper to use (at relatively large quantities). Before the filtration tests, all the sample membranes were filtered with pure water until their fluxes became constant. The pure water flux was recorded first for 30 min and denoted as JW1 in the study. Then, a 1 g/L protein solution prepared in PBS with BSA or LYZ was filtered through the membrane for 3 h and the corresponding permeate flux was recorded against time. After that, the membrane was cleaned by shaking in DI water for 2 h in order to estimate the possibly greatest reversible fouling that can be recovered. The pure water flux of the cleaned membrane was measured again within 30 min and recorded as JW2. The relative flux recovery (RFR) was then calculated by RFR ) (JW2/JW1) × 100% to indicate the extent of the possible reversible fouling. Biofilm Formation Assay. Bacterial Culture Conditions. E. coli (supplied by the Environmental Molecular Biotechnology Lab at the National University of Singapore) was used in the experiments for biofilm formation study. The nutrient solution used for culturing E. coli was tryptone soy broth (TSB, Fluka) aqueous solution with a concentration of 30 g/L. The nutrient solution was autoclaved at 121 °C for 20 min before use. A 1 mL amount of E. coli suspension was added into 30 mL of TSB solution and incubated at 37 °C with shaking at 100 rpm for 24 h. Then, the culturing process continued by adding 1 mL of the E. coli suspension into 30 mL of TSB solution and incubated at 37 °C with shaking at 100 rpm in every 48 h. The concentration of the E. coli in the nutrient solution was at about 108-109 CFU/mL. The prepared membranes for biofilm formation test were immersed into the freshly inoculated E. coli suspension, followed by incubation at 37 °C with shaking at 100 rpm for 48 h. SEM ObserWation. After incubation in the E. coli suspension for 48 h, membranes were taken out, first washed in a 0.1 M NaCl solution for 30 min, and then immersed in a 3 vol % glutaraldehyde PBS solution for 4 h at 4 °C to fix the adhered bacteria. After fixation, the membranes were washed with 0.1 M NaCl solution for three times to remove the remaining glutaraldehyde. The membranes were then dried in air and stored in a desiccator for further use. The dried membranes were coated with gold powder by a vacuum electric sputter coater (JEOL JFC-1300) and observed with SEM (JEOL JEM-6700) for their surface images.

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Figure 2. Dependence of grafting degree (Dg, 0) and water contact angle (O) on the TM:SA ratios in the polymerization system.

QuantitatiWe Analysis. Membrane samples were dried at 40 °C under vacuum to constant weights (denoted as w1). Then, the membrane samples were incubated in the E. coli suspension for 48 h, as described early. The membrane samples were taken out and washed in a 0.1 M NaCl solution for 30 min. Then, the membrane samples were dried again at 40 °C under vacuum to constant weights (denoted as w2). The adhered amount of the bacteria on per unit area of the membrane was given as (w2 w1)/A, where A is the area of the corresponding membrane sample. Confocal Laser Scanning Microscope (CLSM) ObserWation. The distribution and viability of the bacteria adhered on the membrane surfaces were investigated by the CLSM technology. A combination of dyes consisting of propidium iodine (PI) and SYTO 9 (Live/Dead Baclight bacteria viability kits, Molecular Probes, L13152) was used in the study. SYTO 9 is permeable to cell membrane and hence can stain both viable and nonviable bacteria, producing green fluorescence. PI has a higher affinity for nucleic acids but is excluded from viable cells by the cell membrane pumps. The nonviable cells stained by both SYTO 9 and PI would produce red fluorescence. This is in contrast to viable cells that are only stained by SYTO 9 and produce green fluorescence. In the experiments, the bacteria-adhered membranes were stained with the combined dyes for 15 min in dark. The stained membranes were subsequently observed with the CLSM (Nikon A1 Confocal Microscope System) for their fluorescence. Results and Discussion Characteristics of Membranes. Grafting Degree of UVInduced Polymerization. Figure 2 shows the grafting degree (Dg) of polymers on the membranes with TM and SA at varying monomer molar ratios in the UV-induced graft polymerization. It can be found that the Dg values depended on the TM/SA ratios, even though the total concentration of TM and SA was kept constant in the monomer solutions in the polymerization system. For the polymerizations in which only one monomer (TM or SA) was used, the grafting degrees were relatively lower. With the addition of the oppositely charged monomer into the system, the value of Dg increased and the highest Dg (12.4 wt %) was obtained when the concentrations of TM and SA were equal in the system. The increase in Dg may be attributed to the more favorable electrostatic interaction between TM and SA monomers in the grafting process. From Figure 2, it is also observed that the grafting degree in terms of Dg value was almost the same for the single monomer system (TM or SA), which suggests that TM and SA monomers possessed similar polymerization rate under the conditions applied in this study.

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Figure 3. SEM images of the original PP membrane (a) and PP membranes modified with TM:SA at different monomer ratios of (b) 1:0, (c) 2:1, (d) 1:1, (e) 1:2, and (f) 0:1.

Water Contact Angle. The changes in the hydrophilicity of the membrane surfaces after UV-induced graft polymerization, as characterized by the water contact angle values, are also shown in Figure 2. The original PP membrane had a water contact angle value of about 106°, representing a typical hydrophobic surface. The surface grafting of TM/SA copolymer brushes on PP membranes resulted in a significant decrease in the water contact angle values, and all the modified membranes became hydrophilic. It was found that the water contact angles showed a strong dependence on the grafting degree. For example, the water contact angle for membrane surfaces entirely grafted with TM (1:0) or SA (0:1) polymer brushes was 47° (Dg ) 8.7 wt %) or 42° (Dg ) 9.2 wt %), respectively. When the monomer ratio was equal (1:1), the water contact angle of the membrane surface decreased to as low as 27° (Dg ) 12.4 wt %). Surface Morphology. Figure 3 shows the SEM images of the original PP membrane and the modified PP membranes. From Figure 3a, it can be found that the interfaces between the interlaced PP fibers in the original PP membrane are sharp and clear. After grafted with polymer brushes at different TM:SA monomer ratios, the interfaces obviously become obscure, and some of the fibers appear to be buried in other nearby ones; see Figure 3b-f. In particular, the image in Figure 3d for the modified PP membrane with the highest grafting degree shows that a fiber in the original PP membrane was even entirely wrapped by the grafted copolymers. On the basis of the SEM images in Figure 3, one may easily infer that TM/SA polymer brushes had been successfully grafted to the PP membrane surface by the UV-induced polymerization. Surface Compositions. The membrane surfaces were characterized with XPS to determine their characteristic chemical compositions. Figure 4 shows the XPS survey scan spectra of the membrane surfaces modified by TM/SA in varying monomer molar ratios. When single monomer was used in the polymerization, the N 1s peak at 400.8 eV and the S 2p peak at 165.8 eV were observed separately in Figure 4a,e. When mixed monomers were used, the N 1s peak and the S 2p peak appeared simultaneously in the same spectrum for each of the modified membranes (Figure 4b-d), with different N 1s and S 2p intensities corresponding to the TM:SA monomer ratios. From the XPS results, the atomic percentages of nitrogen and sulfur were calculated and used to quantify the TM and SA ratios in the grafted copolymer brushes. The results are summarized in Table 1. It can be seen in Table 1 that the ratio of N/S decreased

Figure 4. XPS survey spectra of membrane surfaces modified by TM: SA with monomer ratio of (a) 1:0, (b) 2:1, (c) 1:1, (d) 1:2, and (e) 0:1.

TABLE 1: N/S Ratios in Grafted TM/SA Copolymer Brushes Calculated from XPS Analysis TM:SA monomer ratio 1:0

2:1

1:1

1:2

0:1

N (mol %) 7.31 ( 0.1 5.22 ( 0.2 3.95 ( 0.3 2.61 ( 0.1 0 S (mol %) 0 2.83 ( 0.1 4.08 ( 0.2 5.32 ( 0.2 7.92 ( 0.1 N/S ratio NA 1.84 ( 0.14 0.97 ( 0.12 0.49 ( 0.04 0

as the ratio of TM:SA reduced from 1:0 to 0:1. At the meantime, the N/S ratios in the copolymer brushes are found to be quite similar to the corresponding TM:SA monomer ratios used in the polymerization process, indicating that the chemical compositions of the grafted copolymer brushes on the membrane surfaces can be readily tuned by adjusting the monomer ratios used in the polymerization. Furthermore, the monomer ratio of 1:1 in the polymerization resulted in the formation of an approximate 1:1 ratio of TM:SA in the grafted copolymer brushes (the calculated N/S ratio from XPS analysis is about 0.97 ( 0.12). Zeta Potential. The change of N/S ratio in the grafted polymer brushes on the membrane surfaces can lead to the alteration of the surface electrical property of the modified membrane. Figure 5 shows the experimentally measured zeta potentials of the membrane surfaces with grafted polymer brushes from various TM/SA monomer ratios. For pH values from around 5.5 to 9.5, the TM:SA ) 1:0 and 2:1 membrane samples showed positive zeta potentials, while the TM:SA ) 1:2 and 0:1 membrane samples showed negative ones. For the TM:SA ) 1:1 membrane

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Figure 5. Zeta potentials of the various modified membranes.

sample, the zeta potentials of the membrane surface showed a change from weakly positive to weakly negative but overall were at about neutral (zeta potential around zero). Thus, the N/S ratios in the grafted polymer brushes on the membrane surfaces have a correlation with the electrical properties of the modified membranes: membrane surfaces with more N contents (e.g., Figure 4a, b) would be overall positively charged, those ones with more S contents (e.g., Figure 4d,e) be negatively charged, and the membrane surface with equal N and S contents (e.g., Figure 4c) have balanced charges and overall be electrically neutral. The different electrical properties of the membranes caused by the grafted copolymer brushes can have an effect on the non-biofouling performance of the prepared membranes. Protein Fouling Behaviors. BSA and LYZ were examined for their fouling behaviors to the original and modified PP membranes. The pH value of the PBS was adjusted to 7.4 in the experiments. At such a pH value, BSA was negatively charged (ζ ∼ -22.3 mV) and LYZ was positively charged (ζ ∼ +3.5 mV).25,26 Figure 6 compares the permeate fluxes of the various membranes and their BSA and LYZ fouling behaviors during the filtration experiments. It can be observed in Figure 6 that the initial pure water fluxes (JW1) decreased slightly when the membranes were grafted with the TM/SA copolymer brushes, which may be attributed to the possible decrease in the pore sizes of the modified membranes due to the grafted polymer brushes. The chemical compositions of the membrane surfaces, however, had a significant effect on the permeate flux of the protein solutions. For BSA solutions, the fluxes declined sharply with time for the TM:SA ) 1:0 and 2:1 membrane samples (Figure 6a), and the decreasing rates were even faster than that of the original PP membrane. On the contrary, the permeate flux for the BSA solution decayed very slowly for the TM:SA ) 1:2 and 0:1 membrane samples (Figure 6a). For LYZ solutions (Figure 6b), opposite trend in the decrease in the permeate fluxes was observed. However, in both parts a and b of Figure 6, the decreasing rates of the permeate fluxes through the TM:SA ) 1:1 membrane sample appeared at the intermediate level and were not remarkably affected by the types of proteins. The permeate flux through the TM:SA ) 1:1 membrane sample was always much higher than that of the original PP membrane. After the filtration of the protein solutions, the membranes were cleaned and pure water fluxes were measured again (JW2). From JW1 and JW2, the relative flux recovery (RFR) rates are calculated and summarized in Table 2. The RFR rate may be used to indicate the extent of reversible (or correspondingly irreversible) fouling effect. With the TM:SA ratio decreasing from 1:0 to 0:1, the RFR of the various membranes after the filtration of BSA solutions increased from 12.2 to 95.5%,

Figure 6. Permeate fluxes of the various membranes in the filtration of BSA solution (a) and LYZ solution (b), demonstrating the protein fouling behaviors.

TABLE 2: Relative Flux Recovery Rates after Filtration of Protein Solutions relative flux recovery (RFR, %) TM:SA

1:0

2:1

1:1

1:2

0:1

pure PP

BSA LYZ

12.2 95.4

23.3 92.6

91.4 90.2

93.2 34.3

95.5 20.9

45.8 44.0

suggesting an improved anti-BSA-fouling property due to the different surface properties of the membranes. The opposite trend is observed in the RFR for the various membranes after the filtration of LYZ solutions. However, for the TM:SA ) 1:1 membrane, high RFR rates were achieved for both the filtration of BSA and LYZ solutions. As the TM:SA ratios decreased from 1:0 to 0:1, the overall zeta potentials of the membrane surface changed gradually from positive to neutral, and eventually negative (Figure 5). The positively charged surfaces (TM:SA ) 1:0 and 2:1) would repel the positively charged protein LYZ and attract negatively charged protein BSA due to the electrostatic interaction. As a result, they incurred reversible fouling by LYZ (high RFR) but irreversible fouling by BSA (low RFR). Similarly, for the negatively charged surfaces (TM:SA ) 1:2 and 0:1), the fouling of LYZ became irreversible (low FRF) and that of BSA was reversible (high RFR), arising from the different types of electrostatic interactions. Considering that both the positively charged and negatively charged membrane surfaces were hydrophilic (Figure 2), one may easily come to a conclusion that a hydrophilic surface is not necessarily always leading to a more effective nonfouling property. In fact, the electrical properties of the membrane and the foulant can play a much greater role than the hydrophilic interaction in the nonfouling performance. For the TM:SA ) 1:1 membrane sample, in which the N/S ratio was determined to be approximately at 1 by the

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Figure 7. SEM images after membranes immersed in E. coli suspensions for 48 h: (a) original PP membrane, and membranes with (b) TM:SA ) 1:0, (c) TM:SA ) 2:1, (d) TM:SA ) 1:1, (e) TM:SA ) 1:2, and (f) TM:SA ) 0:1.

XPS analysis, the different types of charges on the membrane surface were statistically in balance. Since the surface of the TM:SA ) 1:1 membrane was also highly hydrophilic, as indicated by the water contact angle in Figure 2, a hydrate layer could be formed at the membrane surface in the aqueous environment. Hence, the protein molecules were not able to penetrate the hydrate layer to reach the membrane surface because the surface was electrically neutral and there was a lack of attractive force on them. Consequently, the TM:SA ) 1:1 membrane could not show affinity to BSA or LYZ, displaying better nonfouling characteristics when both positively and negatively charged protein species are concerned. These results revealed that, depending on the nature of the proteins, membrane surfaces can be designed by the presented method to have high extent of positive charges, negative charges or balanced charges to obtain the best nonfouling performance for a specific application. Resistance to Bacteria Growth and Biofilm Formation. Due to the abundant existence in water, E. coli, which is a Gramnegative bacterium, was used as a model bacterium to evaluate the non-biofouling performance of the various PP membranes. Figure 7 shows the SEM images of the membranes after immersion in E. coli suspensions for 48 h. It is obvious that the extent of biofilm formation was strongly dependent on the chemical compositions of the membrane surfaces. For the original PP membrane (Figure 7a), the surface was covered by uniformly distributed E. coli bacteria. When the membrane surface was grafted entirely with TM (1:0) polymer brushes (Figure 7b), the E. coli bacteria adhered on the membrane surface were even clustered and the density of the bacteria was much higher than that on the surface of the original PP membrane. When the TM:SA ratios reduced from 1:0 to 2:1 in the polymerization process, the adhesion of the bacteria on the corresponding membrane surface also reduced (Figure 7c). For the membranes grafted with TM/SA copolymer brushes from TM:SA ratio of 1:1 (Figure 7d), 1:2 (Figure 7e), and 0:1 (Figure 7f), very few E. coli bacteria (highlighted with red circles) were observed on the membrane surfaces. It has been reported that two factors, i.e., hydrophobic/hydrophilic interactions and electrostatic interactions, would usually dominate the extent of bacterial adhesion.27,28 It may be reasonable to infer that the adhesion of E. coli bacteria on the original PP membrane surface was mainly induced by the hydrophobic interaction. The high

Figure 8. Amounts of E. coli adhesion on the various membrane surfaces obtained from the quantitative analysis.

adhesion of E. coli bacteria on the TM/SA ) 1:0 and 2:1 membranes (Figure 7b,c) was due to the attractive electrostatic interaction because E. coli is well-known to have negatively charged surfaces. Similar reasons can be used to explain the low adhesion of E. coli on the TM/SA ) 2:1 and 0:1 membranes (Figure 7e,f) due to the repulsive electrical interactions. For the TM/SA ) 1:1 membrane surface, the lack of both hydrophobic and electrostatic interactions probably contributed to the negligible E. coli bacteria on the membrane surface. Figure 8 shows the amounts of the bacteria on the various membrane surfaces obtained from the quantitative analysis. The results clearly support those observed from the SEM images in Figure 7. The membrane surfaces were also observed by CLSM to investigate the viability of the adhered E. coli bacteria. To distinguish the live and the dead bacteria in mixed populations, the samples were dyed with Live/Dead baclight kits, through which live cells fluoresce green and dead cells fluoresce red. Figure 9 shows the CLSM images of the various membranes after immersion in the E. coli suspensions for 48 h. It can be seen that most of the bacteria adhering on the original PP membrane surface were viable, while those on the positively charged membrane surfaces (Figure 9b,c) were mostly dead. When the membrane surfaces had balanced charges (Figure 9d) or were negatively charged (Figure 9e,f), few E. coli bacteria

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Figure 9. Confocal images after membranes immersed in E. coli suspensions for 48 h: (a) original PP membrane, and membranes with (b) TM:SA ) 1:0, (c) TM:SA )2 :1, (d) TM:SA ) 1:1, (e) TM:SA ) 1 :2, and (f) TM:SA ) 0:1.

were observed by CLSM, in good agreement with the SEM observation. Positively charged surfaces that can kill bacteria have been widely reported.29-31 Two mechanisms for the loss of bacterial activity on polymer chains with quaternary ammonium compounds have been suggested. One of these mechanisms would be expected to operate in Gram-negative bacteria: polymer chains with positively charged surfaces displace divalent cations (e.g., Ca2+ and Mg2+), which hold together the negatively charged surface of the lipopolysaccharide network, thereby disrupting the outer membrane of Gram-negative bacteria.32 The other mechanism is likely to operate when the positively charged polymer chains penetrate into the inner cell membrane, leading to cell leakage and eventually inactivation.33 In the case of E. coli, either mechanism would be sufficient to be lethal. Since the EPS excreted by E. coli can help the bacteria adhere strongly, membranes with more positive charges seem to have an advantage in biofilm formation, which is supported by SEM images (Figure 7b,c) and CLSM images (Figure 9b,c). When the E. coli bacteria adhered on the membrane surface were live (Figure 9a), they would reproduce themselves and form biofilm in the end. The biofilm formation on the surfaces of the original PP membrane or the positively charged membranes is certainly undesirable for membrane separation. It is also interesting to mention that experiments on protein fouling behaviors and resistance to biofilm formation were also conducted for membranes with different TM:SA ratios as mentioned before but at a similar grafting degree (Dg ≈ 12%). The obtained results showed similar characteristics to those in Figures 6 and 7; see Supporting Information. The results in the protein solution filtration and bacteria growth and biofilm formation tests clearly demonstrated that the non-biofouling property of the membranes is strongly dependent on the characteristics of the membrane surfaces, including hydrophobicity/hydrophilicity and, perhaps more importantly, the surface electrical charges. Figure 10 illustrates the various possible interactions between foulants (proteins and bacteria) and the membrane surfaces with different charge

Figure 10. Possible interactions between foulants (proteins and bacteria) and membrane surfaces with different charge compositions.

compositions. To achieve a high performance in resisting protein and bacteria fouling with both positively and negatively charged species simultaneously, the membrane surface should be highly hydrophilic and have balanced charges. The results obtained in this study also show that copolymer brushes can be grafted to membrane surfaces from oppositely charged monomers, replacing the necessity in common practice of using zwitterionic polymers, and also provide the advantage of flexibility to modify membrane surfaces easily with different extents of positive or negative charges as may be needed to achieve the best nonbiofouling performance in applications. Conclusions This study examined the non-biofouling properties of PP membranes grafted with copolymer brushes through UV-induced surface polymerization of two oppositely charged monomers.

Non-biofouling Performance for PP Membranes Negtively charged BSA and positively charged LYZ as well as Gram-negtive E. coli were used as the probe proteins and bacteria to evaluate their effects on the non-biofouling performance of the prepared membranes. It was found that the original PP membrane was highly susceptible to both protein adsorption and bacteria adhesion. When grafted with TM/SA copolymer brushes, the non-biofouling properties of the membranes showed a strong dependence on the surface charge compositions of the membranes. A positively charged surface was prone to BSA adsorption and E. coli adhesion, while could effectively resist LYZ adsorption. On the contrary, a negatively charged surface could prevent BSA adsorption and E. coli adhesion, but prompt LYZ adsorption. When the copolymer brushes grafted on the membrane surfaces showed overall balanced charges, a membrane surface that could resist both the protein adsorption and biofilm formation simultaneously was achieved. It was suggested that the excellent non-biofouling properties of the membrane surfaces with balanced charges could be due to the lack of affinity between the membrane surface and the foulants that are either positively or negatively charged. The study also demonstrated a relatively simple and yet versatile approach to modify membrane surfaces from oppositely charged monomers, which provides the flexibility of making membrane surfaces with different extents of positive or negative charges to best meet the non-biofouling needs in applications. Acknowledgment. The financial support from the Ministry of Education Singapore, through R-288-000-046-112, the Academic Research Fund of National University of Singapore, is acknowledged. The CLSM images were acquired in the SBICNikon Imaging Centre at Biopolis, Singapore. The authors thank Dr. Clement Khaw for his help in the CLSM analysis. Supporting Information Available: Experimental results on protein fouling behaviors and resistance to biofilm formation for membranes with different TM:SA ratios as mentioned in the paper but at a similar grafting degree. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Jarusutthirak, C.; Amy, G. EnViron. Sci. Technol. 2006, 40, 969– 974. (2) Asatekin, A.; Kang, S.; Elimelech, M.; Mayes, A. M. J. Membr. Sci. 2007, 298, 136–146. (3) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323–330.

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