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Trypsin-enabled construction of anti-fouling and self-cleaning polyethersulfone membrane
Bioresource Technology 102 (2011) 647–651
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Trypsin-enabled construction of anti-fouling and self-cleaning polyethersulfone membrane Qing Shi, Yanlei Su, Xue Ning, Wenjuan Chen, Jinming Peng, Zhongyi Jiang ⇑ Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 25 March 2010 Received in revised form 2 August 2010 Accepted 9 August 2010 Available online 10 August 2010 Keywords: Surface modification Ultrafiltration membrane Trypsin Protein fouling resistant Self-cleaning
a b s t r a c t Constructing anti-fouling and self-cleaning membrane surfaces based on covalent attachment of trypsin on poly(methacrylic acid)-graft-polyethersulfone (PMAA-g-PES) membrane was reported. The carboxylic acid groups enriched on asymmetric PMAA-g-PES membrane surface were activated with 1-ethyl-(3-3dimethylaminopropyl)-carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) and employed as chemical anchors for the conjugation with amino groups of trypsin. Activity assays showed that such chemically immobilized trypsin was much more active and stable than that of the physically adsorbed counterpart. Trypsin covalently attached on membrane surface could substantially resist protein fouling in dynamic flow process. The considerable enhancement of protein solution permeation flux was observed as a consequence of rapid enzymatic degradation of protein deposited onto membrane surface. The permeation flux of the membrane could be recovered upon simple hydraulic flush after protein filtration, suggesting superior self-cleaning property. After multi-cycle BSA filtration over 15-day period, the active self-cleaning membrane maintained more than 95.0% of its initial flux. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The widespread application of membrane-based technology in water treatment has been greatly restricted by membrane fouling (Shannon et al., 2008; Lo et al., 2005; Phattaranawik and Leiknes, 2009). Protein has been identified as one of the major membrane foulants in wastewater treatment and reclamation applications (She et al., 2009; Shon et al., 2006). The accumulation of protein on membrane surfaces or inside membrane pores decreases flux greatly, affects the quality and quantity of products, and eventually shortens the membrane lifetime. According to previous studies, it was generally accepted that membrane fouling was attributed to the protein adsorption and deposition during flow conditions (Mueller and Davis, 1996). To inhibit protein fouling on membrane surfaces, great efforts have been devoted to modify surfaces with synthetic anti-fouling or nonfouling moieties. Among them, poly(ethylene glycol) (PEG) (Herrwerth et al., 2003; Blattler et al., 2006) and zwitterionic functionalities (Holmlin et al., 2001; Chen et al., 2005) are most extensively studied. Even though PEG and zwitterionic polymer-based materials could reduce the adsorption of proteins on dense surfaces below several ng cm2 (Chelmowski et al., 2007; Zhang et al., 2006), reducing protein fouling on porous ⇑ Corresponding author. Address: Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China. E-mail address: [email protected] (Z. Jiang). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.030
membrane surfaces still represents a major challenge. The existence of the pores greatly restricts the choice of appropriate surface modifying agent and modification method. In addition, when the membranes are operated in dynamic flow process, hydrodynamic forces always drag protein molecules toward the membrane surfaces. The technology for creating self-cleaning surfaces has been developed rapidly in recent years (Parkin and Palgrave, 2005). Basically, the key to producing self-cleaning surfaces lies in the removal of contamination by either surface reaction (chemically break down adsorbed biomolecules) or droplet flow (the Lotus-Effect) (Blossey, 2003; Genzer and Marmur, 2008; Allain et al., 2007). In recent years enzymes have found widespread use in decontamination of waste streams (Lejeune et al., 1998). Kim et al. (2001) and Asuri et al. (2007) incorporated enzymes into a variety of different matrices to create self-cleaning dense surfaces that resist protein adsorption. These active proteolytic enzymes attached on surfaces could break down protein foulants, and the fragments can be subsequently removed through a simple water flashing operation. Therefore, the employment of enzymes may enable the superior protein fouling resistance of membrane surfaces. The idea of using enzymes to construct self-cleaning membranes is not new (Chen et al., 1992), however, only few studies have been conducted till now. Herein, we proposed a facile and general strategy for attaching enzyme onto porous membrane surface to suppress protein fouling in dynamic flow process. Asymmetric membranes with
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Q. Shi et al. / Bioresource Technology 102 (2011) 647–651
well-defined morphology and excellent flow-through property were first prepared via non-solvent-induced phase separation from amphiphilic poly(methacrylic acid)-graft-polyethersulfone (PMAA-g-PES). The model enzyme trypsin was then conjugated to membrane surface through 1-ethyl-(3-3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS)-activation of carboxylic acid groups enriched on the membrane surface. We found that the stable and active trypsin immobilized on membrane surface could prevent and remove protein fouling simultaneously, rendered the membrane with both antifouling and self-cleaning capacities. A flux recovery ratio (FRR) of 100% could be achieved after protein solution filtration. In particular, the membrane displayed long-term protein fouling resistance.
(0.1 M, pH 7.6). After incubated at 37 °C for 10 min, the reaction was terminated by the addition of 5 wt.% trichloroacetic acid. The mixture was subsequently centrifugated at 2000g and the absorbance of the supernatant was measured at 280 nm using a UV– vis spectrophotometer (Hitachi, U-2800). The values obtained in blank reactions performed in the presence of casein sodium salt but without trypsin were discounted from all readings. One enzyme unit (U) was defined as the amount of enzyme that produced an increase of 0.1 in absorbance. The enzymatic activity of physically adsorbed trypsin on PES membrane surface was also measured for comparison. The activity measurement was conducted in triplicate for each assay.
2. Methods
The surface chemical compositions of the membranes were analyzed by XPS (PHI-1600, USA) using Mg Ka as radiation source (the takeoff angle of photoelectron was set to 90o). The survey spectra were collected at fixed analyzer pass energy of 160 eV. XPS analysis was conducted on the side of the membrane facing the water bath during the precipitation step of fabrication.
2.1. Materials Polyethersulfone (PES, 6020P) was purchased from BASF Co. (Germany) and was dried at 110 °C for 12 h before use. Trypsin from porcine pancreas was supplied from Gibco Co. (USA). Casein sodium salt from bovine milk was purchased from the Sigma Chemical Co. (USA). EDC and NHS were purchased from Shanghai Medpep Co. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Institute of Hematology, Chinese Academy of Medical Science (Tianjin, China). Benzoyl peroxide (BPO), 2-morpholinoethanesulfonic acid (MES), N,N-dimethyl formamide (DMF), trichloroacetic acid and all other reagents were purchased from local chemical reagent company and used as received. 2.2. Preparation of the membranes The copolymer PMAA-g-PES was synthesized using our previous reported method (Shi et al., 2010). Briefly, PMAA side chains were graft polymerized directly onto commercial PES by BPO initiated free radical polymerization. The copolymer was then cast into ultrafiltration membranes via phase inversion process in an aqueous medium. In this study, the membrane cast from PMAA-g-PES copolymer with a graft yield of 3.7% was used for trypsin immobilization. According to XPS analysis, the surface coverage of PMAA on membrane surface region was 20.6% (Shi et al., 2010), which indicated remarkable surface enrichment of the hydrophilic MAA segment in the membrane. PMAA-g-PES membranes were activated in an MES buffer solution (50 mM, pH 6.0) containing 50 mM NHS and 100 mM EDC for 4 h at room temperature. The pH of MES buffer solution was adjusted with NaOH. The activated membranes were then rinsed with deionized water to remove unreacted NHS and EDC. The immobilization of trypsin was based on the reaction of activated carboxylic sites on membrane surface with primary amine groups of trypsin. Specifically, the activated membranes were immersed in 2.0 mg/mL trypsin dissolved in phosphate buffer (0.1 M, pH 7.0) at room temperature for 4 h followed by 0 °C for 12 h. The reacted membranes were rinsed with phosphate buffer three times to remove adsorbed trypsin. The efficiency of immobilization was calculated from the protein concentration decrease in solution before and after the contact with the membrane. 2.3. Activity assays of trypsin immobilized on membrane surface The method of Arnon as described by Purcena et al. (2009) was used for trypsin activity measurement. The activity of trypsin immobilized on membrane surface was determined using 1.0 wt.% casein sodium salt as substrate in phosphate buffer
2.4. X-ray photoelectron spectroscopy (XPS) characterization
2.5. Ultrafiltration experiments The anti-fouling and self-cleaning properties were evaluated with dynamic protein fouling experiments. A dead-end stirred cell (Model 8200, Millipore Co., USA) was used for the filtration study. The effective membrane area was 28.7 cm2. Each membrane was initially compacted with deionized water for 30 min at 150 kPa. Then the pressure was lowered to the operating pressure of 100 kPa and the water flux (Jw1) was calculated by the following equation:
J w1 ¼
V ADt
ð1Þ
where V was the volume of permeated water (l), A was membrane area (m2) and Dt was permeation time (h). After switching the feed solution to 1.0 g/L BSA solution, BSA filtration was conducted for 1 h and the flux (Jp) was measured. The flux decline rate (RFD) was calculated by the following equation:
RFD ¼
J 1 P 100% J w1
ð2Þ
Finally, the membrane was cleaned with phosphate buffer (0.1 M, pH 7.0) for 30 min and the pure water flux (Jw2) was measured again. The FRR, Jw2/Jw1, could be employed to represent the antifouling properties of the membranes. For the multi-cycle ultrafiltration experiments, the FRR of the nth cycle was defined as Jwn/Jw1. The membranes were stored at 4 °C in phosphate buffer (0.1 M, pH 7.0) between filtration cycles. 3. Results and discussion Recently, we reported the graft polymerization of MAA onto PES using benzoyl peroxide (BPO) as chemical initiator (Shi et al., 2010). The synthesized PMAA-g-PES copolymers were then cast into membranes via non-solvent-induced phase inversion process. The membrane exhibited asymmetric cross-sectional structure with the thin separation layer on the top, supported from underneath through the main macroporous part of the membrane (Fig. S1). Due to the surface segregation, PMAA graft chains were significantly enriched on membrane surface (Shi et al., 2010), which ensured the sufficient amount of carboxylic acid groups for the subsequent enzyme attachment. A mixture of EDC/NHS was commonly used for the covalent attachment of proteins on
Q. Shi et al. / Bioresource Technology 102 (2011) 647–651
Relative enzymatic activity (%)
100 Before washing After washing
80 60 40 20 0
(a)
(b)
Fig. 1. Relative specific enzymatic activity of trypsin adsorbed (a) and immobilized with EDC/NHS activation (b) on membrane surface. The stabilities were characterized by measuring relative enzymatic activity of the membranes after hydraulic washing.
surfaces (Timkovich, 1977). The conjugation of trypsin with PMAAg-PES membrane surface was achieved via the formation of amide bonds between carboxylic groups and amine groups of trypsin using EDC as the coupling agent and NHS as activator. As shown in Fig. 1, the catalytic activity of trypsin immobilized by EDC/NHS activation procedure was higher than that immobilized by physical adsorption procedure. More importantly, the trypsin immobilized by carbodiimide chemistry was found to be stable and retained large extent of its initial activity after 60 min hydraulic washing (magnetic stirring, 300 rpm), while the physically adsorbed trypsin exhibited significant loss of activity under identical conditions. Obviously, EDC/NHS mediated linkage between enzyme and membrane rendered the stable existence of trypsin, which was of great significance since in practical applications rigorous stirring was always required to eliminate concentration polarization near membrane surface. To investigate the effect of immobilized trypsin on protein adsorption to membrane surface, protein fouling experiments were performed use a three-step ultrafiltration protocol. As shown in Fig. 2, after the immobilization of trypsin on membrane surface, the water flux showed no obvious decline (compared with the pure
120 Water
Flux (l/(m2 h))
100
Protein Solution
Water
80 60 40 20 0
0
20
40
60
80
100
120
Time (min) Fig. 2. The time-dependent flux variation of ultrafiltration operation with PMAA-gPES membrane (}), trypsin-containing membrane prepared by physical adsorption (4), trypsin-containing membrane prepared by covalent bonding (d), and trypsincontaining membrane prepared by covalent bonding pre-treated with pH 2.0 solution (h).
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water flux of PMAA-g-PES membrane). This was not surprising given the fact that the immobilized amount of trypsin (by the use of carbodiimide chemistry) was measured at about 0.7 mg/m2. This value was lower than the theoretical surface concentration of closely packed trypsin molecules (2.1 mg/m2) (Koutsopoulos et al., 2007), which indicated the membrane surface was not fully occupied by trypsin molecules. It was believed that the repulsive lateral interactions between positively charged enzyme molecules may hamper the formation of a fully occupied monolayer (Koutsopoulos et al., 2007). As a result, the well-controlled trypsin immobilization process had no obvious affect to the permeation performance of the membrane. The immobilization of trypsin on membrane surface was further confirmed by the appearance of N and P peaks in XPS results (Fig. S2). Since the membrane surface was only partially covered by trypsin molecules, the peak intensities of N and P were relatively low. It is well-known that, in general, protein fouling on membrane surface leads to sharp flux losses and often limits its useable lifetime. The flux decline rate (RFD) was used as a measurement of protein fouling for the dynamic flow studies. The lower value of RFD represents the higher fouling resistant ability of the membrane. We used bovine serum albumin (BSA) as a model protein for protein fouling studies under dynamic flow conditions. In the process of protein solution filtration, the permeate flux of PMAA-g-PES membrane decreased significantly (RFD = 73.0%), indicated the membrane suffered from drastic fouling. In contrast, the RFD value of trypsin modified membrane, both for EDC/NHS mediated immobilization (RFD = 19.1%) and physical adsorption (RFD = 27.6%), was much smaller. This suggested the active membranes were able to resist a substantial fraction of BSA adsorption in dynamic flow process. The reduced protein fouling to the trypsin-containing membrane was believed due to the proteolytic property of trypsin (Kang et al., 2006), which could affect settlement of protein on the surface. Specifically, the enzymatic cleavage of peptide bonds would increase the conformational entropy of the adsorbing protein. The increased conformational entropy would decrease the Gibbs free energy for adsorption, thus reduce the probability that the protein would adsorb to the membrane surface (Kim et al., 2001). The membrane with immobilized trypsin by carbodiimide chemistry, which was more active and stable, showed lower RFD value compared with that of physically adsorbed. The membrane had almost no drop in flux in the tested time span of protein filtration. Even more striking results were obtained within FRR analysis. FRR is the most commonly used parameter for assessing the effectiveness of a cleaning treatment after protein fouling. In the present study, we use FRR to assess the self-cleaning efficiency of the active membrane. The pure water flux of these samples after protein filtration and hydraulic washing was also plotted in Fig. 2. The flux recovery for PMAA-g-PES membrane was considerably low (22%), resulted from the severe protein fouling. In comparison, the trypsin-containing membranes had significant improved FRR. Particularly, the FRR for the membrane with immobilized trypsin (by the use of carbodiimide chemistry) was as high as 100%, indicating after single-cycle ultrafiltration experiments, the permeation performance was totally recovered within simple hydraulic washing. The active trypsin on membrane could breakdown protein into smaller fragments, which would release subsequently from membrane surface, made the formation of self-cleaning membrane that prevented protein build-up. Even though the membrane surface were not totally occupied by trypsin molecules, the relaxation of the immobilized trypsin molecules would lead to a certain extent of spreading, result in an increase of the ‘‘footprint” of the trypsin at membrane surface (Norde and Giacomelli, 1999). Therefore, it was reasonable to deduce that the high biocatalytic activity can be achieved within the whole membrane surface.
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As expected, the presence of active trypsin immobilized on membrane surface could render the membrane with excellent resistance to protein fouling. To further confirm the anti-fouling and self-cleaning behavior were truly due to the proteolytic activity of trypsin, we inactivated the immobilized trypsin by immersing the membrane in pH 2.0 solution for 10 min. According to Jw1, Jw2, and JP shown in Fig. 2, the RFD value and FRR value of the membrane with the pre-inactivated trypsin was calculated to be 37.4% and 72.0%, respectively. Compared with the active counterpart, the membrane with pre-inactivated trypsin showed relative higher RFD value and lower FRR value. This illustrated that the enzyme activity was of significant importance for the prevention and removal of protein pollutants. Compared with PMAA-g-PES membrane, the membrane with pre-inactivated trypsin showed improved fouling resistance. Recently, many efforts have been made to enhance the fouling resistance of membranes by surface grafting biomacromolecules. For example, Ulbricht and Riedel (1998) and Fang et al. (2009) proposed the modification of PES membrane by grafting BSA on the surface. The increased anti-fouling property was mainly attributed to the improved hydrophilicity of BSA-modified membrane. However, in the present work, hydrophilicity of the membrane with pre-inactivated trypsin was not playing a dominate role in depressing protein fouling. Even though membrane with pre-inactivated trypsin showed better fouling resistance, there was no significant difference of surface hydrophilicity between PMAA-g-PES membrane and the membrane with pre-inactivated trypsin. A possible explanation for the fouling resistance of membrane with pre-inactivated trypsin was that the chemically immobilized trypsin on membrane surface could prevent protein from blocking the pores in dynamic flow processes. Multi-cycle ultrafiltration experiments were used to further investigate the long-term stability and reusability of trypsin modified membrane. The FRR results are shown in Fig. 3. Because of the inherent hydrophobicity of PES, the FRR of pristine PES membrane decreased rapidly. In contrast, the trypsin modified membrane maintained more than 95.0% of its initial flux after multi-cycle BSA filtration over a 15-day period. These results demonstrated the membrane with immobilized active trypsin had much longer lifetime for high-performance protein filtration. Besides removing protein pollutants in wastewater treatment, the as-prepared membranes are also suitable for separation of non-protein products with serious fouling by protein impurities, and comparatively suitable for separation of edible proteins with a variety of resources. However, since the hydrolytic trypsin immobilized on the membrane might also digest part of proteins in
100
Flux Recovery (%)
90 80 70 60 50 40 30 -50
0
50
100 150 200 250 300 350 400
Time (h) Fig. 3. Long-term FRR test for PES control membrane (4) and trypsin-containing membrane prepared by covalent bonding (h).
filtrate, it may be not suitable for separating proteins (Chen et al., 1992). Nevertheless, we offered a novel cost-effective method to suppress dynamic membrane fouling, minimizing the involvement of harmful chemicals. Moreover, this method might be readily extended to the design of anti-fouling membranes by immobilizing a series of enzymes for downstream bioprocessing. For example, membranes with immobilized polysaccharide hydrolases (e.g., dextranases) are capable of breaking down glucans, which are a major constituent of biofilms that offering significant resistance to permeate flow in filtration process (Khalilkova et al., 2005; Azeredo et al., 1998). Therefore, the membraneenzyme composites may provide a general route to the thorough elimination of membrane biofouling. 4. Conclusions In summary, we demonstrated that anti-fouling and self-cleaning membrane could be acquired via the stable covalent attachment of trypsin. The presence of trypsin on membrane surface did not cause obvious appreciable flux decline. The proteolytic activity of trypsin-anchored membrane was exploited in suppressing protein fouling in dynamic flow process. After protein solution filtration, the membrane could recover its initial flux upon simple hydraulic washing. The results suggested that the enzyme-resided membrane displayed desirable anti-fouling and self-cleaning properties. More importantly, the high stability of the membrane surface rendered the membrane with long lifetime for protein filtration. Acknowledgements This work is financially supported by Research Fund for the Doctoral Program of Higher Education of China (20060056032), and the Program of Introducing Talents of Discipline to Universities (No. B06006). The authors would like to thank Fei He for the XPS experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.08.030. References Allain, E., Besson, S., Durand, C., Moreau, M., Gacoin, T., Boilot, J.-P., 2007. Transparent mesoporous nanocomposite films for self-cleaning applications. Adv. Funct. Mater. 17 (4), 549–554. Asuri, P., Karajanagi, S.S., Kane, R.S., Dordick, J.S., 2007. Polymer–nanotube–enzyme composites as active antifouling films. Small 3 (1), 50–53. Azeredo, J., Oliveria, R., Lazarova, V., 1998. A new method for extraction of exopolymer from activated sludges. Water Sci. Technol. 37 (4–5), 367–370. Blattler, T.M., Pasche, S., Textor, M., Griesser, H.J., 2006. High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates. Langmuir 22 (13), 5760–5769. Blossey, R., 2003. Self-cleaning surfaces – virtual realities. Nat. Mater. 2 (5), 301– 306. Chelmowski, R., Prekelt, A., Grunwald, C., Wöll, C., 2007. A case study on biological activity in a surface-bound multicomponent system: the biotin–streptavidinperoxidase system. J. Phys. Chem. A 111 (49), 12295–12303. Chen, J., Wang, L., Zhu, Z., 1992. Preparation of enzyme immobilized membranes and their self-cleaning and anti-fouling abilities in protein separations. Desalination 86 (3), 301–315. Chen, S., Zheng, J., Li, L., Jiang, S., 2005. Strong resistance of phosphorylcholine selfassembled monolayers to protein adsorption: Insights into nonfouling properties of zwitterionic materials. J. Am. Chem. Soc. 127 (41), 14473–14478. Fang, B., Ling, Q., Zhao, W., Ma, Y., Bai, P., Wei, Q., Li, H., Zhao, C., 2009. Modification of polyethersulfone membrane by grafting bovine serum albumin on the surface of polyethersulfone/poly(acrylonitrile-co-acrylic acid) blended membrane. J. Membr. Sci. 329, 46–55. Genzer, J., Marmur, A., 2008. Biological and synthetic self-cleaning surfaces. MRS Bull. 33 (8), 742–746.
Q. Shi et al. / Bioresource Technology 102 (2011) 647–651 Herrwerth, S., Eck, W., Reinhardt, S., Grunze, M., 2003. Factors that determine the protein resistance of oligoether self-assembled monolayers internal hydrophilicity, terminal hydrophilicity, and lateral packing density. J. Am. Chem. Soc. 125 (31), 9359–9366. Holmlin, R.E., Chen, X., Chapman, R.G., Takayama, S., Whitesides, G.M., 2001. Zwitterionic SAMs that resist nonspecific adsorption of protein from aqueous buffer. Langmuir 17 (9), 2841–2850. Kang, K., Kan, C., Yeung, A., Liu, D., 2006. The immobilization of trypsin on soap-free P(MMA-EA-AA) latex particles. Mater. Sci. Eng. C 26 (4), 664–669. Khalilkova, E., Susi, P., Korpela, T., 2005. Microbial dextran-hydrolyzing enzymes: fundamentals and applications. Microbiol. Mol. Biol. Rev. 69 (2), 306–325. Kim, Y.D., Dordick, J.S., Clark, D.S., 2001. Siloxane-based biocatalytic films and paints for use as reactive coatings. Biotechnol. Bioeng. 72 (4), 475–482. Koutsopoulos, S., Patzsch, K., Bosker, W.T.E., Norde, W., 2007. Adsorption of trypsin on hydrophilic and hydrophobic surfaces. Langmuir 23 (4), 2000–2006. Lejeune, K.E., Wild, J.R., Russell, A.J., 1998. Nerve agents degraded by enzymatic foams. Nature 395 (6697), 27–28. Lo, Y.M., Cao, D., Argin-Soysal, S., Wang, J., Hahm, T.-S., 2005. Recovery of protein from poultry processing wastewater using membrane ultrafiltration. Bioresour. Technol. 96 (6), 687–698. Mueller, J., Davis, R.H., 1996. Protein fouling of surface-modified polymeric microfiltration membranes. J. Membr. Sci. 116 (1), 47–60. Norde, W., Giacomelli, C.E., 1999. Conformational changes in proteins at interfaces: from solution to the interface, and back. Macromol. Symp. (145), 125–136. Parkin, I.P., Palgrave, R.G., 2005. Self-cleaning coatings. J. Mater. Chem. 15 (17), 1689–1695.
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Phattaranawik, J., Leiknes, T., 2009. Double-deck aerated biofilm membrane bioreactor with sludge control for municipal wastewater treatment. AIChE J. 55 (5), 1291–1297. Purcena, L.L.A., Caramori, S.S., Mitidierl, S., Fernandes, K.F., 2009. The immobilization of trypsin onto polyaniline for protein digestion. Mater. Sci. Eng. C 29 (4), 1077–1081. Shannon, M.A., Bohn, P.W., Elimelech, M., Georgiadis, J.G., Mariñas, B.J., Mayes, A.M., 2008. Science and technology for water purification in the coming decades. Nature 452 (7185), 301–310. She, Q., Tang, C.Y., Wang, Y.-N., Zhang, Z., 2009. The role of hydrodynamic conditions and solution chemistry on protein fouling during ultrafiltration. Desalination 249 (3), 1079–1087. Shi, Q., Su, Y., Ning, X., Chen, W., Peng, J., Jiang, Z., 2010. Graft polymerization of methacrylic acid onto polyethersulfone for potential pH-responsive membrane materials. J. Membr. Sci. 347 (1–2), 62–68. Shon, H.K., Vigneswaran, S., Kim, I.S., Cho, J., Ngo, H.H., 2006. Fouling of ultrafiltration membrane by effluent organic matter: a detailed characterization using different organic fractions in wastewater. J. Membr. Sci. 278 (1–2), 232–238. Timkovich, R., 1977. Detection of the stable addition of carbodiimide to proteins. Anal. Biochem. 79 (1–2), 135–143. Ulbricht, M., Riedel, M., 1998. Ultrafiltration membrane surfaces with grafted polymer ‘tentacles’: preparation, characterization and application for covalent protein binding. Biomaterials 19 (14), 1229–1237. Zhang, Z., Chen, S., Chang, Y., Jiang, S., 2006. Surface grafted sulfobetaine polymers via atom transfer radical polymerization as superlow fouling coatings. J. Phys. Chem. B 110 (22), 10799–10804.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Trypsin-enabled construction of anti-fouling and self-cleaning polyethersulfone membrane Qing Shi, Yanlei Su, Xue Ning, Wenjuan Chen, Jinming Peng, Zhongyi Jiang ⇑ Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 25 March 2010 Received in revised form 2 August 2010 Accepted 9 August 2010 Available online 10 August 2010 Keywords: Surface modification Ultrafiltration membrane Trypsin Protein fouling resistant Self-cleaning
a b s t r a c t Constructing anti-fouling and self-cleaning membrane surfaces based on covalent attachment of trypsin on poly(methacrylic acid)-graft-polyethersulfone (PMAA-g-PES) membrane was reported. The carboxylic acid groups enriched on asymmetric PMAA-g-PES membrane surface were activated with 1-ethyl-(3-3dimethylaminopropyl)-carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) and employed as chemical anchors for the conjugation with amino groups of trypsin. Activity assays showed that such chemically immobilized trypsin was much more active and stable than that of the physically adsorbed counterpart. Trypsin covalently attached on membrane surface could substantially resist protein fouling in dynamic flow process. The considerable enhancement of protein solution permeation flux was observed as a consequence of rapid enzymatic degradation of protein deposited onto membrane surface. The permeation flux of the membrane could be recovered upon simple hydraulic flush after protein filtration, suggesting superior self-cleaning property. After multi-cycle BSA filtration over 15-day period, the active self-cleaning membrane maintained more than 95.0% of its initial flux. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The widespread application of membrane-based technology in water treatment has been greatly restricted by membrane fouling (Shannon et al., 2008; Lo et al., 2005; Phattaranawik and Leiknes, 2009). Protein has been identified as one of the major membrane foulants in wastewater treatment and reclamation applications (She et al., 2009; Shon et al., 2006). The accumulation of protein on membrane surfaces or inside membrane pores decreases flux greatly, affects the quality and quantity of products, and eventually shortens the membrane lifetime. According to previous studies, it was generally accepted that membrane fouling was attributed to the protein adsorption and deposition during flow conditions (Mueller and Davis, 1996). To inhibit protein fouling on membrane surfaces, great efforts have been devoted to modify surfaces with synthetic anti-fouling or nonfouling moieties. Among them, poly(ethylene glycol) (PEG) (Herrwerth et al., 2003; Blattler et al., 2006) and zwitterionic functionalities (Holmlin et al., 2001; Chen et al., 2005) are most extensively studied. Even though PEG and zwitterionic polymer-based materials could reduce the adsorption of proteins on dense surfaces below several ng cm2 (Chelmowski et al., 2007; Zhang et al., 2006), reducing protein fouling on porous ⇑ Corresponding author. Address: Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China. E-mail address: [email protected] (Z. Jiang). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.030
membrane surfaces still represents a major challenge. The existence of the pores greatly restricts the choice of appropriate surface modifying agent and modification method. In addition, when the membranes are operated in dynamic flow process, hydrodynamic forces always drag protein molecules toward the membrane surfaces. The technology for creating self-cleaning surfaces has been developed rapidly in recent years (Parkin and Palgrave, 2005). Basically, the key to producing self-cleaning surfaces lies in the removal of contamination by either surface reaction (chemically break down adsorbed biomolecules) or droplet flow (the Lotus-Effect) (Blossey, 2003; Genzer and Marmur, 2008; Allain et al., 2007). In recent years enzymes have found widespread use in decontamination of waste streams (Lejeune et al., 1998). Kim et al. (2001) and Asuri et al. (2007) incorporated enzymes into a variety of different matrices to create self-cleaning dense surfaces that resist protein adsorption. These active proteolytic enzymes attached on surfaces could break down protein foulants, and the fragments can be subsequently removed through a simple water flashing operation. Therefore, the employment of enzymes may enable the superior protein fouling resistance of membrane surfaces. The idea of using enzymes to construct self-cleaning membranes is not new (Chen et al., 1992), however, only few studies have been conducted till now. Herein, we proposed a facile and general strategy for attaching enzyme onto porous membrane surface to suppress protein fouling in dynamic flow process. Asymmetric membranes with
648
Q. Shi et al. / Bioresource Technology 102 (2011) 647–651
well-defined morphology and excellent flow-through property were first prepared via non-solvent-induced phase separation from amphiphilic poly(methacrylic acid)-graft-polyethersulfone (PMAA-g-PES). The model enzyme trypsin was then conjugated to membrane surface through 1-ethyl-(3-3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS)-activation of carboxylic acid groups enriched on the membrane surface. We found that the stable and active trypsin immobilized on membrane surface could prevent and remove protein fouling simultaneously, rendered the membrane with both antifouling and self-cleaning capacities. A flux recovery ratio (FRR) of 100% could be achieved after protein solution filtration. In particular, the membrane displayed long-term protein fouling resistance.
(0.1 M, pH 7.6). After incubated at 37 °C for 10 min, the reaction was terminated by the addition of 5 wt.% trichloroacetic acid. The mixture was subsequently centrifugated at 2000g and the absorbance of the supernatant was measured at 280 nm using a UV– vis spectrophotometer (Hitachi, U-2800). The values obtained in blank reactions performed in the presence of casein sodium salt but without trypsin were discounted from all readings. One enzyme unit (U) was defined as the amount of enzyme that produced an increase of 0.1 in absorbance. The enzymatic activity of physically adsorbed trypsin on PES membrane surface was also measured for comparison. The activity measurement was conducted in triplicate for each assay.
2. Methods
The surface chemical compositions of the membranes were analyzed by XPS (PHI-1600, USA) using Mg Ka as radiation source (the takeoff angle of photoelectron was set to 90o). The survey spectra were collected at fixed analyzer pass energy of 160 eV. XPS analysis was conducted on the side of the membrane facing the water bath during the precipitation step of fabrication.
2.1. Materials Polyethersulfone (PES, 6020P) was purchased from BASF Co. (Germany) and was dried at 110 °C for 12 h before use. Trypsin from porcine pancreas was supplied from Gibco Co. (USA). Casein sodium salt from bovine milk was purchased from the Sigma Chemical Co. (USA). EDC and NHS were purchased from Shanghai Medpep Co. (Shanghai, China). Bovine serum albumin (BSA) was obtained from Institute of Hematology, Chinese Academy of Medical Science (Tianjin, China). Benzoyl peroxide (BPO), 2-morpholinoethanesulfonic acid (MES), N,N-dimethyl formamide (DMF), trichloroacetic acid and all other reagents were purchased from local chemical reagent company and used as received. 2.2. Preparation of the membranes The copolymer PMAA-g-PES was synthesized using our previous reported method (Shi et al., 2010). Briefly, PMAA side chains were graft polymerized directly onto commercial PES by BPO initiated free radical polymerization. The copolymer was then cast into ultrafiltration membranes via phase inversion process in an aqueous medium. In this study, the membrane cast from PMAA-g-PES copolymer with a graft yield of 3.7% was used for trypsin immobilization. According to XPS analysis, the surface coverage of PMAA on membrane surface region was 20.6% (Shi et al., 2010), which indicated remarkable surface enrichment of the hydrophilic MAA segment in the membrane. PMAA-g-PES membranes were activated in an MES buffer solution (50 mM, pH 6.0) containing 50 mM NHS and 100 mM EDC for 4 h at room temperature. The pH of MES buffer solution was adjusted with NaOH. The activated membranes were then rinsed with deionized water to remove unreacted NHS and EDC. The immobilization of trypsin was based on the reaction of activated carboxylic sites on membrane surface with primary amine groups of trypsin. Specifically, the activated membranes were immersed in 2.0 mg/mL trypsin dissolved in phosphate buffer (0.1 M, pH 7.0) at room temperature for 4 h followed by 0 °C for 12 h. The reacted membranes were rinsed with phosphate buffer three times to remove adsorbed trypsin. The efficiency of immobilization was calculated from the protein concentration decrease in solution before and after the contact with the membrane. 2.3. Activity assays of trypsin immobilized on membrane surface The method of Arnon as described by Purcena et al. (2009) was used for trypsin activity measurement. The activity of trypsin immobilized on membrane surface was determined using 1.0 wt.% casein sodium salt as substrate in phosphate buffer
2.4. X-ray photoelectron spectroscopy (XPS) characterization
2.5. Ultrafiltration experiments The anti-fouling and self-cleaning properties were evaluated with dynamic protein fouling experiments. A dead-end stirred cell (Model 8200, Millipore Co., USA) was used for the filtration study. The effective membrane area was 28.7 cm2. Each membrane was initially compacted with deionized water for 30 min at 150 kPa. Then the pressure was lowered to the operating pressure of 100 kPa and the water flux (Jw1) was calculated by the following equation:
J w1 ¼
V ADt
ð1Þ
where V was the volume of permeated water (l), A was membrane area (m2) and Dt was permeation time (h). After switching the feed solution to 1.0 g/L BSA solution, BSA filtration was conducted for 1 h and the flux (Jp) was measured. The flux decline rate (RFD) was calculated by the following equation:
RFD ¼
J 1 P 100% J w1
ð2Þ
Finally, the membrane was cleaned with phosphate buffer (0.1 M, pH 7.0) for 30 min and the pure water flux (Jw2) was measured again. The FRR, Jw2/Jw1, could be employed to represent the antifouling properties of the membranes. For the multi-cycle ultrafiltration experiments, the FRR of the nth cycle was defined as Jwn/Jw1. The membranes were stored at 4 °C in phosphate buffer (0.1 M, pH 7.0) between filtration cycles. 3. Results and discussion Recently, we reported the graft polymerization of MAA onto PES using benzoyl peroxide (BPO) as chemical initiator (Shi et al., 2010). The synthesized PMAA-g-PES copolymers were then cast into membranes via non-solvent-induced phase inversion process. The membrane exhibited asymmetric cross-sectional structure with the thin separation layer on the top, supported from underneath through the main macroporous part of the membrane (Fig. S1). Due to the surface segregation, PMAA graft chains were significantly enriched on membrane surface (Shi et al., 2010), which ensured the sufficient amount of carboxylic acid groups for the subsequent enzyme attachment. A mixture of EDC/NHS was commonly used for the covalent attachment of proteins on
Q. Shi et al. / Bioresource Technology 102 (2011) 647–651
Relative enzymatic activity (%)
100 Before washing After washing
80 60 40 20 0
(a)
(b)
Fig. 1. Relative specific enzymatic activity of trypsin adsorbed (a) and immobilized with EDC/NHS activation (b) on membrane surface. The stabilities were characterized by measuring relative enzymatic activity of the membranes after hydraulic washing.
surfaces (Timkovich, 1977). The conjugation of trypsin with PMAAg-PES membrane surface was achieved via the formation of amide bonds between carboxylic groups and amine groups of trypsin using EDC as the coupling agent and NHS as activator. As shown in Fig. 1, the catalytic activity of trypsin immobilized by EDC/NHS activation procedure was higher than that immobilized by physical adsorption procedure. More importantly, the trypsin immobilized by carbodiimide chemistry was found to be stable and retained large extent of its initial activity after 60 min hydraulic washing (magnetic stirring, 300 rpm), while the physically adsorbed trypsin exhibited significant loss of activity under identical conditions. Obviously, EDC/NHS mediated linkage between enzyme and membrane rendered the stable existence of trypsin, which was of great significance since in practical applications rigorous stirring was always required to eliminate concentration polarization near membrane surface. To investigate the effect of immobilized trypsin on protein adsorption to membrane surface, protein fouling experiments were performed use a three-step ultrafiltration protocol. As shown in Fig. 2, after the immobilization of trypsin on membrane surface, the water flux showed no obvious decline (compared with the pure
120 Water
Flux (l/(m2 h))
100
Protein Solution
Water
80 60 40 20 0
0
20
40
60
80
100
120
Time (min) Fig. 2. The time-dependent flux variation of ultrafiltration operation with PMAA-gPES membrane (}), trypsin-containing membrane prepared by physical adsorption (4), trypsin-containing membrane prepared by covalent bonding (d), and trypsincontaining membrane prepared by covalent bonding pre-treated with pH 2.0 solution (h).
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water flux of PMAA-g-PES membrane). This was not surprising given the fact that the immobilized amount of trypsin (by the use of carbodiimide chemistry) was measured at about 0.7 mg/m2. This value was lower than the theoretical surface concentration of closely packed trypsin molecules (2.1 mg/m2) (Koutsopoulos et al., 2007), which indicated the membrane surface was not fully occupied by trypsin molecules. It was believed that the repulsive lateral interactions between positively charged enzyme molecules may hamper the formation of a fully occupied monolayer (Koutsopoulos et al., 2007). As a result, the well-controlled trypsin immobilization process had no obvious affect to the permeation performance of the membrane. The immobilization of trypsin on membrane surface was further confirmed by the appearance of N and P peaks in XPS results (Fig. S2). Since the membrane surface was only partially covered by trypsin molecules, the peak intensities of N and P were relatively low. It is well-known that, in general, protein fouling on membrane surface leads to sharp flux losses and often limits its useable lifetime. The flux decline rate (RFD) was used as a measurement of protein fouling for the dynamic flow studies. The lower value of RFD represents the higher fouling resistant ability of the membrane. We used bovine serum albumin (BSA) as a model protein for protein fouling studies under dynamic flow conditions. In the process of protein solution filtration, the permeate flux of PMAA-g-PES membrane decreased significantly (RFD = 73.0%), indicated the membrane suffered from drastic fouling. In contrast, the RFD value of trypsin modified membrane, both for EDC/NHS mediated immobilization (RFD = 19.1%) and physical adsorption (RFD = 27.6%), was much smaller. This suggested the active membranes were able to resist a substantial fraction of BSA adsorption in dynamic flow process. The reduced protein fouling to the trypsin-containing membrane was believed due to the proteolytic property of trypsin (Kang et al., 2006), which could affect settlement of protein on the surface. Specifically, the enzymatic cleavage of peptide bonds would increase the conformational entropy of the adsorbing protein. The increased conformational entropy would decrease the Gibbs free energy for adsorption, thus reduce the probability that the protein would adsorb to the membrane surface (Kim et al., 2001). The membrane with immobilized trypsin by carbodiimide chemistry, which was more active and stable, showed lower RFD value compared with that of physically adsorbed. The membrane had almost no drop in flux in the tested time span of protein filtration. Even more striking results were obtained within FRR analysis. FRR is the most commonly used parameter for assessing the effectiveness of a cleaning treatment after protein fouling. In the present study, we use FRR to assess the self-cleaning efficiency of the active membrane. The pure water flux of these samples after protein filtration and hydraulic washing was also plotted in Fig. 2. The flux recovery for PMAA-g-PES membrane was considerably low (22%), resulted from the severe protein fouling. In comparison, the trypsin-containing membranes had significant improved FRR. Particularly, the FRR for the membrane with immobilized trypsin (by the use of carbodiimide chemistry) was as high as 100%, indicating after single-cycle ultrafiltration experiments, the permeation performance was totally recovered within simple hydraulic washing. The active trypsin on membrane could breakdown protein into smaller fragments, which would release subsequently from membrane surface, made the formation of self-cleaning membrane that prevented protein build-up. Even though the membrane surface were not totally occupied by trypsin molecules, the relaxation of the immobilized trypsin molecules would lead to a certain extent of spreading, result in an increase of the ‘‘footprint” of the trypsin at membrane surface (Norde and Giacomelli, 1999). Therefore, it was reasonable to deduce that the high biocatalytic activity can be achieved within the whole membrane surface.
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As expected, the presence of active trypsin immobilized on membrane surface could render the membrane with excellent resistance to protein fouling. To further confirm the anti-fouling and self-cleaning behavior were truly due to the proteolytic activity of trypsin, we inactivated the immobilized trypsin by immersing the membrane in pH 2.0 solution for 10 min. According to Jw1, Jw2, and JP shown in Fig. 2, the RFD value and FRR value of the membrane with the pre-inactivated trypsin was calculated to be 37.4% and 72.0%, respectively. Compared with the active counterpart, the membrane with pre-inactivated trypsin showed relative higher RFD value and lower FRR value. This illustrated that the enzyme activity was of significant importance for the prevention and removal of protein pollutants. Compared with PMAA-g-PES membrane, the membrane with pre-inactivated trypsin showed improved fouling resistance. Recently, many efforts have been made to enhance the fouling resistance of membranes by surface grafting biomacromolecules. For example, Ulbricht and Riedel (1998) and Fang et al. (2009) proposed the modification of PES membrane by grafting BSA on the surface. The increased anti-fouling property was mainly attributed to the improved hydrophilicity of BSA-modified membrane. However, in the present work, hydrophilicity of the membrane with pre-inactivated trypsin was not playing a dominate role in depressing protein fouling. Even though membrane with pre-inactivated trypsin showed better fouling resistance, there was no significant difference of surface hydrophilicity between PMAA-g-PES membrane and the membrane with pre-inactivated trypsin. A possible explanation for the fouling resistance of membrane with pre-inactivated trypsin was that the chemically immobilized trypsin on membrane surface could prevent protein from blocking the pores in dynamic flow processes. Multi-cycle ultrafiltration experiments were used to further investigate the long-term stability and reusability of trypsin modified membrane. The FRR results are shown in Fig. 3. Because of the inherent hydrophobicity of PES, the FRR of pristine PES membrane decreased rapidly. In contrast, the trypsin modified membrane maintained more than 95.0% of its initial flux after multi-cycle BSA filtration over a 15-day period. These results demonstrated the membrane with immobilized active trypsin had much longer lifetime for high-performance protein filtration. Besides removing protein pollutants in wastewater treatment, the as-prepared membranes are also suitable for separation of non-protein products with serious fouling by protein impurities, and comparatively suitable for separation of edible proteins with a variety of resources. However, since the hydrolytic trypsin immobilized on the membrane might also digest part of proteins in
100
Flux Recovery (%)
90 80 70 60 50 40 30 -50
0
50
100 150 200 250 300 350 400
Time (h) Fig. 3. Long-term FRR test for PES control membrane (4) and trypsin-containing membrane prepared by covalent bonding (h).
filtrate, it may be not suitable for separating proteins (Chen et al., 1992). Nevertheless, we offered a novel cost-effective method to suppress dynamic membrane fouling, minimizing the involvement of harmful chemicals. Moreover, this method might be readily extended to the design of anti-fouling membranes by immobilizing a series of enzymes for downstream bioprocessing. For example, membranes with immobilized polysaccharide hydrolases (e.g., dextranases) are capable of breaking down glucans, which are a major constituent of biofilms that offering significant resistance to permeate flow in filtration process (Khalilkova et al., 2005; Azeredo et al., 1998). Therefore, the membraneenzyme composites may provide a general route to the thorough elimination of membrane biofouling. 4. Conclusions In summary, we demonstrated that anti-fouling and self-cleaning membrane could be acquired via the stable covalent attachment of trypsin. The presence of trypsin on membrane surface did not cause obvious appreciable flux decline. The proteolytic activity of trypsin-anchored membrane was exploited in suppressing protein fouling in dynamic flow process. After protein solution filtration, the membrane could recover its initial flux upon simple hydraulic washing. The results suggested that the enzyme-resided membrane displayed desirable anti-fouling and self-cleaning properties. More importantly, the high stability of the membrane surface rendered the membrane with long lifetime for protein filtration. Acknowledgements This work is financially supported by Research Fund for the Doctoral Program of Higher Education of China (20060056032), and the Program of Introducing Talents of Discipline to Universities (No. B06006). The authors would like to thank Fei He for the XPS experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.08.030. References Allain, E., Besson, S., Durand, C., Moreau, M., Gacoin, T., Boilot, J.-P., 2007. Transparent mesoporous nanocomposite films for self-cleaning applications. Adv. Funct. Mater. 17 (4), 549–554. Asuri, P., Karajanagi, S.S., Kane, R.S., Dordick, J.S., 2007. Polymer–nanotube–enzyme composites as active antifouling films. Small 3 (1), 50–53. Azeredo, J., Oliveria, R., Lazarova, V., 1998. A new method for extraction of exopolymer from activated sludges. Water Sci. Technol. 37 (4–5), 367–370. Blattler, T.M., Pasche, S., Textor, M., Griesser, H.J., 2006. High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates. Langmuir 22 (13), 5760–5769. Blossey, R., 2003. Self-cleaning surfaces – virtual realities. Nat. Mater. 2 (5), 301– 306. Chelmowski, R., Prekelt, A., Grunwald, C., Wöll, C., 2007. A case study on biological activity in a surface-bound multicomponent system: the biotin–streptavidinperoxidase system. J. Phys. Chem. A 111 (49), 12295–12303. Chen, J., Wang, L., Zhu, Z., 1992. Preparation of enzyme immobilized membranes and their self-cleaning and anti-fouling abilities in protein separations. Desalination 86 (3), 301–315. Chen, S., Zheng, J., Li, L., Jiang, S., 2005. Strong resistance of phosphorylcholine selfassembled monolayers to protein adsorption: Insights into nonfouling properties of zwitterionic materials. J. Am. Chem. Soc. 127 (41), 14473–14478. Fang, B., Ling, Q., Zhao, W., Ma, Y., Bai, P., Wei, Q., Li, H., Zhao, C., 2009. Modification of polyethersulfone membrane by grafting bovine serum albumin on the surface of polyethersulfone/poly(acrylonitrile-co-acrylic acid) blended membrane. J. Membr. Sci. 329, 46–55. Genzer, J., Marmur, A., 2008. Biological and synthetic self-cleaning surfaces. MRS Bull. 33 (8), 742–746.
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