Surface-Engineered Biocatalytic Composite Membranes for Reduced

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Surfaces, Interfaces, and Applications

Surface Engineered Biocatalytic Composite Membrane for Reduced Protein-Fouling and Self-Cleaning Anbharasi Vanangamudi, Daisuke Saeki, Ludovic F Dumée, Mikel C. Duke, Todor Vasiljevic, Hideto Matsuyama, and Xing Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07945 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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ACS Applied Materials & Interfaces

Surface Engineered Biocatalytic Composite Membrane for Reduced Protein-Fouling and Self-Cleaning Anbharasi Vanangamudi1,2*, Daisuke Saeki3, Ludovic F. Dumée2, Mikel Duke1, Todor Vasiljevic4, Hideto Matsuyama3 and Xing Yang1* 1

Institute for Sustainability and Innovation (ISI), College of Engineering and Science, Victoria University, Melbourne, PO Box 14428, Victoria 8001, Australia 2

3

Deakin University, Waurn Ponds, Institute for Frontier Materials, Victoria 3216, Australia

Kobe University, Department of Chemical Science and Engineering, 1-1 Rokkodai-cho, Nada, Kobe, Hyogo, 657-8501 Japan

4

College of Health and Biomedicine, Victoria University, Melbourne, PO Box 14428, Victoria 8001, Australia

KEYWORDS: biocatalysts, ultrafiltration, enzymes, anti-fouling, nanofibers

1

ACS Paragon Plus Environment

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ABSTRACT A new biocatalytic nanofibrous composite ultrafiltration membrane was developed to reduce protein

fouling

interactions

and

self-clean

the

membrane

surface.

The

dual

layer

poly(vinylidenefluoride)/nylon-6,6/chitosan (PNC) composite membrane contains a hydrophobic poly(vinylidenefluoride) cast support layer and a hydrophilic functional nylon-6,6/chitosan nanofibrous surface layer where enzymes were chemically attached. The intrinsic surface chemistry and high surface area of the nanofibers allowed optimal and stable immobilization of trypsin (TR) and α-chymotrypsin (CT) enzymes via direct covalent binding. The enzyme immobilization was confirmed by X-ray photoelectron spectroscopy and visualised by confocal microscopy analysis. The prepared biocatalytic composite membranes were nano-porous with superior permeability offering stable protein anti-adhesion and self-cleaning properties owing to the repulsive mechanism and digestion of proteins into peptides and amino acids which was quantified by gel electrophoresis technique. The trypsin immobilized composite membranes exhibited 2.7-fold higher permeance and lower surface-protein contamination with 3-fold greater permeance recovery, when compared to pristine membrane after two ultrafiltration cycles with model feed solution containing bovine albumin serum/NaCl/CaCl2. The biocatalytic membranes retained about 50% of the enzyme activity after six reuse cycles but were regenerated to 100% activity after enzyme reloading, leading to simple and cost-effective water remediation operation. Such surface and pore engineered membranes with self-cleaning properties offer a viable solution for severe surface-protein contamination in food and water applications.

2


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INTRODUCTION The complexity of effluents used in water treatment poses great challenges associated with surface chemistry leading to irreversible membrane fouling. Proteins have been recognised as one of the key membrane foulants in wastewater treatment. Surface-protein contamination via nonspecific interfacial interactions is the key to the development of progressive cost-effective processes requiring advanced material solutions and chemistry control strategies.1 The accumulation of proteins on membrane surface or into pores causes blockage and forms cake layer leading to rapid decline in membrane permeability, increase in cleaning frequency and eventually diminish membrane performance.2-3 To reduce membrane fouling, membrane surface modifications using synthetic anti-fouling moieties such as hydrophilic4-6 and amphiphilic copolymers7, zwitterionic functionalities8-9, responsive materials10 and metal oxides6, 11 have been extensively studied. However, these moieties mainly work towards reducing continuous adsorption of proteins on to the membrane surface but not mitigating irreversible fouling, which is critical in sustaining membrane integrity. Therefore, there is a need to develop a new class of membranes with versatile structure and interfacial chemistry for tackling solid deposition issues via actively responding to foulant-membrane interactions, and ultimately achieving sustainable performance in long term. Enzymes are macromolecules that catalyse biochemical reactions with specific substrates like proteins to yield respective products. As rapidly degradable natural molecules, biocatalytic enzymes having high specificity attract attention as antifouling compounds.12 Biocatalytic enzymes have been found to degrade and remove the organic contaminants from waste streams, extending their potential to prepare self-cleaning surfaces that can lyse and detach the adsorbed proteins.13 Often, the use of free proteolytic enzymes, which digest proteins, is limited due to instability issues, poor performance recovery and difficile reusability in solution.14 Enzymes 3



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possess a high degree of substrate specificity, which indicates the binding of enzyme active sites only to specific substrates. The nature of substrate including material types, compositions and structures, play an important role in enhancing enzyme loading, activity and stability.15 In order to be used for filtration applications, the enzymes can be immobilized onto two different substrates including conventionally cast membranes and electrospun nanofibers. Various studies have reported enzyme immobilization onto conventionally cast membranes for fouling mitigation via protein digestion.13,

16-18

However, the application of such biocatalytic membranes is typically

associated with unstable enzyme loading, fast deterioration of enzymatic activity and major decline in membrane permeability. On the other hand, nanostructured materials have been used as suitable substrates to immobilize enzymes because of the high surface-to-volume ratio which provides high enzyme loading and improved stability.19 In particular, electrospun nanofibers are reported to be suitable substrates for enzyme immobilization due to material versatility and highly modifiable surface chemistry.20 The high porosity and pore interconnectivity of the nanofiber membranes also provide low hindrance for mass transfer and hence are ideal for filtration. It was demonstrated that the enzymatic activities of nanofiber membranes were higher than that of both free enzymes and enzymes immobilized onto commercial cast films.21-22 Also, the enzyme immobilized nanofibers presented greater reusability compared to enzyme immobilized cast films. For example, trypsin immobilized onto chitosan nanofibers and PET/PLA nanofiber mats showed 97% (five cycles) and 80% (eleven cycles) reusability respectively

23-24

. The tailorable surface of

nanofibers provide apt conditions for hosting the enzymes and the arrangement of nanofibers forms a non-woven mesh that can be used repeatedly as membrane filters. Enzyme immobilized nanofibers were widely used as biosensors, catalysts for treatment of specific substrates, drug delivery and other downstream applications,23-27 but not used for water filtration due to poor selectivity and strength. To treat complex wastewater, nanofibers are mostly used as a surface

4


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functional layer along with a support layer, to have superior selectivity and mechanical strength.28 However, there is lack of studies on nanofibrous membrane with dual functionality of biocatalysis and filtration. To prevent self-hydrolysis of free enzymes,13, 29-30 immobilization strategies are often used to attach enzymes onto substrates or membrane surfaces via the following methods such as physical adsorption, entrapment/encapsulation, cross-linking and covalent binding, each one of which directly impacts the enzymatic activity and stability.31 Although physical adsorption is a simple and easy method offering high enzyme loading, the vital disadvantage is enzyme leaching with time, resulting in loss of activity and operational stability.32 Such high enzyme loading may also decline the membrane permeability due to the dense adsorbed layer. Layer-by-layer adsorption and fouling induced immobilization methods have been widely used to adsorb more enzymes onto the membrane surface and into pores, which however led to substantially impaired membrane permeability.17,

33

Enzyme immobilization via entrapment/encapsulation is another

method to load high amount of enzymes usually onto the nanofiber substrate, which is highly dependent on the availability of enzyme active sites during

encapsulation.25,

27

Enzyme

attachment via glutaraldehyde cross-linking establishes strong bonding between the enzyme and substrate which enhanced enzyme activity and operational stability compared to adsorption.16, 24, 26, 34-35

Among all methods, enzyme immobilization via direct covalent binding was reported to

provide

much

more

efficient

enzyme

retention

and

stability

via

carbodiimide/N-

hydroxysuccinimide (EDC/NHS) reaction forming amide covalent linkages between the available functional groups across the enzyme and carrier substrate.13,

36-40

Although most of the

biocatalytic UF membranes reported thus far showed good recovery of membrane performance and enzyme reusability (i.e. operational stability) via covalent binding, the membranes generally exhibited undesirable low permeability, which tend to result in low fouling and did not truly 5



reflect the advantages of biocatalytic functionality of the membranes.13,

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41-42

Therefore, much

effort is needed to develop biocatalytic antifouling membranes with strong enzyme attachment and engineered porous structure, which offers both long term operational stability and high membrane permeability over long term. In this study, a new nano-porous biocatalytic composite membrane was prepared by covalently immobilizing enzymes onto nano-structured surface to achieve active self-cleaning functionality for water treatment applications. The dual sided composite membrane was made by the integration of a hydrophobic poly(vinylidene fluoride) (PVDF) cast layer and a hydrophilic electrospun nylon-6,6/chitosan nanofiber layer, which offered high surface area for increased enzyme attachment and stability that enhances the digestion of protein contaminants. The intrinsic chemistry of the nanofibers allowed optimal and stable enzyme immobilization through a one-step covalent binding technique. Here, the protein-digestive enzymes, trypsin and α-chymotrypsin were immobilized onto nanofibrous surface to resist the critical surface-protein interaction during ultrafiltration (UF). Trypsin (TR) and α-chymotrypsin (CT) are pancreatic enzymes that breakdown proteins to aid in food digestion. These enzymes are serine proteases with high sequence, structural and functional similarities widely used in liquid applications.13,

43

The

enzyme function is to catalyse the hydrolysis of proteins by circumventing the hindrances of breaking a peptide bond via proper positioning of catalytic triad, proton transfer and forming catalytic intermediate.44 Throughout the work reported here, the pristine PNC nanofiber composite membrane is utilised as the control for comparison. Since proteins have been identified as one of the major membrane foulants in wastewater treatment13, in this study, the proteindigestive enzymes were immobilized onto the composite membranes to cleave peptide bonds and degrade the protein foulants. In Figure 1a, we propose a hypothesis of the biocatalytic function of

6


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enzymatic membrane, where the progressive fouling mechanism is constantly disrupted due to protein degradation and thus a unique self-cleaning interface is created to sustain filtration.

Figure 1. Synthesis and functions of biocatalytic membrane. (a) Schematic concept of selfcleaning biocatalytic membrane – typical membrane fouling in control membrane (left) and proposed active degradation of protein fouling layer through cleavage of protein peptide bonds via hydrolysis (right) . (b) Synthetic scheme for preparation of biocatalytic membranes via EDC/NHS reaction. EXPERIMENTAL: Materials. The polymer PVDF Kynar® 761 was purchased from Arkema Pte. Ltd., Singapore. Trypsin enzyme from porcine pancreas was purchased from Wako pure chemical industries Ltd (Osaka, Japan). α-Chymotrypsin from bovine pancreas was purchased from Tokyo Chemical 7



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Industry (Tokyo, Japan). The following chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) and used as received: Polyamide-6,6 (nylon-6,6), chitosan (190-310 kDa molecular weight), poly(vinyl pyrrolidone) (PVP-K-40), EDC, NHS, bovine serum albumin (BSA), albumin-fluorescein isothiocyanate (BSA-FITC), analytical grade N,N’-dimethyl acetamide (DMAC), >95% formic acid, 99% trichloroacetic acid (TCA), 75% ethanol, sodium chloride (NaCl) and glycerol. Deionized (DI) water used in all experiments was obtained from the Milli-Q plus system (Millipore, Bedford, MA, USA). SDS-PAGE

Preparation of PVDF/nylon-6,6/chitosan (PNC) membrane. The PNC composite membrane was prepared similar to our previous work.45 Briefly, the composite membrane was prepared by three subsequent steps, (1) electrospinning a blend solution of 5 wt% nylon-6,6 and 1 wt% chitosan in formic acid at a voltage of 16 kV, flow rate of 0.2 mL/h and tip to collector distance of 150 mm to obtain the functional nanofiber mat, (2) conventional casting of solution prepared using 18 wt% PVDF and 8 wt% PVP in DMAC on to the as-obtained nanofiber mat and (3) phase inversion of the cast film on nanofiber mat by immersing into a coagulation tank of DI water at 25°C and removing the residual solvent, followed by post treatment by immersing into a mixture of ethanol, glycerol and DI water in the ratio 1:2:2 (vol%). Finally the membrane was dried.

Preparation of biocatalytic PNC-TR and PNC-CT membranes. The immobilization of TR and CT enzymes on to the as-prepared PNC composite membranes were achieved by 1-ethyl-(3-3dimethylaminopropyl)-carbodiimide

hydrochloride

(EDC)/N-hydroxysuccinimide

(NHS)

coupling reaction to form PNC-TR and PNC-CT membranes, respectively. The reaction schematic is given in Figure 1b. Firstly, the enzyme carboxyl groups was activated by reacting 1 mg/mL of enzyme solution with aqueous EDC and NHS in different molar ratios 1:1 (0.5 mM : 0.5 mM), 2:1, 3:1 and 4:1 for 1 h at room temperature, forming O-acylisourea active ester.46 8


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Secondly, the EDC/NHS activated enzymes were allowed to react with the primary amines on PNC membranes for 12 h at 4°C to covalently attach on to the membranes forming amide bonds via reaction between membrane amine groups and enzyme activated carboxyl groups. The reacted membranes were rinsed with DI water to remove the adsorbed TR and CT. The efficiency of immobilization was calculated from the enzyme concentration decrease in solution before and after contact with the membrane.

Membrane characterization. The surface morphology of the TR and CT immobilized PNC membranes was observed using scanning electron microscopy (SEM) (ZEISS SUPRA 55VP, Germany) with an accelerating voltage of 5 kV and working distance of 10 mm. The membrane samples were sputter coated with a 5 nm layer of gold in high vacuum, using a Leica EM ACE600 prior to imaging using SEM. The enzyme immobilization on to the membrane surface via EDC/NHS reaction was confirmed using XPS (JPS-9200, JEOL, Tokyo, Japan). The measurements were performed using an Mg Kα X-ray source (1253.6 eV) operated at 10 kV and 1 mA. Deconvolution of the C1s peaks were performed in order to resolve different functional groups and their composition. The pore size and pore size distribution of the membranes were measured using Porometer 3Gzh from Quantachrome. The membrane samples of 25 mm diameter each were completely wetted in the Porofil™ liquid before analysis and then placed in the sample holder. The sample was subjected to pressures from 6.4 to 34 bar for wet and dry run to measure the mean pore size. The pore size was measured three times for each membrane to obtain the average pore size. The water contact angle of the biocatalytic membranes were determined using an optical contact angle meter CAM101 (KSV instruments, Finland) to investigate the surface hydrophilicity. Each membrane sample was cut into thin strips and pasted on to a glass slide by using sticky tape on the two edges of membrane. A glass syringe filled with DI water was used to dispense about 4 µL droplet through a needle onto the membrane surface. The measurement was 9



recorded in triplicates for each sample. Further, to visualize the capacity of enzyme immobilization on to the composite membrane, BSA-FITC (model enzyme) was immobilized on to the PNC membranes and observed by using a confocal laser scanning microscope (CLSM; FV1000D; Olympus, Tokyo, Japan) at pH 7.0.

Quantification of immobilized enzymes. The surface density of immobilized enzymes TR and CT on the PNC membrane was calculated by measuring the enzyme concentration decrease in solution before and after contact with the membrane using UV-Visible spectrophotometer at 280 nm13. These measurements were carried out for enzyme immobilized membranes fabricated using different EDC concentrations.

Enzymatic activities of biocatalytic membranes against BSA. The enzymatic activities of PNC-TR and PNC-CT membranes were determined by measuring their hydrolytic activities using the method described previously with 1 wt% BSA solution as the substrate in phosphate buffer (0.1 M, pH 7.6).13 Likewise, the hydrolytic activities of free TR and CT was also determined for comparison. The enzymatic activities of biocatalytic membranes were measured prior to a continuous filtration testing. The sampled membranes and free enzymes were allowed to react with the BSA solution through mild shaking at 50 rpm. The reaction was carried out for different time periods up to 1 h at 37°C and terminated by the addition of 5 wt% TCA. Then, the mixture was centrifuged at 2000xg and the absorbance of the supernatant containing hydrolytic products was measured at 280 nm using a UV-Visible spectrophotometer. The blank contained the supernatant of the reaction carried out as above using PNC membranes without TR and CT. One digestion unit (DU) represents an increase of 0.1 in absorbance of the hydrolytic products denoting an increase in the amount of substrate digested by the enzymes via hydrolysis. The enzyme activity here relates to the amount of products formed. 10


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BSA digestion of native and immobilized enzymes via SDS-PAGE method. The BSA digestion into peptides via enzymatic hydrolysis were analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) using the method described elsewhere.47 Here, the BSA digestion by TR and CT immobilized membranes and free enzymes were measured by reacting them with 1 wt% BSA as substrate in phosphate buffer (0.1 M, pH 7.6). The reaction was carried out for 1 h at 37°C and the samples were loaded into respective wells of prepared gel. The gel was then run at 210 V for 1.1 h and the bands were stained with Coomassie Brilliant Blue dye for visualization.

The decrease in the band intensities of BSA were measured via Image J

software to determine the BSA digestion (in percentage) by free and immobilized enzymes using the equation:

% = 1 −

!

"# ∗ 100

(1)

Where BSAinitial and BSAenzyme are the intensities of BSA band before and after the reaction using native and immobilized enzymes.

Fouling studies. The antifouling and self-cleaning properties of the biocatalytic membranes and the pure PNC membrane was evaluated using a cross flow ultrafiltration set up having an effective area of 42x10-4 m2 and flow velocity of 12.6 cm/s. Proteases are specific to peptide bonds present in any protein and can hydrolyse proteins in the absence of inhibitors. While various proteins are present in real wastewater, the as-developed biocatalytic membranes were tested against a model protein foulant (BSA) in this filtration study. The prepared feed solution (10 L) contained 1 mg/mL BSA, 7 mM NaCl and 1 mM CaCl2 in DI water that had a pH of 7.8 which falls within the optimal pH range of TR (pH 7.5-8.5)48 and CT (pH 7.8-8).49 The addition of CaCl2 to the protein feed solution was to simulate a representative fouling environment through forming Ca2+-protein complexes. On the other hand, even though CaCl2 may suppress the self11



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digestion of TR and improve enzyme stability, addition of 1 mM CaCl2 concentration does not make significant effect in improving TR activity.50 Each membrane was initially exposed to 10 min of compaction using DI water at 120 kPa. It was then subjected to DI water containing 7 mM NaCl at 100 kPa for 15 min to measure the clean water permeance (Pw) in L.m-2.h-1 calculated by the following equation:

&' = ( ⁄ ∗ ∗ )

(2)

where V is the volume of permeate in L, A is the membrane area in m2, t is the permeation time in h and p is the constant pressure (1 bar). Each UF experiment had 2 cycles and each cycle included filtration of the prepared feed solution for 1 h followed by DI water for 15 min. The number ‘n’ represented the cycle number. The fouling studies for each membrane sample were performed three times and the permeance during filtration was recorded, averaged and presented as normalized permeance in % by calculating Pw/Pw(t)*100, where Pw(t) is the clean water permeance at time ‘t’. As a measure of protein fouling, the rate of permeance decline (RPD) after each cycle was calculated using the equation,

+&, % = 1 −

- -.

"# ∗ 100

(3)

where Pe(n) is the final feed permeance in nth cycle. Further, to represent the antifouling properties of membranes, the permeance recovery ratio (PRR) after each cycle was worked out using the equation,

&++ % =

-. -.

∗ 100

(4)

where Pw(n) is the clean water permeance in nth cycle. The self-cleaning property of membranes were also studied by computing the fouling parameters namely reversible fouling (RF), irreversible fouling (IF) and total fouling (TF) for each cycle by the following equations:

/0 = [&'234 − &'2 ]/& 12


(5)

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+0 = 7&82 − &92 :/& ;0 = /0 + +0

(6) (7)

where Ps is the initial feed permeance of each cycle and Pe is the final feed permeance of each cycle. Finally, the surfaces of PNC, TR and CT membranes after 2 cycles of filtration were imaged using SEM to visualise and compare the antifouling and self-cleaning properties of the enzyme immobilized membranes with that of the control PNC membranes.

Reusability and storage of immobilized enzymes. The reusability of immobilized enzymes were analysed by measuring the enzyme hydrolytic activity against BSA during six cycles consecutively before and after reloading enzymes. The reloading of enzymes on to the used membranes was achieved by EDC/NHS reaction mentioned earlier in the above section (Figure 1b). Briefly, the enzyme carboxyl groups was activated by reacting 1 mg/mL enzyme solution with aqueous EDC and NHS in different molar ratios 4:1 for 1 h at room temperature. The EDC/NHS activated enzymes were then allowed to react with the used membranes for 12 h at 4°C followed by rinsing with DI water. Furthermore, the biocatalytic membranes was stored under refrigeration at 4°C and at room temperature (RT) and the activity was measured at regular intervals for up to two weeks.

RESULTS and DISCUSSION Characteristics of biocatalytic membranes. The surface morphologies of the as-prepared control and biocatalytic membranes using 0.4 mol/L EDC and 0.1 mol/L NHS were observed by using scanning electron microscopy (SEM), as shown in Figure 2a, 2b and 2c. Basic membrane characterization data is provided in Table S1 of the Supporting information, including the nanofiber diameter and membrane thickness. In Figure 2a the control membrane without the

13



enzymes showed a homogenous morphology with an average nanofiber diameter of 130 nm (Table S1). Also, Figure 2b and 2c show that the biocatalytic PNC-TR and PNC-CT membranes demonstrated similar nanofiber structures with average diameters of 139 ± 3 nm and 143 ± 2 nm (Table S1), respectively. This slight increase in nanofiber diameter of the biocatalytic membranes compared to control membrane may be due to the attachment of enzymes. Enzymes are reported to cause morphological changes such as nanofiber diameter increment and entanglement.51-52 Furthermore, the PNC-CT membrane shows beads and clusters in some nanofibers that could be attributed to a higher enzyme concentration in specific areas (Figure 2c). The thickness of the biocatalytic membranes was measured from the cross sectional SEM micrographs of PNC, PNCTR and PNC-CT, and is also given in Table S1. The immobilization of enzymes slightly increased the thickness of the biocatalytic membranes i.e. PNC-TR and PNC-CT membranes had respective thickness of 244±10 µm and 247±10 µm compared to the 242±7 µm of the PNC membrane. The immobilization of enzymes on to the membrane surface via EDC/NHS reaction was confirmed by X-ray photoelectron spectroscopy (XPS) measurements. Figure S1 (Supporting information) shows the C1s core-level spectra of the PNC (Figure S1 left) and PNC-TR (Figure S1 right) membranes with binding energies at about 284.6 eV, 285.86 eV, 287.4 eV and 288.08 eV, corresponding to C-H, C-O, O=C-NH and C=O groups, respectively. Successful grafting of enzymes on the membrane surface can be deduced from the increase of amide (O=C-NH) groups in the biocatalytic PNC-TR membrane. Specifically, the peak area proportion calculated for amide was found to be 12.4% compared to 3.7% for the PNC membrane, indicating an increase in amide groups attributed to the covalent binding of enzymes on to the amine rich PNC membrane surfaces. The mean pore sizes and overall pore size distributions of the as-prepared membranes were measured using a capillary-flow porometer.45 Figure S2 (Supporting Information) compares the 14


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differential pore distributions of the three membranes in terms of pore diameters. The PNC and PNC-TR membranes exhibit similar narrow distribution curves; while the PNC-CT membrane has slightly wider distribution with double peaks, possibly due to the uneven distribution or clustering of CT enzymes as observed in Figure 2b and 2c. The mean pore size of the PNC, PNC-TR and PNC-CT membranes decreases slightly from 36.4 nm to 34.2 nm and 31.4 nm, respectively, as given in Table S1 (Supporting Information). The reduced pore size of the biocatalytic PNC-TR and PNC-CT membranes is ascribed to the enzyme attachment on to the membrane surface and/or depositing into the pores. PNC-CT membrane has the smallest mean pore size owing to the presence of enzyme clusters at some spots of the membrane. These clusters were formed due to possible aggregation of enzymes by randomized attachment points which implies lack of control on the position of enzyme structures.31 To evaluate the hydrophilicity of the as-prepared biocatalytic membranes, the water contact angle measurements (CAw) were acquired and given in Table S1 (Supporting Information). The immobilization of enzymes onto the membrane was found to influence the surface hydrophilicity owing to the intermolecular hydrogen bonding between the enzymes, bearing carboxyl and amine functional groups, and the water molecules.45 Compared to the PNC membrane with a CAw of 19±1°, the PNC-CT and PNC-TR membranes showed respective CAw of 10±2° and 14±1°. The difference in CAw could be due to the combined effect of enzyme properties and morphological changes due to enzyme immobilization as shown in Figure 2a, 2b and 2c. Although, the PNC membrane itself shows good hydrophilicity due to the presence of chitosan, the incorporation of enzymes increased hydrophilicity further that can offer additional resistance to protein fouling. Similarly, in literature, the addition of CT on to the dipeptide tethered surfaces53 and the immobilization of lysozyme on to the modified polyamide reverse osmosis (RO) membrane36 was

15



reported to influence the CAw. In addition to improved fouling resistance, the enhanced wettability offers high water permeability due to increased material compatibility with water.

Figure 2. Characteristics of biocatalytic membranes. SEM images of: (a) control PNC; (b) PNCTR; and (c) PNC-CT membranes. Cross-sectional SEM images of: (d) surface densities of TR and CT immobilized on to the PNC membranes with varying EDC concentrations. CLSM images of: (e) BSA-FITC adsorbed PNC membrane; and (f) BSA-FITC immobilized PNC membrane. Surface density of immobilized enzymes in biocatalytic membranes. The effect of the concentration of EDC cross-linker on the surface density of immobilized TR and CT enzymes was investigated to optimize the coupling reaction for high enzyme loading. The results are presented in Figure 2d in terms of surface densities of immobilized enzymes in mg/m2. An increasing trend of the surface density was observed, which can be attributed to the increased carbodiimide chemistry via EDC and NHS between the enzymes and membrane surfaces. For example, at an EDC concentration of 0.4 mol/L, the surface densities of immobilized TR and CT were 1.23 mg/m2 and 1.64 mg/m2, which were higher than the reported values of 0.7 mg/m2 of 16


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TR immobilized PES membrane13. Also, these values were within the theoretical limit of surface concentration (2.1 mg/m2) of closely packed monolayer trypsin molecules on the PES cast membrane54. Comparatively, the high surface area of nanofibers will offer greater theoretical limit for surface TR and CT concentrations, indicating that the current membrane surface was not fully covered by enzymes as confirmed by the SEM micrographs (Figure 2b and 2c). It was calculated that one trypsin molecule (MW: 23.3 kDa) occupies 32 nm2 surface whereas one CT molecule (MW: 25 kDa) occupies 25 nm2 surface at 0.4 mol/L EDC concentration. Thus, it is reasonable that the PNC-CT membrane had higher surface density of enzymes. Additionally, the surface immobilized enzymes on the PNC membranes was visualized qualitatively by confocal laser scanning microscopy (CLSM) using bovine serum albuminfluorescein isothiocyanate (BSA-FITC) as a model enzyme. The BSA-FITC adsorbed on to the PNC membrane via physical adsorption was compared against the BSA-FITC immobilized on to the membrane via EDC crosslinking. The results are shown in Figure 2e and 2f. It was observed that the adsorbed BSA-FITC did not show much fluorescence compared to the immobilized BSAFITC. This was due to the leaching effect of adsorbed BSA-FITC during rinsing with water as a result of weak binding with membrane; whereas the immobilized BSA-FITC binds covalently via EDC/NHS reaction on to the membrane thus offering stronger BSA-FITC retention.

Enzymatic activity of native and immobilized enzymes for BSA digestion. The enzymatic activity of native (free) enzymes and biocatalytic membranes were determined by performing the hydrolytic assay using 1 wt% BSA solution which gives the amount of hydrolytic products formed, i.e., peptides. One digestion unit (DU) represents an increase of 0.1 in absorbance of the hydrolytic products denoting an increase in the amount of substrate digested by the enzymes. The DU results are shown in Figure 3 with respect to reaction time. In Figure 3a, based on the DU data, the amount of products formed by immobilized TR and CT were observed to be much 17



greater than that of the native enzymes for all reaction times up to 60 min. For example, at 60 min the respective immobilized TR and CT membranes produced 83% and 94% more peptide products than the corresponding native enzymes. It was also noticed that the activity of immobilized enzymes increased with reaction time; whereas for the native enzymes, the activity increased initially but reached plateau in 10 min. This is due to the autolytic behaviour of the native enzymes, commonly known as self-digestion43,

55-56

; while the increased stability of

immobilized enzymes has greatly enhanced the enzymatic activities13. It is noted that the addition of low dosage of CaCl2 (1 mM to be consistent with fouling study) to the BSA solution was considered insignificant in suppressing the self-digestion of TR.50 The results further revealed that the immobilized CT has superior activity than the immobilized TR, possibly due to high immobilization density (Figure 2d). Although the density of immobilized CT is higher, they are prone to leaching during filtration, which may possible compromise its overall activity. On the other hand, the activity of native TR was found to be greater than the native CT due to substrate specificity as reported in literature.57 The comparison of BSA protein digestion into peptides by native and immobilized enzymes was further studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE).47 Figure 3b shows the respective bands of BSA and digestion products (i.e., peptides) present in the gel. The presence of original BSA in the substrate solution was confirmed by a thick intense band at 66 kDa in Lane 3. It was observed that the bands corresponding to BSA in Lanes 4, 5, 6 and 7 were diminished due to hydrolysis by the native (Lane 6 and 7) and immobilized enzymes (Lane 4 and 5), where the BSA digestion products are different polypeptides with molecular weights of 38 kDa and below. The BSA digestion (in %) was calculated by measuring the decrease in band intensity of BSA in each Lane. Consistent with the results of the enzyme activity essays, the PNC-TR and PNC-CT membranes showed 84.2% and 18


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93.6% BSA digestion, respectively; while the native TR and CT only gave 75.7% and 73.4% BSA degradation, respectively.

Figure 3. Enzymatic activities of TR and CT in biocatalytic membranes. (a) Digestion units of enzymatic hydrolysis by TR and CT enzymes using 1 wt% BSA solution over time. (b) SDSPAGE gel showing bands of BSA and enzymatically cleaved peptides. Protein fouling studies for biocatalytic membranes. The effect of enzyme immobilization on surface-protein interaction of the as-prepared PNC and biocatalytic membranes were investigated by fouling experiments through two filtration cycles, where a model feed was used as described in “methods” with intermediate pure water cleaning step at the start and between cycles. Figure 4 shows the results of two consecutive filtration cycles are presented in terms of the permeance decline rate (RPD) as a measure of protein fouling, and permeance recovery rate (PRR), reversible fouling (RF), total fouling (TF) and irreversible fouling (IF), as measures of self-cleaning ability of the membranes. The biocatalytic membranes PNC-TR and PNC-CT exhibited slightly (≤10%) lower initial water permeance compared to the PNC membrane, i.e., from 411 L.m-2.h-1.bar-1 19



(PNC) to 370 L.m-2.h-1.bar-1 (TR) and 376 L.m-2.h-1.bar-1 (CT), respectively. This is due to the surface attachment of enzyme molecules onto/between the nanofibers and into the pores (Figure 2) and decrease in pore size (Table S1). However, the permeance of PNC-TR and PNC-CT membranes were found to be much higher than that of the other enzyme immobilized UF membranes reported in literature. For example, the PNC-TR membrane exhibited about 4 times higher water permeance than the TR immobilized poly(methacrylic acid)-graft-polyethersulfone (PMAA-g-PES) membrane (88 L.m-2.h-1.bar-1)13 and about 7.5 times higher than the alcohol dehydrogenase (ADH) immobilized commercial GR51PP UF membrane (50 L.m-2.h-1.bar-1).33 As presented in Figure 4a, the normalised permeance for PNC-TR and PNC-CT membranes in the first filtration cycle declined about 14% and 24%, respectively, at the end of cycle. After cleaning, during the second filtration cycle, the permeance decreased further by about 7% and 18% for PNC-TR and PNC-CT membranes respectively. This demonstrates that the decrease in permeance became less significant with increasing filtration cycle, which may be attributed to (1) the intermediate cleaning that removed the reversibly fouled proteins and (2) the protein-digestive nature of the immobilized enzymes that cleaved and removed the fouled proteins from the membrane surface. Thus, the biocatalytic membranes show promising antifouling performance working towards sustainability. However, to claim sustainable membranes, long term fouling experiments need to be performed. The SEM micrographs of the control and biocatalytic membranes were presented in Figure 5. Precisely, compared to the control PNC membrane that showed heavy fouling (Figure 5a), the PNC-TR membrane exhibited much reduced protein deposition presenting clear surface after two filtration cycles (Figure 5b), followed by the PNCCT membrane that showed regional protein accumulation (Figure 5c). Overall, the biocatalytic membranes revealed superior fouling resistance with reduced protein interactions compared to the PNC membranes. This is attributed to the additional hydrophilicity (Table S1) and protein-

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digestive functionality (Figure 3) of the enzyme immobilized membranes, other than the presence of chitosan that was reported to offer good membrane hydrophilicity and antifouling property.45 Although the PNC-CT membrane showed greater activity than the PNC-TR against BSA in solution reaction (as presented in Figure 3a), the latter exhibited much improved filtration performance due to the homogenous distribution of TR on the surface (Figure 2b). On the other hand, the cluster formation of CT during immobilization (Figure 2c) may contain physically adsorbed CT on the membrane surface, which may have been washed away during filtration. Based on the flux patterns observed for all membranes in Figure 4a, the rate of permeance decline (RPD, Equation (3)) was calculated and presented in Figure 4b to indicate the resistance to protein fouling. During the first filtration cycle, the PNC membrane suffered severe fouling as indicated by an RPD of 48%, which is higher than that of the PNC-TR and PNC-CT membranes with respective RPD of 24% and 40%, suggesting that the enzyme active membranes were able to resist BSA attachment to a large extent. The results in this study appear to be promising when compared to the reported TR immobilized poly(methacrylic acid)-g-poly(ether sulfone) (PMAAg-PES) UF membrane having a flux decline rate of 19.1% using a simple 1 g/L BSA solution13; while a more realistic model feed solution was used in this work containing BSA as well as calcium chloride and sodium chloride, that greatly increase potential for surface fouling. It was reported that the calcium-induced protein aggregation leads to thicker and more compact fouling layer on the membrane surface through (1) intermolecular bridging among proteins forming protein-Ca2+-protein complexes and (2) intramolecular electrostatic shielding of negative charges on the proteins by Ca2+.58 Further, during the second filtration cycle, the PNC, PNC-TR and PNCCT membranes exhibited more distinct fouling behaviours leading an RPD of 80%, 31% and 51%, respectively. Combined with the hydrolysis analysis in Figure 3, the much improved flux patterns of the biocatalytic membranes could be due to the proteolytic ability i.e., protein digestive feature 21



of the enzymes.55 Specifically, the enzymatic cleavage of peptide bonds would increase the conformational entropy of adsorbing proteins and hence decrease the Gibbs free energy for adsorption. As a result, there is less interaction of proteins with the membrane surface resulting in reduced fouling.59 This phenomenon supports our hypothesis on self-cleaning mechanisms of the membrane, as depicted in Figure 1a. The lower RPD of the PNC-TR membrane compared to PNCCT membrane was attributed to the higher density of TR than the CT against specific substrate BSA. This is because, in the form of clusters, the immobilized CT was more prone to leaching during filtration and hence the surface density of immobilized CT is eventually reduced. The trend of decreasing RPD with time is strongly related to the self-cleaning ability of the membranes.

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Figure 4. Protein fouling studies for control and biocatalytic membranes. (a) Normalized permeance values for two filtration cycles. (b) RPD after each filtration cycle. (c) PRR after each filtration cycle. (d) TF, IF and RF for 2 filtration cycles. Experimental Conditions: Pressure = 100 kPa, cross-flow velocity = 12.6 cm/s, feed solution = 1 g/L BSA, 1 mM CaCl2 and 7 mM NaCl.

The self-cleaning ability of the biocatalytic membranes was quantified by computing the permeance recovery rate (PRR, Equation (4)) and fouling parameters namely reversible fouling (RF, Equation (6)), irreversible fouling (IF, Equation (5)) and total fouling (TF, Equation (7)). In this study, membrane cleaning using DI water was sufficient to restore the membrane performance, instead of using strong acid and alkaline solutions which may reduce membrane stability. Figure 4c reveals that after the first filtration cycle, the PNC-TR and PNC-CT membranes were able to recover about 80% and 70% of the initial permeance, respectively; while the control membrane only recovered 56%. The improvement in permeance recovery for the biocatalytic membranes is attributed to the breakdown of proteins into smaller polypeptides as revealed from the hypothesis in Figure 1a and results in Figure 3, which would release them subsequently from the membrane surface. In comparison, it was reported in literature that the flux of a TR immobilized PMAA-g-PES ultrafiltration membrane was fully recovered after the first filtration cycle, which was mainly related to the use of a 1 g/L BSA solution and much lower initial water permeance of 88 L.m-2.h-1.bar-1 leading to low fouling.13 Similarly, another TR immobilized PVDF microfiltration (MF) membrane fabricated via a complex electron beam method showed 90% flux recovery after first filtration cycle, also with simple 3 g/L BSA solution after backwashing and cleaning steps.42 However, the flux recovery in subsequent filtration cycles, the impact of more realistic model solution chemistry and choice of initial flux have not been investigated with enzymatic membranes, which will have strong implication on the practical applications.

23



Subsequently, after the second filtration cycle, the PNC-TR and PNC-CT membranes regained 70% and 48% of the initial permeance, respectively. The corresponding reversible and irreversible fouling parameters (RF & IF) are presented in Figure 4d. After the first filtration cycle, the biocatalytic PNC-TR and PNC-CT membranes reduced the IF by 55% and 32%, respectively, compared to the control membrane, explaining the higher PRR presented in Figure 4c. Also, the same trend was observed after the second filtration cycle with 70% and 36% less IF, respectively. This is strong evidence of less permanent fouling featuring the self-cleaning capacity of the biocatalytic membranes. Thus, the biocatalytic membranes exhibited much lower TF, which is corresponding to their higher PRR. However, the higher filtration performance of PNCTR membrane is due to the homogenous immobilization and distribution of active TR on the surface. Overall, the PNC-TR membrane was identified to be the best performing biocatalytic membrane in terms of fouling mitigation and self-cleaning ability. The main advantage of using enzyme approach for fouling control is in reduction of irreversible fouling where strongly attached proteins can be removed by protein digestion into smaller peptides and aminoacids. This approach ensures that the removed proteins would not be able to readsorb further onto the membrane surface unlike the other foulant release approach.60 Hence, the approach of enzyme immobilization onto nanofibrous surface has greater potential including fouling mitigation and self-cleaning beyond membrane separation.

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Figure 5. Protein fouled membranes. SEM micrographs of fouled membranes (a) PNC; (b) PNCTR; and (c) PNC-CT after two filtration cycles. Experimental conditions: pressure = 100 kPa, cross-flow velocity = 12.6 cm/s, feed solution = 1 g/L BSA, 1 mM CaCl2 and 7 mM NaCl. Reusability and storage. The reusability of the biocatalytic membranes was investigated by measuring the hydrolytic activities over six consecutive reaction cycles before and after enzyme reloading. The results are given in Figure 6a. Overall, the hydrolytic activities of immobilized enzymes gradually declined with increasing reuse cycles. The immobilized TR retained about 59% of the initial activity after three cycles; whereas the immobilized CT retained about 63%. However, after six reuse cycles, the immobilized TR retained more enzymatic activity (49%) than the immobilized CT (42%). Enzyme reloading was conducted to ensure the reusability of the enzymatic membranes. The reloaded membranes showed similar trends over another six cycles. This loss of hydrolytic activity of the enzymatic membranes may have occurred due to (a) the 25



release of weakly bound enzymes to the solution, if any, and (b) the gradual change of fibrous morphology because of swelling and disintegration due to high hydrophilicity.61 Nevertheless, reusability data obtained in this work shows significant improvement compared to the literature data, where the immobilized papain retained only 12% of its initial activity after six cycles.61 Other recent works have evaluated the reusability of biocatalytic membranes in terms of water flux % (about 30% flux retained after 5 cycles) during filtration.41-42 The effect of storage time on the hydrolytic activities of the immobilized enzymes at 4°C and room temperature (RT) were analysed and given in Figure 6b. It was revealed that in both refrigerated and RT environment, the immobilized TR and CT retained about 81% and 78% of their initial biocatalytic activities after 7 days, respectively, and about 70% of their initial activities for both enzymes after 14 days of storage. Thus, the prepared membranes may not require inconvenient refrigerated storage conditions. In comparison, this was found to be higher than the literature data where the immobilized trypsin having 64% of its initial activity and immobilized papain having 40% of its initial activity after 14 days of storage at 4°C.61-62 The higher activity retention of immobilized enzymes could be attributed to the intermolecular bonding between the enzymes and the nanofiber layer that prolongs and sustains the biocatalytic behaviour of the immobilized enzymes.61 Furthermore, other recent works have evaluated the stored enzyme activity of biocatalytic membranes indirectly in terms of water flux % (about 40% flux retained after 7 days of storage) during filtration.41-42 However, the stability and reusability of the membranes highly depend on the complexity of treated solutions or substrates. Hence, the asprepared PNC-TR membrane in this work offered ideal engineered structure and stable enzyme activity with high permeability, protein resistance and self-cleaning in practical fouling environment during water treatment. Thus, the originality of this work relates to investigation of the mechanisms of interactions between enzymatic nanofiber surface and the proteins with respect 26


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to the filtration performance behaviour, which is a key aspect to design functional surfaces and porous materials for use in complex effluents.

Figure 6. Stability of biocatalytic membranes. (a) Reusability of membranes in terms of hydrolytic activity for 6 consecutive reuse cycles before and after reloading of enzymes. Error bars are in the range 0.04 to 2.3 %. (b) Hydrolytic activities of biocatalytic membranes for up to 14 days of storage at 4°C and RT. Error bars are in the range 0.06 to 1.7 %.

CONCLUSIONS In summary, highly permeable biocatalytic ultrafiltration membrane was successfully fabricated by immobilizing trypsin and α-chymotrypsin enzymes onto hydrophilic nylon-6,6/chitosan nanofibrous layer supported by hydrophobic PVDF cast layer. The PNC-TR membrane minimized surface-protein interaction on the surface, induced by enzyme proteolytic digestion. Through systematic design and evaluation, we demonstrated that ideal enzyme loading and stability can be achieved owing to the amine-rich and high surface to volume ratio of the nanofibrous structure. The biocatalytic membranes offered outstanding performance in separation and purification applications, where they are more permeable and less fouled than previously 27



reported membranes, and can be used to treat a range of protein contaminant solution. Through a dedicated ultrafiltration study using model feed solution containing BSA, CaCl2 and NaCl, the PNC-TR membrane exhibited lowest fouling propensity with highly recoverable permeance and declined irreversible fouling compared to the PNC-CT and PNC membranes. Also, in response to the decrease in enzymatic activity after every reuse cycle, the enzyme reloading ability of the biocatalytic membranes ensured reoccurrence of enzymatic activity. The strategy where nanofibers are used for enzyme immobilization has great potential beyond the sole scope of membrane separation and hence the biocatalytic membranes had greater prospective in treating complex effluents with surface-protein contamination via digestion and self-cleaning. Further, to treat wastewaters containing other organic contaminants such as lipids, lipolytic enzymes can be immobilized on to the composite membranes for digesting lipid foulants by using similar methodology used in this study. We envision that such highly permeable self-cleaning membranes will contribute to future material engineering and surface functionalised membranes for sustainable food and water supplies.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge via the Internet at http://pubs.acs.org. C1s core-level spectra of the PNC (left) and PNC-TR (right) membranes; properties of biocatalytic membranes; differential pore number (in %) distributions of PNC, PNC-TR and PNC-CT membranes. AUTHOR INFORMATION Corresponding Author *Ms. Anbharasi Vanangamudi, E-mail: [email protected], Tel.: +61 399 197 640. 28


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*Dr. Xing Yang, E-mail: [email protected], Tel.: +61 399 197 690. Author contributions The experimental work was designed and carried out by Ms. Anbharasi Vanangamudi under the guidance of Dr. Saeki, Dr. Ludovic Dumee, Dr. Xing Yang, Prof. Todor Vasiljevic, Prof. Matsuyama and Prof. Mikel Duke. The manuscript was written by Ms. Anbharasi Vanangamudi and reviewed by all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by Victoria India Institute via Victoria India Doctoral Scholarship. Dr. L. DUMEE acknowledges Deakin University for his Alfred Deakin Post-Doctoral Fellowship. Dr Xing Yang would like to acknowledge Victoria University for the Industry Postdoctoral Fellowship.

Notes The authors declare no competing financial interest.

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Surface-Engineered Biocatalytic Composite Membranes for Reduced

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