Novel biodegradation system for bisphenol A using laccase-immobilized hollow fiber membranes

Accepted Manuscript Novel biodegradation system for bisphenol A using laccaseimmobilized hollow fiber membranes

Ashkan Mokhtar, Tomoya Nishioka, Hikaru Matsumoto, Soma Kitada, Nonoka Ryuno, Tadashi Okobira PII: DOI: Reference:

S0141-8130(18)35923-3 https://doi.org/10.1016/j.ijbiomac.2019.03.004 BIOMAC 11834

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

1 November 2018 1 March 2019 1 March 2019

Please cite this article as: A. Mokhtar, T. Nishioka, H. Matsumoto, et al., Novel biodegradation system for bisphenol A using laccase-immobilized hollow fiber membranes, International Journal of Biological Macromolecules, https://doi.org/10.1016/ j.ijbiomac.2019.03.004

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ACCEPTED MANUSCRIPT Novel Biodegradation System for Bisphenol A Using Laccase-Immobilized Hollow Fiber Membranes Ashkan Mokhtar, Tomoya Nishioka, Hikaru Matsumoto, Soma Kitada, Nonoka Ryuno, Tadashi Okobira*

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150 Higashihagio-Machi, Omuta, Fukuoka 836-8585, Japan

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Department of Chemical Science and Engineering, National Institute of Technology, Ariake

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*Author for correspondence and reprint requests ([email protected])

Highlights:

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Graphical Abstract

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1. Multilayer adsorption of enzymes is possible on amine-containing polymer brushes. 2. Space velocity (SV) affects the activity of immobilized laccase. 3. Immobilized laccase showed high stability in organic media. 4. Bisphenol A (BPA) was biodegraded with good efficiency using immobilized laccase. 5. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) reduction by oxidation of BPA indicated the initiation of redox reactions. Keywords: Radiation-induced graft polymerization, Immobilized laccase, Bisphenol A, Biodegradation, Redox mediator 1

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Abstract Radiation-induced graft polymerization was applied to prepare membranes for multilayer immobilization of laccase, which has biodegradation ability for bisphenol A (BPA). Glycidyl methacrylate (GMA) was grafted onto porous polyethylene membranes as the monomer of polymer brushes, and aminoethanol (AE) was introduced to the grafted GMA membrane, creating unfolded polymer brushes that serve as a good support for multilayer immobilization of laccase. The objectives of this study were as follows: adjustment of space velocity (SV) for optimum performance; enhancement of stability in organic media through moisture retention; biodegradation of BPA at continuous operation; and investigation of the effects of redox mediators. Laccase and membrane activities were increased at higher SVs as

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a result of stronger substrate transport. The 1.85% moisture retention as a result of highdensity AE containing polymer brushes demonstrated the improved stability of immobilized laccase over free laccase in methanol-containing solutions. BPA was removed with an activity of 0.11 mol/h/kg-membrane. The effects of three major laccase mediators on BPA oxidation was studied, and only 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was shown to increase the oxidation of BPA to 100% at low SVs. Improved stability of laccase and high removal rates in the continuous biodegradation of BPA were achieved by the presented method.

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1. Introduction Global production of bisphenol A (BPA) is growing annually [1] because of its use in the production of various polymer materials. One of polymer materials is polycarbonate, which is widely used in everyday products varying from food product containers such as milk bottles to electronics such as the protection layer on compact discs. The good mechanical and chemical properties of BPA products are some of the reasons why an even higher growth in its production is expected. However, recent studies indicate an increase in the migration of BPA into the environment, which has received a great deal of attention because of its hazardous properties [2-4]. Studies indicate that it is highly toxic to aquatic life [5], increases liver diseases [6], raises the risk of cancer development [7], etc. Thus, many researchers are now focusing on developing methods for removing BPA from wastewater [8]. Among these studies, the biodegradation of BPA using laccase appears to be the most promising method because it is an efficient and clean process. Laccase is an enzyme in the multicopper oxidase family. It is widely found in some fungi and plants, and has the capability of oxidizing a wide range of phenolic compounds. Laccase oxidizes substrates through the reduction of oxygen to water [9-11]. However, the application of laccase in chemical processes is limited because of its lack of stability in most organic solvents [12]. Moreover, enzymes are expensive and difficult to recover for further reuse [13]. Many studies have been conducted on reusing enzymes and increasing their stability by immobilizing them on supports such as silica [14], cellulose [15], carbon 2

ACCEPTED MANUSCRIPT nanoparticles [16], microspheres [17], and reversed micelles [18]. Such studies on laccase

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immobilization could result in cheaper and more environmentally friendly pollutant treatments. Use of membrane bioreactors is one of the prominent methods in wastewater treatment. A vast variety of materials are available for the preparation of such membranes, among which polymers are easy and cheap to design for specific treatment processes. Graft polymerization can be used to turn different kinds of trunk polymers into functionalized materials. Among numerous methods that could be used for grafting, radiation-induced graft polymerization (RIGP) is superior as it allows the initiation of polymerization to take place at low temperatures and different monomer states. The preparation of active membranes with the

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ability to capture metals or catalyze reactions using this method has been reported [19-22]. One of the benefits of this method is that it enables easy introduction of various functional groups to the polymer brush [23]. The high density of the polymer brush and high mass transfer through the membrane create appropriate environments for many chemical processes [24]. Enzyme immobilization on polymer brushes of membranes is one of the accomplishments of this method. A previous study on immobilized lipase using RIGP for the biosynthesis of biodiesel showed that proper polymer brushes, containing positively charged amine groups, unfold because of electrostatic repulsion, thereby producing an adequate environment for multilayer immobilization of enzymes and enhancing their activity and stability [25]. The application of this method to laccase can result in highly functional

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membranes capable of degrading BPA. Most of the recent studies focus on the improvement of laccase stability and its reuse by immobilization. Significant improvements in the immobilization technique have been achieved. Only few of these studies developed membranes with biodegradation capabilities at continuous operation [26-29]. The present challenge in laccase-immobilized membranes, which is considered in this study, is to further increase the durability and efficiency by improving the support and the reaction environment. The suggested RIGP method benefits from high substrate transport to laccase as a result of multilayer immobilization while increasing stability with dense moisture-retaining functional groups. Such a combination allows efficient utilization of laccase on membranes by the adjustment of polymer brushes and reaction conditions, enhancing the stability and activity of continuous membrane treatment while using commercially available cheaper laccase. Aside from the preparation of membranes, increasing the reaction efficiency is another important challenge. Studies record an increase in the efficiency of both free and immobilized laccase using proper redox mediators. These redox mediators are reported to initiate a cycle of redox reactions by acting as high-potential active intermediates. Different mediators exhibit different mechanisms such as ionic interactions, hydrogen radical transfer, and electron transfer [30,31]. These redox mediators could play an important role in designing a unique laccase membrane bioreactor with high biodegradation efficiency. It is important to choose 3

ACCEPTED MANUSCRIPT the right mediator based on the substrate and reaction conditions to achieve the best results.

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Studying these mediators will provide further insight necessary for the future development of this method. The aim of this study was to design a new system of hollow fiber membranes, functionalized by polymer brushes prepared using RIGP for hosting laccase and BPA biodegradation. The novelty of this study lies in the exploitation of the dense brush structure of polymer brushes to achieve higher laccase loading per unit volume of membrane through multilayer immobilization of laccase, the retention of high moisture content to protect laccase from denaturation in organic media, and the capability of substrate treatment at very high SVs. Studying the effects of SV on membrane activity and the use of redox mediators to

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enhance BPA biodegradation were important goals.

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2. Experimental 2.1 Chemicals A 70% porous polyethylene (PE) hollow fiber membrane having a pore size of 500 nm, an outer diameter of 3.1 mm, and a thickness of 1.1 mm was obtained from Asahi Kasei (Tokyo, Japan) and was used as the base polymer. Laccase from Trametes sp. was sponsored by Amano Enzyme Inc. (Aichi, Japan). 1-Hydroxybenzotryazole (HBT) and 2,2,6,6tetramethyl-1-piperidinyloxyl (TEMPO) were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan) and Sigma-Aldrich Japan (Tokyo, Japan), respectively. All other chemicals

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were purchased from Wako Pure Chemical Industries (Osaka, Japan) and were of analytical grade or higher.

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2.2 Preparation of polymer brushes on the hollow fiber membranes using RIGP Radicals were formed by irradiating membranes with electron beam at 200 kGy (NHV Co., Tosu, Japan) under vacuum. The irradiated membranes were then immersed in 10% (v/v) glycidyl methacrylate (GMA)/methanol solution for 50 s at 313 K to graft GMA. The membranes were then washed with N,N-dimethylformamide and methanol. The resulting grafted membranes are referred to as GMA membranes. The degree of GMA grafting (DG) was defined as: DG (%) = 100 (W1 – W0) / W0

(1)

where W0 and W1 are the masses of membranes prior to and after grafting, respectively. The resulting membranes were immersed in 0.5 M sulfuric acid aqueous solution and aminoethanol (AE) at 80 °C for 24 h in order to introduce hydroxyl groups (OH) and AE, respectively. A Fourier transform-infrared spectrometer (Jasco, FT-IR 4100, Tokyo, Japan) was used to verify the conversion of GMA. The resulting membranes are OH and AE membranes. The molar conversion of GMA groups was defined as: 4

ACCEPTED MANUSCRIPT Molar conversion (%) = 100 (moles of functional groups) / (moles of GMA)

(2)

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where the moles were calculated from changes in the masses of membranes [24]. The moisture retention of the membranes was measured by a Karl-Fischer titration unit (KEM, MKC-501, Kyoto, Japan). A scheme of the process used to design the membrane reactor is shown in Fig. 1.

Fig. 1 Scheme for design of functional membranes using RIGP

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2.3 Immobilization of laccase onto the hollow fiber membrane Hydrochloric acid (0.1 M) was permeated in order to open possibly unreacted epoxy rings and create an electrostatic repulsion force by adding positive charges on amine groups. Laccase solution (0.2 g/L) purified by centrifuging at 3000 rpm for 20 min was permeated through the membrane at 30 mL/h and 293 K. The concentration of laccase in the effluent was calculated by measuring the absorption of protein at 280 nm with a UV-Vis spectrophotometer (Shimadzu, UV-2550, Kyoto, Japan). The pH of the solution was kept at 7.0 using 50 mM aqueous phosphate buffer (This prevents nonspecific opening of GMA epoxy rings.). The amount of adsorbed laccase was defined as: 𝑉′

g (C0 -C) Amount of adsorbed laccase ( ) = ∫ dV kg W

(3)

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where C0 is laccase concentration in the initial feed solution, C is laccase concentration in the effluent, V´ is the volume of the effluent, and W is the mass of the prepared membrane. Immobilization was performed by covalent cross-linkage of the adsorbed laccase. Therefore, the membranes were put in 0.01% (v/v) glutaraldehyde aqueous solution for 24 h. 5

ACCEPTED MANUSCRIPT The laccase molecules that were not successfully immobilized were eluted by canceling the charges through permeation of 0.5 M NaCl aqueous solution at a speed and temperature of 30 mL/h and 293 K, respectively. The amount of the immobilized laccase and immobilization percentage were defined as: Amount of immobilized laccase (g/kg) = Aa - Ae

Amount of immobilized laccase (g) ×100 (5) Amount of laccase in the feed solution (g)

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Immobilization percentage (%)=

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where Aa and Ae are the amount of adsorbed laccase and the amount of laccase in the effluent, respectively [25].

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2.4 Activity assay in batch reactor 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; ABTS) and laccase were mixed in a 100-mL reactor where the concentration was adjusted to create 0.01, 0.05, and 0.1 g/L laccase in 0.5 mM ABTS solution. The 0.05 g/L batch reactor contains the same number of enzymes as the 90 g/kg immobilized laccase membranes. Samples were taken at 30-s intervals and were immediately mixed with 0.625 M oxalic acid aqueous solution to unfold all laccase and terminate further oxidation reactions. The UV-Vis absorption of samples was

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measured at 415 nm, where oxidized ABTS has a molar absorptivity of ε = 36000, and the concentration was calculated using the Lambert-Beer law. The same study was performed in a reactor containing 10% (v/v) methanol. The pH of all reactors was maintained at 5 using a 50 mM acetate buffer (Experimental data provided by the laccase supplier indicated high stability and activity at this pH.). The reaction rate was defined as: Oxidized substrate (mol) ×100 Substrate in feed solution (mol)

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Reaction rate (%) =

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2.5 Activity assay of immobilized laccase ABTS and BPA were used as substrates to compare the activity of different membranes and to determine the biodegradation capability of the membranes, respectively. Aqueous ABTS (0.5 mM, pH 5.0) and 10, 30, 50% (v/v) methanol/water solutions were permeated through the membrane at 30 mL/h and 293 K for 30 min in order to let the substrate transport and oxidation reaction reach steady state. The permeation speed was gradually increased and the samples were collected. The effluent was collected in 0.625 M oxalic acid aqueous solution to unfold any laccase molecules that might leak from the membrane. The UV-Vis absorption of the effluent was then measured at 415 nm. 6

ACCEPTED MANUSCRIPT Solutions of BPA (10 ppm, pH 5.0) and 10 ppm BPA with 0.5 mM redox mediators (pH

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5.0) were permeated by the same methods, where HBT, TEMPO, and ABTS were used as redox mediators and 2 ppm hydroquinone in acetonitrile was used as the reaction terminator. The pH of all reactors was maintained at 5 using a 50 mM acetate buffer. The BPA concentration of the effluent was measured using an ultra-high-performance liquid chromatograph equipped with a photodiode array spectrophotometer unit (PDA) and a mass spectrometer (UPLC/MS, H-Class, Waters Co. Ltd., USA). A reversed phase BEH C18 column (Waters Co. Ltd.) was used as the solid phase. Water (A) and methanol (B) were used as the mobile phase and a gradient was applied to the mobile phase. The applied gradient parameters are shown in Table 1. The PDA unit was run from 200–600 nm and the mass

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spectrometer was operated in negative ion mode by the electron spray method with a capillary voltage of 3 kV and a cone voltage of 40 V. Laccase activity was defined as:

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mol Oxidized Substrate ( h ) mol Immobilized laccase activity ( )= h ∙kg-laccase Immobilization amount (kg-laccase)

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Table. 1 Gradient parameters of the mobile phase for UPLC/MS Time (min) 0

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95% A;

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Composition of mobile phase

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3. Results and Discussion 3.1 Characteristics of immobilization on different membranes OH and AE functional groups were introduced with a conversion rate of 100% into membranes with 40% DG and the change in the amount of immobilization was studied. The disappearance of epoxy ring absorption at 850 cm-1 and the appearance of broad hydroxyl group absorption at 3400 cm-1 in the FT-IR spectrum were observed (FT-IR spectra are presented in Fig. S1 to Fig. S4 of the supplementary data). The variation in the adsorption of laccase as a result of changing the functional groups is shown in Fig. 2 as a function of the dimensionless effluent volume (DEV), defined as: DEV (-) = effluent volume/membrane volume

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Fig. 2 Characteristics of adsorption by different functional groups

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The results indicate low and high amounts of adsorption on OH and AE membranes, respectively. Amine-containing polymer brushes unfold as a result of the electrostatic repulsion force, allowing multilayer adsorption of enzymes. Polymer brushes of OH membranes remained folded by interactions within themselves. GMA membranes showed an amount of adsorption between those of OH and AE membranes, indicating nonspecific chemical adsorption by covalent bonding. Multilayer adsorption on amine-containing

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functional groups, previously reported by the authors [25], was confirmed in this system and nonspecific chemical adsorption on GMA membranes highly suggested the opening of epoxy rings before the immobilization of laccase. Differences in adsorption types are shown in Fig 3. Although membranes with a DG of 40% are appropriate for studying the immobilization behavior, the increase in operating pressure caused by long polymer brushes makes them inappropriate for biodegradation process. Moreover, membranes with a very low DG are not reliable because big errors occur in the measurement of DG and their short polymer brushes will not be able to adsorb a sufficient amount of enzyme. Membranes with a DG of 20% were used for further studies as they showed acceptable behavior. Over 80% of permeated laccase was immobilized on AE membranes (DG = 20%), which indicates that the proposed system is among those with the highest efficiency at the immobilization stage (similar systems with immobilization percentages between 15% and 85% have been reported [28,32]). This allows for easy adjustment of immobilization amounts, which greatly affects the properties of the system. These properties are discussed in the next section. High laccase loading on a smaller unit volume can be achieved using the suggested multilayer immobilization method.

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Fig. 3 Adsorption state of laccase on polymer brushes, (a) mono-layer adsorption on folded polymer brushes of OH membranes, (b) nonselective chemical adsorption on GMA brushes, and (c) multilayer adsorption on unfolded polymer brushes of AE membranes

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3.2 Effect of SV and immobilization amount on the activity of laccase The relations between space velocity (SV) and the activity of AE membranes at different immobilization amounts are shown in Fig. 4 (reaction rates are given in Fig. S5 of the supplementary data). AE membranes with a DG of 20% were used. An increase in SV led to a higher activity of laccase at immobilization amounts between 50 and 180 g/kg, indicating a stronger diffusion force and, therefore, higher mass transfer to the enzyme region. Substrate transport and oxidation are the two factors that determine the rate-limiting step of the whole process. At low SVs, substrate transport is the rate-limiting step, and increasing SV leads to higher laccase activity. However, after a certain SV, the oxidation reaction becomes the rate-

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limiting step, which means enough substrates are bonded to the enzymes at any given time and no more enzymes can bond with the substrate until the reaction is over. Therefore, the observed activity tended to approach an asymptote at very high SVs.

Fig. 4 Effect of SV range and immobilization amount on laccase activity

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defined as a function of laccase weight that differs from the activity of the membrane. Fig. 5 shows the activity of the same membranes defined as a function of membrane weight. An increase in immobilization rate led to higher activity, but after a certain immobilization amount, increasing the immobilization amount resulted in a decrease in activity. Even though increasing the immobilization amount results in a lower substrate transport and decreases the laccase activity, the increasing number of immobilized laccase molecules causes higher membrane activity. At very high immobilization rates, substrate transport is highly hindered, which results in a lower membrane activity. Membranes with an immobilization amount of 90 g/kg were chosen as a model membrane for further studies as they showed high enzymatic

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and membrane activity.

Fig. 5 Effect of SV range and immobilization amount on the membrane activity

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3.3 Stability of immobilized laccase on AE membrane in organic media To evaluate changes in the stability of laccase as a result of immobilization, laccase activity had to be compared to a batch reactor. However, when comparing to batch reactors, unifying SV and the immobilization amount is impossible; therefore, reaction time and reaction rate were compared. The number of laccase molecules immobilized on the AE membranes and the number of laccase molecules within the batch reactor were unified in a 0.05 g/L batch reactor (batch reactors with different laccase concentrations showed similar behavior; data presented in Fig. S6 of the supplementary data). A comparison of reaction rates when 10% methanol/water solution was permeated is shown in Fig. 6. The immobilized laccase membrane showed up to 10 times higher activity than free laccase, indicating the higher stability of laccase when immobilized.

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Fig. 6 Comparison of the stability of (a) free and (b) immobilized laccase

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Laccase denatures because of dehydration within organic media. However, creating a mild environment using proper functional groups such as AE is possible. By introducing AE, two hydroxyl groups are created on each grafted GMA molecule, resulting in the retention of

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moisture by hydrogen bonding. The moisture content increased from 0.20% to 1.85% by the introduction of AE to GMA membranes, which plays an important role in protecting laccase within organic media. Fig. 7 shows the activity assay results when membranes with an immobilization amount of 90 g/kg were used to evaluate stability in 10%, 30%, and 50% methanol/water solutions. Increasing the ratio of methanol lowered the activity to the point that in 50% methanol/water solutions almost no activity was seen, indicating denaturation of all immobilized laccase. Even though protecting laccase from denaturation is possible by moisture retention, at high ratios of methanol, which is an amphiphilic solvent, the moisture content will be completely replaced with methanol, thereby denaturing the enzymes. 11

Effects of methanol concentration on laccase activity

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The stability of immobilized laccase membranes was studied by activity assay within

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organic media (10% methanol) and aqueous solutions, three times. The activity of the immobilized laccase and reaction rates are plotted against SV, shown in Fig. 8 and Fig. 9, respectively. A decrease in activity when 10% methanol solution was permeated for the first time and second time was observed, indicating denaturation of some laccase. Laccase did not gain its initial activity after permeation by an aqueous solution. However, activity was not

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affected by repeating the process for a third time. These results indicate an increase in stability within organic media because of the moisture-retaining AE functional groups. Reaction rates within organic media were observed to increase when SV is increased to an upper limit of 300 1/h. However, if increased further, a decrease in reaction rate was observed. These results indicate that the effect of substrate transport is stronger than that of dehydration at lower SV ranges. However, at higher SVs, the strong current results in a higher rate of moisture being replaced with methanol, dehydrating laccase faster and lowering its activity.

Fig. 8

Activity of immobilized laccase during repeated runs within organic media 12

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Fig. 9 Reaction rate of the system within organic media during repeated runs

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3.4 Removal of BPA using AE membrane By changing the substrate to BPA, the SV was first adjusted at the same range as when ABTS was used as the substrate; however, no activity was observed. Fig. 10 shows the membrane activity when the SV range was lowered. By increasing SV, parabolic behavior was seen, where the activity increased and then, after a certain SV, decreased. When ABTS was permeated as the substrate, the activity grew towards an asymptote, indicating that

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substrate oxidation becomes the rate-limiting step, but when BPA solution was permeated, the activity started to decrease at high SVs. Laccase strongly interacts with ABTS, which results in higher activity at high SVs, but it is thought that enzyme interactions with BPA are weaker than those with ABTS and the enzymes are unable to form complexes with the substrate at high SVs because of the strong current, resulting in lower activity. AE membranes showed high background adsorption at the low SV range (Reaction rates and adsorption behavior are shown in Fig. S8 of the supplementary data.). The mechanism of this adsorption is unclear because when BPA was degraded in the presence of HBT, an even lower activity than the background adsorption was observed. A comparison of the current method with some notable

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systems is given in Table 2.

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Fig. 10 Relation between SV and biodegradation of BPA

Origin of

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Table. 2 A comparison of the current method with similar systems BPA laccase Support

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Enzymatic Total BPA

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BPA removal

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recirculation)

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95

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2

6.7

93

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versicolor Trametes

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versicolor

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Semi-aromatic polyamide

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TiO2 coated PVDF membranes

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90

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Current work

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The data does not show the effect of mediators. Please refer to supplementary data for mediator effects.

By taking several factors into consideration, the current system showed competitive behavior with respect to that of similar systems. These factors include operational flux, substrate concentration, immobilization amount, and the portion of BPA that is biodegraded by laccase. The main issue in comparing such systems arises from the difference in the origin of laccase, which drastically affects the activity. It should be noted that the enzyme used in 14

ACCEPTED MANUSCRIPT this study is cheap and substantially available, making it appropriate for large-scale use. The advantage of the presented system is the achievement of high degradation efficiency by a single permeation cycle at high flux. It also proves that BPA removal in terms of percentage does not indicate the full potential of the system for the biodegradation of BPA, as the presented results showed higher amounts of degraded BPA at higher SVs even though reaction rates were lower.

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3.5 Effect of redox mediators Three main redox mediators of laccase with different known mechanisms were used, and their effects on BPA oxidation were studied. TEMPO is known to form an oxoammonium ion,

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HBT forms an N-O radical and ABTS changes to a radical cation and dication after being oxidized by laccase. Each of these oxidized products works through ionic interaction, hydrogen radical transfer, and electron transfer, respectively, to initiate redox cycles [30,31]. Fig. 11 shows the BPA biodegradation activity of laccase when each of these redox mediators was used. TEMPO and HBT decreased the activity but ABTS increased the activity to more than three times higher than that of the system with no mediator (samples were analyzed within 24 h after collection; activity of immobilized laccase towards mediators and reaction rates of mediators are presented in Fig. S9 and Fig. S10 of the supplementary data). This indicates that the oxidation of BPA at the studied reaction conditions requires mediators with a mechanism close to that of ABTS. The concentration of BPA and the UV-vis spectra of the

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samples were measured within 1 h after permeation and after storage for 5 days (UV-Vis spectra are shown in Fig. 12). ABTS and its oxidized products show strong absorptions at 350 and 415 nm, respectively. The BPA concentration was observed to decrease with time (data not shown) and UV-vis spectra showed an increase in ABTS and a decrease in its oxidized products. These results indicate the initiation of redox reaction cycles by electron transfer as ABTS returned to its unoxidized form and the improvement of BPA removal efficiency. The color of the effluent was observed to change from green to purple with time.

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Fig. 11 Effect of three major redox mediators on BPA biodegradation activity of laccase

Fig. 12 Changes in UV-Vis spectrum of the effluent after storage

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4. Conclusion A polyethylene hollow fiber membrane was used as the base material for the preparation of membrane bioreactors. Polymer brushes of GMA were prepared using RIGP. Aminoethanol was introduced to GMA as the functional group, resulting in unfolded polymer brushes by electrostatic repulsion, an appropriate environment for multilayer adsorption, and

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immobilization of laccase. The effects of space velocity and amount of immobilization on the activity of laccase and membranes were studied. It was observed that higher substrate transport at low immobilization amounts and high SVs lead to higher enzymatic activity. The designed system successfully increased the stability of immobilized laccase in organic media by moisture retention using the hydroxyl groups of aminoethanol. No change in the activity of immobilized laccase was observed after permeating 10% methanol/water for a second time. This indicated that some laccase molecules retain their activity at low concentrations of amphiphilic solvent. The membrane system was successful in efficiently oxidizing BPA, which is a potential environmental pollutant. The obtained results showed the importance of the SV parameter in the efficiency of the system. At an appropriate SV, even though reaction rates decrease, higher amounts of substrate are degradable. The effects of ABTS, TEMPO, and HBT, which are three major redox mediators of laccase, were studied for further improvement of the method. It was observed that only ABTS increased the oxidizing rate, with 100% removal of BPA at low SVs. The reproduction of ABTS and the decrease in the concentration of BPA proved the initiation of electron transfer redox reaction cycles. We will continue exploring the enhancement of the system using the information gained in this study on the effects of redox mediators, which is hoped to allow for an even more efficient biodegradation system. 16

ACCEPTED MANUSCRIPT Acknowledgments The hollow fiber membranes and laccase were kindly sponsored by Asahi Kasei Corporation and Amano Enzyme respectively. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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References [1] E. Burridge, Bisphenol A: Product profile, Eur. Chem. News. 17 (2003) 14-20. [2] J. Maia, J.M. Cruz, R. Sendón, J. Bustos, J.J. Sanchez, P. Paseiro, Effect of detergents in the release of bisphenol A from polycarbonate baby bottles, Food. Res. Int. 42(10) (2009)

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