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Article
Bio-inspired multifunctional membrane for aquatic micro-pollutants removal
Xiaotong Cao, Jianquan Luo, John M. Woodley, and Yinhua Wan
ACS Appl. Mater. Interfaces, Just Accepted Manuscript DOI: 10.1021/acsami.6b10823 Publication Date (Web): 21 Oct 2016
Downloaded from http://pubs.acs.org on October 22, 2016
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Page 1 of 30 ACS Applied Materials & Interfaces
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4 Bio-inspired Multifunctional Membrane for Aquatic
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7 Micro-pollutants Removal
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9 Xiaotong Cao,ab Jianquan Luo,a* John M. Woodleyc and Yinhua Wana*
10 a. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, University of the
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12 Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100190, China.
13 b. Sino-Danish College, University of the Chinese Academy of Sciences, Beijing 100049, China.
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15 c. Department of Chemical and Biochemical Engineering, Technical University of Denmark, 2800, Kgs.
16 Lyngby, Denmark
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18 ABSTRACT
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20 Micro-pollutants present in water have many detrimental effects on the ecosystem. Membrane technology
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22 plays an important role in the removal of micro-pollutants but there remain significant challenges such as
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24 concentration polarization, membrane fouling and variable permeate quality. The work reported here uses
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26 a multifunctional membrane with rejection, adsorption and catalysis functions to solve these problems.
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28 Based on mussel-inspired chemistry and biological membrane properties, a multifunctional membrane
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30 was prepared by applying reverse filtration of a laccase solution and subsequent dopamine coating on a
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32 nanofiltration (NF) membrane support, which was tested on bisphenol A (BPA) removal. Three NF
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34 membranes were chosen for the preparation of the multifunctional membranes based on the membrane
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36 properties and enzyme immobilization efficiency. Compared with the pristine membrane, the
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38 multifunctional membrane exhibited significant improvement of BPA removal (78.211.95%, 84.277.30%
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40 and 97.040.33% for NT103, NF270 and NF90 respectively), all of which are clearly superior to the
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42 conventional Fenton treatment (55.0%) under similar conditions, and comparable to soluble laccase
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44 coupled with NF270 membrane filtration (89.0%). The improvement would appear to be due to a
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46 combination of separation (reducing the enzymatic burden), adsorption (enriching the substrate
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48 concentration as well as prolonging the residence time) and finally, catalysis (oxidizing the pollutants and
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50 breaking the adsorption saturation limits). Furthermore, the synergistic effect of the polydopamine (PDA)
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52 layer on the enzymatic oxidation of BPA was confirmed, which was due to its enhanced adsorption and
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54 electron transfer performance. The multifunctional membrane could be reused for at least 7 cycles with an
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56 acceptable activity loss, demonstrating good potential for micro-pollutants removal.
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ACS Applied Materials & Interfaces Page 2 of 30
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4 KEYWORDS: Multifunctional membrane; laccase immobilization; micro-pollutants; nanofiltration;
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6 adsorption; enzyme
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8 1. INTRODUCTION
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Global water shortages and pollution greatly limit rapid urbanization and modernization of society.
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Thus effective water treatment technologies are required for water purification and restoration. For many
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years, membrane filtration has been regarded as one of the most promising technologies and has been
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widely used in water treatment due to several unique advantages such as energy saving, small plant
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footprint, continuous operation and high quality effluent.1-2 Nowadays, public concern about the
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environmental impact of micro-pollutants, including pharmaceuticals and personal care products, steroidal
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hormones, pesticides, as well as endocrine-disrupting chemicals, has grown due to their high resistance to
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conventional oxidation methods and negative effects on human health even at very low dosages.3-6 This
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not only provides new opportunities for membrane technology, but also brings some specific challenges.7
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For example, Yoon and co-workers applied nanofiltration (NF) and ultrafiltration (UF) membranes to treat
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natural water containing 52 different types of micro-pollutants, and found that their retention was
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unexpectedly low due to the transport phenomenon associated with adsorption.8 Even utilizing a reverse
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osmosis (RO) membrane, the retention was still unsatisfactory because of the presence of hydrophobic
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adsorption between the micro-pollutants and the aromatic polyamide material.9-10 Aside from such
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adsorption of micro-pollutants in the membrane, there is also the problem of membrane fouling
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(associated serious flux decline), while the chemical cleaning process would wash those pollutants out
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from the membrane, resulting in a secondary pollution. Thus, relying on the retention ability of a
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conventional membrane alone would unfortunately never fulfill the stringent emission standards. On this
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basis we reasoned that membranes with multi-functional capacities would be needed in order to achieve a
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more effective removal of micro-pollutants.
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While it has been reported that adsorbents coupled with membranes (or membrane adsorbers)
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produced good micro-pollutant removal in short-term operations,11-13 nevertheless, after the occurrence of
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55 the inevitable adsorption saturation, the micro-pollutants rejection by the membrane were severely
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4 decreased, leading to an unsatisfactory permeate quality. Besides, the regeneration of adsorbents and
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6 membrane adsorbers would cause severe secondary pollution.
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8 In order to improve this situation we considered the integration of enzymatic catalysis with the
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10 membrane to help facilitate the micro-pollutants removal. It was confirmed that ligninolytic enzymes such
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12 as laccase (E.C. 1.10.3.2) and lignin peroxidase (E.C. 1.11.1.14) can eliminate compounds such as
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14 endocrine disruptors and pharmaceuticals effectively.14 Laccase is of great interest to researchers because
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16 it can oxidize both phenolic and non-phenolic compounds, as well as extremely recalcitrant environmental
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18 pollutants within a wide range of pH values, and the final products are usually non-toxic.15-16 However,
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20 since the applications of free enzymes are frequently limited due to inactivation, high costs for enzyme
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22 isolation/purification and serious product inhibition, enzyme immobilization is often favored. Using
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24 filtration membrane to retain enzyme, enzymatic membrane reactor can reuse enzymes in continuous
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26 mode, improve enzyme stability and extract the product to relieve the product inhibition effect.17-18 Indeed,
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28 recently it was reported that continuous oxidation of micro-pollutants (e.g. antibiotics and endocrine
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30 disruptors) by a reactive membrane with immobilized laccase has been achieved.19-21 Nevertheless it was
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32 found that the complex immobilization procedure resulted in a low enzyme activity and a full recycling
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34 mode was required to obtain a high level of micro-pollutant removal.19, 22
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36 More recently, Luo and co-workers proposed a simple approach to immobilize enzyme in membranes
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38 using reverse filtration of the enzyme solution followed by dopamine coating on the membrane
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40 support,23 in order to entrap the enzyme in the membrane and minimize changes in enzyme as well as
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42 membrane structure.24 Dopamine can form a stable self-polymerized coating layer on various substrates
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44 under alkaline conditions in an air atmosphere25, which is inspired by the strong adhesion of mytilus edulis
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46 foot.26-27 This has been widely used for enzyme immobilization,28 and compared with other enzyme
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48 immobilization strategies (e.g. covalent bonding, entrapment, adsorption and crosslinking), the
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50 dopamine-based method is simple, facile, stable and shows less harm to enzyme activity as it is
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52 biocompatible and the non-covalent bonding is dominant.21,23-24 Besides, it was also reported that the
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54 polydopamine (PDA) layer improved the catalytic efficiency by adsorbing and enriching the substrate.29
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56 On the other hand, it is well known that biological membranes are able to control substances entering into
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59 3
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4 the cell for metabolism, and inspired by this, we reasoned that the enzyme of interest could be
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6 immobilized beneath a tight NF membrane since it may effectively reject the micro-pollutants and reduce
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8 the enzymatic burden. Based on the above, we therefore propose a novel concept using a bio-inspired
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10 multifunctional membrane, which combines membrane physical rejection and intrinsic adsorption, PDA
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12 adsorption and enzymatic catalysis functions, as illustrated in Fig. 1. Such a multifunctional membrane
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14 could be prepared using the simple method developed by Luo and co-workers.23 When such a membrane
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16 is applied to micro-pollutant removal, we hypothesized that the micro-pollutants would be partly retained
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18 by the NF membrane, then the residual pollutants would be adsorbed and enriched in the membrane and
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20 PDA layer (close to the enzymes), prolonging the contact time between substrate and enzyme, and finally,
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22 the immobilized enzyme would oxidize those micro-pollutants that enter into the membrane. After
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24 bio-oxidation, the adsorption sites would be liberated, avoiding the adsorption saturation problem. If this
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26 hypothesis could be verified, the micro-pollutants in an aquatic environment could also be removed
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28 effectively and continuously by our multifunctional membrane.
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30 This work describes the preparation of a multifunctional membrane which can separate, adsorb and
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32 catalyze the pollutants simultaneously, and investigates its potential application for removal of bisphenol
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34 A (BPA), an endocrine disruptor widely found in aquatic environments, which has an irreversible
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36 detrimental effect on human.30 In hazardous waste landfill leachates, BPA concentrations can reach up to
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38 17.2 mgL-1.31 Laccase was chosen as the enzyme of interest due to its activity towards various
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40 micro-pollutants including BPA.5, 16 Suitable NF membranes for multifunctional membrane preparation
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42 were selected on the basis of their physical properties (e.g. permeability, rejection and adsorption capacity),
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44 as well as enzyme immobilization efficiency (e.g. enzyme loading and activity). The performance of
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46 nascent and multifunctional membranes was systematically compared in order to emphasize the benefit of
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48 the multi-functions. Moreover, by designing four membranes with different functions, the contribution of
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50 each function (i.e. rejection, adsorption and catalysis) to BPA removal and the synergistic mechanism of
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52 the multifunctional membrane were clarified. Finally, the stability and reusability of the prepared
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54 multifunctional membrane were also assessed. To the best of our knowledge, this is the first work
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56 investigating a multifunctional membrane combining separation, PDA/membrane adsorption and laccase
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59 4
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4 catalysis functions together to remove micro-pollutants. The successful implementation of this work will
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6 open a new direction for multi-functionalization of NF membranes and provide an alternative method for
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8 continuous, stable and effective water treatment.
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23 Fig 1 Schematic diagram of multifunctional membrane preparation and its immobilization/working
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25 mechanisms. Enzyme immobilization was carried out by reverse filtration of enzyme solution and then dopamine
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27 was self-polymerized at alkaline condition, where enzymes were immobilized via entrapment, covalent bonding as
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29 well as hydrophobic adsorption. When this multifunctional membrane was applied in water treatment, rejection,
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31 adsorption and catalysis functions worked simultaneously and a synergistic effect among them would facilitate the
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33 micro-pollutants removal.
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38 2. EXPERIMENTAL
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40 2.1 Materials
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A dead-end filtration cell (Amicon 8050, Millipore, USA) with an effective area of 13.4 cm2 was
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used in all the following filtration experiments. Laccase (EC 1.10.3.2, 60-70 KDa, 0.53Umg-1) from
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Trametes versicolor was purchased from Sigma-Aldrich (Shanghai, China). Bradford reagent used for
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protein assay was supplied by Sigma-Aldrich. 2.6-dimethoxy phenol (DMP) was used as the assay
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substrate for laccase activity. DMP (154.16 gmol-1) and BPA (228.29 gmol-1) was purchased from
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Aladdin (Shanghai, China). All the enzyme solutions and BPA solutions were prepared using a 10 mM
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54 phosphate buffer (pH 5.3) and dopamine hydrochloride were prepared freshly using a 10 mM Tris-HCl
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56 buffer (pH 8.5). DMP solution was prepared using a 100 mM sodium citrate buffer (pH 4.3). All the
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4 chemical reagents used in experiments were of analytical grade and used directly without any further
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6 purification. Six commercial polymeric membranes (Table 1) were used in this work.
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8 Table 1 Basic properties of NF membranes used in this work
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10 Membrane MWCO
11 Skin-layer material Manufacturer Ref.
12 type (Da)
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14 NT101 Polyamide (PA) <500 Microdyn-Nadir
15 www.microd
16 NT102 PA 100-200 Microdyn-Nadir
17 yn-nadir.com
18 NT103 Poly(piperazinamide) 200-300 Microdyn-Nadir
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20 NF270 Semi-aromatic PA 200-300 Dow-Filmtec
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22 NF90 Fully aromatic PA 100-200 Dow-Filmtec
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24 Desal-DL Semi-aromatic PA 300-400 GE-Osmonics
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26 MWCO: molecular weight cut-off.
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30 2.2 BPA retention and permeate flux
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32 Before BPA rejection measurements, the membranes were immersed in a BPA solution (20 mgL-1)
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34 overnight to saturate BPA adsorption. BPA adsorption was saturated after 25 hours immersion (Data is
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36 shown in Fig. SI-1). Subsequently, the membranes were taken out and soaked in deionized water for 20
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38 minutes to remove any non-bound molecules. The membranes were then used to filtrate 55 ml of BPA
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40 solution (20 mgL-1) at 4 bar, 23 C and 500 rpm agitation in flow-through mode. The first 5 mL of
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42 permeate was discarded and was subsequently collected in batches of 2 mL using a measuring cylinder.
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44 After 16 mL of permeate had been collected, the filtration was stopped. A spectrophotometer (UV-9000S,
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46 METASH, China) was used to measure absorbance at 278 nm to assay the BPA concentration. The
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48 detection limit of BPA with this method is 70 gL-1. The observed rejection ( ) was calculated
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50 according to the following equation:
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52 = 1 100% (1)

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where and represented the BPA concentrations in permeate and feed, respectively.
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The permeate flux ( ) was calculated as:
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59 6
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4 = (2)

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6 While the permeability ( ) was calculated as:
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9 =

(3)
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11 where was the permeate volume after certain time, !" was the effective filtration area and TMP
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13 represented the transmembrane pressure.
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16 2.3 Enzyme immobilization
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18 2.3.1 Preparation of the multifunctional membrane
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Enzyme immobilization was performed using a protocol adapted from Luo and co-workers previous
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study.23 Firstly, after soaking in 50% ethanol for ten seconds and ultrapure water overnight, the membrane
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was placed in sandwich mode (support layer facing feed solution with an extra polypropylene support
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beneath the skin layer), and washed using NaOH solution (pH 10) for 20 minutes at 2 bar. The membrane
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permeability was measured using deionized water at 2 bar till the water flux became constant with time.
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Then 20 mL of enzyme solution (containing 8 mg of laccase) was added into the filtration cell for enzyme
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immobilization at 4 bar and 100 rpm agitation. When 18 mL of liquid had passed through the membrane,
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the filtration process was stopped, and the enzyme-loaded membrane was washed using 10 mL deionized
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water at 2 bar. The final retentate (2 mL) and washing residual (10 mL) were combined and stored for
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further analysis. Then 10 mL dopamine hydrochloride (2 gL-1) solution (prepared using 10 mM pH 8.5
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Tris buffer) was poured into the open filtration cell and coating was conducted at 23C with 100 rpm
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agitation for 2 hours. After coating, the membranes were cleaned using deionized water at 2 bar for 20
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minutes, and the permeability was measured again. Subsequently, the coated membranes were soaked in
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10 mL phosphate buffer (pH 7) overnight. Finally, the membrane was mounted in the cell in normal mode
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(skin layer facing feed without extra support) and washed with 10 mL deionized water at 2 bar. The buffer
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used for soaking and the water used for washing (20 mL totally) were collected and analyzed in order to
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measure any enzyme leakage.
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2.3.2 Enzyme loading
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55 The enzyme concentration was measured by protein concentration measurement using the Bradford
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57 assay with a spectrophotometer. 1 mL sample was taken from the enzyme solution and mixed with 1mL
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59 7
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4 Bradford reagent, and the absorbance at 595 nm was recorded. The detection limit of laccase with this
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6 method was 20 gL-1. The immobilized enzyme amount was calculated according to the mass balance
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8 equation:
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10 #$"" = # % % &' &' (' (' (4)
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12 The Enzyme loading was calculated according to equation:
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14 enzyme loading = 100% (5)
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where #$"" and # are immobilized and total enzyme amount, respectively; % , &' and (' are
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the enzyme concentrations in the permeate, in the mixture of retentate and washing residuals (before
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coating), and in the mixture of soaking and washing residuals (after membrane reversal), respectively;
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% , &' and (' are enzyme solution volumes of permeate, mixture of retentate and washing residuals,
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mixture of soaking and washing residues respectively.
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26 2.3.3 Activity assay of immobilized enzyme
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28 Immobilized laccase activity was determined by monitoring the oxidation rate of DMP (10 mM, PH
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30 4.3) over 4 minutes (the measurement interval was 30 seconds) at room temperature (23C). The laccase
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32 loaded membrane was placed in the filtration cell in reverse mode (support layer facing feed solution) and
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34 then 20 mL DMP solution was poured into the open cell at 100 rpm. The absorbance change at 468 nm
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36 was recorded using a spectrophotometer. Laccase activity was expressed in MDMPmin-1 calculated from
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38 the molar absorptivity, 468nm=49.6(mMcm)-1. The cuvette used here has an effective length of 1 cm.
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40 2.4 Membrane characterization
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42 For the membranes before and after enzyme immobilization, their morphology was examined using a
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44 JSM-6700F Field Emission Scanning Electron Microscope (SU8020, Hitachi, Japan) in cross section (5
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46 KV operational voltages). Contact angle measurements were performed using a contact angle system
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48 (OCA20, Dataphysics, Germany) with the sessile drop method to determine hydrophilicity changes.
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50 Fourier transform infrared (FT-IR) spectra were tested for the laccase, PDA layer and membrane to verify
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52 the successful enzyme immobilization and dopamine coating via an FT-IR spectrometer (Nicolet iS50,
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54 USA). The membrane thickness and weight were measured using an Electronic Digital Outside
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56 Micrometer (CT00-521, China) and Electronic Scales (FA2004, China) respectively.
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58 2.5 BPA removal
59 8
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4 A multifunctional membrane was prepared by filtrate 45 mL laccase solution (containing 27 mg
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6 laccase) using the same method described in Section 2.3.1. The optimization of the enzyme dosage for
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8 enzyme immobilization was shown in Fig. SI-2. Pristine and multifunctional membranes were used to
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10 treat 43 mL BPA solution (10 mgL-1, pH=5.3). Transmembrane pressure (TMP) was controlled manually
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12 to keep a constant permeate flow for 5 hours until 40 mL permeate was collected. The permeate was
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14 collected using a measuring cylinder every 4 mL for 30 minutes and each of them was mixed with 2 mL
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16 0.1 M HCL to terminate the reaction. The TMP change was also recorded during filtration to quantify the
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18 development of membrane fouling. Each permeate sample together with the retentate remaining in the
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20 filtration cell was saved for further analysis. The BPA content was measured by High Performance Liquid
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22 Chromatography (HPLC) (Shimadzu, Japan) installed with a UV-visible detector (SPD-20A, Japan) and a
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24 C18 HPLC column (ZORBAX SB-C18, 250 mm4.6 mm i.d.; 5 m; Agilent, USA). Before HPLC
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26 analysis, the solution was filtered with a 0.22 m filter to remove all the insoluble substance. HPLC
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28 analytical conditions were set as follows: isocratic elution with 50% HPLC-grade water and 50%
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30 acetonitrile (V/V); flowrate of 1.0 mLmin-1; injection volume of 50 L; the column oven temperature of
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32 40 C, and detection at 278 nm. The BPA retention time was 5.850.070 min and the BPA detection limit
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34 in this protocol is 10 gL-1.
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36 The BPA stable rejection for these membranes is defined as:
37

6
38 = 1 100% (6)

39
where is the stable BPA concentration in the permeate.
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BPA removal efficiency is measured as the reduction of BPA amount after filtration, which is caused
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by a combination of adsorption and enzymatic oxidation as well as membrane rejection. It was calculated
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according to the following equation:
48 :;
4<:
4
4
49 78 = 1 100% (7)

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51 where $ and $ are the BPA concentration and volume in permeate collected, respectively; is the
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53 BPA concentration in feed solution; and is the total permeate volume during the whole process.
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55 2.6 Synergistic mechanism analysis
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4 The removal of micro-pollutants by the prepared multifunctional membrane was the result of
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6 simultaneous membrane rejection, membrane and PDA adsorptions, as well as laccase catalysis. In order
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8 to distinguish these functions, the micro-pollutants removal efficiency by four differently-designed
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10 membranes (i.e. pristine membrane, adsorption membrane, catalytic membrane, and multifunctional
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12 membrane) with different functions was compared. The adsorption membrane was almost identical to the
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14 multifunctional membrane except the enzyme was deactivated. The membrane loaded with the same
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16 amount of laccase without dopamine coating was referred to as catalytic membrane. The detailed
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18 preparation procedure of the adsorption and catalytic membranes can be found in Supporting Information
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20 III (Fig. SI-3). 43 mL BPA solution with a concentration of 10 mgL-1 (pH=5.3) was treated by these four
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22 membranes using the same procedure described in Section 2.5, and the BPA removal was compared
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24 systematically
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26 2.7 Membrane reusability
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The reusability of the multifunctional and catalytic membranes was investigated by testing BPA
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removal for 7 reuse cycles (5 hours for each experiment). The operational conditions were identical to
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those described previously. After each experiment, the membrane was rinsed with 50% ethanol for 30
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seconds, followed by ultrapure water for 5 minutes to remove the attached/adsorbed BPA/products, and it
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was then stored in 10 mL buffer (pH 7) overnight at 4 oC. As a control, a fresh multifunctional membrane
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was stored in buffer (pH 7) at 4 oC and its activity was measured once a day (the membrane was taken out
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39
and put into the cell for measurement). At the same time, another multifunctional membrane was stored
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for 7 days in the same conditions but without operation, and the enzyme concentration in the storage
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buffer measured every day to determine enzyme loss.
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48 3. RESULTS AND DISCUSSION
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50 3.1 Membrane physical properties
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52 The permeate flux and BPA rejection of six NF membranes are shown in Fig. 2. As the filtration
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54 proceeded, BPA retention initially increased for NF90 and NT102 membranes, which might be due to the
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56 BPA desorption (BPA adsorption by the membranes was saturated before filtration). Specifically, at the
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59 10
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4 beginning of filtration, some detached BPA would be washed away from the membrane, thus increasing
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6 the BPA content in the permeate. This interference only had a noticeable effect on NF90 and NT102
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8 membranes with a high BPA retention (i.e. a low BPA concentration in the permeate). As expected, as
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10 shown in Fig. 2A, the rejection of these six membranes can be roughly divided into three categories which
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12 correspond to their molecular weight cut-off (MWCO) (see Table 1). NT101 membrane showed the
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14 lowest rejection (~10%) due to its largest MWCO while NF90 and NT102 with the lowest MWCO
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16 exhibited the highest rejection (~80%).
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18 Fig. 2B shows the permeate flux of the different NF membranes using pure water and BPA solution as
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20 feeds. It is well known that membrane permeability is mainly governed by hydrophilicity, pore size and
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22 porosity. With respect to hydrophilicity, it is reasonable that NF270 gave the largest water flux since it has
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24 the highest hydrophilicity (Table 2). Although NT103 has a similar MWCO (around 200-300 Da) and
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26 hydrophilicity to NF270, the water flux of NT103 was much lower than that of NF270 since the latter had
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28 higher porosity (in Table 2, NF270 membrane has smaller density). It was also found that the water flux
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30 of NF90 was much higher than that of NT102 (with similar MWCO but better hydrophilicity) also due to
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32 the difference in membrane porosity. In reality, the water flux of all the membranes from Microdyn-Nadir
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34 was relatively low because of their special manufacturing technology. Additionally, the permeate flux of
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36 the BPA solution was always lower than the water flux, implying that the difference in the osmotic
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38 pressure due to BPA across the membrane could not be neglected. It is worth mentioning that NF90
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40 showed the most obvious difference between water and BPA fluxes, which is explained by the fact that it
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42 had highest retention and adsorption capacity (i.e. BPA adsorption was also a kind of membrane fouling
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44 resulting in an additional filtration resistance, see Table 2).
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36 Fig 2 (A) BPA rejection and (B) permeate flux for six NF membranes when filtrating BPA solution (20 mgL-1) and
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38 deionized water under 4 bar, 23 C, 500 rpm.
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40 Table 2 Main physical properties of six NF membranes tested in this work
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42 Membrane Water contact Adsorption capacity Membrane Membrane density
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44 type angle () (mgg-1) thickness (m) (mgcm-3)
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46 NT101 58.851.22 0.720.031 140.001.42 864.143.61
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48 NT102 69.043.76 0.600.043 146.611.06 880.628.58
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50 NT103 33.112.24 0.630.017 136.680.63 894.413.66
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52 NF270 28.402.19 0.730.035 143.770.44 797.583.35
53
54 NF90 80.102.12 0.780.032 148.650.61 806.673.22
55
56 Desal-DL 56.233.18 0.680.021 134.032.57 910.043.63
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59 12
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2
3
4
5
6 Besides BPA retention and permeate flux, the adsorption capacity of the membrane was also important
7
8 for the preparation of the multifunctional membrane. As listed in Table 2, the adsorption capacity of these
9
10 membranes is more than 0.60 mgg-1, and the BPA adsorption of NF90 with the lowest hydrophilicity is
11
12 the highest, implying that hydrophobic adsorption plays an important role in this case. Moreover, the
13
14 membrane thickness and structure could greatly affect the enzyme immobilization efficiency. The
15
16 thickness of these membranes is around 130-150 m with NF90 the thickest, as shown in Table 2, which
17
18 may therefore result in potentially loading more protein/enzyme. Usually NF membranes are composed of
19
20 an ultra-thin polyamide layer, a porous polysulfone layer (sponge-like and finger-like sublayers) and a
21
22 non-woven fabric support, where the sublayer and non-woven fabric act as a mechanical support, while
23
24 the thin active layer determines the separation performance. As can be seen from Fig. 3, the NT series of
25
26 membranes have finger-like sublayers with more space to harbor the enzymes, while the other three
27
28 membranes have a sponge-like sublayer which may not be favorable for enzyme loading.
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54 Fig 3 SEM imagines of the cross-section of the six membranes.
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59 13
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4 3.2 Enzyme immobilization
5
6 3.2.1 Characterization of laccase and dopamine on membrane
7
8
9
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12
13
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20
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25
Fig 4 FT-IR spectra of pure PDA, pure laccase, pristine membrane and multifunctional membrane.
26
27
28
29
The FT-IR spectra of pure PDA, pristine membrane, pure laccase and multifunctional membrane with
30
31
laccase and PDA coating layer were obtained to verify the successful preparation of the multifunctional
32
33
34
membrane (Fig. 4). For laccase, the wide peak around 3366 cm-1 was related to NH and OH stretching
35
vibrations in the protein, and the peaks at 1647 cm-1 were attributed to the characteristic absorption peaks
36
37
38
of amide.33 For PDA, the absorption at 1502 cm-1 was caused by phenolic ring C-C vibration and the
39
40
absorption peak at 1287 cm-1 was assigned to C-O stretching of the phenolic OH.34 Comparing the pristine
41
42
and multifunctional membranes, the occurrence of a peak at 3366 cm-1 and the enhanced peak at 1647
43
44
cm-1 for the latter indicated successful laccase immobilization. Although the characteristic peaks for PDA
45
46
at 1502 cm-1 and 1287 cm-1 were the same as those of the pristine membrane, which could not be used as a
47
48
proof for PDA layer on the membrane, the peaks appeared at 1037 cm-1 for multifunctional membrane due
49
50
to C-N stretching vibration of PDA35 confirming successful dopamine coating. In addition, the dopamine
51
52 coating could also be observed in the images of the pristine and multifunctional membranes (Fig. SI-4),
53
54 where it was clearly seen that a PDA layer covered on the membrane surface.
55
3.2.2 Enzyme loading, activity and permeability of multifunctional membrane
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46
Fig 5 Enzyme immobilization efficiency of six membranes when laccase was immobilized at pH=5.3, 4 bar, 100 rpm
47
48
followed by dopamine coating for 2 hours at pH=8.5. (A) Enzyme loading and (B) activity of six multifunctional
49
50
membranes, and (C) membranes permeability variations in different operating stages. IMM represented the
51
52
immobilized laccase portion in membrane. P represented the enzyme portion in the permeate. RW represented the
53
54
enzyme portion in the mixture of retentate and washing water before dopamine coating. SW meant the enzyme
55
56
portion in the mixture of soaking buffer and washing water after dopamine coating.
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59 15
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2
3
4
5
6 The multifunctional membrane was prepared via reverse filtration of the laccase solution and
7
8 dopamine coating on the membrane support. The enzyme loading and activity, together with membrane
9
10 permeability at different preparation stages were measured. It can be seen from Fig. 5A that NT103 has
11
12 the highest enzyme loading and that the NT series membranes exhibit a relatively good enzyme
13
14 immobilization efficiency, which may be due to their finger-like sublayer with larger space available to
15
16 immobilize enzymes (Fig. 3). Enzyme loading was the lowest for DL membrane because the pores in its
17
18 sponge-like sublayer were the smallest (Fig. 3). This indicates that the membrane sublayer structure plays
19
20 an important role in the efficiency of enzyme immobilization. For NT101 and NT103, the enzyme loss
21
22 during soaking and washing in normal filtration mode (SW), was lower than that with the NF series and
23
24 DL membranes, and especially for NT101, where no enzyme was detected in SW. This might be
25
26 attributed to their finger-like sublayer, which was favorable for enzyme entrapment. For NT102,
27
28 although it had the smallest pore size (skin layer) in NT series, the enzyme loss during reverse filtration
29
30 was relatively high because the retained enzyme by the skin layer possibly leaked from the edges of the
31
32 support layer more easily. More specifically, since it had the lowest flux during immobilization (Fig.
33
34 SI-5), the enzyme had more time to pass through the membrane by diffusion. This also can partly explain
35
36 the result for the DL membrane, which had a relatively large pore size but the fastest flux decline,
37
38 resulting in the lowest enzyme loading. Consequently, NT103 had the highest enzyme loading (85.28%,
39
40 2.55 gcm-2) due to its finger-like sublayer, suitable pore size and immobilization flux.
41
42 As seen in Fig. 5B, the activity of the multifunctional membrane is positively correlated with the
43
44 enzyme loading in general, and the more protein that was loaded in/on the membrane resulted in a higher
45
46 enzyme activity. NT103 displayed the highest DMP oxidation rate and DL showed the lowest,
47
48 corresponding to the relative enzyme loading. A multifunctional membrane should have a high enzyme
49
50 activity, which is desirable for enzymatic oxidation of the micro-pollutants, and from this perspective,
51
52 NT103 is the most suitable for preparation of the multifunctional membrane.
53
54 As shown in Fig. 5C, all the membranes permeability declined dramatically after laccase
55
56 immobilization by reverse filtration due to the formation of a fouling layer as a result of either pore
57
58
59 16
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1
2
3
4 blocking or deposition of enzyme aggregates. After dopamine coating, the permeability of NT101 and
5
6 NF270 recovered to some extent, while for NT102, NT103 and NF90 membranes, the permeability
7
8 decreased even further. It is well known that the membrane permeability mainly depends on filtration
9
10 resistance and surface hydrophilicity. The PDA coating obviously improved the surface hydrophilicity of
11
12 the support layer (the water contact angle decreased from 62.29, 70.21 and 74.48 to 43.69, 40.00 and
13
14 54.48 for NT103, NF270 and NF90 membranes, respectively) since the PDA layer was very
15
16 hydrophilic,36-37 but this coating layer inevitably increased the whole filtration resistance. As reported by
17
18 Luo and co-workers23 when a cake layer of enzyme forms on the sublayer of the membrane, the membrane
19
20 permeability declines after enzyme immobilization due to the reduction of surface hydrophilicity. While
21
22 such hydrophilicity reduction could be mostly recovered by dopamine coating, leading to a permeability
23
24 recovery. When pore blocking was dominant during the enzyme immobilization, such permeability
25
26 recovery was not possible. In reality, enzyme immobilization in/on the membrane was a process of
27
28 membrane fouling formation, and the fouling mechanism for six membranes in this work were found to
29
30 follow the intermediate blocking model or cake layer model according to the established filtration
31
32 model38 (Fig. SI-6, Table SI-1). The high enzyme content in the retentate and washing water before
33
34 coating (RW) for NT101, NF270 and the DL membranes (22.46%, 14.22% and 15.83% respectively) was
35
36 a proof of cake layer formation, because the enzymes on the sublayer were easily removed by washing.
37
38 This was also in accordance with the obvious hydrophilicity recovery after dopamine coating for NT101
39
40 and NF270 membranes. However, permeability recovery after dopamine coating for the DL membrane
41
42 was not significant. Actually, the augmentation of hydrophilicity and filtration resistance caused by PDA
43
44 coating layer was a trade-off, and for the DL membrane with the highest density (Table 2), the increase
45
46 in filtration resistance by the PDA layer was dominant. On the other hand, for NT102, NT103 and NF90,
47
48 since the permeability after dopamine coating was even lower, it was assumed that the intermediate
49
50 blocking model was a more appropriate mechanistic description.
51
52 As for the interface interaction between enzyme, PDA layer and membrane, we hypothesized that
53
54 non-covalent bonding was dominant for laccase immobilization in this case. This hypothesis was verified
55
56 by detecting the enzyme leakage after reverse filtration. Since the membrane, laccase and PDA were
57
58
59 17
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1
2
3
4 almost neutral or little charged at pH 5.3,29, 39 electrostatic interaction among them was negligible. While
5
6 the support layer of NF membrane is normally made from hydrophobic polypropylene, the hydrophobic
7
8 adsorption between laccase and membrane would not be neglected. Because dopamine coating could even
9 40
10 used to fabricate the skin layer of NF membrane , the hydrophilic PDA network in the support layer
11
12 would absolutely wrap the laccase when the modified membrane was placed in normal mode. Therefore,
13
14 hydrophobic interaction, hydrogen bonding and entrapment might therefore be the main mechanisms for
15
16 enzyme immobilization. Moreover, covalent bonding might form between the PDA layer and enzymes by
17
18 a Schiff-base reaction or Michael addition.27 This would provide a tight bonding to firmly immobilize the
19
20 protein, but would likely be detrimental to enzyme activity as the covalent bonding might disturb the
21
22 enzyme function.24
23
24 Since our long-term interest is to apply the prepared multifunctional membranes in practical water
25
26 purification, a higher BPA rejection is preferred for reducing the subsequent enzymatic burden (NF90) and
27
28 a higher enzyme loading/activity is preferred to improve catalysis efficiency (NT103). Besides, higher
29
30 membrane permeability can decrease energy consumption (NF270). Therefore, in the following studies,
31
32 NF90, NT103 and NF270 were chosen for multifunctional membrane preparation and application test.
33
34 3.3 BPA removal
35
Chea and co-workers found that in the presence of laccase and oxygen, phenolic compounds such as
36
37
BPA, were oxidized to phenoxy radicals firstly, then to quinone, and finally polymerized.41 The resultant
38
39
polymers did not show estrogenic activity and could easily be removed by sedimentation or filtration.42
40
41
42
When the BPA solution was treated by pristine and multifunctional NF membranes, different removal
43
44
efficiencies were observed (Fig. 6). For the pristine membranes, the BPA concentration in the permeate
45
46
gradually increased during the filtration, because the adsorption capacity was saturated and concentration
47
48
polarization became more serious (i.e. more and more BPA molecules accumulated on the membrane
49
50
surface and then passed through the membranes by diffusion); while for the multifunctional membranes,
51
52
due to the presence of enzymatic oxidation of BPA during the filtration, the BPA concentration in the
53
54 permeate was much lower than that for the pristine membranes (Fig. 6A). The NF270, NT103 and NF90
55
56
multifunctional membranes displayed around 84.27 7.30%, 78.211.95% and 97.040.33% BPA
57
58 removal (Fig. 6B), respectively, which is superior to the average elimination (55%) by the conventional
59 18
60
1
2
3
4 Fenton treatment31 under similar conditions. Additionally, these results are comparable to, or even better
5
6 than, the results reported by Escalona and co-workers31 where 89% elimination was achieved by soluble
7
8 laccase coupled with NF270 membrane. It is worth noting that all filtration experiments were conducted in
9
10 triplicate and the maximum deviation between experiments was 7.30% (NF270).
11
12 Although all three of the multifunctional membranes exhibited a substantial improvement in BPA
13
14 removal compared with the pristine membranes, the detailed mechanism for these membranes was
15
16 different. For NT103 and NF270, the stable BPA removal efficiency was caused by in-situ bio-oxidation
17
18 with immobilized laccase.42 After that, the adsorption sites are liberated for the next adsorption-oxidation
19
20 cycle. In this way, the immobilization of laccase broke the adsorption saturation limitation, making it
21
22 possible to achieve continuous and stable BPA removal. The outcome is consistent with the results
23
24 reported by Luong and co-workers, where four micro-pollutants were removed successively by granular
25
26 activated carbon-bound laccase.5 On the other hand, the multifunctional NF90 membrane showed the
27
28 highest BPA removal, and it can be seen in Fig. 6B that such high BPA removal efficiency was mainly
29
30 caused by the high rejection of NF90 (RS=74.02%), while laccase oxidation and PDA adsorption only
31
32 contributed 12.57%, much lower than that for NT103 (41.68%) and NF270 (49.46%). First, this was
33
34 attributed to the improved hydrophilicity by dopamine coating which greatly enhanced the physical
35
36 rejection of hydrophobic BPA molecules.43 Secondly, the enzyme entrapment in NF90 membrane narrows
37
38 the pores, thus increases BPA retention. Indeed, this could further decrease the BPA adsorption (i.e.
39
40 enzyme entrapment sites occupied the BPA adsorption sites) and oxidation (i.e. lower substrate
41
42 concentration penetrating the skin layer for enzymatic reaction) in the membrane. However, for other two
43
44 looser membranes (NT103 and NF270 with larger MWCO), the retention increase due to laccase
45
46 immobilization was not so obvious, so that much of the BPA could still pass through the skin layer,
47
48 resulting in stronger BPA adsorption and oxidation in the membrane itself. Moreover, it was also seen that
49
50 the multifunctional NF270 membrane displayed a little better BPA removal than NT103 which showed
51
52 the highest enzyme activity measured without pressure (Fig. 6B). This may be ascribed to the different
53
54 operating pressures for the two membranes in flow-through mode. Under the same permeate flux, TMP
55
56 for NF270 was much lower than NT103 (around 1.63 bar vs. 2.21 bar), and thus NT103 was more
57
58
59 19
60
1
2
3
4 compressed, which might suppress the accessibility of substrates (BPA is larger than DMP) to the
5
6 immobilized enzymes. In addition, the sponge-like sublayer of NF270 membrane had a better
7
8 distribution of enzyme in the membrane and was therefore likely more favorable for BPA interaction with
9
10 the enzyme.
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12
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42
Fig 6 BPA removal of three pristine and multifunctional membranes when 43 mL BPA solution (10 mgL-1) was
43
44
filtrated in 5 hours. (A) BPA concentration in permeate with filtration proceeding and (B) overall BPA removal
45
46
efficiency.
47
48
49
50
3.4 Synergistic mechanism
51
52 As previously described, the superior BPA removal efficiency of the multifunctional membrane is
53
54 due to the combination of membrane rejection, adsorption and laccase oxidation. Aside from membrane
55
56 adsorption, it was also found that the PDA layer itself could adsorb BPA (see Supporting Information
57
58
59 20
60
1
2
3
4 VI, Fig. SI-7), which is consistent with previous studies that PDA coatings could adsorb various
5
6 compounds via covalent or non-covalent bonding.29, 44-46 To clarify the contributions of these functions to
7
8 BPA removal, BPA removal efficiencies of four differently-designed membranes (see Section 2.6 and
9
10 Supporting Information III ) were compared, as shown in Fig. 7. BPA elimination efficiencies for these
11
12 four types of membranes were 34.810.62%, 36.180.77%, 82.200.42% and 91.502.51%, respectively.
13
14 In comparison to the pristine membrane, no obvious improvement was observed for the adsorption
15
16 membrane (the enzymes inside were deactivated), indicating that the PDA coating layer had negligible
17
18 effect on the overall membrane adsorption. PDA is very hydrophilic which is unfavorable for BPA
19
20 hydrophobic adsorption, even though the BET surface area of the membrane increased dramatically after
21
22 coating with the dopamine layer (from 6.79 to 22.93 m2g-1). Thus, the PDA coating layer decreased the
23
24 intrinsic BPA adsorption of the pristine membrane, which offset the additional BPA adsorption resulting
25
26 from the PDA layer itself. The catalytic membrane showed good BPA removal efficiency as a result of
27
28 laccase catalysis. Interestingly, for the multifunctional membrane coated with the PDA layer, the BPA
29
30 removal efficiency was even better. This is not only attributed to the synergistic effect of adsorption and
31
32 catalysis, but also to the enhanced electron transfer by the PDA layer. It was reported that the BPA
33
34 oxidation by laccase involves the four-electron reduction of dioxygen to water and the four single-electron
35
36 oxidations of the reducing BPA molecules, where electron transfer is vital to the reaction.16, 39 PDA, a
37
38 melanin-like substance, displays electrical conductivity properties,47 which is beneficial to electron
39
40 transfer between the BPA, oxygen and the laccase, thus promoting oxidation efficiency. Accordingly, the
41
42 mechanism for BPA removal using the multifunctional membrane is illustrated in Fig. 7B. Specifically,
43
44 the micro-pollutants would be partly retained by the NF membrane, and then the residual pollutants would
45
46 be adsorbed in the membrane and PDA layer (close to the enzymes). Thus, membrane rejection reduced
47
48 the enzymatic burden, and BPA adsorption in/on the membrane enriched BPA molecules near the enzyme
49
50 and also prolonged the contact time between substrate and laccase, as well as PDA enhanced electron
51
52 transfer, thus definitively improving the catalytic efficiency. In return, the laccase oxidation breaks the
53
54 adsorption saturation limitation, making it possible to achieve a continuous and stable BPA removal.
55
56
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59 21
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42
Fig 7 (A) BPA removal efficiency of four differently-designed membranes, (B) illustration of working mechanism of
43
44
multifunctional membrane and (C) synergy analysis and the contribution of each function to overall micro-pollutants
45
46
removal. 43 mL BPA solution (10 mgL-1) was filtrated in 5 hours.
47
48
49
50
51
It is believed therefore that the combination of multi-functions can facilitate overall micro-pollutant
52
53 removal. In this work, we attempted to clarify the synergistic effect among the multi-functions on BPA
54
55 elimination. We assumed that the contribution of each function could be derived by mass balance and
56
57 more details about the calculation can be found in Supporting Information VII. Finally, it was found that
58
59 22
60
1
2
3
4 the NF270 membrane displayed around 21.32% of BPA rejection, and membrane and PDA adsorption
5
6 accounted for 14.85% BPA removal, while laccase catalysis could remove 47.39% of BPA (Fig. 7C).
7
8 These functions facilitate each other and we therefore found as expected a synergistic effect, where the
9
10 PDA coating exhibited an improvement of 7.93% for BPA removal as a result of enhanced BPA adsorption
11
12 and electron transfer during BPA oxidation.
13
14 3.5 Reusability and stability of multifunctional membrane
15
The reusability and stability of the multifunctional membrane is crucial to the practical application of
16
17
the membrane. Therefore, the reusability of both the multifunctional and catalytic membranes in 7 cycles
18
19
was compared, and the results are shown in Fig. 8. The BPA removal efficiency decreased gradually and
20
21
reached 33.21% and 21.04% after 7 reuse cycles for the multifunctional and catalytic membranes
22
23
respectively. This is comparable with other reports where enzymes were immobilized on inert
24
25
supports.48-49 For example, Spinelli and co-workers found that the activity of laccase-loaded Amberlite
26
27
beads reduced to 30% after 7 days reuse.48 As seen in Fig. 8A, the enzyme activity in this case also
28
29
decreased gradually over 7 days. It is possible that the enzyme was leaching and/or denaturation occurred
30
31
during the reaction and caused the low reusability in continuous operation. The enzyme immobilized by
32
33
weak physical bonds (e.g. Van der Waals force and hydrogen bonding) would diffuse away from the
34
35
membrane or be washed away. This could be verified by the fact that after 7 days of storage the enzyme
36
37
38
content in the storage buffer was increased initially, and then reduced (Fig. 8B), which might be due to
39
the covalent reaction and/or re-adsorption of desorbed laccase on the PDA layer (although this would be
40
41
42
avoided in flow-through mode during real application). We conclude that the prevention of enzyme
43
44
leakage is of great importance to improve membrane stability.
45
46
47
48
49
50
51
52
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59 23
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47
48
49 Fig 8 (A) BPA removal efficiency during 7 reuse cycles for the multifunctional and catalytic membranes when 43
50
51 mL BPA solution (10 mgL-1) was filtrated in 5 hours, (B) enzyme portion variation in storage buffer during 7 days
52
53 storage and (C) TMP profile during BPA treatment process using multifunctional membrane.
54
55
56
57
58
59 24
60
1
2
3
4 The improvement due to the PDA coating layer was evident in the 7 cycles where the BPA removal
5
6 efficiency of multifunctional membrane was much higher compared with the catalytic membrane without
7
8 dopamine coating. During storage, enzyme leakage was much more severe from the catalytic membrane
9
10 because of no PDA coating layer was present as a barrier. Likewise, some BPA oxidation products might
11
12 be retained on the sublayer when the reverse mode was employed with the catalytic membrane, leading
13
14 to more serious membrane fouling as well as enzyme denaturation. Furthermore, the PDA layer changes
15
16 the micro-environment in the membrane (increased specific area and higher hydrophilicity), which may be
17
18 desirable to maintain enzyme activity for a long-term operation.
19
20 It was also found that membrane fouling caused by adsorption/accumulation of BPA oxidation
21
22 products was important as the TMP increased with time and even doubled at the end of one cycle (Fig.
23
24 8C). This confirmed the suppression of enzyme activity caused by undesired hydrophobic interaction
25
26 between laccase and products though the increase of osmotic pressure due to buffer salts during filtration
27
28 was responsible for the partial TMP augment. Fortunately, the membrane fouling could easily be
29
30 overcome. After the membrane was immersed in 50% ethanol for 30 seconds and then washed by
31
32 ultrapure water for 5 minutes, the TMP value for the next cycle (after storing in neutral buffer overnight)
33
34 was almost restored to its original value in the previous cycle. The easy cleaning procedure would allow
35
36 the regeneration of the multifunctional membrane in practical application.
37
38
39
40 4. CONCLUSION
41
42
43
In this study, a bio-inspired multifunctional membrane was prepared successfully via reverse
44
45
filtration and dopamine coating. NT103, NF270 and NF90 membranes were suitable for this purpose due
46
47
to their advantages in enzyme loading, enzyme activity, permeate flux and BPA rejection, which were
48
49
greatly influenced by hydrophilicity, pore size, sublayer structure and porosity of the membrane. For
50
51
application in BPA removal, these three multifunctional membranes worked much more efficiently
52
53
compared with the pristine membranes due to laccase in-situ bio-oxidation or pore narrowing effect.
54
55 During the implementation of the multifunctional membrane, membrane rejection, adsorption and
56
57 catalysis functions facilitated each other and provided 21.32%, 14.85% and 47.39% micro-pollutant
58
59 25
60
1
2
3
4 removal, respectively, and the synergistic effect of PDA layer which benefited from the enhanced
5
6 adsorption and electron transfer was proved to be 7.93%. This work clearly confirms the benefits of the
7
8 combination of NF rejection, laccase catalysis and membrane/PDA adsorption in flow-through mode, and
9
10 this novel multifunctional membrane is therefore promising in tertiary wastewater treatment or household
11
12 water purifier.
13
14
15
16 ACKNOWLEDGMENTS
17
18 The authors thank the National Science Foundation of China (No. 21506229) and the National High
19
20 Technology Research and Development Program of China (No.2014AA021006) for the financial supports.
21
22 This work is supported by 100 Talents Program of Chinese Academy of Sciences.
23
24
25
26 ASSOCIATED CONTENT
27
28 Supporting Information. BPA static adsorption capacity of different membranes and determination of
29
30 the optimal enzyme dosage used. Illustration of the preparation of catalytic membrane and adsorption
31
32 membrane. Images of NF270 membrane before and after enzyme immobilization as well as images of
33
34 PDA layer. Filtration blocking model of six membranes during enzyme immobilization. Detailed
35
36 calculation method of different functions and synergistic effect. This material is available free of charge
37
38 via the internet at http://pubs.acs.org.
39
40
41
42 AUTHOR INFORMATION
43 *
44
Corresponding author: E-mail: yhwan@ ipe.ac.cn (Y. Wan); jqluo@ipe.ac.cn (J. Luo)
45
46
47 NOTES
48
49
The authors declare no competing financial interest.
50
51
52
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2. Christian, A.; Frederike, C.; Matthias, W. Membrane Processes in Biorefinery Applications. J. Membr. Sci. 2013,
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11 Biotransformation of Micropollutants by Granular Activated Carbon-Bound Laccase in a Packed-Bed Enzyme
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13 Reactor. Bioresour. Technol. 2016, 210, 108-16.
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53 19. Cazes, M. d.; Belleville, M.-P.; Petit, E.; Llorca, M.; Rodrguez-Mozaz, S.; Gunzburg, J. d.; Barcel, D.;
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