Biomimetic Aquaporin Membranes Coming of Age

Desalination 368 (2015) 89–105

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Desalination

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Biomimetic aquaporin membranes coming of age

Chuyang Tang a, Zhining Wang b, Irena Petrinić c, Anthony G. Fane d, Claus Hélix-Nielsen c,e,⁎ a Department of Civil Engineering, The University of Hong Kong, Pokfulam, Hong Kong b Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education of China, Ocean University of China 238 Songling Road, Qingdao 266100, China c University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova ulica 17, 2000 Maribor, Slovenia d Singapore Membrane Technology Centre, Nanyang Technological University, 1 Clean Tech Loop, CleanTech One #06-08, Singapore 637141, Singapore e Technical University of Denmark, Department of Environmental Engineering, Bygningstorvet 115, 2800 Kgs., Lyngby, Denmark

HIGHLIGHTS

• Development progress of biomimetic aquaporin membranes over the last decade • Comprehensive overview over membrane ﬂux versus rejection performance • Comprehensive overview over the biomimetic aquaporin patent landscape • Discussion of challenges in future membrane development and commercialization

article info abstract

Article history: Membrane processes have been widely used for water purification because of their high stability, efficiency, low Received 4 November 2014 energy requirement and ease of operation. Traditional desalting membranes are mostly dense polymeric films Received in revised form 22 April 2015 with a “trade off” effect between permeability and selectivity. Biological membranes, on the other hand, can per- Accepted 23 April 2015 form transport in some cases with exceptional flux and rejection properties. In particular the discovery of selec- Available online 7 May 2015 tive water channel proteins – aquaporins – has prompted interest in using these proteins as building blocks for new types of membranes. The major challenge in developing an aquaporin-based membrane technology Keywords: Aquaporin membranes stems from the fact that the aquaporin protein spans a membrane only a few nanometers thick. Such ultrathin Biomimetics membranes will not be able to withstand any substantial pressures, nor being industrially scalable without Water purification supporting structures. Incorporating aquaporin proteins into compatible materials, while ensuring membrane Desalination performance, scalability, and cost-effective production, is crucial for a successful technology development. Since the first suggestions for using aquaporins in membrane technology appeared around ten years ago, two main approaches have been suggested based on planar membranes and vesicles respectively. Here we summa- rize the essentials of aquaporin protein function and review the latest progress in this fascinating area of membrane research and development. © 2015 Elsevier B.V. All rights reserved.

1. Introduction around 46% for brackish water RO (similar savings would be expected from reclamation) using ultra-permeable membranes (UPMs). Interest- Water scarcity and climate change are two major challenges in the ingly permeability enhancements beyond three to five times would 21st century. Solutions to these problems present a dilemma, because bring little extra benefit due to limits of thermodynamics and concen- increasing water supply via desalination and water reclamation will in- tration polarization. Another energy saving strategy involves a hybrid crease global energy usage. Therefore membrane technology, a key to forward osmosis (FO)–RO process, where seawater is diluted by low sa- the water scarcity problem needs to achieve improved efficiency and linity impaired water through FO to lower the feed osmotic pressure to energy minimization. One strategy involves the development of mem- RO. When coupled with pressure retarded osmosis (PRO) on the brine branes with enhanced permeability. Recent analysis [1] suggests poten- stream to recover osmotic power the FO–RO–PRO combination could tial energy savings of 15% for seawater reverse osmosis (SWRO) and halve the energy per m3 water produced [2]. UPMs could facilitate these approaches. There are various candidates for enhanced permeability membranes [3], and one of these is incorporation of biological ⁎ Corresponding author at: Department of Environmental Engineering, Building 115, office 140, Technical University of Denmark, DK-2800 Kgs, Lyngby, Denmark. water channels. This review provides an update and prospects for bio- E-mail address: [email protected] (C. Hélix-Nielsen). mimetic water filtration membranes. The primary focus is on RO/NF

List of abbreviations SWRO seawater reverse osmosis TEM transmission electron microscpy AB Diblock copolymer monomer with one hydrophilic A TFC thin film composite segment and one hydrophobic B segment TMC trimesoyl chloride ABA Triblock copolymer with a hydrophobic B segment UPM ultrapermeable membrane flanked by two identical hydrophilic A segments UV ultraviolet AQP aquaporin proteins VEM vesicle encapsulated membrane AQP-0 mammalian aquaporin 0 isoform AQP-1 mammalian aquaporin 1 isoform List of symbols AQP-3 mammalian aquaglyceroporin 3 isoform I(t) light intensity AQP-4 mammalian aquaporin 4 isoform k Time constant in stopped-flow measurements

AqpZ bacterial (E. coli)aquaporinZisoform k0 Hopping rate of water molecules in single ﬁle transport Brij™-98 polyoxyethylene(20) oleyl ether (detergent) N Number of water molecules in channel with single ﬁle CA cellulose acetate transport

CF cell-free (protein production system) pd Diffusional permeability Chol cholesterol pf Osmotic permeability CTRW continuous-time random walk S Vesicle surface area

CYMAL-5 5-cyclohexyl-1-pentyl-β-D-maltoside (detergent) vw Molar volume of water DDM dodecyl-b-D-maltoside (detergent) V0 Vesicle volume DOTAP N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- Δμ Chemical potential trimethylammonium Δosm Difference in osmolarity DRM detergent resistant membranes ΔP Hydrostatic pressure difference DSM detergent solubilized membranes DSP downstream processing (in protein production) FO forward osmosis GA glutaraldehyde biomimetic aquaporin membranes but we will also briefly discuss FO GLP glycerol facilitator like protein (aquaglyceroporin) biomimetic aquaporin membranes, as they may provide an apt pre- GlpF aquaglyceroporin (glycerol uptake facilitator protein) treatment for RO in hybrid FO–RO processes. from E. coli In nature water is cleaned by physico-chemical and biological pro- GPV giant protein vesicles cesses occurring at the air–water interface, within the bulk water, and kDa kilodalton at water–soil/mineral interfaces. In living organisms selective water LbL layer by layer transport across membrane is facilitated by a specialized class of pro- − − LMH liters per square meter per hour (l m 2 h 1) teins – aquaporins (AQPs), also referred to as Major Intrinsic Proteins LPR lipid protein ratio (MIPs) – or water channels, a family of integral membrane proteins LUV large unilamellar vesicle that are expressed broadly throughout the animal and plant kingdoms MD molecular dynamics [4,5]. In higher organisms there are several complex structures capable MIP major intrinsic protein (synonymous with AQP) of selective water transport including the blood–brain barrier, the skin MPD m-phenylenediamine (1,3-diaminobenzene) barrier, and intestinal epithelial walls. These structures share some gen- NF nanofiltration eral features, but are also developed to very specific tasks. In terms of NPA the signature asparagine (N)–proline (P)–alanine water and salt transport the kidney is an exemplary system dealing (A) motif with fluid and electrolyte balance. One of the key features of the kidney OG octyl-β-D-glucopyranoside (detergent) is its remarkable capacity in terms of water filtration. Under normal PAA poly(acrylic acid) conditions the human kidney filters approximately 180 L blood plasma PAH poly(allylamine hydrochloride) and excretes about 1.5 L per day [6]. This implies that most of the water PAI poly(amide-imide) and solutes are re-circulated and this transport is to a large extent me- PAN poly(acrylonitrile) diated by a complex interplay between active and passive facilitators, PC phosphatidylcholine where AQPs play a key role [7]. PCTE polycarbonate tracked-etched Since the discovery of AQPs there has been considerable interest in PDA poly(dopamine) using this protein as a component in membrane technology. Membrane PEG polyethylene glycol processes have been widely used for water purification because of their PEI polyethylenimine high stability, efficiency, relatively low energy requirement, and ease of PG phosphatidylglycerol operation. However, more research efforts are still needed to deal with PLL poly-L-lysine disadvantages, such as the performance decline and the “trade off” ef- PPR polymer protein ratio fect between permeability and selectivity [8]. In this context AQPs rep- PRO pressure retarded osmosis resent a new membrane material in the sense that they provide ideally PSS poly(styrenesulfonate) selective conduits for water with a high osmotic permeability. Thus, one RO reverse osmosis would in principle be able to design semi-permeable membranes with SAM self-assembled monolayer 100% rejection of all other solutes except water, provided that AQPs SDS sodium dodecyl sulfate (detergent) can be embedded in a solute-impermeable stable matrix. Inspired by SLB supported lipid bilayer this, Kumar et al. found that AQPs incorporated into polymersomes SML supported membrane layer show water permeability up to two orders of magnitude greater than SM sphingomyelin the commercial RO membranes and a selectivity of about 100% [9]. SoPIP2;1 spinach aquaporin At a first glance an aquaporin membrane should then be ideally suit- SPM supported polymer membrane ed as a building block for UPMs capable of bringing down the current desalination energy cost of ~2.6 kWh/m3 [10]. Also for seawater RO C. Tang et al. / Desalination 368 (2015) 89–105 91 desalination plant about 20% of the capital cost originates from pressure the mammalian AQP-1, it appears that AQPs exist in the plasma mem- vessels and piping [11]. So, the potential for energy and cost savings ap- brane as homo-tetramers. AQPs show high sequence similarity: se- pears to be significant, if membrane permeability can be increased. In- quence identity ranges from 26% to 49%, increasing to 45–60% in the deed a recent study has suggested that a 3-fold increase in membrane channel region. The three-dimensional structures of the proteins are permeabilitywouldallowfor44%fewerpressurevessels,or15%lessen- very similar. The root mean-square displacements for Cα atoms are be- ergy for a seawater RO plant with a given capacity and recovery ratio [1]. tween 0.17 and 0.23 nm for the whole chain, and 0.07 to 0.13 nm in the For brackish water desalination (i.e. based on low pressure RO) a 3-fold membrane spanning helices reflecting a conserved structure [24].Dif- increase in permeability would result in 63% fewer pressure vessels or ferent aquaporins have different patterns of glycosylation. In the case 46% less energy consumed [1]. However, UPMs will also require new of AQP-1, the peptide backbone is roughly 28 kDa (corresponding to module and system design in order to minimize the effects of concen- around 112 kDa for the tetramer) and the glycosylated forms range tration polarization at the membrane surface due to increased mass- from 40 to 60 kDa (i.e. up to 240 kDa for the tetramer) [25].Mostaqua- transfer. porins have cytoplasmic protein kinase A phosphorylation motifs It is also important to realize that highly water permeable AQP pointing to a general regulatory function [26]. proteopolymersomes (or proteoliposomes) per se do not constitute a The main properties of AQP relevant for biomimetic membrane membrane and the ‘grand challenge’ in biomimetic aquaporin mem- technology for water purification are 1) water flux, 2) gating properties, brane technology is indeed to provide a strategy for efficient upscaling, 3) solute rejection, 4) stability to external conditions, and 5) the ease of while preserving and effectively exploiting the unique functionality of reconstitution into biomimetic membrane matrixes [13]. the aquaporin proteins. Over the last years many new designs for AQP laden filtration membranes have been reported (for earlier reviews 2.2. Water flux see [12,13]), and opened up an exciting direction in separation membranes due to the high AQP water transport and excellent selectivity. Since the first AQP crystal structure was reported, extensive molec- Here we review some of the latest progress and outstanding chal- ular dynamics (MD) simulations describing how water is passing lenges in the fast-moving field of biomimetic AQP membranes. A brief through narrow pores have been conducted; for recent reviews see overview of AQPs and their properties is presented in Section 2. [27,28]. A starting point for understanding how MD simulations can Section 3 reviews the structural design and fabrication of AQP incorpo- provide useful insights into AQP function is to consider a narrow rated biomimetic membranes, while the applications of these mem- water filled pore with water molecules arranged in a single file fashion. branes and their performance are summarized in Section 4. A patent In this simple system translocation occurs, when one water molecule survey is provided in Section 5, and future perspectives are discussed enters from one end and another water molecule leaves the channel in Section 6. at the other end. The model implies that the pore is always filled with water molecules, and that water molecules move in an orderly fashion 2. The fundamental building block: aquaporins along the luminal axis of the pore. This is known as the continuous- time random walk (CTRW) model [29], which was developed to explain Since the discovery of AQPs [14,15], accumulating genomic and the transport of water filling a channel in a single-file arrangement [30]. transcriptomic data from vertebrates and flowering plants as well as For AQPs the key flux parameters are the diffusional (pd)andosmotic 3 from unicellular eukaryotes, fungi, green algae, mosses, and non- (pf) permeability coefficients (both measured in cm /s). As pd describes vertebrates are revealing AQP evolution throughout the diversity of water movement by thermal fluctuations, pd can be determined from life and today more than 1700 AQP sequences are known [16]. Based equilibrium MD simulations, where we can follow individual water on function, AQPs can be classified in two major groups. One group, molecules and also experimentally by radioactively labelled water where all homologues have exclusive water permeability – are often re- molecules [31].UsingtheCTRWmodel,pd can be expressed as ferred to as orthodox aquaporins – or classical AQPs; another group, where the homologues are all both water- and glycerol-permeable re- vw k0 p ¼ ð1Þ ferred to as aquaglyceroporins or glycerol facilitation-like proteins d N þ 1 (GLPs) [5]. The pore diameter of the aquaglyceroporins is slightly greater than that of the orthodox AQPs, and the pore is lined by relatively hy- where vw is the average volume of a single water molecule, k0 is the hop- drophobic residues compared with the pore of an orthodox AQP [17]. ping rate of water molecules that enter or leave the channel, and N is the fi Many eubacteria have one of each kind, thus for example Escherichia number of water molecules that occupy the channel in single le [32]. coli has the orthodox AqpZ and the aquaglyceroporin GlpF channel The osmotic permeability pf describes water molecule translocation Δμ [18]. In addition to water and glycerol, there is evidence, that some under osmotic i.e. chemical potential differences. pf can be calculated from AQPs may allow passage of gases (CO2,NH3, NO, O2), various small solutes such as H O , certain metalloids, and ions [19–21].Non- 2 2 ¼ ; ð Þ transporting functions for some aquaporins have also been suggested, p f vw k0 2 such as cell–cell adhesion, membrane polarization, and regulation of fl interacting proteins, such as ion channels, for recent comprehensive re- where the hopping rate k0 is dependent on the water ux, which in turn views see [19,22]. is determined by the chemical potential difference across the membrane. In simulations chemical potential differences can be mimicked 2.1. Basic structure by changes in hydrostatic pressure as

Δμ ¼ v ΔP ð3Þ The primary sequence of AQP reveals its general unit structure in the w form of two tandem repeats, each containing three transmembrane spanning segments, see Fig. 1a [5]. Biochemical analysis and later crystal Combining Eqs. (1) and (2) one can obtain the pf/pd ratio, which is structure analysis revealed an hour-glass structure with pseudo two- related to the number of steps (N + 1) that a water molecule must fold symmetry where the six transmembrane segments surround a cen- move when passing a channel with an average occupancy of N water tral pore. Thus, each repeat represents a hemi-pore, where the two molecules: hemi-pores fold together to form a water-channel, see Fig. 1b. Each tan- – p dem repeat contains a loop between TM2 and TM3 with an asparagine f ¼ N þ 1 ð4Þ proline–alanine (NPA) signature motif, see Fig. 1c. Based on studies with pd 92 C. Tang et al. / Desalination 368 (2015) 89–105 a)

b) c) d)

N186 V185 F62 N63

Fig. 1. Aquaporin (AqpZ) monomer basic structure. (a): Each monomer is formed by two tandem repeats of three membrane-spanning alpha-helices (green, yellow, and red cylinders) labelled 1–6connectedbyfive loops (A–E) where loops A, C, and E are extracellular and B, and D together with the N- and C-termini are intracellular. Loops B and E are hydrophobic and each contains the highly conserved, asparagine–proline–alanine (NPA) motif at a position corresponding to the lipid bilayer midplane. (b): Schematic cross-sectional view of the monomer showing the water selective pore with a narrow constriction site containing a Histidine (H) and an Arginine (R) generally referred to the ar/R (aromatic/arginine) selectivity filter and a region defined by the two NPA motifs (c): The constriction site has an effective diameter of only 2.8 Å (just enough to let one water molecule pass at the time) and is highly conserved in orthodox AQPs. In AqpZ it is formed by three amino acids: Arginine 189 (blue), Histidine 174 (green) and Phenylalanine F43 (yellow). (c): The NPA motif coordinates the central water molecule by having N63 and N186 each donating a hydrogen bond to water oxygen (red sphere) by projecting their NH2 moieties into the pore. Further (‘second shell’) stability is provided by hydrogen bonds from the carbonyl moieties of V185 and F62, respectively. Panels c & d are generated using PDB accession code 1RC2 [23].

Eq. (4) can be seen as a measure of the single-file propensity where a the dynamics of a short single file transport of water molecules in indi- pf /pd ≈ N corresponds to a perfect single file, while a pf /pd b N indicates vidual AQPs. that water molecules are passing one another in the channel. For the or- Various experimental methods can be used to estimate the osmotic thodox aquaporin AqpZ the ratio has been estimated using molecular water permeability of membranes with AQPs. For ion transport across dynamics (MD) to be 13 ± 6 whereas for the aquaglyceroporin GlpF it membranes single channel events can be easily recorded using standard is 2.9 ± 0.8 [17]. methods (as even a small amount of electric charge across a thin dielec- The use of CTRW models only holds for the narrow part of the AQP tric medium will give rise to mV potential differences across the mem- protein. In order to incorporate molecular shape details of the protein brane). This is not the case for water molecules and water transport lumen Hashido et al. proposed a pf-matrix method, in which pf is studies in AQPs have so far been limited to indirect (ensemble) decomposed into contributions from each local region of the channel measurements. [33]. Generally the lumen geometry can be seen as a central tube A popular method for measuring water permeability is based on os- (with single file transport) connected to the bulk water reservoirs via motically-induced shrinking or swelling of vesicles with incorporated cone-shaped vestibules, see Fig. 1b. The conical entrances are much AQP protein using the fact that light scattering of suspended particles larger in volume compared to the central tube, which implies that con- is dependent on the size of the particles (and the viscosity of the sur- tinuum hydrodynamics should apply to some extent in these cone- rounding media) [38]. Here the main challenge is to measure the initial shaped vestibules. Indeed it has been shown that the Navier–Stokes volume change upon a rapid change in environment (osmolarity), and description is valid down to the nanometer range [34,35]. Using a com- this is implemented in the so-called stopped flow method In a bination of finite element calculations and analytical modeling, Gravelle stopped-flow apparatus the change in intensity of scattered light at a et al. showed that conical entrances with suitable opening angle con- 90-degree angle is measured as a function of time after mixing, see tributes significantly to the overall channel permeability [36].Thisalso Fig. 2. The light scattering changes resulting from osmotically-induced point to the benefits in using AQPs as water conduits in biomimetic changes in vesicle size have been used extensively to estimate the os- membranes as opposed to the approaches, where aligned carbon nano- motic permeability of the vesicle Pf. tubes (i.e. without conical entrances) have been suggested as the build- With S/V0 as the initial vesicle surface area to volume ratio (measuring block for ultrapermeable membranes [37].AlthoughMDcan able by dynamic light scattering) Pf can be expressed as: provide qualitative information on AQP water transport on the sub- nanometer scale, quantitative comparison with experimental data is still problematic due to parameterization, short simulation times (typi- ¼ k ; ð Þ P f = Δ 5 cally less than 1 μs), and the lack of experimental techniques to probe S V 0vw osm C. Tang et al. / Desalination 368 (2015) 89–105 93

2.4. Solute rejection

One of the remarkable properties of orthodox aquaporins is the ability to exclude virtually all solutes – even protons. For large solutes steric and/or electrostatic factors prevent electrolyte passage. AQP interiors are relatively hydrophobic and this results in an incomplete compensa- tion for hydration energy in transferring an ion from bulk water [46]. Thus the de-solvating energies create high-energy barriers at the pore entrances. Further, Monte Carlo kinetic calculations suggest that acidic hydrophilic residues in the selectivity filter and at the NPA region electrostatically destabilize cations, whereas the channel-lining carbonyls electrostatically destabilize anions [47]. For protons, simulations reveal that close to the NPA region the average water dipole orientation is re- versed, see also Fig. 1c. This observation initially led to the suggestion of a proton rejection mechanism based on the disruption hydrogen bonds Fig. 2. Stopped flow principle for measuring osmotic water permeability in vesicles. Two solutions, A (high osmolarity) and B with vesicles (low osmolarity) are mixed. The volume [48]. However, there also may be contributions from the loss of solva- injected is limited by the stop syringe (S) which provides the “stopped-flow”. As the in- tion [49,50] when moving a proton into the narrow partly hydrophobic duced osmotic gradient shrinks the vesicles the 90° light scatter increases with a time con- part of the channel. The result is an impeded proton flux – even in the stant k which can be inserted into Eq. (5) for determination of pf. For a critical review of the presence of an intact water wire. The mechanism is not fully elucidated, methodology seen in relation to biomimetic aquaporin membrane performance see Ref. yet the delicate balance between hydrogen bonding forces in a hydro- [39]. phobic narrow pore combined with electrostatic barriers [51,52], and helical dipolar moments [53] result in a very efficient and highly selective transport of water, although various conducting states are likely to where k (in s−1) is the time constant associated with the volume exist [44,45]. change and Δosm is the difference in osmolarity between the intra- vesicular and extra-vesicular aqueous solutions. Experimental values 2.5. Stability for k are obtained by fitting the scattered intensity I(t)toanexponential function. If the protein content per vesicle is known one can use Eq. (5) A major requirement for the use of AQPs in biomimetic membrane to determine an upper limit for the single channel osmotic permeability applications is protein stability towards external perturbations during pf. protein reconstitution, and membrane function. For the plant orthodox spinach aquaporin SoPIP2;1 Plasencia et al. showed that it can exist as a stable folded protein in octyl-β-D-glucopyranoside (OG) detergent mi- 2.3. Gating celles solutions and that the protein can be transferred from detergent micelle solutions and reconstituted into selected phospholipid mem- Generally aquaporins may be just seen as water conduits, but in fact branes preserving its structural characteristics [54]. This study was water transport may be regulated via mechanisms that temporarily oc- followed by a comparative study revealing that the anionic detergent clude the lumen, thus blocking water transport. This is often referred to sodium dodecyl sulfate (SDS) does not unfold neither SoPIP2;1, nor as gating and gating mechanisms have been investigated for a number AqpZ during transition from a membrane reconstituted form to a deter- of aquaporins. In spinach plasma membrane aquaporin SoPIP2;1, pH gent stabilized state albeit the native folds are changed [55].Thusitis gating is governed by the protonation state of a highly conserved His- evident that AQPs may be isolated, purified, and inserted into a biomi- 193 in the D-loop [40]. MD simulations, based on high-resolution X- metic host matrix, while preserving functionality. ray structures of SoPIP2;1 in closed and open conformations, have provided mechanistic insight into phosphorylation-induced gating [41]. 2.6. Reconstitution Thus, the closed conformation of SoPIP2;1 is achieved by the D-loop occluding the channel entrance from the cytoplasmic side, whereas open- In the early 1990s several studies appeared describing reconstitution ing is achieved by phosphorylation of Ser-115 in cytoplasmic loop B and of functional AQPs (red blood cell AQP-1 and bovine lens AQP-0) into Ser-274 in the C-terminal region. For the mammalian AQP-4 gating like- proteoliposomes (e.g. [38,56,57]). Using a lipid-containing oil phase, ly involves a Serine (Ser-180) present near the cytoplasmic lumen en- formation of giant vesicles from mixing protein-reconstituted large trance. The phosphorylated Ser-180 may interact with positively unilamellar vesicles (LUVs) has been achieved for reconstitution of charged residues present in the C-terminus (Lys-259, Arg-260, Arg- membrane proteins in a controllable fashion [58]. This method makes 261 in the rat AQP-4) thereby blocking the channel [42]. In the yeast it possible to create giant protein vesicles (GPVs) from a variety of Pichia pastoris the AQP Aqy1 may be closed due to the N-terminus fold- lipid compositions, including lipids that previously have posed difficul- ing in such a way that a tyrosine on the N-terminal (Tyr 31) docks into ties for giant lipid vesicle formation for example, pure negatively the channel cytoplasmic entrance, and hydrogen bonds to a water mol- charged lipids and total-lipid extracts [30]. The method supports GPV ecule in the lumen thereby occluding it [43]. For AqpZ gating involves formation at physiological ionic strength, such as phosphate-buffered the Arg-189 in the selectivity filter. MD simulations reveal that Arg- saline, and does not require specialized equipment, specific lipids, syn- 189 can flip between two distinct yet otherwise stable conformations thetic peptides, or dehydration/rehydration steps. GPV could provide a [44,45]. The open and closed states can be directly related to water per- step in creating larger bilayer areas by vesicle collapse (see also meation dynamics through the channel, and the free energy profile ob- Section 3.1). tained implies fast transitions between them with thermal fluctuation In 2006 Stoenescu et al. showed that His-tagged bovine lens AQP-0 [44]. Interestingly, for the triple mutant AqpZ F43W/H174G/T183F, could be inserted into block copolymer membranes and that orientation which has a low permeability (10% average water permeability com- of the protein depended on the block copolymer symmetry [59]. The pared to wildtype AqpZ), this low permeability can be explained by a 2007 study of Kumar et al. [9] also described AqpZ reconstitution in double gate involving a ring stacking between W43 and F183 acting as ABA triblock copolymers and discussed the possibility of creating sepa- a secondary steric gate in the triple mutant with R189, as the primary ration membranes for technological applications. This was followed up steric gate in both mutant and wildtype AqpZ. by a study showing that bovine lens AQP-0 can be functionally 94 C. Tang et al. / Desalination 368 (2015) 89–105 incorporated into both AB di- and ABA triblock membranes at high Table 1 density, and that incorporation of membrane proteins at high polymer Summary of advantages and disadvantages of AQP laden biomimetic membranes with different structures. to protein ratios (PPRs) changed the morphology of the polymer − membrane protein aggregates from vesicular to planar structures. In Structure of Advantages Disadvantages some cases, AQP-0 formed 2D crystals in block copolymer membranes, biomimetic representing the limit of membrane protein packing in bilayer-like membranes membranes. SMLs 1. Good biocompatibility 1. Typically lower mechanical Thus, both lipid and polymers may in principle be used as building 2. Ultrathin bilayer structure stability facilities fast transportation of 2. More difficult to control blocks for creating biomimetic membranes. This could suggest a rather water molecules; water defect formation low sensitivity to the nature of the host membrane. However, mem- molecules only need to pass 3. More difficult to scale up brane–protein interactions may play an important role, since the me- through AQP proteins once 4. May involve more expensive chanical properties (i.e. membrane stiffness and curvature propensity) (i.e., high water permeability) materials and/or fabrication 3. Less chemicals employed methods can affect the function of transmembrane spanning proteins [60–63]. VEMs 1. Mechanically robust structure 1. Chemicals and procedures – The implications of membrane protein interactions for AQPs are evi- with high stability for encapsulation may affect dent in a series of papers describing AQP-0 and AQP-4 reconstitution 2. Polymer matrix protects AQPs the activity of AQPs into lipid bilayers. For example, Tong et al. investigated the sorting of from chemical and biological 2. Water molecules need to bovine AQP-0 into lipid microdomains in lens membranes [64]. Lens attack pass through vesicle at least 3. Scalable twice membranes contain high concentrations of cholesterol (Chol) and 4. Less defects 3. Polymer matrix introduces sphingomyelin (SM), as well as phospholipids, such as phosphatidyl- additional hydraulic resistance choline (PC) with unsaturated hydrocarbon chains resulting in formation of detergent resistant SM/Chol enriched microdomains in these membranes. They found that for both crude membrane fractions and shall be (1) highly permeable, (2) highly selective (and free of defects), proteoliposomes composed of lens proteins in phosphatidylcholine/ (3) mechanically stable to withstand the required pressure for target sphingomyelin/cholesterol lipid bilayers, AQP-0 was found in both de- applications, (4) chemically and biologically stable for long term use, tergent resistant membrane (DRMs) and detergent soluble membranes and (5) easy to scale up at reasonable cost. According to membrane (DSMs). Analysis of purified reconstituted AQP-0 showed that the mi- structural design, AQP incorporated biomimetic membranes can be crodomain location of AQP-0 depended on lipid-to-protein ratio (LPR). classified into two basic types [68–91]: (1) AQP laden supported (lipid AQP-0 was located almost exclusively in DSMs at an LPR of 1200:1, or polymer) membrane layers (SMLs, see Fig. 3a), and (2) AQP laden whereas ~50% of the protein was located in DRMs at an LPR of 100:1. vesicle encapsulated membranes (VEMs), where AQP laden vesicles In another study the same group analyzed how water permeability of (proteoliposomes or proteo-polymersomes) are immobilized in a bovine lens AQP-0 depends on lipid composition [65]. The estimated sin- dense polymer layer (see Fig. 3b). For SMLs [68–70,72,74,75] the ultra- gle channel water permeability of AQP-0 in SM:chol bilayers was found to thin selective layer facilitates the fast transport of water molecules. Al- be about seven times smaller than for AQP-0 in PC:Phosphatidylglycerol though potentially scalable to large membrane area production (PG) bilayers. This indicates that an important additional factor to consid- [76–78], the low stability is an inherent drawback of lipid bilayer er in designing biomimetic membranes is the membrane composition, SMLs. VEMs [79–85] generally provide a mechanically robust mem- which may modify inherent AQP permeability. These findings are mir- brane. The use of a dense polymer matrix can further minimize defects rored in experiments with rat AQP-4 [66]. Here for each of the two (which enables potential industrial-scale production [12]), and protect AQP-4 isoforms investigated, the single channel osmotic water perme- AQPs from chemical and biological attack. Nevertheless, this approach ability pf strongly depended on bilayer composition and systematically will likely scarify membrane water permeability to some degree (de- decreased with increasing bilayer compressibility modulus and bilayer pending on the property of the matrix among other factors [86]). In ad- thickness. A recent MD study on lipid–AQP interactions found similarities dition, the chemicals used to immobilize the AQP laden vesicles may in the binding orientations and interactions of the lipids. However, there potentially affect the activity of the embedded AQP. The key advantages do not appear to be high-specificity lipid binding sites, but a more broadly and disadvantages of the two basic designs are summarized in Table 1, conserved protein/lipid interface [67]. This is also consistent with the and detailed fabrication methods are presented in Sections 3.1 (SMLs) polymeric reconstitution studies mentioned above demonstrating AQP and 3.2 (VEMs). functionality in non-biological host membranes. 3.1. AQP laden supported membrane layers (SMLs) 3. Structure design and fabrication of AQP incorporated membranes There has been an increasing emphasis on transmembrane protein Continuous and robust AQP incorporated membranes are potential incorporated SMLs, because of their promising applications, such as candidates for practical water purification application. Ideal ABMs aqueous separations and biosensors. Two kinds SMLs have been

Fig. 3. Schematic presentation of AQP laden membranes (a): Supported membrane layer (SML) where AQPs (purple) is embedded in a ﬂat bilayer (orange) deposited onto a porous support (gray). (b): and AQP laden vesicles encapsulated membrane (VEM) where vesicles (orange) are immobilized in a polymer layer (yellow) on a porous support substrate (gray). C. Tang et al. / Desalination 368 (2015) 89–105 95

Fig. 4. Schematic presentation of two biomimetic membranes (green) with reconstituted AQPs (purple) on porous substrates. (a): biomimetic membrane formed directly on a porous alumina (blue cross shaded structure); (b): biomimetic membrane formed on a carboxylated PEG cushion (dark blue) chemisorbed on a 60 nm gold layer (yellow) that was deposited on a porous alumina. Reproduced with permission from Ref. [88]. reported: supported lipid bilayers (SLBs) and supported polymer mem- this work, two kinds of substrates were used. One is porous alumina branes (SPMs). with a pore diameter of about 80 ± 10 nm (Fig. 4a), and the other SLBs are popular model for cell membranes, and are promising for substrate is gold-coated porous alumina which is further modified by future application in biomimetic devices and biosensors, see for exam- carboxyl-PEG-SH by self-assembly of thiol groups (Fig. 4b). The ple [87]. It is a facile and effective way to construct AQP incorporated carboxylated-PEG coated substrate significantly enhances the flexibility biomimetic membranes by mimicking the structure of cell membranes, of the SLB, which resulted in improved mechanical strength of the AQP i.e. incorporating AQP in SLBs. To optimize the performance and develop laden SLB. stable biomimetic membranes, several strategies have been studied, in- In view of the electrical properties of phospholipids and polyelectro- cluding vesicle rupture and fusion of bolaamphiphile micelles. lytes, the layer by layer (LbL) assembly strategy is a facile tool for con- In 2010, Kaufman et al. used commercial nanofiltration (NF) mem- struction of composite materials with precise control over the branes as substrates to fabricate SLB membranes [68]. It is an interesting structure and properties. It has advantages in fabrication of stable and attempt to assemble a continuous lipid bilayer using vesicles rupture mechanically robust AQP laden biomimetic membranes under the elec- approach, although AQPs were not incorporated in the vesicles in that trostatic attraction between the oppositely charged SLB and polyelec- study. Li et al. utilized vesicle rupture facilitated by hydraulic pressure trolytes substrates. Wang et al. fabricated a biomimetic NF membrane on a positively charged lipids spin-coated NF membrane to form the by immobilizing the AQP incorporated SLB with positive charges on AQP laden SLB membrane [69]. Recently, Kaufman et al. reported that LbL complex polyelectrolytes substrates with negative charges, see the formation of SLB is governed by the double-layer interactions be- Fig. 5 [89]. These composite membranes exhibit excellent permeability, tween the lipids and substrate membranes via altering the charge of and salt rejection accompanied with a considerable high mechanical lipid head groups [70]. Moreover, substrate membrane with higher stability. dissociation constant of sulfonic groups favors the assembly of higher Other self-assembled structures than vesicles are able to fuse into coverage SLB. supported bilayer membranes. Kaufman et al. prepared stable SLB Wang et al. fabricated AQP laden SLB membranes on porous alumina membranes via a micelle fusion process [72].AsshowninFig. 6, spher- and carboxylated polyethylene glycol (PEG) cushion substrate [88].In ical and elongated thread-like micelles formed by cationic single-chain bolalipid could fuse, and form a continuous SLB membrane on silica surface due to the electrostatic attraction between the positive charge of bolalipid and negative charge of silica substrate. AQP could be successfully incorporated in the bolalipid SLB membrane in a manner similar to SLB prepared by vesicle rupture method.

Fig. 5. Schematic presentation of the formation procedures for AQP embedded LbL membranes by proteoliposomes rupture. The SBL is established on top of a porous support with polyelectrolyte layers: positively charged polyethylenimine (PEI) (yellow) and negatively charged poly(styrenesulfonate) (PSS) (green). Positively charged N-[1-(2,3- Fig. 6. Schematic presentation of possible curved aggregated structures formed by one-tail dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP) lipids electrostatically anchor bolalipids: spherical or thread-like micelles in solutions that fuse into a membrane on sil- the bilayer to the PSS:PEI cushion. The support (gray) is hydrolyzed polyacrylonitrile ica. The membrane has both bilayer features (U-shaped bolalipids) and monolayer fea- (H-PAN). tures (transmembrane bolalipids). Reproduced with permission from Ref. [89]. Reproduced with permission from Ref. [72]. 96 C. Tang et al. / Desalination 368 (2015) 89–105

ABA block AqpZ-DDM copolymer vesicles mixture

B primary amine residues acrylate residues

Fig. 7. Schematic presentation of the design and fabrication of AQP laden SPMs via proteopolymersome rupture on surface modiﬁed polycarbonate tracked-etched (PCTE) substrates. (a): proteopolymersomes are formed by mixing polymersomes made of ABA triblock copolymers (blue) with AqpZ (green) solubilized in n-Dodecyl-β-D-maltopyranoside (DDM) detergent (red) followed by detergent removal. (b): functionalization of the PCTE support material. (c): deposition and fusion of proteopolymersomes onto the support material. Reproduced with permission from Ref. [90].

Supported polymer membranes (SPMs) can also be used as an AQP SPM on gold coated porous alumina substrates [74]. Zhong et al. host matrix to construct biomimetic membranes [74,90,91]. Wang employed ABA copolymers with methacrylate groups to produce biomi- et al. synthesized AQP laden SPM membranes via proteopolymersomes metic NF membranes upon cellulose acetate membrane substrate func- rupture on modiﬁed polycarbonate tracked-etched (PCTE) substrates tionalized with methacrylate groups by UV polymerization [91]. [90]. Fig. 7 presents the preparation procedure. Gold coated PCTE membranes are deposited with a monolayer of cysteamine through chemisorption. Afterward, the acrylate groups are produced by conjugating 3.2. AQP laden vesicles immobilized membranes (VEMs) acrylic acid to the primary amine residues. Then AQP incorporated ABA triblock copolymer vesicles adsorb on the substrates assisted by The major problems of supported bilayer membranes are the fragile hydraulic pressure. The proteopolymersomes rupture through covalent structure of the ultrathin selective layer, and the defects formed during interaction between the methacrylate groups on ABA copolymer and the bilayer structure formation process. To fabricate a mechanically ro- the acrylate residues on PCTE. This strategy improves the stability of bust and defect-free AQP laden biomimetic membrane, AQP incorporat- the supported bilayer structure membranes. Duong et al. utilized ed vesicles were encapsulated in a dense layer that surrounds and disulﬁde-functionalized ABA triblock copolymer to form AQP laden protects the vesicles.

Fig. 8. Schematic showing the interfacial polymerization synthesis of AQP based biomimetic membranes. (a): A thin ﬁlm composite (TFC) layer is created by interfacial polymerization of 1,3-diaminobenzene (MPD) + proteoliposomes in aqueous solution mixed with trimesoyl chloride (TMC) in an organic phase. The TFC layer w is stabilized on a polysulfone substrate. (b): the ﬂux and rejection properties are determined by the combined TFC + proteoliposome matrix. Reproduced with permission from [80]. C. Tang et al. / Desalination 368 (2015) 89–105 97

Fig. 9. Schematic showing the AQP based biomimetic membrane preparation. Proteoliposomes (orange) with AqpZ (blue) are functionalized with PDA and immobilized on a poly(amide- imide) (PAI) substrate (gray). Then PEI (red) is added and PEI primary amine groups crosslink with PAI creating the ﬁnal membrane. Reproduced with permission from Ref. [81].

The VEM design was conceptualized by Zhao et al. [80]. These au- are immobilized on the substrate by the amine-catechol bonding. The thors introduced AQP incorporated liposomes in a thin film composite resulted membrane is further cross-linked with glutaraldehyde (GA) (TFC) rejection layer by using a modified interfacial polymerization to enhance the stability. method. In brief, a polysulfone substrate is soaked with an m- The LbL assembly technique was successfully used to immobilize phenylene-diamine (MPD) aqueous solution containing proteolipo- proteoliposomes on polyelectrolytes substrate. As discussed above, pro- somes. Subsequently, it is exposed to trimesoyl chloride (TMC) to teoliposomes rupture to form SLB because of the electrostatic attraction form a polyamide layer with proteoliposomes embedded in the thin between phospholipids and substrate. Therefore, protection to proteoli- polyamide matrix layer, see Fig. 8. The resulting membrane has an posomes is needed. Sun et al. encapsulated poly-L-lysine (PLL) covered area greater than 200 cm2 [80], and this method can be easily scaled proteoliposomes within a LbL cationic polyelectrolyte/anionic polyelec- up to produce membrane areas at an industrial scale. trolyte film, as illustrated in Fig. 11 [82]. The LbL approach facilitates the In a recent paper reported by Li et al., they progress to prepare a formation of a well-ordered selective layer encapsulated with proteoli- highly permselective AQP based membrane by polymer crosslinking posomes under non-covalent interactions. [81]. The proteoliposomes are initially decorated with polydopamine Unlike liposomes formed by phospholipids, polymersomes formed (PDA) to enhance their affinity, because of the strong adhesive ability by ABA triblock copolymers possess higher stability, which prevents of PDA via formation of strong non-covalent and covalent bonds with vesicles from rupture during the fabrication and filtration process. Re- the substrate. PDA functionalized proteoliposomes are immobilized on cently, Wang et al. presented a biomimetic membrane formed by AQP a poly(amide-imide) (PAI) substrate, and subsequently covered by incorporated polymersomes. The cross-linked proteopolymersomes polyelectrolyte (PEI) with primary amine groups, which can crosslink with disulfide anchors are covalent bonded upon the gold coated sub- with PAI, see Fig. 9. Similarly, Sun et al. prepared biomimetic mem- strate, then encapsulated by PDA and histidine (His) coating, see branes covered with AQP containing liposomes, see Fig. 10 [79].In Fig. 12 [83]. The design and construction of proteopolymersomes encap- brief, porous membranes are coated with polydopamine (PDA). Meth- sulated membranes not only offers the mechanically robust vesicles, but acrylate monomers are used to crosslink the amine-functionalized pro- also provides effective anchoring sites by the functionalized ABA copol- teoliposomes with ultraviolet (UV) illumination. Then proteoliposomes ymers for immobilization of polymersomes in the dense selective layers,

Fig. 10. Schematic presentation of immobilization of cross-linked proteoliposomes on a PDA coated membrane. Vesicles with AqpZ (orange) are stabilized by UV induced cross-linking of methacrylate (red) and functionalized with amine groups covalently linked to distearoylphosphatidylethamonlamine lipids via a PEG linker (DSPE-PEG-NH2). A porous PAN support (blue) is surface-functionalized by PDA. Vesicles are then immobilized by cross-linking the amine groups to the PDA using glutaraldehyde (GA). Reproduced with permission from Ref. [79]. 98 C. Tang et al. / Desalination 368 (2015) 89–105

Fig. 11. Schematic presentation of the formation procedure for the liposome embedded LbL membrane. Hydrolyzed PAN membranes (yellow) are coated with polyallylamine hydrochloride (PAH) polycation layer, and a mixture of polyacrylic acid (PAA) and polystyrene sulfonate (PSS) was used to form the polyanion layer. The positively charged lysine moieties on the liposomes ensure electrostatic coupling to the polyelectrolyte PAH–PSS/PAA layers. Reproduced with permission from Ref. [82].

which prevents vesicles from being peeled off by water flow. In another For all designs presented here, the main engineering challenge is to work, Xie et al. fabricated an“AQP vesicle-imprinted membrane” by transform them into a robust and cost-effective technology (Table 1). similar strategy, see Fig. 13 [84].Briefly, AQP were reconstituted in While the SLM approach (Fig. 3a) is preferable from a flux point of polymersomes formed by ABA copolymer with hydroxyl groups. Then view (i.e. only one aquaporin passage is needed), there are considerable the cross-linked proteopolymersomes were conjugated on amine func- challenges associated with up-scaling nanometer-thick areas to m2 tionalized cellulose acetate (CA) substrate by amidation reaction. sized robust membranes. The VEM approach (Fig. 3b) is appealing Furthermore, a variety of new strategies have increasingly been from a stability and up-scaling point of view and is indeed being com- developed for the construction of AQP incorporated biomimetic mem- mercialized by the Danish cleantech company Aquaporin A/S together branes. Sun et al. developed a simple method to further improve the with its Singaporean subsidiary Aquaporin Asia Pte. Ltd. Here the main embedding efficiency of AQP incorporated vesicles in the biomimetic challenge is to ensure high effective vesicle density in the TFC layer. membrane by magnetic-aided LbL method [85]. As presented in Finally a major challenge in upscaling any biomimetic membrane is Fig. 14, proteopolymersomes containing magnetic nanoparticles to produce large scale (gram to kilogram) quantities of proteins in a were encapsulated in a multilayer polyelectrolyte membrane with cost-effective manner. Bomholt et al. explored the capacity of yeast Sac- the assistance of a magnet to enrich the loading efficiency of AQP in charomyces cerevisiae as host for heterologous expression of human biomimetic membrane, which could be attributed to deposit more AQP-1 [92]. They found that AQP-1 constituted 8.5% of total membrane proteopolymersomes. protein content after expression at 15 °C. A detergent screen for

Fig. 12. Schematic diagram of AqpZ-embedded vesicular membrane design and synthesis route. (a): reconstitution of dodecyl-b-D-maltoside (DDM) solubilized AqpZ into vesicles formed from ABA block copolymers. (b): pressure-assisted vesicle adsorption and immobilization on the gold-coated PCTE membrane support; (c): self-assembled monolayer (SAM) of cysteamine established on the gold-coated surface through chemisorption. (d): the gold surface not occupied by vesicles is further functionalized with a self-assembled monolayer of cysteamine and PDA and His alternative layer-by-layer coating on the top of the vesicular membrane. Reproduced with permission from Ref. [83]. C. Tang et al. / Desalination 368 (2015) 89–105 99

expression. Cell-free (CF) systems is another approach for protein production, and Müller-Lucks et al. recently presented milligram scale production of AQP-3 using a histidine-tagged protein (hAQP-3-6His) in an E. coli extract-based CF system in the presence of the non-ionic detergent Brij™-98 [94]. For a recent review of systems for membrane protein production [95].

4. Biomimetic membranes performance

Most of the early biomimetic membrane performance tests were conducted under pressure-driven (RO and NF) testing conditions, where a hydraulic pressure in excess of the osmotic pressure of the feed solution is applied to drive water through the target membrane under evaluation. NF and RO processes have a wide range of applications, including desalination, wastewater reclamation, and driven water treatment; for a review, see [96]. On the other hand, osmotically-driven membrane processes such as FO and RO are emerging, which have potential application in water recovery and osmotic energy harvesting, respectively, and biomimetic aquaporin FO membranes have shown excellent rejection properties [97]. In fact a promising strategy would be to take advantage of syn- ergistic effects in combining FO and PRO with RO [2]. As one example biomimetic FO can be seen as a pretreatment for RO allowing for the use of impaired feed streams due to the lower inherent fouling propensity of the FO process besides the good rejection properties (see Fig. 16a). Also, FO can be used to pre-dilute feed seawater to an SWRO process, which reduces the osmotic pressure of the feed and thus the speciﬁc energy consumption of SWRO. Meanwhile, the concentrated brine from SWRO provides a high salinity solution whose osmotic energy can be recovered by PRO (see Fig. 16b).

4.1. Pressure driven processes

Fig. 13. Schemes for the fabrication and water puriﬁcation mechanism of the AQP vesicles Table 2 shows water permeability and salt (NaCl and/or MgCl2)re- imprinted membrane. (a): (1) AqpZ-polymer vesicles, (2) CA membrane substrate, jection of pressure driven biomimetic membranes reported in the (3) AqpZ vesicles immobilized on the porous membrane, (4) AqpZ-vesicle-imprinted existing literature. SMLs showed relatively poor solute rejection under membrane, (5) cross-section of the AqpZ-vesicle- imprinted membrane. (b): Pressure- pressure-driven conditions, revealing the general difﬁculty to achieve driven water transport and solute rejection mechanism. Reproduced with permission from Ref. [84]. robust and defect-free biomimetic membranes with the SML design (Table 1). From Table 2 it appears that VEMs generally offered higher

salt rejection (N60% NaCl rejection and N75% MgCl2). The vesicle solubilization revealed that CYMAL-5 was superior in solubilizing re- embedding matrix acts to minimize defects formation and membrane combinant AQP-1 and generated a monodisperse tetrameric protein with high NaCl rejection (~96% at an applied pressure of 5 bar and preparation. The expression level is approximately fourteen times 98% at 10 bar) can be achieved [80]. Further studies by the same higher than in a previous study based on the Pichia pastoris expression group demonstrated that the proteoliposome embedded TFC system [93] indicating the power of S. cerevisiae based heterologous

Fig. 14. Schematic representation of the fabrication process for the magnetic-aided LbL membrane. PAH deposition onto the negatively charged H-PAN substrate (orange) formed a polycation layer (blue). This is followed by deposition of a PAA-PSS mixture to form a polyanion layer (red). Next, PLL-functionalized proteopolymersomes with encapsulated magnetic nanoparticles are deposited onto the polyanion layer, driven by a magnetic Fig. 15. Pilot-production facility for making ﬂat sheet AQP membranes based on the ap- ﬁeld. Finally another PSS/PAA layer is added to ensure the stabilization of the membrane. proach depicted in Fig. 8. Reproduced with permission from Ref. [85]. Photo courtesy: Aquaporin A/S. 100 C. Tang et al. / Desalination 368 (2015) 89–105

Fig. 16. Examples of RO combined with FO and PRO in water production. (a): FO as pre-treatment to the RO process. Here a suitable draw solute is recirculated in the RO step with the feed water being osmotically drawn in across the FO membrane. (b): Combined RO/FO/PRO process where FO pre-dilutes feed seawater to an SWRO process, which reduces the osmotic pressure (energy consumption) during SWRO. Further, the concentrated SWRO brine provides a high salinity solution whose osmotic energy can be recovered from PRO. membranes prepared in this way was able to withstand the high pres- where R is the fractional salt rejection (thus R = 1 corresponds to per- sure (50 bar) that is required for SWRO desalination applications [98]. fect rejection) and J is the water flux (in LMH). Nevertheless, as the dense polyamide matrix played a significant role Fig. 17 shows the combination of water permeability and solute in determining the overall membrane permeability, the available permeability of biomimetic membranes as well as the state-of-the-art water permeability of the resulting composite membranes was some- commercial RO and NF membranes. Membranes with a higher water what limited (~4 LMH/bar), but still about 30% above the bench value permeability and lower solute permeability (data points on the lower of the commercial BW30 membrane. The AqpZ-DOPC VEM from [81] right corner in the figure) suggest better separation performance and and AqpZ-ABA VEM from [84] both had excellent water permeability are thus preferred. From Fig. 17, we can observe a strong tradeoff rela- (36.6 LMH/bar and 22.9 LMH/bar, respectively) and good (~95%) tionship between water permeability and salt permeability for commer-

MgCl2 rejection. This makes these membranes good candidates for NF cial membrane — the more water permeable commercial membranes membranes, making them potentially applicable to both SWRO and also have higher salt permeability (with the trend indicated by the dot- brackish water RO applications. ted line). Compared to commercial membranes, several AQP biomimet- To further access the current development status, the biomimetic ic membranes fall below the tradeoff line, strongly suggesting the great membranes shown in Table 2 are benchmarked against existing com- potential for AQPs, as suitable water permeability enhancers without mercial RO and NF membranes. It shall be noted that salt rejection is adversely affecting solute permeability. not an intrinsic property of a dense membrane, since its value is greatly dependent on the applied pressure (and thus the water flux) [96].For 4.2. Osmotically driven processes this reason, the solute permeability together with water permeability is used for comparison purpose (Fig. 16). The permeability coefficient Table 3 shows the FO performance of some biomimetic membranes B (in units of l m−2 h−1 or LMH) can be defined as: reported in the literature. In general, decent FO water flux of ~20 LMH (and as high as N80 LMH in some cases) are reported in the literature. B ¼ ðÞ1=R−1 J ð6Þ The reported FO water flux and solute flux are comparable or even

Table 2 RO and NF performance of biomimetic membranes.

Approaches RO/NF performance Testing conditions Membrane area (cm2) Remark

SMLs AqpZ-DOPC [69] 3.6 LMH/bar, 1 mM NaCl @1 bar 28.3 DOTAP coated NF270, pressure/charge 20% NaCl rejection assisted sorption AqpZ-ABA [91] 34.2 LMH/bar (pure water), 32.9% NaCl 200 ppm NaCl @5 bar 0.071 Silanized CA substrate, vacuum and UV rejection crosslinking AqpZ-ABA [74] 8.2 LMH/bar (pure water), 45.1% NaCl 200 ppm NaCl @5 bar 0.20 Gold coated porous alumina substrate, rejection disulﬁde crosslinking for enhanced spreading (rupture) of vesicles AqpZ-DOPC/DOTAP [89] 5.5 LMH/bar, 75% NaCl rejection 500 ppm NaCl @4 bar 19.56 DOPC/DOTAP SLBs on PSS/PEI/PAN substrate

VEMs AqpZ-DOPC [80] 4 LMH/bar, 96% NaCl rejection @5 bar 10 mM NaCl @5 bar N200 (highly scalable) TFC composite VEM. Vesicles embedded in (98% rejection @10 bar) (max. testing pressure 10 bar) interfacially polymerized PA layer AqpZ-DOPC [98] 2 LMH/bar, 94% NaCl rejection 10 mM NaCl @ 50 bar N200 (highly scalable) TFC composite VEM. Vesicles embedded in interfacially polymerized PA layer

AqpZ-DOPC [79] 3.8 LMH/bar, 66.2% NaCl rejection, 200 ppm NaCl/MgCl2 @5 bar 0.785 PDA treated PAN substrate, pressure assisted

88.1% MgCl2 rejection sorption, crosslinking between amine functionalized vesicle and PDA

AqpZ-POPC/ ~6 LMH/bar, ~96% MgCl2 rejection 200 ppm MgCl2 @4 bar 0.785 PAN substrate, vesicles embedded in LbL POPG/cholesterol [82] assembly layer

AqpZ-DOPC [81] 36.6 LMH/bar, 85% MgCl2 rejection 100 ppm MgCl2, 1 bar 28.3 PAI substrate, PDA coated vesicles embedded in crosslinked PEI matrix

AqpZ-ABA [84] 22.9 LMH/bar, 61% NaCl rejection, 200 ppm NaCl/MgCl2 @5 bar 0.196 Amine functionalized CA, crosslinking

75% MgCl2 rejection AqpZ-ABA [83] 4.3 LMH/bar, 65.8% NaCl rejection Tested at 1 bar 0.196 Tracked-etched polycarbonate substrates, coated with gold and cyteamine, pressure assisted sorption, UV crosslinking C. Tang et al. / Desalination 368 (2015) 89–105 101

help further to protect the protein against degradation (e.g. in the form of protease activity). This, taken together with the membrane performance data discussed above, indeed points to the use of aquaporin proteins as a viable strategy for making UPMs. Still, the omnipresent issue of membrane fouling must be consid- ered, and long-term operation with minimal fouling and cleaning is a prerequisite for cost-effective operation [101–103]. More specifically membrane fouling is deposition of retained matter (particles, colloids, macromolecules salts, etc.) on the membrane surface, or inside the porous membrane material and can generally be classified into four groups: (1) organic fouling, (2) inorganic (scaling), (3) biofouling, and (4) colloidal/particulate fouling [104]. Membrane fouling is impacted by various factors ranging from hydrodynamic operating conditions to physical and chemical interactions between foulants and the membrane. No systematic studies of aquaporin membrane fouling have yet – Fig. 17. Benchmarking of biomimetic membranes. B versus J (as defined by Eq. (6)) for been conducted but in general pressure-driven (and possibly also os- SMLs (■) and VEMs (♦) and commercial RO and NF membranes (○) for NaCl and VEMs motically driven) UPMs are likely to be more prone to fouling due to the ▲ for MgCl2 ( ) Membranes with a higher water permeability J and lower solute permeabil- increased mass transfer and ensuing interfacial polarization. ity B (data points in the lower right corner) indicate better separation performance. In controlling membrane fouling membrane surface charge plays a Separation properties of commercial membranes are obtained from Refs. [8,96]. key role [105]. Generally heterogeneous systems consisting of solid material in liquid medium the phase interface exhibits an electrical charge. Experimental determination of membrane effective charge density can be carried out analytically by equilibrating the membrane sequentially superior to commercial FO membranes [99]. It is important to note that in acid and salt solution or indirectly by electrokinetic methods, such FO water flux has a strong dependent on membrane substrate pore as streaming potential measurements, electrophoresis or electro- structure, in addition to its selective layer separation properties [99]. osmosis. A useful tool is here to monitor Zeta potentials (calculated Also, most of the existing FO tests on biomimetic membranes were per- from streaming potentials), as a function of pH [106]. formed for very small membrane areas (b1cm2). Therefore, further The challenge in mitigation is that fouling depends on the specific study is still needed to verify FO performance of large area biomimetic membrane application: organic fouling will behave differently from membranes and with more realistic support structure and testing con- particulate fouling, and yet again differently from biological fouling. In- ditions. Furthermore, the long term performance (fouling, biological terestingly, some level of bio-fouling may be helpful to maintain long- stability, etc.) is yet to be investigated. term stable solute rejection albeit with lower water flux [107].One strategy for mitigating fouling is based on the use of polymeric building 4.3. Operational considerations blocks for anti-fouling graft-components, and it appears that having H- bond acceptors, polar groups, and being electro-neutral seem to imbue For all membrane applications overall mechanical stability is an ob- anti-fouling properties in general [108]. Based on the approaches for de- vious requirement. Both the VEM and the SML approaches depend on a signing AQP biomimetic membranes reviewed here (all of which relies good supporting structure. Currently the VEM approach seems the most on an intricate interplay between polymers and AQP proteins) it ap- promising in terms of delivering robust membranes suitable for pears that integrating functional properties presents a viable approach upscaling and use in industrial applications, see Table 1. The robustness to mitigate fouling in these systems. Thus future AQP membrane devel- of the key component – the aquaporin protein – is of course vital for opments may benefit from integrating this in parallel with optimizing aquaporin membrane function. As discussed in Section 2.5 some aqua- other parameters such as flux, rejection, and mechanical robustness – porin protein isoforms are in fact very stable and the VEM design may keeping the final application in mind.

Table 3 FO performance of biomimetic membranes.

Approaches FO performance Testing conditions Membrane area (cm2) Remark

SMLs AqpZ-ABA [90] 16.4 LMH, 90% NaCl rejection 0.3 M sucrose as draw solution, 0.096 Tracked-etched polycarbonate 200 ppm as feed solution substrates, coated with gold and cyteamine, pressure assisted sorption, UV crosslinking

VEMs AqpZ-POPC/ 21.8 LMH, salt ﬂux 2.4 g/m2/h 0.3 M sucrose as draw solution and 0.785 PAN substrate, magnetic enhanced 2 POPG/Cholesterol [85] 83.5 LMH, reverse salt ﬂux ~30 g/m /h 200 ppm MgCl2 as feed solution vesicle incorporation

1.5 M MgCl2 draw solution and ultrapure water as feed AqpZ-ABA [84] 5.6 LMH, 50.7% NaCl rejection 0.3 M sucrose as draw solution and 0.196 Amine functionalized CA, crosslinking 200 ppm NaCl as feed solution AqpZ-ABA [83] 17.6 LMH and 91.8% NaCl rejection 0.8 M sucrose as draw solution and 0.196 Tracked-etched polycarbonate 43.6 LMH, reverse salt ﬂux 8.9 g/m2/h 6000 ppm NaCl feed substrates, coated with gold and 0.5 M NaCl draw solution and cyteamine, pressure assisted sorption, ultrapure water as feed UV crosslinking AIM™ [100] 9 LMH, reverse salt ﬂux 1 M NaCl draw solution and ultrapure 140 (highly scalable) TFC composite VEM. Vesicles 1.9 g/m2/h water as feed embedded in interfacially polymerized PA layer 102 C. Tang et al. / Desalination 368 (2015) 89–105

Table 4 List of published patents related to aquaporin membranes.

Publication Publication No Applicant and country code Title Technology date

28 Aug 2014 WO2014/128293 Aquaporin A/S [DK] Systems for water extraction Water extraction systems using forward osmosis and a LINK biomimetic membrane comprising aquaporin water channels, e.g. for fertigation and up-concentration of organic solutes 17 Jul 2014 WO2014/108827 Aquaporin A/S [DK] A hollow fiber module having Hollow fibers modified with a thin film composite layer LINK TFC-Aquaporin modified membranes comprising aquaporin water channels 26 Jun 2014 WO2014/100412 Robert McGinnis [US] Selective membranes formed by alignment Alignment and orientation of porous materials LINK c/o Nagare Membranes LLC of porous materials (e.g. aquaporin pores) in the membrane surface [US] 15 May 2014 WO2014/075086 Nagare Membranes Llc [US] Methods for reducing ion exchange and Reducing unwanted ion flux effects by modification of the LINK reverse salt flux phenomena in membranes surface charge. for osmotically driven membrane processes The membranes may comprise aquaporins or biomimetic synthetic water selective porous material meant to replicate aquaporin protein function. 16 Apr 2014 CN103721572 Ocean University of China [CN] Preparation method of phospholipid Aquaporin containing vesicles are spread on a LINK biomimetic membrane containing layer-by-layer composite membrane aquaporins 13 Mar 2014 WO2014/039493 Lauren Sciences LLC [US] Bolaamphiphilic compounds, compositions Supported biomimetic membranes with amphiphiles LINK and uses thereof compounds useful for incorporation of transmembrane proteins, e.g. aquaporin, and platforms for e.g. water purification and as biosensors 20 Feb 2014 WO2014/028923 The Penn State Research [US] High density membrane protein membranes A block copolymer-protein membrane with a high density LINK & President and Fellows of of protein, e.g. aquaporin, prepared by slow controlled Harvard College [US] removal of the detergent 15 Dec 2013 CN103463997 Ocean University of China [CN] Composite membrane containing aquaporin A membrane produced by auto polymerization of LINK and manufacturing method thereof dopamine on support followed aquaporins in a phosphatidylethanolamine formulation 30 May 2013 WO2013/078464 Znano LLC [US] Self-assembled surfactant structures A membrane with a stabilized surfactant mesostructure LINK bonded to a surface of a porous support, where the mesostructure is derived from a sol–gel precursor 28 Mar 2013 WO2013/043118 Nanyang Technological Aquaporin based thin film composite A thin film composite membrane having incorporated LINK University [SG] & Aquaporin membranes aquaporin containing amphiphilic vesicles A/S [DK] 29 Nov 2012 WO2012/161662 National University of Pore-spanning biomimetic membranes A biomimetic membrane having a metal coating on the LINK Singapore [SG] embedded with aquaporin surface of the porous substrate 21 Jun 2012 WO2012/080946 Aquaporin A/S [DK] A liquid membrane suitable for water An aquaporin containing bulk liquid membrane matrix LINK extraction 12 Jan 2012 WO2012/004357 Hydrogene Lund AB [SE] Membrane comprising constitutively open A membrane with constitutively open aquaporins to LINK aquaporins provide improved water purification capabilities 24 Nov 2011 WO2011/146936 Adrian Brozell [US] Self-assembled surfactant structures A membrane with a stabilized surfactant mesostructure LINK bonded to a surface of a porous support 13 Apr 2011 US2011/0084026 B G Negev Technologies Ltd Biomimetic membranes, their production A membrane with a lipid bilayer on a dense support layer LINK [IL] and uses thereof in water purification with aquaporins embedded in the bilayer 23 Dec 2010 WO2010/146365 Aquaporin A/S [DK] Biomimetic membranes and uses thereof A liquid membrane system based on vesicles formed from LINK amphiphilic compounds 23 Dec 2010 WO2010/146366 Aquaporin A/S [DK] Assays relating to biomimetic membranes Determining whether the membrane protein, e.g. LINK and their uses aquaporin, is correctly folded in a biomimetic membrane by detecting a fluorescent signal 1 Nov 2010 KR20100116344 LG Electronics Inc [KR] A water purification filter and method for Aquaporins in a layer formed by MPD and TMC LINK fabricating the same cross-linking 27 Oct 2010 EP2243746 LG Electronics Inc [KR] Water purifying filter and method for Aquaporin vesicles or bilayers arranged in a polymer LINK fabricating the same support layer with holes. 12 Aug 2010 WO2010/091078 Danfoss AquaZ, now Applied Nanofabricated membrane using Proteoliposomes having a UV-cross-linkable chemical LINK Biomimetic A/S [DK] polymerized proteoliposomes structure included in the hydrophobic tail of the lipid 15 Apr 2010 WO2010/040353 Danfoss AquaZ, now Applied Biomimetic membrane formed from a Membranes formed by cross-linking conjugates of LINK Biomimetic A/S [US] vesicle-tread conjugate cellulose-threads and aquaporin-containing vesicles 18 Jun 2009 WO2009/074155 Aquaporin A/S [DK] Scaffold for composite biomimetic A planar membrane scaffold with apertures for preparing LINK membrane biomimetic membranes with aquaporins 18 Jun 2009 WO2009/076174 The Board of Trustees of the Highly permeable polymer membranes A flat membrane being an aquaporin-containing film of LINK University of Illinois [US] cross-linkable block copolymer 8 Jan 2009 WO2009/003936 Université Joseph Biomimetic artificial membrane device A device for production of electrochemical energy having a LINK Fourier - Grenoble 1 [FR] biomimetic membrane with a transport protein, e.g. aquaporin 29 Mar 2007 WO2007/033675 Aquaporin Aps, now Biomimetic water membrane comprising Aquaporin containing membrane for osmotic power LINK Aquaporin A/S [DK] aquaporins used in the production of salinity production power 23 Nov 2006 WO2006/122566 Aquaporin Aps, now Membrane for filtering of water Aquaporins reconstituted in lipid vesicles and a membrane LINK Aquaporin A/S [DK] having lipid bilayers in which aquaporins are incorporated 5 Feb 2004 WO2004/011600 MT Technologies Inc [US], now Biomimetic membranes Membrane proteins, e.g. aquaporins, are incorporated in a LINK Applied Biomimetic A/S [DK] block copolymer matrix C. Tang et al. / Desalination 368 (2015) 89–105 103

5. The current biomimetic aquaporin membrane patent landscape

The recent surge in biomimetic membrane research and development reviewed in this paper is also reﬂected in a growing number of patent applications. Since 2004 almost 30 patent applications have been published related to the use of aquaporins in membrane technology. Table 4 lists aquaporin membrane patent publications as of April 2015. The publications are found by searches performed in the patent databases Patentscope, Espacenet and Google Patents. Table 4 presents the publication date, publication number, applicant (by the time of publication), title, and a short description of the core technology presented in the publication. Where the same technology is described in several patent applications (e.g. an international, a European, a US, or a Chinese application), only the earliest publication of each patent family is listed. Fig. 18. Approximated cost in USD per liter treated water in selected market segments The web-link under each publication number refers to the Espacenet [109]. description.

6. Conclusion and prospects All design approaches presented in this review emerge from small- scale laboratory experiments, where the membrane areas are in the When reviewing the last years AQP biomimetic membrane papers, it square micron or square centimeter range. So far the only up-scaled is evident that this field is rapidly advancing and that biomimetic mem- AQP membrane commercially available is the Aquaporin Inside™ membranes clearly are coming of age. It is also obvious that more work is brane from Aquaporin A/S, see Figs. 8 and 15. Still in order to be a tech- needed in order to fully explore the technological potential of AQP pro- nology alternative to existing membranes, AQP membranes will have to teins. In particular the issues of cost, scalability, and robustness remain. be incorporated in modules where hydrodynamic conditions needs to These issues must be seen and addressed as a whole, in order to secure be optimized. Modules for UPM will have to be developed to be able competitiveness with respect to specific application demands. to deal with the ensuing low mass transfer resistance. Historically in Many of the approaches for designing AQP membranes involve poly- module design the overall mass transfer resistance has been dominated meric materials, and although not all of these are currently available in by membrane resistance. With the emergence of UPMs with a much kilogram let alone ton quantities, it is conceivable that they will be once smaller mass transfer resistance, one also must address the resistances the market need has been established. The only – but vital – part of AQP in the feed and permeate streams as well. While established standard membranes not yet a commercial commodity is the AQP protein itself. form factors exist for RO membranes (e.g. spiral wound modules), Production of AQP proteins (and any other membrane protein) is a there is no widely accepted FO module standard yet, and further work non-trivial task, as the amphiphilic properties, intracellular transport on hydrodynamic FO module designs are clearly needed [110,111]. and modification pathways, and the potentially detrimental effects on Some application areas such as FO, which could act as pretreatment the production host are significant challenges in heterologous overex- to RO, may be particularly interesting for the introduction of AQP mem- pression of membrane proteins. Recent progress using yeast-based sys- branes. FO membranes are operated in the absence of applied pressure, tems may point the way to overcome some of these limitations. Also CF and the issue of robustness may be less severe than for pressure driven expression systems may be a way to diminish or eliminate toxic or in- processes exemplified by RO desalination. However, any AQP mem- hibitory effects of the recombinant proteins on the host cell physiology, brane for FO still faces the same challenges as any other FO membrane and to avoid issues with having complicated transportation or translo- in terms of minimizing concentration polarization issues. Any encapsu- cation systems for the synthesized proteins. Still there are challenges as- lation of a b5 nm thick biomimetic AQP containing membrane may po- sociated with CF such as the DNA degradation by endogenous nucleases tentially impede the osmotic gradient needed to drive the FO process. in the cell extract. Finally, an intriguing aspect of AQP based biomimetic is that AQPs in However, no matter how the membrane proteins are produced, they nature in fact do perform FO (and not RO) transport of water Thus, must be solubilized (detergent stabilized) and concentrated to the while AQPs can be seen as the immediate bottom-up inspiration for de- needed purity. Detergents used in downstream processing (DSP) carry signing ‘rejection layers’ for UPMs, integrated organs such as the kidney from fairly low to very high costs – and the match between detergent may provide top-down inspiration with respect to membrane operation and protein must be optimized for each protein isoform. Also DSP and module design. often involves the use of ultracentrifugation, and other unit operations that are not (yet) compatible with ton-scale production. All these chal- Acknowledgments lenges must be addressed in order to secure cost-effectiveness in the production of AQP for biomimetic membrane technology. We thank our colleagues for the valuable discussions and Intellectual The ability to scale up AQP membranes is closely related to the cost of Property Manager Michael Abildgren, Aquaporin A/S for assistance in – the goods used to produce the membranes and the need to scale up compiling Table 4. We also thank the reviewers for their constructive production of AQP membranes (and reduce cost) is related to the appli- comments. CHN was supported by IBISS: Industrial Biomimetics for cation. Fig. 18 shows the approximate cost for treating water in selected Sensing and Separation, a platform funded by the Danish National Ad- applications. It is evident that for seawater desalination the AQP vanced Technology Foundation (97-2012-4). membrane cost and scale (i.e. membrane area) must be comparable with existing RO based technology. 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