1

Supporting Information

Ultrafast Ion Sieving from Honeycomb-like

Polyamide Membranes Formed Using Porous

Protein Assemblies

Liangliang Gui, ‡§† Jinchen Dong, ‡† Wangxi Fang, ‡* Shenxiang Zhang, Kun Zhou, ‡*

Yuzhang Zhu,‡ Yatao Zhang, and Jian Jin‡§*

‡i-Lab and CAS Key Laboratory of Nano-Bio Interface, CAS Center for Excellence in

Nanoscience, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of

Sciences, Suzhou, 215123, China.

§School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210,

China.

School of Chemical Engineering and Energy, Zhengzhou University, Zhengzhou, 450001,

China

†These authors contributed equally

*Address correspondence to: wxfang2017@sinano.ac.cn; kzhou2011@sinano.ac.cn;

jjin2009@sinano.ac.cn.

2

METHODS

Materials. Trimesoyl choride (TMC), piperazine (PIP), BSA and humic acid (HA)were

purchased from Aladdin Co., Ltd. (Shanghai, China). Polyethylene glycol (PEG, Mw=200, 400,

600 and 1000 Da), Potassium dihydrogen phosphate and disodium hydrogen phosphate are

provided by Sinopharm Chemical Reagent (Shanghai, China). Mixed cellulose esters (MCE)

microfiltration membranes (pore size: 0.45μm) were provided by Yibo Co., Ltd. (Haining,

Zhejiang province, China). Commercial ultrafiltration membrane PES-50000 was purchased

from Microdyn-Nadir. AAO (pore size: 0.45μm) were obtained from Whatman plc. Graphene

oxide (GO) nanosheets were obtained from Qingdao Henglide Graphite Co., Ltd, and reduced

graphene oxide (rGO) nanosheets were converted from GO by chemical reduction using

hydrazine as the reducing agent.

Preparation of the TMV coat protein variants. In this paper, T103C-TMV-4his was based

on a 103rd cysteine mutation and four histidine residues insert that extended from the carboxy

terminus of the wild type TMV coat protein (WT-TMVP-CP). T103C-TMV-4his contains a

cysteine mutation at 103 site of TMV coat protein to form S-S bond in the cavity, which has

been verified to dramatically improve the assembly efficiency and structural stability of the

TMV coat protein in our early work [1-3]. The first mutagenesis of TMV coat protein variants

was made by routine polymerase chain reaction (PCR) to form T103C-TMV using two pairs

of primers (P1: 5′-AGATATACATATGAGCTATAGCAT-3′ and P2: 5′-

GTTTCCGCGGTACACGGATTGGCCT-3′; P3: 5′-AGGC

CAATCCGTGTACCGCGGAAAC-3′ and P4: 5′-GGAATTCT- CAGGTCG CCGGGCC-3′).

The second mutagenesis of TMV coat protein variants was made by routine (PCR) to form

T103C-TMV-4his coat protein based on T103C-TMV using one pair of primers (P5: 5'-

GAGATATACATATGAGCTATAGCAT-3' and P6: 5'-GGA

ATTCTCAATGATGATGATGGGTCGCCGGGCCG-3'). The gene of the T103C-TMV-4his

3

mutant was then cloned into the pET32a(+) vector (Novagen) at NdeI-EcoRI restriction sites

using T4 DNA ligase. The construct was verified by sequencing (Shanghai Invitrogen

Biotechnology Co., Ltd.) and then transformed into E. coli BL21 (DE3) strain (Novagen).

Expression of the T103C-TMV-4his subunits. As a general procedure, E. coli BL21 (DE3)

containing the desired expression plasmid was cultured in Luria−Bertani medium containing

100 μg/mL ampicillin at 37 °C with constant shaking at 200 rpm. When cultures reached an

optical density of 0.5 to 0.7 determined by OD at 600 nm, isopropyl β-D-1-

thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. Then the cultures

were grown for 8~12 h at 27 °C, harvested by centrifugation, and stored at −80°C for further

purification. Cells from a 500 mL culture were thawed, resuspended in 40 mL lysis buffer (20

mM Tris-HCl, 20 mM NaCl, 20 mM EDTA, pH 7.2) containing 15 mM dithiothreitol (DTT)

and then lysed by sonication for 1 h. The resulting lysate was cleared by centrifugation for 30

min at 12000 rpm. The cleared lysate was first purified by ammonium sulfate fractionation.

Then the T103C-TMV-4his precipitate was resuspended in phosphate buffer (PB) (100 mM,

pH 9.5) containing 20 mM DTT, followed by dialyzing against Bis-Tris buffer (20 mM, pH

6.6). The resulting protein solution was applied to a strong anion exchange column (HiTraP Q

HP, GE Healthcare) on an AKTA prime plus FPLC system (GE Healthcare) and eluted with a

0−800 mM NaCl gradient. The elution buffer containing target protein was dialyzed against

PB (100 mM, pH 9.5) and added with 20 mM DTT to prevent thiol oxidation. the T103C-TMV-

4his subunits was stored at 4 °C.

Assembly and purification of T103C-TMV-4his disks. For in vitro self-assembly in certain

conditions, the resulting T103C-TMV-4his subunit solution was dialyzed against phosphate

buffer (PB) (50 mM, pH 8.0) for 1 d at RT to form disks. Then, sucrose density gradient

centrifugation (SDGC) was employed to separate discrete disks and from the other assemblies,

such as TMVCP oligomers, short TMV rod. Briefly, the samples were loaded onto a 10–50%

4

sucrose density gradient, and then centrifuged at 38000 rpm min-1 for 3.5 h. Gradient was

collected at the third fraction of SDGC tube which was averagely divided into ten fractions

from top to bottom, and dialyzed against PB (50 mM, pH 8.0) to remove sucrose. The T103C-

TMV-4his disks were stored at 4 °C.

Preparation of single-layer T103C-TMV-4his nanosheets. A piece of prepared Cu(OH)2

nanowire-haired mesh was initially immersed into the 5 mg/mL T103C-TMV-4his disks

solution for overnight at 4 °C. Macroscopic crystal sheet on the surface of mesh was peeled

by gently shaking in the PB (5 mM, pH 8.0). The crystal sheets were further subjected to mild

ultrasonic processing until the solution becomes crystalline. The solution was centrifuged at

800 g min-1 for 5 min. The supernatant solution was collected and further centrifuged at 3500

g min-1 for 5 min. Finally, the supernatant was collected as small TMV nanosheet and the

precipitate was dispersed in PB (5 mM, pH 8.0) as large TMV nanosheet. Both TMV

nanosheets were stores at 4 °C and subjected to TEM examination.

Modification of Au nanoparticles in TMV nanosheet. T103C-TMV-4his sheets were mixed

with excess 3.5 nm BSPP-AuNPs in PB (5 mM, pH 7.0). Then the mixture was further shaken

at room temperature for 2 h. After that, the solution was centrifuged at 5000 rpm min-1 for 5

min and then the supernatant was carefully removed and the assembly produce was dispersed

in ddH2O.

Preparation of TFC nanofiltration membrane. The detailed procedure of the fabrication of

polyamide nanofiltration membrane was reported in our previous work. Specifically, a certain

amount of TMV nanosheets dispersion was diluted with water and vacuum-filtrated onto the

MCE microfiltration membrane at 0.01 MPa vacuum pressure. Then, interfacial polymerization

was conducted at 25 °C and relative humidity of about 60%. Typically, a piece of TMV

nanosheets modified MCE membrane was placed on the surface of a glass plate where a few

drops of PIP PB buffer solution (∼0.5 ml) was preloaded. 2 ml of PB (PH=7) buffer solution

5

containing 5 mg/ml PIP was dripped onto the membrane and stood for 30 s. The excess PIP

solution was drained by placing the glass plate vertically until no obvious water spot can be

observed on the membrane surface. 2 ml hexane containing 2 mg/ml TMC was then dropped

onto the above wetted membrane where the edge of the membrane was sealed by a filter cup

and stood for 30 s. After removing the excess TMC solution, the membrane was immersed into

hexane for 30 s to remove unreacted TMC and then preserved at room temperature for 30 min.

The resulting membrane was stored in water at 4 °C prior to further usage.

Membrane performance test. The separation performance of the TFC membrane was tested

according to the methodology reported in our early work. A crossflow nanofiltration apparatus

with a separation area of 7.1 cm2 was used to test the membrane performance at room

temperature. Rejection (R) was calculated from conductivity of feed and permeate solutions

using Eq. (1), where Cf and Cp represent conductivity of feed and permeate solution,

respectively.

𝑅 = (1 −𝐶𝑝

𝐶𝑓) × 100% (1)

The selectivity factor between two different species are calculated in Eq. (2), where R1 and R2

are rejections of the two species achieved from the same membrane by applying identical

concentration of the species in the respective feed solutions.

𝑆𝑒𝑙𝑒𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝐹𝑎𝑐𝑡𝑜𝑟 =1−𝑅1

1−𝑅2 (2)

Flux J is determined by Eq. (3), where Δw is the weight increase of permeate during filtration

time Δt, A is the effective separation area of the cell, ρ is the density of permeate.

𝐽 =∆𝑤

𝜌𝐴∆𝑡 (3)

Here, water permeance is defined as flux per unit applied pressure, see in Eq. (4), where ΔP is

the applied transmembrane pressure for the nanofiltration experiment.

6

𝑃𝑒𝑟𝑚𝑒𝑎𝑛𝑐𝑒 =𝐽

∆𝑃 (4)

The flux Jcalculated is defined in Eq. (5), where Ppure is pure water permeance, ΔP is the applied

transmembrane pressure, and Δπ is salt osmotic pressure.

𝐽𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 = 𝑃𝑝𝑢𝑟𝑒(Δ𝑃 − Δ𝜋) (5)

Characterization. SEM images were taken from a Hitachi S4800 cold field emission scanning

electron microscopy. TEM images were acquired from a FEI Tecnai G2 F20 S-TWIN 200KV

field-emission transmission electron microscopy. AFM images were obtained in tapping mode

of a Bruker Dimension Icon atomic force microscopy. Surface zeta potential was tested on an

Anton Paar Surpass solid surface analysis equipment. Water CA was measured on a Data-

Physics OCA 20 setup with 2μl water droplet. Total organic carbon (TOC) was detected by OI

Analytical Aurora Model 1030. The effective membrane pore size was determined via the

separation of polyethylene glycol (PEG) molecules with different molecular weights (200, 400,

600, and 1000 Da, respectively). 200 ppm PEG aqueous solutions were used as feed solutions

at applied pressure of 2 bar. Rejection was calculated from TOC contents of feed and filtrate

solutions, respectively. MWCO was obtained according to the molecular weight where the

rejection is 90%. Stokes radius of PEG can be calculated according to its average molecular

weight based on the following equation (Eq. S1):

𝑟𝑝 = 16.73 × 10−12 × 𝑀0.557 (5)

where M is the molecular weight, and rp is the Stokes radius.

7

1. Morphology of MCE microfiltration membrane with or without TMV nanosheets

Figure S1 is AFM images of MCE microfiltration membrane before and after deposited

with TMV nanosheets in its cavities. After deposited with TMV nanosheets, the root mean

square (RMS) roughness of MCE microfiltration membrane decreased from 596 nm to 260 nm.

Figure S1. AFM images of MCE microfiltration membrane (a) before and (b) after deposited

with TMV nanosheets in its cavities.

2. Honeycomb-like structure of polyamide selective layer

After soaking the TFC nanofiltration membrane fabricated from small TMV nanosheets in

DMF solvent to remove the MCE support, we assessed the morphology of freestanding

polyamide thin film by TEM, indicating a novel morphology with 3D honeycomb-liked spatial

structure formed.

Figure S2. TEM image of freestanding polyamide thin film (PA-1) after removing the MCE

porous support membrane.

3. Polyamide membranes fabricated from original MCE membrane support without

TMV nanosheets

8

To investigate how the TMV nanosheets influence the polyamide layer formation and the

corresponding structure and separation performance, we started by conducting interfacial

polymerization on the original MCE microfiltration membrane directly. Although the

polyamide layer is formed on the sponge-like MCE porous support surface (Figure S3a), a

rather flat freestanding polyamide thin film (denoted as PA-0) is obtained after removing the

MCE support (Figure S3b). It reveals that the MCE support itself is not responsible for the

formation of 3D honeycomb-liked structured polyamide layer, but the existence of TMV

nanosheets interferes the interfacial polymerization process and results in the construction of

such layer morphology, which explains why the Figure 2b and Figure 2c present similar

morphology. TMV nanosheets in cavities of MCE microfiltration membrane form a continue

tortuous protein layer which can localize the water-oil interface with evenly distributed

monomers for interfacial polymerization. During interfacial polymerization, a uniform thin

polyamide selective layer forms along the surface of TMV nanosheets, which contributes to

the similar surface morphology of TMV nanosheets modified MCE microfiltration membrane

(Figure 2b) and corresponding polyamide membrane after (Figure 2c) interfacial

polymerization.

Figure S3. (a) Surface SEM image of TFC nanofiltration membrane fabricated without TMV

nanosheets (PA-0/MCE). (b) SEM image of freestanding polyamide thin film (PA-0) after

removing the MCE porous support.

9

In terms of separation performance, with the involvement of TMV nanosheets, both water

permeance and Na2SO4 rejection of the polyamide membrane (PA-1/MCE) increase from 18 L

m-2 h-1 bar-1 and 82.1% to 62 L m-2 h-1 bar-1 and to 99.0% respectively (Figure S4). Meanwhile,

according to XPS analysis results on the surface chemical composition of PA-1/MCE as well

as the control PA-0/MCE membrane listed in Table S1, the O/N ratio of PA-0/MCE and PA-

1/MCE membranes are 3.33 and 2.21, respectively, indicating higher cross-linking degree of

the PA-1/MCE polyamide membrane. The enhancement of water permeance could be

attributed to increased effective filtration area of the polyamide selective layer, while the

improved salt rejection and crosslinking degree of the PA-1/MCE membrane suggest that the

TMV nanosheets facilitate the formation of a high-quality polyamide layer. We believe that the

uniform pores and appropriate hydrophilicity of TMV nanosheets are beneficial in providing a

steady water-oil interface with evenly distributed monomers during interfacial polymerization,

so that highly regulated reaction of monomers along such interface results in a defect-free

polyamide selective layer.

Figure S4. Na2SO4 rejection and water permeance of TFC nanofiltration membrane fabricated

with and without preloaded TMV nanosheets. (2 mg/ml TMC, 5 mg/ml PIP)

10

Table S1. Surface composition and O/N ratio of the MCE substrate, PA-0/MCE and PA-

1/MCE nanofiltration membranes from XPS analysis.

Sample C O N O/N

MCE 49.44 42.71 7.84 5.45

PA-0/MCE 57.47 32.70 9.83 3.33

PA-1/MCE 66.62 22.98 10.39 2.21

11

4. TFC membranes with TMV protein disks or TMV protein nanosheets incorporated

into the polyamide layer

Two sets of control experiments are conducted by adding certain amount of TMV protein

disks or TMV protein nanosheets into the PIP aqueous phase solution prior to interfacial

polymerization, so that TMVs are expected to be incorporated into the polyamide layer atop of

the PES ultrafiltration substrate. Comparing to blank polyamide membranes without TMV, no

significant change in Na2SO4 rejection or water permeance is observed by adding 0.2-0.4

mg/ml of TMV proteins into the system. Further increasing the amount of TMV proteins leads

to slight increase of water permeance but drop in salt rejection for both the cases of TMV disks

and TMV nanosheets (Figure S5). These two sets of control experiments reveal that the

incorporation of TMV proteins into the polyamide layer could hardly bring positive influence

on the membrane rejection and permeance.

Figure S5. Na2SO4 rejection and water permeance of polyamide membranes with the

incorporation of (a) TMV protein disks and (b) TMV protein nanosheets.

5. Effect of nanosheet hydrophilicity on the polyamide layer morphology

The effect of nanosheet hydrophilicity on the polyamide layer morphology is verified by

replacing the TMV nanosheets with graphene oxide (GO) and reduced graphene oxide (rGO)

nanosheets for the TFC nanofiltration membrane fabrication. As shown in Figures S6b and S6c,

12

the GO or rGO nanosheets modified MCE membranes present similar surface morphologies

comparing to the small TMV nanosheets modified membrane (Figures S6a). Since the GO and

rGO nanosheets used are with size smaller than 500 nm, they are expected to deposit inside the

membrane cavities as well. Upon removing the support membrane after the interfacial

polymerization, the two freestanding polyamide thin films present different morphologies. The

polyamide film fabricated from the highly hydrophilic GO nanosheets modified support

possesses a rather flat surface (Figure S6e), whereas a honeycomb-like morphology similar to

PA-1 is observed for the polyamide thin film fabricated from the relative hydrophobic rGO

nanosheets modified support (Figure S6f). We can therefore conclude that the TMV nanosheets

possess appropriate hydrophilicity to provide localized water-oil interface inside the MCE

membrane cavities for the polymerization reaction to occur, and guide the formation of 3D

honeycomb-like spatial structured polyamide membrane.

Figure S6. Morphology comparison of freestanding polyamide thin films fabricated from TMV,

GO and rGO nanosheets modified MCE membranes. SEM images of MCE support membrane

deposited with (a) TMV nanosheets, (b) GO and (c) rGO. (d-f) are SEM images of the

corresponding freestanding polyamide thin film after removing the MCE support.

13

6. The location relationship between TMV nanosheets and polyamide thin film

The role of TMV nanosheets on the construction of 3D honeycomb-like polyamide

membrane is further explored using a batch of TMV nanosheets that are modified by Au

nanoparticles prior to deposition onto the MCE support. By comparing with the original TMV

nanosheet (Figure S7a), discrete black dots representing the Au nanoparticles are found to be

attached to the TMV nanosheet (Figure S7d). Multiple Au nanoparticles also appear in the

enlarged TEM image of freestanding honeycomb-like polyamide thin film with the underlying

MCE support removed by DMF soaking (Figure S7f), revealing that the TMV nanosheets are

integrated with the polyamide layer. This is another solid proof that the TMV nanosheets

localize the polymerization reaction interface and even involve themselves in the thin film

formation process to yield an integrated TMV-polyamide layer.

Figure S7. TEM images of (a) single-layered TMV nanosheet, (b) freestanding polyamide thin

film PA-1 after removing the underlying MCE support, (c) enlarged freestanding polyamide

thin film PA-1. (d, e and f) are the corresponding TEM images of samples made with Au

nanoparticles modified TMV nanosheets (black dots in d and f are the Au nanoparticles).

14

7. The influence of the size of TMV nanosheets on TFC nanofiltration membrane

Figure S8a shows the SEM image of MCE microfiltration membrane modified by the larger

TMV nanosheets. Different from the case of small TMV nanosheets, a firm and smooth layer

of TMV nanosheets appears on the surface of MCE microfiltration membrane support, and

cavities of the underlying MCE support can only be observed through discrete surface pores of

the layer. As the surface SEM image shown in Figure S8b, the polyamide membrane fabricated

from the large TMV sheets modified MCE support (denote as PA-2/MCE) presents a rather

rough surface, but cavities of the underlying MCE support are not observed. The surface SEM

image reveals a relatively flat freestanding polyamide thin film (PA-2) after removing the MCE

support (Figure S8c), and the cross-sectional SEM image of freestanding thin film PA-2 shows

that the layer thickness is about 550 nm (Figure S8d). The thickness, root mean square (RMS)

roughness (nm) and surface area increase of PA-1 and PA-2 thin films are listed in Table S2.

The C1s, O1s, and N1s peaks of the XPS spectra (Figure S9) provide more detailed information

of the chemical compositions of polyamide layer, which are further summarized in Table S3.

The unreacted carboxylic acid groups and amine groups of the polyamide layer fabricated with

small TMV nanosheets modified support are found to be 47.03% and 3.93%, respectively. With

large TMV nanosheets involved, the portion of unreacted function groups reduces to 26.82%

and 4.12%, indicating a higher crosslinking degree and denser polyamide layer structure

formed.

15

Figure S8. Morphology of polyamide nanofiltration membrane fabricated from large TMV

nanosheets modified MCE support. (a) Surface SEM image of MCE support after depositing

large TMV nanosheets. (b) Surface SEM image of TFC nanofiltration membrane based on large

TMV nanosheets deposited MCE support (PA-2/MCE). (c) Surface SEM image and (d) cross-

sectional SEM image of freestanding polyamide thin film (PA-2) placed on an AAO filter

membrane.

Table S2. Properties of freestanding polyamide films (PA-1 and PA-2) based on AFM

analysis.

Sample Thickness (nm) Root mean square

roughness (nm)

Surface area

increase (%)

PA-1 350±250 127 25.7

PA-2 500±100 34.1 3.5

16

Figure S9. (a and b) C1s, (c and d) O1s, and (e and f) N1s XPS spectra of polyamide

membranes prepared from small and large TMV nanosheets modified MCE membrane

supports, respectively.

17

Table S3. Surface chemical components of polyamide membranes PA-1/MCE and PA-2/MCE

prepared from small and large TMV nanosheets deposited MCE membranes, respectively.

Samples Surface chemical species from C1s

COOH(%) NH2(%)

B.E. (eV) Species Content(%)

PA-1/MCE

284.8 C-H/C-C 66.25

47.03 3.93 286.0 C-N 18.16

287.9 O-C=O/O-C=N 15.59

PA-2/MCE

284.8 C-H/C-C 62.15

26.28 4.12 286.0 C-N 20.20

287.9 O-C=O/O-C=N 17.65

8. Surface area comparation between PA-1 and PA-2 via adsorption of dye molecules

A dye adsorption-desorption experiment is conducted to compare the specific surface area

of PA-1 and PA-2. Considering that the surface of PA-1 and PA-2 exhibit negative charges,

we chose a positive charged methylene blue (MB) as the adsorbate. The top surfaces of PA-1

and PA-2 membrane samples with identical dimensions were brough into contact with 10 ml

MB solution (10 ppm) for 12 h respectively to allow the adsorption of MB molecules to reach

equilibrium. After thorough wash with deionized water, the membrane sample was then soaked

in 10 ml ethanol solution for desorption. The UV–vis absorption spectra of corresponding MB

solutions during the adsorption-desorption process are presented in Figure S10. The lower peak

of PA-1 in Figure S10a demonstrates that larger amount of MB are adsorbed to PA-1 during

the adsorption process. Meanwhile, the significantly higher peak of PA-1 in Figure S10b

indicates that larger amount of MB desorbs from PA-1 during the desorption process. Since

adsorption capacity is proportional to surface area, we can conclude that PA-1 possesses much

higher specific surface area compared to PA-2.

18

Figure S10. (a) UV–vis absorption spectra of methylene blue (MB) solutions after in contact

with PA-1 and PA-2 membranes for 12 h; (b) UV–vis absorption spectra of ethanol solutions

after soaking with MB adsorbed PA-1 and PA-2 membranes.

9. Separation performance variation of TFC nanofiltration membranes with respect to

concentration of PIP

When the concentration of PIP reaches 0.4 mg/ml (TMC concentration of 2 mg/ml),

nanofiltration membrane exhibits high salt rejection and water permeance, but water

permeance decreased obviously with further increased PIP concentration.

Figure S11. Na2SO4 rejection and water permeance variation of TFC nanofiltration membranes

with respect to concentration of PIP (TMC: 2 mg/ml).

19

10. Separation performance benchmarking with other state-of-the-art nanofiltration

membranes reported in literature

Comparing to various TFC nanofiltration membranes reported so far, the herein 3D

honeycomb-like spatial structured polyamide nanofiltration membrane reported in this work

has the highest water permeance, and the Na2SO4 rejection is also on the high side (Figure S12).

In addition, the corresponding graphs using log(1-R) as the y axis are shown in Figure S13,

which distinguish differences among high salt rejections more clearly and provide very good

benchmarking on where the current work stands. Specifically, our membrane clearly breaks off

the permeance-rejection trade-off relationship of nanofiltration membranes as indicated from

the figure.

Figure S12. Summary of the separation performance of state-of-the-art nanofiltration

membranes reported in literature in terms of water permeance and rejection to Na2SO4. The

corresponding refences of circle dot of 1-23 are Ref. 4-26 listed in Supplementary References.

20

Figure S13. The variation of log(1-R) with respect to permeance of the TFC nanofiltration

membrane. (a) R = MgSO4 rejection, and (b) R = Na2SO4 rejection.

11. Separation performance of TFC nanofiltration membrane under different applied

pressure and feed salt concentration

With increasing applied pressure from 1 to 4 bar in Figure S14a, water flux of the

membrane is found to increase proportionally with the applied pressure, while the rejection to

Na2SO4 of the membrane decreases slightly, likely due to the enhanced concentration

polarization at high permeating flux. According to the figure, the membrane exhibits a very

high permeation flux around 200 L m-2 h-1 at 3-4 bar applied pressure. Along with high salt

rejection, such high water flux could magnify the influence of concentration polarization

significantly, and the therefore concentrated feed near the membrane surface would result in

the decrease of apparent salt rejection. The effect of concentration polarization is presented

more clearly by a new graph plotting the experimental flux and calculated flux of the membrane

against applied pressure (Figure S14c). As shown in the figure, the experimental water flux is

lower than the calculated flux (calculated by adjusting the pure water flux according to the

effective pressure difference) when the applied pressure exceeds 3 bar, indicating significant

influence of concentration polarization. As for different feed salt concentration in Figure S14b,

water flux decreases quickly with the increasing Na2SO4 concentration, and the membrane also

21

experiences a rejection decrease when concentration of Na2SO4 feed solution is higher than

3000 ppm. Since a low applied pressure of 2 bar is used for the test, such water flux decline is

attributed to the much-increased osmotic pressure and the decreased effective driving force

with the elevated feed salt concentration and rejection decline is attributed to the weakened

Donnan effect under high salt concentration. We also provide calculated flux vs. salt

concentration graph obtained from adjusting the pure water flux according to effective pressure

difference (Figure S14d). As shown in the figure, the experimental flux is in good accordance

with the calculated value, which indicates the membrane performance stability against feed salt

concentration variations.

Figure S14. Nanofiltration performance of the TFC membrane against different applied

pressure and feed salt concentration. Rejection and water flux with respect to (a) applied

pressure (with 1000 ppm Na2SO4 as feed) and (b) feed salt concentration (with 2 bar applied

pressure). Experimental flux and calculated flux with respect to (c) different applied pressure

and (d) feed salt concentration.

22

12. Anti-fouling experiment

To investigate the anti-fouling capability of nanofiltration membrane with 3D honeycomb-

liked structure, we examined the fouling behavior of the membrane by using bovine serum

albumin (BSA) and humic acid (HA) respectively in a multi-cycled test. It is found that the

membrane water permeance drops from 80 to 60 L m-2 h-1 bar-1 when challenged by 1000 ppm

HA feed solution, but is almost fully recovered by crossflow water wash. Whereas the BSA

challenged membrane fails to recover the water permeance during the first washing cycle

probably due to stronger adsorption of BSA protein onto the polyamide membrane surface, but

its recoverable water permeance maintains at 60 L m-2 h-1 bar-1 for the subsequent cycles. The

fouling resistance of the membrane appears comparable to other polyamide-based

nanofiltration and reverse osmosis membranes27-29, probably due to the fact that most

polyamide TFC membranes especially membranes made from MPD-TMC chemistry also

possess a highly crumpled rough surface feature.

Figure S15. Variation of membrane permeance during the anti-fouling test (feed: 1000 ppm

BSA or 1000 ppm HA for multi-cycled test).

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