Subscriber access provided by FLORIDA INTL UNIV
Article
Mitigation of Biofilm Development on Thin-Film Composite Membranes Functionalized with Zwitterionic Polymers and Silver Nanoparticles Caihong Liu, Andreia Fonseca de Faria, Jun Ma, and Menachem Elimelech Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03795 • Publication Date (Web): 02 Dec 2016 Downloaded from http://pubs.acs.org on December 6, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
Environmental Science & Technology
1 2
Mitigation of Biofilm Development on Thin-Film
3
Composite Membranes Functionalized with
4
Zwitterionic Polymers and Silver Nanoparticles
5 6
Environmental Science and Technology
7
Revised: November 12, 2016
8
Caihong Liu1, Andreia F. Faria2, Jun Ma1*, and Menachem Elimelech2, 3* 1
9
State Key Laboratory of Urban Water Resource and Environment,
10
Harbin Institute of Technology, Harbin 150090, China
11 12
2
13
Yale University, New Haven, Connecticut 06520-8286, USA
Department of Chemical and Environmental Engineering,
14 15
3
Nanosystems Engineering Research Center for Nanotechnology-Enabled Water Treatment
16
(NEWT), Yale University, New Haven,Connecticut 06520-8286, USA
17 18
*Corresponding author. E-mail:
[email protected] (J.M.);
[email protected] 19
(M.E.)
ACS Paragon Plus Environment
Environmental Science & Technology
20
ABSTRACT
21
We demonstrate the functionalization of thin-film composite membranes with zwitterionic
22
polymers and silver nanoparticles (AgNPs) for combating biofouling. Combining hydrophilic
23
zwitterionic polymer brushes and biocidal AgNPs endows the membrane with dual functionality:
24
anti-adhesion and bacterial inactivation. An atom transfer radical polymerization (ATRP) reaction
25
is used to graft zwitterionic poly(sulfobetaine methacrylate) (PSBMA) brushes to the membrane
26
surface while AgNPs are synthesized in situ through chemical reduction of silver. Two different
27
membrane architectures (Ag-PSBMA and PSBMA-Ag TFC) are developed according to the
28
sequence AgNPs and PSBMA brushes are grafted on the membrane surface. A static adhesion
29
assay shows that both modified membranes significantly reduced the adsorption of proteins, which
30
served as a model organic foulant. However, improved antimicrobial activity is observed for
31
PSBMA-Ag TFC (i.e., AgNPs on top of the polymer brush) in comparison to Ag-PSBMA TFC
32
membrane (i.e., polymer brush on top of AgNPs), indicating that architecture of the antifouling
33
layer is an important factor in the design of zwitterion-silver membranes. Confocal laser scanning
34
microscopy (CLSM) imaging indicated that PSBMA-Ag TFC membranes effectively inhibit
35
biofilm formation under dynamic cross-flow membrane biofouling tests. Finally, we demonstrate
36
the regeneration of AgNPs on the membrane after depletion of silver from the surface of the
37
PSBMA-Ag TFC membrane.
38 39
TOC Art
40 41
1 ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
Environmental Science & Technology
42
INTRODUCTION
43
Membrane-based processes are widely considered as sustainable technologies to supply clean
44
water and address the problem of water scarcity that afflicts millions of people worldwide.1, 2 Thin-
45
film-composite (TFC) membranes, the current state-of-the-art membrane technology for
46
desalination processes, are the most robust membranes for water purification and desalination.2
47
However, TFC membranes suffer from the problem of organic and biological fouling.3, 4 Biological
48
fouling, or biofouling, involves complex mechanisms in which adhesion of organic molecules and
49
microorganisms plays a crucial role.5-7 The growth of attached bacterial cells to biofilms leads to
50
increased use of chemicals for cleaning, higher operation costs, and shorter membrane lifetime.8, 9
51
Modification of membrane surfaces with hydrophilic polymers, such as polyethylene glycol10-
52
12
53
and reduce bacterial adhesion. Recently, zwitterionic polymers, including poly(sulfobetaine
54
methacrylate) (PSBMA), have been applied as a promising class of antifouling agents in a range
55
of industrial and biomedical applications.15-17 The chemical structure of betaine zwitterionic
56
polymers contains negatively and positively charged residues at the same monomer unit.18, 19
57
Because these opposite charges are evenly distributed throughout the polymer chain, zwitterionic
58
polymers are considered as zero-charge molecules.15, 18 One of the most important characteristics
59
of the zwitterionic polymer brush layer is its inherent ability to form a tight hydration layer via
60
ionic solvation with the surrounding water molecules. This hydration layer serves as a steric and
61
energetic barrier against the adsorption of organic and biological entities.17, 20
62
and oligo ethylene glycol13, 14, has been a common strategy to improve organic fouling resistance
Zwitterionic polymers may delay or even prevent microbial attachment to the membrane
63
surface, but they cannot inactivate bacteria cells.4,
13, 21, 22
64
establishing a “defensive” strategy, where very hydrophilic polymers offer protection against
65
bacterial adhesion, several studies have proposed the fabrication of dual-function membranes
66
through the combination of “offensive” and “defensive” strategies.23, 24 Offensive approaches rely
67
on the use of strong antimicrobial agents, such as cationic quaternary ammonium (QACs) or
68
biocidal nanoparticles, to inhibit bacterial proliferation.13,
69
functionalization may emerge as a promising solution to biofouling in membrane-based
70
technologies.
Therefore, contrary to simply
21, 25
2 ACS Paragon Plus Environment
This new concept of surface
Environmental Science & Technology
71
Page 4 of 28
Over the past decades, silver nanoparticles (AgNPs) have received heightened attention due
72
to their efficacy and broad-spectrum antimicrobial activity.12,
28, 29
73
antimicrobial agents, such as QACs, that inactivate bacterial cells through a contact-mediated
74
mechanism, the toxicity of AgNPs is driven by the release of Ag+ ions. Although the toxicity of
75
silver-modified membranes depends on the durability of AgNPs, the use of leachable nanoparticles
76
has some advantages over contact-dependent antimicrobial agents.26-28 The physicochemical
77
properties of AgNPs can be tailored to achieve improved reactivity. Because their mechanism of
78
toxicity is dissolution-dependent, the antimicrobial properties of AgNPs are unlikely to be affected
79
by the presence of other chemical foulants in the feed stream. Even though the dissolution property
80
is occasionally considered a disadvantage, especially in terms of long-term efficiency, the
81
regenerative capability of AgNPs has proven to be a key element of the preferential design of
82
silver-modified membranes for biofouling mitigation.27
In contrast to organic
83
Combining highly hydrophilic zwitterionic polymer brushes and biocidal AgNPs is an
84
innovative route to optimize the surface chemistry of membranes to minimize the adhesion of
85
organic foulants and bacteria and maximize the inactivation of bacterial cells. On a dual
86
functionality surface, AgNPs can inactivate bacteria while zwitterionic polymer brushes shield the
87
surface from adsorption of organic foulants. Such an approach can enable the fabrication of
88
multifunctional membranes that can overcome technical challenges that have hampered the
89
advancement of membrane-based processes. Despite efforts to develop membranes with
90
antifouling or biofouling properties,24, 29 studies regarding the fabrication of membranes with
91
simultaneous antiadhesive and bactericidal capabilities are still scarce.
92
This paper demonstrates a new pathway for the fabrication of anti-biofouling TFC membranes
93
by grafting zwitterionic polymer brushes and AgNPs to the membrane surface. We investigated
94
the role of membrane surface functionalized layer architecture on the membrane antiadhesive and
95
antimicrobial properties. Membrane surface architecture was found to strongly affect the
96
antimicrobial property and the biofouling behavior of the functionalized TFC membranes. Our
97
results suggest that functionalization of TFC membranes with zwitterionic polymer brushes and
98
biocidal nanoparticles is an attractive strategy to mitigate biofouling in membrane-based processes.
3 ACS Paragon Plus Environment
Page 5 of 28
99
Environmental Science & Technology
MATERIALS AND METHODS
100
Materials and Chemicals. Dopamine hydrochloride, α-bromoisobutyryl bromide (BiBBr)
101
(98%), N,N-dimethylformamide (DMF), tris(hydroxymethyl)aminomethane (Tris) (>99.8%),
102
triethylamine (TEA) (>99%), [2-(methacryloyloxy)-ethyl]dimethyl-(3-sulfopropyl)ammonium
103
hydroxide (also called sulfobetaine methacrylate, SBMA), copper-(II) chloride, tris(2-
104
pyridylmethyl)amine (TPMA), L-ascorbic acid, isopropanol (IPA), silver nitrate (AgNO3) (≥99%),
105
sodium borohydride (NaBH4) (99.99%), and phosphate buffered saline (PBS, pH 7.4) were
106
purchased from Sigma-Aldrich. Fluorescein-conjugated BSA (FITC-BSA) (Life Technologies,
107
A23015) was purchased from Thermo Fisher Scientific. Commercial thin-film composite (TFC)
108
forward osmosis (FO) membranes were kindly provided by Porifera (Porifera, Inc., CA). Prior to
109
use, the membranes were wetted for 30 minutes in 25% isopropanol solution, rinsed with deionized
110
(DI) water for approximately four hours, and stored at 4 °C until use. A Milli-Q ultrapure water
111
purification system (Millipore, Billerica, MA) was used to supply DI water.
112
TFC Membrane Functionalization Pathways. ARGET-ATRP (activators regenerated
113
by electron transfer-atom transfer radical polymerization) was employed to modify the TFC
114
membranes with zwitterionic polymers. PSBMA was grafted on the TFC membrane as previously
115
reported.4, 30 First, dopamine hydrochloride (800 mg, 2.10 mmol) was dissolved in DMF (40 mL)
116
and the mixture was transferred to a sealed amber bottle which was bubbled with nitrogen gas.
117
After 20 minutes, BiBBr (0.26 mL, 1.05 mmol) and TEA (0.3 mL, 1.05 mmol) were added. The
118
BiBBr-initiator-dopamine solution was then left stirring under nitrogen atmosphere for three hours
119
at room temperature. The pristine TFC membranes were placed on a stirring plate at 60 rpm after
120
being sandwiched between a clean glass and a rubber frame (inner hole size of 10 cm × 6.5 cm)
121
with the membrane active layer facing up. The prepared BiBBr-initiator-dopamine solution was
122
diluted with 200 mL of aqueous Tris buffer (pH 8.5, 2.0 mmol) and then immediately exposed to
123
the active layer of the membrane for 10 minutes. After exposure, the membrane was thoroughly
124
rinsed with DI water to remove excess reactants.
125
For the polymer binding, SBMA monomer (15.64 g, ~ 56 mmol) was dissolved in 200 mL of
126
an isopropanol-water solution (1:1 v/v) in a sealed glass bottle covered with aluminum foil. After
127
bubbling the dispersion with nitrogen gas for 10 minutes, a mixture of CuCl2 (0.004 g, ~5.92 µmol)
128
and TPMA (0.056 g, ~0.038 mmol) in isopropanol aqueous solution (1:1 v/v, 8 mL) was 4 ACS Paragon Plus Environment
Environmental Science & Technology
129
introduced into the sealed bottle using a syringe. Next, the membranes (previously exposed to
130
BiBBr-initiator-dopamine) were placed into the glass bottle and kept in contact with the SBMA
131
dispersion for another 10 minutes under nitrogen atmosphere. Then, 12 mL of an ascorbic acid
132
solution (1 g in 10 mL of 1:1 isopropanol/water) were syringed into the glass bottle to initiate the
133
polymerization. After one hour of polymerization, the bottle was opened to air to terminate the
134
ARGET-ATRP reaction and the membrane was excessively rinsed with DI water. The PSBMA
135
modified membrane (PSBMA TFC hereafter) was stored in aqueous isopropanol (10% v/v) at 4°C.
136
After functionalizing the membranes with PSBMA polymers, silver nanoparticles (AgNPs)
137
were nucleated on the surface through an in situ synthesis previously described by Ben-Sasson et
138
al.28 Briefly, the membranes were immobilized between a clean glass and rubber frame (inner hole
139
size of 10 cm × 6.5 cm) so that only the active layer of the membrane was available for
140
functionalization. AgNO3 solution (15 mL, 5 mmol) was allowed to contact the membrane surface
141
for 10 minutes. Subsequently, the AgNO3 solution was removed, leaving a thin layer of AgNO3
142
solution on the membrane surface. NaBH4 solution (15 mL, 5 mmol) was then added to the
143
membrane surface to react with the nucleated Ag+ ions, leading to the formation of AgNPs. After
144
five minutes of reaction, the NaBH4 solution was discarded and the AgNPs-modified membranes
145
were rinsed with DI water for approximately 10 seconds. This procedure was used for the
146
preparation of PSBMA-Ag TFC membranes. Similarly, the Ag-PSBMA TFC membranes were
147
prepared by first modifying TFC membranes with AgNPs and subsequently grafting PSBMA
148
using the same conditions described above. A schematic diagram for the preparation of the Ag-
149
PSBMA TFC and PSBMA-Ag TFC membranes is shown in Figure 1.
150
FIGURE 1
151
Membrane Surface Characterization. Scanning electron microscopy (SEM Hitachi SU-
152
70 FE-SEM, Hitachi High Technologies America, Inc.), atomic force microscopy (AFM, Bruker
153
Dimension Fastscan AFM, Bruker Corp., Santa Barbara, CA), and contact angle measurements
154
(OneAttension contact angle meter, Biolin Scientific, Finland) were used to characterize the
155
surface properties of pristine and modified membranes. For SEM imaging, membrane samples
156
were dried and sputter coated with 16 nm of chrome. Surface roughness was obtained from AFM
157
by using a Scanasyst-air silicon probe (Bruker Nano, Inc., Camarillo, CA) in a peak force tapping
158
mode. The probe has a spring constant of 0.4 N·m-1, resonance frequency of 70 KHz, tip with a 5 ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
Environmental Science & Technology
159
radius of 2 nm, and length of 115 µm. Water contact angle measurements were performed to
160
evaluate the surface hydrophilicity of the pristine and modified membranes. To obtain
161
representative measurements, water contact angles were acquired from at least twelve random
162
locations on each membrane surface. All membranes were air-dried prior to the measurements.
163
The intrinsic membrane transport properties, namely water permeability coefficient (A), salt
164
permeability coefficient (B), and structural parameter (S), were determined in a laboratory-scale
165
cross-flow unit operated in FO mode, as reported elsewhere.31 Observed values are presented in
166
Figure S1. Specifically, for pristine membrane, A = 2.26 ± 0.2 L·m-2·h-1·bar-1 and B = 0.64 ± 0.07
167
L·m-2·h-1, while for PSBMA-Ag TFC membrane, A = 1.55 ± 0.06 L·m-2·h-1·bar-1 and B = 1.30 ±
168
0.05 L·m-2·h-1. Streaming potential measurements (EKA, Brookhaven Instruments, Holtsville, NY)
169
were performed to determine the zeta potential of the membrane surface using an electrolyte
170
solution (1 mM KCl and 0.1 mM KHCO3) at a pH range that varied between 3 and 9. Zeta
171
potentials for pristine and PSBMA-Ag TFC membrane are shown in Figure S2.
172
Protein Adhesion Assay. The antiadhesive properties of the modified membranes were
173
evaluated by a static protein adsorption assay using bovine serum albumin (BSA) as a model
174
organic foulant. A protein solution (0.05 mg·mL-1) was prepared by dissolving fluorescein-
175
conjugated BSA (FITC-BSA) in PBS solution (pH 7.4). Membrane coupons (2.1 cm in diameter)
176
were cut and mounted in custom-made membrane cell holders. FITC-BSA solution (3 mL) was
177
then added into each cell holder and left to contact the membrane surface for three hours under a
178
mild stirring in the dark. Next, the solution was discarded and the membrane surfaces were rinsed
179
twice with PBS buffer to remove unbounded proteins. The membrane coupon was then removed
180
and placed on a glass slide with a droplet of PBS buffer on top, covered by a cover glass and sealed
181
with nail polish. The samples were imaged using an inverted Axiovert 200 M epifluorescence
182
microscopy (Carl Zeiss Inc., Thornwood, NY, USA).
183
Antimicrobial Activity Experiments. The antimicrobial properties of the pristine and
184
modified TFC membranes were evaluated using Pseudomonas aeruginosa (P. aeruginosa, ATCC
185
BW27853, American Type Culture Collection) as a model microorganism. Bacteria were
186
cultivated overnight in Lysogeny broth (LB) at 37 °C. The bacteria suspension (1 mL) was
187
transferred to fresh LB media (24 mL) and grew for two to three hours to exponential phase. The
188
culture was centrifuged and the pellet was washed three times with sterile saline solution (NaCl, 6 ACS Paragon Plus Environment
Environmental Science & Technology
189
0.9 wt %) to remove the excess macromolecules. After washing, the microbial cells were re-
190
suspended in saline solution to reach a concentration of 108 colony-forming units per milliliter
191
(CFU·mL-1).
192
Membrane coupons with area of ~3.5 cm2 were cut and the active side of the membrane was
193
placed in contact with the bacterial suspension (3 mL) for three hours at room temperature. After
194
incubation, the bacteria suspension was discarded and the membrane surface was rinsed twice with
195
saline solution. To detach deposited bacteria from the membrane surface, membrane coupons were
196
transferred into 50 mL falcon tubes containing 10 mL of saline solution, and the tubes were bath-
197
sonicated for 10 minutes. Serial dilutions of the cell suspension were plated on LB plates and
198
incubated overnight at 37 oC. After 24 hours of growth, the colonies were counted to evaluate the
199
number of viable cells on silver-functionalized membranes compared to that on the pristine TFC
200
membrane.
201
Assessing Biofouling Behavior in a Membrane Cross-flow System. Dynamic
202
biofouling experiments were carried out to evaluate the resistance of PSBMA-Ag TFC membranes
203
against biofilm formation by P. aeruginosa, as previously described.9, 32, 33 Prior to the experiments,
204
the FO system was sequentially cleaned and disinfected with bleach solution (10%, v/v), EDTA
205
solution (5 mM, pH 7), and pure ethanol. Each of the cleaning solutions was circulated throughout
206
the entire unit for one hour. To remove any trace of these chemical compounds, the FO system
207
was thoroughly rinsed three times with DI water.
208
P. aeruginosa was cultivated in LB medium overnight at 37 oC to an optical density (OD600)
209
of 0.6. The bacterial culture (50 mL) was centrifuged at 4000 rpm, 4 oC for 20 minutes and the
210
pellet was re-suspended in a sterile synthetic wastewater (10 mL). The synthetic wastewater was
211
prepared according to previous studies (8 mM NaCl, 0.15 mM MgSO4, 0.5 mM NaHCO3, 0.4 mM
212
NH4Cl, 0.2 mM CaCl2, 0.2 mM KH2PO4 and 0.6 mM glucose, with ionic strength of 16 mM, pH
213
7.6 ± 0.2) 9, 33 and used as feed solution while a NaCl solution (stock solution: 5M) was applied as
214
draw solution. After stabilization, the NaCl stock solution was used to adjust the initial water flux
215
at 20 L·m-2·h-1, and the initial bacteria concentration was ~ 2 × 106 CFU·mL-1. Temperature was
216
kept constant at 25 oC. Feed wastewater was monitored periodically for number of viable bacteria
217
cells, pH, and conductivity. Before each biofouling run, a baseline experiment at the same
7 ACS Paragon Plus Environment
Page 8 of 28
Page 9 of 28
Environmental Science & Technology
218
condition (without the addition of bacteria) was performed. The baseline curve was used to subtract
219
the dilution effect of the draw solution and reverse salt diffusion of FO itself.32, 34
220
Biofilm Characterization. At the end of each biofouling experiment (500 mL of permeate
221
water accumulation), subsections of the membrane coupons were cut and characterized for the
222
composition of biofilm using confocal laser scanning microscopy (CLSM) and total organic
223
carbon (TOC) measurements.
224
For CLSM analysis, membrane subsections (1 cm × 1 cm) were cut from the center of the
225
biofouled membranes and stained with SYTO 9, propidium iodide (PI) (LIVE/DEAD BacLight,
226
Invitrogen), and concavalin A (Con A, Alexa Flour 633, Invitrogen). SYTO 9, PI, and Con A are
227
known to specifically stain live cells, dead cells, and polysaccharides–extracellular polymeric
228
substances (EPS), respectively. After 40 minutes of contact in the dark, the membrane coupons
229
were rinsed three times with sterile wastewater to remove unbound stains.
230
The stained samples were placed on a custom-built chamber for CLSM imaging.9, 35 A CLSM
231
microscopy (Zeiss LSM 510, Carl Zeiss, Inc.) equipped with a plan-apochromat was used to
232
capture confocal images of the biofilm. SYTO 9, PI, and Con A were excited with 488 nm argon,
233
561 nm diode-pumped solid state, and 633 nm helium-neon laser, respectively. At least six Z stack
234
random fields (635 µm × 635 µm) with a slice thickness of 2.14 µm were collected from each
235
sample to obtain a representative biofilm orthogonal image. At least eight smaller stack regions
236
(90 µm × 90 µm) with a slice thickness of 1.2 µm were captured for biofilm dimension calculation.
237
Confocal images were analyzed using Auto-PHLIP-ML, Image-J, and MATLAB software, as
238
suggested by previous publications.9, 32, 33, 35 Biovolume and thickness were determined for the live
239
cells, dead cells, and EPS of the biofilm for all the samples. Average biofilm thickness was
240
calculated by averaging the thicknesses of these three components.
241
For TOC measurements, membrane subsections (1cm × 1cm) were cut from the biofouled
242
membranes and transferred to 25 mL glass vials containing 20 mL of DI water and 4 µL of a 1 M
243
HCl solution. The vials were probe-sonicated for ~3 minutes to remove the organic content from
244
the membrane surface and TOC measurements were carried out at a TOC analyzer (TOC-V,
245
Shimadzu). TOC concentrations were normalized according to the membrane coupon size (TOC
246
mass per membrane area). 8 ACS Paragon Plus Environment
Environmental Science & Technology
Page 10 of 28
247
Silver Release and Regeneration Capacity. The experiments for the release of silver
248
was performed via a reservoir method.28 PSBMA-Ag TFC membrane coupons (2 cm2) were
249
incubated in glass vials containing 10 mL of 5mM NaHCO3 solution (pH 8.3, without HNO3). The
250
vials with the membrane coupons were placed on a stir plate at 60 rpm. At specific intervals of
251
time, the membrane coupons were withdrawn and transferred to fresh DI water acidified with 0.1
252
mL of HNO3 (70%). The content of silver on the membrane coupons was thoroughly dissolved in
253
the acid solution after 24 hours of agitation. Then the supernatant was collected and the silver ion
254
concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS, ELAN
255
DRC-e ICP Mass Spectrometer, Perkin Elmer). The experiments were conducted in duplicates.
256
To demonstrate the regeneration of AgNPs, we proposed the deposition of AgNPs on the
257
membrane surface after a vigorous leaching process. Specifically, after a thorough release of silver
258
ions in DI water, AgNPs were regenerated on the surface of PSBMA Ag-TFC membranes using
259
the same in situ method described in this section earlier. The recharged PSBMA-Ag TFC
260
membranes were re-characterized by SEM imaging, contact angle measurements, and static
261
protein adsorption assay. In addition, the antimicrobial properties of PSBMA-Ag TFC membranes
262
were re-evaluated after silver regeneration.
263 264
RESULTS AND DISCUSSION
265
Membrane Surface Characteristics. TFC membrane surface morphologies were investigated
266
by SEM imaging (Figure 2A). A smooth film was observed on the membrane surface after PSBMA
267
zwitterionic polymer brushes were grafted on the polyamide layer (PSBMA TFC). This
268
observation indicates that PSBMA brushes were successfully polymerized on the membrane
269
surface. The immobilization of PSBMA polymer brushes on the membrane surface was conducted
270
by ATRP, a versatile technique that allows the growth of monomers into polymer chains using
271
transition metal complexes as catalysts.36 Compared to conventional strategies for surface
272
modification, such as chemical crosslinking,37 ATRP results in the growth of a denser and more
273
uniform layer of polymer brushes with controllable thickness and architecture.38-40
274
FIGURE 2
9 ACS Paragon Plus Environment
Page 11 of 28
Environmental Science & Technology
275
After the in situ formation of AgNPs, different morphological structures were visualized. For
276
the Ag-PSBMA TFC membrane, where AgNPs were loaded before the grafting of PSBMA
277
brushes, SEM images show a thin polymer film on the membrane top surface. Because the particles
278
are covered by the polymer brush layer, we could not attribute the observed morphology to the
279
AgNPs. Conversely, when AgNPs were formed after PSBMA polymerization (PSBMA-Ag TFC),
280
the presence of AgNPs on the top surface was noticeable, whereas the PSBMA film was less
281
distinguishable compared to the Ag-PSBMA membrane. The AgNPs displayed a round-like
282
morphology and were well distributed throughout the membrane surface.
283
To investigate changes in surface roughness due to surface modification with PSBMA or
284
AgNPs, the membranes were characterized by AFM imaging. Figures 2B and 2C show
285
representative 3D AFM images and calculated roughness parameters for pristine and modified
286
membranes, respectively. After functionalization with zwitterionic PSBMA brushes (PSBMA
287
TFC), the membrane surface became smoother than the pristine TFC membrane, exhibiting a ~44%
288
decrease in mean-square roughness (Rq) and ~51% reduction in average roughness (Ra) (Figure
289
2C). However, after impregnation of AgNPs, the surface roughness significantly increased for both
290
Ag-PSBMA and PSBMA-Ag TFC membranes. For instance, Rq parameters for Ag-PSBMA and
291
PSBMA-Ag TFC membranes were increased by more than 30% and 50%, respectively, compared
292
to the pristine TFC membrane. Grafting of PSBMA polymer brushes provided smoother
293
membrane surfaces, while AgNPs contributed to increased surface roughness.
294
Membrane surface hydrophilicity was evaluated by sessile water contact angle measurements
295
(Figure 2D). The membrane contact angle was reduced from 74° ± 10° to 21° ± 7° after
296
functionalization of pristine TFC membrane with PSBMA polymer. The marked decrease in
297
contact angle is attributed to the strong electrostatic interaction of zwitterionic PSBMA brushes
298
with water molecules.15,
299
hydrophilicity compared to the pristine TFC membrane. These observations suggest that the
300
sequence of AgNPs deposition on the membrane surface did not have a significant impact on
301
membrane hydrophilicity. In contrast, membranes modified only with AgNPs did not exhibit
302
change in contact angle compared to pristine TFC membranes (Figure S3), in agreement with a
303
previous investigation.28
41
Similarly, Ag-PSBMA and PSBMA-Ag also showed improved
10 ACS Paragon Plus Environment
Environmental Science & Technology
Page 12 of 28
304
Grafted Zwitterionic Brushes Impart Antiadhesive Property to the Membrane.
305
Antiadhesive properties of the modified membranes were determined via a static protein assay
306
using bovine serum albumin (BSA) protein as a model organic foulant. Proteins are ubiquitous in
307
wastewater effluent and surface waters, and their accumulation on the membrane surface
308
deteriorates membrane performance. Proteins also play an important role during biofouling by
309
forming a conditioning film7 and providing a source of carbon and nitrogen for the growth of
310
microorganisms.42
311
For the protein adhesion assay, a fluorescein-conjugated BSA (FITC-BSA) solution was
312
allowed to contact the active layer of the pristine and modified membranes for three hours in the
313
dark. The acquired fluorescence images for the pristine and functionalized TFC membranes are
314
presented in Figure 3. The fluorescence intensity is directly related to the amount of proteins
315
attached on the membrane surface after exposure to FITC-BSA. High fluorescence intensity was
316
observed for the pristine TFC membrane, indicating significant adsorption of proteins. PSBMA
317
TFC membranes, on the other hand, showed nearly no fluorescence intensity, demonstrating that
318
the membrane successfully prevented the adsorption of BSA. This observation is attributable to
319
the thick hydration layer on the zwitterionic polymer brushes that prevents the adsorption of
320
organic foulants, including proteins.15, 43-46 Surprisingly, both Ag-PSBMA and PSBMA-Ag TFC
321
membranes suppressed BSA adsorption, regardless of the membrane architecture. Contrary to
322
what would be expected, the grafting of AgNPs over the PSBMA brushes did not interfere with
323
the antiadhesive properties of PSBMA-Ag membranes.
324
FIGURE 3
325
Contact angle measurements and fluorescence microscopy images demonstrated that all three
326
membranes functionalized with zwitterionic PSBMA brushes exhibited increased hydrophilicity
327
and excellent antiadhesive properties against the adsorption of BSA protein (Figure 2D and Figure
328
3). In contrast, TFC membranes modified only with AgNPs (without polymer grafting) did not
329
show significant changes in contact angle or protein adsorption (Figure S3), thus proving that
330
PSBMA brushes impart enhanced membrane hydrophilicity and antiadhesive property.
331
Antimicrobial Activity is Affected by the Functionalized Layer Architecture. To
332
evaluate the antimicrobial property of fabricated membranes, the membrane surface was exposed
11 ACS Paragon Plus Environment
Page 13 of 28
Environmental Science & Technology
333
to a bacterial suspension for three hours and the number of attached viable cells was determined
334
by the plate counting method (Figure 4A). PSBMA TFC membrane showed an inhibition rate of
335
17.4% compared to pristine TFC membrane. As zwitterionic polymer brushes are not toxic to
336
bacteria, this slight reduction in cellular attachment is probably associated with the intrinsic
337
antiadhesive property of PSBMA polymers. As discussed earlier, PSBMA polymer brushes
338
promote the formation of a tightly bound hydration layer that reduces the adhesion of bacteria.13,
339
24
340
FIGURE 4
341
Ag-PSBMA and PSBMA-Ag TFC membranes, on the other hand, revealed a significant
342
decrease in bacterial cell viability compared to either PSBMA or pristine TFC membranes.
343
Notably, PSBMA-Ag displayed a greater antimicrobial activity than Ag-PSBMA TFC membranes.
344
For instance, Ag-PSBMA and PSBMA-Ag exhibited 39% and 95% inactivation rates, respectively,
345
compared to the control PSBMA TFC membrane. It is widely accepted that the toxicity mechanism
346
of AgNPs occurs via surface oxidation and subsequent release of Ag+ ions.24, 26, 28 The resulting
347
Ag+ ions can damage the cell membrane and cause leakage of intracellular components.2, 32, 33
348
The discrepancy in antimicrobial activity between Ag-PSBMA and PSBMA-Ag TFC
349
membranes is attributed to the differences in the membrane surface architecture. AgNPs on
350
PSBMA-Ag TFC membranes are significantly more available for contact with bacterial cells,
351
which leads to a superior toxicity. In contrast, AgNPs on Ag-PSBMA TFC membranes are
352
partially covered by the PSBMA brush layer, thus hindering direct contact between bacteria cells
353
and the biocidal nanoparticles. Therefore, the combination of PSBMA with AgNPs in a very
354
specific morphological architecture is crucial for the fabrication of TFC membranes with both
355
antiadhesive and antimicrobial properties. Rather than adopting a random procedure, the sequence
356
of surface modification with AgNPs and PSBMA brushes has been found to affect the
357
antimicrobial properties and should be considered an important parameter during membrane
358
functionalization.
359
The morphological characteristics of the bacterial cells attached to the pristine TFC, Ag-
360
PSBMA TFC, and PSBMA-Ag TFC membrane surfaces were examined by SEM imaging (Figure
361
4B). Compared to the pristine TFC membrane, the cells attached to the functionalized TFC
362
membranes, particularly the PSBMA-Ag TFC membrane, exhibited expressive losses in 12 ACS Paragon Plus Environment
Environmental Science & Technology
Page 14 of 28
363
morphological integrity, showing flattened and wrinkled characteristics. The significant
364
morphological damage with the PSBMA-Ag TFC membrane is likely caused by the abundance of
365
Ag+ ions near the cell surface due to the direct contact between bacterial cells and AgNPs on the
366
membrane surface.28 Owing to its enhanced antimicrobial performance (Figure 4), the PSBMA-
367
Ag TFC membrane was selected for the dynamic biofouling experiments.
368
PSBMA-Ag TFC Membrane Exhibits Reduced Biofouling. Because PSBMA-Ag
369
TFC membranes showed both strong antiadhesive and antimicrobial activities, we further
370
investigated the impact of the functionalization with PSBMA and AgNPs on membrane biofouling
371
by P. aeruginosa. The dynamic biofouling experiments were conducted in FO mode for
372
approximately 20 hours using glucose as a carbon source. Baseline experiments were performed
373
prior to each biofouling experiment to subtract the effect of dilution of draw solution and reverse
374
salt diffusion as described in our previous publications.34, 47
375
The normalized water flux during the accumulation of 500 mL of permeate water is shown in
376
Figure 5. The growth of biofilm on pristine TFC membrane reduces water permeation, thus leading
377
to an approximate 16% of flux decline. On the other hand, PSBMA-Ag showed only 8% of flux
378
decline, implying that PSBMA-Ag membranes exhibited improved resistance to biofouling
379
compared to the pristine TFC membrane. The modification with zwitterionic PSBMA brushes and
380
AgNPs conferred upon the membrane surface a great ability to control biofouling under a dynamic
381
cross-flow condition. The reduced adhesion of bacteria combined with the remarkable killing
382
properties of AgNPs explain the mechanism by which PSBMA-Ag TFC membranes are able to
383
hinder the development of biofilm.
384
FIGURE 5
385
To further characterize the biofouling behaviors of pristine and PSBMA-Ag TFC membranes,
386
the biofilm structure and total organic carbon content were analyzed by CLSM and TOC
387
measurement, respectively. Figure 6 presents representative CLSM orthogonal images for the
388
biofouled pristine and PSBMA-Ag TFC membranes. Live cells, dead cells, and EPS were stained
389
in green, red, and blue, respectively. The amount of dead cells is remarkably higher for PSBMA-
390
Ag TFC membrane, while pristine TFC membranes exhibit a greater concentration of live cells,
391
EPS, and nearly no dead cells.
392
FIGURE 6 13 ACS Paragon Plus Environment
Page 15 of 28
Environmental Science & Technology
393
Table 1 depicts the biofilm properties calculated from CLSM images. The average biofilm
394
thickness decreased from 33.7 µm to 23.0 µm after membrane modification with PSBMA brushes
395
and AgNPs. Compared to pristine TFC, the PSBMA-Ag TFC membrane exhibits a 48% reduction
396
in the live cells biovolume, a 46% increase in dead cells biovolume, and a 60% decrease in EPS
397
content. In addition, TOC measurements revealed that the total biofilm biomass was significantly
398
decreased by 43% after membrane functionalization. Similar anti-biofouling performance has been
399
previously described for TFC membranes modified with either AgNPs or zwitterionic polymers. 4,
400
28
401
membrane indicate that the functionalized membrane is able to efficiently suppress biofilm
402
formation. Therefore, functionalization of TFC membranes with PSBMA brushes and AgNPs in a
403
proper architecture can become an attractive strategy to mitigate biofouling in membrane-based
404
processes.
The increased content of dead cells and decreased biomass concentration on the PSBMA-Ag
405
TABLE 1
406
Membranes can be Regenerated after Silver Depletion. Surface oxidation and
407
subsequent release of Ag+ ions are the main toxicity mechanism of AgNPs. Therefore, the
408
dissolution of AgNPs over time remains one of the biggest challenges for the long-term application
409
of silver-modified membranes.13,
410
membrane was investigated by measuring the silver remaining on the membrane surface at specific
411
time intervals during one week of dissolution (Figure S4). It can be observed that the residual silver
412
on the membrane surface decreased continuously during the first four days and thereafter remained
413
almost unchanged at ~ 4.8 µg·cm-2; this observation indicates that silver dissolution was
414
significantly faster in the beginning given its large quantity, and then slowed down to a constant
415
plateau. We also note that under dynamic cross-flow conditions, AgNPs dissolution rate was
416
significantly higher due to enhanced mass transfer caused by the shear flow (data not shown).
48
The dissolution of AgNPs from the PSBMA-Ag TFC
417
We have demonstrated the regeneration of AgNPs on the PSBMA-Ag TFC membrane using
418
the same in situ nanoparticle formation method to initially form the AgNPs on the polymer brush
419
layer. Figures 7A and 7B display SEM images of PSBMA-Ag TFC membranes before and after
420
the regeneration of AgNPs. Figure 7A shows the original PSBMA-Ag TFC membrane after a
421
thorough release of silver. After the leaching procedure, AgNPs were no longer observed on the
422
membrane surface. Afterwards, the membrane surface underwent the in situ method to re14 ACS Paragon Plus Environment
Environmental Science & Technology
423
synthesize the AgNPs on the membrane surface. The regenerated AgNPs were homogeneously
424
distributed throughout the membrane surface, demonstrating that the membrane reactivity can be
425
reestablished after consecutive cycles of silver depletion (Figure 7B).
426
Page 16 of 28
FIGURE 7
427
In order to investigate the overall performance of the silver-regenerated membrane, static
428
protein adsorption assay (Figure 7C), antimicrobial experiments, and contact angle measurements
429
(Figure 7D) were performed. BSA adsorption assay was conducted to assess the antiadhesive
430
property of PSBMA-Ag TFC membrane after AgNPs regeneration. As we discussed earlier, the
431
high intensity of fluorescence for the pristine TFC membrane indicates significant adsorption of
432
BSA, whereas no fluorescence is observed for the regenerated PSBMA-Ag TFC membrane
433
(Figure 7C). The results in Figures 7A-C suggest that the process of silver regeneration does not
434
compromise the antiadhesive property imparted by the zwitterionic PSBMA brushes.
435
After exposure to P. aeruginosa for three hours, the viability of the adhered bacteria cells on
436
silver-regenerated the PSBMA-Ag TFC membrane was decreased by 97% relative to that on
437
pristine TFC membrane (Figure 7D). It is noteworthy that the silver-regenerated membrane has
438
shown a very similar toxicity to P. aeruginosa compared to the original PSBMA-Ag TFC
439
membrane, thereby confirming that the regenerated membrane preserved its original antimicrobial
440
performance after silver regeneration. Similarly, contact angle measurements were conducted to
441
demonstrate that the hydrophilicity conferred by the zwitterionic polymers was not changed after
442
the regeneration of AgNPs (Figure 7D). A slightly increased but not statistically different contact
443
angle is observed for the silver-regenerated membrane (Figure 7D). Based on these results, we
444
surmise that the membrane can undergo sequential processes for silver regeneration without
445
affecting its antiadhesive and antimicrobial properties.
446
ASSOCIATED CONTENT
447
Supporting information available: membrane transport parameters (Figure S1); zeta potentials of
448
pristine and PSBMA-Ag TFC membrane (Figure S2); contact angle measurements and static
449
protein adsorption assay of pristine and silver-modified TFC membrane (Figure S3); silver release
450
profile of PSBMA-Ag TFC membrane (Figure S4). This material is available free of charge via
451
the internet at http://pubs.acs.org. 15 ACS Paragon Plus Environment
Page 17 of 28
Environmental Science & Technology
452 453
AUTHOR INFORMATION
454
Corresponding Author
455
*Jun Ma, Phone: +86 451 86283010; fax: +86 451 86283010; email:
[email protected].
456
*Menachem Elimelech, Phone: +1 203 432 2789; fax: +1 203 432 4387; email:
457
[email protected].
458
Author Contributions
459
The manuscript was written through contributions of all authors. All authors have given approval
460
to the final version of the manuscript.
461 462
ACKNOWLEDGMENT
463
We acknowledge financial support from the U.S. Department of Defense through the Strategic
464
Environmental Research and Development Program (SERDP, ER-2217) and from the National
465
Science Foundation through the Engineering Research Center for Nanotechnology-Enabled Water
466
Treatment (ERC-1449500). We also acknowledge the China Scholarship Council (CSC) for
467
providing a graduate fellowship (to C.L.). A.F.F thanks the Program “Science without Borders”
468
through the Brazilian Council of Science and Technology for their financial support. We would
469
like to thank Dr. Joseph Wolenski from the Molecular, Cellular, and Developmental Biology
470
Department at Yale University for technical assistance using the CLSM. The authors also
471
acknowledge the Yale Institute of Nanoscale and Quantum Engineering (YINQE) and Dr. Michael
472
Rooks for their support on the SEM and AFM analyses.
473
16 ACS Paragon Plus Environment
Environmental Science & Technology
Page 18 of 28
474
REFERENCES
475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518
1. Shannon, M. A.; Bohn, P. W.; Elimelech, M.; Georgiadis, J. G.; Marinas, B. J.; Mayes, A. M., Science and technology for water purification in the coming decades. Nature 2008, 452, (7185), 301-310. 2. Werber, J. R.; Osuji, C. O.; Elimelech, M., Materials for next-generation desalination and water purification membranes. Nature Reviews Materials 2016, 1, 16018. 3. Perreault, F.; Tousley, M. E.; Elimelech, M., Thin-Film Composite Polyamide Membranes Functionalized with Biocidal Graphene Oxide Nanosheets. Environmental Science & Technology Letters 2014, 1, (1), 71-76. 4. Ye, G.; Lee, J.; Perreault, F.; Elimelech, M., Controlled Architecture of Dual-Functional Block Copolymer Brushes on Thin-Film Composite Membranes for Integrated "Defending" and "Attacking" Strategies against Biofouling. ACS applied materials & interfaces 2015, 7, (41), 23069-23079. 5. Davies, D., Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2003, 2, (2), 114-122. 6. Liao, B. Q.; Bagley, D. M.; Kraemer, H. E.; Leppard, G. G.; Liss, S. N., A Review of Biofouling and its Control in Membrane Separation Bioreactors. Water Environment Research 2004, 76, (5), 425-436. 7. de Kerchove, A. J.; Elimelech, M., Impact of Alginate Conditioning Film on Deposition Kinetics of Motile and Nonmotile Pseudomonas aeruginosa Strains. Applied and Environmental Microbiology 2007, 73, (16), 5227-5234. 8. Ben-Sasson, M.; Zodrow, K. R.; Genggeng, Q.; Kang, Y.; Giannelis, E. P.; Elimelech, M., Surface Functionalization of Thin-Film Composite Membranes with Copper Nanoparticles for Antimicrobial Surface Properties. Environmental science & technology 2014, 48, (1), 384-393. 9. Kwan, S. E.; Bar-Zeev, E.; Elimelech, M., Biofouling in forward osmosis and reverse osmosis: Measurements and mechanisms. Journal of Membrane Science 2015, 493, 703-708. 10. Sagle, A. C.; Van Wagner, E. M.; Ju, H.; McCloskey, B. D.; Freeman, B. D.; Sharma, M. M., PEG-coated reverse osmosis membranes: Desalination properties and fouling resistance. Journal of Membrane Science 2009, 340, (1–2), 92-108. 11. Romero-Vargas Castrillón, S.; Lu, X.; Shaffer, D. L.; Elimelech, M., Amine enrichment and poly(ethylene glycol) (PEG) surface modification of thin-film composite forward osmosis membranes for organic fouling control. Journal of Membrane Science 2014, 450, 331-339. 12. Yeh, C.-C.; Venault, A.; Chang, Y., Structural effect of poly(ethylene glycol) segmental length on biofouling and hemocompatibility. Polymer Journal 2016, 48, (4), 551-558. 13. Banerjee, I.; Pangule, R. C.; Kane, R. S., Antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms. Advanced materials 2011, 23, (6), 690-718. 14. He, Y.; Chang, Y.; Hower, J. C.; Zheng, J.; Chen, S.; Jiang, S., Origin of repulsive force and structure/dynamics of interfacial water in OEG-protein interactions: a molecular simulation study. Physical Chemistry Chemical Physics 2008, 10, (36), 5539-5544. 15. Schlenoff, J. B., Zwitteration: coating surfaces with zwitterionic functionality to reduce nonspecific adsorption. Langmuir : the ACS journal of surfaces and colloids 2014, 30, (32), 962536. 16. Cloutier, M.; Mantovani, D.; Rosei, F., Antibacterial Coatings: Challenges, Perspectives, and Opportunities. Trends in Biotechnology 2015, 33, (11), 637-652.
17 ACS Paragon Plus Environment
Page 19 of 28
519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562
Environmental Science & Technology
17. Jiang, S.; Cao, Z., Ultralow-fouling, functionalizable, and hydrolyzable zwitterionic materials and their derivatives for biological applications. Advanced materials 2010, 22, (9), 92032. 18. Chen, S.; Li, L.; Zhao, C.; Zheng, J., Surface hydration: Principles and applications toward low-fouling/nonfouling biomaterials. Polymer 2010, 51, (23), 5283-5293. 19. He, M.; Gao, K.; Zhou, L.; Jiao, Z.; Wu, M.; Cao, J.; You, X.; Cai, Z.; Su, Y.; Jiang, Z., Zwitterionic materials for antifouling membrane surface construction. Acta Biomaterialia 2016, 40, 142-152. 20. Leng, C.; Hung, H.-C.; Sun, S.; Wang, D.; Li, Y.; Jiang, S.; Chen, Z., Probing the Surface Hydration of Nonfouling Zwitterionic and PEG Materials in Contact with Proteins. ACS applied materials & interfaces 2015, 7, (30), 16881-16888. 21. Carmona-Ribeiro, A. M.; de Melo Carrasco, L. D., Cationic antimicrobial polymers and their assemblies. International journal of molecular sciences 2013, 14, (5), 9906-46. 22. Marré Tirado, M. L.; Bass, M.; Piatkovsky, M.; Ulbricht, M.; Herzberg, M.; Freger, V., Assessing biofouling resistance of a polyamide reverse osmosis membrane surface-modified with a zwitterionic polymer. Journal of Membrane Science 2016, 520, 490-498. 23. Hu, R.; Li, G.; Jiang, Y.; Zhang, Y.; Zou, J.-J.; Wang, L.; Zhang, X., Silver–Zwitterion Organic–Inorganic Nanocomposite with Antimicrobial and Antiadhesive Capabilities. Langmuir : the ACS journal of surfaces and colloids 2013, 29, (11), 3773-3779. 24. Yu, Q.; Wu, Z.; Chen, H., Dual-function antibacterial surfaces for biomedical applications. Acta Biomaterialia 2015, 16, 1-13. 25. Xu, J.; Feng, X.; Chen, P.; Gao, C., Development of an antibacterial copper (II)-chelated polyacrylonitrile ultrafiltration membrane. Journal of Membrane Science 2012, 413–414, 62-69. 26. de Faria, A. F.; Perreault, F.; Shaulsky, E.; Arias Chavez, L. H.; Elimelech, M., Antimicrobial Electrospun Biopolymer Nanofiber Mats Functionalized with Graphene Oxide– Silver Nanocomposites. ACS applied materials & interfaces 2015, 7, (23), 12751-12759. 27. Soroush, A.; Ma, W.; Cyr, M.; Rahaman, M. S.; Asadishad, B.; Tufenkji, N., In Situ Silver Decoration on Graphene Oxide-Treated Thin Film Composite Forward Osmosis Membranes: Biocidal Properties and Regeneration Potential. Environmental Science & Technology Letters 2016, 3, (1), 13-18. 28. Ben-Sasson, M.; Lu, X.; Bar-Zeev, E.; Zodrow, K. R.; Nejati, S.; Qi, G.; Giannelis, E. P.; Elimelech, M., In situ formation of silver nanoparticles on thin-film composite reverse osmosis membranes for biofouling mitigation. Water research 2014, 62, 260-270. 29. Matin, A.; Khan, Z.; Zaidi, S. M. J.; Boyce, M. C., Biofouling in reverse osmosis membranes for seawater desalination: Phenomena and prevention. Desalination 2011, 281, 1-16. 30. Blok, A. J.; Chhasatia, R.; Dilag, J.; Ellis, A. V., Surface initiated polydopamine grafted poly([2-(methacryoyloxy)ethyl]trimethylammonium chloride) coatings to produce reverse osmosis desalination membranes with anti-biofouling properties. Journal of Membrane Science 2014, 468, 216-223. 31. Tiraferri, A.; Yip, N. Y.; Straub, A. P.; Romero-Vargas Castrillon, S.; Elimelech, M., A method for the simultaneous determination of transport and structural parameters of forward osmosis membranes. Journal of Membrane Science 2013, 444, 523-538. 32. Xie, M.; Lee, J.; Nghiem, L. D.; Elimelech, M., Role of pressure in organic fouling in forward osmosis and reverse osmosis. Journal of Membrane Science 2015, 493, 748-754.
18 ACS Paragon Plus Environment
Environmental Science & Technology
563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607
Page 20 of 28
33. Xie, M.; Bar-Zeev, E.; Hashmi, S. M.; Nghiem, L. D.; Elimelech, M., Role of Reverse Divalent Cation Diffusion in Forward Osmosis Biofouling. Environmental science & technology 2015, 49, (22), 13222-13229. 34. Tiraferri, A.; Kang, Y.; Giannelis, E. P.; Elimelech, M., Superhydrophilic thin-film composite forward osmosis membranes for organic fouling control: fouling behavior and antifouling mechanisms. Environmental science & technology 2012, 46, (20), 11135-44. 35. Bar-Zeev, E.; Perreault, F.; Straub, A. P.; Elimelech, M., Impaired Performance of Pressure-Retarded Osmosis due to Irreversible Biofouling. Environmental science & technology 2015, 49, (21), 13050-13058. 36. Matyjaszewski, K., Atom Transfer Radical Polymerization (ATRP): Current Status and Future Perspectives. Macromolecules 2012, 45, (10), 4015-4039. 37. Laurent, B. A.; Grayson, S. M., Synthesis of Cyclic Dendronized Polymers via Divergent “Graft-from” and Convergent Click “Graft-to” Routes: Preparation of Modular Toroidal Macromolecules. Journal of the American Chemical Society 2011, 133, (34), 13421-13429. 38. Lee, S. H.; Dreyer, D. R.; An, J.; Velamakanni, A.; Piner, R. D.; Park, S.; Zhu, Y.; Kim, S. O.; Bielawski, C. W.; Ruoff, R. S., Polymer Brushes via Controlled, Surface-Initiated Atom Transfer Radical Polymerization (ATRP) from Graphene Oxide. Macromolecular rapid communications 2010, 31, (3), 281-8. 39. Król, P.; Chmielarz, P., Recent advances in ATRP methods in relation to the synthesis of copolymer coating materials. Progress in Organic Coatings 2014, 77, (5), 913-948. 40. Ran, J.; Wu, L.; Zhang, Z.; Xu, T., Atom transfer radical polymerization (ATRP): A versatile and forceful tool for functional membranes. Progress in Polymer Science 2014, 39, (1), 124-144. 41. Laughlin, R. G., Fundamentals of the zwitterionic hydrophilic group. Langmuir : the ACS journal of surfaces and colloids 1991, 7, (5), 842-847. 42. Fletcher, M., The Effects of Proteins on Bacterial Attachment to Polystyrene. Microbiology 1976, 94, (2), 400-404. 43. Yue, W.-W.; Li, H.-J.; Xiang, T.; Qin, H.; Sun, S.-D.; Zhao, C.-S., Grafting of zwitterion from polysulfone membrane via surface-initiated ATRP with enhanced antifouling property and biocompatibility. Journal of Membrane Science 2013, 446, 79-91. 44. Hucknall, A.; Rangarajan, S.; Chilkoti, A., In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins. Advanced materials 2009, 21, (23), 2441-2446. 45. Weng, X.-D.; Ji, Y.-L.; Ma, R.; Zhao, F.-Y.; An, Q.-F.; Gao, C.-J., Superhydrophilic and antibacterial zwitterionic polyamide nanofiltration membranes for antibiotics separation. Journal of Membrane Science 2016, 510, 122-130. 46. Zhang, Z.; Chen, S.; Chang, Y.; Jiang, S., Surface Grafted Sulfobetaine Polymers via Atom Transfer Radical Polymerization as Superlow Fouling Coatings. The Journal of Physical Chemistry B 2006, 110, (22), 10799-10804. 47. Shaffer, D. L.; Jaramillo, H.; Romero-Vargas Castrillón, S.; Lu, X.; Elimelech, M., Postfabrication modification of forward osmosis membranes with a poly(ethylene glycol) block copolymer for improved organic fouling resistance. Journal of Membrane Science 2015, 490, 209219. 48. Yin, J.; Yang, Y.; Hu, Z.; Deng, B., Attachment of silver nanoparticles (AgNPs) onto thinfilm composite (TFC) membranes through covalent bonding to reduce membrane biofouling. Journal of Membrane Science 2013, 441, 73-82.
608 19 ACS Paragon Plus Environment
Page 21 of 28
Environmental Science & Technology
609
610 611 612
Figure 1. Illustrative scheme demonstrating the preparation of the two different membrane architectures.
613
One membrane architecture (top) was prepared by first synthesizing AgNPs on the membrane polyamide
614
layer followed by the grafting of zwitterionic polymer brushes (PSBMA) via ATRP method. The second
615
membrane design (bottom) was fabricated by first grafting PSBMA brushes via ATRP followed by
616
consecutive in situ synthesis of AgNPs.
617 618
20 ACS Paragon Plus Environment
Environmental Science & Technology
Page 22 of 28
619 620 621
Figure 2. (A) Scanning electron microscopy (SEM) images of the polyamide active layer of pristine TFC,
622
PSBMA, Ag-PSBMA, and PSBMA-Ag TFC membranes. (B) Atomic force microscopy (AFM) 3D images
623
of pristine TFC, PSBMA, Ag-PSBMA, and PSBMA-Ag TFC membranes. (C) Surface roughness
624
determined by AFM for pristine TFC, PSBMA, Ag-PSBMA, and PSBMA-Ag TFC membranes. The
625
roughness values of root mean-square (Rq) and average roughness (Ra) were calculated from AFM images
626
using at least six different locations on each membrane sample. (D) Water contact angles for pristine TFC,
627
PSBMA, Ag-PSBMA, and PSBMA-Ag TFC membranes. Contact angle measurements were obtained from
628
at least twelve random locations on each membrane sample. Error bars represent standard deviations.
629
21 ACS Paragon Plus Environment
Page 23 of 28
Environmental Science & Technology
630 631 632
Figure 3. Epifluorescence microscopy images of pristine and modified TFC membranes after adhesion of
633
fluorescein-labeled BSA (FITC-BSA). Pristine TFC, PSBMA, Ag-PSBMA, and PSBMA-Ag TFC
634
membranes were exposed to FITC-BSA in a PBS buffer solution (pH 7.4) for three hours at room
635
temperature.
636
22 ACS Paragon Plus Environment
Environmental Science & Technology
Page 24 of 28
637 638 639
Figure 4. (A) Antimicrobial properties of pristine TFC and modified membranes after exposure to P.
640
aeruginosa bacterial cells for three hours. The antimicrobial activity was expressed as the percentage of
641
colony-forming units (CFU) relative to that on the pristine TFC membrane (control). The horizontal dashed
642
line in the figure indicates the CFU value for PSBMA TFC membrane. Standard deviation error bars were
643
calculated from twelve independent replicates. (B) SEM images displaying the morphological
644
characteristics of P. aeruginosa cells on the surface of pristine, PSBMA-Ag, and Ag- PSBMA TFC
645
membranes after exposure.
646
23 ACS Paragon Plus Environment
Page 25 of 28
Environmental Science & Technology
647 648 649
Figure 5. Normalized permeate water flux due to biofouling by P. aeruginosa. Standard deviations
650
indicated by the error bars are the results of three independent biofouling experiments. Biofouling runs
651
were conducted at an initial water flux of 20 L·m-2·h-1. Cross-flow velocities of feed and draw solutions
652
were 4.25 and 9.56 cm·s-1, respectively. The initial bacterial concentration was fixed at 2×106 CFU·mL-1.
653
The synthetic wastewater solution medium (described in Materials and Methods) had an initial ionic
654
strength of 15.9 mM, electric conductivity of 1142 ± 50 μS, and pH 7.6 ± 0.2. Temperature was kept at 25.0
655
± 0.5 °C.
656 657 658
24 ACS Paragon Plus Environment
Environmental Science & Technology
Page 26 of 28
659 660 661
Figure 6. Confocal laser scanning microscopy (CLSM) orthogonal images of P. aeruginosa biofilm
662
structures developed on pristine TFC and PSBMA-Ag TFC membranes after biofouling (500 mL of
663
permeate volume). Top panels represent enlargements of the biofilm layer inside view. Biofilms were
664
stained with SYTO 9 (green), PI (red), and ConA (blue) dyes that specifically stain live cells, dead cells,
665
and polysaccharide–EPS, respectively.
666 667
25 ACS Paragon Plus Environment
Page 27 of 28
Environmental Science & Technology
668 669 670
Figure 7. Properties of silver-regenerated PSBMA-Ag TFC membranes. SEM images of PSBMA-Ag TFC
671
membranes (A) before and (B) after the regeneration of AgNPs. Prior to the regeneration, the original
672
PSBMA-Ag TFC membranes were soaked in water to allow a thorough leaching of silver. After silver
673
depletion, AgNPs were recharged on the surface of PSBMA-Ag membranes using the in situ formation
674
method described earlier. (C) Antiadhesive characteristics of pristine TFC and PSBMA-Ag TFC
675
membranes before and after regeneration of AgNPs. The epifluorescence microscopy images display the
676
adsorption of FTIC-BSA protein on membrane surfaces after exposure for three hours. (D) Antimicrobial
677
properties and water contact angle measurements of pristine TFC and PSBMA-Ag TFC membranes before
678
and after silver regeneration. The viability of P. aeruginosa was expressed as the percentage of CFU relative
679
to the pristine TFC membrane. Standard deviation error bars are the results of twelve independent replicates.
680 681 682 683 684 685
26 ACS Paragon Plus Environment
Environmental Science & Technology
Page 28 of 28
686
Table 1. Characteristics of P. aeruginosa biofilms grown on pristine TFC and PSBMA-Ag TFC membranes.
687
All parameters were calculated from confocal laser scanning microscopy (CLSM) images. Data are the
688
average of six random samples analyzed in triplicates. Average
“Live” cell
“Dead” cell
EPS
TOC
biofilm
biovolume
biovolume
biovolume
biomass
thickness (µm)
(µm3·µm-2)
(µm3·µm-2)
(µm3·µm-2)
(pg·µm-2)
TFC
33.7 ± 0.8
28.3 ± 1.6
16.5 ± 4.2
27.0 ± 3.4
1.69 ± 0.46
PSBMA-Ag
23.0 ± 5.3
14.8 ± 2.5
30.6 ± 5.6
10.7 ± 2.1
0.96 ± 0.38
689 690
27 ACS Paragon Plus Environment