Mitigation of Biofilm Development on Thin-Film Composite

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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

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Environmental Science & Technology

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Mitigation of Biofilm Development on Thin-Film

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Composite Membranes Functionalized with

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Zwitterionic Polymers and Silver Nanoparticles

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Environmental Science and Technology

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Revised: November 12, 2016

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Caihong Liu1, Andreia F. Faria2, Jun Ma1*, and Menachem Elimelech2, 3* 1

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State Key Laboratory of Urban Water Resource and Environment,

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Harbin Institute of Technology, Harbin 150090, China

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2

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Yale University, New Haven, Connecticut 06520-8286, USA

Department of Chemical and Environmental Engineering,

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Nanosystems Engineering Research Center for Nanotechnology-Enabled Water Treatment

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(NEWT), Yale University, New Haven,Connecticut 06520-8286, USA

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*Corresponding author. E-mail: [email protected] (J.M.); [email protected]

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(M.E.)

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ABSTRACT

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We demonstrate the functionalization of thin-film composite membranes with zwitterionic

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polymers and silver nanoparticles (AgNPs) for combating biofouling. Combining hydrophilic

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zwitterionic polymer brushes and biocidal AgNPs endows the membrane with dual functionality:

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anti-adhesion and bacterial inactivation. An atom transfer radical polymerization (ATRP) reaction

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is used to graft zwitterionic poly(sulfobetaine methacrylate) (PSBMA) brushes to the membrane

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surface while AgNPs are synthesized in situ through chemical reduction of silver. Two different

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membrane architectures (Ag-PSBMA and PSBMA-Ag TFC) are developed according to the

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sequence AgNPs and PSBMA brushes are grafted on the membrane surface. A static adhesion

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assay shows that both modified membranes significantly reduced the adsorption of proteins, which

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served as a model organic foulant. However, improved antimicrobial activity is observed for

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PSBMA-Ag TFC (i.e., AgNPs on top of the polymer brush) in comparison to Ag-PSBMA TFC

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membrane (i.e., polymer brush on top of AgNPs), indicating that architecture of the antifouling

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layer is an important factor in the design of zwitterion-silver membranes. Confocal laser scanning

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microscopy (CLSM) imaging indicated that PSBMA-Ag TFC membranes effectively inhibit

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biofilm formation under dynamic cross-flow membrane biofouling tests. Finally, we demonstrate

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the regeneration of AgNPs on the membrane after depletion of silver from the surface of the

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PSBMA-Ag TFC membrane.

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TOC Art

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INTRODUCTION

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Membrane-based processes are widely considered as sustainable technologies to supply clean

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water and address the problem of water scarcity that afflicts millions of people worldwide.1, 2 Thin-

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film-composite (TFC) membranes, the current state-of-the-art membrane technology for

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desalination processes, are the most robust membranes for water purification and desalination.2

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However, TFC membranes suffer from the problem of organic and biological fouling.3, 4 Biological

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fouling, or biofouling, involves complex mechanisms in which adhesion of organic molecules and

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microorganisms plays a crucial role.5-7 The growth of attached bacterial cells to biofilms leads to

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increased use of chemicals for cleaning, higher operation costs, and shorter membrane lifetime.8, 9

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Modification of membrane surfaces with hydrophilic polymers, such as polyethylene glycol10-

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12

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and reduce bacterial adhesion. Recently, zwitterionic polymers, including poly(sulfobetaine

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methacrylate) (PSBMA), have been applied as a promising class of antifouling agents in a range

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of industrial and biomedical applications.15-17 The chemical structure of betaine zwitterionic

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polymers contains negatively and positively charged residues at the same monomer unit.18, 19

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Because these opposite charges are evenly distributed throughout the polymer chain, zwitterionic

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polymers are considered as zero-charge molecules.15, 18 One of the most important characteristics

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of the zwitterionic polymer brush layer is its inherent ability to form a tight hydration layer via

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ionic solvation with the surrounding water molecules. This hydration layer serves as a steric and

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energetic barrier against the adsorption of organic and biological entities.17, 20

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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

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surface, but they cannot inactivate bacteria cells.4,

13, 21, 22

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establishing a “defensive” strategy, where very hydrophilic polymers offer protection against

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bacterial adhesion, several studies have proposed the fabrication of dual-function membranes

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through the combination of “offensive” and “defensive” strategies.23, 24 Offensive approaches rely

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on the use of strong antimicrobial agents, such as cationic quaternary ammonium (QACs) or

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biocidal nanoparticles, to inhibit bacterial proliferation.13,

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functionalization may emerge as a promising solution to biofouling in membrane-based

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technologies.

Therefore, contrary to simply

21, 25


This new concept of surface


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Over the past decades, silver nanoparticles (AgNPs) have received heightened attention due

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to their efficacy and broad-spectrum antimicrobial activity.12,

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antimicrobial agents, such as QACs, that inactivate bacterial cells through a contact-mediated

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mechanism, the toxicity of AgNPs is driven by the release of Ag+ ions. Although the toxicity of

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silver-modified membranes depends on the durability of AgNPs, the use of leachable nanoparticles

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has some advantages over contact-dependent antimicrobial agents.26-28 The physicochemical

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properties of AgNPs can be tailored to achieve improved reactivity. Because their mechanism of

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toxicity is dissolution-dependent, the antimicrobial properties of AgNPs are unlikely to be affected

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by the presence of other chemical foulants in the feed stream. Even though the dissolution property

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is occasionally considered a disadvantage, especially in terms of long-term efficiency, the

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regenerative capability of AgNPs has proven to be a key element of the preferential design of

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silver-modified membranes for biofouling mitigation.27

In contrast to organic

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Combining highly hydrophilic zwitterionic polymer brushes and biocidal AgNPs is an

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innovative route to optimize the surface chemistry of membranes to minimize the adhesion of

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organic foulants and bacteria and maximize the inactivation of bacterial cells. On a dual

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functionality surface, AgNPs can inactivate bacteria while zwitterionic polymer brushes shield the

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surface from adsorption of organic foulants. Such an approach can enable the fabrication of

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multifunctional membranes that can overcome technical challenges that have hampered the

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advancement of membrane-based processes. Despite efforts to develop membranes with

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antifouling or biofouling properties,24, 29 studies regarding the fabrication of membranes with

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simultaneous antiadhesive and bactericidal capabilities are still scarce.

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This paper demonstrates a new pathway for the fabrication of anti-biofouling TFC membranes

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by grafting zwitterionic polymer brushes and AgNPs to the membrane surface. We investigated

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the role of membrane surface functionalized layer architecture on the membrane antiadhesive and

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antimicrobial properties. Membrane surface architecture was found to strongly affect the

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antimicrobial property and the biofouling behavior of the functionalized TFC membranes. Our

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results suggest that functionalization of TFC membranes with zwitterionic polymer brushes and

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biocidal nanoparticles is an attractive strategy to mitigate biofouling in membrane-based processes.


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MATERIALS AND METHODS

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Materials and Chemicals. Dopamine hydrochloride, α-bromoisobutyryl bromide (BiBBr)

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(98%), N,N-dimethylformamide (DMF), tris(hydroxymethyl)aminomethane (Tris) (>99.8%),

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triethylamine (TEA) (>99%), [2-(methacryloyloxy)-ethyl]dimethyl-(3-sulfopropyl)ammonium

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hydroxide (also called sulfobetaine methacrylate, SBMA), copper-(II) chloride, tris(2-

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pyridylmethyl)amine (TPMA), L-ascorbic acid, isopropanol (IPA), silver nitrate (AgNO3) (≥99%),

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sodium borohydride (NaBH4) (99.99%), and phosphate buffered saline (PBS, pH 7.4) were

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purchased from Sigma-Aldrich. Fluorescein-conjugated BSA (FITC-BSA) (Life Technologies,

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A23015) was purchased from Thermo Fisher Scientific. Commercial thin-film composite (TFC)

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forward osmosis (FO) membranes were kindly provided by Porifera (Porifera, Inc., CA). Prior to

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use, the membranes were wetted for 30 minutes in 25% isopropanol solution, rinsed with deionized

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(DI) water for approximately four hours, and stored at 4 °C until use. A Milli-Q ultrapure water

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purification system (Millipore, Billerica, MA) was used to supply DI water.

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TFC Membrane Functionalization Pathways. ARGET-ATRP (activators regenerated

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by electron transfer-atom transfer radical polymerization) was employed to modify the TFC

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membranes with zwitterionic polymers. PSBMA was grafted on the TFC membrane as previously

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reported.4, 30 First, dopamine hydrochloride (800 mg, 2.10 mmol) was dissolved in DMF (40 mL)

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and the mixture was transferred to a sealed amber bottle which was bubbled with nitrogen gas.

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After 20 minutes, BiBBr (0.26 mL, 1.05 mmol) and TEA (0.3 mL, 1.05 mmol) were added. The

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BiBBr-initiator-dopamine solution was then left stirring under nitrogen atmosphere for three hours

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at room temperature. The pristine TFC membranes were placed on a stirring plate at 60 rpm after

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being sandwiched between a clean glass and a rubber frame (inner hole size of 10 cm × 6.5 cm)

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with the membrane active layer facing up. The prepared BiBBr-initiator-dopamine solution was

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diluted with 200 mL of aqueous Tris buffer (pH 8.5, 2.0 mmol) and then immediately exposed to

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the active layer of the membrane for 10 minutes. After exposure, the membrane was thoroughly

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rinsed with DI water to remove excess reactants.

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For the polymer binding, SBMA monomer (15.64 g, ~ 56 mmol) was dissolved in 200 mL of

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an isopropanol-water solution (1:1 v/v) in a sealed glass bottle covered with aluminum foil. After

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bubbling the dispersion with nitrogen gas for 10 minutes, a mixture of CuCl2 (0.004 g, ~5.92 µmol)

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and TPMA (0.056 g, ~0.038 mmol) in isopropanol aqueous solution (1:1 v/v, 8 mL) was 4 ACS Paragon Plus Environment


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introduced into the sealed bottle using a syringe. Next, the membranes (previously exposed to

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BiBBr-initiator-dopamine) were placed into the glass bottle and kept in contact with the SBMA

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dispersion for another 10 minutes under nitrogen atmosphere. Then, 12 mL of an ascorbic acid

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solution (1 g in 10 mL of 1:1 isopropanol/water) were syringed into the glass bottle to initiate the

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polymerization. After one hour of polymerization, the bottle was opened to air to terminate the

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ARGET-ATRP reaction and the membrane was excessively rinsed with DI water. The PSBMA

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modified membrane (PSBMA TFC hereafter) was stored in aqueous isopropanol (10% v/v) at 4°C.

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After functionalizing the membranes with PSBMA polymers, silver nanoparticles (AgNPs)

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were nucleated on the surface through an in situ synthesis previously described by Ben-Sasson et

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al.28 Briefly, the membranes were immobilized between a clean glass and rubber frame (inner hole

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size of 10 cm × 6.5 cm) so that only the active layer of the membrane was available for

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functionalization. AgNO3 solution (15 mL, 5 mmol) was allowed to contact the membrane surface

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for 10 minutes. Subsequently, the AgNO3 solution was removed, leaving a thin layer of AgNO3

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solution on the membrane surface. NaBH4 solution (15 mL, 5 mmol) was then added to the

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membrane surface to react with the nucleated Ag+ ions, leading to the formation of AgNPs. After

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five minutes of reaction, the NaBH4 solution was discarded and the AgNPs-modified membranes

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were rinsed with DI water for approximately 10 seconds. This procedure was used for the

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preparation of PSBMA-Ag TFC membranes. Similarly, the Ag-PSBMA TFC membranes were

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prepared by first modifying TFC membranes with AgNPs and subsequently grafting PSBMA

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using the same conditions described above. A schematic diagram for the preparation of the Ag-

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PSBMA TFC and PSBMA-Ag TFC membranes is shown in Figure 1.

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FIGURE 1

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Membrane Surface Characterization. Scanning electron microscopy (SEM Hitachi SU-

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70 FE-SEM, Hitachi High Technologies America, Inc.), atomic force microscopy (AFM, Bruker

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Dimension Fastscan AFM, Bruker Corp., Santa Barbara, CA), and contact angle measurements

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(OneAttension contact angle meter, Biolin Scientific, Finland) were used to characterize the

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surface properties of pristine and modified membranes. For SEM imaging, membrane samples

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were dried and sputter coated with 16 nm of chrome. Surface roughness was obtained from AFM

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by using a Scanasyst-air silicon probe (Bruker Nano, Inc., Camarillo, CA) in a peak force tapping

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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

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radius of 2 nm, and length of 115 µm. Water contact angle measurements were performed to

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evaluate the surface hydrophilicity of the pristine and modified membranes. To obtain

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representative measurements, water contact angles were acquired from at least twelve random

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locations on each membrane surface. All membranes were air-dried prior to the measurements.

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The intrinsic membrane transport properties, namely water permeability coefficient (A), salt

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permeability coefficient (B), and structural parameter (S), were determined in a laboratory-scale

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cross-flow unit operated in FO mode, as reported elsewhere.31 Observed values are presented in

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Figure S1. Specifically, for pristine membrane, A = 2.26 ± 0.2 L·m-2·h-1·bar-1 and B = 0.64 ± 0.07

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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 ±

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0.05 L·m-2·h-1. Streaming potential measurements (EKA, Brookhaven Instruments, Holtsville, NY)

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were performed to determine the zeta potential of the membrane surface using an electrolyte

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solution (1 mM KCl and 0.1 mM KHCO3) at a pH range that varied between 3 and 9. Zeta

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potentials for pristine and PSBMA-Ag TFC membrane are shown in Figure S2.

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Protein Adhesion Assay. The antiadhesive properties of the modified membranes were

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evaluated by a static protein adsorption assay using bovine serum albumin (BSA) as a model

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organic foulant. A protein solution (0.05 mg·mL-1) was prepared by dissolving fluorescein-

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conjugated BSA (FITC-BSA) in PBS solution (pH 7.4). Membrane coupons (2.1 cm in diameter)

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were cut and mounted in custom-made membrane cell holders. FITC-BSA solution (3 mL) was

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then added into each cell holder and left to contact the membrane surface for three hours under a

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mild stirring in the dark. Next, the solution was discarded and the membrane surfaces were rinsed

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twice with PBS buffer to remove unbounded proteins. The membrane coupon was then removed

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and placed on a glass slide with a droplet of PBS buffer on top, covered by a cover glass and sealed

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with nail polish. The samples were imaged using an inverted Axiovert 200 M epifluorescence

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microscopy (Carl Zeiss Inc., Thornwood, NY, USA).

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Antimicrobial Activity Experiments. The antimicrobial properties of the pristine and

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modified TFC membranes were evaluated using Pseudomonas aeruginosa (P. aeruginosa, ATCC

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BW27853, American Type Culture Collection) as a model microorganism. Bacteria were

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cultivated overnight in Lysogeny broth (LB) at 37 °C. The bacteria suspension (1 mL) was

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transferred to fresh LB media (24 mL) and grew for two to three hours to exponential phase. The

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culture was centrifuged and the pellet was washed three times with sterile saline solution (NaCl, 6 ACS Paragon Plus Environment


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0.9 wt %) to remove the excess macromolecules. After washing, the microbial cells were re-

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suspended in saline solution to reach a concentration of 108 colony-forming units per milliliter

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(CFU·mL-1).

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Membrane coupons with area of ~3.5 cm2 were cut and the active side of the membrane was

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placed in contact with the bacterial suspension (3 mL) for three hours at room temperature. After

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incubation, the bacteria suspension was discarded and the membrane surface was rinsed twice with

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saline solution. To detach deposited bacteria from the membrane surface, membrane coupons were

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transferred into 50 mL falcon tubes containing 10 mL of saline solution, and the tubes were bath-

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sonicated for 10 minutes. Serial dilutions of the cell suspension were plated on LB plates and

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incubated overnight at 37 oC. After 24 hours of growth, the colonies were counted to evaluate the

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number of viable cells on silver-functionalized membranes compared to that on the pristine TFC

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membrane.

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Assessing Biofouling Behavior in a Membrane Cross-flow System. Dynamic

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biofouling experiments were carried out to evaluate the resistance of PSBMA-Ag TFC membranes

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against biofilm formation by P. aeruginosa, as previously described.9, 32, 33 Prior to the experiments,

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the FO system was sequentially cleaned and disinfected with bleach solution (10%, v/v), EDTA

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solution (5 mM, pH 7), and pure ethanol. Each of the cleaning solutions was circulated throughout

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the entire unit for one hour. To remove any trace of these chemical compounds, the FO system

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was thoroughly rinsed three times with DI water.

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P. aeruginosa was cultivated in LB medium overnight at 37 oC to an optical density (OD600)

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of 0.6. The bacterial culture (50 mL) was centrifuged at 4000 rpm, 4 oC for 20 minutes and the

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pellet was re-suspended in a sterile synthetic wastewater (10 mL). The synthetic wastewater was

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prepared according to previous studies (8 mM NaCl, 0.15 mM MgSO4, 0.5 mM NaHCO3, 0.4 mM

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NH4Cl, 0.2 mM CaCl2, 0.2 mM KH2PO4 and 0.6 mM glucose, with ionic strength of 16 mM, pH

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7.6 ± 0.2) 9, 33 and used as feed solution while a NaCl solution (stock solution: 5M) was applied as

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draw solution. After stabilization, the NaCl stock solution was used to adjust the initial water flux

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at 20 L·m-2·h-1, and the initial bacteria concentration was ~ 2 × 106 CFU·mL-1. Temperature was

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kept constant at 25 oC. Feed wastewater was monitored periodically for number of viable bacteria

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cells, pH, and conductivity. Before each biofouling run, a baseline experiment at the same


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condition (without the addition of bacteria) was performed. The baseline curve was used to subtract

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the dilution effect of the draw solution and reverse salt diffusion of FO itself.32, 34

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Biofilm Characterization. At the end of each biofouling experiment (500 mL of permeate

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water accumulation), subsections of the membrane coupons were cut and characterized for the

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composition of biofilm using confocal laser scanning microscopy (CLSM) and total organic

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carbon (TOC) measurements.

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For CLSM analysis, membrane subsections (1 cm × 1 cm) were cut from the center of the

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biofouled membranes and stained with SYTO 9, propidium iodide (PI) (LIVE/DEAD BacLight,

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Invitrogen), and concavalin A (Con A, Alexa Flour 633, Invitrogen). SYTO 9, PI, and Con A are

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known to specifically stain live cells, dead cells, and polysaccharides–extracellular polymeric

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substances (EPS), respectively. After 40 minutes of contact in the dark, the membrane coupons

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were rinsed three times with sterile wastewater to remove unbound stains.

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The stained samples were placed on a custom-built chamber for CLSM imaging.9, 35 A CLSM

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microscopy (Zeiss LSM 510, Carl Zeiss, Inc.) equipped with a plan-apochromat was used to

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capture confocal images of the biofilm. SYTO 9, PI, and Con A were excited with 488 nm argon,

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561 nm diode-pumped solid state, and 633 nm helium-neon laser, respectively. At least six Z stack

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random fields (635 µm × 635 µm) with a slice thickness of 2.14 µm were collected from each

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sample to obtain a representative biofilm orthogonal image. At least eight smaller stack regions

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(90 µm × 90 µm) with a slice thickness of 1.2 µm were captured for biofilm dimension calculation.

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Confocal images were analyzed using Auto-PHLIP-ML, Image-J, and MATLAB software, as

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suggested by previous publications.9, 32, 33, 35 Biovolume and thickness were determined for the live

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cells, dead cells, and EPS of the biofilm for all the samples. Average biofilm thickness was

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calculated by averaging the thicknesses of these three components.

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For TOC measurements, membrane subsections (1cm × 1cm) were cut from the biofouled

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membranes and transferred to 25 mL glass vials containing 20 mL of DI water and 4 µL of a 1 M

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HCl solution. The vials were probe-sonicated for ~3 minutes to remove the organic content from

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the membrane surface and TOC measurements were carried out at a TOC analyzer (TOC-V,

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Shimadzu). TOC concentrations were normalized according to the membrane coupon size (TOC

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mass per membrane area). 8 ACS Paragon Plus Environment


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Silver Release and Regeneration Capacity. The experiments for the release of silver

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was performed via a reservoir method.28 PSBMA-Ag TFC membrane coupons (2 cm2) were

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incubated in glass vials containing 10 mL of 5mM NaHCO3 solution (pH 8.3, without HNO3). The

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vials with the membrane coupons were placed on a stir plate at 60 rpm. At specific intervals of

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time, the membrane coupons were withdrawn and transferred to fresh DI water acidified with 0.1

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mL of HNO3 (70%). The content of silver on the membrane coupons was thoroughly dissolved in

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the acid solution after 24 hours of agitation. Then the supernatant was collected and the silver ion

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concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS, ELAN

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DRC-e ICP Mass Spectrometer, Perkin Elmer). The experiments were conducted in duplicates.

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To demonstrate the regeneration of AgNPs, we proposed the deposition of AgNPs on the

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membrane surface after a vigorous leaching process. Specifically, after a thorough release of silver

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ions in DI water, AgNPs were regenerated on the surface of PSBMA Ag-TFC membranes using

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the same in situ method described in this section earlier. The recharged PSBMA-Ag TFC

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membranes were re-characterized by SEM imaging, contact angle measurements, and static

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protein adsorption assay. In addition, the antimicrobial properties of PSBMA-Ag TFC membranes

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were re-evaluated after silver regeneration.

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RESULTS AND DISCUSSION

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Membrane Surface Characteristics. TFC membrane surface morphologies were investigated

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by SEM imaging (Figure 2A). A smooth film was observed on the membrane surface after PSBMA

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zwitterionic polymer brushes were grafted on the polyamide layer (PSBMA TFC). This

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observation indicates that PSBMA brushes were successfully polymerized on the membrane

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surface. The immobilization of PSBMA polymer brushes on the membrane surface was conducted

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by ATRP, a versatile technique that allows the growth of monomers into polymer chains using

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transition metal complexes as catalysts.36 Compared to conventional strategies for surface

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modification, such as chemical crosslinking,37 ATRP results in the growth of a denser and more

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uniform layer of polymer brushes with controllable thickness and architecture.38-40

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FIGURE 2


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After the in situ formation of AgNPs, different morphological structures were visualized. For

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the Ag-PSBMA TFC membrane, where AgNPs were loaded before the grafting of PSBMA

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brushes, SEM images show a thin polymer film on the membrane top surface. Because the particles

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are covered by the polymer brush layer, we could not attribute the observed morphology to the

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AgNPs. Conversely, when AgNPs were formed after PSBMA polymerization (PSBMA-Ag TFC),

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the presence of AgNPs on the top surface was noticeable, whereas the PSBMA film was less

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distinguishable compared to the Ag-PSBMA membrane. The AgNPs displayed a round-like

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morphology and were well distributed throughout the membrane surface.

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To investigate changes in surface roughness due to surface modification with PSBMA or

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AgNPs, the membranes were characterized by AFM imaging. Figures 2B and 2C show

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representative 3D AFM images and calculated roughness parameters for pristine and modified

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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

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2C). However, after impregnation of AgNPs, the surface roughness significantly increased for both

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Ag-PSBMA and PSBMA-Ag TFC membranes. For instance, Rq parameters for Ag-PSBMA and

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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

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membrane surfaces, while AgNPs contributed to increased surface roughness.

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Membrane surface hydrophilicity was evaluated by sessile water contact angle measurements

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(Figure 2D). The membrane contact angle was reduced from 74° ± 10° to 21° ± 7° after

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functionalization of pristine TFC membrane with PSBMA polymer. The marked decrease in

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contact angle is attributed to the strong electrostatic interaction of zwitterionic PSBMA brushes

298

with water molecules.15,

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hydrophilicity compared to the pristine TFC membrane. These observations suggest that the

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sequence of AgNPs deposition on the membrane surface did not have a significant impact on

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membrane hydrophilicity. In contrast, membranes modified only with AgNPs did not exhibit

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change in contact angle compared to pristine TFC membranes (Figure S3), in agreement with a

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previous investigation.28

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Similarly, Ag-PSBMA and PSBMA-Ag also showed improved



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Grafted Zwitterionic Brushes Impart Antiadhesive Property to the Membrane.

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Antiadhesive properties of the modified membranes were determined via a static protein assay

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using bovine serum albumin (BSA) protein as a model organic foulant. Proteins are ubiquitous in

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wastewater effluent and surface waters, and their accumulation on the membrane surface

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deteriorates membrane performance. Proteins also play an important role during biofouling by

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forming a conditioning film7 and providing a source of carbon and nitrogen for the growth of

310

microorganisms.42

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For the protein adhesion assay, a fluorescein-conjugated BSA (FITC-BSA) solution was

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allowed to contact the active layer of the pristine and modified membranes for three hours in the

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dark. The acquired fluorescence images for the pristine and functionalized TFC membranes are

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presented in Figure 3. The fluorescence intensity is directly related to the amount of proteins

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attached on the membrane surface after exposure to FITC-BSA. High fluorescence intensity was

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observed for the pristine TFC membrane, indicating significant adsorption of proteins. PSBMA

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TFC membranes, on the other hand, showed nearly no fluorescence intensity, demonstrating that

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the membrane successfully prevented the adsorption of BSA. This observation is attributable to

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the thick hydration layer on the zwitterionic polymer brushes that prevents the adsorption of

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organic foulants, including proteins.15, 43-46 Surprisingly, both Ag-PSBMA and PSBMA-Ag TFC

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membranes suppressed BSA adsorption, regardless of the membrane architecture. Contrary to

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what would be expected, the grafting of AgNPs over the PSBMA brushes did not interfere with

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the antiadhesive properties of PSBMA-Ag membranes.

324

FIGURE 3

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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


Page 13 of 28


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


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

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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


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


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



Page 18 of 28

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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.



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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.

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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



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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


Page 23 of 28


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



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


Page 25 of 28


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



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


Page 27 of 28


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



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


Mitigation of Biofilm Development on Thin-Film Composite

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