Antibiofouling Polyvinylidene Fluoride Membrane Modified by

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Anti-biofouling Polyvinylidene Fluoride Membrane Modified by Quaternary Ammonium Compound: Direct Contact-killing versus Induced Indirect Contact-killing Xingran Zhang, Jinxing Ma, Chuyang Y. Tang, Zhiwei Wang, How Yong Ng, and Zhichao Wu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00902 • Publication Date (Web): 22 Apr 2016 Downloaded from http://pubs.acs.org on April 25, 2016

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Environmental Science & Technology

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Anti-biofouling Polyvinylidene Fluoride Membrane Modified by

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Quaternary Ammonium Compound: Direct Contact-killing versus

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Induced Indirect Contact-killing

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Xingran Zhang1, Jinxing Ma1, Chuyang Y. Tang2, Zhiwei Wang*,1, How Yong Ng3, Zhichao

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Wu1

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1

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Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China

State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental

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2

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China

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3

13

University of Singapore, 1 Engineering Drive 2, Singapore 117576, Singapore

Department of Civil Engineering, The University of Hong Kong, Pokfulam, Hong Kong,

Centre for Water Research, Department of Civil and Environmental Engineering, National

14 15 16 17 18

Revised Manuscript submitted to: Environmental Science & Technology

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April 11, 2016

20 21 22 23 24

*Corresponding author: Zhiwei Wang, E-mail: [email protected], Tel./fax: +86(21)65980400

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ACS Paragon Plus Environment


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ABSTRACT

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Widespread applications of membrane technology call for the development of anti-biofouling

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membranes. For the traditional contact-killing strategy, the antibacterial action is restricted to

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the surface: the membrane loses its anti-biofouling efficacy once its surface is completely

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covered with a fouling layer. However, in this study, polyvinylidene fluoride (PVDF)

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microfiltration membranes blended with quaternary ammonium compound (QAC) exhibited

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a surprisingly lasting antimicrobial activity in the vicinity of the membrane surface. The

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results indicated that QAC was capable of driving surface segregation with a high structural

34

stability, and the QAC modified membrane shows clear antibacterial effects against both

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Gram-positive and Gram-negative bacteria. Covering the modified membrane surface by an

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abiotic alginate layer resulted in a loss of antibacterial efficiency by 86.2%. In contrast, the

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antibacterial efficiency was maintained after developing a biofilm of Staphylococcus aureus

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of 30 µm in thickness. The current study may suggest that bacteria affected by contact-killing

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might interact with other bacteria in the vicinity, resulting in retarded biofilm growth. The

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anti-biofouling effect and associated mechanism of the QAC modified membrane were

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further validated in a membrane bioreactor during long-term operation.

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INTRODUCTION

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In the last two decades, membrane separation technology has been widely used in

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advanced water and wastewater treatment.1,2 However, membrane fouling, particularly

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biofouling, results in frequent membrane cleaning and/or replacement and thus significantly

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increases the overall treatment cost.3-5 Biofouling, i.e., the bacterial colonization on

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membrane surface and the development of a cohesive biofilm, is a thorny practical

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problem.6,7 Biofouling can be mitigated through the pretreatment of feed solution and the

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control of membrane operational conditions (e.g., operating a membrane under moderate

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flux). A more attractive alternative is to develop anti-biofouling membranes, since this

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approach has a less significant impact on the cost and operation of the membrane process.8

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Current strategies for preparing anti-biofouling membranes involve graft polymerization

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on the membrane surface and in situ membrane surface modification under fabrication, while

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the latter is more efficacious because the antimicrobial layers or antimicrobial agent on the

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membrane surfaces have higher structural stability and lower partial blocking of surface

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pores.9-11 Numerous studies have shown that antimicrobial polymers, nanoparticles (e.g.,

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silver nanoparticles) and enzymes embedded on the membrane surfaces are capable of

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inhibiting bacterial proliferation by disrupting the cell membranes of microorganisms that are

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contacting, and leading to cell death (termed contact-killing).12-16 Meanwhile, there is another

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possibility that these bound agents can leak out into the boundary layer above the membrane

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surface to form an inhibition zone (termed release-killing).17-19Although continuous release of

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biocides can cause an inhibition of microbial adhesion in the vicinity of membrane surfaces,

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the difficulties in triggering and controlling the release process in the microfiltration or

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ultrafiltration processes are further complicated with the released agents being able to readily

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pass through the large pores, resulting in (i) lower concentrations of biocides in the vicinity of

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membrane surface, (ii) shorter lasting period and (iii) severe concerns that biocides in the 3



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permeate are likely to cause an environmental risk.18,20 Therefore, particular attention has

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recently been given to developing and advancing membrane matrix and antimicrobial agents

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to bring about better contact-killing properties such as stable and lasting anti-biofouling

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activities.21-24

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With respect to contact-killing of bacteria, the primary mechanisms for the antimicrobial

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agents have been well addressed, i.e., bacterial disorganization begins with the agents binding

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to cell membranes by ionic and/or hydrophobic interactions25-27. Therefore, it is always taken

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for granted that contact-killing is restricted to membrane surfaces and may lose its efficacy

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once the membranes are covered with a fouling layer.12 However, in this study, we

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surprisingly found that the modified polyvinylidene fluoride (PVDF) microfiltration

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membranes, which were blended with quaternary ammonium compound (QAC, a kind of

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biocide) through a phase inversion method, exhibited a lasting antibacterial activity even

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when biofouling layers were formed on the membrane surfaces. Despite the possibility that a

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small amount of QAC might be released into the solution attaining release-killing effect, the

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results enabled us to put forward a hypothesis that the affected bacteria on the surface might

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further interact with those in the vicinity, finally retarding cell deposition and biofilm growth.

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We elucidate this hypothesis in the present work by examining the anti-biofouling

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properties of the modified PVDF microfiltration membranes using model bacterial

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suspensions containing Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and

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also in a membrane bioreactor. In the meantime, QAC release and its contribution to the

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overall antibacterial activity were monitored. Key questions addressed in this study include:

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(i) which strategy, e.g., contact-killing or release-killing, accounts for the anti-biofouling

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behaviours of QAC blended PVDF membranes? (ii) how will the antibacterial activity change

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once the membrane surface is covered with different foulants? and (iii) what scenario is likely

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prevailing in the vicinity of membrane surface if contact-killing dominates? 4


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MATERIALS AND METHODS

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Reagents. All chemicals used in this work were of analytical reagent grade unless

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otherwise stated. Commercial grade PVDF was purchased from Solvay Corporation (Solef®

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6020, Mw = 670~700 kDa). Dimethyl acetamide (DMAC) and dimethyl sulfoxide (DMSO)

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used as solvents and polyvinyl pyrrolidone (PVP, Mw = 40 kDa) as a pore-forming additive

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were obtained from Sinopharm (Shanghai, China). Dodecyl dimethyl benzyl ammonium

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chloride (DDBAC) was used as the model QAC due to its efficient antibacterial activity and

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low toxicity28, which was purchased from Sigma Aldrich. Sodium alginate was received from

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Sigma Aldrich. All solutions were prepared using deionized (DI) water. Solution pH was

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adjusted by the addition of 1 M NaOH and 1 M HCl when necessary. McFarland bacterial

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suspensions of Gram-negative Escherichia coli (E. coli, ATCC25922) and Gram-positive

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Staphylococcus aureus (S. aureus, ATCC6538) were prepared by dispersing the colonies from

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agar slants (Shanghai Weike Biotechnology Corporation) in sterile saline.

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Membrane Preparation. Membranes used in this study were prepared by a phase

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inversion method via immersion precipitation. The detailed chemical composition for

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membrane preparation can be found in Supporting Information (SI) Table S1. Briefly, upon

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drying at 80 °C for 24 h to eliminate moisture, a predetermined amount of PVDF and PVP

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were dissolved in a solvent mixture comprised of 50 vol.% DMAC and 50 vol.% DMSO. The

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suspension was agitated at 80 °C for 48 h to obtain a homogeneous solution. Meanwhile, a

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QAC solution was prepared by completely dissolving a pre-weighted amount of DDBAC in a

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DMAC/DMSO solvent mixture. Subsequently, the two solutions were mixed and stirred at 5



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80 °C overnight followed by degassing under ultrasonication for at least half an hour. Finally,

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the dope solution was casted uniformly on a non-woven support (Shanghai Tianlue Advanced

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Textile Co., Ltd.) with a casting knife gap of 250 µm. The casted films were immersed in a

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deionized water bath at room temperature. The resulting pristine and QAC (0.2 wt.%)

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blended PVDF membranes were denoted as MP and MQ, respectively.

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Membrane Characterization. To determine the surface composition of membranes,

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X-ray photoelectron spectroscopy (XPS, AXIS UltraDLD, Kratos Analytical Ltd., U.K.) was

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performed with the calibration using C 1s = 284.6 eV as a reference. Zeta potential of the

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membrane surface was measured by a streaming potential analyzer (EKA 1.00, Anton-Paar,

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Swiss) at a controlled temperature (25 ± 1 °C) in 10 mM KCl according to a procedure as

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described elsewhere.29 Surface hydrophilicity of the membranes was determined by sessile

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drop contact angle measurement of water on membranes.30 Each value is shown as the

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average of at least seven measurements. Measurements of water permeability using DI water

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were conducted according to the protocol described previously.31 Each reported value was

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expressed by averaging three measurements.

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Antibacterial Behaviour Evaluation. A schematic illustrating the antibacterial test

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procedures for clean membrane coupons can be found in SI Figure S1. Membrane coupons

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(MP and MQ, active surface area = 2.8 cm2) were rinsed with sterile phosphate buffered

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saline (PBS) (0.01 M, pH = 7.4) thrice followed by UV sterilization for 30 min. The coupons

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were then placed in a 12-well plate with the addition of 2 mL of nutrient broth (Sinopharm)

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and 20 µL of bacterial suspension (about 107 cells mL-1). Subsequently, the plate was

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incubated in a dark rotary shaker at 100 rpm at 37 °C. Growth curves of E. coli and S. aureus 6


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were measured by quantifying the optical density values of culture solutions at 600 nm (i.e.,

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OD600)32 using a spectrophotometer (TU-1810, PERSEE, China) after different incubation

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time (t = 0, 3, 6, 18 and 24 h). Each reported value was obtained by averaging 24 individual

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measurements. Data of the exponential growth phases (3 to 24 h for E. coli and 6 to 24 h for S.

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aureus) was fitted by the pseudo-first-order kinetics equations (Eqs. 1 and 2): ln

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Xt = µt X0

(1)

ln 2 µ

(2)

τ d=

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where Xt and X0 are the OD600 values representing the bacterial concentrations at t h and 0 h

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respectively, µ (h-1) the specific growth rate and τd (h) the doubling time. After 24 h of

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incubation, the membrane coupons were withdrawn and rinsed three times with sterile PBS to

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remove any non-adhesive or loosely adhesive bacteria. Visualization of the attached bacteria

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was performed using a scanning electron microscope (SEM, SU8010, Hitachi, Japan) as

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described previously.33

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Additional tests were performed to determine whether release-killing is the prevailing

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mechanism responsible for the antibacterial behaviours of MQ. Briefly, sterile membrane

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coupons (active area of 2.8 cm2) were placed in 12-well plates containing 2 mL of nutrient

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broth in each well. At predetermined time intervals, the membrane was removed from the

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well and 20 µL of S. aureus bacterial suspension (about 107 cells mL-1) was added with

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subsequent incubation in a dark shaker at 100 rpm at 37 °C for 24 h to test the antibacterial

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activity of solution phase (which had prior exposure to the membrane, see further details in SI

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Figure S1). In addition, antibacterial activity of these pre-soaked membrane coupons was also 7



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assessed by re-immersing the samples in the solution containing 2 mL of nutrient broth and

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20 µL of bacterial suspension, which was then incubated under the same conditions as

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described above (SI Figure S1). The antibacterial efficiencies (Ea, %) of the solution phase

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and the pre-soaked membrane coupons were calculated as follows:

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E a (%) =

∆OD MP − ∆ODt × 100 ∆OD MP

(3)

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where ∆ODMP represents the increase of OD600 value in the blank culture solution in which

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MP sample (without QAC, i.e., the control) was used, following 24 h of incubation. ∆ODt

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indicates the increase of OD600 value in the culture solution either (i) upon contact with MQ

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for t or (ii) containing MQ coupon that has been pre-soaked in nutrient broth for t, following

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24 h of incubation.

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The production of intracellular reactive oxygen species (ROS) by bacteria upon

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exposure to aqueous QAC and QAC blended membrane (MQ) was measured by a ROS

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detection kit containing H2DCF-DA (Life Technologies, U.S.).34 The specific procedure is

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shown in SI Section S1.

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Antibacterial activity of pre-fouled MQ membranes was further evaluated, using sodium

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alginate and S. aureus as the model foulants representing organic and biological contaminants,

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respectively (see the determination procedures in SI Figure S1). After UV irradiation and

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immersion in sterile water for 4 h to remove any unbound QAC, MQ coupons of 1.1 cm2

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were subject to the deposition of 0.1 g L-1 alginate solution (5 mL) or 2×108 S. aureus cells

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mL-1 (1 mL) bacterial suspension through dead-end filtration, respectively. Confocal laser

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scanning microscopy (CLSM, Nikon A1, Japan) was employed to characterize the thickness

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of cake layers.35 The viable and dead cells were stained using LIVE/DEAD® BacLightTM 8


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Bacterial Viability Kits (Molecular Probes, Inc.) as described by He et al.36 Concavalin A

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(Con A, Molecular Probes, Inc.), a kind of fluorescent markers, was used to label

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α-glucopyranose polysaccharides.37 Ea of the MQ covered with different cake layers was then

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evaluated and calculated according to Eq. 3.

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Long-term Anti-biofouling Performance. The long-term performance of MP and MQ

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were evaluated in a submerged membrane bioreactor (MBR) with an effective volume of 5 L.

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Details about this reactor can be found in SI Section S2 and Figure S2. Prior to the

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experiments, the MQ membrane was subject to 4-h soaking in sterile DI water to remove any

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unbound QAC. At the end of each operation cycle, cake layers were carefully scraped off

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from the fouled membrane modules and mixed well with DI water before analysis.38 The

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amount of solids deposited on membrane surface was determined according to the protocol

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reported in literature by quantifying the suspended solids on membranes of a certain surface

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area.39 Quantitative real-time polymerase chain reaction (qRT-PCR) was also carried out to

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characterize the total amount of bacteria on the membrane surface according to the method

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reported by Zhang et al.40. The primers for amplification of 16S rRNA genes were EUB338F

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(5’-ACTCCTACGGGAGGCAGCAG-3’) and EUB518R (5’-ATTACCGCGGCTGCTGG-3’).

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Further information on amplification procedure can be found in SI Section S3. Visualization

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of the spatial structures of the cake layers was performed by CLSM using LIVE/DEAD®

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BacLightTM Bacterial Viability Kits as probing stains.

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RESULTS AND DISCUSSION Intrinsic Membrane Properties. Figure 1A presents the typical XPS spectra of MP, MQ 9



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and pure QAC, respectively. Strong signals at 685 eV in the spectra of MP and MQ were

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associated with F 1s orbital, which is solely contributed by PVDF. Elemental balance analysis

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showed that PVDF accounted for 76.7 and 72.4 wt.% of the surface compositions of MP and

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MQ, respectively. Presence of other elements including carbon (C 1s), nitrogen (N 1s) and

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oxygen (O 1s) was generally expected due to their omnipresence in solvents, additives and

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the QAC biocide. In view of the chemical composition of casting solution (SI Table S1), the

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XPS spectrum of the QAC-loaded membrane MQ can be considered as the superposition of

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those of MP and QAC. Based on XPS data (see SI Table S2), the mass concentration of QAC

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at the surface of MQ was approximately 5.4 wt.% (see detailed calculation in SI Section S4).

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The corresponding QAC to PVDF mass ratio of 0.075 was 3-fold of the bulk ratio of 0.025

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(SI Section S4). The major difference in the surface and bulk composition suggests that

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surface segregation of QAC occurred, possibly because the hydrophobic alkyl chains of

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amphiphilic quaternary ammonium compounds were capable of driving surface-concentrating

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via segregation during the casting process (exposure to air) and the hydrophilic head induced

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segregation during the following phase separation process.41

10


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N+

F 1s Cl-

MQ

C 1s N 1s O 1s

MP QAC

0

100

200 400 600 Binding energy (eV)

800

50

(B)

40 30 20 10 0 0

(C)

MP

MQ

(D)

-3 Zeta potential (mV)

80 Contact angle (° )

2

Normalized XPS spetra

(A)

Water permeability (L/m h kPa)

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60 40 20 0

MP

-6 -9 -12 -15 -18

MQ

MP

MQ

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Figure 1. Blending of QAC into the PVDF membrane and its influence on membrane properties. (A) XPS

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spectra for pristine membrane (MP, black line), QAC blended membrane (MQ, red line) and pure QAC

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(blue line); (B) Water permeability (n = 3); (C) Contact angle of DI water on membranes (n = 7); (D) Zeta

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potential (n = 3). The inset figure on Figure 1A is the chemical structure of DDBAC.

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QAC blending did not significantly affect the intrinsic membrane permeability (37.2 ±

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1.8 and 34.1 ± 3.2 L m-2 h-1 kPa-1 for MP and MQ, respectively, see Figure 1B). Besides, the

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membrane hydrophilicity was slightly decreased (Figure 1C), with the measured contact

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angles of 71.0 ± 1.1 and 79.3 ± 2.8 for MP and MQ, respectively. The increase of

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hydrophobicity of the membrane surface may be attributed to the alkyl chains of QAC and

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the increase of roughness for MQ (Figure S3A), consistent with the surface-concentrating of

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QAC based on the XPS analysis. Figure 1D shows that membrane surface charge was

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significantly altered by QAC blending: -14.5 ± 0.8 mV for MP and -5.7 ± 3.2 mV for MQ at

o

o

11



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pH = 7.4. The less negative charge of MQ may be attributed to the coverage of the membrane

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surface by the positively charged QAC.42-44 With the introduction of QAC, porosity and pore

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size of MQ was also increased compared to those of MP (Figure S3B and Figure S3C).

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Antibacterial Activity of QAC Blended Membrane. Model bacteria strains of E. coli

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(Gram-negative) and S. aureus (Gram-positive) were chosen to test the membrane

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antibacterial activity by loading membrane coupons (MQ or MP) into their culture solutions.

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The OD600 values during 24-h incubation are shown in SI Figure S4. Specifically, cell

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proliferation modeling was conducted for the exponential growth phases45 (i.e., 3 to 24 h for

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E. coli and 6 to 24 h for S. aureus), and the results indicate that specific growth rates (µ) of E.

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coli and S. aureus subject to MQ were retarded dramatically (Figures 2A and D), leading to a

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substantial increase in doubling time (τd, SI Figure S5). In contrast, experimental results

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showed no obvious difference between the culture solutions with and without MP (denoted as

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NM in Figure 2) in terms of the bacterial growth rates. This implied that the presence of QAC

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in MQ should be the primary reason causing inhibition of microbial growth. Consistent to the

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OD600 measurements, SEM micrographs (Figures 2B, C, E and F) show significantly reduced

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bacteria attachment/growth on MQ in comparison to MP after exposing the membranes to E.

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coli or S. aureus bacterial culture suspensions for 24 h. The antibacterial and cell attachment

248

experiments clearly demonstrated that the QAC blended membrane, MQ, presented an

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effective antibacterial activity for both Gram-positive and Gram-negative bacteria, which is

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consistent with the observations in using QAC for material surface coatings reported by

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previous literature.46-48

12


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Figure 2. Antibacterial activity of MP and MQ. (A) µ of E. coli upon contact with MP and MQ [with

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NM (no membrane) as a control]; SEM images of the surfaces of (B) MP and (C) MQ following the

255

exposure to E. coli for 24 h; (D) µ of S. aureus upon contact with MP and MQ [with NM as a control];

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SEM images of the surfaces of (E) MP and (F) MQ following the exposure to S. aureus for 24 h. The white

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bars in Figures 2B, C, E and F indicate the length of 10 µm. Asterisks (*) denote a statistically significant

258

difference between MP and MQ (p < 0.01).

259 260

Antibacterial tests were further performed for pre-soaked MQ coupons and the

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supernatant (with pre-determined soaking of MQ) to better understand the detailed

262

antibacterial mechanisms (see Figure 3A). It can be observed that Ea for supernatant

263

increased in the first 4 h, indicating that release-killing was at least partially responsible for

264

the antibacterial effect. Nevertheless, Ea of the supernatant reached a stable value after 4 h.

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On the other hand, the pre-soaked MQ membrane remained highly effective in suppressing 13



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bacteria growth over the entire experimental course of 8 days. The Ea value for MQ

267

membrane was rather stable after the first 2 h, revealing that its antibacterial efficiency could

268

last for a long duration (8 d in the batch test and over 110 d in a membrane bioreactor (MBR)

269

as described in the following MBR test). 120

(A)

Membrane Supernatant

100 Ea (%)

80 60 40 20

3

ROS response value (×10 )

0

8.0

0.5h1h 2h 4h 8h 1d 2d 4d 8d Time Free Immobilized

(B)

6.0 4.0 2.0 0.0

100

0

20 40 Time (min)

60

(C)

Ea (%)

80 60 40 20 0 270

Alginate

S. aureus

271

Figure 3. Antibacterial efficiencies (Ea) of (A) MQs and the supernatant where MQs were soaked as a

272

function of elapsed time, (B) Intracellular ROS production of S. aureus upon exposure to free and

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immobilized QAC, and (C) MQs covered with abiotic alginate and S. aureus fouling layers. In Figures 3B

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and 3C, MQ coupons were firstly rinsed in sterile DI water for over 4 h to remove any unbound QAC. The

275

blue dashed line in Figure 3C indicates the maximum Ea (about 90%) (after removal of unbound QAC). 14


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Antibacterial Mechanisms. To further elucidate the mechanisms involved, the

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production of intracellular ROS was determined for S. aureus upon exposure to free and

279

immobilized QAC (pre-soaked MQ membrane) (see Figure 3B and detailed test procedure in

280

SI Section S1). For the free QAC, significant amount of ROS was detected. Furthermore, it

281

showed an increase rate of 73.8 unit min-1 with the increase of exposure time. It suggests that

282

the QAC in the solution phase may interact with the cells, inducing the generation of ROS

283

that can cause acute disruption to bacteria. This observation is consistent with the results in

284

Figure 3A and is also supported by existing literature that the presence of benzalkonium

285

chloride (a kind of QAC) can induce enhanced ROS production for Chang conjunctival

286

cells49. For S. aureus exposed to the pre-soaked MQ, the production of ROS was not detected

287

(Figure 3B) despite of their persistent high antibacterial effectiveness. This observation may

288

suggest that contact-killing is a prevailing mode of action for the pre-soaked MQ membranes.

289

According to previous studies, QAC has a strong affinity with polymeric matrix and could be

290

effectively immobilized at the membrane surface.41,50,51 The immobilized QAC and its

291

associated contact-killing mechanism ensure a long-term effectiveness against biofouling.

292

One crucial drawback of contact-killing mode is that the antibacterial action may be

293

restricted to the membrane surface. Its effectiveness can be dramatically decreased when the

294

membrane surface is covered with foulants.22 Consequently, we investigated the antibacterial

295

mechanisms in the vicinity of pre-fouled MQ surface by depositing two model foulants on

296

membrane surfaces: abiotic alginate and S. aureus (MQ was pre-soaked for 4 h to remove

297

unbound QAC based on Figure 3A before the pre-fouling process, see Figure S1). Based on

298

the measurements using CLSM (SI Figure S6 and Figure S7), the thickness of both model 15



299

cake layers was about 30 µm, providing a sufficient coverage of the alkyl chains of the QAC.

300

Ea values of the covered membranes are shown in Figure 3C. It is evident that the

301

antibacterial activity of MQ was decreased significantly due to the surface fouling of abiotic

302

alginate and the residual Ea was only 13.8% (i.e., a loss of 86.2%). In contrast,

303

biofilm-covered MQ exhibited a persistent antibacterial activity against the microorganism.

304

The proposed mechanism of MQ’s antibacterial activity is shown in Figure 4. According to

305

previous studies, QAC involving disruption of the integrity of the cell wall are also capable of

306

causing induced feedback such as inhibition of respiratory enzymes and dissipation of the

307

proton motive force, which is likely to trigger SOS response via cell regulation or

308

signaling.52,53 The hypothesis of the interaction between the affected microorganisms at

309

membrane surfaces and those in the vicinity resulting in consequent inhibition of population

310

growth and colonization (as illustrated in Figure 4) is not unwarranted. For example, it has

311

been reported that bacteria can use contact-dependent signaling, notably to limit their

312

growth.54 Another relevant evidence for the antibacterial activity in the vicinity of membrane

313

surface is the built-in bacterial programmed cell death pathway mediated by quorum sensing

314

peptides using toxin-antitoxin system such as mazEF.55,56 In order to further confirm the

315

antimicrobial mechanisms involved, long-term experiments in a membrane bioreactor (MBR)

316

were then carried out to assess the effectiveness of MQ against microorganisms as a function

317

of the development of cake layers.

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Figure 4. A schematic representation of the antibacterial behaviours at MQ membrane surface and in the

320

vicinity. SOS response as a result of contact with immobilized QAC is especially presented in the inset.

321 322

Long-term anti-biofouling performance in MBR. Prior to the experiment, the MQ

323

membrane was pre-soaked in sterile DI water for 4 h (based on the result shown in Figure 3A)

324

to remove unbound QAC. Figure 5A shows the variations of trans-membrane pressure (TMP)

325

of MQ as a function of MBR operation time, with MP examined for as a control. The two

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kinds of membranes were operated in the same bioreactor to ensure that they encountered the

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same operating conditions and mixed liquor. In line with the results of batch experiments,

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MQ exhibited a much better performance against bio-fouling. Notably, the TMP increase rate

329

of MQ was milder when the biofilm thickness was less than ~45 µm (blue dashed line in

330

Figure 5A57) compared to that of MP. However, similar slopes of TMP were observed at

331

biofilm thickness of > ~45 µm possibly due to the shielding of QAC by the dead cells.

332

Interestingly, the antimicrobial effectiveness of MQ was restored upon membrane cleaning

333

(0.5 wt.% NaOCl soaking for 2 h), suggesting that surface antimicrobial modification in

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combination with a carefully designed cleaning strategy can effectively ensure long-term

335

biofouling abatement. Figure 5B indicates the abundances (copies cm-2) of 16S rRNA genes 17



336

in the cake layers of MP and MQ. For qRT-PCR analysis, it has been assumed that the

337

abundance of amplified DNA product should be positively correlated to cell counting.40 As

338

can be seen from Figure 5B, the amount of bacteria was substantially reduced when QAC

339

were blended into the matrix. A similar conclusion can be drawn with regard to the

340

measurement of solids (biomass) of the cake layers of MP and MQ (SI Table S3): i.e., the

341

cake layer on MP surface had significantly greater amount of volatile suspended solids (1.08

342

± 0.36 mg cm-2) compared to that of MQ (0.18 ± 0.02 mg cm-2).

343 344

Figure 5. Long-term experiments revealing the antimicrobial behaviours in an MBR. (A) Variations of

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TMP for MP and MQ as a function of time; (B) Abundances of 16S rRNA gene in the cake layers of MP

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and MQ revealed by qRT-PCR; CLSM images of the cake layers of (C) MP and (D) MQ at the end of the

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4th and 2nd operation cycle, respectively. In Figure 5A, the dashed circles indicate membrane cleaning

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points, and the blue dashed line represents the cake layer thickness of ~45 µm.57 In Figures 5C and 5D, red

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color represents dead cells, and green color suggests viable and/or dead cells.

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Furthermore, visualization of the bacteria in the vicinity of membrane surface was

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carried out using CLSM with SYTO 9 and propidium iodide (PI) to stain all bacteria and

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dead bacteria respectively.58 As can be seen from Figures 5C and D, most microorganisms in

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the cake layer of MP were alive with only a few bacteria dead. In contrast, the relative

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abundance of viable cells in the biofilm of MQ was quite low. Considering the surface

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roughness of membranes (

Antibiofouling Polyvinylidene Fluoride Membrane Modified by

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