Accepted Manuscript Regular article Imparting Antimicrobial and Anti-Adhesive Properties to Polysulfone Membranes through Modification with Silver Nanoparticles and Polyelectrolyte Multilayers Li Tang, Khanh An Huynh, Margaret L. Fleming, Mathieu Larronde-Larretche, Kai Loon Chen PII: DOI: Reference:

S0021-9797(15)00335-5 http://dx.doi.org/10.1016/j.jcis.2015.03.051 YJCIS 20362

To appear in:

Journal of Colloid and Interface Science

Received Date: Accepted Date:

22 January 2015 27 March 2015

Please cite this article as: L. Tang, K.A. Huynh, M.L. Fleming, M. Larronde-Larretche, K.L. Chen, Imparting Antimicrobial and Anti-Adhesive Properties to Polysulfone Membranes through Modification with Silver Nanoparticles and Polyelectrolyte Multilayers, Journal of Colloid and Interface Science (2015), doi: http:// dx.doi.org/10.1016/j.jcis.2015.03.051

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Imparting Antimicrobial and Anti-Adhesive Properties to Polysulfone Membranes through Modification with Silver Nanoparticles and Polyelectrolyte Multilayers

Journal of Colloid and Interface Science Revised: March 26, 2015 Li Tang a, Khanh An Huynh a, b, Margaret L. Fleming a, Mathieu LarrondeLarretche a, c, and Kai Loon Chen a,* a

Department of Geography and Environmental Engineering, Johns Hopkins University, Baltimore, Maryland 21218-2686, USA

b

Current Address: NRC Associate, U.S. Environmental Protection Agency, National Exposure Research Laboratory, Environmental Sciences Division, Las Vegas, Nevada 89119, USA c

School of Engineering, University of Glasgow, Glasgow G12 8LT, United Kingdom

* Corresponding author: Kai Loon Chen, E-mail: [email protected], Phone: (1) 410-5167095

Abstract The antimicrobial and bacterial anti-adhesive properties of polysulfone (PSU) membranes modified with silver nanoparticles (AgNPs) and polyelectrolyte multilayers (PEMs) composed of poly(allylamine hydrochloride) and poly(acrylic acid) were investigated.

The membranes’

antimicrobial properties were evaluated using a colony forming unit (CFU) enumeration method, while the anti-adhesive properties of the membranes were examined using a direct microscopy observation membrane filtration system. The AgNP mass loading required for the inhibition of bacterial growth on the AgNP/PEM-modified membranes was significantly lower than the AgNP loadings reported in other studies for membranes with the nanoparticles dispersed within the membrane matrix. The immobilization of AgNPs on the membrane surface maximized the opportunities for bacteria–nanoparticle contact, which allowed for effective bacteria inactivation. Furthermore, in comparison to unmodified PSU membranes, the bacterial deposition kinetics on all the modified membranes were reduced by ca. 50 % and the bacterial removal efficiencies were significantly increased from close to 0 % to as high as over 90 %. Three-cycle filtration and rinsing experiments were also performed to evaluate the effectiveness of the surface modification over an extended time period of use.

Keywords: Antimicrobial membrane; Bacterial anti-adhesive membrane; Biofouling; Layer-bylayer adsorption; Polyelectrolyte multilayers; Silver nanoparticles.

1. Introduction Membrane technology, such as microfiltration (MF), ultrafiltration, nanofiltration, and reverse osmosis (RO), is rapidly becoming one of the most popular technologies for drinking water and wastewater treatment because of improvements in membrane filtration performance and decreasing membrane cost [1-4]. Nevertheless, one major obstacle that continues to impede the application of membrane technology is biofouling, or the formation of biofilms on membrane surfaces or within the membrane matrices [5-9]. It is difficult to completely remove biofilms from membranes using biocide solutions as the protective structure of the biofilms’ extracellular polymeric substances protects the embedded microbial cells from biochemical attack [10-12]. The development of anti-biofouling membranes over the last decade has centered on the modification of membrane surfaces through the enhancement of surface charge and/or

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hydrophilicity to render them more resistant to bacterial and colloidal adhesion [13-22]. The assembly of polyelectrolyte multilayers (PEMs) on membrane surfaces through layer-by-layer (LbL) adsorption is an emerging membrane surface modification technique to inhibit or retard biofouling [17, 22-25]. Polyethersulfone (PES) membranes modified with PEMs comprising 1.5 bilayers of poly(styrene sulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDADMAC) were found to be more resistant to the adhesion of Escherichia coli bacteria compared to the unmodified membranes due to the increase in surface charge and hydrophilicity of the modified membranes [22]. Polyamide thin-film composite (TFC) RO membranes were modified with 10 bilayers of polyethylene amine (PEI) and poly(acrylic acid) (PAA) and further functionalized through the grafting of hydrophilic poly(sulfobetaine) [25].

This surface

modification was shown to result in a considerable reduction in E. coli cell adhesion on the membranes because of the increased hydrophilicity [25]. Similarly, our previous study showed that the assembly of 2 bilayers of poly(allylamine hydrochloride) (PAH) and PAA on polysulfone (PSU) membranes can significantly enhance the membranes’ bacterial anti-adhesive properties due to the hydrated, swollen nature of the PAH/PAA PEMs [17]. More recently, the use of PEMs as a sacrificial layer for the easy cleaning of fouled membranes has also been explored [26]. While the surface modification of membranes with PEMs can impart some antifouling properties to the membranes, PEMs alone cannot completely prevent bacterial adhesion to the membranes since the drag forces caused by the permeate flow may be strong enough to immobilize the bacteria on the membrane surface [27]. Therefore, it is also desirable to impart antimicrobial properties to PEM-modified membranes to inactivate bacteria that deposit on the membrane surfaces. Recently, several studies have shown that PEMs can be used to immobilize antimicrobial nanomaterials, such as silver nanoparticles (AgNPs), on membrane surfaces [22, 25, 28-30]. PES membranes that were modified with 1.5 bilayers of PSS and PDADMAC together with AgNPs showed no cell growth when the nanocomposite membranes were exposed to E. coli [22]. PES membranes coated with 1.5 bilayers of chitosan and poly(methacrylic acid) also showed a considerable inhibition of E. coli cell growth when AgNPs were dispersed in each polyelectrolyte layer [29]. In another study, polyamide TFC RO membranes coated with PEMs which were composed of PEI and PAA together with AgNPs exhibited a E. coli inactivation efficiency of over 95% [25]. While these studies have demonstrated that AgNP/PEM assemblies

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can impart antimicrobial properties to the membranes, not many studies, to date, have been conducted to systematically evaluate these membranes’ bacterial anti-adhesive properties. Furthermore, no studies have been performed to examine the effectiveness of AgNP/PEM- and PEM-modifications over multiple cycles of filtration. The objective of this research is to evaluate the antimicrobial and bacterial anti-adhesive properties of PSU MF membranes that are modified with AgNPs and PEMs composed of PAH and PAA. Three different AgNP mass loadings are employed to investigate the effect of AgNPs on the PEM-modified membranes’ antimicrobial and anti-adhesive properties. The antimicrobial properties of the modified membranes are examined through the use of a colony forming unit (CFU) enumeration method. The bacterial anti-adhesive properties of the modified membranes are assessed by comparing the kinetics and reversibility of E. coli deposition on the membranes using a direct microscopic observation membrane filtration system. Finally, the membranes’ antimicrobial and anti-adhesive properties are examined over three cycles of filtration and rinsing.

2. Materials and Methods 2.1

Base membranes PSU MF membranes (Pall Corporation, Ann Arbor, MI) were used as the base

membranes in this study.

According to the manufacturer, the nominal pore size of the

membranes is 0.2 µm on the active side.

Membrane coupons were cut from flat sheet

membranes, rinsed, and then stored in deionized (DI) water (Millipore) at 4 ºC for at least three days before use. More information about the physicochemical properties of the PSU membranes was provided in our previous publication [17].

2.2

Silver nanoparticles and polyelectrolytes AgNPs were synthesized through the reduction of a Tollen’s reagent with the use of

glucose and then cleaned and suspended in a citrate solution [31-33]. The detailed procedure is provided in the Supporting Information (SI). The average hydrodynamic diameter of AgNPs was measured to be 47.3–55.0 nm through dynamic light scattering (DLS, BI-200SM and BI9000AT, Brookhaven). The total and dissolved silver concentrations of the citrate-coated AgNP stock suspensions (three batches) were determined to be 6.66–7.14 and 0.11–0.16 mg/L,

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respectively, through inductively coupled plasma mass spectrometry (ICP-MS) (details in SI). PAH (Mw = 15,000, Sigma-Aldrich, St. Louis, MO) and PAA (Mw = 50,000, Polysciences, Inc., Warrington, PA) solutions were prepared in DI water. The PAH and PAA solutions were used for membrane modification within 5 days of preparation. The concentrations of both the PAH and PAA solutions were 5 mM (based on the repeat unit molecular weight). The ionic strength of both solutions was adjusted to 150 mM using NaCl and the pH of the solutions was adjusted to 3.0 using 1 M HCl.

2.3

Membrane modification with AgNPs and PEMs A custom-made polycarbonate flow cell was used to modify the PSU membrane surfaces

with AgNPs and PAH/PAA multilayers. The flow cell comprises a top plate and a bottom plate. The dimensions of the cross-flow channel in the cell are 76.0 mm in length, 25.0 mm in width, and 3.0 mm in height. The membrane to be modified was clamped between the top and bottom plates and sealed with double O-rings. The flow cell can be operated either in the dead-end filtration or cross-flow mode. The inlet valve is in the top plate while the outlet valves are in both the top and bottom plates. In the dead-end mode, the outlet valve of the top plate was closed and the outlet valve of the bottom plate was opened. In the cross-flow mode, the outlet valve of the bottom plate was closed and the outlet valve of the top plate was opened. To modify the membrane surface, a diluted AgNP suspension of a desired concentration (150 mL) was filtered through a PSU membrane under the dead-end mode at a filtration rate of 15 mL/min. Following that, PEMs comprising two bilayers of PAH and PAA were assembled on the surface of the AgNP-modified membrane under the cross-flow mode in the absence of permeation. PEMs were assembled through the LbL adsorption technique using an approach similar to that described in our previous study [17]. In that study, it was verified through X-ray photoelectron spectroscopy that the PEMs can be assembled on the PSU membranes using the LbL adsorption approach. Briefly, the membrane was first rinsed with a PAH solution for 10 min. After that, the membrane was rinsed with a 150 mM NaCl solution (pH 3.0) for 10 min to wash away the loosely-bound polyelectrolytes. Following that, the membrane was rinsed with a PAA solution for 10 min and then with a 150 mM NaCl solution (pH 3.0) for 10 min. This process was then repeated in order to form two bilayers on the membrane. The cross-flow velocity used for the assembly of PEMs was 2.2 mm/s. The mass loading of AgNPs on the

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membranes was determined by measuring the total silver concentrations of the permeate, polyelectrolyte solutions, and rinse solutions collected from the flow cell during membrane modification using ICP-MS and by performing mass balance. Surface morphologies of the base, PEM-modified, and AgNP/PEM-modified membranes were acquired using scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR). The membrane samples used for SEM analysis were vacuum-dried overnight in a desiccator and examined under the low-vacuum mode.

2.4

Model bacteria The model bacteria used in this study was E. coli K12 MG 1655 [27]. This bacterial

strain is labeled with the green fluorescent protein which allows the bacterial cells to be observed under an epifluorescent microscope. The E. coli cells were incubated in a culture solution containing 25 g/L Luria Bertani (LB) broth (Fisher Scientific) and 50 mg/L kanamycin (SigmaAldrich) at 37 ºC for ca. 3 hours and then harvested at the exponential growth phase. The cleaning procedure of the bacterial cells to be used for the bacterial deposition and release experiments has been described in our previous study [17]. For these experiments, the cell concentration in the feed suspension was ca. 1.4 × 107 cells/L.

2.5

Evaluation of antimicrobial properties of membranes A CFU enumeration method was used to evaluate the antimicrobial properties of the

membranes modified with AgNPs and PEMs [34-36]. Specifically, the membrane coupon was first placed on top of the glass support of a vacuum filtration setup (Millipore, Billerica, MA) with the active side facing up. A 0.1 mL E. coli suspension was serial-diluted to 4.0 × 104 cells per mL with a 154 mM NaCl solution. The diluted bacterial suspension (0.5 mL) was further diluted with 25 mL of a 154 mM NaCl solution to ca. 500 cells per mL and was then gently filtered through the membrane. The cell concentration was determined through optical density (wavelength 600 nm) measurements and using a calibration curve that was obtained for this E. coli strain. The membrane coupon with the deposited bacterial cells was placed on an agar plate (25 g/L LB broth, 15 g/L agar, and 50 mg/L kanamycin) with the active side facing up (i.e., support side attached to the agar) and the agar plate was placed in an incubator (VWR, Radnor, PA) and incubated at 37 °C for ca. 15 hours. The bacterial colonies on the membranes that were modified with AgNPs and PEMs were counted and compared with the colonies on the

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membranes modified with PEMs alone. This test was carried out at least three times with different membrane coupons and with a different bacterial culture for each membrane.

2.6

Direct microscopic observation of bacterial deposition and release A direct microscopic observation membrane filtration system was used in this study to

observe bacterial deposition and release during a filtration process. This system has been described in our previous studies [17, 36] and is similar to the systems used in other studies [10, 27]. Briefly, the membrane to be tested was held between the top and bottom plates of a crossflow membrane filtration (CMF) cell with the active side facing the cross-flow channel. A 3-mm thick glass window was inserted into the top plate of the CMF cell, enabling the florescent bacterial cells to be observed under an epifluorescence microscope.

The CMF cell was

incorporated into a closed-loop filtration system, which was operated under the cross-flow mode. A stainless steel pressure vessel (Alloy Products, Waukesha, WI) containing 2 L of the feed bacterial suspension was pressurized to ca. 170 kPa and the suspension was circulated through the CMF cell with the use of a gear pump (Cole-Parmer, Vernon Hills, IL) at a cross-flow velocity of 10 cm/s.

The permeate flux was maintained constant at 30 µm/s during the

deposition experiment using an 8-roller digital peristaltic pump (Cole-Parmer, Vernon Hills, IL) and the permeate was circulated back into the pressure vessel. The CMF cell was placed on the stage of an epifluorescence microscope (Nikon Eclipse E600W, Japan). The microscope is equipped with a 10× objective lens (Nikon Plan Fluor, Japan) and an emission filter (Nikon CFL Endow GFP HYQ, EX 450-490, DM 495, BA 500-550). The digital images of E. coli cells on the membrane surface were acquired with a CCD camera (Roper Scientific, Photometrics CoolSnap ES, Germany) in real time during the filtration experiment. The E. coli cells deposited on the membrane surface within the field of view of the microscope were enumerated after each experiment in order to obtain the deposited cell densities as a function of time. The bacterial deposition experiments were conducted in 10 mM NaCl and at pH 7.0 (buffered with 0.15 mM NaHCO3) using a procedure similar to that in our previous study [17]. The solution chemistry represents that of a typical tertiary wastewater effluent [37, 38]. Briefly, the membrane was equilibrated at a permeate flux of 30 µm/s for ca. 40 min. Following that, E. coli cells were introduced into the pressure vessel to initiate the deposition experiment. The bacterial deposition experiment was carried out for 20 min and an image of the central part of the

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membrane surface was acquired every 3 min. The deposition rate coefficient of the E. coli cells, kobs, was calculated by dividing the rate of bacterial deposition by the product of the image area and cell concentration in the suspension [10, 17]. A bacterial release (detachment) experiment was conducted in two stages after each deposition experiment. In Stage 1, the membrane with deposited bacteria was rinsed with a solution of 10 mM NaCl and pH 7.0 for 30 min at a cross-flow velocity of 10 cm/s and in the absence of permeate flow. In Stage 2, the membrane was rinsed with a solution of 1 mM NaCl and pH 7.0 for another 30 min, also in the absence of permeate flow. The solution with a lower ionic strength was used in Stage 2 to increase the electric double layer repulsion between the deposited bacteria and membrane [39]. The removal efficiencies for Stage 1 and Stage 2 were calculated by dividing the numbers of bacteria removed during Stage 1 and during Stage 2, respectively, by the number of bacteria deposited on the membrane immediately before Stage 1. All the salts used in the experiments were ACS grade (Fisher Scientific) and electrolyte stock solutions were prepared by dissolving the salts in DI water. All the bacterial deposition and release experiments were conducted at room temperature (24 °C) and were carried out three times for each type of membrane using different membrane coupons and different bacterial cultures.

2.7 Direct microscopic observation during three cycles of filtration and rinsing The E. coli cells were allowed to undergo three cycles of deposition and release on the modified membranes at 10 mM NaCl and pH 7.0 (buffered with 0.15 mM NaHCO3). All of the three-cycle filtration and rinsing experiments were conducted using the direct microscopic observation membrane filtration system.

Specifically, bacterial deposition took place at a

permeate flux of 30 µm/s and cross-flow velocity of 10 cm/s for 20 min in the first cycle of filtration. After filtration, the reversibility of bacterial deposition was evaluated by turning off the peristaltic pump for 5 min to stop the permeate flow while the cross flow was maintained. After that, the peristaltic pump was turned on again to initiate the second cycle of bacterial deposition. The filtration and rinsing process was then repeated twice. The removal efficiency for each cycle was calculated by dividing the number of the bacteria removed during each release process by the number of the bacteria deposited on the membrane immediately before the start of the same release process. In addition, the antimicrobial properties of the membranes

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were evaluated after three cycles of filtration and rinsing with 10 mM NaCl solutions using the method described in Section 2.5.

2.8 Silver leaching tests An initial silver leaching test was conducted on the AgNP/PEM-modified membranes with the highest AgNP mass loading to examine the dissolution and release of AgNPs from the modified membranes.

This test was performed using the direct microscopic observation

membrane filtration system under the same solution chemistry as that for the bacterial deposition experiments (10 mM NaCl and pH 7.0). The membrane to be tested was held between the top and bottom plates of the CMF cell with the active side facing the top plate. A 10 mM NaCl solution was then circulated through the CMF unit at a cross-flow velocity of 10 cm/s in the absence of permeation for 1 hour. The total silver concentration of the circulated solution was measured using ICP-MS to determine the degree of silver leaching from the membrane. In order to assess the amount of silver that passed through the membrane in the permeate, a second leaching test was conducted on the modified membranes with the highest AgNP mass loading with the use of a cross-flow membrane filtration system that allows for the separate collection of samples from the retentate and permeate. The membrane filtration system was operated under constant pressure mode with a modified membrane held in the CMF cell (76.0 mm in length, 25.0 mm in width, and 3.0 mm in height). A 10 mM NaCl feed solution (pH 7.0) was driven through the cell with the use of a gear pump at a cross-flow velocity of 10 cm/s. The initial permeate flux was adjusted to 30 µm/s by adjusting the backpressure regulator (GO Regulator, Spartanburg, SC) that was mounted on the retentate line to ca. 19 kPa. The retentate and permeate solutions were not circulated back into the feed tank to avoid silver accumulation in the reservoir. Triplicate tests were carried out at room temperature (24 ºC). The retentate and permeate solutions were collected during the first 10 min of the filtration experiment and the silver concentrations were analyzed through ICP-MS.

3 Results and discussion 3.1

Characterization of membranes modified with PEMs and AgNPs PSU membranes were modified with PEMs only, as well as PEMs with three different

AgNP mass loadings. The AgNP loadings of the membranes modified with both AgNPs and

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PEMs were determined to be 3.6, 14.7, and 31.4 µg (0.005, 0.020, and 0.043 wt. %, respectively) over a membrane surface area of 19.4 cm2. The membrane designations and modification conditions are summarized in Table 1. TABLE 1 The SEM images of Membranes P, P20, and P43, as well as the PSU base membrane, are shown in Figure 1. Membranes P, P20, and P43 exhibited similar morphologies as that of the base membrane. However, several pores on Membranes P, P20, and P43 appeared to be covered by the PEM film, confirming the successful assembling of PEMs on the membrane surface. The pKa of NH3+ groups in PAH has been reported to be ca. 9.0 [40] while the isoelectric point of the PSU membrane has been reported to be ca. 3.0 [41-43]. Therefore, the first layer of PAH adsorbed on the PSU membrane surface through electrostatic attraction since PAH is positively charged at pH 3.0 while the PSU membrane surface is slightly negatively charged. Additionally, in the case of Membranes P20 and P43, we observed several white spots sparsely distributed on the membrane surfaces which were likely AgNPs and AgNP aggregates that had deposited on the membranes and were trapped in the membrane pores. Based on the SEM images, the AgNP coverages on Membranes P20 and P43 were determined (using ImageJ software, National Institutes of Health) to be 0.6 % and 1.1 %, respectively. It is noteworthy that some AgNPs may have penetrated into the membranes and deposited deeper within the membrane pores and thus were not observed through SEM imaging. FIGURE 1 The hydraulic resistances of Membranes P, P20, and P43 were determined to be 1.4 × 1011, 1.3 × 1011, and 1.0 × 1011 m-1, respectively. These values were within the typical range of hydraulic resistances for MF membranes (between 1 × 1011 and 1 × 1012 m-1) [44]. The hydraulic resistances of the membranes modified with both PEMs and AgNPs (Membranes P20 and P43) were comparable to that of the membranes modified with only PEMs (Membrane P), indicating that the deposited AgNPs’ contribution to the hydraulic resistance of the AgNP/PEMmodified membranes was insignificant. This finding confirms that the nanoparticle coverage on the membrane was extremely low even at the highest AgNP loading, which is consistent with our observations through SEM imaging. In comparison, we had shown in our previous study [17] that the hydraulic resistance of a membrane can be increased by over three times after PEM modification. The increase in hydraulic resistance after PEM modification was likely due to the

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partial coverage of the membrane pores by the film of PEMs, as observed through SEM imaging in that study [17]. The degree of silver leaching from Membrane P43 after being rinsed with a 10 mM NaCl solution at a cross-flow velocity of 10 cm/s for 1 hour using the direct microscopic observation membrane filtration system was determined to be 14.5 %. In a study by Diagne et al. [22], a silver leaching test was performed by filtering DI water through AgNP/PEM-modified membranes under a dead-end mode for 150 min and 50 % of the AgNPs were lost after filtration. Even though the surface-immobilized AgNPs in our study experienced a much larger shear force (cross-flow velocity = 10 cm/s) compared to that in the study of Diagne et al. [22] (no cross flow), the degree of silver leaching from Membrane P43 in our study was noticeably lower. This observation may imply that the 2 bilayers of PAH and PAA are more robust than the 1.5 bilayers of PSS and PDADMAC employed in the study of Diagne et al. [22], possibly due to stronger electrostatic interactions between the PAH and PAA layers compared to those between the PSS and PDADMAC layers. Additionally, it is plausible that the method used to incorporate AgNPs into the PEMs may influence the degree of leaching of AgNPs. In our study, the AgNPs were first deposited on the membrane surface through filtration under a dead-end mode before the membranes were coated with the PEMs which can form protective thin films over the deposited AgNPs. In the study of Diagne et al. [22], the AgNPs were dispersed in the PSS solution before the PSS–AgNP mixture was used to form the top layer of the PSS/PDADMAC PEMs. This approach was likely to result in the deposited AgNPs being fully exposed to the aqueous environment and thus more prone to detachment and dissolution. In order to assess the amount of silver that passed through the membrane in the permeate, an additional leaching test was conducted on Membrane P43 with the use of a cross-flow membrane filtration system that allows for the separate collection of samples from the retentate and permeate. The silver concentrations in the retentate and permeate solutions were 1.6 ± 2.0 and 6.6 ± 3.1 µg/L, respectively, which were significantly lower than the maximal contaminant limit of silver in drinking water (i.e., 100 µg/L) established by the U.S. Environmental Protection Agency [45] and also by the World Health Organization [46].

3.2

Effect of AgNP/PEM-modification on membranes’ antimicrobial properties

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In order to assess the antimicrobial properties of the AgNP/PEM-modified membranes, the bacterial colonies formed on the membrane surfaces were enumerated after the E. coli suspensions were filtered through the membranes by vacuum filtration and after ca. 15 hours of incubation at 37 °C. The numbers of colonies on the surfaces of Membranes P, P5, P20, and P43 are presented in Figure 2. While 223 colonies were observed on Membrane P, only 144 and 14 colonies were found on Membranes P5 and P20, respectively. Furthermore, no CFUs were observed on Membrane P43. This result clearly shows that the number of colonies on all the AgNP/PEM-modified membranes was smaller than the PEM-modified membranes. Also, the number of colonies on the AgNP/PEM-modified membranes was shown to decrease as the mass loading of AgNPs was increased. Therefore, it can be concluded that the incorporation of AgNPs into the PEMs on the membrane surfaces can impart antimicrobial properties to the PSU membranes. Furthermore, the membranes’ antimicrobial properties are demonstrated to have a direct dependence on the mass loading of AgNPs. FIGURE 2 In this study, the minimum AgNP mass loading that resulted in the complete inhibition of cell growth (i.e., no colonies observed on membranes) was 0.043 wt. % (Membrane P43). This loading was much lower compared to the loadings reported in other studies that applied similar methods to evaluate the antimicrobial activities of AgNP-impregnated membranes that were cast using PSU mixtures with AgNPs dispersed within the mixtures [34, 35]. Zodrow et al. [34] showed that their AgNP-impregnated PSU membranes enabled a 99 % reduction in E. coli cell growth when the AgNP concentration in the membranes was 0.9 wt. %. In the study of Liu et al. [35], the authors reported a similar AgNP mass loading of 0.88 wt. % in their AgNPimpregnated PSU membranes to achieve an antibacterial efficiency of 99 % with E. coli cells. In comparison, the AgNP mass loading (0.043 wt. %) that led to the complete inhibition of bacterial colony growth on the membrane surface in our current study was about two orders of magnitude lower than the values reported in the studies described above [34, 35]. This large difference in AgNP concentrations implies that the location of AgNPs on the membranes (either immobilized on the membrane surface or embedded in the membranes) plays a crucial role in controlling the membranes’ antimicrobial properties. Recently, several studies have provided evidence that the direct contact or the close proximity between AgNPs and bacterial cells can greatly enhance the toxicity effects of the

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AgNPs [47-52]. The direct contact or close proximity between AgNPs and bacteria allows the cells to be exposed to lethal concentrations of Ag+ ions that are released from dissolving AgNPs [49, 51]. The casting of membranes using a polymer mixture with AgNPs dispersed within the mixture, as employed by Zodrow et al. [34] and Liu et al. [35], was likely to result in most of the AgNPs to be embedded inside the membrane matrix and therefore unavailable for direct contact with deposited bacterial cells.

In contrast, our approach of immobilizing AgNPs on the

membrane surface through the use of PEMs dramatically enhanced the opportunities for the direct contact or close proximity between the AgNPs and deposited bacteria. Therefore, a much lower AgNP mass loading is required for the inhibition of bacterial growth on a membrane when AgNPs are immobilized on the membrane surface using PEMs compared to the incorporation of AgNPs within the membrane matrix.

3.3

Influence of AgNP/PEM-modification on kinetics and reversibility of bacterial deposition In order to evaluate the anti-adhesive properties of the AgNP/PEM-modified membranes,

bacterial deposition experiments were conducted by using the direct microscopic observation membrane filtration system. Figure 3a presents a representative plot of the number density of deposited E. coli cells on the surface of Membrane P43 as a function of time during a deposition experiment. Figure 3b presents the deposition rate coefficients, kobs, of the base, PEM-modified, and AgNP/PEM-modified membranes when bacterial deposition took place at 10 mM NaCl. The experimental results showed that the AgNP/PEM-modified membranes (Membranes P20 and P43), as well as the PEM-modified membrane (Membrane P), exhibited considerably lower kobs values (ca. 15 µm/s) than that of the base membranes (ca. 33 µm/s). Clearly, AgNP/PEMmodifications are demonstrated to be as effective as PEM-modifications in reducing the bacterial deposition rates on the membrane surfaces and the presence of AgNPs on the membrane surface does not affect the deposition rates. FIGURE 3 Since the kobs values for the AgNP/PEM-modified membranes (Membranes P20 and P43) were independent of the AgNP mass loadings and comparable to that of the PEM-modified membrane (Membrane P), it is likely that the enhancement of the AgNP/PEM-modified membranes’ resistance to bacterial adhesion was controlled by the PAH/PAA PEMs. The

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bacterial deposition kinetics during filtration are governed by the drag forces due to the permeate flow, as well as the interfacial interactions between the bacteria and membranes [10, 27]. These interfacial interactions include van der Waals, electrostatic, and hydrophobic interactions. Since the drag forces exerted by the permeate flow were the same for the base membrane and the PEM- and AgNP/PEM-modified membranes, the reduced bacterial deposition kinetics observed on the modified membranes indicates that the approaching bacterial cells experienced stronger repulsive forces with the modified membranes compared to the base membrane. Our previous study demonstrated that the repulsive interaction between bacteria and PEM-modified membranes resulted from the highly hydrated and swollen structure of the PEM film [17]. When the PEM film composed of PAH and PAA is assembled at low pH (pH 3.0) and high ionic strength (150 mM) conditions, there are relatively few crosslinks between COOgroups of PAA and NH3+ groups of PAH due to the protonation of COO- groups and charge screening of COO- and NH3+ groups. Hence, the PAA polyelectrolytes in the PEM film take a loopy conformation [17, 23, 24]. Subsequently, when the PEM film is exposed to a higher pH (pH 7.0) and lower ionic strength (10 mM NaCl) solution, COO- groups become fully deprotonated and the charges of the COO- and NH3+ groups are not as highly screened as before. Thus, the PAA polyelectrolytes within the PEMs begin to repel each other due to electrostatic repulsion and take an extended conformation [17, 23, 24]. This change of PAA conformation results in a considerably swollen and highly hydrated PEM structure [23, 24]. Interfacial force measurements conducted with the use of an atomic force microscope (AFM) in our previous study also confirmed that the interactions between carboxylate-modified latex colloidal probes, which were used as surrogates for bacterial cells, and PAH/PAA PEM-modified membranes were highly repulsive compared to that between the colloidal probes and base membranes due to the hydrated, swollen structure of the PEM film [17]. Therefore, the strong repulsion exerted by the PEM films was expected to inhibit the strong adhesion of the bacteria to the PEM- and AgNP/PEM-modified membranes. In addition to bacterial deposition kinetics, the reversibility of bacterial deposition on the membrane surface was examined by comparing the removal efficiencies between the membranes modified with PEMs and AgNPs and the base membranes. After bacterial deposition had taken place at 10 mM NaCl, the membranes were subsequently rinsed with a 10 mM NaCl solution, followed by a 1 mM NaCl solution (Figure 3a). Figure 3c shows the removal efficiencies

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obtained from both release stages, Stage 1 and Stage 2, for the base membranes, PEM-modified membranes (Membrane P), and AgNP/PEM-modified membranes (Membranes P20 and P43). The AgNP/PEM- and PEM-modified membranes exhibited very similar removal efficiencies for Stage 1 and Stage 2, which were ca. 80 % and 90 %, respectively. Conversely, almost no removal of deposited bacteria was observed for the base membranes. The significant increase in the removal efficiencies through the AgNP/PEM-modification of the membranes clearly demonstrates that the PEMs can substantially weaken the adhesion of bacteria to membrane surfaces [17] and that the incorporation of AgNPs in the PEMs does not impact the anti-adhesive properties of the membranes.

Depending on the AgNP concentrations on the modified

membranes, the bacteria that remain on the membrane surface will be inactivated by the AgNPs. The combinatorial anti-adhesive and antimicrobial properties of the modified membranes are expected to retard the development of biofilms on the membrane surface.

3.4

Antimicrobial and anti-adhesive properties over three cycles of filtration and rinsing Three cycles of membrane filtration and rinsing with 10 mM NaCl solutions were

conducted to evaluate the long-term effectiveness of AgNP/PEM-modifications on the membranes’ antimicrobial properties. The CFU enumeration tests showed that the numbers of colonies on Membranes P and P43 after the three cycles of filtration and rinsing were 228 ± 5 and 142 ± 6, respectively. Membrane P43 after three cycles of filtration and rinsing inactivated 38 % of deposited bacteria (Membrane P as negative control), while a freshly prepared Membrane P43 achieved complete inactivation (Figure 2).

The diminished antimicrobial

property of the AgNP/PEM-modified membranes was attributed to silver leaching that might have occurred during the repeated filtration and rinsing of the membranes. Furthermore, the multiple filtration processes might have resulted in the penetration and deposition of AgNPs deeper into the membrane matrix, thus reducing the opportunities for deposited bacteria to come into contact with AgNPs. The long-term effectiveness of PEM- and AgNP/PEM-modifications on the membranes’ anti-adhesive properties was evaluated through three-stage filtration and rinsing experiments with bacterial suspensions. The number density of deposited bacteria on the base membrane throughout the three cycles of filtration and rinsing was presented in Figure 4a, while the number

15

densities of deposited bacteria on the PEM- and AgNP/PEM-modified membranes (Membranes P and P43, respectively) were presented in Figure 4b. The result showed that there were fewer bacteria deposited on the PEM- and AgNP/PEM-modified membranes at any time compared to the base membrane. At the end of the three cycles of bacterial deposition and release, the number density of deposited bacteria on the base membrane, Membrane P, and Membrane P43 was 8685, 5323, and 5062 per mm2, respectively. FIGURE 4 For the base membranes, no removal of deposited bacteria was observed at the end of each of the three release processes. In contrast, the removal efficiencies for Membrane P at the end of the first, second, and third release processes were 61.3 %, 40.2 %, and 21.6 %, respectively. Similarly, the removal efficiencies for Membrane P43 at the end of the first, second, and third release processes were 59.4 %, 40.0 %, and 26.0 %, respectively. Therefore, the PEMand AgNP/PEM-modified membranes exhibited considerably higher bacterial removal efficiencies compared to the base membranes over the three cycles of bacterial deposition and release, demonstrating that both PEM- and AgNP/PEM-modifications have the potential to impart bacterial anti-adhesive properties to the membranes over multiple cycles of filtration and rinsing. The bacterial removal efficiencies on Membrane P and P43 were noted to decrease over the three cycles of filtration and rinsing, probably due to the deterioration of the PEM films over the repeated filtration and rinsing processes. In order to explore the possibility of a further enhancement in bacterial removal efficiencies for the PEM-modified membranes, the procedure for the assembly of PEMs was slightly modified.

More concentrated PAH and PAA solutions were used during the

modification, which likely resulted in the formation of a thicker PEM film on the membrane (Membrane PM). Additionally, during the rinsing process, the deposited bacteria were flushed with a 1 mM NaCl rinse solution instead of a 10 mM NaCl solution in order to maximize the swelling of the PEM and to enhance the electric double layer repulsive interaction between the deposited bacteria and the membrane [39]. The detailed procedures for PEM modification and for the rinsing of Membrane PM are provided in SI. In this three-cycle filtration and rinsing experiment, the number density of deposited bacteria on Membrane PM at the end of three cycles of bacterial deposition and release was 688 per mm2 (Figure 5), which was significantly lower than those on the base membrane (Figure 4a), as well as Membranes P and P43 (Figure 4b).

16

Furthermore, the removal efficiencies at the end of the first, second, and third release processes were 97.6 %, 96.3 %, and 86.9 %, respectively, which were substantially higher than the removal efficiencies for Membranes P and P43. These results imply that the PEM modification of membranes can allow for high bacterial removal efficiencies over multiple cycles of filtration and cleaning through the optimization of the conditions for PEM assembly and membrane rinsing. FIGURE 5

4 Conclusions The antimicrobial and bacterial anti-adhesive properties of AgNP/PEM-modified PSU membranes were evaluated in this study. The immobilization of low concentrations of AgNPs (0.043 wt. %) on the membrane surface with the use of PEMs completely inhibited the growth of bacteria colonies on the membranes. This AgNP loading was about two orders of magnitude lower than the reported loadings for nanocomposite membranes with AgNPs incorporated in the membrane matrix since the surface immobilization of AgNPs with PEMs dramatically enhanced the opportunities for the direct contact or close proximity between the AgNPs and deposited bacteria. Furthermore, the modification of membranes with AgNPs and PEMs was shown to reduce the bacterial deposition kinetics by about 50 % and increase the reversibility of bacterial deposition to over 90 %, likely due to the strong repulsive forces exerted by the hydrated and swollen PEMs on the depositing bacteria. Additionally, the PEM- and AgNP/PEM-modified membranes exhibited considerably higher bacterial removal efficiencies compared to the unmodified membranes over the three cycles of bacterial deposition and release, demonstrating that both PEM- and AgNP/PEM-modifications have the potential to impart bacterial antiadhesive properties to the membranes over multiple cycles of filtration and rinsing. Since the anti-biofouling properties of the membranes can diminish with time due to the deterioration of PEM and AgNP/PEM thin films, further studies on methods to improve on the robustness and longevity of the nanocomposite layers on membranes, such as cross-linking of PEMs [53, 54], are required. Additional studies should also be conducted to explore the regeneration of PEM and AgNP/PEM films on membranes after the films have deteriorated or fouled [26].

17

Acknowledgements This work was funded by the National Science Foundation (CBET-1133559) and the Johns Hopkins Water Institute. L.T. acknowledges funding support from the Dean Robert H. Roy and Gordon Croft fellowships. M.L-L. is supported by the U.S. NSF and the University of Glasgow. We acknowledge Ji Yeon Hong from the Department of Geography and Environmental Engineering at Johns Hopkins University (JHU) for her assistance with the experiments. The SEM images of the membranes are taken by Michael McCaffery from the Integrated Imaging Center, Department of Biology (JHU).

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Table 1. Designations and modification conditions of PEM- and AgNP/PEM-modified membranes. Membrane ID

Membrane Modification

Surface Density of AgNPs (μg/cm2)

Membrane P

PEMs only

0

Membrane P5

0.005 wt. % AgNPs + PEMs

0.19

Membrane P20

0.020 wt. % AgNPs + PEMs

0.76

Membrane P43

0.043 wt. % AgNPs + PEMs

1.62

21

(a)

(b)

(c)

(d)

Fig. 1. SEM images of (a) PSU base membrane, (b) Membrane P, (c) Membrane P20, and (d) Membrane P43.

22

CFU/membrane

250 200 150 100 50

*

6

8

M em br an e M P em br an M e em P5 br an e M P2 em 0 br an e P4 3

0

Fig. 2. Number of bacterial colonies (or CFUs) on Membrane P, Membrane P5, Membrane P20, and Membrane P43. Error bars represent standard deviations. Membrane P43.

23

* No colonies were present on

Flush with 1 mM NaCl

4000

(a)

40

Right before flushing

(b)

kobs (μ m/s)

3000 2000 1000

60

80

20 10 0

100

5

6

Ba se

Time (min)

10 mM NaCl 1 mM NaCl

Removal Efficiency (%)

100

(c) 80 60 40 20 0

5

6

7

7

8

M em br an M e em br an M e em P br an e M P2 em 0 br an e P4 3

40

30

8

9

10

11

M em br an M e em br an M e em P br an e M P2 em 0 br an e P4 3

20

se

0 0

Ba

2

Deposited Bacteria (per mm )

Flush with 10 mM NaCl

24

12

Fig. 3. (a) Number of bacteria on Membrane P43 during the deposition and release stages. The deposition experiment was conducted at 10 mM NaCl and a permeate flow rate of 30 µm/s. The membrane was subsequently rinsed with a 10 mM NaCl solution, followed by a 1 mM NaCl solution, in the absence of permeate flow. For the deposition and release stages, the pH was maintained at 7.0. (b) Bacterial deposition rates, kobs, for base membrane, Membrane P, Membrane P20, and Membrane P43. (c) Bacterial removal efficiencies for base membrane, Membrane P, Membrane P20, and Membrane P43 after deposition when rinsed with 10 mM NaCl and 1 mM NaCl solutions. Error bars represent standard deviations.

25

2

Deposited Bacteria (per mm )

10000

Cycle 3

(a) 8000

Cycle 1

Cycle 2

6000 4000 2000 0 0

10 20 30 40 50 60 70 80

Membrane P Membrane P43

2

Deposited Bacteria (per mm )

Time (min)

10000 8000

(b) Cycle 1

Cycle 3 Cycle 2

6000 4000 2000 0 0 10 20 30 40 50 60 70 80

Time (min) Fig. 4. Number of bacteria on (a) base membrane and (b) Membranes P and P43 over threecycles of bacterial deposition and release. For each cycle, bacterial deposition took place at 10 mM NaCl in the presence of a permeate flow rate of 30 µm/s. The membrane was subsequently rinsed at 10 mM NaCl in the absence of permeate flow. The pH was maintained at 7.0 over the three cycles of deposition and release.

26

2

Deposited Bacteria (per mm )

10000 Cycle 1

Cycle 2

Cycle 3

8000 6000 4000 2000 0 0

20

40

60

80

100

Time (min)

Fig. 5. Number of bacteria on Membrane PM over three-cycles of bacterial deposition and release. For each cycle, bacterial deposition took place at 10 mM NaCl in the presence of a permeate flow rate of 30 µm/s. The membrane was subsequently rinsed at 1 mM NaCl in the absence of permeate flow. The pH was maintained at 7.0 over the three cycles of deposition and release.

27

Graphical abstract

28

Highlights •

Silver nanoparticles are immobilized on membranes with polyelectrolyte multilayers.

•

Complete inhibition of bacterial growth can be achieved at low silver loadings.

•

Nanoparticles on membrane surface maximize chances for nanoparticle–cell contact.

•

Membranes modified with multilayers and nanoparticles can resist bacterial adhesion.

•

Multiple cycles of filtration and rinsing are conducted to test long-term performance.

29