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Journal of Membrane Science 351 (2010) 112–122

Contents lists available at ScienceDirect

Journal of Membrane Science

journa l homepage: www.e lsev ier .com/ locate /memsci

valuation of surface properties of reverse osmosis membranes on the initialiofouling stages under no filtration condition

onil Leea, Chang Hoon Ahna, Seungkwan Hongb, Seunghyun Kimc, Seockheon Leed,oungbin Baeka, Jeyong Yoona,∗

World Class University (WCU) Program of Chemical Convergence for Energy & Environment (C2E2), School of Chemical and Biological Engineering, Seoul National University,an 56-1, Silim-dong, Gwanak-Gu, Seoul, Republic of KoreaDepartment of Civil Engineering, Korea University, Seoul, Republic of KoreaDepartment of Civil Engineering, Kyungnam University, Masan, Republic of KoreaCenter for Environmental Technology Research, Korea Institute of Science and Technology, Cheongryang, Seoul 130-650, Republic of Korea

r t i c l e i n f o

rticle history:eceived 29 September 2009eceived in revised form 14 January 2010ccepted 15 January 2010vailable online 22 January 2010

eywords:everse osmosis (RO) membraneembrane biofouling

seudomonas aeruginosa biofilm

a b s t r a c t

In order to evaluate the effect of membrane surface properties on the initial stage of biofouling, in thereverse osmosis (RO) membrane process, initial bacterial adhesion and biofilm formation experimentswere performed under no filtration condition. In this study, five commercialized polyamide thin-filmcomposite RO membranes (SW30HRLE, SW30HR (Dow FilmTec Co., USA), TM820 (Toray Co., Japan), RE-BE, RE-FE (Woongjin Chemical Co., Korea)) were chosen and their surface properties such as surfacecharge, roughness, hydrophobicity and surface morphology were measured. For examining initial bac-terial adhesion, a flow channel reactor was employed for 3 h, while for examining a biofilm formation,the CDC reactor was employed for 48 h. Pseudomonas aeruginosa PAO1 tagged with GFP was selected as amodel bacterial strain. Major findings in this study indicate that although the initial bacterial cell adhe-

ydrophobicityxtracellular polymeric substances (EPS)

sion in a flow channel reactor indicated more bacterial cells attachment on the membrane surface withhigher hydrophobicity, the extent of biofilm grown in CDC reactor for 48 h became similar regardlessof the difference of the membrane surface properties, indicating that the membrane surface propertiesbecome a less important factor affecting the biofilm growth on the membrane surface. This finding willbe helpful in improving the understanding of biofouling issue occurring in the real RO membrane system,although practical implication is somewhat limited since this study was performed under no filtration

condition.

. Introduction

During last decade, reverse osmosis (RO) process has beenighlighted in the water purification and wastewater reclama-ion systems. However, undesirable abiotic and biotic materials arerone to deposit on the membrane surface, called as membraneouling, which may alleviate the system efficiency [1,2]. It is wellocumented that the membrane biofouling is one of the major con-erns in the RO process, resulting in the decline of permeate fluxnd salt rejection efficiency [3,4].

Biofouling phenomenon on membrane surfaces may be

receded through two processes, the initial adhesion of microor-anisms and biofilm formation by reproduction of attached cells [5].n initial adhesion of microorganisms, it is likely that organics couldave more chance to be attached on the membrane surface than

∗ Corresponding author. Tel.: +82 2 880 8927; fax: +82 2 876 8911.E-mail address: jeyong@snu.ac.kr (J. Yoon).

376-7388/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.memsci.2010.01.035

© 2010 Elsevier B.V. All rights reserved.

bacteria cells due to the overwhelming numbers of organics. Diffu-sion, gravitational settling, van der Waals force, electrostatic force,steric interaction, Lewis acid–base interaction, hydration pres-sure, hydrogen bonding, and hydrophobic effects will be played animportant role [6–9]. After initial attachment of cells, the attachedcells will be colonized on the surface of membrane to form a biofilm.Then, the attached cells are supposed to produce extracellular poly-meric substances (EPS) and develop into microcolonies [10,11].EPS is metabolic products that are mainly composed of organics,including protein, carbohydrates, DNA, and RNA [12].

Membrane surface can be characterized by physical and chem-ical properties. Physical properties of the membrane surface aremorphology of steepness and evenness, topology of peak and valley,whereas chemical ones include the degree of hydrophobicity (or

hydrophilicity), surface charge with many functional groups, and soon. It was reported that the permeate flux tends to be more declinedas membrane surface becomes rougher [13,14], more hydrophobic[15] and neutral or negative charge [16]. Also, membrane foulingincluding biofouling is influenced by various parameters, such as

W. Lee et al. / Journal of Membrane Science 351 (2010) 112–122 113

Table 1Operational specifications and surface characteristics of five types of RO membranes.

Model (Manufacturer) SW30HRLE (DowFilmTec Co.)

SW30HR (DowFilmTec Co.)

TM820 (Toray Co.) RE-BE (WoongjinChemical Co.)

RE-FE (WoongjinChemical Co.)

Fluxa (L m−2 h−1) 31.5 31.9 28.2 48.0 41.7Stabilized salt rejectiona (%) 99.75 99.75 99.75 99.5 99.5Max. operating pressurea (bar) 83 83 69 41 41RMSb roughness (nm) 108 ± 12 75 ± 13 64 ± 4 55 ± 2 70 ± 2

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Dynamic contact angle (◦) 33.0 ± 0.4 42.8 ± 2.3

a Data provided by manufacturers.b RMS = root mean square.

eed solution composition (e.g., pH, ionic strength, and divalentation concentrations), fluid flow, EPS [17] and interaction betweenoulants [18,19]. Up to now, only a few researches reported on theirect microscopic observation of initial microbial cell attachmentn RO membrane [20,21], and biofilm formation on RO membranes barely found. Fundamental mechanisms of the initial foulingtages including cell attachment and biofilm formation still needo be investigated further.

The objectives of this paper are to evaluate the effect of majorhysico-chemical properties of the membrane surface on the bac-erial adhesion and to investigate the features of the biofilmormed by attached bacterial cells under no filtration condition.n the present study, five commercialized polyamide (PA) thin-lm composite (TFC) RO membranes were used and characterizedystemically. Pseudomonas aeruginosa PAO1 tagged with GFP waselected as a model bacterial strain to observe initial bacteria adhe-ion directly in a flow channel and to form biofilms in a Center forisease Control (CDC) reactor.

. Materials and methods

.1. Operational specifications of five selected RO membranes

Table 1 summarizes operational specifications of five types ofO membranes that were recommended by manufacturers. All ofhem are made of polyamide thin-film composite (<1 �m) that isupported by polysulfone layer (∼140 �m). All the membranes areonfigured by spiral-wound type with high salt rejection efficiency>99.5%) and their manufacturing aims are described briefly as fol-ows:

SW30HRLE and SW30HR (Dow FilmTec Co., USA) are madefor the seawater desalination with high salt rejection rate andSW30HRLE is for low energy requirements.TM820 (Toray Co., Japan) is for the seawater desalination withhigh permeate flux.RE-BE and RE-FE (Woongjin Chemical Co., South Korea) is forbrackish feed water and low fouling potential, respectively.

The membranes were kindly received from the manufacturerss flat sheets and stored in deionized water (Barnsted NANO Pure,SA) at 4 ◦C. The water was replaced regularly prior to all experi-ents in order to remove the protected coating chemicals and/or

umectants.

.2. Membrane characterization

The surface roughness of the RO membranes was measured byn atomic force microscopy (AFM, SIS, Pucostation, German) in a

apping mode, of which probe is made of silica coated with gold. Inrder to measure accurately, five points of membrane surface wereeasured and averaged. Morphological characteristics of the virgin

O membranes were analyzed by AFM images. For all RO mem-ranes, root mean square (RMS) roughness ranged 55–108 nm;

91.0 ± 0.4 68.8 ± 2.1 54.1 ± 1.2

RE-BE is the least at 55 ± 2 nm, whereas SW30HRLE is the highestat 108 ± 12 nm (Table 1).

In order to identify the functional groups on virgin membranesurfaces, the attenuated total reflection—Fourier transform infrared(ATR-FTIR) analyses were performed using a Nicolet spectropho-tometer 5700 (Thermo Electron Corp., USA). Thirty two ATR-FTIRspectra were obtained for every membrane type, with each spec-trum averaged from 128 scans collected from 1100 to 4000 cm−1

at 1 cm−1 resolution.Membrane surface charge was evaluated by the zeta potential

at the polyamide side of RO membrane and was calculated fromstreaming potential measurements (Anton Parr, GmbH, Ostfildern,Germany). pH of the electrolyte (KCl, 0.1 mM) was adjusted in orderto obtain zeta potential values between pH 2 and 9.

Hydrophilicity/hydrophobicity of the membrane was deter-mined by a dynamic contact angle (Sigma 701, KSV InstrumentsLtd., Helsinky, Finland). The value of dynamic contact angle indi-cates the surface wettability; the higher contact angle, and the morehydrophobic [22]. After drying samples, three replicate measure-ments were done per sample.

In addition, macro morphology of the membrane was analyzedby using a field emission scanning electron microscope (FE-SEM)(JSM-6700 F, JEOL Ltd., Tokyo, Japan).

2.3. Bacterial cell adhesion experiments

2.3.1. Preparation of the bacterial cellSince P. aeruginosa is an opportunistic human pathogen and pre-

dominantly found in biofilms [1,18,19], P. aeruginosa PAO1 taggedwith green fluorescent protein (GFP) was selected as a modelbacterial strain and GFP includes plasmid with only carbenicillinresistant gene. A derivative of P. aeruginosa PAO1 tagged with GFPwas received from the Center for Biofilm Engineering (MontanaState University, USA). To obtain a single pure strain, PAO1 waspre-cultured on a tryptic soy agar (TSA, Difco, Franklin Lakes, NJ)plate supplemented with 150 �g mL−1 of carbenicillin (Aldrich, St.Louis, MO) for 18 h at 37 ◦C in an incubator (IB 25G, Lab Campan-ion, Korea). A fresh, single colony from the plate was inoculated in3 g L−1 of tropic soy broth (TSB, Bacto, Franklin Lakes, NJ) contain-ing 150 �g mL−1 of carbenicillin and cultured for 18 h at 110 rpmat 37 ◦C to reach a mid-exponential growth phase. Pure bacterialcells were harvested by centrifugation at 4000 rpm for 10 min. Thesupernatant was discarded and the remaining pellet was washedtwice in 20 mM phosphate buffer solution (KH2PO4, pH 7.1) toremove nutrients. After washing, the cells were re-suspended indeionized water and the bacterial populations in the suspendedsolution were calculated from the optical density of cells measuredby a UV–vis Spectrophotometer (Agilent 8453, Agilent Technolo-gies, Santa Clara, USA) at a wavelength of 600 nm (OD600).

2.3.2. Flow channel experimentA flat sheet (74 mm in length × 24 mm in height) of the con-

cerned RO membrane was carefully placed into the flow channelreactor (FC 281, BioSurface Technologies, Bozeman, MT). Both

114 W. Lee et al. / Journal of Membrane

Fig. 1. Changes of zeta potential of five types of RO membranes against solution pH.

Fig. 2. FE-SEM images of five types of virgin RO membranes with 30,000 times magnificTM820 (Toray Co., Japan), (d) RE-BE (Woongjin Chemical Co., Korea), and (e) RE-FE (Woon

Science 351 (2010) 112–122

surface and bottom of the membrane specimen were coveredtightly with a microscope slide glass (Superior Marienfeld, Ger-many) and a cover glass (VWR, USA) to monitor in situ bacterialadhesion under a fluorescent microscope (Eclipse 80i, Nikon Instru-ments Inc., Melville, NY). The silicon gaskets were placed betweenthe flow channel cover plates and the top/bottom covered glasses,and all holes were thoroughly fastened with screws. The flow ratewas set at 1.3 mL min−1, providing fluids shear rate of 1.11 s−1

through the flow channel by a peristaltic pump (Miniplus 3, Gilson,France). The shear rate was calculated using the following equationas described by Busscher and Van der Mei [23]:

� = 3Q

2(

h02

)2w0 · t

(1)

where � is the shear rate (s−1), Q is the volumetric flow rate(m3 s−1), h0 is the height of channel (m) and w0 is the width ofchannel (m). The height and width of the flow chamber are 3 mmand 13 mm, respectively.

ation: (a) SW30HRLE (Dow FilmTec Co., USA), (b) SW30HR (Dow FilmTec Co.), (c)gjin Chemical Co., Korea). Scale bar = 100 nm.

brane Science 351 (2010) 112–122 115

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W. Lee et al. / Journal of Mem

First, 20 mM of cell-free phosphate buffer solution (pH 7.1)as flowed for 30 min to remove any impurities and to stabi-

ize the system. Then, the bacterial suspension was flowed for80 min while stirring at 240 rpm with magnetic bar at room temp.20 ± 2 ◦C). In the flow channel experiment, the bacterial suspen-ion was prepared at an initial cell concentration of about 1 × 107

olony forming unit (CFU) mL−1 approximately in deionized water.mages at the designated times (for 30, 60, and 180 min) wereaptured simultaneously from three equidistant locations on theembrane surface by using a digital camera (DS-2U, Nikon Inc.,

okyo, Japan). The average number of bacterial cells adhered to theembrane specimen per unit area (CFU cm−2) and calculated using

mage processing software (i-solution, IMT Technologies Co., Ltd.,SA).

.4. Examination of biofilm development in a CDC reactor

.4.1. Biofilm development assayBiofilms of P. aeruginosa PAO1 tagged with GFP were grown in

CDC reactor (Biosurface Technologies Inc., Bozeman, MT). Theeactor contains eight rods, each of which houses three holes forampling coupons. Each membrane sample sheet was attached onhe polycarbonate coupon.

The pure bacterial cell (GFP-tagged PAO1) suspension wasrown in 10% TSB for 18 h at 37 ◦C, as described in Section 2.3.1,nd inoculated in the CDC reactor containing 350 mL of 1% TSBulture medium. The initial PAO1 cell concentration was about× 106 CFU mL−1 and the reactor was operated in a batch mode for4 h at room temperature (20 ± 2 ◦C) in 1% of TSB medium whiletirring at 100 rpm. After 24 h, a 10-l carboy containing 0.3% of TSBedium was connected to the CDC reactor that has working vol-

me of 350 mL. Then, the operation of the CDC reactor was changedo a continuous flow mode by feeding 0.3% TSB with at a flow rate of1 mL min−1, with stirring at 100 rpm for the subsequent 24 h. TSAnd TSB in the present study contained carbenicillin for antimicro-ial agent. After 48 h, each rod was taken out from the CDC reactor.o remove the TSB solution left on the membrane coupon, eachembrane coupon was gently rinsed with deionized water, as not

isturbing the biofilm attached on the membrane surface.In order to detach cells and EPS in the biofilm matrix, membrane

oupons were soaked in DI water and the biofilm was re-suspendedy ultrasonication for 2 min and vortexing for 3 min. Total numberf cells in detached biofilm from the CDC coupons was enumer-ted by a spread plate count method, in which diluted biofilmolution was spread on TSA plate and incubated at 37 ◦C for 18 h.fter incubating, the numbers of viable colonies were counted.he bound EPS extracted from the re-suspended biofilm solution,ncluding polysaccharides and proteins, was analyzed by a cationxchange resin (CER) method [24]. The extracted polysaccharidesnd proteins from the detached biofilms were analyzed by using ahenol/sulfuric method [25] and a Lowry method [26], respectively.

Dissolved organic carbon (DOC) concentration was measuredsing carbon analyzer (TOC-V, Shimadzu, Japan). The non-urgeable organic carbon (NPOC) method was employed. Allamples were filtered through 0.45 �m membrane prior to the DOCeasurement and were acidified with the addition of 2N HCl to

emove inorganic carbon by sparging hydrocarbon free air prior toOC measurement.

.4.2. Fluorescent stains and confocal laser scanning microscopyThe biofilms grown on membrane coupons, obtained from the

DC reactor experiment, were immediately stained with SYTO9BacLight Live/Dead bacterial viability kit, Molecular Probes, USA)nd Concanavalin A (ConA, Molecular Probes, Eugene, OR) lectinonjugated with tetramethyl rhodamine isothiocyannate (TRITC),or the probing of live cells and EPS, respectively [27]. 100 �L of

Fig. 3. ATR-FTIR spectra of the virgin polyamide RO membranes: (a) wavenumberranged between 1100 and 1800 cm−1, and (b) wavenumber ranged between 2400and 4000 cm−1.

SYTO9 solution (3 �L was dissolved in 1 mL of distilled water) wasadded to each of biofilm samples, which were then incubated in thedark at room temperature for 1 h. Unbound SYTO9 was carefullyrinsed by distilled water. Following this, 100 �L of ConA solution(3 �L was added in 1 mL of distilled water) was added to the SYTO9-stained biofilm specimens, which were then incubated in the darkat room temperature for 1 h. The excessive ConA solution was thenremoved by distilled water in the same manner as the SYTO9 stain-ing.

Subsequently, biofilm image observation, acquisition and anal-ysis were performed on the stained biofilm specimen using aconfocal laser scanning microscopy (CLSM, Eclipse 90i, Nikon,Japan) equipped with an argon–krypton laser and Bio-Rad Radi-ance scanning confocal system. The CLSM was equipped withspecific detectors and filter sets for monitoring the stained cells:SYTO9 (excitation wave length (ex) = 488 nm; emission wavelength (em) = 522/32 nm) and ConA conjugated TRITC (ex = 568 nm;em = 605/32 nm). Biofilm images were observed with a waterimmersion lens (60× object and numerical aperture of 1.4) anda series of z-axis images were generated through optical section-ing at a sliced thickness of 1 �m. Each membrane specimen with

adhered biofilm was scanned randomly between 4 and 5 positions.The observed images covered an area of 202 �m × 202 �m at aresolution of 512 × 512 pixels (256 gray-values). The CLSM imageswere reconstructed using a digital images analysis program, IMARIS

116 W. Lee et al. / Journal of Membrane Science 351 (2010) 112–122

F on fi( er 30e

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ig. 4. Time-lapse fluorescent microscopic images of P. aeruginosa PAO1 adhesionj–l), and RE-FE (m–o). The fluorescent microscopic micrographs were acquired aftxperiment.

version 6.1.5, Bitplane AG, Zurich, Switzerland) and presented asD structure.

. Results and discussion

.1. Surface morphology of RO membranes

Fig. 1 plotted changes of zeta potential (ZP) for five differentypes of RO membranes against solution pH. In spite of the sameomposite materials, the profiles of ZPs are different from eachther. Three membranes (TM820, RE-BE and RE-FE) show a typi-

al surface charge variation in the literature [28]; under lower pH<3), ZP values are positive, which become negative at higher pH>4). Such amphoteric behavior with isoelectric points around pH 4s ascribed to both amine and carboxylic functional groups, whichan be observed typically for uncoated polyamide membrane sur-

ve types of RO membranes: SW30HRLE (a–c), SW30HR (d–f), TM820 (g–i), RE-BE(left column), 60 (middle column) and 180 (right column) min in the flow channel

faces [29]. In contrast, SW30HR is negative for the entire testedrange, indicating that the membranes are less sensitive against thesolution pH. Peculiarly, SW30HR has the least negative zeta poten-tial (−9.7 mV) at neutral pH. It seemed that the surface layer ofSW30HR is fully coated. According to Tang et al. [29] who examinedthe same membrane, the reduced surface charge of the SW30HRcould be attributed to both significant amount of alcoholic (–COH)groups and fewer amounts of amine groups of the coated layer.In addition, from the results of XPS measurement, Tang and hisgroup further explained that SW30HR composed of N-rich regionand N-lean region with the non-uniformity of the coating layers.A high N content polyamide layer of SW30HR membrane had ele-

mental composition with 20% oxygen (O), 10% N, and 70% carbon(C), whereas a low N content of SW30HR had 34% O, 1% N, and 65%C.

FE-SEM images of five types of RO membranes are representedin Fig. 2. All the images show pebble-style surfaces with peak-and-

W. Lee et al. / Journal of Membrane Science 351 (2010) 112–122 117

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dtmstai3satl[3

ig. 5. Bacterial adhesion of P. aeruginosa PAO1 tagged with GFP on five types of Rontact angle), (c) surface charge (measured by zeta potential), and (d) surface rougll the cases. Points represent data mean and arrow are standard deviations.

alley structures, which are attributed to the top facial polyamideayers of several hundred nanometers thick [30].

In order to verify functional groups on the membrane sur-aces, ATR-FTIR spectra of the five virgin RO membranes arebtained. Fig. 3a represents ATR-FTIR spectra of wave numberanged between 1100 and 1800 cm−1. The peaks around 1240,290 and 1320 cm−1 can be assigned to aromatic amines I, II and

II stretching, respectively [31]. The peaks at 1500 and 1600 cm−1

an be assigned to polysulfonyl group in the porous polysulfoneayer, whereas the broad bands around 1540 and 1680 cm−1 cane assigned to amides I and II. Below 2000 cm−1 of wave number,hin-film polyamide and polysulfone support layer can be ana-yzed because of greater penetration depth [32]. Although aminesnd polysulfonyl groups of RO membranes are confirmed, all thepectra look similar and are not discernible in this range of spec-ra.

Higher range ATR-FTIR spectra between 2400 and 4000 cm−1

isplay interesting differences (Fig. 3b). Tang et al. [29] dis-inguished the peaks between the coated and the uncoated

embranes, and found that the coated layer is aliphatic with aignificant amount of OH groups. For the uncoated membranes,he peaks around 2930–2970 cm−1 and 3040–3080 cm−1 can bessigned to aliphatic (C–H) stretching and aromatic ( C–H) stretch-ng, respectively [31]. On the other hand, the peaks around300–3400 cm−1 can be assigned to free and hydrogen bonded N–Htretching [33,34]. SW30HR has the most outstanding broad band of

round 3300 cm−1, which may be attributed to an stretching vibra-ion of N–H and carboxyl groups (–COOH) groups of the polyamideayer, and potential alcoholic (–COH) groups in the coating layers29]. Both RE-BE and RE-FE have the similar broad band around300 cm−1 with reduced intensity, which may be due to the over-

branes in flow channel: (a) time-lapse profiles, (b) hydrophobicity (measured by. The initial concentrations of bacterial solution were adjusted to 107 CFU mL−1 for

lapping of stretching vibration of N–H and carboxylic groups of thepolyamide layer.

3.2. Real time observation of the initial bacteria adhesion on ROmembranes

Fig. 4 illustrates time-lapse fluorescent microscopic images of P.aeruginosa PAO1 adhesion on five types of RO membranes. Imagesof GFP-tagged PAO1 cells were acquired after relatively short periodof time (30, 60, and 180 min) in a flow channel. Even at a short expo-sure time (30 min), considerable amount of bacterial cells wereattached on the RE-BE (Fig. 4j), the second greatest hydrophobic.As time passed, the number of cells attached on RE-BE and RE-FE increased steadily, whereas those on SW30HRLE and SW30HRalmost stayed at the same level. Apparently, the largest number ofcells was attached on the TM820, which has the greatest hydropho-bic surface. For all the membrane surfaces, the attached cells werespread out and micro-colonized.

Fig. 5 shows the bacterial cell concentrations expressed as bac-terial coverage with time in the flow channel system (5a), androughness, zeta potential, and contact angle (5b–d). As shown inFig. 5a, after 30 min of elapsed time, a considerable number ofPAO1 cells were attached on the RE-BE with steepest peaks and val-leys among tested membranes. As time passed 60 min, the amountof bacterial cell attached on the TM820 and RE-FE increased,both of which have hydrophobic surfaces as well. By the end of

experiment, the bacterial cell concentration of the RE-BE reached2 × 106 CFU cm−2, while the other three membranes (SW30HRLE,SW30HR and RE-FE) were less than half (∼1 × 106 CFU cm−2)(Fig. 5a). Intriguingly, the bacterial cell concentration of TM820kept increasing over 5 × 106 cm−2 till 180 min, suggesting that the

1 brane

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18 W. Lee et al. / Journal of Mem

acterial deposition did not reach a saturation point yet. In addition,lthough RE-FE which was reported to be made for the purpose ofow fouling potential has less bacterial cells accumulation than RE-E, it has still more bacterial cells accumulation than SW30HRLEnd SW30HR.

Fig. 5b plotted hydrophobicity (measured by contact angle)ersus bacterial cell concentration at 180 min of elapsed time. Mea-ured contact angle ranged from 33◦ to 91◦; SW30HRLE is the leastydrophobic (or the most hydrophilic) with 33.0 ± 0.4◦, whereasM820 is the most hydrophobic (or the least hydrophilic) with1.0 ± 0.4◦ (Table 1). Clearly, it was shown that the bacterial celloncentration was linearly proportional to the hydrophobicity ofembrane surface in a short-term experiment. TM820 accumu-

ated the greatest bacterial cell concentration with the highestydrophobicity, whereas SW30HRLE had the least bacterial con-entration with the greatest hydrophilicity.

Fig. 5c shows the relationship between the surface chargesmeasured by zeta potential) versus bacterial cell concentra-ions. As shown in Fig. 5c, the greater numbers of cells werettached on the less negatively charged surface except SW30HR.his trend is likely if the repulsive interaction between the sur-

ace of bacterial cells and the membrane surface is considered,oting that zeta potential of the bacterial cell (PAO1) was nega-ive (−22 mV at pH 7). For instance, SW30HRLE had the greatestegative surface charge at −30 mV with the smallest number of

ig. 6. AFM images of the virgin RO membranes: (a) SW30HRLE, (b) SW30HR, (c) TM820

Science 351 (2010) 112–122

bacterial cells (∼0.5 × 106 CFU cm−2) and TM820 had the secondleast negative at −17 mV with the greatest number of attachedcells (∼5 × 106 CFU cm−2). However, fully coated SW30HR whichhad the least negative surface charge at −6 mV had the small-est bacterial concentration (∼0.5 × 106 CFU cm−2), which deviatesfrom the general trend. No good explanation is available forSW30HR.

Fig. 5d compares surface roughness versus bacterial cell con-centration. It is believed that both abiotic and biotic depositionsare prone to increase, as the membrane surface gets rougher[13,35]. In the present study, the bacterial cell concentration showsa weakened reverse relationship to the surface roughness of ROmembrane. For instance, although RMS values for SW30HR andTM820 were close to each other (75 ± 13 and 64 ± 4 nm, respec-tively), the attached cell number of TM820 (∼5 × 106 CFU cm−2)was almost one order of magnitude greater than that of SW30HR(∼0.5 × 106 CFU cm−2).

In order to determine the morphological characteristics, AFMimages of the virgin RO membranes were obtained (Fig. 6), whichcan be used for observing the degree of steepness according to theshape of peaks. As the peaks get higher than the base level of mem-

brane surface, resulting in greater degree of steepness, the bacterialadhesion may be intensified. Then, the surface morphology couldinfluence on the initial bacterial adhesion. According to Tansel etal. [35], the bacterial adhesion could be attributed to the active

, (d) RE-BE and (e) RE-FE. Each side of plane is 10 �m with the height of 400 nm.

brane

d‘rRE

m

Fr�

W. Lee et al. / Journal of Mem

eposition by functional groups and the passive deposition by acompatible pocket’ phenomenon. Even with the similar surface

oughness of the membranes, the steep morphologies of RE-BE andE-FE could provide a ‘hook and crook’ effect for bacterial cells andPS than SW30HR.

Our experimental results suggest that many properties of theembrane surfaces influence the bacterial adhesion in a time-

ig. 7. CLSM 3D images (top row) and side-view projection (bottom row) showing theeactor: (a) SW30HRLE, (b) SW30HR, (c) TM820, (d) RE-BE and (e) RE-FE. Green spots locam, Y = 200 �m and Z = 10 �m (bottom row). (For interpretation of the references to colo

Science 351 (2010) 112–122 119

dependent manner. At the very initial stage of the experiment(Fig. 5a), RE-BE that has steep morphology accumulated a consid-

erable amount of bacterial cells. As time passed, all the membranesurfaces were covered with the attached bacterial cells. Then, theinfluence of surface morphology became lessened on account of theaccumulated bacterial layer, while chemical properties still workon to a certain degree.

spatial structure of P. aeruginosa biofilm on five types of RO membranes in a CDClize P. aeruginosa PAO1 and red locales are EPS. Scale bar = 20 �m (top row); X = 200r in this figure legend, the reader is referred to the web version of the article.)

120 W. Lee et al. / Journal of Membrane Science 351 (2010) 112–122

(Conti

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3

4Il

Fig. 7.

As shown in Fig. 5, hydrophilicity/hydrophobicity showedtronger relationship than surface roughness and surface charge athe initial bacterial adhesion stage. The most hydrophobic mem-rane (TM820) accumulated the highest number of cells, whilehe least hydrophobic membrane (SW30HRLE) and the secondydrophilic/fully coated membrane (SW30HR) accumulated the

owest number of cells.In addition, it is also claimed that surface charge would provide a

trong effect on the bacterial cell attachment [36,37]. The measuredeta potentials ranged between −9 and −30 mV at neutral pH andhe bacterial adhesion increased in order of SW30HR < TM820 < RE-E < RE-BE < SW30HRLE. The bacterial cell concentrations of four ROembranes (SW30HRLE, TM820, RE-BE and RE-FE) followed the

eneral trend according to the surface charge. However, SW30HRhich is assumed to have the fully coated layer had the least bacte-

ial concentration even with the least negatively charged surface.he surface conditioning by macromolecules (e.g., EPS) in a sus-ended solution might change the zeta potential values to a great ormall extent [38]. Thus, surface charge would provide a weakenedffect on the bacterial adhesion compared to the hydrophobicity.

.3. Biofilm formation on the RO membranes

A relatively long-term test was conducted in the CDC reactor for8 h in order to examine biofilm formation on the RO membranes.

t was noted that in general, over 50% flux loss occurs within 30 h inab-scale RO cross-flow test unit with P. aeruginosa PAO1 GFP, sig-

nued).

naling RO membrane cleaning [4,17]. However, since this study wasdone under no filtration condition, the direct comparison requiresa caution. For the better interpretation of the biofilm formation, itwas necessary to localize the live cells and EPS distribution.

Fig. 7 displays images of viable cells (green spots) and polysac-charides (red locales) bound to ConA with TRITC that indicates thelocations of EPS. As shown in the left column of the figure, theplane view of biofilm formed on each membrane looks different. Anoverview reveals that live cells are spread out ubiquitously, whileexcreted EPS are lumped in stacks on the membrane surface. Alongwith the greatest cell accumulation in the flow channel experiment,TM820 has abundant EPS at different locations, which might dragmore cells as time passed. By closer look, RE-BE and RE-FE havelive cells distributed evenly in a lawn style with EPS stacks at acouple of locales. On the other hand, SW30HRLE had EPS along thelive cells. Side-view projections in the right column show that thevertical distribution of live cells and EPS. It is noticeable that thelayers of EPS are located at rather high altitudes for all the cases,indicating relatively young biofilms. With a view to cell aggrega-tion and extension, Type IV pili-dependent twitching motility ofP. aeruginosa should contribute distribution of EPS on the floor ofmembrane surface [39]. On the other hand, mushroom shapes of

cell stacks, which might be a good indicator of the mature biofilmat a second hand, were not observed for all the membranes [11].We believed that the adhered cells were in the early stage of biofilmformation and still in the way of processing biofilm developmentor maturization.

W. Lee et al. / Journal of Membrane

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ig. 8. Concentration of polysaccharide and protein as well as cell concentration of. aeruginosa PAO1 accumulated on five types of RO membranes in a CDC reactor.oints and columns represent data mean and arrows are standard deviations.

After the biofilm formation experiments were finished, the con-tituents of the bulk solution in the CDC reactor were analyzed.he solution pH was neutral (6.9 ± 0.1) and the dissolved organicarbon (DOC) concentration was measured at 23.5 ± 0.6 mg C L−1.acterial cell concentration in the CDC reactor increased rapidly

rom ∼106 CFU mL−1 to ∼109 CFU mL−1 for 48 h. Polysaccharidend protein concentrations were 4.8 ± 0.8 and 7.7 ± 0.7 �g mL−1,espectively.

Fig. 8 plotted the concentrations of polysaccharide and pro-ein as well as bacterial cell concentrations accumulated on fiveypes of RO membranes in the CDC reactor. The bacterial cell con-entrations ranged from ∼109 to ∼1010 CFU cm−2 regardless ofembrane types. The bacterial cell concentrations show no sig-

ificant differences among biofilms in the CDC reactor, indicatinghat the membrane surface properties become a less importantactor affecting the biofilm growth on the membrane surface. How-ver, this trend is in contrast with recent study reported thatifferences in surface properties of the membrane contributedo variations in biofilm formation [40]. Among three membranesmployed in his study, one was BW30 (Dow FilmTec Co., USA)nd the others were the experimental membranes developed byow FilmTec Co. One explanation is that some of the mem-ranes used in Khan’s paper could be biocide coated and itsurface property may affect the development of biofilm thick-ess. However, the exact explanation is not available at present.urthermore, the polysaccharides concentration of the P. aerugi-osa PAO1 biofilm on the RO membranes was higher than protein.or example, the concentrations of polysaccharide ranged from.3 ± 1.1 (SW30HRLE) to 3.9 ± 2.6 �g cm−2 (RE-BE), whereas thosef protein varies from 0.8 (SW30HR and RE-BE) to 1.3 ± 0.2 �g cm−2

SW30HRLE). This trend was consistent with the previous study17]. This preferential adsorption/accumulation of polysaccharidesccurs more membrane fouling due to enhancing mechanical sta-ility of biofilms with polysaccharide-calcium specific interactions41].

The ratio of polysaccharide (PC) to protein (PN) varies dependingn the functioning microbial consortium and operating condition.n the present study, the ratio of PC/PN of the bulk solution was

−1

.6 �g �g . On the contrary, the ratio of PC/PN of RE-BE was thereatest (5.4 �g �g−1), whereas that of SW30HRLE was the small-st (1.9 �g �g−1). It is indicated that the amounts of secreted EPSould be different in membrane surface properties although theoncentrations of attached cells onto membrane surface are alike.

[

[

Science 351 (2010) 112–122 121

Mucosity of P. aeruginosa is mainly attributed to the synthesis ofexopolysaccharide alginate [39]. Considering the higher ratio of thebiofilm, the bacterial cell attached on the membrane surface couldproduce more polysaccharide than protein to adhere themselvesand to aggregate together.

4. Conclusions

Fundamental mechanisms of biofouling were investigated withfive types of polyamide thin-film composite RO membranes byemploying a model bacterial strain of P. aeruginosa PAO1 taggedwith GFP. The followings are deduced from a series of laboratoryscale experiments:

• In a real time observation of cell adhesion using a flow channelreactor, the bacterial cell concentrations increased proportionallyto the hydrophobicity. In general, less negatively charged surfacesaccumulated more bacterial cells except SW30HR.

• In a relatively long-term experiment using a CDC reactor, theeffects of physico-chemical properties of membrane surface werelessened, showing no significant differences of biofilm formationamong five types of RO membranes.

• Practical implication of this study is somewhat limited since thisstudy was performed under no filtration condition, requiring fur-ther study.

Acknowledgements

This research was supported by a grant (code# C106A1520001-06A085500121) from Plant Technology Advancement Programfunded by Ministry of Construction & Transportation of Korean gov-ernment and the WCU (World Class University) program throughthe Korea Science and Engineering Foundation by the Ministryof Education, Science and Technology (400-2008-0230). We areappreciated to Dow FilmTec Co. (USA), Toray Co. (Japan) andWoongjin Chemical Co. (Korea) for kind providing the membranesfor this study.

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