Bacterial biofilm formation on ion exchange membranes

Journal Pre-proof Bacterial biofilm formation on ion exchange membranes Moshe Herzberg, Soumya Pandit, Meagan S. Mauter, Yoram Oren PII:

S0376-7388(19)32087-3

DOI:

https://doi.org/10.1016/j.memsci.2019.117564

Reference:

MEMSCI 117564

To appear in:

Journal of Membrane Science

Received Date: 10 July 2019 Revised Date:

10 October 2019

Accepted Date: 11 October 2019

Please cite this article as: M. Herzberg, S. Pandit, M.S. Mauter, Y. Oren, Bacterial biofilm formation on ion exchange membranes, Journal of Membrane Science (2019), doi: https://doi.org/10.1016/ j.memsci.2019.117564. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Bacterial Biofilm Formation on Ion Exchange Membranes

Moshe Herzberg1*, Soumya Pandit1, Meagan S. Mauter2, and Yoram Oren1

Revised version submitted to Journal of Membrane Science October 10, 2019

1

Ben-Gurion University of the Negev

The Jacob Blaustein Institutes for Desert Research Zuckerberg Institute for Water Research Sede Boqer Campus, Midreshet Ben-Gurion, 84990, ISRAEL 2

Carnegie Mellon University

Department of Civil & Environmental Engineering 5000 Forbes Ave., Pittsburgh, PA, 15213, USA

*

Corresponding author:

Phone + 972 8 6563520; Fax + 972 8 6563503; E-mail: [email protected]

1

1

Abstract

2 3

Ion exchange membranes (IEMs) often suffer from biofouling, which reduces ion exchange rates

4

and increases energy consumption in water treatment processes, such as electrodialysis, reverse

5

electrodialysis, membrane capacitive deionization, and Donnan dialysis, and in energy devices,

6

such as microbial fuel cells. In the present study, microbial biofilm formation was studied on

7

anion exchange membranes (AEMs) and a cation exchange membranes (CEMs) of the

8

homogeneous and heterogeneous types. Biofilm formation of Pseudomonas aeruginosa PAO1

9

on the IEMs was higher on the CEMs than on the AEMs, although more dead cells were found

10

on the AEMs, likely due to the presence of quaternary ammonium moieties on the AEM surface,

11

which are bactericidal. An XTT assay and NPN uptake tests confirmed the antimicrobial

12

properties of the AEM surface. The results also suggested that the surface roughness of the

13

membranes affected interactions between bacteria and the IEMs, being more pronounced on the

14

heterogeneous IEMs than on the homogeneous IEMs. Counter-ion transport properties were

15

studied under the Donnan exchange regime for both pristine and biofouled IEMs. The reduction

16

of counter-ion transport due to biofouling was more pronounced for heterogeneous CEMs and

17

AEMs than for their homogeneous counterparts, while it was more noticeable for the AEMs than

18

for the CEMs. The latter result is explained based on the preferential adsorption of the negatively

19

charged EPS components to the positively charged AEMs.

20 21 22 23 24 25 26 27 28 29 30 31 32

Keywords: ion exchange membranes; biofilm; XTT assay; specific conductivity; Donnan

33

exchange; intracellular reactive oxygen species 2

34 35 36

1. Introduction

37

treatment-related processes, such as electrodialysis and Donnan dialysis and devices such as fuel

38

cells, which have a plethora of industrial applications [1]. They have been applied widely in

39

advanced engineering tools for desalting, concentrating and modifying products in seawater

40

desalination, industrial wastewater treatment, and beverage and food engineering processes [2].

41

Recently, IEMs have been utilized in energy conversion and storage technology, including

42

reverse electrodialysis for blue energy harvesting [3], membrane capacitive deionization [4],

43

contaminant removal and wastewater treatment in ion exchange membrane bioreactors[5] and

44

microbial desalination cells [6] as examples of promising cutting-edge technologies. Biofouling

45

of IEMs poses serious concerns, when the membranes are exposed to aquatic environments.

46

Thus, reducing biofouling of IEMs is essential to achieve optimal process performance as

47

microbial biofilms and their components present a major obstacle for maintaining the IEMs’

48

long-term effectiveness [7].

Ion exchange membranes (IEMs) are important components in energy generation and water-

49

Owing to the presence of fixed ionic charges in their interior, the IEMs are utilized to separate

50

ionic species from solutions containing neutral components by applying electrical fields or

51

concentration gradients [8]. Suspended solids carrying positive or negative electrical charges and

52

colloidal matter, such as polyelectrolytes, humic acids, surfactants, biological materials and

53

multivalent salts, near the saturation level, can cause severe problems when using IEMs (e.g., in

54

electrodialysis) due to precipitation on the membrane surface or by partial penetration into the

55

membranes. When this occurs, it may be followed by a dramatic increase in the membrane

56

electrical resistance. Since most of the colloids present in natural waters are negatively charged,

57

the anion exchange membranes are mostly affected by their presence [7]. Lindstrand

58

demonstrated that negatively charged solutes, such as octanoic acid and anionic surfactants

59

(sodium octanoate and sodium dodecylbenzene sulfonate), are responsible for a higher degree of

60

fouling on anion exchange membranes (AEMs), while cation exchange membranes (CEMs) are

61

marginally affected by those foulants [9]. Bukhovets et al. studied biofouling of the

62

heterogeneous anion exchange membrane MA-41 in an electrodialysis stack in the presence of

63

amino acids and observed maximum fouling, close to the limiting current density [10].

64

Consequently, membrane electrical conductivity decreased significantly in the presence of

65

phenylalanine.

66

Bacteria-mediated biofouling is caused by attachment of planktonic bacteria, followed by

67

the proliferation of sessile colonies on the membrane surface and maturation of the microbial 3

68

biofilm [11]. Previous work on bacterial attachment and biofilm formation has identified key

69

surface properties that promote biofilm growth: Hydrophobic rough surfaces, which include H-

70

bond donors and lack H-bond acceptors with low toxicity, usually promote bacterial attachment

71

and biofilm [12,13]. There is a lack of information on bacteria-mediated biofouling of various

72

categories of IEMs, as well as the effects on membrane performance, related transport

73

mechanisms, and particularly, the influence of the fixed charged groups of the IEM surface on

74

biofilm formation. In this study, commercially available homogeneous and heterogeneous IEMs

75

were investigated for their biofouling propensity using mono-culture biofilm experiments.

76

Variations of biofilm components and the related production of reactive oxygen species (ROS)

77

were analyzed in response to the membrane type. The mode of action by which biofilm

78

formation was affected was related to a comprehensive characterization of the IEM surface. The

79

effect of biofilm formation on counter-ion transport under the Donnan exchange regime was

80

tested as well.

81 82

2. Materials and Methods

83 84

2.1 Membrane conditioning.

85

The IEMs were conditioned prior to their use according to the following procedures.

86

Heterogeneous membranes were cleaned with acetone in order to remove wax/oily coatings from

87

the membranes. This step was followed by immersing the dry membrane in 50% (v/v) ethanol

88

for at least 6–8 h. Further, both homogeneous and heterogeneous IEMs were kept in a saturated

89

NaCl solution (300 g/L) for 12 h with gentle stirring. Before use, the membranes were washed

90

for 30 min with double distilled water (DDW) followed by exposing the membranes to a 10%

91

LB medium in the flow cell device.

92 93

2.2 Characterization of the anion and cation exchange membranes

94

The types of the commercial ion exchange membranes and their prime characteristics are

95

presented in Table 1. All the membranes were analyzed for surface roughness topology, surface

96

charge, and hydrophobicity [14].

97 98 99 100 101

4

102 103

Table 1: Specification of the commercially available IEMs used in the present study Type

Heterogeneous

Commercial name

Company, Country

MA40

MK40

Polyethylene Shchekinoazot, Russia

Thickness

RR'NH,

0.51

(mm)

Polyethylene

–SO3H

0.54

Polyethylene

R (CH3)3N+

0.72

Polyethylene

R - SO3-

0.64

N.A.

R- SO3-

0.34

N.A.

RR'R"N

0.37

Polyethylene

R (CH3)3N+

0.21

CMT

Polyethylene

R–SO3H

0.2

AMV

N.A.

N.A

0.14

AMH-PES

Mega,

RALEX®

Czechs

CMH-PES

Republic

Excellion I-200 Excellion I-100

SnowPure USA

LLC,

AMT Asahi Glass, Japan

CMV

Selemion, Japan

N.A.

N.A

0.16

AEX

Neosepta, Alstom, Japan

N.A.

N.A

0.18

N.A.

N.A

0.17

CMX 104

Fixed ionic group

RR'R"N

RALEX®

Homogeneous

Inert binder

N.A.: Not available

105 106

Surface hydrophobicity/hydrophilicity: Hydrophobicity of the different surfaces was determined

107

by the captive bubble contact angle method using the OCA 20 (DataPhysics, Filderstradt,

108

Germany) instrument. The IEMs were immersed in DDW overnight for conditioning at room

109

temperature prior to measurement. The analysis was carried out by placing a 10-µL air bubble

110

onto the surface; contact angles were measured using SCA-20 software (DataPhysics) by

111

drawing the surface baseline and drop profile and calculating the angle at the line of the three-

112

phase contact. At least five measurements were taken for each sample. In this case, a higher

113

contact angle indicates higher hydrophilicity. 5

114

Zeta potential: Zeta potentials of the IEM surfaces were determined at different pH values using

115

a Zeta potential analyzer (SurPass Elektrokinetic Analyzer, Anton Paar, Austria). As an

116

electrolyte, 0.1 mM KCl was used, and 0.1 N NaOH and 0.1 N HCl were used for adjusting the

117

pH.

118

Surface topography: Atomic force measurements were performed on the pristine IEM surfaces

119

for a surface roughness comparison. Imaging of the surface topography was performed using a

120

Nanoscope IIID MultiMode AFM microscope (Veeco-DI, Santa Clara, CA, USA) using an NP-S

121

cantilever with a spring constant of 0.06 N/m in tapping mode. The AFM image scanning area

122

was 25 µm2, and scans were carried out in air[14]. The temperature of the sample was monitored

123

during the scans. Roughness indices were estimated using the method of root-mean-square for

124

the Z-plane at a resolution of 5 µm2.

125

2.2 Biofilm growth on different IEM surfaces in a flow cell

126

Twelve different IEMs (Table 1) were placed in a flow cell for determination of biofilm

127

formation. For this purpose, an FC 81-PC transmission flow cell (BioSurface Technologies

128

Corporation, MT, USA) was used. The flow cell was sterilized with 70% ethanol for 30 min

129

followed by a thorough rinse with sterile DDW for 60 min [14].

130

Preparation of Pseudomonas aeruginosa PAO1 inoculum: The P. aeruginosa PAO1 strain, a

131

well-characterized Gram-negative bacterium that has become the most accepted model organism

132

for studying biofilm formation, was used in this study. Here, three independent biofilm

133

experiments were initiated from a stationary phase overnight culture of P. aeruginosa PAO1.

134

Each culture originated from one distinct bacterial colony. After 8.5 h of incubation at 30°C and

135

150 rpm stirring, the liquid culture was diluted 100 × in LB broth and incubated overnight. A

136

volume sample of 80 mL of the overnight culture was washed three times in 100 mM of NaCl

137

solution, and the optical density of the bacterial suspension was adjusted to OD600 nm of 0.1.

138

This suspension was used as the inoculum for biofilm growth in the flow cells and the Donnan

139

exchange experiments.

140

Biofilm formation studies: The washed bacterial suspension was injected into the flow cell at a

141

rate of 2 mL/min (shear rate of 27 s-1) for 40 min [14]. After the bacterial deposition phase, a

142

bacterial growth medium was injected for 24 h at 2 mL/min. The growth medium (10% LB

143

solution) consisted of 1.0 g/L of Bacto Tryptone (Becton, Dickinson and Company), 0.5 g/L of

144

yeast extract (Becton, Dickinson and Company), and 100 mM of NaCl (Merck), and adjusted to

6

145

pH 7.0 ± 0.1. The average biovolumes of dead cells, live cells and extracellular polymeric

146

substances (EPS) for four different types of IEMs (homogeneous CEM, homogeneous AEM,

147

heterogeneous CEM, and heterogeneous AEM) were calculated and plotted for comparison.

148

Confocal laser scanning microscopy (CLSM) and imaging: Fouled membranes were carefully

149

removed from the flow cell and cut into pieces of around 5 mm × 5 mm from a similar location

150

in the middle of the membrane coupon. A biofilm staining solution (Molecular Probes, Inc.) was

151

prepared by mixing 5 µM of SYTO 9™ (live cell stain), 3 µM of propidium iodide (PI, dead cell

152

stain), and 0.1 mg/mL of Concanavalin A conjugated to Alexa Fluor 633 (binds to alpha-linked

153

mannose residues of EPS) in a phosphate buffer saline (PBS) solution at pH 7.2 (Invitrogen).

154

Biofilms were incubated in the staining solution for 30 min in the dark. The stained biofilm

155

samples were visualized using a CLSM ZeissMeta510 (Carl ZEISS, Inc., USA) equipped with

156

Zeiss dry objective LCI Plan-Neo Fluor (20 × magnification and numerical aperture of 0.5).

157

Images were analyzed, and the specific biovolume (µm3/µm2) in the biofouling layer was

158

determined by COMSTAT, an image processing software, written as a script in Matlab 6.5 (The

159

Math Works, Inc., Natick, MA, USA) and equipped with an image-processing tool box [15]. For

160

every sample, six positions on each IEM were chosen and microscopically observed, acquired,

161

and analyzed. A three-dimensional reconstruction of the CLSM image stacks was carried out

162

using Imaris software (Imaris Bitplane, Zurich, Switzerland). Averages of the biovolumes of live

163

and dead cells and EPS for four different IEM categories (each category has three different

164

commercial IEMs; Table 1) were calculated and compared.

165

2.3 Antimetabolic activity of IEM surface on P. aeruginosa PAO1

166

In order to investigate the antimetabolic activity of the IEM surface on attached biofilm, XTT, a

167

colorimetric 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino) carbonyl]-2H-

168

tetrazolium hydroxide (XTT) reduction assay was carried out [16]. The electron transport system

169

in the cellular membrane of live bacteria reduces the XTT tetrazolium salt to formazan, which

170

can be measured spectrophotometrically. N-methyl dibenzo pyrazine methyl sulfate (PMS) was

171

used as an electron mediator transferring electrons from the bacterial outer membrane to

172

XTT[17]. On the other hand, nonviable bacteria are unable to reduce tetrazolium salt. By virtue

173

of the XTT assay, the antimetabolic activity of the IEM surface on attached biofilm can be

174

determined. In the present study, the biofouled IEM was removed from the flow cell after 18 h

175

of the experiment and cut into three pieces. Each piece of 1.6 cm × 1.2 cm covered with P.

176

aeruginosa PAO1 biofilms was gently washed with PBS to remove non-adherent bacteria. Each 7

177

IEM sample was added to a 15-ml centrifuge tube (Falcon™ 15 mL Conical Centrifuge Tubes)

178

containing XTT/PMS buffered solution [16]. The XTT stock solution was prepared by dissolving

179

10 mg of XTT (Thermo Fisher Scientific) in 10 ml of 10% LB solution. Ten mM of the (N-

180

methyl dibenzo pyrazine methyl sulfate) PMS solution in phosphate-buffered saline (PBS) was

181

prepared by dissolving 3 mg PMS (AppliChem, Darmstadt, Germany) into 1 mL PBS (Sigma-

182

Aldrich). The XTT/PMS mix solution was prepared by supplementing the 10 mL of XTT

183

solution with 25 µL of the PMS solution. Each falcon tube containing one IEM piece was filled

184

with 1.5 ml of autoclaved PBS buffer and 1 mL of XTT/PMS mix solution. Falcon tubes were

185

then incubated at 30 ºC for 2 h. Then, 150 µl from each sample was taken to a 96-well

186

transparent plate (Greiner 96 Flat Bottom Transparent Polystyrol). The results of the colorimetric

187

change due to formazan production were measured at 450 nm with multimode reader device [14]

188

(Infinite 200 PRO, Tecani-control).

189

An additional set of control experiments for this test was done with a similar size of different

190

pristine IEMs (namely, in the absence of bacteria) to ensure that XTT does not adsorb on IEM

191

surfaces and that the IEM surfaces do not oxidize XTT to formazan. A calibration curve for the

192

reduction of XTT to formazan by P. aeruginosa PAO1 was provided elsewhere [14]. The

193

average absorbance values for four different categories of IEMs (homogeneous CEM,

194

homogeneous AEM, heterogeneous CEM, and heterogeneous AEM) were calculated, and a

195

graph was plotted.

196

2.4 The effect of IEM surface exposure to the bacteria on cell membrane permeation

197

P. aeruginosa PAO1 membrane permeation assays were performed by using fluorescent

198

hydrophobic probe 1-N-phenylnaphthylamine (NPN) uptake as a tool to investigate whether the

199

exposed fixed ionic groups of the IEM have any impact on the bacterial cell membrane. The

200

ability of any chemical substance to cause damage to the bacterial outer cell surface has been

201

extensively quantified by NPN uptake assay [18]. Owing to its hydrophobic nature, the NPN is

202

unable to percolate through intact bacterial membranes and therefore exhibits a weak

203

fluorescence emission. On the contrary, NPN uptake should increase with damaged (functionally

204

invalid) outer membranes. Hydrophobic chemical agents such as NPN cannot be absorbed to the

205

bacteria due to the orientation of the LPS present on the outer side of the bacterial membrane

206

[18]. Therefore, bacteria with impaired cell membranes emit high fluorescence compared to non-

207

damaged ones [19]. In the present experiments, four different IEMs from each category were

208

chosen for biofilm formation in NPN uptake assays; these were CMV for homogeneous CEMs, 8

209

AMV for homogeneous AEMs, MK40 for heterogeneous CEMs, and MA40 for heterogeneous

210

AEMs, respectively. NPN was added to the 12-h-old biofilm, on the different IEM surfaces at a

211

concentration of 10 µM. After 30 min of incubation, the excess amount of NPN was washed

212

gently with 0.85% NaCl solution. The uptake of NPN was determined by measuring the

213

fluorescence emitted at 420 nm after excitation at 350 nm using an argon laser in the CLSM

214

[19]. CLSM images were generated using the Zeiss LSM Image Browser. Colored images were

215

analyzed with COMSTAT to determine the biovolume (µm3/µm2) as described in the previous

216

section, and imaging was done with IMARIS v7.5 software (IMARIS Bitplane, Zurich,

217

Switzerland).

218

2.5 Assessment of bacterial intracellular oxidative stress

219

The bacterial intracellular oxidative stress was assessed by quantification of reactive oxygen

220

species (ROS) inside the bacterial cell. The ROS-sensitive green fluorescent dye 2′, 7′-

221

dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, Israel), which is converted to

222

dichlorodihydrofluorescein in the presence of intracellular ROS [20], was used. This test was

223

conducted because it was hypothesized that the presence of a charged surface close to bacterial

224

biofilm can cause oxidative stress, which is a reason for the overproduction of intracellular free

225

radicals [21]. The amount of intracellular ROS was estimated from dichlorodihydrofluorescein

226

(DCF) production measured at 480-nm excitation/530-nm emission [22]. The four different

227

IEMs (CMV, AMV, MK40 and MA40) were considered for this test. The biofilm was allowed to

228

grow for 12 h on the surface of the IEMs. The DCFH-DA was added immediately to the IEMs

229

with biofilms at a concentration of 5.0 µg/ml (dissolved in ethanol) after which the cells were

230

incubated for 30 min. Next, the excess amount of DCFH-DA dye was carefully washed with

231

phosphate buffer (pH 7.2), followed by CLSM imaging performed on the biofilms.

232

2.6 Performance of biofouled IEMs during Donnan exchange

233

Counter-ion transport ability in terms of ion diffusion flux was determined and compared

234

between pristine and biofouled IEMs in a customized Donnan dialysis setup [23]. In the Donnan

235

exchange, the concentration gradients across the studied membrane separating two solutions with

236

different compositions are the driving force for counter-ion transport. A rectangular Plexiglas

237

cell, with feed and receiving compartments (9.1 cm long × 4.5 cm wide) and a channel height of

238

0.44 cm separating a single ion exchange membrane (IEM) with an active area of 41 cm2, was

239

used for these experiments. The solutions were delivered to each compartment from 2.5-L

240

vessels and circulated by two-headed centrifugal pumps (MRC Ltd., Israel) at flow rates of 3.2 9

241

L/h (linear velocity 1.13 cm/sec) from/to the feed/receiving compartments. All experiments were

242

performed at 26 ± 10 ºC, controlled by an aquarium heater (Aqua One, Chung Xing. Co. Ltd.).

243

The different solutions and counter-ions determined for their fluxes are listed in Table 2.

244 245

Table 2: Description of the solutions in the feed and receiving compartments for the Donnan

246

exchange experiments Studied membranes CEMs (CMV, MK40) AEMs (AMV, MA40)

Receiving compartment NaCl (1M)

Feed compartment KCl (1M)

Counter-ion measured K+

NaCl (1M)

Na2SO4 (1M)

SO4-2

247 248

A customized cylindrical flow cell (height 21 cm, radius = 8 cm) of ≈ 4-L volume was used for

249

biofilm formation on the surface of four different categories of IEMs (CMV, homogeneous

250

CEM; AMV, homogeneous AEM; MK-40, heterogeneous CEM; and MA-40, heterogeneous

251

AEM) (Figure S-1, SI). The same growth medium was used and the same inoculum preparation

252

technique was followed as mentioned earlier (biofilm growth on different IEM surfaces in a flow

253

cell). Feed solution was injected to the flow cell without recirculation using a peristaltic pump

254

(Masterflex, Cole-Parmer) at a flow rate of 2 mL/min for 6 d (144 h). After this period, IEMs

255

were collected aseptically, gently washed with the background solution, and used for the Donnan

256

exchange experiment (Figure S-2, SI).

257

Counter-ion flux was determined by tracking the change in sulfate and potassium concentrations

258

as a function of time in the receiving compartment for AEMs and CEMs, respectively [23]. For

259

the Donnan dialysis experiment with AEMs, the Standard 4500E Turbidimetric Method was

260

used for determining sulfate concentration, where sulfate is precipitated as BaSO4 by the

261

addition of barium chloride. The turbidity of the BaSO4 suspension was then measured using a

262

spectrophotometer (Lambda EZ201 Perkin Elmer). For the CEMs, potassium samples were

263

analyzed by ICP (Varian 720-ES, Australia) for determining sulfate and potassium

264

concentrations. All the samples were diluted (20 ×) prior to ICP analysis due to the high sodium

265

concentration of the samples [23]. Counter-ion transport ability in terms of ion diffusion flux was

266

determined and compared between pristine and biofouled IEMs.

267 10

268 269 270

3. Results and Discussion

271

3.1 IEM surface characterization

272

3.1.1 Evaluation of IEM surface hydrophobicity

273

Surface hydrophobicity was estimated by the contact angle of a captive air bubble under the

274

aquatic conditions applied during the biofilm formation experiments [24]. Prior to the contact

275

angle measurement experiments, IEMs were soaked overnight in 10% LB solution under aseptic

276

conditions. The captive air bubble contact angle test results, presented in the supplementary

277

information, show that the AEM and CEMs’ surfaces are relatively hydrophilic according to

278

their large captive air bubble contact angle range of 128–150º (Table S1 and Figure S-2). Note

279

that although most of the results show that AEM surfaces are more hydrophilic than the CEM

280

surfaces, the differences in contact angles, in most cases, were not significant.

281 282

3.1.2 Roughness estimation with AFM

283

AFM contact mode scans were carried out on the various IEMs. Roughness indices were

284

estimated at a resolution of 5 µm2, using the method of root-mean-square for the Z-plane. The

285

3D and 2D visualization of the AFM scans are provided in Figure 1 and Figure S-4

286

(supplementary information), respectively. Previous studies showed that larger surface roughness

287

facilitates more biofilm formation: Nano- and micro-scale surface roughness commonly

288

enhances the adhesion of bacteria to substrates during the initial steps of colonization as it

289

provides more surface area for cell attachment [25]. Accordingly, the higher average roughness

290

values of the heterogeneous IEMs likely play a role in their biofouling propensity (Figure 1).

291 292 293

11

294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324

Figure 1: Roughness of different IEMs: Top panel- Average roughness values of different

325

categories of IEMs; Bottom panel- 3D visualization of the commercial IEMs: (A) Heterogeneous

326

AEM (MA-40); (B) Heterogeneous CEM (MK-40); (C) Homogeneous AEM (AMV); (D)

327

Homogeneous CEM (CMV) at a resolution of 5 µm2.

328 329 330

3.1.3 Evaluation of zeta potential of IEM surface

331

In order to determine the possible impact of electrostatic interactions between the IEM surface

332

and the bacterial cell membrane, the surface zeta potential of the IEMs was analyzed, while the

333

zeta potential of the cells under similar aquatic conditions was studied elsewhere [26]. Zeta

334

potential values of the different IEMs used in this study are depicted in Figure 2 for a pH range 12

335

of 4 to 10. Larger negative zeta potential values were detected for both the homogeneous and

336

heterogeneous CEM surfaces than for the AEM surfaces, indicating that a relatively stronger

337

electrostatic repulsion is expected between the bacterial cells and the CEM surface. Owing to the

338

presence of carboxylate groups in the lipopolysaccharides (LPS) of the outer cell membrane of

339

P. aeruginosa PAO1, these cells possess a negative surface charge (as expressed by negative

340

zeta potential values) between a pH of 2.2 and 11 [27]. On the contrary, zeta potential values of

341

both homogeneous and heterogeneous AEMs were at a positive magnitude (approximately

342

within the range of +5 mV to +35 mV), indicating the presence of positively charged fixed

343

groups on the surface of the AEM. Owing to the presence of the positive charge, a higher degree

344

of bacterial and EPS attachment is expected to take place due to electrostatic attraction [28]. The

345

CEM displayed a slightly negative charge (approximately within the range of -10 mV to -85

346

mV), in 0. 1 mM KCl, correlating to the charge of the sulfonate functional groups, which may

347

result in the repulsion of the negatively charged bacterial cell surfaces and EPS.

348 349 350 351 352 353 354 355 356 357

Figure 2: Zeta potentials of different commercial IEMs as a function of pH: CEMs (left panel)

358

include homogeneous (CMV and CMT) and heterogeneous (MK-40 and CMX) surfaces. AEMs

359

(right panel) include homogeneous (AMV and AMT) and heterogeneous (MA-40 and AEX)

360

surfaces. All samples were measured in 1 mM of KCl solution and titrated with 100 mM of HCl

361

or 100 mM of NaOH.

362 363 364

3.2 Quantification of biofilm formation

365

In this part of the study, we evaluated the effect of IEM surface on the magnitude of biofilm

366

formation, while the hydrodynamic conditions in the flow cell were maintained as constant.

367

Figure 3 shows representative CLSM images of biofilm formed on different IEM types. A 13

368

quantitative analysis of the dead and live cells is presented in Figure 4. The IMARIS 3D images

369

in Figure 3 show a presence of predominantly dead cells on the AEMs (Figures 3 A and C),

370

while EPS and live cells were more visible on the heterogeneous CEM (Figure 3 D). Further, it

371

can be inferred that none of the IEMs were capable of inhibiting biofilm development. Likely,

372

once bacterial cells and EPS overrode the charged functional group of the underlying IEMs,

373

biofilm could grow [29,30].

374

375 376 377

Figure 3: P. aeruginosa PAO1 biofilms formed on different types of IEMs as observed with

378

CLSM: (A) Homogeneous AEM (AMV); (B) Homogeneous CEM (CMV); (C) Heterogeneous

379

AEM (MA-40); and (D) Heterogeneous CEM (MK-40) after 24 h of biofilm formation. The red,

380

green, and blue clusters indicate dead cells, live cells, and EPS, respectively. Orange clusters

381

indicate an overlapping zone of dead cells and live cells. Each image (A–D) is a perspective of a

382

600 µm × 600 µm image.

383 384 385 386 387

14

388

389 390

Figure 4: Biofilm formation on IEMs: Left panel – Average biovolumes of dead and live cells

391

on the different commercial IEMs after 24 h. Right panel – Ratio of biovolumes of dead/live

392

cells in biofilms formed on the different commercial IEMs after 24 h. X-axis legend: (A)

393

Homogeneous AEMs include AMV, Neosepta AEX and AMT; (B) Heterogeneous AEMs

394

include Ralex AEM, MA-40 and Excellion I-200; (C) Homogeneous CEMs include CMV,

395

Neosepta CMX and CMT; (D) Heterogeneous CEMs include Ralex CEM, MK-40 and Excellion

396

I-100. Each error bar represents one standard error.

397 398

A significantly higher biomass of cells was observed on the heterogeneous CEM than on

399

all the other IEMs. The lowest and highest biovolumes of the cell layers (live and dead) of 16.49

400

µm3/µm2 (± 0.84) and 25.44 µm (±2.5) were measured for the homogeneous AEM and the

401

heterogeneous CEM, respectively. No significant difference in the cell biovolumes was found

402

between the heterogeneous AEM and the homogeneous CEM (≈18.7 µm3/ µm2). Interestingly,

403

the biovolume ratio of dead to live cells was significantly higher for AEMs (≈0.6) than for CEMs

404

(0.27) (Figure 4B). The larger dead cell biomass detected on the AEMs suggested that the AEM

405

surface provides a higher surface toxicity towards the attached bacteria. In addition, the contact

406

angle analysis showed that the AEM surface is slightly more hydrophilic than that of the CEM,

407

which may result in reduced biofilm formation. At the stationary growth phase of P. aeruginosa

408

PAO1, the bacterial membrane surface was observed to increase its hydrophobicity [26].

409

Therefore, hydrophobic interaction should be important for biofilm formation, especially on the

15

410

CEM surface [29]. This higher surface roughness of the heterogeneous membranes also

411

contributes to elevation in biofilm formation for both CEM and AEM types.

412

A higher amount of EPS was detected on the CEMs than on the AEMs as shown in

413

Figures 3 and 5A. The significantly low EPS amount observed on the homogeneous AEM could

414

be the result of a low percentage of viable cells in the biofilms and a relatively low surface

415

roughness (Figures 4 and 1). Both homogeneous and heterogeneous CEMs provided a better

416

surface for EPS formation with ~ 1.1–1.2 µm3/ µm2 specific biovolume. On the contrary, the

417

lowest amount of EPS (< 0.2 µm3/µm2) was present on the homogeneous AEM with a higher

418

amount of EPS on the heterogeneous AEM of ~ 0.5 µm3/µm2. Interestingly, a higher ratio of

419

EPS biovolume (EPS on CEM/EPS on AEM) was observed for the homogeneous IEMs than for

420

the heterogeneous ones (Figure 5B). While the lower cell viability observed for the AEMs

421

compared to the CEMs, which will be further analyzed in this study, could explain the lower

422

EPS amounts, surface roughness provides a secondary effect of retaining the EPS matrix under

423

shear. The combination of low surface roughness for the homogeneous IEM surface and

424

cytotoxic effects owing to quaternary ammonium moieties on the AEM surface is likely the

425

reason for the lowest amount of EPS and the highest ratio of its EPS amount to the EPS amount

426

on its CEM counterpart (Figure 5 B). It should be mentioned that the CEMs in this study provide

427

a better surface for biofilm colonization despite the repulsive interactions that are expected

428

between negatively charged bacteria/biopolymers and the surface (negatively charged by

429

sulfonate groups) [31]. Reduced bacterial attachment to negatively charged surfaces usually

430

reduces biofilm formation only in the short term [15], and commonly, adsorption of dissolved

431

organic matter and EPS , via hydrophobic interactions, provides a conditioning film, which

432

enables sessile microbial growth on the surface and biofilm formation [30,32]. In addition,

433

elevated biofilm growth and EPS production on the CEM surface was also attributed to the

434

higher viability of the attached cells compared to the AEM surface.

435 436 437 438 439 440 441 442 443 444

16

445 446 447

Figure 5: EPS accumulated on the different commercial IEMs after 24 h of biofilm growth: Left

448

panel – Biovolume of EPS on the IEMs (µm3/µm2): X-axis legend (A) Homogeneous AEMs

449

include AMV, Neosepta AEX and AMT; (B) Heterogeneous AEMs include Ralex AEM, MA40

450

and Excellion I-200; (C) Homogeneous CEMs include CMV, Neosepta CMX and CMT; (D)

451

Heterogeneous CEMs include Ralex CEM, MK40 and Excellion I-100; Right panel – The

452

average ratio of EPS amounts generated on different CEMs to AEMs for both homogeneous and

453

heterogeneous membrane types. Error bars represent one standard error.

454 455 456

3.3 Antimetabolic activity of IEM surface on P. aeruginosa

457

The measurement of the metabolic activity of sessile P. aeruginosa cells by the XTT assay

458

provided a clear indication of the possible antimicrobial activity of the membrane surface [33].

459

The viable cells on the surface will convert XTT to soluble formazan salt, and therefore, the

460

absorbance of formazan at 450 nm is indicative for the viability of the sessile bacteria. The

461

results of the XTT tests are summarized in Figure 6. The higher absorbance of formazan

462

originated from the biofouled CEMs indicates that the metabolic activity of the bacteria attached

463

to the CEM was not hampered by the exposed -SO3- ionic group of the CEM. On the contrary,

464

the magnitude of absorbance was lower for the case of both homogeneous and heterogeneous

465

AEMs, suggesting that the viability of the sessile bacteria was reduced while growing on these

466

surfaces. The quaternary ammonium ion in the AEM likely inhibits proliferation and reduced the

467

viability of the attached bacteria. Interestingly, a higher absorbance was observed for the

468

heterogeneous than for the homogeneous AEM. In the heterogeneous AEM, the ion exchange

469

groups are clustered and unevenly distributed in the membrane matrix unlike in the

470

homogeneous AEM where the charged groups are distributed uniformly over the membrane

17

471

polymer matrix [34]. Hence, the lower magnitude of absorbance value for the homogeneous

472

AEM indicates greater antimicrobial activity on the surface owing to the more uniform

473

distribution of charged quaternary ammonium resin. The lower absorbance value with the blank

474

sample suggested that none of the charged groups (quaternary ammonium or sulfonate) in the

475

IEMs interacted with XTT, and they were also unable to reduce it to formazan. The XTT assay

476

was thus useful for measuring the effect of the charged groups in the IEM on the viability of P.

477

aeruginosa PAO1 biofilms.

478

Possible effects of EPS or dead cells covering the quaternary ammonium groups should

479

be considered for longer term, more realistic biofouling scenarios. In this study, well developed

480

biofilm was formed providing opportunity to the EPS/dead cells to cover the quaternary

481

ammonium groups. However, these effects, which may alter the antimicrobial effectiveness of

482

the surface, should be further considered. Indeed, the time range as well as the aquatic conditions

483

promoting adsorption of foulants, which may consequently affect the antimicrobial activity of

484

the surface, is a subject for a continuing study.

485 486 487

Figure 6: The effect of the different IEM types on cell viability analyzed with XTT: Average

488

absorbance value at 450 nm attributed to the production of soluble formazan salts by biofilms

489

developed after 18 h on the different membranes. X-axis legend (A) Heterogeneous AEMs:

490

Ralex AEM, MA40 and Excellion I-200; (B) Heterogeneous CEMs: Ralex CEM, MK40 and

491

Excellion I-100; (C) Homogeneous AEMs: AMV, Neosepta AEX and AMT; (D) Homogeneous

492

CEMs: CMV, Neosepta CMX and CMT. Blank samples denote the average absorbance value

493

obtained using pristine IEMs. Error bars represent one standard error. 18

494 495

3.4 Cell membrane integrity assays upon cell exposure to different IEM surfaces

496

The effect of exposing P. aeruginosa PAO1 sessile cells to different IEM surfaces on the cell

497

membrane integrity was analyzed by NPN permeation tests. Enhanced uptake of NPN occurs in

498

a bacterial population containing cells whose outer membrane is damaged and functionally

499

inactive [35]. In the present study, an increased fluorescence was detected in cells on the AMV

500

(homogeneous AEM) and the MA-40 (heterogeneous AEM), which indicated the detrimental

501

effect of quaternary ammonium groups present on these membranes' surfaces (Figure 7). The

502

NPN uptake was found to be much less for the CEMs. Average contents of cells stained with

503

NPN as determined by specific biovolumes of 1.006 µm3/ µm2 (± 1.03), 9.32 µm3/µm2 (± 1.004),

504

2.93 µm3/µm2 (± 0.92) and 28.3 µm3/µm2 (± 1.65) were measured for the homogeneous CEM

505

(CMV), homogeneous AEM (AMV), heterogeneous CEM (MK40) and heterogeneous AEM

506

(MA40), respectively. A possible cell-surface electrostatic interaction could be promoted by the

507

positive charge of quaternary ammonium and the negatively charged LPS of the P. aeruginosa

508

PAO1 cell surface. The quaternary ammonium groups are known to display an electrostatic

509

interaction with anionic LPS, which leads to alteration of the cell membrane architecture, thus

510

enhancing membrane permeability, leakage of cell components, and subsequent cell death[28].

511

These results corroborate the biofilm formation results observed in Section 3.2 and delineate the

512

antimetabolic activities of the AEMs observed in Section 3.3.

513

19

514

515 516

Figure 7: Analysis of bacterial outer membrane permeability according to the uptake assay of 1-

517

N-phenylnaphthylamine (NPN): Cells tagged with NPN, after 12 h of biofilm growth, were

518

observed in the CLSM (blue clusters) and reconstructed images were made with IMARIS

519

software (A-D); The images present the following: (A) Homogeneous AEM (AMV); (B)

520

Homogeneous CEM (CMV); (C) Heterogeneous AEM (MA40); and (D) Heterogeneous CEM

521

(MK40). Each image (A-D) is a perspective of 600 µm × 600 µm image. (E) A quantitative

20

522

analysis of the specific biovolume attributed to the NPN-tagged cells. Performed with

523

COMSTAT Matlab script. Error bars represent one standard error.

524 525

The results presented so far are in accordance with earlier reports where two different

526

antibacterial modes of actions were deduced for positively charged surfaces by quaternary

527

amines[36]. One of the mechanisms is involved in the displacement of divalent cations (e.g. Ca2+

528

and Mg2+) present on the bacterial outer surface by polymer chains with positively charged

529

surfaces[37]. Displacement of these divalent ions, which hold together the negatively charged

530

surface of the lipopolysaccharide network of Gram-negative bacteria, leads to disruption of the

531

outer membrane of these bacteria. The second mechanism deals with the penetration of

532

positively charged polymer chains into the inner bacterial membrane, which leads to cell leakage

533

and eventually inactivation[38]. In our case, the first suggested mechanism is possible, while the

534

second one is less likely, as it requires penetration of positively charged polymers, which in this

535

case, are affixed to the IEM matrix and not accessible to the bacterial cell membranes.

536 537

3.5 Effect of IEM surface charge on bacterial intracellular ROS generation

538

An excessive liberation of bacterial intracellular ROS in response to a harsh surrounding

539

environment (for example, the presence of nanoparticles [29], antibacterial agents, and positively

540

charged compounds) was previously shown by Terada et al [28]. In the present study, we

541

hypothesized that exposure of bacterial cells to the charged IEM surface may affect the redox

542

potential across the cell membrane and accelerate ROS production in the sessile bacteria.

543

Overproduction of intracellular/endogenous ROS can commence due to exposure to exogenous

544

stimulation (both physically and chemically), and consequently, cellular damages occur that may

545

become irreversible and cause cell death [39]. For the above mentioned purpose, the oxidation of

546

DCFH-DA was carried out to detect and quantify the intracellular ROS when bacterial cells were

547

exposed to differently charged IEM surfaces [27]. It was evident from the CLSM results that

548

AEMs, irrespective of their types, induce production of more intracellular/endogenous ROS than

549

CEMs (Figure 8), and that the presence of quaternary ionic groups on AEMs likely accelerate

550

ROS overproduction.

551

21

552 553

Figure 8: IMARIS 3-D images of 2′, 7′-dichlorodihydrofluorescein (DCFH) stained bacterial

554

cells (for ROS generation) on the surface of different categories of IEM after 12 h of biofilm

555

growth; (A) Homogeneous AEM (AMV); (B) Homogeneous CEM (CMV); (C) Heterogeneous

556

AEM (MA-40); (D) Heterogeneous CEM (MK-40); each image (A-D) is a perspective 600 µm ×

557

600 µm.

558 559 560

3.6 Effects of biofouling on transport properties of IEMs during the Donnan exchange

561

In the Donnan dialysis process, counter-ions carrying the same electrical charge are exchanged

562

between two solutions through an ion exchange membrane. The driving force for the transport of

563

ions through the membranes is their concentration gradients across the membrane. It is expected

564

that the transport of ions will be hampered when the membrane is biofouled due to mass

565

transport limitations. In the present study, sulphate and potassium passage through AEMs and

566

CEMs, respectively, was investigated in a customized Donnan dialysis cell. The results for

567

sulphate and potassium accumulation in the receiving compartment as a function of time are

568

shown in Figure 9. Fluxes are expressed by the slopes of the respective curves and summarized

569

in Table 3. The percentage of flux decline shown in the rightmost column of Table 3 was

570

calculated as 100 × [flux(pristine membrane)-flux(fouled membrane)]/flux (pristine membrane). 22

571

From the data presented in Table 3, it is evident that the extent of flux decline is larger for

572

the heterogeneous IEMs than for the homogeneous IEMs and for the AEMs than for the CEMs.

573

The differences between the heterogeneous and the homogeneous membranes correlate well with

574

the results described in Section 3.2 that show the difference in the amount of biomass

575

accumulated on the two classes of membranes. The larger biovolumes collected on the surfaces

576

of the heterogeneous membranes is attributed to the larger diffusion limitations at the solution-

577

membrane interface, which emerge from the enhanced concentration polarization in this

578

region[40].

579

Based on the differences between the behavior of the CEMs and AEMs as a substratum

580

for biofilm development discussed in the previous sections, the differences in the mass transfer

581

properties for these types of membranes are, in a way, counterintuitive. While the AEMs support

582

biofilm formation to a lower extent than the CEMs, the ion flux decline was more pronounced

583

for the biofouled AEM membrane (Table 3). It should be noted that most types of EPS are

584

negatively charged at pH values exceeding 4, mostly due to deprotonation of carboxylic,

585

hydroxyl, or sulfonic functional groups [41–43]. As a result, depending on the pH and the other

586

aquatic conditions, EPSs are adsorbed to positively charged surfaces, such as those of the AEMs,

587

or are prone to bind to positively charged organic molecules and multi-valent cations [42–46]. In

588

this respect, also other studies confirm the fouling propensity of AEMs towards organic fouling

589

[47–49].

590

Based on the above properties, we hypothesize that under the current experimental

591

conditions for biofilm growth followed by the Donnan exchange studies, the negatively charged

592

EPS components are preferentially bound, mainly via electrostatic interactions, to the positively

593

charged fixed ionic groups of the AEMs at the membrane-solution interface. This interaction will

594

likely form an electrostatically neutral layer on the membrane surface, which will become

595

partially blocked, and thus, interfere with the ion exchange process. Such an adsorbed EPS layer

596

is minute and does not have to exceed the density of the fixed ionic groups on the membrane

597

surface that can be estimated to be as low as 1.5–2.5 × 10-9 equivalents/cm2 (supplementary

598

information).

599 600 601

23

602 603 604 605 606 607 608 609 610 611 612 613

24

614

Figure 9: Sulphate (A) and potassium (B) diffusion to the receiving compartments per unit

615

membrane area as a function of time, for pristine and biofouled membranes. Linear lines

616

represent the slope of the ions' passage versus time, from which ion flux was calculated.

617 618 619

Table 3: Summary of ion fluxes for pristine and fouled membranes. Conditions, flux Membrane

Membrane Type

Analyzed

Pristine

Fouled

ion

(eq.m-2.h-1)

(eq.m-2.h-1)

Flux Decline (%)

MA-40

Heterogeneous

MK-40 AMV CMV

Homogeneous

SO42-

0.123

0.032

74

K+

0.232

0.0975

58

SO42-

0.48

0.284

40.8

K+

0.35

0.252

28

620 621 622

4. Conclusions

623

The present study investigates the formation of mono-culture bacterial biofilm on different

624

commercially available IEM surfaces. It should be mentioned that mono-culture biofouling

625

studies commonly provide reproducible and robust approach, in which involved mechanisms are

626

more easily elucidated as concluded in this paper, while under more realistic conditions of mixed

627

environmental consortia, interactions between microbial species and environmental conditions

628

effects on the different microbes would make it hard to draw conclusive mechanisms. The results

629

suggest that IEM type affects the electrostatic interactions with bacteria; however, the CEM

630

surface provides a better carrier for biofilm formation despite the possible repulsion from

631

bacteria and most negatively charged EPS components. The specific biovolumes of cells and

632

EPS, used for quantifying biofilm growth, were larger on the CEM than on the AEM surface.

633

The antimicrobial characteristics of the AEM were demonstrated, in which the lower cell

634

viability on the AEM surfaces was due to quaternary amine groups on the surface facilitating the

635

disruption of bacterial cell membranes, while the CEM surface did not facilitate such an effect. 25

636

Hence, the CEM provides a better platform for sessile microbial growth. The Donnan exchange

637

studies also confirmed the impact of bacterial biofilm growth on the IEMs, in which a

638

consequent sharp decline in counter-ion passage was documented for all types of membranes,

639

with heterogeneous IEMs and AEMs dominating. The overall study revealed that the charged

640

groups and the surface roughness determine the biofouling propensity of IEMs.

641 642

Acknowledgments

643

This research was supported by the United States-Israel Binational Science Foundation

644

under award number 2012142 and the Planning and Budgeting Committee (PBC) of the

645

Council for Higher Education for the Postdoctoral Fellowship Award provided to Dr. Soumya

646

Pandit.

647 648

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649

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31

Highlights •

Mechanisms of biofilm formation ion exchange membranes (CEM and AEM) were tested.

•

Anti-metabolic activity of AEM surface affecting membrane integrity was confirmed.

•

Biofilms reduced the counter-ion transport mainly for heterogeneous AEMs.

Ben-Gurion University of the Negev Blaustein Institutes for Desert Research Zuckerberg Institute for Water Research Dept. of Desalination and Water Treatment Assoc. Prof. Moshe Herzberg Phone: 972-8-6563520, 972-50-2029608 Fax: 972-8-6563503 E-mail: [email protected]

Journal of Membrane Science Editorial Office

Dear Editor: In our revised manuscript “Bacterial Biofilm Formation on Ion Exchange Membranes”, which is submitted for possible publication in Journal of Membrane Science, there are no declarations of interest.

"Declarations of interest: none'. We thank you very much for your consideration, Sincerely, Moshe Herzberg

Sede Boqer Campus, Tel: 086563520` Fax: 086563503

,‫קמפוס שדה בוקר‬ 8086563503 :‫ ; פקס‬086563520:'‫טל‬