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Viscoelastic properties of extracellular polymeric substances can strongly affect their washing efficiency from reverse osmosis membranes Diana Lila Ferrando Chavez, Ali Nejidat, and Moshe Herzberg Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01458 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016

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Environmental Science & Technology

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Viscoelastic properties of extracellular polymeric substances can strongly affect

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their washing efficiency from reverse osmosis membranes

3 4 5 6

Diana Lila Ferrando Chavez, Ali Nejidat, and Moshe Herzberg*

7 8 9

The Jacob Blaustein Institutes for Desert Research

10

Zuckerberg Institute for Water Research

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Ben Gurion University of the Negev

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Sede Boqer Campus, 84990 Midreshet Ben Gurion, Israel

13 14 15 16 17

Revised Version Submitted to

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Environmental Science and Technology

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July 03, 2016

20 21 22 23 24 25 26 27 28 29 30

*Corresponding author Tel.: +972 8 6563520, fax: +972 8 6563503. E-mail address: [email protected]

ACS Paragon Plus Environment


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Abstract

32

The role of the viscoelastic properties of biofouling layers in their removal from the

33

membrane was studied. Model fouling layers of extracellular polymeric substances (EPS)

34

originated from microbial biofilms of Pseudomonas aeruginosa PAO1 differentially

35

expressing the Psl polysaccharide were used for controlled washing experiments of

36

fouled RO membranes. In parallel, adsorption experiments and viscoelastic modeling of

37

the EPS layers were conducted in a quartz crystal microbalance with dissipation (QCM-

38

D). During the washing stage, as shear rate was elevated, significant differences in

39

permeate flux recovery between the three different EPS layers were observed. According

40

to the amount of organic carbon remained on the membrane after washing, the magnitude

41

of Psl production, provide elevated resistance of the EPS layer to shear stress. The

42

highest flux recovery during the washing stage was observed for the EPS with no Psl. Psl

43

was shown to elevate the layer's shear modulus and shear viscosity but had no effect on

44

the EPS adhesion to the polyamide surface. We conclude that EPS retain on the

45

membrane as a result of the layer viscoelastic properties. These results highlight an

46

important relation between washing efficiency of fouling layers from membranes and

47

their viscoelastic properties, in addition to their adhesion properties.

48 49 50

Keywords: Reverse Osmosis; Extracellular Polymeric Substances; Biofouling, QCM-D,

51

Viscoelasticity

52 53


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Introduction

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microorganisms including bacteria attach to the membrane surface, resulting in the

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formation of a biofilm. Although biofilms that spontaneously form on membrane surfaces

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may not significantly interfere with filtration processes, relatively small changes in the

60

feed solution conditions can result in significant biofilm growth that will impair

61

membrane performance1, 2. Despite many on-going efforts, there is no threshold measure

62

that can function as an early warning system for the formation of such biofilms, and

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therefore, biofouling remediation generally occurs only after plant operation has been

64

significantly impaired3, 4. Biofilms prevent effective mixing in the solution adjacent to the

65

surface, which enhances concentration polarization (CP), impacting selectivity and

66

increasing osmotic pressure in RO processes5-7. Additionally, CP facilitates biofouling

67

due to the more uniform access to nutrients and oxygen for the biofilm8. Furthermore,

68

increased resistance to water flow by deposition and adsorption of self-produced

69

extracellular polymeric substances (EPS) to the membrane usually elevates membrane

70

hydraulic resistance9.

During the process of reverse osmosis (RO) desalination, organic compounds and

71

Microbial biofilms on RO membranes are embedded in a slime matrix, through

72

which the cells adhere to the surface10, 11. This matrix is comprised of EPS excreted by

73

the sessile microorganisms, which facilitates both adhesion of the microorganisms to

74

inanimate surfaces and cohesion of the biofilm layer12-14. The EPS includes carbohydrates

75

and proteins as major constituents, and lipids, nucleic acids and other various hetero-

76

polymers in smaller portions13, 15. Life embedded in the EPS matrix offers important

77

advantages for the biofilm microorganisms. One important role of the EPS is to provide

78

mechanical stability to the microbial biofilm through physico-chemical interactions,

79

mainly via electrostatic forces, hydrogen bonds, and van der Waals interactions8. In

80

addition, the EPS acts as a protective layer for bacterial cells by retarding penetration of

81

and sequestering antimicrobial agents16.

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The viscoelastic properties of the matrix, which is affected by the EPS

83

composition, have a major contribution to the cohesion of biofilms17, 18. These properties

84

are affected by chemical and physical interactions within the biofilm matrix19.

85

Additionally, cohesion of the EPS matrix is an important characteristic for biofilm



86

resistance to shear forces20. Previous studies suggest that the most significant impairment,

87

as well as the challenges of membrane cleaning efforts, arise from the presence of the

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EPS matrix and its attachment to the membrane and not from the microorganisms

89

themselves9, 21. Clearly, effective minimization and cleaning strategies of biofilms are

90

critical to optimize membrane processes. Quartz crystal microbalance with dissipation

91

monitoring (QCM-D) allows real-time monitoring of changes in the frequency and

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dissipation of an adhering mass onto a piezoelectric quartz sensor to assess EPS-surface

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interactions and accordingly, the critical EPS properties – adhesion and viscoelasticity –

94

can be determined22-26.

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Model bacterial strains are commonly used in biofilm research in order to ensure

96

controlled conditions throughout the study. Thus, even though Pseudomonas aeruginosa

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PAO1 is normally found in medical systems rather than wastewater, it is widely used in

98

EPS biosynthesis research27. In the present study we selected this bacterium as our model

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EPS producing strain. P. aeruginosa produces at least three extracellular polysaccharides

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important for biofilm development: In mucoid strains, alginate polysaccharide will be

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most commonly produced; whereas non-mucoid strains express primarily two

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biosynthetic loci, pel and psl, which are in charge of the production of the two

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polysaccharides, Pel and Psl28. Both have been identified to play important roles in

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initiation and formation of biofilms and each are capable of functioning as a structural

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adhesin involved in maintaining biofilm integrity29. Specifically, P. aeruginosa PAO1

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relies primarily on Psl polysaccharide for biofilm development, although it is able to

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produce Pel polysaccharides as well29-31. In a previous study, during biofilm growth, a

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PAO1 ∆psl mutant strain was capable of upregulating Pel production to compensate for

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the loss of Psl; suggesting that the advantage of having both Pel and Psl is to reduce the

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impact of deleterious mutations on an important survival mechanism, such as biofilm

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formation29. Therefore, the main extracellular polysaccharides of the ∆psl mutant of P.

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aeruginosa PAO1 will be Pel29 with minor amount of alginate. In the overexpressed Psl

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and the wild type strains of PAO1, main extracellular polysaccharides will be Psl27, 32, 33

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and Psl with minor amounts of alginate27,

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polysaccharide was identified as a neutral polysaccharide that not only functions as a

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scaffold for biofilm development (holding biofilm cells together in the matrix), but also

29, 34

, respectively. Recently, the Psl


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serves as a signaling molecule for the subsequent events leading to the initiation and

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maintenance of biofilms27-29, 35. In addition, Psl plays an important role in adhesion by

119

promoting cell-surface and intercellular (cell-cell) interactions to both abiotic and biotic

120

surfaces32,

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polysaccharide that is required for both solid surface-associated biofilm formation and

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pellicle formation at the air–liquid interface of a standing liquid culture27, 31.

34

. The Pel polysaccharide has been suggested to be a cationic exo-

123

Recently, the production of Psl was shown to elevate biofilm's Young’s modulus

124

and to elevate the peripheral stiffness of the microcolonies developed. An elevated

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Young modulus was observed for bigger colonies and attributed to later stages of

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maturation for hemispherical or mushroom shaped microcolonies36,

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study, we analyzed the effects of down and overexpression of Psl in EPS layers, on their

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resistance to shear stress under flow with respect to their adherence and viscoelastic

129

properties. Adherence experiments of P. aeruginosa EPS variants were conducted on

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polyamide and gold-coated QCM-D sensors, in which an EPS layer of nanometer scale

131

was formed and the effects of the relative Psl polysaccharide content on the extent of EPS

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adherence and the resulting EPS layer viscoelastic properties (shear modulus and shear

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viscosity) and layer thickness were determined25, 26. The viscoelastic properties of the

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EPS layers were related to their removal from fouled RO membranes and the associated

135

permeate recovery.

37

. In the present

136 137

Materials and Methods

138 139

Bacterial strains and growth media

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P. aeruginosa PAO1 - a well-characterized bacterial strain commonly used for

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biofilm research- was used in this study. In addition, two P. aeruginosa PAO1 mutants

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were used: ∆psl, which has a full deletion of the psl (PA2231 to PA2245)32, 38 gene

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cluster, and WFPA801 (∆psl pBAD-psl), a psl-inducible PAO1 strain32.The latter strain

144

was generated by replacing the psl promoter with an araC-pBAD cassette so that the psl

145

expression is induced by the addition of 0.2% arabinose to the growth medium, such that

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Psl polysaccharide is essentially over-produced32. A fresh single colony of either of the

147

strains, pre-grown on Luria Bertani (LB) agar and supplemented with arabinose (in the



148

case of WFPA801), was used as an inoculum for an overnight culture grown in LB broth

149

at 30 oC on a 150 rpm shaker. This overnight culture was re-diluted in 100 mL LB broth

150

(ratio of 1:100) and allowed to grow to stationary phase for 16 hours for use as inoculum

151

for biofilm growth in the flow-through columns.

152 153

EPS preparation and extraction

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For EPS extraction, biofilms of each of the variants of P. aeruginosa PAO1 were

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grown in a flow-through column. The 100 mL packed bed column (~2.5 cm in diameter)

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contained acid washed glass beads (425-600 µm in diameter, cat. #G8772 Sigma-Aldrich,

157

St. Louis, MO). The column was wet-packed, sterilized with 70% ethanol and washed

158

with sterilized deionized water, prior to inoculation with a 100 mL of the stationary phase

159

culture for 50 minutes (2 mL·min-1). At the end of the inoculation stage, pure LB was

160

injected to the column for 48 hours to allow biofilm growth on the beads. EPS was

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extracted from each of the variants of PAO1 biofilms grown on glass beads in the

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columns, using a modified version of the Liu and Fang method39 as described

163

previously40-42. Protein concentration in the extracted EPS was determined using the Bio-

164

Radª Protein Assay (Bio-Rad, Hercules, CA) according to Bradford43. Polysaccharide

165

content was determined according to Dubois et al.44 using glucose and alginic acid as

166

standards. EPS extracted was expressed as dissolved organic carbon (DOC) concentration

167

measured by using an Apollo 9000 TOC Analyzer (Teledyne Tekmar, Mason, OH).

168 169

Coating QCM-D sensors with a linear aromatic polyamide (Nomex)

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A solution of 10 mL of dimethyl formamide (DMF) containing 2.5% (w/v)

171

lithium chloride was mixed and filtered through a 0.2 µm syringe filter to remove any

172

dust particles. This solution was later used to dissolve 30 mg of linear aromatic

173

polyamide fibers (Nomex, which was kindly received from the laboratory of Professor

174

Freger at the Israel Institute of Technology, see Figure S1, Supplementary Information)

175

in a 50 mL flask connected to a reflux chamber, where the flask containing DMF and the

176

Nomex material was submerged in a heated oil bath at 90 °C overnight. Prior to the

177

coating procedure, the sensors were placed in dichloromethane (DCM) for 1 hour, then

178

washed with deionized water, and later exposed to UV in an Ozone chamber (BioFORCE


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nanoscience, Ames IA) for 10 minutes. Lastly, the sensors were dried with 99.99% pure

180

nitrogen for the removal of any moisture or accumulated dust particles. The spin coating

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technique was used to coat the sensors, where 45 µL of the polyamide solution was added

182

to the middle point of a gold-coated sensor rotating at 40 rounds per second (rps). Spin

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coating rotation time of the sensor was one minute. Finally, the coated sensor was

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carefully vacuum dried for 2 hours at 45 °C.

185 186

QCM-D adsorption experiments

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The adherence and adsorption kinetics of the extracted EPS were determined by

188

using AT-cut quartz crystals mounted in E4 modules of QCM-D (Q-sense, Gothenburg,

189

Sweden)

190

(Nomex), were used for the adherence experiments. The latter one mimics the active

191

layer of the RO membrane on the QCM-D sensors. Before each measurement, the

192

crystals were soaked in a 5% ethylenediaminetetraacetic acid (EDTA) solution for 30

193

minutes, rinsed thoroughly with double distilled water and dried with 99.99% pure

194

nitrogen. The EPS adsorption onto the sensor surface was characterized by the change in

195

the oscillation frequency of the quartz crystal sensor during the parallel flow of aqueous

196

medium with flow rate of 150 µl·min-1 above the sensor surface. The variations of

197

frequency, f (Hz) and dissipation factor, D were measured for the five overtones (n = 3, 5,

198

7, 9 and 11). Solutions were injected into the QCM-D flow cell in 5 sequential stages of

199

30 minutes each, at a constant temperature (22 0C). The solutions were injected in the

200

following order: double distilled water (DDW), 50 mM NaCl aqueous solution, 20 mg·L -

201

1

202

The results from these QCM-D experiments were used to calculate the thickness and the

203

viscoelastic properties (shear modulus and shear viscosity) of the adsorbed EPS layers

204

using the Q-Tools program provided by Q-Sense. This software is based on the Voigt

205

model according to Voinova et al.25 as previously reported for different added EPS layers

206

to the QCM-D sensors40,

207

modulus (µ), and Voigt thickness of the adsorbed layer were obtained by modeling the

208

experimental data of ∆f and ∆D for two overtones (5 and 7).

45

. Gold coated crystals and crystals coated with a linear aromatic polyamide

of EPS as DOC dissolved in 50 mM NaCl, 50 mM NaCl aqueous solution, and DDW.

42, 46

. The best-fit values for the shear viscosity (η), shear

209



210

RO membrane and crossflow test unit

211

A commercial thin film composite RO membrane ESPA-1 (Hydranautics,

212

Oceanside, CA) was used as a model membrane for the biofouling experiments. The

213

hydraulic resistance was determined to be 1.53 (±0.012) x 1014 m-1 at 25 oC. The

214

membrane was received as a flat sheet and stored in DDW at 4 C. A flat-sheet, plate and

215

frame, RO laboratory system, similar to that described in previous publications40, 47, 48

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(Figure S2, Supplementary Information), was used to study the extent of RO membrane

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fouling by different types of extracted EPS. This system is a closed loop system designed

218

for operation at low pressure (10 bars) and small volumes (0.4 – 1.5 L). Detailed

219

description of the RO unit is reported in our previous studies40, 49, 50.

220

Flux recovery experiments of membranes fouled with different types of EPS

221

RO fouled membranes with different EPS of the three Psl variants were tested for

222

their permeate flux recovery after washing the EPS matrix from the membrane under two

223

different shear rate conditions using the RO feed solution. The fouling experiments were

224

carried out with the EPS extracted from biofilms of the different P. aeruginosa Psl

225

variants, grown in a flow through column. Similar amounts of the different EPS layers

226

were accumulated on the membrane at the beginning of all experiments. Thus, the fouling

227

experiments were carried out in a three stage procedure: Firstly, the membrane was

228

compacted with distilled water. Once the compaction of the membrane was achieved, the

229

plate and frame flow cell containing the compacted membrane was removed from the RO

230

unit and connected to a dead-end high-pressure system. Then, the fouling stage was

231

carried out by injecting 100 mL of dissolved EPS (5 mg·L-1 as DOC) in deionized water

232

at a pressure of 17 bars. Finally, the RO plate and frame flow cell, accommodating the

233

fouled membrane, was taken back to the RO unit (Figure S2) and the resistance to shear

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stress of the different EPS layers was tested at shear rates of 185.2 s-1 (low) and 666.7 s-1

235

(high) sec-1 (flow velocities of 9.3 and 33.3 cm·sec-1, respectively), exposed to 50 mM

236

NaCl solution under pressure of 10 bars. After the EPS washing stage, the fouled

237

membrane was carefully removed from the RO flow cell and the EPS deposited on the

238

membrane was re-dissolved in NaOH solution. After a dialysis stage of the EPS solutions

239

against deionized water, the EPS was characterized by measuring DOC, total protein and

240

polysaccharide contents. The fouling and consequent washing experiments were


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performed in triplicates. A scheme of the completely mixed RO desalination unit for the

242

washing experiment of fouled membranes with EPS is presented in Figure S2.

243 244

Results

245 246

The effect of Psl polysaccharides on the EPS washing from fouled RO membranes

247

The fouling layers of three different EPS solutions containing different quantities

248

of the Psl polysaccharide were tested for their resistance to shear stress during parallel

249

retentate flow with the original feed water. Figure 1 represents the recovered normalized

250

permeate flux over time during EPS washing by the retentate flow. During the washing

251

stage at low shear rate, no differences in the flux recovery of the fouled RO membrane

252

were observed between the three different types of EPS layers (Figure 1A). An increase

253

in permeate flux, likely due to removal of some amount of EPS right at the beginning of

254

the washing stage was observed, at the highest magnitude for the ∆psl mutant, while

255

later, permeate flux remained stable until the end of experiment. One may claim a re-

256

fouling of the membrane may occur, since the retentate solution is recycled back to the

257

feed tank in this system. Even if the entire fouling layer would have been washed

258

immediately, this would contribute a 0.05 mg/L of DOC to the retentate flow, a

259

negligible DOC in this case.

260

In contrast, when high shear rate was applied, significant differences in permeate

261

flux recovery between the three different EPS layers were observed as follow (Figure

262

1B): No flux recovery was observed for the wild type EPS, a flux recovery of about 10%

263

was observed for the EPS extracted from the Psl overexpression mutant (similar for both

264

shear rate conditions) and the highest flux recovery rate (~25%) was observed for the

265

∆psl mutant. Notably, a weaker resistance to shear was detected for the EPS with the

266

highest amount of Psl polysaccharide, as observed by its higher flux recovery during the

267

washing stage compared to the wild type strain, but higher resistance to shear with a

268

corresponding lower flux recovery rate compared to the ∆psl mutant strain. It should be

269

mentioned that for all experiments, as in low shear condition, during the first 20 minutes,

270

a transient period of elevated flux was observed for all cases at different magnitude.



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Possible explanation for this behavior could be some removal of the EPS and further

272

rearrangement of the fouling layer.

273 1.4 Wild type

Washing stage

Fouling stage

100 ml solutrion (5 mg/L of EPS as DOC)

Permeate

1.2

1.1

RO plate and frame desalination lab system

1.0 1.4

0

2

4

6

8

B 1.3

High-pressure system

Normalized flux (J/Jo)

Dead end

Normalized flux (J/Jo)

1.3

Low shear rate -1 (185.2 s )

A

∆psl mutant ∆psl PBAD-psl mutant

1.2

High shear rate -1 (666.7 s )

1.1

1.0 0

2

4

Time (hrs)

6

8

274 275

Figure 1: Normalized permeate flux over time during washing procedure of the fouled

276

RO membrane with EPS extracted from P. aeruginosa wild type, ∆psl mutant and ∆psl/

277

PBAD-psl (Psl overexpression) strains (similar amount at t=0). During the washing

278

procedure, the applied shear rates were 185.2 (A) and 666.7 s-1 (B) using 50 mM NaCl

279

solution. Experimental conditions were as follows: constant transmembrane pressure of

280

10 bars; temperature: 25 oC; initial permeate flux: 24.3 ±2.8 L m-2 h-1.

281 282

EPS characterization before and after the washing procedure

283

Initially, the total amounts of proteins and polysaccharides loaded on the

284

membrane were estimated according to their analysis in the feed solution and assuming

285

their contribution to the DOC rejection is similar (Table 1). It should be mentioned that

286

each of the psl variants in this study expressed different amounts of extracellular proteins

287

as observed at the beginning of each of the washing experiments (Table 1). Likely, total

288

cell transcript is different between the different mutants and pleiotropic effects regarding

289

expression of extracellular proteins may take place. During the fouling stage, the


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permeate solution was collected with an average of 0.6 (±0.1) mg·L-1 as DOC for all

291

types of EPS, showing that the RO membrane surface was loaded with fouling layers (for

292

all types of EPS) of ~25 µg DOC·cm-2 (accurate values in Table 1).

293

The dissolved EPS layers, remaining on the membrane after the washing stage,

294

either at low or high shear, were characterized by measuring the polysaccharide, proteins

295

and DOC concentrations (Table 1). Under low shear conditions there was almost no

296

change in the amount of DOC removed, between the different types of EPS (69-73%

297

removal). Still, under low shear, an elevated removal of polysaccharides was detected for

298

the ∆psl mutant EPS (Table 1, 62.4 ±6.6 vs 43.5 ±4.3 and 49.9 ±5.3 % removal of the Psl

299

overexpression and wild type strains, respectively). The DOC content was not affected,

300

probably due to the presence of other EPS components that were not analyzed here such

301

as extracellular DNA and lipids. Under high shear conditions, the main differences in the

302

removal efficiency of the organic components (measured as DOC) from the membrane

303

were between the ∆psl mutant EPS (76.9 ±9.6 %) and the EPS overexpressed with Psl

304

(60.3 ±1.8 %), probably due to stronger selection for EPS material that was either more

305

adhesive, less elastic or both (further to be tested in this work). In the case of the wild

306

type strain, the amount of DOC remaining on the membrane (7.66 µg DOC·cm-2 ± 0.32)

307

was in between the two Psl variants (intermediate removal of 69.4 ±8.5 %). The amount

308

of protein remaining on the membrane after the washing stage was close to zero

309

regardless of the initial amount present on the membrane under either of the two shear

310

stress conditions of the washing stage applied. The entire proteins removal after the

311

washing stage (~98% for all EPS types and shear conditions) showed their insignificant

312

role in membrane performance.

313

Previous studies have reported that the presence of Psl polysaccharide in the EPS

314

of P. aeruginosa led to enhanced cell-surface and intercellular adhesion30, 32. It is also

315

known that the EPS matrix provides the biofilm with its cohesion and viscoelastic

316

properties. We questioned whether the highest resistance to wash of the EPS extracted

317

from the Psl overexpression mutant, compared to the EPS originated from the ∆psl

318

mutant, can be explained by the reported improved adhesion that this EPS gained due to

319

Psl overexpression32 or elevated stiffness36, 37 . Therefore, in addition to adhesion analysis

320

of the EPS matrix, we sought to determine the role of the Psl polysaccharide in the



321

rigidity of the EPS layer, i.e., elevation in its shear and viscosity moduli. Thus, adsorption

322

experiments of the three different EPS, dissolved in the feed solution, were carried out in

323

the QCM-D.

324 325


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326 327

Table 1. Proteins, polysaccharide and DOC content in the feed solution and re-suspended EPS of fouled RO membranes after washing

328

process for the three strains: P. aeruginosa wild type, ∆psl mutant and psl overexpression. The results for both shear rates are shown

329

and the standard deviation is presented in brackets. P. aeruginosa variants used for EPS extraction

Before washing

After Removal washing Eff. (%) -2 DOC (µg·cm )

Before washing

After Removal washing Eff. (%) -2 Proteins (µg·cm )

Before After Removal washing washing Eff. (%) Polysaccharides (µg·cm-2)

Psl overexpression Wild type

25.03 (±0.7) 25.04 (±3.3)

7.7 (±0.1) 6.9 (±0.3)

69.1 (±2.3) 72.3 (±9.1)

9.7 (±0.9) 1.7 (±0.3)

Low Shear 0.14 (±0.05) 98.6 (±26.3) 0.05 (±0.08) 97.0 (±19.0)

∆psl mutant

25.07 (±3.2)

6.9 (±0.1)

72.6 (±8.3)

4.9 (±1.0)

0.04 (±0.01)

99.2 (±22.7)

38.0 (±4.6)

14.3 (±0.1)

62.4 (±6.6)

29.4 (±2.8) 31.6 (±4.3)

16.9 (±0.8) 18.2 (±0.3)

42.5 (±4.6) 42.5 (±4.9)

38.0 (±4.6)

12.6 (±0.3)

66.8 (±7.3)

Psl overexpression Wild type

25.03 (±0.7) 25.04 (±3.3)

9.9 (±0.1) 7.7 (±0.3)

60.3 (±1.8) 69.4 (±8.5)

9.7 (±0.9) 1.7 (±0.3)

High Shear 0.27 (±0.02) 97.2 (±11.4) 0.07 (±0.12) 95.8 (±18.8)

∆psl mutant

25.07 (±3.2)

5.8 (±0.3)

76.9 (±9.6)

4.9 (±1.0)

0.10 (±0.08)

330 331 332 333


97.9 (±18.7)

29.4 (±2.8) 31.6 (±4.3)

16.6 (±0.3) 15.8 (±0.7)

43.5 (±4.3) 49.9 (±5.3)


334 335

The effect of Psl expression on EPS adherence and viscoelastic properties

336

Figure 2 displays the amount of adsorbed EPS to the polyamide-coated sensors

337

(for full adsorption experiments of the EPS to QCM-D sensors the reader is referred to

338

the supplementary information, Figures S4 and S5) measured toward the end of the fourth

339

injection stage (background solution free of EPS). As observed in Figure 2, no significant

340

difference (p>0.05) was observed between the adsorbed amount (expressed as a

341

frequency shift) of EPS extracted from the ∆psl mutant and the wild type strain, while

342

both of them showed a significantly higher (p

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