Physicochemical Interactions between Rhamnolipids and

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Physicochemical Interactions between Rhamnolipids and Pseudomonas aeruginosa Biofilm Layer Lan Hee Kim, Yongmoon Jung, Hye-Weon Yu, Kyu-Jung Chae, and In S. Kim Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es505803c • Publication Date (Web): 10 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Environmental Science & Technology

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Physicochemical Interactions between Rhamnolipids and

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Pseudomonas aeruginosa Biofilm Layer

3

Lan Hee Kim,† Yongmoon Jung,† Hye-Weon Yu,‡ Kyu-Jung Chae,§ and In S. Kim*,†,⊥

4 5 †

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School of Environmental Science & Engineering, Gwangju Institute of Science and Technology

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(GIST), 123 Cheomdan-gwagiro, Buk-gu, Gwangju, 500-712, Republic of Korea ‡

Department of Soil, Water and Environmental Science, Department of Chemical and Environmental

9 10

Engineering, University of Arizona, Tucson, AZ85721, United States §

Department of Environmental Engineering, Korea Maritime and Ocean University, 727 Taejong-ro,

11 12

Yeongdo-Gu, Busan 606-791, Republic of Korea ⊥

Global Desalination Research Center (GDRC), Gwangju Institute of Science and Technology

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(GIST), 123 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea

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*Corresponding author (E-mail: [email protected], Tel: +82-62-715-2436, Fax: +82-62-715-2434)

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1

ACS Paragon Plus Environment


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ABSTRACT

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This study investigated the physicochemical interactions between a rhamnolipid biosurfactant

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and biofilm layer. A concentration of 300 µg mL-1 of rhamnolipids, which is around the

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critical micelle concentration value (240 µg mL-1), showed great potential for reducing

20

biofilm. The surface free energy between the rhamnolipids and biofilm layer decreased, as

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did the negative surface charge, due to the removal of negatively charged humic-like, protein-

22

like, and fulvic acid-like substances. The carbohydrate and protein concentrations composed

23

of extracellular polymeric substances decreased by 31.6% and 79.6%, respectively, at a

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rhamnolipid concentration of 300 µg mL-1. In particular, rhamnolipids can interact with

25

proteins, leading to a reduction of N source and amide groups on the membrane. For

26

carbohydrates, the component ratio of glucosamine was decreased, but the levels of glucose

27

and mannose that form the majority of the carbohydrates remained unchanged. To our

28

knowledge, the present study is the first attempt at studying the interactions of the two phases

29

of rhamnolipids and the biofilm layer, and as such is expected to clarify the mechanism by

30

which rhamnolipids lead to a reduction in biofilm.

31

2


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INTRODUCTION

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Rhamnolipids are biosurfactant produced by Pseudomonas species.1 The rhamnolipid

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is composed of a rhamnose moiety (hydrophilic glycon part) head group and a lipid moiety

35

(hydrophobic glycon part) tail group that are linked via an O-glycosidic linkage (Figure S1).

36

Endogenous rhamnolipids act as virulence factor and mediate maintaining the biofilm

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structure or inducing biofilm dispersal through the formation of internal cavities in a mature

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biofilm.2-4 Exogenous rhamnolipids serve many functions, including as an antimicrobial

39

agent in a broad spectrum of Gram-positive and Gram-negative bacteria,5 affecting the

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solubility of hydrophobic hydrocarbons,2, 6 and anti-adhesive activity in bacteria and fungus.7

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With their antimicrobial and biofilm disruption properties, rhamnolipids have been applied

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for reduction of biofilm which was formed by Gram positive bacteria (Bacillus pumilus,

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Listeria monocytogenes and Staphylococcus aureus), Gram-negative bacteria (Salmonella

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Enteritidis) and fungus (Yarrowia lipolytica) strains.3, 8-10 Despite the biofilm reduction by

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rhamnolipids has been observed, still the reduction mechanism need to be elucidated.7

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Biofouling on membrane has been considered a major factor causing the formation of

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fouling that leads to the acceleration of flux decline, membrane biodegradation, a decrease in

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boron rejection, and an increase in concentration polarization and energy consumption in

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membrane water treatment systems.11-13 Biofouling consists of bacterial communities

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embedded

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biomacromolecules such as polysaccharides, proteins, lipids, and DNA accelerate the biofilm

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formation rate and render biofouling more complex and resistant to environmental stress.15, 16

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Finding an effective method for removing the fouling layer from biofouled membrane has

54

been a key issue for membrane-based water treatment systems.

55

in

extracellular

Rhamnolipids

has

polymeric

advantageous

substances

properties

(EPS).14

such

3


as

EPS

low

composed

toxicity,

of

high


56

biodegradability and stability at wide range of temperatures, pH and salinity.17-19 Based on

57

these functions, rhamnolipids have recently been used as a cleaning agent in membrane-based

58

water treatment systems.20, 21 Long et al. (2014)20 noted the potential for rhamnolipids to

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reduce the amount of protein found in organic foulants on ultrafiltration membranes, and

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utilized them to degrade large oil droplets on the membrane surface during the treatment of

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frying oil wastewater in a submerged membrane bioreactor.21 Research on the ability of

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rhamnolipids to mediate fouling reduction has focused on organic fouling. Therefore, our

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group evaluated the rhamnolipids as a biofilm reducing agent on reverse osmosis (RO)

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membrane by confirmation of biofilm reduction at various concentrations and exposure time

65

of rhamnolipid, comparative biofilm reduction with synthetic surfactant and increase of flux

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in dead-end filtration system (will be published elsewhere). However, no reports exist that

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explore the effect of rhamnolipids on the physicochemical interactions between a membrane

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surface and the biofilm layer.

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In this study, physicochemical interactions between exogenous rhamnolipids, a

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biofilm formed reverse osmosis (RO) membrane, and EPS were investigated. The reduction

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of biofilm according to micelle formation was then determined.

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MATERIALS AND METHODS

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Measurement of critical micelle concentration and micelle aggregation number.

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Rhamnolipid used in this study was purchased from Urumqui Unite Bio-Technology Co., Ltd.

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(Xinjiang, China) which is comprised of 48.9% mono-rhamnolipid and 51.1% di-rhamnolipid.

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The 0.1% rhamnolipid solution had a surface tension of 30 mN m-1 (The data was provided

78

by the supplier). The critical micelle concentration (CMC) was measured using a pyrene

79

fluorescent probe (Sigma-Aldrich, MO, USA), under rhamnolipid concentrations ranging 4


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from 0 µg mL-1 to 500 µg mL-1 in deionized water or Dulbecco’s phosphate-buffered saline

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(PBS; Life Technologies, CA, USA) at an ionic strength of 0.16 M. The effect of pH on the

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CMC was also investigated using PBS at a pH of 3, 5, 7, and 9. The fluorescence intensities

83

were measured using a F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The

84

excitation wavelength was 335 nm and the slit widths for emission and excitation were fixed

85

at 2.5 nm and 10 nm, respectively. To calculate the ratio of the fluorescence intensity (I3/I1),

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the first (I1) and third (I3) vibronic bands of pyrene were measured at emission spectra

87

wavelengths of 373 nm (I1) and 384 nm (I3). The CMC value was obtained from the

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intersection point between the sharp increase and stabilization of the I3/I1 ratio. The micelle

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aggregation number (Nagg) was determined using a steady-state fluorescence quenching

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technique.22 Benzophenone (Sigma-Aldrich, MO, USA) was used as a quencher, and

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concentrations were varied from 0 mM to 0.1 mM. The pyrene and rhamnolipid

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concentrations were fixed at 1 µM and 300 µg mL-1, respectively. The fluorescence intensity

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with (I) and without (I0) quencher were determined using a fluorescence spectrophotometer

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having an excitation wavelength of 335 nm and an emission wavelength of 384 nm. The Nagg

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value was calculated using the slope of the ln(I0/I) plot according to the quencher

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concentration.23 The equations used to calculate the results are described in Supporting

97

Information-1 (SI-1).

98 99

Preparation of model bacterium. Pseudomonas aeruginosa P60 (Korea

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Environmental Microorganism Bank; KEMB 9006-001) was used as a model bacterium to

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form the biofilm on the RO membrane. The bacteria were incubated in a Luria-Bertani (LB)

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medium (BD, NJ, USA) for 16 h and centrifuged at 8,000 rpm for 10 min. The pellet was

103

washed three times with PBS in order to remove any remaining nutrients. The optical density 5



104

of the washed cells was adjusted to 1.0 at 600 nm (OD600).

105 106

Biofilm formation on RO membrane. A 3 cm × 3 cm SHN-RE8040 RO membrane

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(Toray Chemical Korea Inc., Seoul, Korea) which was soaked in deionized water overnight

108

was attached to a culture flask (SPL, Pocheon, Korea) and 20 mL of the LB medium was

109

added. And then, 20 µL of cells (OD600 1.0) were inoculated in a culture flask and incubated

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for 4 days at 37 °C in an incubator at 90 rpm in order to allow the biofilm to form.

111 112

Treatment of biofilm formed RO membrane by rhamnolipid. After formation of

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biofilm, the membranes were washed with PBS three times and added 10 mL PBS containing

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0 µg mL-1, 100 µg mL-1, 300 µg mL-1, and 500 µg mL-1 of rhamnolipids to culture flasks. All

115

flasks were incubated for 2 h at room temperature. To analyze membrane surface, the

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membranes were removed from the flasks and dried in a desiccator for 2 days and to quantify

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and characterize EPS, the rhamnolipid treated membrane put in 9 mL PBS.

118 119

Measurement of contact angles and surface charge of RO membrane. To analyze

120

the contact angles and surface charge, four RO membranes with or without rhamnolipid

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treatment were prepared: 1) a virgin RO membrane, 2) a biofouled membrane, and

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rhamnolipid-treated 3) virgin and 4) biofouled membranes. Prior to analysis of the membrane

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surface, the virgin RO membrane, biofilm-formed membrane, and rhamnolipid-treated

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membranes were dried for 2 days in a desiccator.

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The surface free energies of the membrane samples were calculated based on the

126

results of the contact angle measurements made using a Phoenix-300 Touch contact angle

127

measurement system (SEO Co., Ltd., Suwon, Korea) employing a sessile drop method using 6


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two polar liquids (deionized water and formamide) and one non-polar liquid (diiodomethane)

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as the diagnostic liquids. The Lifshitz-van der Waals (LW) (γLW), acid-base (γAB), electron-

130

donor (γ-), and electron-acceptor (γ+) surface energy components were calculated based on

131

the results of the contact angles using SurfaceWare 8 software (SEO Co., Ltd., Suwon,

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Korea). The total surface tension (γTotal) was calculated as the sum of the γLW and γAB

133

components. The surface free energies (∆GLW, ∆GAB) of the samples were then calculated

134

using equations (1) and (2), based on the value of the surface energy components.24

135 136

∆GLW = 2(γwLW − γmLW )×(γcLW − γwLW )

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∆GAB = 2√γw + (√γm − + √ − − √ −) + 2√γw − (√γm + + √γc +− √γw +) −

138

2(√γm+γc − + √γm − γc +)

(1)

(2)

139 140

where γwLW, γmLW, and γcLW are the LW components of the surface tension for water, the

141

virgin membrane, and the biofouled or rhamnolipid-treated membrane, respectively, and γ+

142

and γ- are the electron acceptor and electron donor components of the free energy. The total

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surface free energy (∆GTotal) was calculated as the sum of ∆GLW and ∆GAB.

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The surface charge (mV) of the membrane was measured using an ELS-Z Zeta-

145

potential & Particle size analyzer (Otsuka Electronic Co., Ltd., Osaka, Japan) at pH 7, and a

146

10 mM NaCl solution was used as the background electrolyte solution.

147 148

Extraction and quantification of EPS. To analyze the EPS, the biofouled

149

membranes were put into 9 mL PBS and the biomass (bacteria and EPS) was separated from

150

the membranes using 2 min of vortexing and 5 min of sonication. The EPS was extracted

151

following the method established by Liu and Fang (2002).25 In brief, the whole 10 mL EPS 7



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suspended in PBS was treated using 0.06 mL formaldehyde (36.5%; Sigma-Aldrich, MO,

153

USA) at 4 °C for 1 h and incubated with 4 mL 1 N NaOH at 4 °C for 3 h. The formaldehyde

154

prevented bacterial lysis by interacting with functional groups of proteins and nucleic acids of

155

the cell membrane. The NaOH was used for increase of pH to dissociate acidic groups in EPS

156

and the repulsion between the negative charged EPS.25 After treatment, the samples were

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centrifuged for 20 min at 20,000 × g. The supernatant was filtered through a 0.2 µm

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membrane and dialyzed using a 3,500 Da dialysis membrane (Pierce, IL, USA) for 24 h. The

159

dialyzed samples were lyophilized and the excitation-emission matrix (EEM) measured using

160

a F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) under excitation of 220 nm

161

to 450 nm and emission of 250 nm to 600 nm at a speed of 1500 nm min-1, a voltage of 700 V,

162

and a response time of 2 s.

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The carbohydrates were measured following a previous reference.26 In brief, 80 µL of

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the sample was mixed with 160 µL of an anthrone reagent (0.125% anthrone (w/v) in 94.5%

165

(v/v) H2SO4). These samples were then reacted at 100 °C for 14 min and cooled at 4 °C for 5

166

min. The absorbance at 625 nm was measured using a µQuant microplate reader (BioTek

167

Instruments, Inc., VT, USA) and the protein concentrations measured using a BCA assay kit

168

(Thermo Scientific Inc., NH, USA) according to the manufacture’s guidelines.

169 170

Characterization of functional groups and atomic composition of EPS. The

171

Fourier transform infrared (FT-IR) spectra of the biofouled and 300 µg mL-1 of rhamnolipid-

172

treated RO membranes were recorded between 4000 cm-1 and 800 cm-1 using an Agilent Cary

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660 spectrometer (Agilent Technologies, CA, USA) equipped with KBr beam splitters and

174

deuterated L-alanine-doped triglycine sulfate (DLATGS) detectors. All data were collected

175

and analyzed using the Agilent Resolution Pro software (Agilent Technologies, CA, USA). 8


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The surface of the biofouled membrane and rhamnolipid-treated membranes were analyzed

177

and air was used as the background signal. The membranes were each analyzed 32 times per

178

point at 5 different points per membrane (n=3).

179

The atomic composition of the lyophilized EPS samples was analyzed using a K-

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ALPHA X-ray photoelectron spectroscope (Thermo Scientific Inc., NH, USA) having a

181

monochromated AlKα X-ray source and a penetration depth of 400 µm. The components of

182

sugars in the carbohydrates of the extracted EPS were analyzed using a ICS-5000 high-

183

performance anion exchange chromatograph (Dionex Corp., CA, USA) equipped with a 4

184

mm × 250 mm CarboPac PA10 column (Dionex Corp., CA, USA). The lyophilized EPS

185

samples were hydrolyzed in 2 M trifluoroacetic acid (TFA) for 4 h at 100 °C for neutral

186

sugars; for amino sugars, the EPS samples were hydrolyzed in 6 N HCl for 4 h at 100 °C. The

187

mobile phase was 16 mM NaOH, and the flow rate was 1.0 mL min-1.

188 189

RESULTS AND DISCUSSION

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Rhamnolipid CMC and Nagg. Figure 1A shows the CMC value of rhamnolipid

191

measured in deionized water or PBS under various pH conditions (pH 3, 5, 7, and 9). The

192

CMC value for the rhamnolipids was 240 µg mL-1. The CMC value can be affected by buffer

193

pH, temperature, and ionic strength;27 however, the rhamnolipids were stable for the various

194

pH and ionic strength conditions. It has been reported that the surfactant concentration level

195

can affect both the adsorption of surfactants on the membrane and the degree of fouling

196

reduction.28 Surfactant concentrations below a certain CMC value form smaller aggregations

197

(pre-micelle) than micelles and cause pore blocking on the ultrafiltration (UF) membrane

198

whereas, at a higher surfactant concentration CMC value, the hydrophilic micelle surface has

199

a higher affinity to the solvent than to the UF membrane.28 The number of detergent 9



200

monomers per micelle, i.e., the aggregation number (Nagg), was determined using a

201

benzophenone quencher probe that moves freely between the aqueous and micellar phases.

202

The ratio ln(I0/I) according to the added concentration of benzophenone is shown in Figure

203

1B. The Nagg value calculated using the slope value (1.5281) was 39.2, which indicates that

204

rhamnolipids can form relatively small micelles when compared to those created by

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surfactants such as SDS (59 ± 5),29 Triton X-100 (121 ± 1),30 and Tween 80 (133 ± 1)30.

206 207

Decrease in surface free energy and negative surface charge of membranes by

208

rhamnolipids. Based on the results of the contact angles of the membranes with three liquids

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(Table 1), the membrane surface free energy was calculated (Table 2). The LW component

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representing the energy between the membrane and rhamnolipids was positive and did not

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change as the concentration of the applied rhamnolipids was varied. The positive value of the

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LW component indicates that adhesion to both the virgin and biofilm formed RO membranes

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is unfavorable.31 The negative value of the AB component implies that rhamnolipids and

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bacteria adhere strongly to the RO membrane surface. Therefore, the positive value of the AB

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component at 300 µg mL-1 of rhamnolipids on the virgin RO membrane indicates that

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rhamnolipids adhere weakly to the membrane surface at that concentration.

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The higher value of γ- than γ+ means that both rhamnolipids and bacteria are

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predominantly electron-donating. The cohesive free energy (∆GTotal) denotes the interaction

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energy between the virgin membrane and rhamnolipids or biofouled membrane and

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rhamnolipids. The sample rhamnolipid-treated biofouled membranes showed a slight

221

decrease in free energy at rhamnolipid concentrations of 300 µg mL-1 and 500 µg mL-1. The

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decreasing value of ∆GTotal indicates a lower hydrophilic repulsion between the membrane

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and bacteria, which may be caused by a reduction in the biomass at the RO membrane 10


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surface. It was reported that less amount of bacteria adhere to low surface energy substratum

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and cleaning of the substratum is more easy because of weaker binding at the interface.32

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The surface charges of the virgin and biofouled membranes were -38.3 (±3.0) mV and

227

-17.4 (±5.6) mV, respectively. After treatment with 100 µg mL-1, 300 µg mL-1, 500 µg mL-1 of

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rhamnolipids, the negative surface charge decreased to -5.8 (±2.5) mV, -5.9 (±2.6) mV, and -

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11.1 (±6.7) mV, respectively at pH 7, whereas the rhamnolipids themselves did not affect the

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membrane surface charge (Table 1). The results differed from those in literature in that the

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SDS caused an increase in the negative charge on the RO membrane because of the

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negatively charged sulfate functional groups.33, 34 Al-Amoudi et al. (2007)35 reported that

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SDS can readily absorb into the membrane surface and that the negatively charged functional

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SDS group dominates the membrane surface. The lower negative charge may cause an

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increase in bacterial adhesion; however, it has been reported that the cell adhesion rate has no

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relation to the surface charge when the charge is negative.36 The negative charge of the

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membrane attracts positive constituents such as divalent ions (Ca2+), which make a bridge

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between the negatively charged hydrophilic part of humic acid and the membrane.35

239 240

Effect of rhamnolipids on EPS concentrations and distribution. Figure 2 presents

241

the reduction of carbohydrate and protein concentrations caused by rhamnolipid treatment.

242

Interaction with rhamnolipids reduced the amount of carbohydrates and proteins. EPS is

243

comprised mainly of proteins and polysaccharides (75–89%), some lipids, and DNA.37 After

244

100 µg mL-1, 300 µg mL-1, and 500 µg mL-1 rhamnolipid treatments, the carbohydrate

245

concentrations were reduced by 26.7%, 31.6%, and 31.4%, and protein concentrations were

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reduced by 75.5%, 79.6%, and 78.1%, respectively, when compared to the control sample.

247

The results showed that when applying 100 µg mL-1 rhamnolipid at lower than CMC reduced 11



248

EPS and the efficiency of reduction was maintained even rhamnolipid concentrations were

249

increased. This can be caused by the decrease of total cell numbers on the membrane surface

250

as well as the reduction of organic matters. The images of Figure S2 shows reduction of

251

biofilm density in biofouled and rhamnolipid (100 µg mL-1, 2 h)-treated biofouled

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membranes.

253

Figure 3 shows the EEM plot of the biofouled and rhamnolipid-treated RO

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membranes. The high affinity of rhamnolipids to amphiphilic proteins through electrostatic

255

and hydrophobic interactions leads to reduction of protein. The area was divided into four

256

regions: I (humic-like; Ex > 280 nm, Em > 380 nm), II (protein-like; Ex = 250–280 nm, Em
380 nm), and IV (tyrosine-like protein;

258

Ex = 220–250 nm, Em = 330–380 nm).38 In terms of relative intensities of EEM peaks for all

259

regions, the order of reduction was: protein-like > tyrosine-like > humic-like > fulvic acid-

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like substances. When compared to the control sample at rhamnolipid treatments of 100 µg

261

mL-1, 300 µg mL-1, and 500 µg mL-1, the intensities were reduced for the protein-like (83.3%,

262

80.9%, 78.7%), tyrosine-like (54.6%, 62.2%, 53.8%), humic-like (53.0%, 61.8%, 54.9%),

263

and fulvic acid-like (31.9%, 46.7%, 36.5%) regions, respectively.

264 265

Effect of rhamnolipids on functional groups and EPS compositions. In Figure 4,

266

the infrared spectrum of a given biofouled and 300 µg mL-1 of rhamnolipid-treated membrane

267

was calculated using the ratio of the signal obtained by scanning air to the signal obtained

268

from samples. EPS have several charged groups (carboxyl, phosphoric, sulfhydryl, phenolic,

269

and hydroxyl groups) and apolar groups (aromatics and aliphatics in proteins, and

270

hydrophobic regions in carbohydrates).12 The peak assignments were made according to

271

literature (Table 3).39 The peak absorbance of the hydroxyl group (3000–3400 cm-1) 12


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decreased by 46.5%. The absorbance of stretching fatty chains (νCH3, νCH2, νCH) positioned

273

at the cellular membrane was reduced by more than 35% after rhamnolipid treatment. Among

274

the stretching fatty chains, the CH2 stretching bands at 2925 cm-1 and 2854 cm-1 are present

275

because of lipids at the periphery of the bacterial cells.39 The reduction of CH2 peaks after

276

rhamnolipid treatment might be due to the release of lipopolysaccharides (LPSs) from the

277

outer membrane of the bacterial cells. Rhamnolipids remove LPS from the outer membrane

278

and form a complex with the magnesium in order to maintain the LPS-LPS interactions.40

279

The absorbance peaks of amide I (1693–1627 cm-1) and amide II (1568–1531 cm-1), which

280

are composed of EPS, were reduced by 30.6% and 16.2% compared to the control sample.

281

Figure 5 shows the sugar compositions of carbohydrates extracted from the biofouled

282

and rhamnolipid-treated RO membranes. Glucose and mannose were major components of

283

the carbohydrates in all samples; however, fucose was not detected in any of samples. Among

284

the sugar compositions found in the EPS, glucosamine decreased as according to the

285

rhamnolipid concentration increased. P. aeruginosa can synthesize at least three kinds of

286

extracellular polysaccharides: alginate, polysaccharide synthesis locus (Psl), and pellicle (Pel).

287

Alginate is composed of non-repetitive monomers of β-1,4 linked to L-guluronic and D-

288

mannuronic acids and allows P. aeruginosa to be a mucoid. The Psl and Pel operons encode

289

Psl (mannose- and galactose-rich) and Pel (glucose-rich) polysaccharides.41 These

290

polysaccharides play an important role in the biofilm development by non-mucoid bacteria.

291

Mannose is ubiquitous in biofilms containing exopolysaccharides of broad gram negative

292

bacteria including P. aeruginosa; therefore, breaking the common bonds of mannose can be

293

an effective method for biofilm dispersal.16 In addition, cellulose, which is a kind of

294

polysaccharide, can be produced by Pseudomonas and is composed of monosaccharide

295

glucose.42 In this study, the mannose- and glucose-rich EPS compositions were found in the 13



296

rhamnolipid-treated and non-rhamnolipid-treated RO membrane samples.

297

Compared to the control sample, glucosamine was reduced by 38.1%, 66.1%, and

298

69.9% in the 100 µg mL-1, 300 µg mL-1, and 500 µg mL-1 of rhamnolipid-treated EPS

299

samples, respectively. Glucosamine is a component of cell wall peptidoglycan and has been

300

observed in the polysaccharides of Pseudomonas fluorescence.43 Poly-N-acetyl-glucosamine

301

(PNAG) has a crucial adhesin function as an intercellular adhesive, and the dispersin B

302

enzyme can help break down the biofilms of different bacteria species through the hydrolysis

303

of β-1,6-N-acetyl-D-glucosamine.44 PNAG can electrostatically attract negatively charged

304

teichoic acid at the bacterial cell surface; therefore, the deacetylation of PNAG allows the

305

polysaccharide to be positively charged.45

306

Table 4 shows the composition of the atomic elements found in the EPS of the

307

control sample and the 100 µg mL-1, 300 µg mL-1, and 500 µg mL-1 rhamnolipid-treated

308

samples. The percent of C decreased after rhamnolipid treatment, whereas the amount of O

309

increased, possibly due to the conversion of CH2 or CH3 into C=O or C-O functional groups.

310

The peak intensity of N having a binding energy of 399 eV, which comprises the amine or

311

amide groups (RHN-C=O) that are composed of proteins,46 was greatly reduced by 20.6%,

312

31.4%, and 71.1% after the addition of 100 µg mL-1, 300 µg mL-1, and 500 µg mL-1 of

313

rhamnolipids, respectively. This decrease corresponds with the EEM results, which also show

314

that the amount of protein-like substances in the EPS was significantly reduced.

315

In conclusion, rhamnolipids have great potential to reduce biofilm on RO membrane.

316

The presence of rhamnolipids at the CMC level led to a significant reduction in biofilm and

317

demonstrated high reduction efficiency, with an accompanying decrease in surface free

318

energy on the membrane. The rhamnolipids reduced the amount of EPS present and

319

selectively interacted with different EPS proteins. A subsequent investigation of the 14


Page 14 of 32

Page 15 of 32


320

physicochemical interactions between rhamnolipids and the biofilm layer could help to better

321

understand the exact mechanisms at work in rhamnolipid-mediated biofilm reduction. Further

322

study of the effects of rhamnolipids on the bacteria in the biofilm layer is also needed.

323 324

ACKNOWLEDGEMENTS

325

This research was supported by a grant (13IFIP-C071144-01) from the operation and

326

application of the management system for SeaHERO program products funded by the

327

Ministry of Land, Transport and Maritime Affairs of the government of Korea, and in part by

328

the Basic Research in High-tech Industrial Technology Project through a grant provided by

329

the Gwangju Institute of Science and Technology in 2014.

330 331

Supporting Information. The rhamnolipid structure (Figure S1), SEM images of biofouled

332

and rhamnolipid treated biofouled membranes (Figure S2) and description of micelle

333

aggregation number (Nagg) calculation (SI-1) are provided.

334 335

REFERENCES

336 337

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membranes. J. Membr. Sci. 2014, 454, 82-96.

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control strategies. LWT - Food Sci. Technol. 2010, 43, 573-583.

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Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environ. Sci.

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Technol. 2003, 37, 5701-5710.

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(39) Quilès, F.; Franç ois Humbert, A. D., Analysis of changes in attenuated total reflection

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FTIR fingerprints of Pseudomonas fluorescens from planktonic state to nascent biofilm state. 19



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Spectrochim. Acta Part A. 2010, 75, 610-616.

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(40) Al-Tahhan, R. A.; Sandrin, T. R.; Bodour, A. A.; Maier, R. M., Rhamnolipid-Induced

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Removal of Lipopolysaccharide from Pseudomonas aeruginosa: Effect on Cell Surface

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Properties and Interaction with hydrophobic Substrates. Appl. Environ. Microbiol. 2000, 66,

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aeruginosa biofilm development. Curr. Opin. Microbiol. 2007, 10, 644-648.

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(42) Vu, B.; Chen, M.; Crawford, R.; Ivanova, E., Bacterial Extracellular Polysaccharides

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Involved in Biofilm Formation. Molecules 2009, 14, 2535-2554.

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(43) Kives, J.; Orgaz, B. E.; SanJosé, C., Polysaccharide differences between planktonic and

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biofilm-associated EPS from Pseudomonas fluorescens B52. Colloid surf. B, Biointerfaces

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2006, 52, 123-127.

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(44) Lu, T. K.; Collins, J. J., Dispersing biofilms with engineered enzymatic bacteriophage.

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Proc. Nat. Acad. Sci. 2007, 104, 11197-11202.

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(45) Heilmann, C.; Götz, F. Cell–Cell Communication and biofilm formation in Gram-

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positive bacteria. In Bacterial Signaling ; Krämer, R. Jung, K.; Wiley-VCH Verlag GmbH &

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457

(46) Omoike, A.; Chorover, J., Spectroscopic Study of Extracellular Polymeric Substances

458

from Bacillus subtilis: Aqueous Chemistry and Adsorption Effects. Biomacromolecules 2004,

459

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460 461 462 463 20


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464


Figure captions

465 466

Figure 1. (A) CMC values of rhamnolipids in deionized water or PBS at pH 3, 5, 7, and 9.

467

The ratio of I3/I1 was obtained by measuring the fluorescence intensity at emission

468

wavelengths of 373 nm (I1) and 384 nm (I3). (B) Effects of benzophenone concentration

469

(quencher) on the fluorescence intensity of pyrene in a micellar solution of rhamnolipids,

470

both with (I) and without (I0) quencher.

471 472

Figure 2. Concentrations of carbohydrates and proteins extracted from biofouled and

473

rhamnolipid (0 µg mL-1, 100 µg mL-1, 300 µg mL-1, and 500 µg mL-1)-treated biofouled

474

membranes (n=3).

475 476

Figure 3. EEM plots of organic matter extracted from biofouled membrane samples after

477

treatment with 0 µg mL-1, 100 µg mL-1, 300 µg mL-1, and 500 µg mL-1 rhamnolipids. The

478

plots show the presence of (A) humic-like matter, (B) protein-like matter, (C) fulvic acid-like

479

substances, and (D) tyrosine-like proteins (n=3).

480 481

Figure 4. FT-IR spectra on biofouled membrane (control) and the membrane treated with 300

482

µg mL-1 rhamnolipids for 2 h. The absorbance and assignments for peaks are presented in the

483

figure, while the inset figure shows the spectra band from 1531 cm-1 to 1700 cm-1 (n=3).

484 485

Figure 5. Sugar composition of EPS purified from the biofilm-formed and rhamnolipid-

486

treated (100 µg mL-1, 300 µg mL-1, and 500 µg mL-1) biofilm-formed membranes. The

487

proportion of mannose, glucose, galactose, glucosamine, and galactosamine are shown. 21



488

489

Figure 1. (A) CMC values of rhamnolipids in deionized water or PBS at pH 3, 5, 7, and 9.

490

The ratio of I3/I1 was obtained by measuring the fluorescence intensity at emission

491

wavelengths of 373 nm (I1) and 384 nm (I3). (B) Effects of benzophenone concentration

492

(quencher) on the fluorescence intensity of pyrene in a micellar solution of rhamnolipids,

493

both with (I) and without (I0) quencher. 22


Page 22 of 32

Page 23 of 32


494

495

496

Figure 2. Concentrations of carbohydrates and proteins extracted from biofouled and

497

rhamnolipid (0 µg mL-1, 100 µg mL-1, 300 µg mL-1, and 500 µg mL-1)-treated biofouled

498

membranes (n=3).

499

500

501

502

23



503 504

Figure 3. EEM plots of organic matter extracted from biofouled membrane samples after

505

treatment with (A) 0 µg mL-1, (B) 100 µg mL-1, (C) 300 µg mL-1, and (D) 500 µg mL-1

506

rhamnolipids. The plots show the presence of (I) humic-like matter, (II) protein-like matter,

507

(III) fulvic acid-like substances, and (IV) tyrosine-like proteins (n=3). 24


Page 24 of 32

Page 25 of 32


508 509

Figure 4. FT-IR spectra on biofouled membrane (control) and the membrane treated with 300

510

µg mL-1 rhamnolipids for 2 h. The absorbance and assignments for peaks are presented in the

511

figure, while the inset figure shows the spectra band from 1531 cm-1 to 1700 cm-1 (n=3).

512

513

514

515

516

25



517

518

519

Figure 5. Sugar composition of EPS purified from the biofilm-formed and rhamnolipid-

520

treated (100 µg mL-1, 300 µg mL-1, and 500 µg mL-1) biofilm-formed membranes. The

521

proportion of mannose, glucose, galactose, glucosamine, and galactosamine are shown.

522 523 524 525 526

26


Page 26 of 32

Page 27 of 32

527


Table 1. Effects of rhamnolipids on membrane hydrophobicity and surface charge. Rhamnolipid

Contact angle (θ)

Substratum concentration

a

potential

(µg mL )

Water

0

22.1(±1.2)a

34.1(±3.7)

19.3(±1.6)

-38.3(±3.0)

Virgin RO

100

43.8(±3.8)

43.1(±2.6)

26.3(±3.1)

-31.0(±10.7)

membrane

300

47.1(±3.5)

44.2(±0.2)

36.2(±2.0)

-39.5(±3.8)

500

48.4(±3.1)

45.3(±0.8)

24.0(±1.1)

-38.5(±7.4)

0

35.7(±2.2)

45.6(±7.1)

35.2(±0.8)

-17.4(±5.6)

Biofouled

100

41.5(±1.9)

50.5(±5.9)

34.0(±2.4)

-5.8(±2.5)

membrane

300

41.9(±6.4)

50.7(±5.6)

33.3(±1.9)

-5.9(±2.6)

500

44.5(±2.6)

52.7(±2.0)

36.5(±0.6)

-11.1(±6.7)

-1

528

Zeta

Formamide Diiodomethane

Standard deviation (±) (n=3)

529

27


(mV)


530

Table 2. Surface energies of rhamnolipid-treated virgin RO and biofouled membranes.

Substratum

Rhamnolipid concentration (µg mL-1)

Surface energy componentsa γ

LW

γ

AB

γ

+

γ

-

γ

∆GLW

∆GAB

∆GTotal

total

0

48.00

-5.27

0.14

61.10

42.73

-10.21

50.65

40.44

100

45.63

-3.63

0.10

41.53

42.00

-9.42

36.75

27.33

300

41.50

0.60

0.02

37.25

42.10

-7.99

38.99

29.01

500

46.50

-4.63

0.15

36.73

41.87

-9.71

35.32

25.61

0

41.90

-6.47

0.33

56.10

35.43

-5.50

48.37

42.87

Biofouled

100

42.95

-10.70

0.77

52.80

32.25

-8.49

51.32

42.83

membrane

300

42.77

-10.47

0.63

52.27

32.30

-8.45

44.45

36.00

500

41.33

-9.57

0.47

50.03

31.77

-7.95

43.27

35.32

Virgin RO membrane

531

Page 28 of 32

a

Units of surface energy components, surface energy, ∆GLW, ∆GAB, ∆GTotal are mJ/m2.

28


Page 29 of 32


Table 3. Assignments of FT-IR spectra of biofouled membranes after treatment with 300 µg mL-1 rhamnolipids. Wavenumber (cm-1) 3000–3400

Principal compounds and/or functions

Assignment Hydroxyl (-OH)

-

Main corresponding cellular compounds -

Absorbance

Reduction efficiency 0 300 -1 -1 (%) (µg mL ) (µg mL ) 0.036

0.019

46.5

2961

νaCH3

0.027

0.015

43.6

2925

νaCH2

0.025

0.011

53.9

2897

νCH tertiary

0.019

0.012

35.9

2874

νsCH3

0.019

0.009

52.6

2854

νsCH2

0.016

0.010

40.6

1736

νC=O

Esters from lipids

Cellular membranes

0.012

0.010

17.0

1713

νC=O

Esters, carboxylic acids

Nucleoid, ribosomes

0.018

0.072

-303.4

νC=O, νC=N, νC=C, δNH

DNA/RNA bases

Nucleoid, ribosomes

0.095

0.066

30.3

0.095

0.066

30.6

0.104

0.088

16.2

0.060

0.056

6.7

0.060

0.052

13.3

0.059

0.044

24.9

1700–1580 1693–1627 1568–1531 1468

Amide I (νC=O coupled with δN-H), δH2O Amide II (δN-H coupled with νC-N)

Fatty chains

Cellular membranes

Proteins, water

Membranes, cytoplasm, flagella, pili, ribosomes

Proteins

δCH2, δaCH3 Lipids

1455

δCH2, δaCH4

1400

νsCOO-

Cellular membranes

Amino acids, fatty acid chains

Capsule, peptidoglycan

29



1317

τCH2, ρCH2, Amide III (νCN coupled with δN-H)

Cellular membranes, cytoplasm, flagella, pili, ribosomes

Fatty acid chains, proteins

1281 Phosphodiester, phospholipids, LPS, nucleic acids, ribose

1238

νaPO2-

1220

νC-O-C νC-O, νC-C, δC-O-H, δC-OPolysaccharides C

1200–900 1172

νsC-OH, νC-O

Cellular membranes, nucleoid, ribosomes Capsule, storage inclusions

Page 30 of 32

0.079

0.079

0.1

0.063

0.059

6.0

0.227

0.262

-15.6

0.098

0.110

-12.0

0.078

0.074

4.6

0.107

0.119

-10.7

0.184

0.210

-14.0

1153

νsC-OH, νC-O

Proteins, carbohydrates, esters

1118

νsCC

DNA, RNA

Nucleoid, ribosomes

0.089

0.083

7.2

1086

νsPO2-

Phosphodiester, phospholipids, LPS, nucleic acids

Cellular membranes, nucleoid, ribosomes

0.111

0.102

8.0

Capsule, peptidoglycan

0.082

0.064

21.4

Polysaccharides

1041

νsC-O-C, νsP-O-C (R-O-P-O-R') νO-H coupled with δC-O

0.063

0.045

28.5

1026

CH2OH

Carbohydrates

Storage inclusion

0.049

0.035

28.8

993

-

Ribose skelet

Ribosomes

0.048

0.039

19.9

970 νC-C, νP-O-P RNA backbone Ribosomes 0.046 *Abbreviation: ν; stretching, δ; bending, τ; twist, a; antisymmetric, s; symmetric, LPS; lipopolysaccharides

0.036

20.9

1058

-

30


Page 31 of 32


Table 4. XPS analysis of the measurement of mass percentage in EPS after cleaning with rhamnolipids. Rhamnolipid concentration (µg mL-1)

Important elements (%)

Control

100

300

500

P2p

5.3

9.8

5.4

7.2

C1s

53.5

39.7

52.3

47.1

N1s

9.0

5.4

5.8

5.8

O1s

28.2

37.0

31.9

33.5

Na1s

4.1

8.2

4.6

6.3

31



TOC/Abstract Art

32


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