Physicochemical Interactions between Rhamnolipids and

Feb 10, 2015 - •S Supporting Information. ABSTRACT: This study investigated the physicochemical interactions between a rhamnolipid biosurfactant 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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Physicochemical Interactions between Rhamnolipids and

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Pseudomonas aeruginosa Biofilm Layer

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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

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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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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

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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-

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

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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

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

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

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

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

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

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which rhamnolipids lead to a reduction in biofilm.

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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

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

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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

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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

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

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in

extracellular

Rhamnolipids

has

polymeric

advantageous

substances

properties

(EPS).14

such

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EPS

low

composed

toxicity,

of

high

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

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

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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

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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

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

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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

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

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

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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

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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

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Information-1 (SI-1).

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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

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

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

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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

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

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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.

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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

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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.

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

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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

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

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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-

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

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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

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

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

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∆GLW = 2(γwLW − γmLW )×(γcLW − γwLW )

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

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2(√γm+γc − + √γm − γc +)

(1)

(2)

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

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

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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-

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

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

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

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

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

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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,

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

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

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

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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

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

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

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

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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%

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

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

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

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

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Characterization of functional groups and atomic composition of EPS. The

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

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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

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

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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

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

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

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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

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

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

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

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

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

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

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

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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.

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

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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

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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

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-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

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

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

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

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

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

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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.

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

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

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

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

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

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membranes.

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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

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

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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;

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

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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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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Mannose is ubiquitous in biofilms containing exopolysaccharides of broad gram negative

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

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

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

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

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

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

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

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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

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

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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

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

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polysaccharide to be positively charged.45

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

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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

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

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

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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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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

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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

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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

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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

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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

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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

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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

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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

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a

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

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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

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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

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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

-

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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

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