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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
20
biofilm. The surface free energy between the rhamnolipids and biofilm layer decreased, as
21
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
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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
25
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
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
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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%
165
(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
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
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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
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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
203
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.
206 207
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
210
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
219
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
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
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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.
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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
251
biofilm density in biofouled and rhamnolipid (100 µg mL-1, 2 h)-treated biofouled
252
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
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-
260
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%,
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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.
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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
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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
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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
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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.
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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.
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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
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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
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
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physicochemical interactions between rhamnolipids and the biofilm layer could help to better
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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
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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
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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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Article
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 †
6
School of Environmental Science & Engineering, Gwangju Institute of Science and Technology
7 8
(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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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
19
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
21
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
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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
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
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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
83
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
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
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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
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 +) −
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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+
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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.
144
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
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10 mM NaCl solution was used as the background electrolyte solution.
147 148
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
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
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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
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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
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.
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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
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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
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
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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
210
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
215
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
219
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
227
-17.4 (±5.6) mV, respectively. After treatment with 100 µg mL-1, 300 µg mL-1, 500 µg mL-1 of
228
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
235
increase in bacterial adhesion; however, it has been reported that the cell adhesion rate has no
236
relation to the surface charge when the charge is negative.36 The negative charge of the
237
membrane attracts positive constituents such as divalent ions (Ca2+), which make a bridge
238
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
246
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
249
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
251
biofilm density in biofouled and rhamnolipid (100 µg mL-1, 2 h)-treated biofouled
252
membranes.
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Figure 3 shows the EEM plot of the biofouled and rhamnolipid-treated RO
254
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-
260
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
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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
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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
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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
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
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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
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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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