Impact of Higher Alginate Expression on Deposition of Pseudomonas

Sep 2, 2009 - Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev, Sede Boqer ...
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Environ. Sci. Technol. 2009, 43, 7376–7383

Impact of Higher Alginate Expression on Deposition of Pseudomonas aeruginosa in Radial Stagnation Point Flow and Reverse Osmosis Systems M O S H E H E R Z B E R G , * ,† TESFALEM ZERE REZENE,† CHRISTOPHER ZIEMBA,‡ OSNAT GILLOR,§ AND KALAI MATHEE| Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev, Sede Boqer Campus 84990, Israel, Department of Chemical Engineering, Environmental Engineering Program, Yale University, New Haven, Connecticut 06520-8286, Department of Hydrology and Microbiology, Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev, Sede Boqer Campus 84990, Israel, and Department of Molecular Microbiology and Infectious Diseases, College of Medicine, Florida International University, University Park, Miami, Florida 33199

Received April 10, 2009. Revised manuscript received August 3, 2009. Accepted August 10, 2009.

Extracellular polymeric substances (EPS) have major impact on biofouling of reverse osmosis (RO) membranes. On one hand, EPS can reduce membrane permeability and on the other, EPS production by the primary colonizers may influence their deposition and attachment rate and subsequently affect the biofouling propensity of the membrane. The role of bacterial exopolysaccharides in bacterial deposition followed by the biofouling potential of an RO membrane was evaluated using an alginate overproducing (mucoid) Pseudomonas aeruginosa. The mucoid P. aeruginosa PAOmucA22 was compared with its isogenic nonmucoid prototypic parent PAO1 microscopically in a radial stagnation point flow (RSPF) system for their bacterial deposition characteristics. Then, biofouling potential of PAO1 and PAOmucA22 was determined in a crossflow rectangular plate-and-frame membrane cell, in which the strains were cultivated on a thin-film composite, polyamide, flat RO membrane coupon (LFC-1) under laminar flow conditions. In the RSPF system, the observed deposition rate of the mucoid strain was between 5- and 10-fold lower than of the wild type using either synthetic wastewater medium (with ionic strength of 14.7 mM and pH 7.4) or 15 mM KCl solution (pH of 6.2). The slower deposition rate of the mucoid strain is explained by 5- to 25-fold increased hydrophilicity of the mucoid strain as compared to the isogenic wild type, PAO1. Corroborating with these results, a significant delay in the onset of biofouling of * Corresponding author phone +972 8 6563520; fax +972 8 6563503; e-mail: herzberg@bgu,.ac.il. † Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev. ‡ Yale University. § Department of Hydrology and Microbiology, Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev. | Florida International University. 7376

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the RO membrane was observed when the mucoid strain was used as the membrane colonizer, in which the observed time for the induced permeate flux decline was delayed (ca. 2-fold). In conclusion, the lower initial cell attachment of the mucoid strain decelerated biofouling of the RO membrane. Bacterial deposition and attachment is a critical step in biofilm formation and governed by intimate interactions between outer membrane proteins of the bacteria and the surface. Shielding these interactions by a hydrated and hydrophilic alginate capsule is shown to dramatically lessen the biofouling potential of the membrane colonizers.

Introduction Extracellular polymeric substances (EPS)sthe metabolic products of active bacterial secretionsaccumulate on the bacterial cell surface, thereby contributing to the formation of a biofilm (1). EPS affect the immediate heterogeneous microenvironment of living cells in microbial biofilms, producing changes in their porosity, density, water content, charge, sorption properties, hydrophobicity, and mechanical stability. The biofilm, in turn, acquires from the EPS mechanical stability, mainly as a result of electrostatic and van der Waals interactions and of hydrogen bonding (2). The EPS also play a role in protecting the bacterial cells comprising the biofilm by retarding penetration and sequestering of antimicrobial agents (3). Although EPS are rich in polysaccharides, it is usually the other componentssproteins, lipids, and even DNAsthat influence the physical properties of the biofouling layer (4). In P. aeruginosa biofilms, EPS comprise alginate and other nondefined polysaccharides, glucose- and mannose-rich components, as well as DNA and proteins that play an important role in biofilm maturation (5, 6). Bacterial deposition on solid surfaces is the first step in the biofilm formation process. To gain insight into the mechanisms leading to bacterial deposition, the interaction energies between bacterial cell surface and a solid surface are often predicted using Derjaguin-Landau-VerweyOverbeek (DLVO) theory (7-11). The presence of EPS, lipopolysaccharide (LPS), and membrane proteins (10, 12) was reported to cause large discrepancies between deposition experiments and theory. Better approximation using the extended DLVO calculations (13, 14) also takes into account thermodynamics that include other interaction forces, such as Lewis acid-base interactions, hydrophobic attraction, and hydrophilic repulsion (15). Nevertheless, specific interactions between a small proportion of sites of the microbial and the solid surfaces can control bacterial adhesion. Cell surface polymers may interact directly with the surface through intermolecular interactions attributed to various types of appendages such as fimbriae and flagella. Bos et al. (15) demonstrated how even negatively charged appendages can pierce the electrostatic energy barrier of the classical DLVO prediction. Hence, prediction of bacterial adhesion is impossible even when the extended DLVO approach is being carried out: for example, when EPS is overexpressed, not only may cell hydrophilicity and charge change, but cell appendages may be covered and their exposure to the solid surface will be reduced (16). The influence of EPS on deposition and attachment of bacteria to solid surfaces is not decisive: while most often EPS is reported to enhance bacterial adhesion to solid surfaces (17-19), Gomez-Suarez et al. showed that higher EPS production in P. aeruginosa strain as well as precoating of the surface with EPS discouraged bacterial adhesion to both 10.1021/es901095u CCC: $40.75

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Published on Web 09/02/2009

hydrophilic and hydrophobic surfaces (20). Hence, according to the extended-DLVO theory, higher cell hydrophilicity and negative electrophoretic mobility, attributed to higher EPS production (21), are likely to reduce bacterial adhesion to negatively charged surfaces (22). Once cell hydrophilicity increases, electrophoretic mobility becomes the more influential condition in the adhesion process (23). A further step toward biofouling control of engineered aquatic systems can be carried out by linking bacterial adhesion to biofouling propensity. Hence, bacterial adhesion and attachment rate is strongly related to biofilm formation rate (2). It is clear that biofouling potential varies widely among the different bacterial strains because of variations in their attachment efficiency. In addition, growth rate, EPS production, physicochemical properties of the EPS, and biofilm structure and porosity also affect biofouling propensity of solid surfaces and of membranes, in particular. For reverse osmosis (RO) membranes the most common adverse effects of biofouling include permeate flux decline and decrease in salt rejection (24, 25). The different contributions of EPS to biofouling of RO membranes and the effects on membrane performance are not well understood. On one hand, as the adhesive and cohesive matrix of biofilms, EPS contribute significantly to flux decline by increasing the hydraulic resistance to water permeation (26). On the other hand, the effect of higher expression of EPS in the primary colonizers on bacterial adhesion is unknown. For better understanding of fouling effects of EPS (or effects of model component in the EPS mixture), the effects of increased EPS expression on cell adherence need to be defined. Therefore, the following inter-related characteristics: bacterial adhesion mechanisms, EPS components, and decrease in membrane performance, need to be considered for biofouling control. In this study, the effect of alginate, a model EPS component of P. aeruginosa, on bacterial adhesion and biofouling rate was analyzed, assuming that cell surface characteristics will govern initial cell deposition and biofouling rate in real RO laboratory system. The effects of a constitutive high alginate expression in P. aeruginosa on the primary physicochemical characteristics were defined under simulative wastewater conditions and correlated to initial adhesion rate to quartz surface in a radial stagnation point flow (RSPF) system. Then, further enhanced biofouling experiments conducted under similar conditions supported our supposition, in which the higher alginate expression in the inoculated mutant model strain, reduced RO fouling rate, mainly due to an increased cell hydrophilicity. This is the first study that correlates bacterial physicochemical characteristics (attributed to changes in EPS components), adherence properties, and RO membrane fouling rate.

Materials and Methods Model Bacterial Strains and Media. Pseudomonas aeruginosa PAO1 and its isogenic mucoid mutant PAOmucA22, PDO300 (27), were used as the model bacteria for deposition and enhanced biofouling experiments. A fresh single colony of each of those strains was grown in LB medium and harvested at either exponential (O.D.600 nm ) 0.4) or stationary growth phase (24 h of inoculation, O.D.600 nm ) 2.5) for further physicochemical characterization. For the deposition experiments, the stationary phase cultures were used after washing with either 15 mM KCl or synthetic wastewater solutions (25). For the biofouling experiments both exponential and stationary phase cultures were used as inoculum after a washing step with synthetic wastewater solution. The synthetic wastewater solution was relatively enriched with nutrients and used as the bacterial growth medium to accelerate the biofilm growth on the RO membranes. This medium was supplemented with a relatively high concentration of carbon (as citrate) and with a small amount of LB

broth (1:1000 dilution). The chemical composition of the synthetic wastewater was based on secondary effluent quality from selected wastewater treatment plants in California as described in our previous publication (25). The final pH of the synthetic wastewater was 7.4 and the calculated ionic strength was 14.6 mM. For microscopic analysis of the biofouling layer, the wild type and its isogenic mucA22 mutant were chromosomally tagged with green fluorescent protein (gfp) gene constitutively expressed. The strains used were P. aeruginosa KMD226 (PAO1-mini-Tn5-PA1/04/03 sgfpmut3*s T0sT1, TelR) and KMD230 (PDO300-mini-Tn5-PA1/04/03s gfpmut3*sT0sT1, TelR). Construction of the GFP reporter strains is reported elsewhere (28). Laser Scanning Confocal Microscopy and Image Analysis. At the end of the biofouling experiment with the strains tagged with GFP, the membrane coupon was carefully removed and cut into pieces of 5 mm × 5 mm for staining with concanavalin A (ConA) conjugated to tetramethylrhodamine isothiocyanate (TRITC) (Invitrogen Co.) for probing alginate expression in the biofilms. Microscopic observation and image acquisition were performed using an LSCM (ZeisssAxiovert 10) equipped with Zeiss dry objective Plan-NeoFluar (×10 magnification and a numerical aperture of 0.3). The LSCM was equipped with detectors and filter sets for monitoring TRITC-stained cells and GFP (excitation wavelengths of 568 and 488 nm, respectively). LSCM images were generated using the Bio-Rad confocal assistant software (version 4.02). Gray scale images were analyzed and specific biovolume (µm3/µm2) in the biofouling layer was determined by COMSTAT, an image-processing software (29, 30), written as a script in Matlab 5.1 (The MathWorks, Natick, MA) and equipped with an image-processing toolbox. Thresholding was fixed for all image stacks. For each sample, between six and nine positions on the membrane were chosen and microscopically observed and analyzed. Planktonic Cell Surface Characterization: Relative Hydrophobicity, Zeta Potential, Alginate Expression, and EPS Acidity. Cultures of P. aeruginosa PAO1 and PAOmucA22 were incubated in LB medium at 150 rpm and 30 °C and collected at either exponential or stationary growth phase for cell surface characterization. Surface characterization was repeated either three or four times in different experiments. Resuspended colonies from LB agar plates (24 h of incubation at 30 °C) were also analyzed for their relative hydrophobicity and electrophoretic mobility. The bacterial suspension was centrifuged and washed three times with the relevant medium in which the deposition and biofouling experiments were performed: KCl solution of 15 mM and synthetic wastewater solution (25). Cell surface characterization analysis was carried out also in KCl and synthetic wastewater solutions adjusted with NaCl to ionic strength of 150 mM. In both cases calcium was not added the medium. Measurements of bacterial electrophoretic mobility were performed with a zeta potential analyzer (ZetaPlus 1994, Brookhaven Instruments Co., Holtsville, NY) according to de Kerchove and Elimelech (31). All cultures were washed in the relevant solution tested and diluted to OD600 nm of 0.1 prior to analysis. In fact, 40 measurements were used to get an average of electrophoretic mobility, since for each analysis 10 measurements were taken. Electrophoretic mobility measurements were converted into zeta potentials by using the Smoluchowski equation. This equation was applicable because of the relatively large cells and the ionic strengths used (32). The relative levels of hydrophobicity of the PAO1 and PAOmucA22 strains were measured using the microbialadhesion-to-hydrocarbons (MATH) test with n-dodecane (Sigma-Aldrich, Israel) (33). This analysis was repeated four times. Hydrophobicity is defined here as the fraction of total cells partitioned into the n-dodecane phase. VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Alginate expression in the planktonic cells under different conditions was done in triplicate. ConA conjugated to Alexa Fluor 633 fluorescent dye (Invitrogen Co.) was used for probing alginate expression in the planktonic cultures under different growth conditions. Five dilution series of the respective solution were added to 96-well microtiter plates (Grainer) and equal volumes (300 µL) of cells were added. The plates were incubated in an automicroplate reader (Infinite M200, Tecan) with the monochromators set for excitation at 633 nm and emission at 664 nm. The detection limit of this analysis was defined here for optical density at 600 nm (O.D.600) of the cells that is below 0.08. In two dilutions out of the five dilution series used, the detection was above the detection limit and the normalized fluorescence of the ConA-Alexa Fluor 633 was defined as the fluorescent units divided by the O.D.600. The cell cultures (adjusted to O.D.600 of around 0.4) were incubated in a 100 µg/mL of ConA-Alexa Fluor 633 solution for 20 min in dark and washed three times in 0.85% NaCl solution (3000 g, 20 min, 4 °C) prior to the analysis. The acidity of the EPS extracted from stationary cultures of each strain and of pure sodium alginate (Sigma-Aldrich, St. Louis, MO) was determined by titration using hexadimethrine bromide (Sigma-Aldrich, Israel) and poly(vinyl sulfate) potassium salt (Sigma-Aldrich, Israel) as cationic and anionic standards, respectively (34). Bacterial Deposition Procedure. The effect of alginate expression on bacterial deposition was studied in both KCl (at ambient pH of 6.2 and IS ) 15 mM) and synthetic wastewater media (pH 7.4 and IS ) 14.7 mM) in the absence of calcium cations (35) using reagent-grade salt (Fisher Scientific). After washing steps of stationary phase cultures with either the KCl or with the synthetic wastewater solution, bacterial suspension was diluted in the selected medium. The deposition experiments were carried out in an RSPF system according to previous studies (10, 36, 37). Ultrapure quartz coverslips measuring 25 mm in diameter and 0.1 mm thick (Electron Microscopy Sciences, PA) were used as the solid substrate in the deposition experiments. Coverslips were cleaned according to de Kerchove and Elimelech (37). Bacterial deposition onto the ultrapure quartz substrate was investigated using a RSPF system with contrast phase microscopy (Axiovert 200m, Zeiss). Deposition was recorded in a rectangular viewing area of 220 µm × 165 µm (captured in a 140 µm-radius circle) with a DP70 digital camera (Olympus). Details on the hydrodynamic properties of the flow chamber can be found elsewhere (38). Bacterial transfer rate to the surface at average parallel flow velocity of 2.65 cm/s was measured at 15 s intervals for 30 min. For each culture, the cell concentration was determined in a Buerker-Tuerk cytometer chamber (Marienfield Laboratory Glassware, Germany). For each bacterial deposition experiment, the bacterial transfer rate coefficient, kRSPF, was calculated as the ratio between the bacterial deposition flux (the observed deposited bacteria per time normalized by the camera viewing area) and the initial bacterial bulk concentration (18). All bacterial transfer rate coefficients are averages taken from three experiments conducted using different cell cultures. Counting procedure of the deposited cells was adopted from de Kerchove and Elimelech (37). RO Membrane and Crossflow Test Unit. The model membrane used for the fouling experiments is a commercial thin-film composite RO membrane, LFC-1 (Hydranautics, Oceanside, CA). The hydraulic resistance of the membrane was 1.06 ((0.018) × 1014 m-1 at 25 °C and the observed salt rejection was 97.89 ( 0.44%, determined using the synthetic wastewater solution at an applied pressure of 180 psi (12.4 bar) and a crossflow velocity of 8.5 cm/s. The physical and 7378

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chemical properties of the LFC-1 membrane and detailed description of the RO test unit are described elsewhere (25, 39). EPS Extraction from Planktonic Cultures for Acidity and Component Analysis. EPS were extracted from planktonic cultures of P. aeruginosa PAO1 and PAOmucA22 at different growth stages, i.e., exponential (O.D.600 nm ) 0.4) or stationary growth phase (24 h of inoculation, O.D.600 nm ) 2.5). Triplicate cultures in 20 mL LB broth were shaken at 150 rpm and 30 °C and harvested by centrifugation at 2500g and 4 °C and washed with 0.85% NaCl (2500g, 20 min 4 °C). Then, bacterial cells were resuspended in 10 mL of 0.85% NaCl and adjusted to O.D.600 nm of 0.4 and EPS was extracted from that suspension using a modified version of the Liu and Fang method (26, 40). After the extraction, the EPS solution was filtered through a 0.2-µm hydrophilic nylon filter (Millipore) and dialyzed through a membrane with a molecular weight cutoff of 3500 Da (Spectra/Por). Finally, a 10-mL sample of EPS solution was lyophilized at -50 °C under vacuum (0.01 mbar) to facilitate determination of dry weight concentration. EPS Component Analysis. Polysaccharide content in the triplicate EPS samples was analyzed colorimetrically according to Dubois et al. (41) with sodium alginate as the standard (10-100 mg/L). Total protein content in the EPS was analyzed by using a modified Lowry protein assay kit (Pierce, Rockford, IL) according to Frolund et al. (4). Biofouling Protocols. Biofouling experiments were done using synthetic wastewater solution described previously (25). The RO unit was disinfected and cleaned prior to the biofouling experiments with either the wild type PAO1 or the mucoid PAOmucA22. Both exponential and stationary cultures of both strains were used as inoculum for the biofouling experiments. Biofilm growth on the RO membrane was initiated according to our previously published biofouling protocol (25, 35) and experiments were done in duplicate.

Results and Discussion Characteristics of P. aeruginosa PAO1 and its Isogenic Mucoid PAOmucA22 Strain. To define the effect of alginate expression on P. aeruginosa deposition and fouling potential as well as on its cell surface characteristics, the effect of growth phase on alginate expression in both the wild type PAO1 and the isogenic mucoid PAOmucA22 was analyzed. The presence of alginate was fluorescently analyzed by bonding the conjugated lectin, ConcanavalinA-Alexa 633 fluorescent dye, to the alginate R-D-mannuronate and R-D-guluronate residues as described in Materials and Methods. An evident increase in alginate expression is observed in planktonic culture under stationary growth conditions, while no significant change in alginate expression was detected under the exponential phase of growth (Figure 1a). As expected, the amount of alginate produced was significantly higher in the mucoid PAOmucA22 as compared to the wild type PAO1. Then, the effect of alginate expression in the wild type PAO1 and the mucoid mutant on the physicochemical properties of the cell surface were delineated. A more robust effect of alginate expression on cell surface hydrophilicity and zeta potential was observed for suspended colonies with “wild type” and mucoid morphologies: similar changes in hydrophilicity and zeta potential were analyzed for the mucoid colonies compared to the stationary growth mucoid culture. (a) Changes in EPS Composition Due to Higher Alginate Expression. Total protein and polysaccharide analysis of the EPS extracted from the wild type PAO1 and the mucoid strain showed that under exponential growth conditions total protein and polysaccharide concentration is similar, whereas in stationary growth phase more EPS was produced in both the wild type and its mucoid mutant (Figure 1b). A small increase in the produced extracellular polysaccharides was observed in the wild type strain during stationary growth

FIGURE 1. EPS characterization in exponential (OD600 nm of 0.4) and stationary (24 h incubation, OD600 nm of 2.5) cultures of P. aeruginosa PAO1 and mucoid PAOmucA22: (A) Total alginate produced and analyzed by bonding the conjugated lectin, ConcanavalinA-Alexa 633 to alginate residues. (B) EPS components: Polysaccahrides, proteins, and dry weight concentrations in the extracted solution. Inset figure of (A): Acidity of alginate and EPS extracted from stationary phase cultures. phase compared to the wild type exponential culture. A more noticeable change is observed in the mucoid culture that under stationary growth conditions produced a 3-fold higher amount of extracellular polysaccharides compared to the mucoid exponential culture (Figure 1b). This change in the extracellular polysaccharide content is consistent with the higher constitutive expression of alginate (Figure 1a) in the mucoid strain. (b) Contribution of Higher Alginate Expression to EPS Acidity. The acidity of the EPS extracted from stationary cultures is higher (Figure 1, inset graph), most likely due to a higher fraction of alginate that contains higher density of carboxylate groups than phosphoryl and carboxylate groups attributed to LPS located in the leaflet of the outer membrane of both strains. As expected, alginate acidity was significantly higher than the EPS, even higher than EPS originated from the mucoid strain. A possible explanation for this phenomenon is the presence of proteins in the EPS that contribute a large amount of amine positively charged groups to the EPS mixture. Hence, a significant amount of proteins was analyzed in the EPS extracted from both the wild type PAO1 and its mucoid mutant (Figure 1b). As discussed in the following sections, the higher acidity of EPS extracted from the mucoid PAOmucA22 corroborates with more negative zeta-potential values calculated for this strain as compared to the PAO1 wild type (Figure 2). In addition, due to higher alginate expression, more polar functional groups are exposed on the mucoid capsulated cell surface and increase cell hydrophilicity. (c) Contribution of Higher Alginate Expression to Cell Zeta Potential. The surface potential of the bacteria was studied by analyzing the electrophoretic mobility of the cells that originated from different growth conditions: planktonic cells at exponential and stationary growth phase and resuspended colonies (in both synthetic wastewater and KCl solutions adjusted to either ionic strength of 15 or 150 mM) that were cultivated for 24 h at 30 °C on LB agar plates. As expected,

both modes of growth and ionic strength affect zeta-potential values (Figure 2). Overall, the wild type PAO1 zeta potential did not vary between stationary phase, exponential phase, and plates. However, a significant increase in PAOmucA22 zeta potential was observed in stationary phase cells and those cells that were resuspended from colonies. Under higher ionic strength in KCl solution, a decrease in zetapotential is observed in both strains (Figure 2). This is likely due to compression of the particle diffuse layer and charge neutralization (32). Also, at ionic strength of 15 mM, zetapotential was reduced in the synthetic wastewater media compared to KCl, most likely due to the presence of 0.6 mM magnesium cations in the synthetic wastewater media that may contribute to specific interactions with the cell surface including complexation with negatively charged groups and increased neutralization. Thus, it is likely that under higher ionic strength this observation is obscured due to effective charge shielding with a higher amount of potassium and sodium cations. Interestingly, comparing the growth conditions at both ionic strengths of 15 and 150 mM, the most negative zetapotential values were observed for the mucoid PAOmucA22 that were isolated from sessile bacteria originated from the colonies incubated for 24 h and resuspended in KCl. The growth of sessile cells in colonies induces secretion of polysaccharides, in which production of mucoid colony morphologies was observed (results not shown) and is due to the overproduction of alginate (42). The induced mucoidity under sessile mode of growth elevates the negative cell surface electrophoretic mobility in the surrounding of monovalent cations. In the absence of divalent cations, more carboxylated functional groups (from alginate) are available and contribute a significant negative charge to the mucoid cell surface. These results corroborate with the higher acidity of EPS from the mucoid strain under stationary growth conditions, when alginate expression is induced (Figure 1, inset graph). (d) Contribution of Higher Alginate Expression to Cell Hydrophilicity. The induced polarity under higher expression of alginate is likely contributing to cell hydrophilicity. In all conditions where alginate expression was induced, a significant increase in cell hydrophilicity was observed. The most significant reduced partitioning of the cells in ndodecane was observed for stationary growth cultures and suspended colonies of the PAOmucA22 strain (Figure 2). Hence, overexpression of alginate and a higher density of carboxylated functional groups increases cell hydrophilicity. It should be mentioned that almost no change was observed in cell hydrophilicity under exponential growth conditions, when alginate expression was not significantly different in the mucoid mutant versus the wild type strain (Figure 1a). Deposition Characteristics of P. aeruginosa PAO1 and PAOmucA22. To analyze the effects of the higher expression of alginate under stationary growth conditions on bacterial deposition rates, mass transfer rates of PAO1 and PAOmucA22 to quartz surface were analyzed and calculated in an RSPF system. The mucoid PAOmucA22 showed significant reduction in its mass transfer rate to the surface as compared to the parent strain PAO1 (Table 1). In both 15 mM KCl and synthetic wastewater media (IS of 14.7 mM), the mass transfer coefficient in the RSPF cell (10) was between 4- and 5-fold lower for PAOmucA22. It is presumed that the induced hydrophilicity of the mucoid strain reduces cell deposition to the negatively charged quartz surface. The effect of the minor increase in surface potential of the mucoid strain in 15 mM KCl solution (Figure 2) compared to the wild type strain, on cell deposition is insignificant, since a decrease in mass transfer rate to the surface is also observed with synthetic wastewater solution (Table 1) where surface cell zeta potential was similar to that of the wild type strain (Figure 2). VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Hydrophilicity and Zeta potentials obtained for PAO1 and PAOmucA22. The measurements conducted on exponential and stationary cultures as well as on resuspended colonies grown for 24 h on LB plates. The cells were washed with either KCl or with synthetic wastewater (SW) media adjusted to ionic strength of 15 and 150 mM with sodium chloride.

TABLE 1. Deposition of P. aeruginosa PAO1 and the Mucoid Strain, P. aeruginosa PAOmucA22, on Quartz Surface in an RSPF System synthetic wastewater ionic strength ) 14.7 mM

15 mM KCl PAO1

PAOmucA22

PAO1

PAOmucA22

-1

deposition rate, [cells · sec ] 0.33 ( 0.08 0.20 ( 0.05 0.73 ( 0.11 0.33 ( 0.05 cell concentration, 4.0 × 1007 ( 3.1 × 1006 1.0 × 1008 ( 9.1 × 1006 8.6 × 1007 ( 7.1 × 1006 1.0 × 1008 ( 8.4 × 1006 -1 [cells · mL ] RSPF transfer rate 2.3 × 10-07 ( 5.8 × 10-06 5.4 × 10-08 ( 1.4 × 10-08 2.36 × 10-07 ( 4.0 × 10-08 8.9 × 10-08 ( 1.5 × 10-08 coefficient [m/s]

Negative zeta-potential values were analyzed for quartz particles as surrogates for the quartz slides under aquatic conditions similar to those applied during the bacterial deposition experiments. The values were -24.1 ( 2.9 and -22 ( 3.1 mV in 15 mM KCl and synthetic wastewater media, respectively. Extreme hydrophilic properties were observed for the quartz slide surface under all aquatic conditions with an air bubble contact angle of almost 0° (results not shown). It has been shown that bacteria with hydrophilic properties show reduced adhesion preferring hydrophilic substrata whereas hydrophobic bacteria adhere to both hydrophobic and hydrophilic substrata (43, 44). However, the role of EPS, especially alginate on P. aeruginosa deposition is controversial. In our study, we delineate a clear difference between the interactions of mucoid and nonmucoid bacteria with quartz surface, in which alginate production reduces cell affinity to the substrata. In contrast, de Kerchove and Elimelech (37) reported an increased efficiency in bacterial transfer rate to quartz surface when sodium alginate was used as conditioning film. The difference between this study and the one conducted by de Kerchove and Elimelech is the type of interactions being studied: while de Kerchove and Elimelech compared the interactions between alginate-cell surface and quartz-cell surface, in this study the interactions investigated are between quartz and cell surface that is covered with different quantities of alginate. Indeed, due to strong repulsion between quartz surface and alginate, preconditioning of the quartz surface with alginate requires a positively charged intermediate layer of poly-L-lysine (37). This repulsion, between alginate and 7380

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quartz surface, also plays a role in decelerating the deposition of PAOmucA22. The role of alginate expression on bacterial transfer rate and deposition to glass surface in a packed column using PAOmucA22 was also explored by Liu et al. (18). In contrast to the results presented here, the latter study showed higher retention of PAOmucA22 as compared to the nonmcoid parent PAO1. The reason for the discrepancy presented in the Liu et al. study is not clear. Possible reasons may include the use of glass beads by Liu et al., having local heterogeneities of their surface charge. Also, the constitutive expression of EPS may cause an increased straining due to filling gaps between the glass beads with EPS and creating additional physical barrier for the cells. Unfortunately, lack of information on the hydrophilicity of the strains and the properties of the glass beads used by Liu et al. make the comparison impossible (18). In another study of Liu et al. (45), decreased deposition of Erwinia chrysanthemi to biofilm of PAOmucA22 was observed as compared to deposition to the nonmcoid parent biofilm. Liu et al. explained this result by a higher hydrophilicity of the PAOmucA22 biofilm (45). Hence, the results presented here corroborate with these late findings of Liu et al. (45). It should be mentioned that to minimize specific interactions between alginate and either quartz or cross-linked polyamide membrane surfaces, calcium was not added to either the deposition or fouling experiments described in the following section. Specific interactions are much more strongly induced with calcium than with magnesium cations and include cationic bridges between alginate carboxylic

FIGURE 3. RO permeate flux decline upon biofouling with exponential (A) and stationary (B) phase inoculums. Fouling experiments were conducted in a synthetic wastewater medium (ionic strength of 14.6 mM and pH 7.4) with initial cell concentration of 1.5 ((0.08) × 107 cells/mL. Crossflow velocity in all experiments was 8.5 cm/s, initial permeate flux was 1.2 ((0.03) × 10-5 m/s (42 L/m2 h or 25 gal/ft2 day), and the temperature was fixed at 25 °C. groups and the silanol groups of the quartz surface as well as induced gelation of alginate guluronic acid blocks (46). These effects are likely to increase cell deposition and may obscure the effect of alginate overexpression that in the absence of calcium decreases cell deposition. Effect of Higher Alginate Expression on Biofouling Rate of RO Membranes. Reduction in the permeate flux was delayed when stationary culture of PAOmucA22 was used as an agent for biofouling experiments of high flux RO membranes (Figure 3a). The difference is not due to growth rate variation as both strains exhibit similar growth properties in a synthetic wastewater media, with 0.29 ( 0.03 and 0.30 ( 0.02 hs1 for the wild type PAO1 and the mucoid PAOmucA22, respectively (corresponding to doubling time 2.4 ( 0.29 and 2.33 ( 0.14 h, respectively). The similar growth rate under these aquatic conditions refutes possible changes in fouling behavior that are due to changes in biofilm growth rate. In contrast, when exponential cultures of the wild type and the mucoid mutant were used, similar reduction of permeate flux was observed (Figure 3b). The similar permeate flux decline when exponential cultures of the wild type PAO1 and the mucoid PAOmucA22 were inoculated (Figure 3a) in the RO unit is in agreement with their similar hydrophilicity and surface potential. As shown previously in this study, in exponential growth phase, no significant changes in alginate expression were observed (Figure 1A) and also, EPS component analysis showed similar proteins and polysaccharides concentration (Figure 1B). Under the conditions applied in this study, LFC-1 RO membrane is moderately hydrophobic (air bubble contact angle of 51.8°) with a low negative charge that corresponds to a surface potential of -13.5 mV (39, 47). Hence, the conditions of this study strongly suggest that the increase in alginate production which had direct effect on lessening cell deposition, eventually decelerated flux decline via increase in cell hydrophilicity. Confocal microscopy was further used for analysis of the biofouling layers formed by wild type PAO1 and mucoid PAOmucA22 strains. Increased alginate production in the biofilm of PAOmucA22 is observed in Figure 4. Visualizing TRITC fluorescence by binding ConA to alginate R-Dmannuronate and R-D-guluronate residues (48), shows more than 5-fold increase in alginate production by the mucoid mutant biofouling layer. Interestingly, a reduction in salt rejection, a consequence of another RO biofouling phenomena called “biofilm enhanced osmotic pressure” (BEOP) (24, 25), was observed at higher extent with biofouling layer from the wild type strain in comparison to the biofouling layer that consists of higher fraction of alginate produced using the mucoid strain as the biofouling agent (Figure 4). Salt rejection was decreased from 98.5 ( 0.3 to 94.6 ( 0.5% and from 98.7 ( 0.4 to 98.1 ( 0.2%

FIGURE 4. Three-dimensional reconstruction using Imaris software (Bitplane, Zurich, Switzerland) of LSCM images taken from biofouling runs that were carried out with stationary inoculums of (A) nonmucoid P. aeruginosa PAO1 and (B) mucoid PAOmucA22. The figures were reconstructed from planar images acquired at depth intervals of 1 µm (the field of view for each figure is a perspective of 750 µm × 750 µm). The inoculums used are chromosomally tagged with stable variant of GFP (mut3*), and EPS was stained with concanavalin A conjugated to TRITC (red color). Specific biovolume of cells (GFP) calculated for 10 images with COMSTAT (30, 56) were 19.3 ( 6.1 and 15.1 ( 3.9 µm3/µm2 for PAO1 and PAOmucA22, respetively. Specific biovolumes of EPS (red) calculated for 10 images with COMSTAT were 0.75 ( 0.4 and 4.3 ( 1.6 µm3/µm2 for PAO1 and PAOmucA22, respectively. for biofouling experiments with the wild type and the mucoid strain, respectively. We have recently shown that reduction in permeate flux of RO membranes by fouling with EPS is mainly due to an increase in hydraulic resistance and hydrodynamic restriction of water flux through the EPS fouled membrane (26). The small decrease in salt rejection when the biofouling layer consisted of a higher fraction of alginate was due to “concentration effect” of the salts in the reduced permeate flux. Nevertheless, the high decrease in salt rejection for the case of biofouling with the wild type strain is mainly due to BEOP as shown in previous studies (24-26). The smaller VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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change in salt rejection only for the biofouling experiment with the mucoid biofilm may be due to formation of additional “barrier” of EPS that reduced salt diffusivity through the fouled membrane. This effect was also shown during organic fouling in a similar RO laboratory unit by Ang and Elimelech (49). The higher amount of EPS in the mucoid fouling layer implies that the main mechanism in charge for permeate flux decline, in this case, is an induced hydraulic resistance by the EPS layer as reported in our recent study (26). Since hydraulic resistance is the prominent reason for permeate flux decline during the biofouling experiment with the mucoid strain, the delay in flux decline can be attributed to the time necessary for EPS accumulation on the membrane surface that restricts water permeability. The time for reduction in membrane performance when the membrane is fouled with the biofilm of the wild type strain is achieved by BEOP phenomenon after bacterial deposition and growth of porous biofilm layer. Also, the formation of additional “barrier” by the mucoid EPS is apparently observed by the stable salt rejection during the run. Therefore, reduced effects of BEOP during the experiment with the mucoid biofilm will decelerate the decline in permeate flux. In this case, hindered back-diffusion of salts from RO membrane surface will be less pronounced. Nevertheless, we can speculate that the quality differences in the EPS are responsible for the delay in permeate flux decline and not the reduced cell deposition rate. Still, both deposition experiments and physicochemical characteristics of the inoculated model bacteria support that reduced colonization of the mucoid strain on the membrane is the main reason for the reduced fouling behavior. Role of EPS Expression in Biofouling Phenomena. In general, surfaces that are most likely to resist protein adhesion are characterized as hydrophilic, H-bond acceptors, non H-bond donors, and neutrally charged (50). The induced hydrophilicity of bacterial surface, in addition to a possible steric repulsion caused by extracellular polysaccharides that prevent intimate interaction between bacterial cell membrane and the studied surfaces (quartz or cross-linked polyamide) are shown here to lessen bacterial deposition and biofouling of RO membranes. This steric repulsion shields the necessary interactions needed between the dynamic appendages on bacterial surface and the substratum that eventually enable the transition of the bacteria from planktonic to sessile state. In P. aeruginosa, flagellar and twitching motility are necessary for initial fruitful surface-bacteria interaction and further mature biofilm formation (51). Hence, the mucoid mutant, PAOmucA22, lacks flagella and also, as already suggested for Escherichia coli by Schembri et al. (16), polysaccharide capsule can shield the function of bacterial adhesins. In mucoid variants of P. aeruginosa, alginate capsule may also shield the interaction between bacterial adhesins, such as type IV pili and the surface (52). The role of EPS in biofouling processes of RO membranes has been recently delineated by Herzberg et al. (26, 35). EPS extracted from P. aeruginosa PAO1 biofilms were shown in controlled fouling experiments to induce RO permeate flux decline by increasing membrane hydraulic resistance. Therefore, it is clear that when biofilm is formed on RO membranes an enhanced production of EPS is an undesirable phenotype as that can impair membrane performance. However, bacterial species that produce higher quantities of EPS in their planktonic state are not necessarily better candidates for biofouling of membranes or any other surface. It is known that EPS are an adhesive and cohesive matrix of biofilms, and induced EPS production, in most cases, takes place after cell attachment, at later stages of the biofilm formation process (53-55). In this study, we revisit the notion that EPS 7382

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production enhances bacterial adhesion, and we show that the role of EPS is complex and the timing of EPS production is not coincidental. Biofilm formation is a well-established developmental process, and biofouling is an undesireable developmental phenomena, in which the timing of different steps plays an important role. Here, premature EPS expression in the primary colonizers decelerate bacterial deposition and RO biofouling process.

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