Biofouling of Reverse Osmosis Membranes ... - ACS Publications

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Biofouling of Reverse Osmosis Membranes: Positively Contributing Factors of Sphingomonas Jenia Gutman,† Moshe Herzberg,*,† and Sharon L. Walker‡ †

Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, Albert Katz International School for Desert Studies, Jacob Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Sede Boqer Campus 84990, Israel ‡ Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, United States S Supporting Information *

ABSTRACT: In the present study, we investigate the possible contribution of Sphingomonas spp. glycosphingolipids (GSL) and its extracellular polymeric substances (EPS) to the initial colonization and development of biofilm bodies on reverse osmosis (RO) membranes. A combination of an RO cross-flow membrane lab unit, a quartz crystal microbalance with dissipation (QCM-D), and a rear stagnation point flow (RSPF) system with either model bacteria (Sphingomonas wittichii, Escherichia coli, and Pseudomonas aeruginosa) or vesicles made of the bacterial GSL or LPS was used. Results showed noticeable differences in the adhesion LPS versus GSL vesicles in the QCM-D, with the latter exhibiting 50% higher adhesion to polyamide coated crystals (mimicking an RO membrane surface). A similar trend was observed for EPS extracted from S. wittichii, when compared to the adhesion tendency of EPS extracted from P. aeruginosa. By applying the whole-cell approach in the RO lab unit, the cumulative impact of S. wittichii cells composing GSL and probably their EPS reduced the permeate flux during bacterial accumulation on the membrane surface. Experiments were conducted with the same amount of Sphingomonas spp. or Escherichia coli cells resulting in a two times greater flux decline in the presence of S. wittichii. The distinct effects of Sphingomonas spp. on RO membrane biofouling are likely a combination of GSL presence (known for enhancing adhesion when compared to non-GSL containing bacteria) and the EPS contributing to the overall strength of the biofilm matrix.

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INTRODUCTION

membrane of Gram negative bacteria were investigated through the past decade as bacteria adhesion-related factors.16,18,19 In addition to cell outer membrane composition, bacterial attachment and subsequent biofilm growth are also attributed to the cell self-produced extracellular polymeric substances (EPS), providing the biofilm matrix with an entangled polymeric network. This network includes carbohydrates and proteins as major constituents and lipids, nucleic acids, and other various heteropolymers in smaller portions.21 EPS are mainly responsible for the structural and functional integrity establishing the unique traits of the biofilm.22 The extracellular polymers create bridges between adjacent cell surfaces stabilizing the biofilm and forming a scaffold of threedimensional architecture.23 These interactions are mediated by electrostatic and van der Waals interactions as well as hydrogen bonds.24 During the past five years, several studies have reported the substantial presence of Sphingomonas species (spp.) in biofilms in diverse water treatment and supply systems.25−30 Sphingo-

Bacterial adhesion and subsequent biofouling is a widespread phenomenon that affects the performance and economic feasibility of a variety of engineered systems including water distribution systems1,2 wastewater treatment,3,4 and desalination plants utilizing pressure-driven membrane technologies.5,6 Considerable effort has been invested into fundamental studies of bacteria adhesion to surfaces in natural and engineered systems. Physico-chemical factors found to effect bacterial adhesion include cell and surface hydrophobicity and charge,7−9 surface roughness,10 and solution chemistry parameters such as pH, ionic strength, and divalent cations.11,12 Additionally, biological factors such as motility,13 presence of flagella,14 pili,15 and bacterial polymers16,17 were thoroughly studied. The bacterial outer membrane also plays a major role in cell adhesion followed by biofilm formation.18,19 The outer membrane of Gram negative bacteria is composed of an inner and outer leaflet. These leaflets are asymmetric in their content. While the inner leaflet is composed of simple phospholipids, the outer leaflet is composed of lipopolysaccharides (LPS), consisting of an O-specific chain, a core oligosaccharide, and a lipid component, termed lipid A.20 LPS molecules along with their variations and presence in the outer © 2014 American Chemical Society

Received: Revised: Accepted: Published: 13941

August 12, 2014 October 25, 2014 October 29, 2014 October 29, 2014 dx.doi.org/10.1021/es503680s | Environ. Sci. Technol. 2014, 48, 13941−13950

Environmental Science & Technology

Article

Bertani (LB) medium supplemented with 150 μg/mL Carbenicillin for selection of the GFP expression plasmid, when needed. Bacteria were grown in a shaker−incubator at 150 rpm at 30 °C (ZHWY-1102C, ZHWY). The cells were diluted in LB or meat extract broth (1:100) and incubated for 8 h (E. coli), 16 h (P. aeruginosa), or 24 h (S. wittichii) to achieve early stationary growth phases. Bacteria were harvested by centrifugation (4K15, Sigma) (4000g and 4 °C for 10 min), and the pellet was washed three times using experimental solution. After harvesting the bacteria, the optical density (OD600) of the suspension was adjusted to 0.1 or 0.01, corresponding to 5 × 107 ± 5 × 105 and 5 × 106 ± 5 × 104 cells/mL for RSPF and RO biofouling experiments, respectively. Lipids. As in other studies, due to experimental restrictions, most of the LPS investigations are limited to deep rough mutants with notably shortened sugar moieties on the LPS (termed as Re LPS)37 (Sigma-Aldrich Israel Cat.# L9764). The chemical structures of GSL-1 and LPS Re were depicted by Wiese et al.36 GSL and LPS vesicles were prepared as described by Gutman et al.39 Quartz Crystal Microbalance with Dissipation (QCMD). QCM-D experiments were performed using AT-cut quartz crystals coated with an ∼50 nm polyamide layer, having a fundamental resonance frequency of ∼5 MHz mounted in an E4 system (Q-sense AB, Gothenburg, Sweden). For the polyamide used for coating the sensors in this study, at pH 7, a zeta potential of 10−15 mV is reported47 and a contact angle of 55.7 ± 0.9 was measured in our lab.48 Before each measurement, the crystals were soaked in 2% (wt) sodium dodecyl sulfate (SDS) solution for 30 min, thoroughly rinsed with double distilled water, and dried with N2 gas. All QCM-D experiments were performed at flow-through conditions using a digital peristaltic pump (IsmaTec, IDEX) operating in a sucking mode. The flow rate of the working solution in the QCM-D flow cell was 100 μL/min. For adhesion of lipid vesicles, the following solutions were injected sequentially into the QCM-D system: (i) double distilled water baseline, 20 min; (ii) background solution of 100 mM NaCl, 10 min; (iii) GSL or LPS vesicles, 0.2 mg/mL, in background solution, flow rate 10 μL/min, 150 min; (iv) background solution, flow rate 10 μL/ min, 180 min. The adsorption kinetic curves were generated by Q-Tools software (Q-SENSE, Sweden). The variations of the acquired frequency shifts (Δf, Hz) and dissipation factors (ΔD) were measured for five overtones n = 3, 5, 7, 9, and 11. For adhesion of EPS experiments, the background solution used was 8.5 mM NaCl supplemented with 0.5 mM CaCl2 and stage (iii) contained EPS, at a concentration of 10 mg/L as TOC dissolved in the background solution at a flow rate of 100 μL/ min for 40 min. Stage (iv) was performed at the same flow rate of 100 μL/min. EPS Extraction and Analysis. EPS extraction was performed in an Erlenmeyer flask containing 1 mL of bacteria in 100 mL of medium, to which 10 g of 0.5 mm diameter acidwashed glass beads (Sigma-Aldrich Israel) were added. The Erlenmeyer flask was shaken at 30 °C for 48 h. The EPS extraction step was carried out according to Liu and Fang50 and our previous work.49 Total organic carbon (TOC), proteins, and polysaccharides concentrations were determined in the sample using standard methods. Radial Stagnation Point Flow System (RSPF). The methods for the RSPF system are explained in detail in previous work.51 For favorable deposition experiments (i.e., in the absence of repulsive electrostatic interactions), the slides

monas spp. were found to compose 27% of the total biofilm community in a water reclamation plant using RO (reverse osmosis)31 and 35% in RO lab scale unit desalinating tertiary effluents originated from a membrane bioreactor (MBR).32 A low pressure RO membrane can successfully remove a wide range of contaminants including inorganic salts and trace organic chemicals; thus it is an important, yet prone to biofilm development, step in water-treatment processes toward potable-water quality.33 Bacterial species which succeed to proliferate and thrive in such environments are assumed to be adapted to different environmental conditions such as the substratum (cross-linked aromatic polyamide in the case of RO desalination), scarce amount of nutrients, and high shear stress and pressure. Unlike other Gram-negative bacteria, Sphingomonas spp. lack the LPS but contain a different type of phosphoglycolipid, referred to as glycosphingolipids (GSL). GSL, like LPS, is composed of a lipid anchor covalently linked to a sugar moiety. The main differences between the two molecules is the number of acyl chains in the membrane-anchoring region, two for GSL and six for LPS, and the length of the sugar moiety, when the longest chain known so far for the GSL contains four sugar molecules, while for LPS it can contain a tenth of such molecules. Detailed images of LPS19 and GSL can be found elsewhere.34 Since the full chemical structure of prokaryotic GSL was determined in 1991,35 most studies were focused on the physicochemical and functional properties of GSL in the general context of pathogenicity: the role of GSL as an endotoxin, in the frame of bacterial−mammalian cells interaction.36−38 Notably, only few recent studies focus on the role of GSL in the context of environmental microbiology and engineering.39,40 Since several recent studies have attributed biofouling to the presence of Sphingomonas spp.,26,31,41 there is a considerable need for further work to gain an understanding of the fundamental interactions between Sphingomonas spp. and engineered surfaces in water. The current study was developed to elucidate mechanisms of Sphingomonas fouling in four phases: (i) quantifying attachment of lipid vesicles composed of GSL in comparison to vesicles composed of LPS in a QCMD;42 (ii) quantifying attachment of EPS extracted from S. wittichii versus EPS extracted from P. aeruginosa PAO1 in QCM-D; (iii) evaluating the deposition rate of P. aeruginosa PAO1 cells onto coatings of GSL, LPS, and amino-silanized (favorable) glass surfaces in the RSPF system;43 and (iv) assessing biofouling of an RO membrane with Sphingomonas spp. versus E. coli as the initial biofilm colonizer under high pressure and shear conditions.44

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EXPERIMENTAL PROCEDURES Model Bacterial Strains and Media. Sphingomonas wittichii RW1 (GenBank #NC 00951) was obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH) culture collection, Germany. The bacterium was grown on nutrient agar medium containing peptone (5 g/L) and meat extract (3 g/L) adjusted to pH 7 and supplemented with 200 μg/mL streptomycin (SigmaAldrich, S6501). The E. coli DH10B strain45 was hosting the plasmid pDsRed2Cm which expresses the red fluorescent protein (PlacZ:: DsRed2) as well as resistance to ampicillin and chloramphenicol (Clonetech Laboratories, Inc.). Pseudomonas aeruginosa PAO1 and Pseudomonas aeruginosa hosting a GFP expression vector pMRP9-1 plasmid46 were grown in Luria− 13942

dx.doi.org/10.1021/es503680s | Environ. Sci. Technol. 2014, 48, 13941−13950


Article

period of 12 days. The RO unit was assembled, disinfected, and prepared for the experiment as was previously specified by Herzberg and Elimelech (2007). Detailed chemical characterization of the MBR effluent appears elsewhere.58 For the second RO biofouling tests, the membrane was stabilized and equilibrated with a bacteria-free electrolyte solution for 4 h. After attaining a stable flux, the initial permeate flux and crossflow velocity were adjusted to 41.6 L/m2 h and 25.9 cm/s, respectively. These values were used for all the subsequent biofouling runs. The relatively high initial permeate flux ensured the attachment of the conditioning layer of bacteria, and its subsequent effect on later stages of biofilm formation was delineated. To assess the contribution of the type of the conditioning bacteria, biofouling runs were performed in two subsequent steps. During the first, “conditioning” step, either E. coli or S. wittichii cells at OD600, diluted to a value of 0.001, were recirculated in the system for 7 h. The system was drained, and the feedwater was replaced by E. coli K12 strain DH10B expressing red fluorescent protein (RFP), at an initial cell concentration of 5 × 106 ± 5 × 104 cells/mL for all experiments in background solution of 8.5 mM NaCl and 0.5 mM CaCl2 supplemented with 50 mg/L ampicillin and 20 mg/ L chloramphenicol to prevent further proliferation of the “conditioning” bacteria. E. coli K12 strain DH10B expressing a red fluorescent protein were recirculated in the system for 16 h. ESPA2 (Hydronautics Inc., CA, USA) membrane was used. Confocal Microscopy. At the end of the biofouling experiments, the membrane was carefully removed from the flow cell and cut into pieces of 1 cm × 3 cm for staining with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen Co.) for probing all bacterial cells in the biofilm. Microscopic observation and image acquisition were performed using Zeiss-Meta 510, a CLSM equipped with Zeiss dry objective LCI Plan-NeoFluar (40× magnification). The LSCM was equipped with detectors and filter sets for monitoring DAPIstained cells and RFP (excitation wavelengths of 350 and 495 nm, respectively). Three-dimensional images were obtained using IMARIS software (Bitplane, Zurich, Switzerland) using a fixed threshold. Atomic Force Microscopy (AFM). Surface topographies of the QCM-D coated sensors and the RO membrane were imaged using a Nanoscope IIID MultiMode AFM microscope (Veeco-DI, Santa Clara, CA) using an NP-S cantilever with a spring constant of 0.06 N/m in contact mode with a tip speed 40 μm/s. The AFM scanning area was 25 μm2. Ra and Rq, arithmetic and root-mean-square average roughness coefficients, were calculated based on 10 measurements on different locations of the surface. Representative AFM figures can be found in the Supporting Information, Figure S1.

were chemically modified with aminosilane. Details of the procedure as well as the cleaning steps are described elsewhere.16 To study bacterial adhesion to lipid-vesicle coating, vesicles of Re LPS and GSL-1 were prepared as described in our previous study.39 The adsorbed vesicle layer was created on a PEI-coated quartz surface following the method developed by Tong and McIntosh for vesicle fusion.52 A vesicle suspension (500 μL) was applied onto the PEI-coated quartz surface and allowed to adsorb for 30 min at room temperature. Bacterial deposition onto GSL 1, Re LPS, or silanized quartz substrate was recorded in a rectangular viewing area of 220 μm × 165 μm with a DP70 digital camera (Olympus). Details on the structure of the RSPF51 and on the hydrodynamic properties of the flow chamber can be found elsewhere.53 A constant flow rate of 1 mL/min was fixed by syringe pumps (KD Scientific Inc., PA) during the deposition experiments. All experiments were conducted under the same hydrodynamic conditions, at ambient pH and at 25 °C (±1 °C). Deposition was measured at 20 s intervals for 30 min, and images were subsequently processed in Matlab (Mathworks, Natick, MA). The bacterial deposition flux is the observed deposition rate of bacteria normalized by the camera viewing area. All deposition rate coefficients are averages taken from four experiments conducted using different collector surfaces. For each bacterial deposition experiment, the bacterial transfer rate coefficient, kRSPF, was calculated as the ratio between the bacterial deposition flux (J) and the initial bacterial bulk concentration (C0) .54 The attachment efficiency, αRSPF, is determined by normalizing the deposition rate at a given ionic strength by the deposition rate under “favorable” electrostatic attraction. Bacterial cell deposition experiments in the RSPF system were conducted using different chemistries upon the quartz slide, with a cell concentration of ∼5 × 108 cells/mL. The influent cell concentration for each deposition run was determined directly by visualizing cells in a counting chamber (Buerker-Tuerk chamber, Marienfield Laboratory Glassware, Lauda-Königshofen, Germany). RO Membrane and Crossflow Test Unit. The two types of biofouling experiments were performed with a laboratoryscale crossflow test unit, depicted elsewhere,55,56 using commercial RO membrane, ESPA2 (Hydranautics Inc., CA, USA). The membrane was first compacted with deionized water for 12 h until the permeate flux became constant, followed by the initial baseline performance for 2 h. Following the initial baseline performance, the two types of biofouling experiments were conducted. The first type of biofouling experiments were continuously performed either with S. wittichii or P. aeruginosa, inoculated at OD600 = 0.01. First, the bacteria were introduced to the lab scale RO system for 8 h in a recirculation mode of operation. This inoculation step was followed by a “feed-bleed” run in which the membrane bioreactor effluent57 system treating municipal sewage of Midreshet Ben Gurion (Israel) was used as RO feed solution, although supplemented with 1 mg/L of streptomycin and 150 mg/L carbenicillin for 11 days, for S. wittichii and P. aeruginosa hosting the vector pMRP9-1 plasmid,46 respectively. By following the recommended membrane operational conditions by the manufacturer, in these experiments the initial permeate flux was 31.2 L/m2 h. The crossflow velocity was set on 15 cm/s. Control experiments without bacterial inoculation were performed with a flux decline of less than 5% during an experimental

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RESULTS AND DISCUSSION Sphingomonas wittichii as a Biofouling Agent. To demonstrate the effect of S. wittichii biofilms on the permeate flux decline of RO membranes during desalination of tertiary wastewater, initial biofouling experiments were carried out in a lab-scale RO unit fed with tertiary effluents originating from membrane bioreactors48 under selective conditions for two model bacteria strains, the P. aeruginosa PAO1 and S. wittichii RW1. These experiments confirmed the significant biofouling potential of S. wittichii compared with one of the most common model strain used in biofouling studies, P. aeruginosa PAO1. P. aeruginosa PAO1 is widely prevalent in the environment,59,60 and its biofilms can grow under limited nutritional conditions 13943



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with high resistance to a variety of antimicrobial agents.61,62 EPS of P. aeruginosa PAO1 have profound adhesive characteristics providing the main cause of infection related to the Cystic Fibrosis genetic disorder.63 Surprisingly, a less investigated strain common to RO membrane biofouling layers showed stronger detrimental effects than P. aeruginosa biofilms during RO filtration of MBR effluent. As seen in Figure 1, the first 4 days of biofilm formation of both bacterial species resulted in the same flux decline in the Figure 2. Decline in frequency of QCM-D polyamide-coated sensor upon adhesion of (A) LPS (gray line) and GSL (black line) vesicles and (B) S. wiitichii (●) and P. aeruginosa (◇) EPS (10 mg/L as DOC). Time periods 0−120 and 120−480 min, in (A), are adsorption and desorption (washing) steps, respectively. Time periods 0−40 and 40−60 min, in (B), are adsorption and desorption (washing) steps, respectively. The bound mass is deduced from changes in the resonance frequency, Δf, of the piezoelectric sensor.

washing step (120−480 min) is desorption of the vesicles from the polyamide surface. When the frequency reaches a plateau, e.g. when the loosely bounded vesicles leave the surface, 80% of the GSL mass remains adsorbed, compared with ∼30% of the LPS mass. This finding suggests not only a higher quantitative adhesion of GSL but also stronger attachment between the GSL vesicles and the polyamide surface, possibly due to hydrophobic interactions.7,8 This finding is supported by a study of Williams and Fletcher,67 where increased adhesion and retention of Pseudomonas f luorescence mutants lacking the O antigen component in their LPS was observed in porous media. This LPS structure resembles the short carbohydrate moieties of GSL. Increased adhesiveness in the absence of the O-antigen was also shown, among other studies, for LPS mutants of P. aeruginosa.19,68,69 Apparently, a similar pattern of enhanced adhesion was observed for EPS. As appears in Figure 2B, after 40 min, twice as much mass adhered to the sensor in the case of S. wittichii EPS (Δf = −3.8 versus −8.3 Hz/10 ppm DOC for P. aeruginosa and S. wittichii, respectively) as compared to EPS extracted from P. aeroginosa. In both cases, most of the EPS adhered irreversibly to the surface with 89% and 83% of the mass remaining on the sensor, for P. aeruginosa and S. wittichii EPS, respectively, after the washing step. Earlier studies analyzed the hydrophobicity and chemical composition of Sphingomonas spp. as well as Pseudomonas spp. EPS. In line with our findings, EPS of Sphingomonas paucimobilis was found to decrease the polarity of a glass-collector surface, therefore turning it into a hydrophobic surface. This type of conditioning, altering surface hydrophobicity, resulted in enhanced deposition of the EPS producing strain, S. paucimobilis, onto the surface.70 It should be noted that the carbohydrate portion of EPS is largely dependent on the bacterial outer membrane components, in this study, either LPS or GSL.71,72 Degradation products originating from carbohydrates found in the O-antigen of LPS as well as glucuronic acid originating from GSL are likely to be found in the EPS matrix. Glucuronic acid is a gellan building block, and being a negatively charged molecule, it might act as a surface active compound and bridge between surfaces and foulants.61,70 Indeed, the presence of such acidic polysaccharides as glucuronic acid was reported for EPS of S. paucimobilis,70,73 yet, for P. aeruginosa, glucuronic acid presence was detected only for a certain mucoid derivative of PAO1.62,74

Figure 1. Normalized flux decline during biofouling of the RO membrane with P. aeruginosa PAO1 (◇) and S. wittichii RW1 (●). Experimental conditions were as follows: initial permeate flux of 31.2 L/m2 h, crossflow velocity of 15 cm/s, initial cell concentration of 5 × 106 ± 5 × 104 cells/mL, inoculation time 8 h, ambient pH of 7.2, and background solution of UF permeate of tertiary wastewater treated by membrane bioreactor (MBR) supplemented with antiscalant and antibiotics.

system. A significant difference was detected from day 6 onward, where the flux reduction for P. aeruginosa levels off at 85−80% of the initial flux, while, for S. wittichii, the permeate flux decline remains nearly constant at about 4% flux reduction per day of operation. These initial trends led to the development of the subsequent experiments to evaluate the cause of the behavior. Possible reasons for these outstanding biofouling properties of Sphingomonas wittichii might be due to two unique features of Sphingomonas family: (i)tThe unique composition of the outer membrane-GSL64 and (ii) extracellular polymeric substances (EPS) of outstanding strength and adhesiveness.65 Adhesion of GSL and EPS to a Polyamide Model Surface. A single-component approach was applied to test the adhesion patterns of GSL, LPS, and EPS onto a model polyamide surface within a QCM-D to mimic the RO membrane surface. Figure 2 shows the adhesion of GSL and LPS vesicles. The adhesion method to quartz crystals in the QCM-D flow cell provides real-time, label-free monitoring of the formation of close-packed layers of intact lipid vesicles as well as of EPS.57The bound mass is deduced from changes in the resonance frequency, Δf, of the piezoelectric sensor (Figure 2, Y-axis). The adhesion to a polyamide surface of Re LPS was compared to that of the corresponding GSL with the shortest sugar moiety, GSL-1 (images of Re LPS and GSL-1 can be found elsewhere37). Also, the adhesion of EPS originating from S. wittichii was compared to the EPS of P. aeruginosa, wellknown for its adherence characteristics to various surfaces.66 Two major phenomena are reflected from Figure 2A. The first occurs during the presence of the vesicle flux over the polyamide surface (0−120 min) with stronger adsorption of GSL vesicles, Δf = −4.5 Hz, in comparison with the adsorption of LPS, Δf = −3 Hz. The second phenomenon, during the 13944



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Table 1. Influence of Vesicle Composition Covering the Collector Surface on the Deposition Rate Coefficient, kRSPF, and the Corresponding Attachment Efficiency, αRSPF, of P. aeruginosa in the RSPF System (Background Solution of 10 mM NaCl; Cell Concentration in the Inlet Is 5 × 108 cells/mL, for an Injection Period of 30 min) kRSPFd αRSPFe

favorable (silanized)a

GSL-1b

LPS Rec

(4.6 ± 1.2) × 10−10 1

(3.8 ± 0.4) × 10−10 0.64 ± 0.1

(8.3 ± 0.7) × 10−11 0.18 ± 0.05

a

Favorable conditions were achieved by the addition of silane. bGlycosphingolipids extracted from Sphingomonas paucimobilis. cLipopolysacharides (rough strains) extracted from Salmonella enterica serotype minnesota Re 595 (Re mutant). dkRSPF = (J/C0), cell transfer rate coefficient, m/s. eαRSPF = (kRSPF/kRSPF,fav), attachment efficiency.

The notable adhesiveness of GSL and EPS of S. wittichii to the polyamide coated QCM-D sensors (Figure 2) suggests that the profound biofouling phenomenon of the model Sphingomonas spp. strain, S. wittichii RW1, as seen in Figure 1, is originated from the stronger molecular bonding of GSL and glucuronic acid components. The similar surface charge and hydrophobicity of the QCM-D sensor coated material compared to the RO membrane support this hypothesis. As expected, a higher surface roughness of the RO membrane compared to the QCM-D sensor, Rq and Ra, was detected: 60.3 ± 6.16 and 47.3 ± 4.96 nm compared to 2.0 ± 0.24 and 1.5 ± 0.16 nm (Figure S1). Hence, even the higher surface roughness of the membrane did not obscure the stronger physicochemical interactions between GSL producing bacteria and the polyamide surface, which apparently play an important role in RO fouling, causing a faster permeate flux decline. The adhesive EPS excreted by S. wittichii (Figure 2B), which likely contains an abundant amount of glucuronic acid, may serve as a bridging compound between surface and cells and between cells, protecting them from the shear stress of the RO system. Subsequent Attachment of LPS Containing Bacteria to Sphingomonas spp. GSL. To investigate the contribution of a conditioning layer further, experiments using single outer membrane components were performed in the absence of perpendicular flux toward the surface to estimate the possible contribution of GSL and LPS molecules on the adhesiveness of bacteria bearing LPS in their outer membrane. GSL extracted from S. paucimobilis and LPS extracted from Salmonella enterica were used to coat quartz surfaces, and consequent bacterial deposition was conducted in the RSPF system. The RSPF system is an ideal system to investigate bacterial adhesion in the absence of an external perpendicular force, as it focuses on the chemical interactions occurring between the bacteria and collector surface. In this experiment, a known Gram negative LPS containing model bacteria, P. aeruginosa PAO1, was used for deposition experiments. The results, summarized in Table 1, indicate that the cell transfer rate coefficient, kRSPF, was the same order of magnitude for GSL coated surfaces as a silanized surface. The silainzed surface simulates a chemically favorable surface and a promoter for organic and biological molecules on inorganic surfaces.75,76 This result suggests that the previously deposited GSL contributes to P. aeruginosa adhesion. The enhanced bacterial adhesion to the GSL layer as shown in the RSPF system is well correlated with the significantly higher hydrophobicity of the surface coated with GSL-vesicles than the surface coated with LPS-vesicles: water-drop contact angles of 70.1° ± 2.5° and 30.9° ± 2.8° were measured for the GSL and LPS coated surfaces, respectively. Also, previous studies showed the higher hydrophobicity of GSL containing bodies (bacterial cells and vesicles) and GSL molecules.34,36,37 In addition, less repulsion between GSL and the negatively charged surface is expected compared with LPS due to smaller

elementary charges found on GSL compared with LPS (1.35 and 3.33 elementary charges/nm2, in 50 mM KCl and 5 mM MgCl2 at pH 7.4, for GSL and LPS, respectively).37 Representative images of P. aeruginosa PAO1 deposited on the different slides are shown in the Supporting Information, Figure S2. Influence of S. wittichii Presence in Biofilm on Flux. Since biofilms in natural environments as well as in engineered processes, such as membrane−water treatment, are never monoculture communities, a series of questions regarding Sphingomonas spp. impact on consequential bacterial adhesion arise. These questions include the following: would a pioneering layer of Sphingomonas spp. significantly affect the subsequent attachment of other, non-GSL containing bacteria, under permeate flux conditions? Is the consequent RO membrane biofouling process affected differently if the primary colonizing bacteria contain GSL vs LPS? GSL molecules, vesicles, and GSL-containing bacterial cells are more hydrophobic and less negatively charged than LPS molecules, vesicles, and LPS-containing bacteria.77,78 Owing to these properties that are previously reported as positively contributing to bacterial adhesion8,9 and the findings in Table 1, S. wittichii is likely playing a major role as a conditioning layer enhancing attachment of other bacterial species. In the subsequent biofouling experiment (Figure 3), E. coli K12 was chosen as the LPS possessing bacteria to be tested as a candidate strain for attachment to the S. wittichii conditioning layer. In addition, a conditioning layer of E. coli K12 bacteria on the RO membrane was compared with the S. wittichii conditioning layer and the consequent effects of these layers on the attachment of E. coli and reducing RO permeate flux were tested. The selection of E. coli K12 as the secondary layer of the RO biofilm over P. aeruginosa (selected in the previous parts of this study) was due to the generally weak environmental biofilm-forming characteristics of the E. coli strain. The pronounced ability of Pseudomonas spp. to form biofilm is a feature that might mask the stepwise conditioning effect and result in a similar flux decline for all setups, regardless of the characteristics of the conditioning bacterial layer. The conditioning step with the bacterial layer of either S. wittichii or E. coli in the background solution was performed for 7 h followed by 16 h of the subsequent biofouling step, with freshly harvested E. coli, in the background solution supplemented with two types of antibiotics, ampicillin and chloramphenicol, to prevent proliferation of the S. wittichii added during the conditioning step and allow the analysis of biofouling affected by the attachment of the secondary E. coli layer. To define the intrinsic flux decline attributed to the salts and antibiotics present in the system, a control experiment without addition of bacteria was performed. As the experiment progressed, the flux decline for the bacteria-free solution reached values that resembled the experiment of the S. wittichii 13945



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coli fouling layer (Figure 3 shows a drop of 8% in permeate flux for pure E. coli layer and a drop of 12.3% for the mixed S. wittichii and E. coli cells layer). This suggests that the contribution of S. wittichii cells to the flux decline is not linearly proportional to their fraction in the fouling layer, but rather its influence is greater than in the case of the other cell type. This substantial effect of S. wittichii on RO permeate flux is further supported by comparing the two experiments of S. wittichii conditioning followed by a wash with salts and antibiotics and the E. coli conditioning followed by attachment of E. coli: While a similar flux decline was observed for these two experiments, for the latter, the amount of E. coli biomass was approximately 2-fold higher than that in the former (Figures 3 and 4B). These additional control experiments suggest that the qualitative factor of S. wittichii presence in the biofilm is more important than the quantitative amount of its biomass. Even though the initial phase of this study found GSL vesicles to be highly adhesive compared with LPS vesicles (Figure 2A), in the comparable whole cell fouling experiments, the S. wittichii conditioning layer on the RO membrane did not result in a higher subsequent deposition of bacteria (Figure 4B); rather, it resulted in a greater permeate flux decline (Figure 3). The considerable level of adhesion between the QCM-D polyamide coated sensor (mimicking a RO membrane surface) and the outer membrane GSL and EPS of Sphingomonas (Figure 2) strongly suggests that similar amounts of biomass (Figure 4B) on RO membranes results in different rates of permeate flux decline. As reported previously, GSLcontaining vesicles and bacteria form a rigid layer on the surface in general and more specifically on polyamide.39,78 A positive correlation between GSL content and rigidity was also found in studies investigating the role of eukaryotic GSL on the biophysical properties of fluid membranes.80,81 Since Sphingomonas spp. contain GSL in their outer membrane, this feature may contribute to enhanced adhesion of other bacteria and the overall rigid structure might result in greater hydraulic resistance. Additionally, S. wittichii EPS was more adhesive than EPS of P. aeruginosa PAO1, leading to greater cohesiveness of the biofilm and fouled layers in which Sphingomonas is a part. As suggested by James et al. for mixed biofilms,82 the presence of one species producing copious amounts of EPS may enhance the stability of other cell types even if they do not themselves synthesize EPS. Additionally, for dual-species biofilms, James et al. found that some EPS types contribute a larger share to the stability of the biofilm structure. Certain polymers may provide rigid scaffolding onto which other polymers attach, and a synergetic effect was observed for dual-species biofilms, where mixtures of the EPS components resulted in a higher overall viscosity.83 In conclusion, we have observed Sphingomonas spp. cells to have four unique properties, which contribute to RO biofouling: (i) increased interaction by GSL facilitating the bond between the cells and the pristine surface; (ii) enhanced bacterial adherence with GSL molecules leading to stronger intercellular interactions; (iii) GSL providing the biofilm matrix with strong rigidity; and (iv) stronger EPS adhesion to the pristine surface. Altogether, Sphingomonas spp. GSL and EPS likely contribute to a more “cemented” structure for the biofilm. The strong interaction between GSL and EPS to other bacteria and to the membrane surface likely provides an enhanced hydraulic resistance and maintains biofilm survival under shear.

Figure 3. Normalized flux decline during biofouling induced by (i) E. coli K12 as a conditioning layer, followed by further inoculation of E. coli (◆); (ii) S. wittichii as a conditioning layer, followed by further inoculation of E. coli (●); (iii) S. wittichii as a conditioning layer, followed by injection of background solution without bacteria (○); (iv) no conditioning layer of cells being added with only the background solution injected (×). Experimental conditions were as follow: initial permeate flux of 41.6 L/m2 h, crossflow velocity of 25.9 cm/s, initial cell concentration of 5 × 106 ± 5 × 104 cells/mL for all experiments, pH of 6.8, and background solution of 8.5 mM NaCl supplemented with 0.5 mM CaCl2 during the conditioning stage (in graph termed as salts). Background solution was supplemented with 50 mg/L ampicillin and 20 mg/L chloramphenicol (in graph termed as salts + antibiotics).

“conditioning” step followed by bacteria-free solution as well as the experiment of the E. coli “conditioning” step followed by the E. coli fouling step (∼91.5−92.5% from the initial flux, at the end of 22 h). These findings, alongside emphasizing the intrinsic flux decline due to adsorption of antibiotics to the membrane, suggest that the S. wittichii “conditioning” layer does not proliferate in substantial amounts when supplied with the antibiotics (which are in charge of selection for growth of the red fluorescent protein, RFP, tagged E. coli) and that the single layer of E. coli deposited on the RO membrane has a negligible effect on the flux decline. Possible reasons for the minor effect of E. coli on the RO permeate flux may be low E. coli attachment to its own conditioning layer as well as the bacterial layer structure with low hydraulic resistance and high porosity. In contrast, the S. wittichii conditioning layer resulted in the most profound flux reduction after the attachment of E. coli. The S. wittichii layer followed by E. coli deposition led to a 64% higher flux decline compared with the bacteria-free background solution and a 48% higher flux decline compared with the E. coli conditioning layer followed by further E. coli deposition. Analysis of the total amount of cells using the COMSTAT biofilm program79 in the two cases of conditioning the membrane with either E. coli or S. wittichii and the subsequent effects on E. coli deposition showed similar calculated biovolumes for the two treatments. For these two cases of E. coli and S. wittichii conditioning bacterial layers, 13 ± 0.9 and 13.5 ± 0.27 μm3/μm2 of specific biovolumes were calculated, respectively. By subtracting the values of the RFP (E. coli cells) from the total DAPI stain (S. wittichii and E. coli cells) values for the subsequent E. coli deposition over the S. wittichii conditioning layer, it is evident that the fraction of E. coli cells attached to the S. wittichii layer was about 70% of the total biomass. In other words, the fouling layer was composed of 70% E. coli and 30% S. wittichii cells, causing a permeate flux decline that was ∼50% higher than that caused by the pure E. 13946



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Figure 4. CLSM analysis of the biofouling layer on the membrane surface (A) at the end of the runs with (i) S. wittichii as the conditioning layer followed by washing with a solution of 8.5 mM NaCl, 0.5 mM CaCl2, and antibiotics (top image; ○ in Figure 3); (ii) E. coli as the conditioning layer followed by further deposition of E. coli tagged with RFP (middle image; ◆ in Figure 3); and (iii) S. wittichii as the conditioning layer followed by further deposition of E. coli tagged with RFP. Blue, red, and purple spots represent S. wittichii, RFP tagged E. coli, and RFP tagged E. coli stained with DAPI, respectively. All CLSM pictures were analyzed with COMSTAT biofilm software (B).

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Figures S1 and S2 containing AFM and fluorescent microscopy images are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Israel Science Foundation (Grant No. 1360/10).

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