Role of Pseudomonas aeruginosa Biofilm in the Initial Adhesion

Dec 13, 2007 - Corresponding author phone: (414) 229-6891 ; fax: (414) 229-6958 ; e-mail: [email protected]. Cite this:Environ. Sci. Technol. 42, 2, 443-449 ...
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Environ. Sci. Technol. 2008, 42, 443–449

Role of Pseudomonas aeruginosa Biofilm in the Initial Adhesion, Growth and Detachment of Escherichia coli in Porous Media YANG LIU AND JIN LI* Department of Civil Engineering and Mechanics, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201

Received July 26, 2007. Revised manuscript received October 24, 2007. Accepted October 31, 2007.

This study systematically investigated the impact of Pseudomonas aeruginosa biofilm on the initial adhesion, growth, and detachment of indicator bacteria Escherichia coli JM109 in porous media. Two P. aeruginosa strains, the mucoid PDO300 and wide type PAO1 with different extracellular polymeric substance (EPS) composition and secretion capability, were used to grow biofilm in packed beds. Results from the column breakthrough curves and retained JM109 profiles show that the amount and composition of P. aeruginosa biofilm EPS have a profound impact on the deposition and retention of E. coli in porous media. PAO1 biofilm coating improved E. coli retention in the column, whereas PDO300 biofilm coating had only a small impact on E. coli removal. Biofilm surface hydrophobicity and polymeric interactions between the biofilm and E. coli cell surfaces were found to play important roles in controlling the distribution of E. coli along the columns. After initial attachment, E. coli bacteria were able to survive and grow at similar growth rates in columns coated with either PAO1 or PDO300 biofilms with a relatively low nutrient supply. Biofilm detachment was the major mechanism that introduced E. coli bacteria to the bulk fluid long after the contamination event when E. coli cells became an integral part of the biofilm. Findings of this study suggest that biofilm plays a significant role in controlling the initial attachment, growth, and survival of bacteria in porous media, and that the interaction between bacteria and biofilm surfaces should be considered when predicting bacterial and pathogen migration in the environment.

Introduction Biofilm is an assemblage of microorganisms embedded in a matrix of extracellular polymeric substances (EPS), which are comprised of polysaccharides, proteins, lipids, and nucleic acids (1–3). Found on almost every surface in contact with water, biofilm represents a majority of the bacterial biomass in natural and engineered systems (4–6). The impact of biofilm on the physical, chemical, and hydrodynamic characteristics of porous media, as well as the potential of bacteria to survive and grow in biofilm-coated porous media, are of great importance in various environmental, industrial, and health contexts, e.g., pathogen contamination in aquifers, bioremediation, and water and wastewater filtration. * Corresponding author phone: (414) 229-6891; fax: (414) 2296958 ; e-mail: [email protected]. 10.1021/es071861b CCC: $40.75

Published on Web 12/13/2007

 2008 American Chemical Society

The fate and transport of bacteria in porous media are controlled by a series of events, e.g., the initial attachment, colonization, growth, decay, and detachment (6, 7). The initial adhesion of bacteria to a solid surface is known to be controlled by (i) transport processes, including advection, diffusion, interception, and gravitational settling, that permit collisions between bacteria and collector grains, and (ii) chemical factors controlling attachment, e.g., van der Waals and electrostatic forces (8, 9). More recently, long-range forces, including steric interactions, Lewis acid–base interactions, hydration forces, hydration pressure, hydrogen bonding, and the hydrophobic effects also have been recognized to affect bacterial adhesion (10, 11). In porous media coated with biofilm, the transport and retention of bacteria may be influenced by the presence of a biofilm bacterial community and the physical and chemical nature of the biofilm (12–14). After initial adhesion and colonization, the attached bacteria must compete with indigenous organisms for nutrients and space to survive and grow in the biofilm. Once incorporated into the biofilm matrix, spontaneous detachment of the biofilm may release bacteria back into the bulk fluid, which poses a significant risk to water safety if the microbes are pathogenic (15). Biofilm detachment can be caused by several mechanisms, including erosion, sloughing, abrasion, predator grazing, and human intervention (15, 16). Detachment may redistribute the biofilm biomass and significantly increase the transport distance of bacteria and pathogens over a large time scale. Although various studies have suggested that pathogens associated with biofilm can survive for a prolonged period after the contamination event (17, 18), to date no study has been done to systematically investigate the role of biofilm in the initial attachment, growth and detachment of bacteria in porous media. In this study, the retention, survival, and detachment of Escherichia coli JM109 were investigated in laboratory packed beds coated with two types of Pseudomonas aeruginosa biofilms, i.e., the nonmucoid PAO1 wild type and the mucoid PDO300 mutant strain. The dominant EPS of PAO1 is carbohydrate, including glucose, rhamnose, and mannose, and the main EPS of PDO300 is alginate, a linear copolymer of mannuronic and guluronic acids (uronic acids C6H10O7) joined by β 1–4 linkage (19). E. coli JM109, with 75% lipopolysaccharides (LPS) and 25% proteins (20) on its surface, was frequently used as a model organism in bacterial adhesion studies (10, 21–23). Our results indicate that biofilm EPS plays a significant role in mediating the transport of E. coli bacteria from the carrying solution to porous media, and the deposited E. coli can survive and grow in the biofilm matrix and be released back to the bulk fluid upon biofilm detachment.

Methodology Filtration System Setup. The porous medium used in this study was spherical glass beads with an average diameter of 550µm (MO-SCI Specialty Products, MO). The glass beads were thoroughly cleaned with acid and base (24) and wet packed into the columns (length ) 25 cm and internal diameter ) 2.54 cm). The porosity of the packed columns was determined gravimetrically to be 0.4. Prior to each experiment, the entire filter system was sterilized with 0.5% sodium hypochlorite solution, followed by a thorough rinse with autoclaved distilled water until all residual chlorine was removed. Biofilm Establishment and Characterization. PAO1 and PDO300 bacterial strains were provided by Matthew Parsek at the University of Washington. PAO1 is a wild type P. VOL. 42, NO. 2, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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aeruginosa strain and PDO300 is an isogenic mucoid alginateoverproducing strain of the prototypic PAO1 with a mucA22 mutation (19, 25). The stored bacteria were streaked onto Luria–Bertani (LB) agar plates and incubated at 37 °C overnight. A single colony was then transferred into a LB broth and grown in a shaker incubator at 200 rpm at 37 °C until the stationary phase (20 h) was reached. The inoculation was conducted by injecting 450 mL P. aeruginosa suspension in a LB broth with a cell concentration of 108 colony forming unit (CFU)/mL, into the column. Bacteria were allowed to attach to the glass beads by recycling 100% of the cell suspension for 12 h using a peristaltic pump. After 12 h, the bacterial suspension was replaced with a synthetic nutrient solution, whose composition was reported in a previous paper (25), to facilitate biofilm growth. The system was operated continuously for five days under a constant flow rate of 3.0 mL/min. The flow injection direction was switched between upflow and downflow directions every 12 h to ensure uniform biofilm distribution inside the column. The biofilm was grown at room temperature (20–25 °C) in the dark, and its establishment was monitored daily by measuring the concentration of suspended bacteria in the column effluent. After initial inoculation, bacterial counts in the column effluent were as high as 1010 CFU/mL during the first two days for both types of P. aeruginosa biofilms. After five days, bacterial concentration in the column effluent reached a pseudosteady state and remained at 105–106 CFU/ mL. To analyze the biofilm bacterial and EPS distribution in the column, column media were evenly dissected into five segments and each segment of the glass beads was placed into a beaker containing 50 mL NaCl-MOPS (3-(N-morpholino)-propanesulfonic acid) buffer, composed of 100 mM NaCl and 2.2 mM MOPS in deionized water. This buffer was chosen because it is free of phosphate, which is known to interfere with cell deposition. The pH was adjusted to 6.9–7.1 using NaOH. The bacterial suspension was then sonicated in an ultrasonication bath (FS20H, Fisher Scientific, IL) for 10 min, followed by vigorous shaking by hand for several seconds. Ultrasonication is commonly used to dislodge bacterial cells from surfaces (26, 27) and less than 0.1% cell lysis was found after 10 min sonication in our control study. The biofilm bacterial count was measured by the drop plate method. The biofilm EPS was further extracted using the highspeed centrifugation method described by Brown and Lester (28). PAO1 biofilm EPS carbohydrate content was quantified using the phenol-sulfuric acid method with glucose as the standard and PDO300 biofilm EPS uronic acid concentration was determined using the m-hydroxydiphenyl sulfuric acid method with D-glucuronic acid as the standard (24). The zeta-potentials of JM109, PAO1, PDO300 bacteria and crushed glass beads were measured 10 times using 20 cycles per analysis (ZetaPALS analyzer, Brookhaven Instruments Corp., Holtzville, NY). E. coli Cell Preparation. E. coli JM109 carrying a plasmid pGFPuv that constitutively expresses green fluorescent protein (GFP) were grown in a LB broth supplemented with 100µg/mL ampicillin at 37 °C and shaken at 200 rpm in a shaker incubator until reaching the stationary phase (14 h). Cells were harvested by centrifugation at 3000g and 4 °C for 10 min. After decanting the growth medium, the pellets were resuspended in the NaCl-MOPS buffer. The centrifugationresuspension process was repeated three times to remove traces of growth medium. A final cell density of 106 -107 cell/mL was obtained by measuring the optical density (OD) at 600nm wavelength using a UV/Visible spectrophotometer (Varian, CA). E. coli Transport Study. After biofilm establishment, JM109 transport and survival experiments were conducted 444

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in a total of six packed beds (two clean columns, two columns coated with PAO1 biofilm, and two columns coated with PDO300 biofilm). For each set of experiments, E. coli cells were simultaneously injected into two identical columns; one used for bacterial deposition analysis and the other used for bacterial survival and detachment study. Approximately two pore volumes (PV) of E. coli suspension were injected after equilibrating each column for at least 20 PV of bacteriafree background 100 mM NaCl-MOPS buffer in the upflow direction at a constant approach velocity of 0.011 cm/s. Following bacterial injection, the column was eluted with additional 8 PV of the background buffer solution. Every half PV of the column effluent was collected in a 50 mL polystyrene tube and immediately placed on ice prior to microbiological analysis. After completing the transport experiment, porous media from one of the two columns were evenly dissected into five segments and the retained JM109 cells were recovered by sonication in the same manner as biofilm extraction from glass beads surfaces. Quantitative assessment of E. coli concentrations in the column effluents and cell distribution within the column media was carried out by viewing samples on a Leitz Diaplan microscope fitted with a Leitz Plemopak fluorescence attachment with a 20× objective (Leitz Microsystems, Wetzlar). The GFP-conferred fluorescence was visualized under illumination with a blue laser (excitation 488 nm; suppression 515 nm). The number of cells in a minimum of 20 randomly chosen fields of view was determined for each sample. Colloid filtration theory (CFT) was used to obtain the theoretical particle retention pattern in the packed columns based on E. coli breakthrough curves. The particle distribution S(X), i.e., the number of deposited bacteria per mass of the granular collector, was calculated using the following equation (29, 30): S(X) )

(

kd × X ×  t 0 ×  × k d × C0 exp Fb U

)

(1)

Here, X is the column depth, t0 is the duration of continuous particle injection,  is the bed porosity, C0 is the initial cell concentration, Fb is the porous medium bulk density, U is the approach (superficial) velocity, and kd is the deposition rate coefficient, which was estimated based on the steady state breakthrough concentration (31). E. coli Survival and Regrowth Study. Upon completion of the transport experiment, the second column was continuously fed with a low-nutrient solution containing 23 mg/L NaC2H3O2; 21.8 mg/L K2HPO4; 8.5 mg/L KH2PO4; 17.7 mg/L Na2HPO4; 2.7 mg/L NaNO3; 22.5 mg/L MgSO4; 0.25 mg/L FeCl3; and 36.4 mg/L CaCl2, in the upflow direction. The ionic strength of the nutrient solution is 2.5 mM. The effluent E. coli and biofilm bacterial count were analyzed every 12 h. To obtain an accurate cell count, the effluent bacterial suspension was placed in an ultrasonication bath for five minutes prior to microscopy tests. After six days, the E. coli and biofilm bacterial distribution in the columns was examined by both direct count and plate count methods, and the results were compared with the E. coli distribution immediately after the transport experiment and the biofilm bacterial distribution prior to E. coli injection.

Results Biofilm Characteristics. Figure 1 shows the bacteria count and EPS concentration of P. aeruginosa biofilms along the columns. The results show that the main EPS contents, i.e., carbohydrate for PAO1 biofilm (Figure 1A) and uronic acid for PDO300 biofilm (Figure 1B), had relatively even distribution along the columns before the E. coli JM109 injection based on statistical estimates of the 95% confidence intervals.

FIGURE 1. Distribution of retained biofilm bacteria and biofilm EPS (carbohydrate is the main EPS component for PAO1 biofilm, uronic acid is the main EPS component for PDO300 bioflim) in PAO1 bioflim coated column (1A) and PDO300 biofilm coated column (1B). Error bars represent standard deviations of trilicate measurements. Arrows indicate the column section close to the six day low-nutrient solution injection inlet. It is also noted that the PDO300 biofilm contained more cells than the PAO1 biofilm, which maybe attributed to the higher PDO300 initial attachment compared with the PAO1 bacteria, considering their similar growth rates (24). Six days after the E. coli transport experiment, the distribution of biofilm bacteria in the columns remained the same except for a slight increase of bacterial count near the bottom of the column where the nutrient solution was injected. E. coli Breakthrough-Elution Curves. Figure 2 presents the breakthrough-elution curves showing the normalized effluent concentration, C/C0, during the injection and elution of E. coli cells. The breakthrough plateau was constant at 2.5–3 PV, indicating a steady-state condition in all three types of columns. The magnitude of the steady-state breakthrough plateau for the PAO1 biofilm was lower compared with those for the PDO300 biofilm and clean column, indicating that a higher number of E. coli cells were retained in the PAO1 biofilm-coated column. The PDO300 biofilm, by contrast, had a very small impact on the E. coli bacterial removal, despite that the PDO300 biofilm had a higher bacterial count and EPS content than the PAO1 biofilm. During the MOPS-NaCl buffer elution, tailing of E. coli was observed in all columns with similar magnitudes despite different column media types and breakthrough concentrations. The detachment of previously attached bacteria likely contributed to the tailing (16). A control experiment using a conservative dye tracer did not exhibit any skewed breakthrough or tailing, indicating that physical nonequilibrium was not significant for the tracer in the columns. Retained E. coli profiles immediately after the transport experiment. Figure 3 shows the retained E. coli profiles compared with those predicted by the CFT. A log-linear relationship was observed between the number of retained bacteria and the transport distance in the clean column,

FIGURE 2. E. coli bacterial breakthrough-elution curves in the clean column (2A); PAO1 biofilm-coated column (2B); and PDO300 biofilm-coated column (2C). Error bars represent standard deviations of triplicate measurements. which indicates a constant deposition rate coefficient as implicated in the “clean-bed” theory (9). In the biofilm-coated columns, the retained E. coli concentrations decreased hyper-exponentially as a function of transport distance (Figure 3B, C), indicating that the deposition rate coefficients decreased with increasing transport distance. Recently, various studies examining the retained colloid profiles in porous media reported decreasing deposition rate coefficients (24, 32–37). The apparent change in deposition rate coefficients with distance has been attributed to the heterogeneity in bacterial surface properties, distribution in the interaction energies between bacteria and porous media, and particle deposition dynamics (31, 33, 36). In our study, the occurrence of an energy barrier associated with the negatively charged biofilm surface may have caused the monotonic derivation from CFT (38). Another possibility is that the biofilm surface roughness amplified the distribution heterogeneity among the E. coli population (32, 35–37). Transport Experiment Mass Balance. Table 1 shows the total E. coli bacteria recovered in the effluent and in the column immediately after the E. coli transport experiment. Mass recoveries (total from effluent and column media) for E. coli bacterial transport experiments were between 96.2 and 100.5%. VOL. 42, NO. 2, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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relatively constant four days after the cell injection. Overall, the effluent E. coli count was about 2 orders of magnitude less than, but with a similar pattern to that of the P. aeruginosa. The extended tailing of E. coli also demonstrated that cells could remain in the biofilm matrix for a prolonged period of time following the passage of a pulse injection. E. coli Retention and Survival in Porous Media. The retained E. coli profiles six days after the injection are shown in Figure 3. Compared with the E. coli distribution immediately after the transport experiment, the E. coli cell number decreased in all layers in the clean column (Figure 3A), but increased in most layers in both biofilm-coated columns (Figure 3B and C). Table 1b and 1d compare the total E. coli bacterial cells extracted from the column media immediately and six days after E. coli transport experiment. Despite the low nutrient supply, only 3% of the total injected E. coli cells were recovered from the clean column media after six days compared with the 20.1% recovery rate immediately after the E. coli transport experiment. As shown in the table, the total E. coli recovery rate (column media plus effluent) was 100.5% immediately after the cell transport experiment and 84.4% after Day 6 in the clean column, indicating that some of the E. coli lost their fluorescent signal after six days. Both biofilm-coated columns had more of the E. coli bacteria six days after the pulse injection, which led to the conclusion that the injected E. coli bacteria were able to grow in biofilms. The specific growth rates (Table 1) for E. coli were virtually identical in both types of biofilm (P ) 0.01 by paired-sample t test), indicating that the amount and composition of P. aeruginosa biofilm EPS had little effect on the survival and growth of E. coli bacteria.

Discussion

FIGURE 3. Retained E. coli JM109 profiles in the clean column (3A); PAO1 biofilm-coated column (3B); and PDO300 biofilm-coated column (3C) immediately and six days after the E. coli transport experiment. Error bars represent standard deviations of triplicate measurements. Arrows indicate the column section close to E. coli cells and low-nutrient solution injection inlet after the E. coli transport experiment. Column Effluent Pattern during the E. coli Survival Experiment. The column effluent biofilm and E. coli bacterial densities were monitored every 12 h for six days after the E. coli injection. As shown in Figure 4A, the number of E. coli bacteria in the effluent decreased rapidly and dropped below the sensitivity limit of the analytical method (102 cells/mL) four days after the transport study in the clean column. Extended tailing with low E. coli concentrations following the breakthrough-elution was observed in both columns with biofilm (Figures 4B and 4C). In both columns, the effluent E. coli concentration experienced fluctuation, which occurred concurrently with biofilm detachment. Detachment is known to be the primary process that balances biofilm growth in the filter media through the release of cells and aggregates into the bulk liquid. Our observation implies that the E. coli bacteria became an integral part of the biofilm and that biofilm detachment was the phenomenon that most likely influenced the fate of E. coli bacteria long after the contamination event. Despite the fluctuation of bacterial counts, the average E. coli bacterial number in the column effluent decreased by approximately 55 and 66% in the PDO300-coated column and the PAO1-coated column, respectively, and became 446

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Role of Biofilm Coating on E. coli Initial Adhesion. Biofilm formation is known to affect porous media hydrodynamic and physical properties, and interfere with bacterial adhesion and transport. As biomass accumulates, the reduced bed porosity provides an additional surface area for bacterial deposition; thereby enhancing bacterial removal (39). Increased porous media surface roughness with biofilm coating also is known to encourage bacterial adhesion (40, 41). In our study, fewer E. coli bacteria were captured by the mucoid PDO300 biofilm compared with the wild type PAO1 biofilm despite that PDO300 biofilm is thicker and has a higher degree of roughness (42, 43), indicating that biofilm architecture and reduced bed porosity were not important factors controlling the E. coli bacterial deposition under our current experimental condition. The presence of biofilm EPS may also alter porous media surface electrostatic charge, therefore changing the electrostatic and van der Waals interactions between E. coli bacterial surface and porous media, as described by the DLVO (Derjaguin, Landau, Verwey, Overbeek) theory (44, 45). Our results show that PAO1 and PDO300 bacteria had similar surface zeta potentials (PAO1 -28.57 ( 3.99 mV and PDO300 -29.05 ( 2.63 mV) and both are more negatively charged than crushed class beads (-21.72 ( 1.74 mV). Compared with the clean glass bead surface, more negatively charged biofilm surfaces would pose higher repulsion to the deposition of negatively charged JM109 (-21.86 ( 0.79 mV), according to the DLVO theory. Thus, the transport of E. coli JM109 was governed at least in part by non-DLVO mechanisms, since the presence of biofilm did not impair E. coli deposition in our study. Extended DLVO theory has been developed to account for hydrophobicity and surface free energy. Hydrophobic groups on the porous media surfaces are known to improve bacterial adhesion because they remove water adsorbed to surfaces (44). The main composition of the PAO1 biofilm

TABLE 1. Normalized Percent Recovery of E. coli JM109 (Recovered Cell Concentration/Initial Cell Concentration)

a

transport experiment survival experiment E. coli growth rate (day-1)

column effluent column mediab recovery ratea,b column effluentc column mediad recovery ratea,c,d

no biofilm

PAO1 biofilm

PDO300 biofilm

80.4 ( 0.7% 20.1 ( 1.2% 100.5 ( 1.9% 1.0 ( 0.8% 3.0 ( 1.2% 84.4 ( 2.6% -0.35 ( 0.07

53.6 ( 0.9% 42.7 ( 1.2% 96.2 ( 2.1% 76.7 ( 2.3% 81.9 ( 1.7% 212.1 ( 5.0% 0.11 ( 0.01

81.4 ( 1.3% 15.2 ( 1.0% 96.6 ( 2.3% 12.7 ( 2.6% 31.3 ( 3.5% 125.4 ( 7.3% 0.12 ( 0.01

a

Column effluent during the E. coli bacterial injection and elution. b Column media immediately after the E. coli transport experiment. c Column effluent over six days after the transport experiment; and d Column media six days after the transport experiment, in PAO1 biofilm-coated columns, PDO300 biofilm-coated columns, and clean columns. Bacterial growth rate in column media were calculated by µ ) 1 / (t2 - t1)ln( n2 / n1 ) , where n2 is the total JM109 cell number six days after the transport experiment (time t2), and n1 is the total JM109 cell number immediately after the transport experiment (time t1). Data represent the average recovery of triplicate experiments.

FIGURE 4. Effluent biofilm bacterial and E. coli JM109 concentrations over six days of low-nutrient injection from the clean column (A); the PAO1 biofilm-coated column (B); and the PDO300 biofilm-coated column (C). Error bars represent standard deviations of triplicate measurements. EPS is neutral sugars, which shield the negatively charged surface functional groups, e.g., LPS and protein, located on cell membranes, whereas the PDO300 biofilm EPS is composed of negative charged uronic acid (19, 42). The highly hydrated anionic PDO300 biofilm EPS on the porous media

surfaces may cause greater resistance to E. coli bacterial adhesion by incorporating a large amount of water into its structure through hydrogen bounding. We speculate that the hydration effect may account for the lower adhesion of E. coli in the PDO300 biofilm-coated column than in the column covered with the PAO1 biofilm. Additionally, polymeric bridging between the biofilm surface EPS and E. coli cells may interfere with E. coli bacterial removal. Although steric interactions cannot be directly measured, the presence of steric interactions was assumed to be responsible for large energy barriers and was found especially important at low ionic strength (11, 24, 46). Also, bacterial adhesion may be significantly enhanced by polymer bridging between E. coli cells and the biofilm-coated solid surface (10, 24). PAO1 biofilm greatly increased E. coli bacterial adhesion compared with bare glass beads, possibly suggesting that DLVO type interactions were overcome with the presence of the biofilm surface polymers, which may possess high affinities to the E. coli cell surface. Role of Biofilm Coating and Substrate Concentration on E. coli Survival. Coexistence between two or more bacterial populations is governed by their competition for common nutrients and space. Bacterial competition strategies may include quorum sensing, biofilm formation, motility, and the secretion of antimicrobial compounds that kill or impair other species (1, 4). In our study, E. coli bacteria were able to grow in the P. aeruginosa biofilm with a low nutrient supply. Prolonged survival of pathogenic bacteria within the biofilm matrix has been observed previously in drinking water distribution pipes using culture methods (47–49) and molecular techniques (18, 50). It was believed that better access to the localized nutrients and being metabolically more active than their freeliving counterparts (50, 51) were the main factors promoting bacterial and pathogen survival and regrowth in the biofilms. It is noted that the E. coli bacterial density on the column media and in the column effluent was two to three orders less than the P. aeruginosa biofilm bacteria, which implies that P. aeruginosa bacteria were still the dominant species in the cocultured biofilm under the experimental condition. In addition, the results show that E. coli bacteria had similar growth rate in both types of biofilm-coated columns, implying that biofilm structure and composition had little impact on E. coli bacterial survival after their initial attachment to the biofilm. Implications. The attachment and growth of bacteria on biofilm-covered porous media is the norm rather than the exception and has important implications in environmental and man-made filtration processes. Information on the interaction between bacterial cells and biofilm surfaces may be utilized in designing biologically active filters, improving bioremediation, and predicting pathogen transport. Findings VOL. 42, NO. 2, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of this study suggest that the composition of P. aeruginosa biofilm EPS plays a pivotal role in controlling the initial attachment and deposition profiles of E. coli JM109 in porous media through non DLVO forces, including hydrophobicity, hydration forces, and polymeric bridging. The presence of biofilm is also essential for the survival and growth of E. coli after their initial adhesion and biofilm detachment is the main mechanism through which E. coli was released back to the bulk fluid after the contamination event.

Acknowledgments We thank three anonymous reviewers for their helpful comments on the manuscript. This study was supported in part by the Wisconsin Groundwater Research and Monitoring Program.

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