Fecal Indicator Bacteria Transport and Deposition in Saturated and

Article pubs.acs.org/est

Fecal Indicator Bacteria Transport and Deposition in Saturated and Unsaturated Porous Media Gexin Chen†,‡ and Sharon L. Walker*,‡ ‡

Department of Chemical & Environmental Engineering, University of California, Riverside, California 92521, United States S Supporting Information *

ABSTRACT: Beach sediment and sand are recognized as nonpoint fecal indicator bacteria (FIB) sources capable of causing water quality and health risks for beach-goers. A comprehensive understanding of the key factors and mechanisms governing the migration and exchange of FIB between beach water column and sediment is desired to better predict FIB concentration variations and assess the associated risk. The transport and retention behavior of two model FIB Enterococcus faecalis (E. faecalis) and Escherichia coli (E. coli) was examined using packed-bed columns in both saturated and unsaturated porous media to evaluate FIB migration potentials at conditions simulating the coastal aquatic environment. Additionally, complementary cell characterization techniques were conducted to better understand the migration behaviors of both FIB strains observed in the column experiments. The mobility of the gram-positive species E. faecalis was much more sensitive to solution chemistry and column saturation level than that of the gram-negative species E. coli. Interaction energy calculations suggest that E. faecalis retention was largely governed by the combination of DLVO (Derjaguin−Landau−Verwey−Overbeek) and non-DLVO (most likely hydrophobic and/or polymer bridging) interactions in saturated porous media, while the combination of DLVO and steric interactions controlled the deposition of E. coli cells. The measured surface properties of the two FIB strains supported the distinct bacteria transport behaviors and the differences of the identified mechanisms for each strain. As a result, E. faecalis showed the least affinity to sand in freshwater and appeared to be irreversibly attached in primary energy minima at elevated salt conditions; whereas the retained E. coli cells were reversibly attached and mostly associated with the secondary energy minima at both freshwater and seawater conditions. In unsaturated porous media, E. faecalis cells seemed to prefer to attachment at air/ water interface rather than sand surface, while E. coli showed a similar affinity to the two interfaces. It was proposed that the different surface characteristics of the two FIB strains resulted in the distinct transport and retention behavior in porous media. These results highlight the need for FIB management to consider variations in transport behavior between model FIB when assessing water quality and associated risks.

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INTRODUCTION Coastal beaches represent an important recreational resource for millions of people all over the world and provide local economic benefits with billions of U.S. dollars each year.1,2 To protect recreationists from waterborne illnesses, beach water quality standards for pathogens and indicator organisms have been set by such as the U.S. Environmental Protection Agency (EPA).3 Meanwhile, beach-monitoring programs have been implemented using fecal indicator bacteria (FIB), i.e. Escherichia coli (E. coli) for freshwater and enterococci for marine water, to assess risk through state and local beach management.4 As a result, more than 24,000 beach closing and health advisory days were issued in the United States in 2010.5 However, microbial contamination at recreational beaches still causes over 120 million gastrointestinal and 50 million severe respiratory illnesses per year around the world,6 due in part to the extreme complexity of coastal environments causing the current beach-monitoring practices insufficient to protect beach-goers’ health. For instance, Boehm observed that © 2012 American Chemical Society

enterococci concentrations were extremely variable over short time scales and changes in enterococci concentrations between consecutive samples (1 or 10 min apart) could exceed the EPA’s water quality standards.7 Traditional techniques for detecting FIB levels in beach water take considerably longer to conduct than the time scale of such variations of FIB concentrations.7 With these considerations in mind, there is a clear and pressing need to thoroughly understand FIB sources and their transport and fate in coastal aquatic environments to develop better beach management practices.2 Previous research efforts to elucidate the mechanisms influencing beach water FIB concentration variability have revealed that beach sediment and sand are important bacterial sources. Collective evidence indicates that elevated FIB Received: Revised: Accepted: Published: 8782

April 11, 2012 June 29, 2012 July 18, 2012 July 18, 2012 dx.doi.org/10.1021/es301378q | Environ. Sci. Technol. 2012, 46, 8782−8790

Environmental Science & Technology

Article

environments. For this purpose, Enterococcus faecalis (ATCC 11420) (gram-positive) and E. coli (ATCC 12014) (gramnegative) were selected as representative FIB for this study. Packed column experiments with clean sand were performed for both bacterial strains at two column saturation levels (70% and 100% saturation) in NaCl solutions (with ionic strength (IS) ranging from 1−1000 mM to simulate freshwater and seawater salinity). The initial deposition and transport behavior of FIB cells under a wide range of solution conditions was determined on the basis of column experiments. In addition, cell properties such as electrophoretic mobility, cell size, hydrophobicity, and charge density were measured for each FIB strain. Subsequently, the impact of different factors to the retention and transport behavior of FIB cells in the packedcolumn was evaluated and is discussed below. It must be noted that coastal aquatic environments are extremely complex. Many other factors such as the presence of biofilm, FIB population distribution, sediment and sand characteristics, the presence of natural organic matter, and flow conditions that were beyond the scope of this particular study can also significantly affect the FIB migration process and could be likely responsible for the distinct transport behaviors of enterococci observed by Boehm10 and Phillips16 as discussed above. The present study is also limited by the use of laboratory strains of bacteria as FIB surrogates, as naturalized bacteria tend to be acclimated and may respond differently. All these limitations warrant further comprehensive investigation.

concentration associates with both freshwater and marine beach sediment and sand.8−15 This fact suggests that beach sediment and sand can be an important FIB reservoir and may serve as a potentially important nonpoint source of FIB to coastal waters. It is worth noting that 52% of all closing and advisory days were attributed to unknown sources of microbial contamination in 2010.5 It has been proposed that beach sediments could likely be responsible for a large fraction of these closing days.16 Yamahara et al. observed that the number of enterococci that entered the water column was nearly equivalent to the number lost from exposed sand when it was submerged by seawater during a tide event at a beach located in Lovers Point, CA,10 which corroborates the likelihood. The water-sediment exchange of FIB can be induced by tide-forced infiltration,17 submarine groundwater discharge,18 precipitation related subsurface water flow,19 or sand resuspension due to wave action and recreational activities.20 Unfortunately, none of these processes are well understood in a quantitative manner. Previously, Boehm illustrated employing a column repacked with sand collected from the field that over 96% enterococci dispersed in Huntington Beach groundwater broke through the column, which provided evidence to support the possibility of exchange of FIB between beach sand and water column through groundwater discharge.18 Similarly, limited retention of enterococci in the column was observed when the column repacked with beach sand (that was naturally contaminated with enterococci) was exposed to seawater flow to simulate the scenario of beach-sea interactions dominated by tide events, which demonstrated that beach sand can act as a diffuse source of FIB to coastal waters.10 In contrast, Phillips et al. recently showed that the majority of enterococci remained in beach sediments as the contaminated sand core was exposed to seawater flow.16 The observed discrepancy of the enterococci retention in sediments was attributed by the authors to the methods of sand column preparation and the distinct characteristics of sand collected from different beaches.16 Obviously, a better mechanistic understanding of interactions controlling the adhesion of FIB cells to sediments would provide useful information and insight regarding the migration of FIB cells in the above processes. The transport and deposition behavior of microbes in porous media has been extensively studied in the past decades due to its significance relevant to drinking water supply protection,21 in situ bioremediation,22 water and wastewater treatment,23 and so on. The physical and chemical factors influencing the mobility of bacteria in porous media include solution chemistry,24,25 fluid velocity,26,27 grain collector size, surface roughness28 and charge heterogeneity,29 and saturation of porous media,30 as well as biological factors such as cell type,31 cell growth phase,32 extracellular polymeric substance (EPS),33 cell surface lipopolysaccharide (LPS),34 and the presence of biofilm.35 Despite these and other efforts, studies attempting to specifically understand the interactions between FIB cells and solid or air/water interfaces that exist in actual sediment and the resulting mechanisms governing the transport and retention of FIB cells in coastal sediment environments are limited. Such knowledge can be integrated into models developed to predict FIB variability of beach waters based on observations of hydrometeorological and biological variables, which is a promising alternative for managing beaches.11 The main objective of this study was thus to gain more insight into the fate and transport of FIB cells in porous media under idealized conditions relevant to coastal sediment

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MATERIALS AND METHODS Bacterial Cell Selection. E. faecalis (ATCC 11420) and E. coli (ATCC 12014) were selected as model FIB for this study and were obtained from ATCC (American Type Culture Collection, Rockville, MD). E. faecalis is a nonmotile, grampositive bacteria36,37 and was shown to be a dominant species in water and sediment at the Dana Point Baby Beach in southern California.38 E. coli has been reported to be a nonmotile, rod-shaped, gram-negative bacteria.36 E. faecalis and E. coli are two types of bacteria that are ubiquitous in the human gut and commonly used as FIB.36,38 Both laboratory strains were recently used as FIB representatives to evaluate FIB’s survival in overlying water and sediment in laboratory microcosm experiments.14 Detailed methods of bacterial cell preparation are provided in the Supporting Information (SI). To better understand the mechanisms governing the FIB bacteria transport and deposition in porous media, complementary characterization of E. faecalis and E. coli under the same range of conditions tested in column experiments was conducted. This included the cell size measurement, the hydrophobicity analysis using the microbial adhesion to hydrocarbons (MATH) test,24 the electrophoretic mobility, and the potentiometric titration of the two FIB model strains. Details of these techniques are given in the SI. In addition, DLVO interaction energy profiles were determined for cell interacting with a sand or air/water interface upon close approaching. Details of the DLVO interaction calculations are presented in the SI. Transport Experiments. Quartz sand (Unimin Corporation, Spruce Pine, NC) having an average sand diameter of approximately 275 μm was utilized as the packing material for column transport experiments. Prior to use, the sand was cleaned thoroughly to remove any metal oxide and organic contaminants. 8783

dx.doi.org/10.1021/es301378q | Environ. Sci. Technol. 2012, 46, 8782−8790


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Figure 1. Characteristics of bacterial cells and sand. (a) Electrophoretic mobilities and zeta potentials of E. faecalis and E. coli cells and zeta potentials of quartz sand as a function of IS (NaCl). (b) Potentiometric titration of E. faecalis and E. coli cells. Acidities are presented in milliequivalents per 108 bacterial cells, as determined from the amount of NaOH consumed during titration. Electrophoretic mobility experiments were conducted at pH 5.6−5.8 and temperature 25 °C. Error bars represent one standard deviation for three replicate measurements. Titrations were performed in 10 mM NaCl solution and at a temperature of 25 °C.

addition of NaCl. All experiments were conducted at room temperature and were at least duplicated.

An aluminum alloy column with internal diameter and length of 5 and 10 cm was wet-packed uniformly with clean quartz sand. The resulting porosity of the porous media was gravimetrically determined to be ca. 0.43. Once packed, the column was operated in a downward direction using a peristaltic pump and equilibrated by sequentially pumping DI water and background electrolyte solution through the column. Under saturated conditions, a pulse of bacteria suspension was immediately injected into the column for approximately 4 pore volumes (PVs), followed by a bacteria-free electrolyte solution (ca. 4 PVs) and DI water (ca. 10 PVs) to examine release trends of the previously deposited bacteria. To achieve unsaturated conditions (70% saturation), the saturated columns were gradually drained after equilibration. The desired water saturation level was reached by reducing the injection flow rate and slowly adjusting the elevation of the drip point on a hanging water column to change the suction at the bottom of the column to achieve unit gradient conditions. The average water saturation level of 70% in the column was achieved and monitored gravimetrically with an electronic balance (Sartorius Master Series, LP model, Germany). The sand in the column was then equilibrated by flushing it with approximately 8 PVs of background electrolyte solution, followed by a pulse of 4 PVs of bacteria suspension, 4 PVs of bacteria-free background electrolyte solution, and 10 PVs of DI water under steadystate flow conditions. Additional information about the column experiment operations is provided in the SI. Bacterial transport experiments were conducted under both saturated and unsaturated conditions by the initial injection of 4 × 107 cells/mL. The bacterial cell concentrations in the column effluent were determined every 1 min by measuring the optical density of the effluent (C) at a wavelength of 280 nm with a spectrophotometer (BioSpec-mini, Shimadzu Corp.). The initial bacteria injection concentration (C0) was measured as well. The average interstitial fluid velocity in the column was maintained to be 0.021 cm/s for all transport experiments to maintain a consistent hydrodynamic impact, which is similar to values measured for the natural pore water outflow from sediments.39 The IS of the background electrolyte solutions ranged from 1 to 1000 mM and was adjusted through the

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RESULTS AND DISCUSSION Electrokinetic Properties of Bacterial Cells and Sand Surfaces. The electrophoretic mobilities and zeta potentials of E. faecalis and E. coli cells are presented in Figure 1a. The results indicate that both FIB strains used in this study were negatively charged over the range of IS and pH (5.6−5.8) conditions tested, except that E. coli cells were slightly positively charged (1.77 mV) in the 100 mM solution. The absolute magnitude of the zeta potentials decreased with an increase in salt concentration due to the charge screening effect and electrostatic double layer compression for cell surfaces. In addition, E. faecalis was considerably more negative than E. coli at the same solution conditions. The zeta potentials of E. faecalis cells ranged from −50.01 to −21.65 mV with increasing IS from 1 to 100 mM. Comparable values were reported for E. faecalis at similar conditions.40,41 In contrast, E. coli cells were near neutrally charged over the range of IS tested, with zeta potentials changing from −9.46 to 1.77 mV when IS increased from 1 to 100 mM. At high salt concentrations, the counterions accumulate at close proximity of the cell surfaces and are suspended amidst the cell outmost EPS structures, which was likely leading to the charge reversal of E. coli cells at 100 mM.42,43 The zeta potentials of the quartz sand grains were similar to E. faecalis cells at the same solution conditions (Figure 1a). As shown, the sand was negatively charged at the examined conditions. Therefore, it is expected that the effect of electrostatic repulsion on E. faecalis cells will be much more substantial than that on E. coli in the bacterial transport and deposition experiments. Other Bacterial Cell Characterization. The potentiometric titration was employed to further examine the charge density and distribution on both FIB cell surfaces. The results of the titration are presented in Figure 1b as the acidity or titrated charge (in milliequivalents per 108 cells) as a function of pH.32 The surface charge density was calculated from the acidity and accounting for the surface area of a cell and provides a measure of the total charged functional groups not only on 8784



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Figure 2. Representative breakthrough curves for (a, b) E. faecalis and (c, d) E. coli as a function of solution IS under (a, c) saturated and (b, d) unsaturated (70% saturation) conditions. Experimental conditions were as follows: average interstitial fluid velocity = 0.021 cm/s, mean sand grain diameter = 275 μm, and temperature = 22−23 °C.

test. The analysis established 86.3% and 72.5% of cells in 10 mM NaCl solution partitioned into dodecane for E. faecalis and E. coli, respectively (Table S1, SI). The data indicate that the two FIB cells were quite hydrophobic in NaCl solution, and E. faecalis was slightly more so than E. coli. Transport and Retention of FIB Cells in Saturated Porous Media. To evaluate the deposition and transport behavior of E. faecalis and E. coli cells in sediment under saturated conditions relevant to coastal aquatic environments, the FIB cell transport breakthrough curves were obtained for a wide range of solution IS, i.e. 1−1000 mM (NaCl) at pH 5.6− 5.8. DI water injection was followed upon the completion of a regular bacterial transport experiment to examine the release potential of the deposited bacterial cells from the column due to a dramatic salt concentration drop, which would occur in events such as rainfall infiltration and groundwater recharge. The representative results are shown in Figure 2a and 2c for E. faecalis and E. coli, respectively. In these breakthrough curves, the normalized effluent bacteria concentration (C/C0) is plotted as a function of pore volume. Furthermore, the eluted mass fractions from the effluent of a regular column experiment and DI water flushing for both strains were summarized in Table 1 for each condition. It shows that both bacterial strains exhibited varying extent of mobility in porous media at the conditions tested. As seen in Figure 2a and 2c, distinct effects of solution IS on the transport behavior of E. faecalis and E. coli cells were observed. Specifically, the normalized effluent concentrations of E. faecalis cells were reduced as IS increased from 1 to 100 mM

the outer membrane surface but also within the extracellular polymeric matrix as well. The characterization results indicate that E. faecalis cells are substantially more acidic than E. coli cells. Specifically, E. faecalis has almost 4 times the density of charged groups as E. coli in NaCl solution. As indicated in Table S1, the corresponding titrated surface charge densities are 4947.7 and 1252.0 μC/cm2 for E. faecalis and E. coli cells, respectively. These measured charge densities for the FIB cells are in line with the zeta potential data discussed above, which E. faecalis was considerably more negative than E. coli at the same solution conditions. Interestingly, the titrated surface charge density for the FIB E. coli tested in this study compares well to the value reported for E. coli D21g harvested at midexponential stage (796.8 μC/cm2)32 that is known to produce little or no EPS.44 However, the zeta potentials or the mobility of the FIB E. coli cells are substantially less than the reported values for the D21g cells32 at comparable aqueous conditions, presumably due to a significant fraction of the dissociable function groups of the FIB E. coli cell “hiding” within or shielded by the extracellular polymeric matrix which are on the outer cell membrane surface. This prevents these functional groups from contributing to the mobility of cells in aqueous solution under an electric field due to the soft particle structure.44,45 It also implies that the FIB E. coli cells are likely covered by a thick layer of EPS, which may have important implications to mediate the deposition behavior of E. coli cells in porous media through steric interactions between cell and sand surfaces.46 The final characterization performed on the two FIB cell lines was the measurement of hydrophobicity by the MATH 8785



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Table 1. Transport Results of E. faecalis and E. coli Cells Obtained from Column Experimentsa strain

saturation (%)

IS (mM)

effluent before DI flushing (%)b

recovered from DI flushing (%)c

E. faecalis

100

1 100 1000 1 100 1000 1 100 1000 1 100 1000

85.1 ± 0.4 3.0 ± 0.7 3.6 ± 1.0 55.8 ± 0.5 1.6 ± 0.0 0.8 ± 0.1 11.7 ± 0.3 15.4 ± 6.6 28.1 ± 1.1 18.1 ± 2.9 15.4 ± 0.4 8.8 ± 0.5

22.9 ± 7.9 4.6 ± 1.0 3.6 ± 0.2 16.5 ± 2.4 10.3 ± 0.0 19.2 ± 2.3 36.7 ± 11.6 33.8 ± 3.2 53.8 ± 4.3 43.5 ± 1.0 38.7 ± 1.5

70

E. coli

100

70

profiles presented in Figure 3a, in that increasing the IS substantially lowered the energy barriers that prevent bacterial cells from attaching to sand surface and also deepened the secondary energy minimum wells that may retain cells in the column. Similar transport behavior was observed for E. faecalis and other bacterial strains.32,41,47 In contrast, the effluent concentrations of E. coli remained the same as the IS increased from 1 to 100 mM and slightly increased at 1000 mM (Figure 2c and Table 1). Such transport behavior of E. coli is inconsistent with the DLVO interaction energy profiles shown in Figure 3c. The interaction energy calculations predict an energy barrier of 95 kT at 1 mM and no energy barrier at 100 mM for E. coli cell interacting with sand surface. One would expect more cells retained in the column when the IS increased from 1 to 100 mM if only the DLVO interactions dominated the transport and deposition of E. coli, which is not the case herein. Comparable transport characteristics were observed for other E. coli strains such as O157:H7 at similar aqueous conditions.25,48 It was attributed to steric stabilization of macromolecules on the surface of cells that may hinder bacterial attachment in the primary minimum as the DLVO interaction energy calculations suggested favorable deposition conditions.25 Therefore, it is hypothesized that a combination of the DLVO interactions and a steric repulsion dominated the deposition and transport behavior of E. coli cells observed in this study. In addition, a large fraction of deposited E. coli cells was likely retained in the secondary energy minimum which

a

The percentages presented in the table were based on the bacterial cell mass balance. bThe percentage of injected cells that were recovered by integration of the breakthrough curves in Figure 2 during the initial bacterial transport stage. cThe percentage of retained cells during the initial transport stage that were recovered due to the DI water flushing as shown in Figure 2.

and essentially remained the same as at the condition of 100 mM with further increasing IS up to 1000 mM (Table 1). Overall, the transport and retention behavior of E. faecalis cells is in qualitative agreement with the DLVO interaction energy

Figure 3. Calculated DLVO interaction energy profiles for (a, b) E. faecalis and (c, d) E. coli cells as a function of solution IS interacting with (a, c) water/sand interface and (b, d) water/air interface existing in the column under unsaturated conditions. The profiles at IS of 10 mM are included to better demonstrate the general trend of interaction energy profiles varying with IS. The inset presents a closeup of the secondary energy minimum region of the interaction energy profile. Values used for the calculations are given in the text. 8786



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deposition in porous media observed in this study, elution experiments were performed. Upon the completion of a regular column experiment, the elution experiments were conducted by introducing DI water to the column with the same flow velocity at pH 5.6−5.8. The results of these elution experiments are presented in Figure 2a and 2c. As observed, a rinse of the column with DI water resulted in a considerable amount of deposited cells to release under all conditions for both FIB species. The observed elution breakthrough curves were similar to those reported in the literature,51−54 which provided experimental evidence that the mechanism of the secondary energy minimum played an important role on the deposition of bacterial cells in porous media at conditions studied herein. The experimental observations also corroborate the results of the interaction energy calculations discussed above. For E. faecalis, 22.9% of the deposited cells were eluted out from the column at 100 mM, and the corresponding percentage at 1000 mM was only 4.6% even though similar amount of cells retained in the column at the two conditions (Table 1). The values suggest that a small amount of the retained E. faecalis cells was captured in a secondary energy minimum even when the IS was raised up to 1000 mM. It also indicates that a large fraction of bacteria could attach at a primary energy minimum due to bacterial polymer bridging55 or an additional attractive hydrophobic interaction and/or the charge heterogeneity and surface roughness of both of the bacterial and sand grain surfaces at 100 mM since the percentage of released bacteria was only 22.9%, not 100%.47 As seen in Figure 2c and Table 1, 19.2 to 36.7% E. coli cells were washed out when the columns were subject to DI water flushing at IS values of 1, 100, and 1000 mM, which confirms the earlier hypothesis that the secondary energy minimum plays an important role in the capture of E. coli cells in the column at the conditions tested. Transport and Retention of FIB Cells in Unsaturated Porous Media. The exchange of FIB between beach sand and water column may not only be limited in submerged sediments. Studies revealed that a substantial amount of FIB cells resides in intertidal zone sand. The concentrations of FIB cells harbored in 100 g sand in these areas are often 2 to 38 times higher than the concentrations in 100 mL adjacent beach water, occasionally up to 460 times.9,12 These FIB cells can potentially move into water columns through tide events or rainfall filtration during which the sand porous media in these zones may be partially saturated. To assess the mobility of FIB cells in unsaturated porous media where air exists as an extra phase, unsaturated column experiments were conducted for both FIB strains as a function of IS, i.e. 1−1000 mM (NaCl) at pH 5.6− 5.8. The saturation level was controlled to be 70% to ensure FIB cell being able to migrate in porous media, while a significant air phase was maintained in porous media. The representative breakthrough curves and the resulting mass fractions eluted from the column at different stages are shown in Figure 2b, 2d, and Table 1 for each condition. In addition, to better understand the influence of the existence of an air/water interface on the FIB cell transport and deposition behavior, the DLVO interaction energy profiles were calculated for a bacterium approaching to an air/water interface and the results are presented in Figure 3b and 3d for E. faecalis and E. coli, respectively. As shown in Figure 2, distinct effects of the presence of an air phase in porous media are observed for E. faecalis and E. coli cells. Specifically, the transport of E. faecalis in unsaturated

likely exists through the interplay of the DLVO and steric interactions occurring simultaneously. It is well established that many gram-negative cells exude EPS, which typically consists of proteins, polysaccharides, and nucleic acids.49 In addition, the outer membrane of E. coli is a lipid bilayer, which primarily contains LPS and proteins.49 The existence of such a layer of macromolecular matrix on the outer surface of E. coli cells was also implied by the measurements of the electrophoretic mobilities and the titration tests for the bacteria as discussed above. Such macromolecules associated with cell surface extrude into medium and thus likely execute an influence on the interactions between bacterial cell and sand surfaces upon closely approaching. 32 In addition, the conformation of the polymeric matrix is subject to variations depending on factors such as the surrounding solution chemistry and the nature of the macromolecules; as a result, it may promote bacterial deposition or impede its attachment.46 For instance, at low IS, the macromolecules can enhance cell deposition due to the cell surface polymers “bridging” between the cell and collector surfaces.50 However, at these low-ionicstrength conditions, electrostatic repulsion often dominates. Above a certain IS, the polymers can overcome the electrostatic repulsion and contribute to cell deposition, which depends on solution chemistry and the functionality of the cell polymers.50 Finally, the macromolecules may generate a repulsive interaction and impede further cell deposition at higher ISs which are often greater than 0.1 M.24,46 This is due to the presence of excess ions suspended among the polymers, which may increase the intramolecular electrostatic interactions between individual polymer units and lead to the polymers being more rigid. This rigidity minimizes the ability of the polymers to reconform and interact directly with the collector surface to produce a steric repulsion. The experimental results discussed herein are in line with these bacteria-collector interaction features. As seen in Figure 2c and Table 1, the retention of E. coli was insensitive to IS between 1 and 100 mM, while the DLVO interactions transitioned from unfavorable to favorable deposition conditions as suggested in Figure 3c. It is likely that the macromolecules on E. coli surface adapted different conformations to respond the surrounding solution IS change from 1 to 100 mM, as a result, the contributed interaction of the macromolecules transferred from “bridging” at 1 mM to repulsion at 100 mM. A combination of interactions induced by the macromolecular layer and the DLVO interactions then rendered the insensitivity of E. coli deposition behavior to the IS change. Further increasing the IS to 1000 mM resulted an enhanced steric repulsion and consequently increased bacterial transport (15.4% exited the column at 100 mM versus 28.1% at 1000 mM). The potential contribution of steric interactions was is illustrated in the SI. Role of Secondary Energy Minimum on FIB Retention and Transport. As discussed above, the secondary energy minimum may be an important mechanism for both FIB strain cells retained in the column. It is widely accepted that following the completion of a regular transport experiment, the elution of retained particles by introducing a lower IS solution to the column can be attributed to the particles released from a secondary energy minimum.51,52 A drastic decrease in IS can substantially reduce or even eliminate the secondary energy well to cause a release of the particles retained in the well. To experimentally verify if the secondary energy minimum is an important mechanism governing the FIB cell transport and 8787



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water chemistry change resulted in significantly more E. coli cells eluted out of the column upon DI water flushing in unsaturated porous media (Table 1). Environmental Implications. Overall, this study investigated the transport and retention behavior of E. faecalis and E. coli cells, two FIB strains commonly found in beach environments, in saturated and unsaturated porous media under conditions of coastal aquatic environment interest. The experimental results have shown distinct effects of solution chemistry and saturation level on the transport and deposition kinetics of the two strains in porous media. The transport and deposition of the gram-positive species E. faecalis appeared to be much more sensitive to these factors than that of gramnegative species E. coli. It was the different surface characteristics of the two FIB strains that resulted in the distinct transport and retention behavior in porous media. In general, gram-positive E. faecalis cell wall only has a single lipid bilayer which primarily consists of peptidoglycan, whereas besides peptidoglycan, gram-negative E. coli cells contain an additional cell wall layer made of LPS and also possibly a thick EPS coating.59 Our current understanding of the potential exchange of FIB between water column and beach sediment and sand is primarily based on macroscopic observations. The present study aimed to better understand the specific interactions between FIB cells and sand and air−water interfaces and the resulting effect on the fate and transport of FIB at different conditions, which might provide an effective approach to obtain mechanistic insights for those macroscopic observations. Results from this study suggest that FIB migration potentials can be determined by the microscopic interactions between FIB cells and sand grains in aquatic environments. For instance, Alm et al. observed that enterococci were most abundant in the 5−10 cm sand stratum and E. coli in the 0−5 cm stratum in a freshwater sediment.12 This study demonstrated much less affinity of enterococci to sediment grains than E. coli cells at low IS, which might facilitate the exchange of enterococci between the water column and top layer of sediment. Such results are consistent with the previous trends reported by Alm et al.12 and may provide mechanistic insights for these macroscopic observations. In addition, Russell et al. recently demonstrated that enterococci in unsaturated surface sand subject to transient infiltration of seawater due to the rising tide and wave uprush could be mobilized and transported to the groundwater table.60 Once present in groundwater, Boehm suggested that enterococci could be potentially transported to seawater through submarine groundwater discharge.18,60 This conceptualized enterococci transport pathway is in line with the results reported herein in that enterococci preferred to be associated with air/water interface which would naturally occur in transient infiltration process and were quite mobile in saturated porous media at low IS conditions such as groundwater. Furthermore, this study suggests that E. coli mostly associated with sand surface in secondary energy minima at both freshwater and seawater conditions, which the cells would be easily dislodged from sand. Ge et al. indicated the significance of nearshore sediment resuspension and swash on the beach surface to influence E. coli loading from sediment and sand to sea and subsequently the variability of E. coli concentration in both knee-deep and offshore waters.11,61 The results and approaches presented can be useful to obtain mechanistic insights for the fate and transport of FIB in coastal aquatic

columns was retarded at all conditions as compared to the transport in the corresponding saturated conditions (Table 1). For instance, the percentage of cells showing up in effluents decreased from 85.1% to 55.8% at 1 mM as the saturation level was lowered from 100% to 70%. In contrast, the mobility of E. coli cells was hardly affected under unsaturated conditions, except at 1000 mM in which the transport was slightly hindered as compared to the saturated condition. Furthermore, fewer E. faecalis cells were eluted out when the column was subject to DI water flushing under unsaturated conditions as compared to the corresponding saturated conditions. The opposite was observed for E. coli whereas more cells were eluted out under unsaturated conditions (Table 1). Previous studies have established that bacteria accumulation at the air/water interface is largely governed by a combination of the DLVO and hydrophobic interactions between approaching cells and air/water interfaces.56,57 Moreover, the hydrophobic interaction often dominates the attachment of bacteria to an air/water interface, in that the attachment is inhibited by the repulsive van der Waals interaction at the air/ water interface and the repulsive electrostatic interaction due to the negative charges of both bacterial surface and the interface. It is further proposed that two additional mechanisms likely contribute to increased particle retention in unsaturated porous media, including film straining and air/water interfacial capture.58 It is not likely that film straining was responsible for the increased E. faecalis and E. coli cell retention in unsaturated porous media as compared to the saturated experiments. In an unsaturated system, film straining characterizes as a physical mechanism to retain particles and happens when thickness of water film on sand grains becomes too narrow to allow particle passing.58 In this study, the saturation level was maintained at 70% and the transport of E. coli remained unaffected at low IS in unsaturated porous media. It is therefore proposed that air/water interfacial capture was responsible for the observed cell retention increase of E. faecalis at all IS conditions and E. coli at 1000 mM in unsaturated porous media. The influence of the air phase seems more profound for E. faecalis than E. coli, likely due to the different affinity of the two species to air/water interface. As seen in Figure 3, E. faecalis cells experienced a lower DLVO interaction energy barrier for approaching an air/water interface compared to that of sand surface at 1 mM. Considering the air/water interface is more hydrophobic than the sand surface and E. faecalis cells being hydrophobic as suggested by the MATH tests discussed earlier, it is likely that a combination of the hydrophobic and DLVO interactions promoted E. faecalis irreversible attachment at air/ water interface in unsaturated porous media. In other words, E. faecalis cells preferred to deposit at air/water interface rather than sand surface in unsaturated experiments. In contrast, Figure 3 suggests substantial greater DLVO energy barrier for E. coli cells to overcome to be captured by an air/water interface than a sand surface (Similar steric interactions may be assumed when E. coli approaches a sand surface or an air/water interface. Therefore, its contribution is neglected in the following discussion.). In addition, E. coli cells were relatively less hydrophobic than E. faecalis cells. Therefore, the affinity of E. coli cells to an air/water interface and sand surface was likely similar. As a result, the transport behavior of E. coli was hardly varied in unsaturated porous media compared to at saturated conditions. In addition, it was likely that the different response of a water/sand interface and an air/water interface to pore 8788



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environments, which is critical to understanding and predicting microbial water quality in coastal waters.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Additional text, figures, and tables. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Phone: (951) 827-6094. Fax: (951) 827-5696. E-mail: [email protected]. Present Address †

U.S. EPA, Kerr Environmental Research Center, 919 Kerr Research Drive, Ada, OK 74820. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research work was supported by the University of California Marine Council. The authors are grateful to the five anonymous reviewers for their constructive comments that helped improve the quality of the paper.

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REFERENCES

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