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Sep 12, 2012 - Influence of Bentonite Particles on Representative Gram Negative and Gram Positive Bacterial Deposition in Porous Media. Haiyan Yang ...
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Influence of Bentonite Particles on Representative Gram Negative and Gram Positive Bacterial Deposition in Porous Media Haiyan Yang, Meiping Tong,†,* and Hyunjung Kim‡,* †

The Key Laboratory of Water and Sediment Sciences, Ministry of Education, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, People's Republic of China ‡ Department of Mineral Resources and Energy Engineering, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do 561-756, Republic of Korea S Supporting Information *

ABSTRACT: The significance of clay particles on the transport and deposition kinetics of bacteria in irregular quartz sand was examined by direct comparison of both breakthrough curves and retained profiles with clay particles in bacteria suspension versus those without clay particles. Two representative cell types, Gram-negative strain E. coli DH5α and Gram-positive strain Bacillus subtilis were utilized to systematically determine the influence of clay particles (bentonite) on cell transport behavior. Packed column experiments for both cell types were conducted in both NaCl (5 and 25 mM ionic strengths) and CaCl2 (5 mM ionic strength) solutions at pH 6.0. The breakthrough plateaus with bentonite in solutions (30 mg L−1 and 50 mg L−1) were lower than those without bentonite for both cell types under all examined conditions, indicating that bentonite in solutions decreased cell transport in porous media regardless of cell types (Gram-negative or Gram-positive) and solution chemistry (ionic strength and ion valence). The enhanced cell deposition with bentonite particles was mainly observed at segments near to column inlet, retained profiles for both cell types with bentonite particles were therefore steeper relative to those without bentonite. The increased cell deposition with bentonite observed in NaCl solutions was attributed to the codeposition of bacteria with bentonite particles whereas, in addition to codeposition of bacteria with bentonite, the bacteria−bentonite−bacteria cluster formed in suspensions also contributed to the increased deposition of bacteria with bentonite in CaCl2 solution.

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INTRODUCTION Understanding the factors affecting transport and deposition of bacteria in porous media is essential for processes such as riverbank filtration,1,2 protection of drinking water supplies from bacterial contamination,3,4 and in situ bioremediation.5,6 Bacteria transport in porous media is affected by a number of physical, chemical, and biological factors such as grain shape and size,7,8 fluid conditions,8,9 solution ionic strength and ionic composition,10,11 nutrient conditions,12,13 bacterial growth phase,14 bacterial cell type,15,16 motility,9,17 lipopolysaccharides (LPS),18,19 and extracellular polymeric substances (EPS).20,21 Colloids such as natural organic matter present in solutions have also been shown to have great influence on the transport and deposition kinetics of bacteria in porous media.22,23 Clay particles, one of the most abundant inorganic colloids present in aquatic systems, have been regarded as one of the best natural adsorbents to adsorb pollutants such as metal ions,24,25 pesticides,26 and other chemicals from aqueous solutions.26,27 Due to their high specific surface area and high cation exchange capacity, clay particles have also been found to interact with microbes such as viruses28−30 and bacteria.31,32 While many studies have been conducted to investigate the fate and transport of microbes in porous media (e.g., refs 7, 9, 10, © 2012 American Chemical Society

18, and 21), the study regarding the role of clay particles on the transport behavior of microbes is relatively little33−35 and contradictory results have been reported.33,34 For example, Jin et al.33 reported that the clay particles could facilitate the transport of bacteriophages MS2 in porous media by investigating the transport of viruses with various clay particles. Unlike the observation from Jin et al.,33 Vasiliadou and Chrysikopoulos35 very recently showed that kaolinite particles significantly inhibited the transport of Pseudomonas putida in spherical glass beads. The authors proposed that the attachment of bacteria onto kaolinite particles that retained onto the solid matrix of the column contributed to the increased deposition of Pseudomonas putida in porous media. Since the contradictory observations were reported in these few previous studies, the role that clay particles play on the bacterial transport in porous media is still not fully clear and needs more attention. In addition, investigation regarding the cotransport of clay particles with bacteria in porous media under different Received: Revised: Accepted: Published: 11627

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into 5 mL supernatant samples, and the mixture was centrifuged at 1000 ×g for 3 min as suggested by Vasiliadou and Chrysikopoulos35 and Jiang et al.38 The Histodenz solution was used as a density gradient separation reagent to separate the suspended bacteria from bentonite particles. The supernatant samples from recovery of retained bacteria (after treated with Histodenz solution) and the effluent samples were counted using a counting chamber with an inverted fluorescent Ti-E microscope under bright field to yield cell concentration in each sample. Detailed information for determining mass balance was provided in the Supporting Information. SEM Images. After finishing the clay and bacteria (with bentonite in solutions) transport experiments, the quartz sand in the segment near the column inlet was checked for deposited colloids (bentonite particles and cells) by SEM analysis (FEI Nova Nano SEM 430) after air-dried. SEM images of bacteria with bentonite both in NaCl and CaCl2 solutions were also taken. Prior to the SEM analysis, the cell suspension was dropped onto a small piece of cleaned glass and dried in air. SEM micrographs were taken at a magnification of ∼20 000X.

solution composition, which has been known as one of the major factors significantly affecting bacterial transport in porous media,10,11 has not been systematically performed and thus requires examination. Furthermore, the influence of clay particles on the retained bacteria profiles, from which the mechanisms controlling the retention/deposition can be derived, has not been previously investigated. Hence, this study was designed to fully understand the role of clay particles on the transport and deposition behavior of bacteria by monitoring both column breakthrough and retained bacteria profiles in packed porous media under environmentally relevant solution environments. Specifically, two representative cell types that are known to be widely present in aquifer and soil,36,37 Gram-negative strain E. coli DH5α and Gram-positive strain Bacillus subtilis, were utilized to determine the influence of clay particles on bacteria transport. Bentonite, three layer (TOT) clay, which is ubiquitously present in groundwater, was employed as a model clay particle in this study. Packed column experiments were performed both with and without bentonite in bacterial suspension in both monovalent and divalent salt solutions. The breakthrough curves and retained concentration profiles with bentonite were compared with those without bentonite in solutions. Possible mechanisms by which clay particles affect the transport behavior of bacteria were proposed and discussed.

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RESULTS AND DISCUSSION Influence of Clay Particles on Breakthrough Curves. The transport behavior of a Gram-negative bacterium strain E. coli DH5α in packed quartz sand with and without bentonite were examined at two ionic strengths (5 and 25 mM) in NaCl solution. Flat breakthrough plateaus were observed under both ionic strengths with (both at concentrations of 30 and 50 mg L−1) and without bentonite (Figure 1a,c). The breakthrough plateaus acquired at high ionic strength (25 mM) (Figure 1c) were slightly lower than those at low ionic strength (5 mM) (Figure 1a) regardless of the presence or absence of bentonite in solutions, which was consistent with less negative ζ potentials observed at high ionic strength (Table S2 of the SI) and thus agreed with classic DLVO theory. The increase in deposition with ionic strength was consistent with previous studies.20,21 More important observation was that at the same ionic strength, breakthrough plateaus for E. coli DH5α with bentonite in solutions for both clay concentrations examined (Figure 1a,c, circle and triangle) were lower than those without bentonite (Figure 1a,c, square). This observation was true for both clay concentrations at both ionic strengths examined in NaCl solutions. The results clearly demonstrated that for Gramnegative E. coli DH5α, the presence of bentonite particles in solutions (both 30 mg L−1 and 50 mg L−1) increased cell deposition (decreased cell transport) in packed quartz sand regardless of ionic strength conditions examined in NaCl solutions. Close comparison of breakthrough curves obtained with high clay concentrations (50 mg L−1) versus those obtained at low clay concentrations (30 mg L−1) showed that for both ionic strength conditions examined, breakthrough plateaus for high clay concentrations (Figure 1a,c, triangle) were lower than those for low clay concentration (Figure 1a,c, circle), indicating that the enhancement of cell deposition was more significant when greater concentrations of clay particles were present in bacteria suspensions. Vasiliadou and Chrysikopoulos35 also found that the deposition of Gram-negative Pseudomonas putida increased with increasing concentration of kaolinite in cell suspensions. To test whether the increased cell deposition (reduced cell transport) observed for Gram-negative bacteria strain E. coli DH5α with bentonite particles in bacteria suspensions also held

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MATERIALS AND METHODS Cell Culture and Preparation. Two bacterial cell types, a Gram-negative strain E. coli DH5α (2.01 ± 0.34 μm × 0.98 ± 0.06 μm) and a Gram-positive strain Bacillus subtilis (2.50 ± 0.20 μm × 0.71 ± 0.13 μm), were used in this study. The bacteria were grown and harvested according to the protocols described in the Supporting Information. The target influent bacteria concentration was 1.0 × 108 ± 10% cells mL−1. The information to determine bacterial concentration and the stock suspension concentration was provided in the Supporting Information. Bentonite Suspension Preparation. Bentonite (SigmaAldrich, St. Louis, MO) was employed as a model clay to study the influence of clay particles on the transport behavior of bacteria in this investigation. The clay stock solution was prepared by dissolving 20 mg of clay powders in 100 mL MilliQ water (Q-Gard1, Millipore Inc., MA) and stirring the solution for 24 h. Prior to use, the stock suspension was sonicated for 5 min with a sonicating probe (Ningboxinzhi Biotechnology Ltd., P.R. China). More information of bentonite suspension was provided in the Supporting Information. Column Experiments. The porous media used for bacteria transport experiments were quartz sand (ultrapure with 99.80% SiO2) (Hebeizhensheng Mining Ltd., Shijiazhuang, P.R. China) with median diameter of 280 μm. Quartz sand cleaning protocol as well as detailed procedures for bentonite and bacteria (in the absence/presence of bentonite) transport experiments were provided in the Supporting Information. Following the bacterial transport experiment, the sediment was extruded from the column under gravity and dissected into 10 segments (each 1 cm long). To release the retained bacteria and bentonite from quartz sand and to separate bacteria from bentonite, specified volumes of 0.1 M NaOH solution was first added into each sediment segment and shaking at 300 rpm for 30 min and then sonicating for 5 min. After that, 0.75 mL of Histodenz solution (40% by weight, Sigma D2158) was added 11628

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Figure 2. Breakthrough curves (a) and retained profiles (b) for E. coli DH5α without (solid square) and with the presence of bentonite at concentrations of 30 mg L−1 (solid circle) and 50 mg L−1 (solid triangle) in bacteria suspensions at 5 mM ionic strength in CaCl2 solutions at pH 6.0. Error bars represent the range of two replicate experiments (n = 2). The lines are meant to guide the eye. Figure 1. Breakthrough curves (left) and retained profiles (right) for E. coli DH5α (a−d) and Bacillus subtilis (e and f) without (solid square) and with the presence of bentonite at concentrations of 30 mg L−1 (solid circle) and 50 mg L−1 (solid triangle) in bacteria suspensions in NaCl solutions (5 and 25 mM for E. coli DH5α, 5 mM for Bacillus subtilis) at pH 6.0. Error bars represent the range of two replicate experiments (n = 2). The lines are meant to guide the eye.

mg L−1 bentonite particles (Figures 2a and S1a of the SI, triangle) were also lower than those with 30 mg L−1 clay particles (Figures 2a and S1a of the SI, circle). The observations demonstrated that the enhancement of cell deposition by bentonite also held true in divalent Ca2+ solutions. Close comparison of breakthrough curves obtained in NaCl solutions (Figure 1, left) versus those acquired in CaCl2 solutions (Figures 2a and S1a of the SI) yielded that at the same ionic strength (5 mM) for both cell types, the presence of bentonite particles induced greater decreases of breakthrough plateaus in CaCl2 solutions (Figures 2a and S1a of the SI) relative to those in NaCl solutions (Figure 1, left). For example, breakthrough plateau for E. coli at 5 mM ionic strength in NaCl solutions decreased (15%) from 0.85 without clay particles to 0.74 with 30 mg L−1 bentonite particles, whereas, plateau for E. coli at 5 mM ionic strength in CaCl2 solution decreased (95%) from 0.79 without bentonite to 0.04 with 30 mg L−1 bentonite particles. This observation demonstrated that effects of bentonite particles on the transport of bacteria were more significant in divalent CaCl2 solutions. The above results showed that the presence of bentonite in solutions decreased cell transport in packed quartz sand regardless of cell type (Gram-negative or Gram-positive) and solution chemistry (ionic strength and ion valence). Moreover, the effects of bentonite particles on the transport of bacteria were more significant with the higher clay concentrations and with divalent Ca2+ in solutions. The decreased bacteria transport by the presence of clay particles in solutions has also been observed for bacteriophages MS234 and Gramnegative Pseudomonas putida.35 Influence of Clay Particles on Retained Profiles. To examine whether the presence of bentonite particles in bacterial suspensions would affect the profiles of retained bacteria, the retained profiles for both cell types with and without clay

true for Gram-positive bacteria, the transport behavior of a Gram-positive Bacillus subtilis in packed quartz sand was examined both with (at concentrations of 30 mg L−1 and 50 mg L−1) and without bentonite particles in bacteria suspensions. Similar to the observations for Gram-negative E. coli DH5α, the breakthrough plateaus for Bacillus subtilis with bentonite at two concentrations (30 mg L−1 and 50 mg L−1) (Figure 1e, circle and triangle) were also lower than those without bentonite (Figure 1e, square). Moreover, the breakthrough plateau obtained with high clay concentration in bacteria suspensions (Figure 1e, triangle) was also lower than that with low clay concentration (Figure 1e, circle). These observations showed that the presence of bentonite in solutions also reduced Grampositive bacteria transport in quartz sand and the increase of clay concentration increased cell deposition. To investigate whether the presence of background bentonite particles could also increase cell deposition (reduce cell transport) when divalent ions were present in solutions, the transport behavior of Gram-negative E. coli DH5α and Grampositive Bacillus subtilis were examined both with (30 and 50 mg L−1) and without bentonite particles at 5 mM ionic strength in CaCl2 solutions. Similar to the observations in NaCl solutions (Figure 1, left), flat breakthrough plateaus obtained for both bacteria types in CaCl2 solutions with bentonite particles were also lower than those without clay particles (Figures 2a and S1a of the SI). In CaCl2 solutions, the breakthrough plateaus for both bacteria types acquired with 50 11629

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Figure 3. Scanning electron microscopy images of Bacillus subtilis with the presence of 30 mg L−1 bentonite at 5 mM ionic strength in NaCl solution (a), and in CaCl2 solution (b), bentonite particle deposited on the quartz sand for bentonite transport experiment (c), and Bacillus subtilis−bentonite attached onto quartz sand for transport experiment of Bacillus subtilis in the presence of bentonite.

the same ionic strength (5 mM) in NaCl solutions (Figure 1, right) showed that the retention of bacteria was greater in CaCl2 solutions relative to that in NaCl solutions, which was true for both cell types. Moreover, with bentonite particles in bacteria suspensions, the observed excess retention in CaCl2 solutions relative to that in NaCl solutions also mainly located at segments near the column inlet, as a result, retained profiles with bentonite particles in CaCl2 solutions were relatively steeper relative to those in NaCl solutions. The above results showed that the presence of bentonite in solutions increased cell retention and affected the shape of profiles of retained bacteria in packed quartz sand for both cell types under all examined solution conditions. More importantly, the excess retention observed with clay particles mainly located at segments near the column inlet. Mechanisms of Enhanced Cell Retention by Bentonite Particles. Although the overall ζ potentials of bentonite particles were negative (Table S2 of the SI), previous studies39−41 have shown that charge heterogeneity was present on the surface of clay particles, as a result, the interaction between bacteria and clay particles have been observed.31,32,38 In fact, bentonite particles were observed to attach onto the surface of bacteria in both NaCl (Figure 3a) and CaCl2 solutions (Figure 3b), and even greater degree of aggregation was observed in CaCl2 solution (Figures 3b and S3 of the SI). To understand whether the interaction between cells and clay particles would affect the electrokinetic properties of bacteria, ζ potentials of E. coli DH5α and Bacillus subtilis were measured both with and without bentonite particles in suspensions both

particles in both NaCl (Figure 1, right) and CaCl2 (Figures 2b and S1b of the SI) solutions were obtained. The magnitudes of the retained profiles for both cell types under all examined conditions varied oppositely to the breakthrough plateaus (Figures 1, left; 2a; and S1a of the SI), as expected from mass balance consideration (Table S1 of the SI). Specifically, under all examined conditions, the amounts of bacteria retained in porous media for both cell types with bentonite particles (Figures 1 right; 2b; and S1b of the SI, circle and triangle) were greater relative to those without clay particles (Figures 1 right; 2b; and S1b of the SI, square). Moreover, the increase of concentrations of bentonite particles from 30 to 50 mg L−1 induced greater cell retention in packed quartz sand (Figures 1 right;2b; and S1b of the SI, circle and triangle). Close inspection of retained profiles obtained when bentonite particles were present in bacteria suspensions (Figures 1 right; 2b; and S1b of the SI, circle and triangle) versus those without bentonite particles (Figures 1 right; 2b; and S1 b of the SI, square) showed that the increased retention of bacteria due to the presence of bentonite in solutions mainly occurred at segments near the column inlet, which induced relatively steeper retained profiles of bacteria for both cell types with clay particles relative to those without clay particles in both NaCl and CaCl2 solutions. Likewise, the increased cell retention observed with high concentration of bentonite (50 mg L−1) relative to those acquired with 30 mg L−1 clay also mainly occurred near the column inlet. Comparison of retained profiles with bentonite obtained in CaCl2 solutions (Figures 2b and S1b of the SI) versus those at 11630

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in NaCl and CaCl2 solutions (Table S2 of the SI). It can be clearly found that ζ potentials for both cell types under all examined conditions were negative. More importantly, for both cell types, ζ potentials for bacteria with bentonite particles (both 30 mg L−1 and 50 mg L−1) in solutions were comparable to those without bentonite. This observation held true in both NaCl and CaCl2 solutions (Table S2 of the SI). The results demonstrated that the presence of bentonite did not have obvious influence on the electrokinetic properties of the cell surfaces for both bacterial types. Since the attachment of clay particles onto sand surface has also been previously reported,42,43 the interaction between suspended bentonite particles and quartz sand is expected during bacteria transport with bentonite. It can be clearly seen from the SEM image of Figure 3c, bentonite particles were attached onto quartz sand as expected. The results for bentonite (30 mg L−1) transport in both NaCl (5 and 25 mM ionic strengths) and CaCl2 solutions (5 mM ionic strength) also showed that a large number of bentonite particles (more than 90%) were retained in packed quartz sand in both NaCl and CaCl2 solutions (Figure S2 of the SI). The attachment of bentonite particles onto quartz sand was significant both in NaCl and CaCl2 solutions under examined conditions. Consequently, surface charges of quartz sand might be altered. Measurement results for ζ potentials of quartz sand both with and without bentonite showed that ζ potentials of quartz sand with bentonite particles were less negative relative to those without bentonite in both NaCl and CaCl2 solutions (Table S2 of the SI), indicating that the attachment of bentonite onto quartz sand decreased the surface charge of quartz sand. Less electrostatic repulsive force is therefore expected between bacteria and quartz sand with bentonite in solutions comparing to that without bentonite, which theoretically explained the observed greater cell deposition with bentonite in solutions relative to those without bentonite particles. To further understand the role of bentonite attached onto quartz sand and suspended bentonite (including bentonite attached to bacteria) on the enhanced cell deposition, additional transport experiments were also performed for cell suspension (with and without bentonite) in the column preequilibrated with 30 mg L−1 bentonite suspension for 5 pore volumes. Direct comparison of breakthrough curves for columns pre-equilibrated with bentonite versus those without pre-equilibration yielded that for both E. coli DH5α and Bacillus subtilis without bentonite in NaCl solutions, the breakthrough plateaus for columns pre-equilibrated with bentonite (Figure 4a,c, open square) were lower than those without bentonite pre-equilibration (Figure 4a,c, solid square). This also held true for both cell types in CaCl2 solutions (Figure 5, left). The results showed that bentonite attached onto quartz sand during pre-equilibration process did favor the following bacteria deposition. Additionally, when bentonite particles were present in bacteria suspension, deposition sites on the surfaces of quartz sand would be more readily occupied by bentonite particles compared to bacteria since bentonite particles were less negatively charged and contained charge heterogeneity.39−41 These bentonite particles attached onto quartz sand would lead to larger deposition of bacteria due to the less negative ζ potentials of bentonite particles relative to bare quartz sand. Accordingly, greater bacteria deposition was observed with bentonite for both cell types examined in our study (Figures 4 and 5). Figure 3d showed that the bacterial cell was deposited

Figure 4. Breakthrough curves (left) and retained profiles (right) for E. coli DH5α (a,b) and Bacillus subtilis (c,d) at 5 mM ionic strength in NaCl with the presence (circle) and absence (square) of 30 mg L−1 bentonite in bacteria suspension with (open symbol) and without (solid symbol) pre-equilibration with 5 pore volumes of 30 mg L−1 bentonite. Error bars represent the range of two replicate experiments (n = 2). The lines are meant to guide the eye.

Figure 5. Breakthrough curves (left) and retained profiles (right) for E. coli DH5α (a,b) and Bacillus subtilis (c,d) at 5 mM ionic strength in CaCl2 in the presence (circle) and absence (square) of 30 mg L−1 bentonite in bacteria suspension with (open symbol) and without (solid symbol) pre-equilibration with 5 pore volumes of 30 mg L−1 bentonite. Error bars represent the range of two replicate experiments (n = 2). The lines are meant to guide the eye.

onto bentonite particles, which appear to have been deposited onto quartz sand prior to the cell deposition, indicating that codeposition of bentonite and bacteria did occur when they were present together. The attachment of suspended bacteria onto kaolinite that was injected together and already attached onto glass beads has recently also been observed by Vasiliadou 11631

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and Chrysikopoulos.35 The above results suggested that the attachment of bentonite onto quartz sand attracted the deposition of bacteria and thus contributed to the increased cell deposition with clay particles. Meanwhile, comparison of the profiles of retained bacteria with bentonite versus those of retained bentonite showed that the shape of profiles of retained bacteria for both cell types (Figures 4 and 5, right) was similar to that of retained clay particles (Figure S2 of the SI, right). This was true in both NaCl and CaCl2 solutions. The observations further indicated that bentonite attached onto quartz sand via pre-equilibration or codeposition process had effects on the deposition of bacteria. More noteworthy observation is that for both E. coli DH5α and Bacillus subtilis at 5 mM ionic strength in NaCl solutions, the breakthrough plateaus without pre-equilibration with bentonite yet with bentonite in solutions (Figure 4 left, solid circle) were higher than those with pre-equilibration with bentonite yet without bentonite in solutions (Figure 4 left, open square), indicating that the increased cell deposition was more significant with predepositing bentonite particles onto quartz sand relative to the suspended bentonite in solutions. Whereas, in 5 mM CaCl2 solution, the opposite trend was observed for both cell types. Specifically, the breakthrough plateaus for columns solely with bentonite in solutions (Figure 5 left, solid circle) were lower than those with pre-equilibration with bentonite yet without bentonite in solutions (Figure 5 left, open square), indicat bentonite in suspension had more significant influence on the enhancement of bacteria attachment relative to the process of predeposition of bentonite onto quartz sand. This observation suggested the role of suspended bentonite on the enhanced bacteria deposition led by bentonite in NaCl solutions might be different from those in CaCl2 solutions, and plausible cell retention mechanisms in NaCl and CaCl2 solutions are discussed below in detail. The lower plateaus for bentonite predepositing sand relative to the presence of bentonite (without pre-equilibration) observed in NaCl solutions indicated that when bentonite was present in bacteria suspension, the increased bacteria deposition observed in NaCl solutions was highly possible to be solely caused by the codeposition of cell with bentonite. Since a portion of suspended bentonite were attached onto suspended bacteria (illustrated in Figure 3 a), the amount of bentonite that was available for depositing onto quartz sand would be limited when 30 mg L−1 bentonite particles were present in bacteria suspension. Thus, the sites, which were provided by the bentonite deposited on quartz sand, for the deposition of bacteria, would be fewer than those of pre-equilibration with bentonite, contributing to less deposition of bacteria observed with bentonite in solutions yet without pre-equilibration. Straining44 and colloid−colloid interaction (i.e., interaction between suspended colloids and colloids already attached onto sand)45 could be also responsible for hyper-exponential decrease of cell retention with bentonite (Figure 4, right). However, comparable size distribution of bacteria with bentonite versus those without bentonite (Figure S3 of the SI) was observed in NaCl solution, indicating that straining unlikely occurred. Additionally, no ripening was observed in breakthrough curves obtained for columns with bentonite in suspension (Figure 4 left, solid circle), suggesting that hyperexponential decrease of cell retention was not induced by colloid−colloid interaction. The above results also provided additional information that bentonite attached onto bacteria in NaCl solutions did not have large contribution to increased

bacteria deposition. This was further confirmed by the observation that comparable breakthrough curves were obtained for columns with pre-equilibration with (Figure 4 left, open circle) and without bentonite in solutions (Figure 4 left, open square). In contrast with observations in NaCl solutions, for both cell types in CaCl2 solutions, the breakthrough plateaus for columns with bentonite predepositing sand (Figure 5 left, open square) were higher than those for columns with bentonite (without pre-equilibration) (Figure 5 left, solid circle). The observation indicated that when bentonite was present in CaCl2 solutions, the increased bacteria deposition was not solely caused by codeposition of cell with bentonite, and other mechanisms resulted from suspended bentonite in bacteria suspension were likely involved. Although ζ potentials of suspended bacteriabentonite particles were found to be similar as those of bare bacteria (Table S2 of the SI), size distribution of both cell types with bentonite in suspension was much wider than those of bacteria without bentonite, and the size of bacteria with bentonite was much larger. The SEM image of Figure 3b showed bacteria−bentonite−bacteria clusters formed in CaCl2 solutions, indicating that bentonite particles acted as binding bridges between cells. The formation of colloid−colloid clusters or aggregates in divalent electrolytes has also been previously reported.46−49 For example, the aggregation of alginate-coated hematite nanoparticles was enhanced due to alginate forming bridges between hematite-alginate gel clusters in Ca 2+ solution.47 Since the flat breakthrough plateaus (no ripening) were also observed with bentonite (Figure 5 left), the contribution of the interaction between suspended and attached bacteria−bentonite−bacteria clusters to the enhanced cell retention and the hyper-exponential decrease in retained profiles was minimal. However, the cluster size (ca. 3.8 μm for E. coli and 2.2 μm for Bacillus subtilis) was found to be much larger than initial cell size (ca. 1 μm for both cells) (Figure S3 of the SI), and the corresponding size ratio of bacteria− bentonite cluster and sand was ca. 0.014 and 0.008 for E. coli and Bacillus subtilis, respectively. The values were over the minimum threshold value (0.005) for straining,50 suggesting that the cluster would be easier to be retained by quartz sand especially at pore throat located near column inlet relative to individual cell. Consistent with this hypothesis, the observed excess retention of bacteria for both cell types due to the presence of bentonite in solutions (Figure 5 right, circle) relative to that without bentonite yet with predepositing bentonite onto sand (Figure 5 right, open square) mainly located at the segment near the column inlet. The above results suggested that bentonite in bacteria suspension enhanced bacteria retention for both Gram-negative and Gram-positive bacteria types in both NaCl and CaCl2 solutions. The codeposition of bacteria with bentonite particles was found to mainly contribute to the excess cell deposition observed when bentonite was present in NaCl solutions, whereas, in addition to codeposition of bacteria with bentonite, the bacteria−bentonite−bacteria cluster formed in suspensions also played an important role on the retention of bacteria in CaCl2 solutions via straining. The results of this study suggest that clay particles greatly inhibit the transport of bacteria in porous media. For places where clay particles are abundantly present, bacteria will be more readily retained in the porous media near to the discharge sites and thus their travel distance will be shortened. 11632

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 10 62756491 (M.T.), +82 63 2702370 (H.K.); Fax: +86 10 62756526 (M.T.), +82 63 2702366 (H.K.); E-mail: [email protected]. (M.T.), [email protected] (H.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China under Grant No. 21177002. The authors wish to acknowledge the editor and reviewers for their helpful comments.

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