Environ. Sci. Technol. 2010, 44, 2393–2399
Contribution of Extracellular Polymeric Substances on Representative Gram Negative and Gram Positive Bacterial Deposition in Porous Media M E I P I N G T O N G , * ,† G U O Y U L O N G , † XUJIA JIANG,† AND HYUNJUNG N. KIM‡ The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Department of Environmental Engineering, Peking University, Beijing 100871, P. R. China, and Department of Environmental Science, University of California, Riverside, California 92521
Received September 17, 2009. Revised manuscript received February 4, 2010. Accepted February 24, 2010.
The significance of extracellular polymeric substances (EPS) on cell transport and retained bacteria profiles in packed porous media (quartz sand) was examined by direct comparison of the overall deposition kinetics and retained profiles of untreated bacteria (with EPS) versus those of treated cells (without EPS) from the same cell type. Four representative cell types, Pseudomonas sp. QG6 (gram-negative, motile), mutant Escherichia coli BL21 (gram-negative, nonmotile), Bacillus subtilis (gram-positive, motile), and Rhodococcus sp. QL2 (gram-positive, nonmotile), were employed to systematically determine the influence of EPS on cell transport and deposition behavior. Packed column experiments were conducted for the untreated and treated cells in both NaCl (four ionic strength ranging from 2.5 mM to 20 mM) and CaCl2 (5 mM) solutions at pH 6.0. The breakthrough plateaus of untreated bacteria were lower than those of treated bacteria for all four cell types under all examined conditions (in both NaCl and CaCl2 solutions), indicating that the presence of EPS on cell surfaces enhanced cell deposition in porous media regardless of cell type and motility. Retained profiles of both untreated and treated cells for all four cell types deviated from classic filtration theory (log-linear decreases). However, the degree of deviation was greater for all four untreated cells, indicating that the presence of EPS on cell surfaces increased the deviation of retained profiles from classic filtration theory. Elution experiments demonstrated that neither untreated nor treated cells preferentially deposited in secondary energy minima. Furthermore, the release of previously deposited cells in the secondary energy minima did not change the shape of retained cell profiles, indicating that deposition in secondary energy minima did not produce the observed deviations of retained profiles from classic filtration theory.
* Corresponding author phone: +86 10 62756491; fax: +86 10 62756526; e-mail:
[email protected]. † Peking University. ‡ University of California. 10.1021/es9027937
2010 American Chemical Society
Published on Web 03/04/2010
Introduction Understanding the transport and deposition behavior of bacteria in porous media is of great interest in a wide range of applications, including protection of drinking water supplies from bacterial contamination (1, 2), in situ bioremediation of contaminated soil (3, 4), and riverbank filtration (5, 6) as well as land disposal of effluents from treated sewage and wastewater (7, 8). There are many factors which have been known to influence the bacteria transport in porous media. For instance, physical and chemical factors such as grain shape and size (e.g., refs 9 and 10), surface coating on grain collector (e.g., ref 11), fluid velocity (e.g., ref 12), solution ionic strength and composition (e.g., refs 13 and 14), and bacteria concentration (e.g., ref 15) have been demonstrated to affect transport behavior of bacteria in porous media. Biophysical and biochemical factors such as bacterial cell type (e.g., gram negative or gram positive) (e.g., refs 16 and 17), motility (e.g., refs 15 and 18), cell size and shape (e.g., refs 19 and 20), outer membrane proteins (e.g., refs 21 and 22), lipopolysaccharides (LPS) (e.g., refs 23 and 24), and extracellular polymeric substances (EPS) (e.g., refs 25-27) have also been shown to have great influence on the transport and deposition kinetics of bacteria in porous media. Although previous investigations have examined the effects of a wide of factors on bacteria transport behavior in porous media, far less attention has been directed toward understanding the mechanisms governing the profiles of retained bacteria in porous media following transport experiment. Therefore, investigations of bacteria transport in porous media have recently focused more on the retained profiles. This focus has yielded new insights on the prevalence of deviation from expectations based on classic filtration theory (28) assuming spatially invariant deposition rate coefficient, which is expected to yield log decreases in retained bacterial concentrations with transport distance. One of the most frequently reported deviations is the hyperexponential decrease in retained bacteria concentrations, which indicates deposition rates of bacteria decrease with travel distance. This hyperexponential decrease in retained concentrations with distance has been attributed to a variety of factors such as heterogeneity in collector grain (charge differences or roughness) (e.g., refs 29 and 30), distributions in surface property among bacteria population (e.g., refs 2, 31, and 32), and physical straining (e.g., refs 35 and 36). The influence of EPS on the bacteria transport behavior (especially on the retained profiles) has recently drawn increasing attention. By examining the deposition behaviors of three genetic mutant Pseudomonas aeruginosa strains with different EPS secretion capability and EPS composition in packed glass bead columns, Liu et al. (26) recently demonstrated that the deposition rate coefficients of the EPSproducing strains were greater than those of the EPS deficient strain under the same conditions. Furthermore, the authors found that the retained profiles of the EPS-deficient strain were different from those of the EPS-producing strains, and, more importantly, the retained profiles for both EPSproducing strains obtained under all examined conditions deviated from the classic filtration theory. These authors concluded that the presence of EPS had profound influence not only on the overall cell deposition but also on the deviation of retained bacteria profiles from the classic filtration theory. Although this study clearly showed that the enhancement of cell deposition in packed porous media was due to the presence of EPS, a few other studies (e.g., refs 27 and 35-38) have yet found that the presence of EPS on cell VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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surfaces could hinder cell deposition. For example, by comparing the removal efficiency of untreated and proteinase K treated Escherichia coli O157:H7 on quartz sand in a batch system, Kim et al. (38) recently reported that the greater cell removal occurred for EPS-partially removed cells when ionic strength g1 mM, suggesting that the presence of EPS hindered cell deposition. It should be noted that the relevance of the significance of EPS on cell deposition to different cell types has not been systematically explored previously. By employing a quartz crystal microbalance with dissipation (QCM-D), our recent study (39) compared the deposition behavior of untreated cells (with EPS) on flat silica surfaces with those of treated cells (without EPS) for four different cell types (gram-negative and motile, gram-negative and nonmotile, gram-positive and motile, and gram-positive and nonmotile). It was found that the removal of EPS from cell surfaces led to the decrease in the deposition of bacteria on silica surfaces for all four cell types, indicating that the enhancement of cell deposition due to the presence of EPS on cell surfaces was relevant to all four bacterial species examined regardless of cell type and motility. However, the significance of EPS on cell deposition kinetics for bacteria of different cell types has not been systematically investigated in packed porous media and requires examination. Furthermore, direct examination regarding the influence of EPS on the retained bacteria profiles has not been previously performed by comparing the retention profiles between untreated cells (with EPS) and treated (without EPS) cells for different cell types. Hence, this study was designed to fully understand the influence of EPS on cell deposition and retained bacteria profiles in packed porous media. To achieve the objective, four representative cell types, Pseudomonas sp. QG6 (gramnegative, motile), mutant Escherichia coli BL21 (gramnegative, nonmotile), Bacillus subtilis (gram-positive, motile), and Rhodococcus sp. QL2 (gram-positive, nonmotile), were employed in this study. Packed column experiments were performed for untreated (with EPS) and treated bacteria (without EPS) in both NaCl (with four ionic strengths ranging from 2.5 mM to 20 mM) and CaCl2 solutions (5 mM) for all cell types. The overall deposition kinetics and retained concentration profiles for the untreated bacteria were compared with those for treated cells for all four cell types.
Materials and Methods Cell Culture and Preparation. Four bacterial cell types, a gram-negative and motile strain Pseudomonas sp. QG6, a gram-negative and nonmotile strain Escherichia coli BL21, a gram-positive and motile strain Bacillus subtilis, and a grampositive and nonmotile strain Rhodococcus sp. QL2, were used in this study. These four types of bacteria were grown and harvested according to the protocols described in our previous publication (39) as well as in the Supporting Information. After harvest, the growth medium was decanted, and the cell pellets were then resuspended in Milli-Q water. The centrifugation-resuspension process was repeated one more time. After that, each bacterial cell suspension was divided into two portions with the same volume. One portion of cell suspension was then repeated the centrifugationresuspension process once more (8000 g for 20 min at 4 °C). This portion of cell suspension was used as stock of untreated bacteria (with EPS). The other portion of cell suspension was used to prepare stock of treated bacteria (without EPS). Cells without EPS were achieved via the employment of cation exchange resin (CER) treatment technique developed by Frolund et al. (40). The detailed CER treatment procedure was provided in our previous publication (41) as well as in the Supporting Information. The effectiveness of the EPS removal from cell surfaces via CER treatment has been clearly demonstrated in the previous study (39). 2394
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The prepared stock cell concentration (both untreated and CER treated bacterial cell suspension) was determined using a counting chamber (Buerker-Tuerk Chamber, Marienfeld Laboratory Glassware, Germany) with an inverted fluorescent Ti-E microscope under bright field. The stock concentration was typically at approximate 109-1010 cells per mL, which was diluted to obtain the target influent concentration of 1.0 × 107 ( 25% cells mL-1. Viability of all cells (both untreated and CER treated cells) was determined by use of the BacLight Viability Kit (Molecular Probes, Eugene, OR) on the Ti-E microscope with the appropriate filter set right before each column experiment. The percentage of viable cells for each experiment was found to be greater than 85%. Detailed information of viability for both untreated and CER treated bacteria for each cell types can be found in Table S1. Porous Media and Column Experiments. The porous media used for bacteria transport experiments were quartz sand (ultrapure with 99.80% SiO2) (Hebeizhensheng Mining Ltd., Shijiazhuang, China) with sizes ranging from 417 to 600 µm (the median diameter of 510 µm). The procedure used for cleaning the quartz sand is provided in the previous publication (41) as well as in the Supporting Information. Prior to packing, the cleaned quartz sand was rehydrated by boiling in pure water for at least one hour. The cylindrical Plexiglas columns (20 cm in length and 4.0 cm in inner diameter) were wet-packed after the rehydrated quartz sand was cooled. Packing was performed by adding wet quartz sand in small increments (∼2 cm) with mild vibration of the column to minimize any layering or air entrapment. Two 60 mesh stainless steel screens were placed at each end of the column. To spread the flow upon entry into the column, ∼3.5 g of quartz sand was added to the top of the influent screen, forming a ∼2 mm-thick layer that was covered by another screen. The porosity of packed column is approximately 0.42. After packing, the columns were pre-equilibrated with at least 10 pore volumes of bacteria-free salt solutions at desired ionic strength and pH. Following pre-equilibration, 3 pore volumes of suspended bacteria were injected into the column, followed by elution with 5 pore volumes of salt solution (without bacteria) at the same ionic strength and pH. Selected experiments were eluted first by salt solution (without bacteria, 2 pore volumes) and then by low ionic strength solution (∼0.1 mM, 3 pore volumes). The low ionic strength solution was introduced to eliminate the secondary energy minima. The suspensions and solutions were injected into the columns in up-flow mode using a syringe pump (Harvard PHD 2000, Harvard Apparatus Inc., Holliston, MA). The transport experiments were conducted at four ionic strengths (2.5, 5, 10, and 20 mM) in NaCl solutions at pH 6.0 (adjusted with HCl). Selected experiments were also performed in CaCl2 solutions (5 mM) at pH 6.0 (adjusted with HCl). This wide range of solution chemistries examined in this study can be commonly found in groundwater (e.g., refs 42 and 43). The pore water velocity of all experiments was set to be 8 m day-1 (2.93 mL min-1) to represent fluid velocities in coarse aquifer sediments, forced-gradient conditions, or engineered filtration systems. All the water used in the experiments had been autoclaved for sterility. Samples from the column effluent were collected in sterile 5 mL glass culture tubes using a fraction collector (CF-1, Spectrum Chromatography, Houston, TX). The collected bacteria samples and reservoir samples were preserved using formaldehyde (1%) and were kept at refrigerator at 4 °C until the measurement of cell concentration (finishing within two weeks). Following the transport experiment, the sediment was extruded from the column under gravity and dissected into 10 segments (each 2 cm long). The retained bacteria were desorbed from the sediment segments into specified
volumes of sterilized Milli-Q water with 4% formaldehyde and manual shaking for a few seconds. The effluent samples and supernatant samples from recovery of retained bacteria were directly counted using a counting chamber with an inverted fluorescent Ti-E microscope under bright field to yield cell concentration in each sample. Every two days, standards for both untreated and treated cells (preserved with 1% formaldehyde) were also counted for monitoring the possible decay in the samples. Results from monitoring the standards were used to create a decay-rate (
