Environ. Sci. Technol. 1999, 33, 2958-2964
Motility-Enhanced Bioremediation of Carbon Tetrachloride-Contaminated Aquifer Sediments M I C H A E L E . W I T T , †,| MICHAEL J. DYBAS,‡ R . M A R K W O R D E N , ‡,§ A N D C R A I G S . C R I D D L E * ,†,‡ Department of Civil and Environmental Engineering, Department of Chemical Engineering, and Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824
Pseudomonas stutzeri KC is an aquifer isolate capable of denitrification and cometabolic transformation of carbon tetrachloride (CT) under anoxic conditions. Capillary experiments established that strain KC is chemotactic toward nitrate. A model aquifer column was used to evaluate the effects of motility in the presence of groundwater flow, and a second column was used to isolate motility in the absence of flow. Both columns contained CTsaturated sediments and groundwater containing CT and nitrate. The flow column was inoculated near its upstream end with strain KC, base, acetate, and phosphate and flushed continuously thereafter with contaminated groundwater. Cells migrated through the column at a velocity exceeding that of the groundwater, removing both aqueous and sorbed CT. The no-flow column was inoculated near its midpoint and maintained as a static incubation. Motile KC cells migrated over a 0.3 m distance within 5 days, giving a cell velocity of >5 cm/day. Over 94% of the CT in the column was removed in 26 days. The results support the hypothesis that localized depletion of nitrate during denitrification creates nitrate gradients that trigger a chemotactic response. The results also indicate that motile KC cells can degrade CT.
Introduction Chemotaxis is the ability of motile microorganisms to bias their movement toward higher concentrations of chemoattractants. Although researchers have documented migration and/or transport of bacteria through soil and sediment at pore velocities of up to 30 cm/day in the laboratory (1-3) and in the field (4), the significance of chemotaxis for bioremediation remains an open question. Those who have studied the factors affecting bacterial transport and deposition in laboratory-scale columns have generally not addressed the role of chemotaxis (5-7). Chemotaxis researchers have * Corresponding author present address: Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 943054020; e-mail:
[email protected]; phone: (650) 723-9032; fax: (650) 725-9474. † Department of Civil and Environmental Engineering. | Present address: The Dow Chemical Company, Health and Environmental Research Laboratory, 1803 Building, Midland, MI 48674. ‡ Center for Microbial Ecology. § Department of Chemical Engineering. 2958
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typically focused on the genetic and biochemical basis of chemotaxis (8) or on laboratory and modeling tools needed to quantify chemotaxis (9, 10). In one of the few studies examining chemotaxis in sand cores, Reynolds et al. (11) concluded that motile and chemotactic strains of bacteria have higher penetration rates than nonmotile and nonchemotactic mutants. Under nutrient-saturated conditions, however, chemotaxis did not facilitate transport through sand, with chemotactic strains exhibiting slower penetration rates than nonchemotactic mutants. A possible explanation is that the chemotactic strains sensed nutrient gradients in three dimensions and migrated throughout the entire pore volume of the porous media. Nonchemotactic strains failed to sense these gradients and were thus more likely to move through higher velocity macropores. In contaminated aquifers, however, contaminants are typically distributed in a nonuniform fashion due to variable spatial and temporal inputs and physical heterogeneity. Uniform removal of contaminants may depend on the ability of bacteria to enter regions of reduced hydraulic conductivity or elevated contaminant concentration. Chemotaxis is one mechanism whereby these regions might be made accessible to bacteria. The speed of bacterial dissemination may also be affected by chemotaxis: in swarm plates, the spreading rate of chemotactic cells can be several times that of nonchemotactic mutants that move by random motility only (12, 13). Pseudomonas stutzeri strain KC is a highly motile rod (1.6 µm long × 0.6 µm wide when grown aerobically) and has a single polar flagellum. It grows well under both aerobic and denitrifying conditions. A distinguishing characteristic is its ability to degrade carbon tetrachloride (CT) without producing chloroform (14). Under iron-limiting conditions, strain KC produces a small (∼500 Da) secreted factor that can degrade CT in the absence of oxygen (15). Normally, this factor must be activated for CT transformation, apparently by reduction at a cell membrane, and many cell types can mediate this activation step (16). Products of the transformation are carbon dioxide, formate, and other nonvolatile products (17). Motility could potentially provide a means for more rapid and uniform dissemination of strain KC in bioremediation applications. Previously, Widman (13) established that acetate is a chemoattractant for strain KC under denitrifying conditions. In this paper, we describe capillary experiments establishing that nitrate is also a chemoattractant. Consumption of a chemoattractant can create a gradient of chemoattractant, triggering formation of a chemotactic band (18). To determine whether motility would facilitate enhanced remediation of CT-contaminated sediments, we introduced strain KC and acetate into model aquifer columns containing aquifer sediment and nitrate-contaminated groundwater. Acetate served as the electron donor and nitrate as the electron acceptor. Of interest was whether strain KC motility would favor movement up the acetate or nitrate gradient. Widman et al. (10) showed that metabolism can result in coupled gradients in both the electron donor and the electron acceptor and that chemotaxis can be significant in response to both gradients. Also of interest was whether motile cells would transform CT. This was uncertain because CT transformation by this strain is cometabolic and energy-consuming (16), and it depends on a secreted factor that is only known to be produced under iron-limiting conditions. 10.1021/es981280+ CCC: $18.00
1999 American Chemical Society Published on Web 07/20/1999
Materials and Methods Capillary Assays. Capillary assays (19) were used to evaluate chemotaxis toward nitrate and oxygen. All glassware and buffer solutions were sterilized by autoclaving. Cells were grown in 100 mL of medium D (without vanadium and cobalt trace minerals) to late exponential phase in 250-mL shake flasks at 150 rpm (Lab Line Orbit shaker) (17). Two hours before the capillary assay, the culture was diluted to an OD590 of 0.4 with fresh liquid medium. A cell suspension was prepared for the capillary assay by centrifuging 1.0 mL of culture at room temperature for 10 min, washing the pellet twice with 1.0 mL of buffer (2.0 g of KH2PO4 and 3.5 g of K2HPO4 per liter of deionized water, adjusted to pH 8.2), and resuspending the cells in 4 mL of buffer in a 10-mL test tube. Glass capillaries (9.5 cm long with an inner diameter of 0.16 cm) were filled with one of the following test solutions: 0.5 g/L sodium nitrate in anaerobic buffer, 5.0 g/L sodium nitrate in anaerobic buffer, 50 g/L sodium nitrate in anaerobic buffer, buffer equilibrated with pure oxygen, buffer equilibrated with air, and anaerobic buffer. Oxygen levels were adjusted by bubbling filter-sterilized oxygen, air, or nitrogen gas through the buffer solution (25 mL in a 75-mL test tube) for 30 min. An oxygen electrode confirmed oxygen equilibration between the gas and liquid phases. A filled capillary tube was mounted in the test tube containing washed cell suspension so that the open end of the capillary was immersed in the suspension. After a 2-h incubation at room temperature, capillaries were removed from the test tubes and the contents diluted by a factor of 500 with sterile buffer. Five-microliter aliquots of the diluted cell suspension were spread on sterile Luria Broth agar plates. Plates were incubated aerobically at 20 °C for 2 days and the colonies counted. The cell suspensions in the test tubes were examined under 400× magnification for motility at the beginning and end of the 2-h incubation period. Construction and Operation of Model Aquifer Columns. To investigate motility and CT transformation in aquifer sediments with flowing groundwater, a model aquifer column was constructed using a 2-m-long section of clear polycarbonate pipe, 5.2 cm in diameter (i.d.), with 25 sampling ports spaced at 7.6-cm intervals (Figure 1). A manifold for delivery of organisms and nutrients was constructed near port 5. The delivery manifold consisted of four 8-in. stainless steel pipes (3/16-in. o.d.) spaced to penetrate the polycarbonate column at regular intervals around its circumference, each pipe passing through one side of the column, through its center, and out its opposite side. Sections of the manifold located on the inside of the column interior were slotted to create well screens, allowing free flow of water into and out of the manifold while preventing plugging by sand grains. Sections of the manifold located outside the column were connected together to create injection and extraction lines. The region of the column containing the manifold piping was designated the “slug injection zone”. For chemical and organism delivery into the slug injection zone, a peristaltic pump (WatsonMarlow model 502E) delivered fluid at a flow rate of 20 mL/ min from a reservoir (500-mL Erlenmeyer flask) into the manifold delivery lines. The column was wet-packed with aquifer sediments from Schoolcraft aquifer borings MLS2 and MLS3 (20). Two Harvard Apparatus syringe pumps (model 22) pumped groundwater through the column at an average linear flow velocity of 10 cm/day, the estimated velocity of groundwater at the Schoolcraft site. One pump delivered unfiltered CTfree groundwater. All groundwater was transported to the laboratory from Schoolcraft in presterilized sealed Nalgene carboys, transferred to 1-L Wheaton bottles, stripped by bubbling a N2/CO2 gas mixture through each bottle for 45 min, and sealed with Teflon-lined caps. Nitrogen/CO2 purging
was used to remove residual volatile organics and oxygen and to restore pH to its normal background level (pH 7.1). A second pump delivered filter-sterilized CT-spiked groundwater. The flows from both syringe pumps were combined in a 1:10 ratio of CT-spiked to CT-free groundwater, giving an influent CT concentration of 130 µg/L. The column and both syringe pumps were incubated in a constant-temperature room at 12 °C. To investigate motility and CT transformation in the absence of groundwater flow, a column was constructed using a 1-m-long section of clear polycarbonate pipe, 5.2 cm in diameter (i.d.), with 10 sampling ports spaced at 7.6-cm intervals (Figure 2). A slug injection zone was constructed at the center point of the column. The column was wet-packed with aquifer sediments from Schoolcraft aquifer boring MLS7 (20). CT-spiked Schoolcraft groundwater was pumped through the column to saturate the sediments with CT. Pumping continued until all sampling ports had a CT concentration of 100 ( 5 µg/L. The influent and effluent valves were then closed, and the column was incubated at 12 °C. Prior to inoculation, the flow and no-flow columns were characterized for porosity and dissolved oxygen. Tracer studies with tritiated water indicated a porosity of 0.3 for both the flow and no-flow columns. Denitrifying conditions were achieved by pumping oxygen-free groundwater through both columns for 4 weeks. Sampling and Analysis of Volatile Organics. For analysis of CT and chloroform (CF), 200-µL samples were obtained from sampling ports of the model aquifer columns on alternate days. Aqueous samples were withdrawn using a 500-µL Pressure-Lok gastight syringe (Alltech Associates) equipped with a 1.5-in. sideport needle. Both the syringe needle and sampling port septum were sterilized before sampling with an ethanol-soaked cotton swab. Each 200-µL sample was dispensed into a 1.5 mL glass vial (Sun Brokers, Inc.) sealed with a Teflon-lined crimp top (Sun Brokers, Inc.). Between sampling events, the interior of the gastight syringe was rinsed with 0.5 mL of methanol, followed by 0.5 mL of autoclaved deionized water. CT and CF were assayed by removing 100 µL of headspace from the 1.5-mL glass vials using a 500-µL Precision gastight syringe (Alltech Associates) and injecting the sample into a Perkin-Elmer model 8500 gas chromatograph equipped with a 100/120-mesh column (10% Alltech CS-10 on Chromsorb W-AW) and an electron capture detector with nitrogen carrier (40 mL/min). External standard calibration curves were prepared by addition of a primary standard in methanol to secondary solutions using the same gas-to-water ratios and incubation temperatures as assay samples. A four-point calibration curve was prepared. Anion Analysis. Anions (acetate, bromide, nitrate, nitrite, phosphate, and sulfate) were assayed in 200-µL samples using a Dionex model 2000i/SP ion chromatograph equipped with a Dionex AS4A IonPac column and operated with suppressed conductivity detection and mobile phase containing 1.8 mM bicarbonate/1.7 mM carbonate (3 mL/min). Fivepoint calibration curves were prepared by diluting primary anion standards into secondary water standards. Each sample was diluted into 400 µL of deionized water, filtered through a 0.22-µm nylon filter (Scientific Resources Inc.), and dispensed into a polypropylene sample vial (Alcott Chromatography). Column Inoculation. P. stutzeri KC (DSM deposit number 7136, ATCC deposit number 55595), originally isolated from aquifer sediments from Seal Beach, CA (14), is routinely maintained in our laboratory on R2A agar plates. To prepare inocula, a culture of strain KC was prepared by removing one colony from an R2A agar plate, placing it in an Erlenmeyer flask containing 25 mL of sterile nutrient broth, and shaking the flask overnight. Four milliliters of this culture was VOL. 33, NO. 17, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Changes in the concentrations of key parameters following one-time bioaugmentation of a model aquifer column operated with groundwater flow: (a) planktonic strain KC at different times and distances (port number) along the column; (b) acetate; (c) nitrate; (d) carbon tetrachloride. The days when samples were removed are listed above each plot. The bromide transport profile was similar to that of the acetate. A region centered around port 5 was inoculated on day 0. 2960
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FIGURE 2. Changes in the concentrations of key parameters following one-time bioaugmentation of a model aquifer column operated without groundwater flow: (a) planktonic strain KC at different times and distances (port number) along the column; (b) acetate; (c) nitrate; (d) carbon tetrachloride. The bromide profile was similar to that of the acetate. The days when samples were removed are listed above each plot. The midpoint of the column was inoculated on day 0. transferred to 400 mL of filter-sterilized Schoolcraft groundwater containing acetate (1600 mg/L) and phosphate (10 mg/L), previously adjusted to a pH of 8.2, and then shaken overnight. The flow column was inoculated in the slug injection zone with 320 mL of 24-h-old culture containing strain KC at (7.1 ( 1.3) × 107 colony-forming units (CFU)/mL and supplemented with CT, acetate, nitrate, and bromide to final concentrations of 100 µg/L, 1650 mg/L, 264 mg/L, and 47 mg/L, respectively. The no-flow column was inoculated in the slug injection zone with 320 mL of a 25-h-old culture containing strain KC at (1.2 ( 0.1) × 108 CFU/mL and supplemented with CT, acetate, nitrate, and bromide to final
concentrations of 100 µg/L, 1533 mg/L, 217 mg/L, and 25 mg/L, respectively. The experiment with the no-flow column was repeated using 0.952-µm-diameter Fluoresbrite PC Red latex microspheres dyed with phycoerythrin (Polysciences, Inc.) as an additional inoculum tracer. Microspheres were assayed using a Perkin-Elmer model LS 50 luminescence spectrometer. A five-point calibration curve was prepared. Calibration standards and samples were analyzed in the LS 50 using a 1.4-mL semimicro fluorometer (Buck Scientific) at an excitation maximum of 591 nm and an emission maximum of 657 nm. Enumeration of Planktonic and Attached Bacteria. For enumeration of planktonic bacteria, 200-µL groundwater VOL. 33, NO. 17, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Number of Strain KC Colony-Forming Units (CFU) on Agar Plates in Capillary Tube Assays test solution
CFU (av ( SD)
buffer sparged with nitrogen buffer sparged with air buffer sparged with pure oxygen buffer containing 0.5 g/L nitrate buffer containing 5 g/L nitrate buffer containing 50 g/L nitrate
26 ( 4 18 ( 7 22 ( 16 50 ( 47 287 ( 146 308 ( 59
samples were obtained from all sampling ports every third or fourth day and dispensed into sterile culture tubes containing 1.8 mL of 50 mM phosphate buffer (pH 8.0). Serial dilutions were prepared in phosphate buffer. One hundred microliters from each dilution was spread on R2A agar plates incubated at room temperature for 5 days and then scored for strain KC and indigenous microflora. Strain KC was differentiated by the unique “fried-egg” appearance of strain KC colonies. This method was previously tested by assaying the CT transformation capacity of 50 colonies isolated from KC-inoculated Schoolcraft groundwater: 25 colonies classified by colony morphology as “strain KC” degraded CT to nondetectable levels, and 25 colonies classified as not KC failed to degrade CT (21). As further confirmation of strain KC identity, a strain KC-specific DNA probe (20) was used to confirm the presence of strain KC throughout the no-flow column on day 26. Attached bacteria were enumerated by removing 200700 mg of sediment from each sampling port. The samples were placed in sterile Eppendorf tubes, weighed, and supplemented with 1 mL of autoclaved cell extraction buffer (22). Samples (200 µL) were removed from each tube and serially diluted six times. Samples were spread on R2A agar and incubated for 5 days and the colonies counted. The dry weight of sediment was determined after drying at 105 °C for 24 h.
Results and Discussion Chemotaxis in the Capillary Assays. The capillary assays provided strong evidence that nitrate is a chemoattractant for strain KC. The concentration of strain KC that accumulated in glass capillaries in the presence of 5 and 50 g/L nitrate was approximately an order of magnitude higher than the concentration that accumulated in the control capillaries containing buffer sparged with nitrogen gas, air, or oxygen (Table 1). This difference was statistically significant at the 95% confidence level as determined by a Student t test. The t test also indicated that the two higher nitrate concentrations were not statistically different from each other, but they were statistically different from the lowest nitrate concentration. These data support the conclusion that nitrate is a chemoattractant. However, because nitrate is also an electron acceptor for strain KC, it could be argued that nitrate increased the energy level of the cells, increasing motility and the likelihood of random movement into the capillary. This view was not supported by microscopy showing high motility at the beginning and the end of the 2-h incubation or by capillary experiments with oxygen. Even though oxygen also serves as an electron acceptor for strain KC and should therefore exhibit a similar effect on cell energy level and random motility, there was no increase in the concentration of strain KC cells in capillaries containing buffer saturated with pure oxygen or in capillaries containing buffer saturated with air at 21% oxygen, a concentration of oxygen known to induce chemotactic activity in Escherichia coli (18). The inability of oxygen to stimulate chemotaxis is consistent with a recent study of strain KC growth patterns on soft agar plates in air (23). These patterns included stable spots containing 2962
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high levels of strain KC. The interior of these spots was anaerobic, indicating use of oxygen as an electron acceptor. Despite the higher oxygen concentrations in surrounding regions, cells did not migrate away from the spots. Effects of Inoculum Addition in the Column with Flow. To evaluate motility in a column with flow, we augmented model aquifer columns containing nitrate-contaminated groundwater and sediment from the Schoolcraft site with inocula containing strain KC, acetate, and bromide. Aquifer conditions were simulated using aquifer sediment, a representative groundwater velocity, and an incubation temperature typical of the Schoolcraft aquifer. Bromide and acetate added with the inoculum moved through the column at the velocity of the groundwater, 10 cm/day (Figure 1b). Bromide was conserved, but acetate was not. Acetate was consumed while nitrate was simultaneously present, and its consumption decreased as nitrate concentrations declined. A comparison of the data for nitrate and strain KC indicates that strain KC cells consumed nitrate while moving through the porous media at a velocity exceeding that of the groundwater. On the first day after inoculation, nitrate concentrations decreased at ports 5 and 6 (Figure 1c). By day 4, the region devoid of nitrate extended from port 6 to port 12. By day 8, little nitrate remained downstream from port 10, even though the center of mass of the bromide was near port 15. A similar pattern was observed for planktonic KC: cells were detected at port 9 on day 3 (Figure 1a), but bromide was not detected until a day later. By day 7, strain KC was detected at port 23, but the center of mass of the bromide (and acetate) had only arrived at port 13 or 14 (Figure 1a,b). Strain KC traveled ∼114 cm in one week (an average velocity of ∼15 cm/day), while the bromide traveled only 70 cm (∼10 cm/day). Strain KC is attracted to acetate (13), and, as shown by the capillary assays, it is also attracted to nitrate. In these studies, the cells moved away from the acetate toward the nitrate, indicating a “preference” for the electron acceptor. Nitrate consumption in the absence of acetate is not readily explained. Denitrification requires an electron donor. Stored energy reserves may have served this function. Alternatively, a suitable electron donor may be present in the sediment. Schoolcraft sediments contain ∼0.03% organic carbon (20). This latter explanation seems doubtful given that nitrate persists in the Schoolcraft groundwater microcosms in the laboratory and it persists in the field despite the presence of indigenous denitrifying populations. Nitrate continued to enter the flow column throughout the experiment, and breakthrough at the inoculated region occurred on day 8 (Figure 1c). Breakthrough occurred because no acetate was introduced into the column beyond that present in the original inoculum. Repeated weekly addition of acetate can support a large population of attached strain KC cells and continuous, long-term removal of both nitrate and CT (24). Inoculation of the continuous flow column resulted in limited long-term colonization. Prior to inoculation, strain KC was not detected on sediment samples. After motile strain KC cells had exited the column (after day 11), cells were detected at ports 5 (1 × 107 CFU/g) and 7 (1 × 105 CFU/g), and unattached cells were also at these ports (Figure 1a). This is important because it indicates persistence of cells in the zone of inoculation 20 days after inoculation, despite sustained input of groundwater at pH 7.1. In previous studies with strain KC grown as dispersed cells in pasteurized Schoolcraft groundwater, a rapid die-off occurred when groundwater pH was reduced from 8.3 to 7.5. This was not observed in the column. Taken together with the evidence for motile cells, we conclude that the inoculum contained two cells types: some that attached and persisted in the region of inoculation and others that moved downstream.
As shown by comparison of parts a and d of Figure 1, motile KC cells retained the ability to degrade CT. CT transformation coincided with the detection of strain KC and preceded the arrival of bromide (and acetate). Transformation was observed near port 21 by day 8, but the center of mass of the bromide (and acetate) had only arrived at port 15 (Figure 1b). An overall mass balance on CT in the model aquifer column indicated that motile cells removed ∼55% of the CT in the aqueous phase. No chloroform was detected. After motile strain KC cells exited the column (day 20), sediment samples were analyzed for CT. CT concentrations downgradient from the slug injection zone were ∼ 29% lower than concentrations prior to inoculation. Given the above result and the results of the capillary assays, it is reasonable to conclude that nitrate gradients created by denitrification induced a chemotactic response, although random motility and growth likely also contributed to the observed response. An alternative explanation for the accelerated cell transport is size exclusion, which has been reported for both microorganisms (4) and colloids (25) in the presence of advective flow. To eliminate size exclusion as a contributing factor, we performed experiments without groundwater flow. Effects of Inoculum Addition in the Column without Flow. The no-flow column experiments demonstrated that rapid movement of strain KC cells occurs even in the absence of advective transport, a necessary condition for size exclusion. The only factors affecting movement of bromide and acetate in the no-flow experiments were diffusion and the removal of groundwater samples for analysis. Bromide and acetate moved slowly from the slug injection zone (Figures 2b); after 26 days, these solutes had reached only ports 3 and 8. By contrast, strain KC moved rapidly, reaching ports 3 and 8 in only 2 days (Figure 2a). By day 5, strain KC was detected in samples from all ports. Distribution of strain KC throughout the column was confirmed on day 26 using a DNA probe specific to strain KC (20). The distance traveled was a minimum of 30 cm, giving a mean cell velocity of ∼5 cm/ day, close to the value observed for the flow column. For the no-flow column, the concentration of cells measured at the end of the column was only 103-104 cells/mL. This is in contrast to the flow column, where cell concentrations downstream of the tracer ranged from 104 to 105 cells/mL. This difference suggests growth of motile cells in the flow column, probably because acetate was advectively transported with the bromide. Data for nitrate (Figure 2c) support the conclusion that an actively denitrifying population migrated from the slug injection zone toward both ends of the no-flow column. By day 5, nitrate had disappeared throughout most of the column, with low residual concentrations at ports 3 and 9 (the port 10 sample was lost). After 26 days, only ports 9 and 10 contained detectable nitrate. As observed in the flow column, nitrate removal was observed in regions of the column in which no added electron donor (acetate) was present. In both the flow and no-flow experiments, degradation of CT was unexpected. This is because CT transformation by this organism is energy-dependent (16) and is known to be induced only under iron-limiting conditions (14, 15). For the experiment with flow, it might be argued that that the inoculum contained sufficient CT-degrading factor to account for the CT transformation observed downstream of the slug injection zone or that attached cells secreted sufficient factor to transform CT as it was swept downstream. The experiment without flow eliminated these possibilities. CT removal in the no-flow column followed the same pattern as nitrate removal, except lagging in time, probably because of retarded desorption of CT from the sediment (Figure 2d). Because gradients were created for CT and nitrate,
it could be proposed that movement of strain KC was the result of chemotaxis toward CT, rather than nitrate. However, this hypothesis is not supported by data for ports 3 and 4, where strain KC was detected prior to a CT decrease. We conclude that CT transformation was mediated by motile cells and that these cells were most likely responding to a nitrate gradient. This conclusion is based on the detection of strain KC ahead of tracer, the correlated removal of nitrate and detection of strain KC, the correlated patterns of CT removal and strain KC migration, final removal of >94% of the CT throughout the column, and the absence of chloroform production. Random motility and growth may also have contributed to the observed transport patterns, especially in the experiment with flow, in which acetate was available to support growth. The experiment without flow was repeated using fluorescent microspheres to test movement of 1-µm particles due to sampling and diffusion (data not shown). Microspheres moved at a rate similar to that of a conservative tracer (bromide), and other results of the no-flow experiment with bromide were confirmed.
Acknowledgments Partial support for this work was provided by the Michigan Department of Environmental Quality through Grant Y40386. Partial support was also provided by the Great Lakes and Mid-Atlantic Hazardous Substance Research Center under Grant R-81570 from the Office of Research and Development, U.S. Environmental Protection Agency, and by the Institute for Environmental Toxicology under Grant P42ES 04911-08 from the NIEHS Department of Health and Human Services Superfund Basic Research Program. The content of this paper does not necessarily represent the views of any of the above agencies.
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Received for review December 9, 1998. Revised manuscript received June 1, 1999. Accepted June 8, 1999. ES981280+
