PB91-164277 Movement of Bacteria through Soil and Aquifer Sand Cornell Univ., Ithaca, NY Prepared for: Robert S. Kerr Environmental Research Lab., Ada, OK Mar 91 U.S. DEPARTMENT OF COMMERCE National Technical Information Service NTIS ------- EPA/600/2-91/010 March 1991 MOVEMENT OF BACTERIA THROUGH SOIL AND AQUIFER SAND by M. Alexander, R. J. Wagenet, P. C. Baveye, J. T. Gannon, U. Mingelgrin, and Y. Tan Cornell University Ithaca, New York 14853 Number CR-814487 Project Officer John T. Wilson Processes and Systems Research Division Robert S. Kerr Environmental Research Laboratory Ada, Oklahoma 74820 ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTAL PROTECTION AGENCY ADA, OKLAHOMA 7482 0 ------- TECHNICAL REPORT DATA (Please read Instructions on the reverse before complet' 1. REPORT NO. 2. EPA/600/2-91/010 3- PB91- 1 64277 4. TITLE AND SUBTITLE MOVEMENT OF BACTERIA THROUGH SOIL AND AQUIFER SAND 5. REPORT DATE March 1991 6. PERFORMING ORGANIZATION CODE 7. AUTHORIS) M. Alexander, R.J. Wagenet, P.C. Baveye, J.T. Gannon, U. Mingelgrin, and Y. Tan 8. PERFORMING ORGANIZATION REPORT NO. 9. PERFORMING ORGANIZATION NAME AND ADDRESS Cornell University Ithaca, NY 14853 10. PROGRAM ELEMENT NO. CEWD1A 11. CONTRACT/GRANT NO. CR-814487 12. SPONSORING AGENCY NAME AND ADDRESS Robert S. Kerr Environmental Research Lab. -Ada, OK U.S. Environmental Protection Agency P.O. Box 1198 Ada, OK 74820 13. TYPE OF REPORT AN D PE R IOD COVE RED Proiec.f. Report 10/01/87-12/31, 14. SPONSORING AGENCY CODE EPA/600/15 15, SUPPLEMENTARY NOTES Project Officer: John T. Wilson FTS: 743-2259 16. ABSTRACT The transport of microorganisms in soils is of- major importance for bioremediation of subsurface polluted zones. A procedure for evaluating the relative mobility and recovery of bacteria in the soil matrix was developed. Nineteen bacterial strains were selected that differed in their ability to be transported through soils. Measurements were made of sorption partition coefficient, hydrophobicity, net surface electrostatic charge, zeta potential, cell size, encapsulation, and flagellation of the cells. Only sorption and cell length were correlated with transport of the bacteria through soil. The breakthrouqh curves for Pseudomonas so. KL2 moving through a column packed with a sandy aquifer material were determined. Ionic strength of the inflowing solution, bacterial density, and velocity of water flow were found to have an effect on breakthrough. 17 KEY WORDS AND DOCUMENT ANALYSIS a DESCRIPTORS b. 1 DENTI FlERS/OPEN ENDED TERMS e, COS ATI Field/Group 18. DISTRIBUTION STATEMENT RELEASE TO THE PUBLIC — — 19. SECURITY CLASS (This Report) UNCLASSIFIED 21. NO. OF PAGES 43 20. SECURITY CLASS (This page) UNCLASSIFIED 22. PRICE EPA Form 2220-1 (Re*. 4-77) previous eoition isobsolete ------- DISCLAIMER The information in this document has been funded wholly or in part by the U.S. Environmental Protection Agency under assistance agreement CR-814487 to Cornell University- It has been subjected to the Agency's peer and administrative review, and it has been approved for publication as an EPA document. ii ------- FOREWORD EPA is charged by Congress to protect the Nation's land, air, and water systems. Under a mandate of national environmental laws focused on air and water quality, solid waste management and the control of toxic substances, pesticides, noise and radiation, the Agency strives to formulate and implement actions which lead to a compatible balance between human activities and the ability of natural systems to support and nurture life. The Robert S. Kerr Environmental Research Laboratory is the Agency's center of expertise for investigation of the soil and subsurface environment. Personnel at the Laboratory are responsible for management of research programs to: (a) determine the fate, transport and transformation rates of pollutants in the soil, the unsaturated and the saturated zones of the subsurface environment; (b) define the processes to be used in characterizing the soil and subsurface environment as a receptor of pollutants; (c) develop techniques for predicting the effect of pollutants on ground water, soil, and indigenous organisms; and (d) define and demonstrate the applicability and limitations of using natural processes, indigenous to the soil and subsurface environment, for the protection of this resource. There is a widely held perception that bacteria cannot move appreciable distances through geological materials. This view is based on experience with human enteric microbes such as E^_ coli. which are effectively sorbed and inactivated in the subsurface. This view has inhibited research on inocula to degrade organic wastes in the deeper subsurface. This research establishes that there are organisms that can be readily transported in the subsurface and opens up a new strategy for the development and use of inocula for hazardous waste in the deeper subsurface. Clinton W. Hall Director Robert S. Kerr Environmental Research Laboratory iii ------- ABSTRACT The transport of microorganisms in soils is of major importance for bioremediation of subsurface polluted zones. A procedure for evaluating the relative mobility and recovery of bacteria in the soil matrix was developed. In the method devised, movement of bacteria along the walls of the column of soil and channeling were prevented. Changes in population size during the test period were minimal because temperatures of 2 to 5°C were maintained during the test period and predators and parasites were eliminated by 60Co irradiation. The 19 strains of bacteria tested had markedly different degrees of transport. From 0.01 to 15% of the added cells passed through a 5-cm long column of Kendaia loam with four pore volumes of water. From 4.3% to essentially all of the added bacteria were recovered. The marked differences in the mobilities of the various bacteria and the high recoveries of most of the isolates suggested that the procedure developed is a useful means for selecting bacteria according to their mobilities in soils, aquifer materials, and other porous media. Measurements were made of hydrophobicity, net surface electrostatic charge, zeta potential, cell size, encapsulation, and flagellation of cells of 19 bacterial strains that differed in their ability to be transported through soil. A wide range of hydrophobicities was noted among the bacterial strains. Determinations by electrostatic-interaction chromatography showed that 5 to 47% of the cells were retained by a cation-exchange resin. The zeta potentials of the various isolates ranged from -8 to -3 6 mV. Eight of the bacteria had capsules, and the cell lengths varied from 0.7 to 2.6 /Jm. Some of the mobile strains were flagellated. Sorption and cell length were correlated with transport of the bacteria through soil. The breakthrough curves for Pseudomonas sp. KL2 moving through a column packed with a sandy aquifer material were determined. Ionic strength of the inflowing solution, bacterial density, and velocity of water flow were found to have an effect on breakthrough. iv ------- CONTENTS Pages Disclaimer ii Foreword iii Abstract iv Figures vi Tables vii INTRODUCTION 1-3 MATERIALS AND METHODS 3-10 RESULTS 10-23 DISCUSSION 23-30 References 31-35 v ------- FIGURES Page Figure 1. Experimental setup. 4 Figure 2. Breakthrough of Pseudomonas sp. KL2 with 0.01 M NaCl (flow rate 1.0 X 10"4 m s"1 equivalent to 3.5 pore volume h"1). 19 Figure 3. Breakthrough of Pseudomonas sp. KL2 with deionized water (flow rate 1.0 X 10~4 m s"1, equivalent to 3.5 pore volumes h"1). 2 0 Figure 4. Breakthrough of Pseudomonas sp. KL2 with 0.01 M NaCl solution and flow rate of 2.0 X 10"4 m s"1 equivalent to 6.9 pore volumes h'1. 21 Figure 5. Breakthrough of Pseudomonas sp. KL2 with deionized water at flow rate of 2.0 X 10"4 m s~1 equivalent to 6.9 pore volumes h"1. 22 Figure 6. Breakthrough of Pseudomonas sp. KL2 with 0.01 M NaCl at 1.0 X 109 cells mL"1 (flow rate 1.0 X 10-4 m s"1 equivalent to 3.5 pore volumes h"1). 2 4 Figure 7. Breakthrough of Pseudomonas sp. KL2 with deionized water at 1.0 X 109 cells mL"1 (flow rate 1.0 x 10-4 m s"1 equivalent to 3.5 pore volumes h*1) . 25 Figure 8. Breakthrough of Pseudomonas sp. KL2 at 1.0 X 109 cells mL"1 using 0.01 M NaCl for 1 h followed by deionized water (flow rate 1.0 X 104 m s"1 equivalent to 3.5 pore volumes h"1) . 2 6 vi ------- TABLES Table 1. Distribution of Pseudomonas KL2 in columns of soil after passage of four pore volumes of water. Table 2. The numbers and percentages of bacteria transported and recovered. Table 3. Adsorption coefficients of bacteria. Table 4. Relationship between characteristics of bacteria and their transport through soil. vii ------- INTRODUCTION Approximately 50% of the United States population depends upon ground water as a source of drinking water (Bitton and Gerba, 1984). Consequently, the contamination of ground water by organic chemicals is widely recognized as a critical environmental problem. The transport and mobility of bacteria in soil and subsurface materials has been a subject of interest for the past few decades because of the environmental importance of these organisms. The bioremediation of underground waste-disposal sites by the use of introduced bacteria requires that the microorganisms move from the point of their introduction to the site of contamination. Such inoculation is necessary if microorganisms degrading the chemical contaminants are not present in the hazardous waste site or adjacent groundwaters. Many toxic organic chemicals persist at underground hazardous waste sites despite being readily biodegradable under laboratory conditions. When this occurs, introduced bacteria selected for their capacity to degrade target contaminants, and capable of surviving and proliferating after injection in the aquifer, might be used to promote biodegradation. The introduction of bacteria to degrade toxic wastes in soil and subsurface has generated renewed interest in the transport and mobility of bacteria (Baveye and Valocchi, 1989; Borden and Bedient, 1986; Gannon et al. , 1991). However, the mobility of the added bacteria may determine their effectiveness for in situ bioremediation. If biodegradative bacteria are not present at the site of contamination, then bioremediation will only be effective if the added bacteria have both the capacity to reach the contaminated zone and to move through porous materials with the contaminant plume. Considerable attention has been given to the mobility of bacteria and other microorganisms in soil and subsurface materials. These studies were conducted primarily because of concern with the dissemination of pathogens from land spreading operations, groundwater recharge, or the disposal of manure or municipal sludge (e.g., Gerba et al., 1975; Brown et al. , 1979; Bell and Bole, 1978) . Several studies have shown poor mobility of the investigated species of bacteria through soil (Bitton et al., 1974; Dazzo et al., 1973; Madsen and Alexander, 1982). However, considerable movement of some bacteria was observed in field studies (Schaub and Sorber, 1977; Viraraghavan, 1978). It is unclear whether the movement of bacteria that has been observed occurred through the soil matrix or through the macropores or channels that afford the organisms a relatively unhindered passage (Hagedorn et al., 1981; Rahe et al., 1978). It is, however, generally acknowledged that the movement of bacteria in a homogeneous porous medium is poor because of both adsorption (Hattori and Hattori, 1976; Marshall, 1980) and mechanical filtration (Pekdeger and Matthess, 1983; Smith et al., 1985) of 1 ------- bacterial cells, which have been suggested as mechanisms for their retention in soils. In a study by Wollum and Casell (1978) , streptomycete conidia and a bacterium were displaced through a sand column; only 0.2% of the bacterial cells and 6% of the conidia were recovered in the effluent after passage of four pore volumes of water, but over 90% of the organisms were recovered in the sand within 3 cm of the inlet surface. When bacteria were injected into a sandy aquifer using a forced gradient, the relative breakthrough of cells into a sampling well located 1.7 m from the injection well was less than 1% (Harvey et al., 1989). Smith et al. (1985) found nearly a 16-fold greater breakthrough of Escherichia coli through intact cores compared to disturbed cores of a sandy loam. They concluded that soil structure and the velocity of water flow are critical in determining movement of bacteria through soil. Baveye and Valocchi (1989) reviewed a number of mathematical models describing the concurrent growth of bacteria and transport of biodegradable substrates in saturated porous media; these models assume that bacteria form either continuous biofilms or discrete microcolonies on surfaces of solid particles and hence are immobile (Rittmann et al., 1980; McCarty et al., 1984; Molz et al., 1986). However, many environmental factors greatly affect bacterial transport. Ionic strength is particularly important. Krone et al. (1958) and Tan (1989) showed that infiltrating solutions with low ionic strength decrease retention of bacteria in sand. In acid- treated sand, the efficiency of coliform retention was higher when the bacteria were suspended in tap water than in distilled water, and no retention occurred when the bacteria were suspended in triple-distilled water (Goldshmid et al., 1973). On the basis of the electrical double layer developing in the vicinity of charged surfaces, Marshall (1975) concluded that the number of bacteria attracted to surfaces lessened with decreased electrolyte concentrations, whereas adsorption of cells to surfaces increased with higher electrolyte concentrations. Duboise et al., (1976), Gerba and Lance (1978), and Sosbey et al. (1980) reported that the application of rain water or distilled water will result in the desorption of viruses from soil particles. Vinten and Nye (1985) reported that complete blockage of pores due to soil dispersion does not occur in sodic soils leached with water of low electrolyte content, and that dispersed clay may be transported considerable distances before deposition. Despite these findings on the effects of ionic strength, recent models for bacterial transport in porous media do not include ionic strength as a factor in determining the movement of cells (Corapcioglu and Haridas, 1984, 1985; Germann et al., 1987; Taylor and Jaffe, 1990). The effect of ionic strength on movement of bacteria through subsurface earth materials seems to have been largely ignored in terms of promoting bioremediation with introduced bacteria. The objectives of this study were to develop a reproducible procedure that would yield consistent measurements of relative 2 ------- mobility of bacteria in soil by avoiding uncontrolled variations in bacterial behavior and to relate transport to efficiency of recovery and adsorption of the cells. In the procedure that was developed, flow through macropores did not occur. In addition, a study was conducted to determine the influence of certain properties of bacterial cells on their movement through soil. The traits investigated were net surface electrostatic charge, hydrophobicity, cell size, and presence of capsules. Disturbed columns of aquifer sand were used to study the breakthrough of bacteria and of a chloride tracer. The effect of variations in the ionic strength of an inflowing solution, bacterial cell density, and flow velocity on the transport of bacteria through the earth materials was evaluated. MATERIALS AND METHODS TRANSPORT THROUGH SOIL Bacteria able to degrade benzene, chlorobenzene, or toluene were isolated by enrichment culture in solutions containing 100 mg of the organic compound, 1.6 g K2HP04, 0.4 g KH2P04, 0.5 g (NH4)2S04, 25 mg CaS04-2H20, 0.2 g MgSO^- 7H20, and 2.3 mg FeSO^-7H^O per L of deionized water. The pH was adjusted to 7.0. Each enrichment was transferred at least four times into fresh medium of the same composition before plating the bacteria on enrichment medium supplemented with 1.5% agar. Biodegradation was determined by spectrophotometric measurement of the loss of UV absorbance of the added organic compound. To obtain bacteria by a method that presumably favored mobile organisms, diesel-fuel contaminated soil, Kendaia loam, and liquid from the primary settling tank of the Ithaca, NY sewage treatment plant were placed on the soil column, and bacteria that passed through the soil were isolated on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, MD). Bacillus sp. CU 4519 was obtained from S.A. Zahler, and Arthrobacter sp. Lula D from an underground aquifer was provided by J. L. Sinclair, both from Cornell University. The bacteria were grown in Trypticase soy broth at 30°C for 24 to 48 h, the cells were harvested by centrifugation and washed twice, and the organisms were suspended and diluted in 0.9% NaCl solution. The cell suspensions were cooled to 0° for 1 h prior to their addition to columns of soil. Bacterial counts were made on Trypticase soy agar using triplicate samples from the effluent of each soil column and triplicate plates per dilution. The plates were incubated at 30°C for 24 to 48 h. The soil was air-dried, ground, passed through a 2-mm sieve, and then sterilized by 60Co irradiation (2.5 Mrad). Aseptic conditions were maintained during the experiments. The experimental setup (Fig. 1) was as follows. The soil was packed in 3 ------- 10 c /r,- Deionized h2o Buchner Funnel 600 ml . Petrolatum Coating Soil Fritted Disc (40-60 /^m) Hg Column Row Regulator Hg 500 ml Erlenmeyer Flask Collected Effluent Figure I. Experimental Setup ------- 600-mL Buchner funnels, 10-cm diam, containing a fritted-glass disc with pore sizes of 4 0 to 60 jL/m. Preliminary tests indicated that the pore sizes in the disc permitted passage essentially only of bacteria and water-dispersible clay, and during experiments, the flow rate remained constant for each of the four pore volumes passed through the soil column. Thus, the filters did not become clogged, and this allowed for free passage of bacteria. The funnel was attached to a 500-mL Erlenmeyer flask fitted with a sidearm containing Hg in a bulb to control the flow rate. The Hg in the bulk restricted the rate of flow by controlling the release of air displaced as drops of effluent passed from the column into the attached Erlenmeyer flask. The walls of the funnel were coated with a thin layer of sterile petrolatum added in liquid form at >60°C. The petrolatum sealed the interface between the funnel walls and the soil. The soil was pressed down with a fitted brass compacting plate to a depth of 5 cm to give a bulk density of 1.08±0.062 g cm"3 and porosity of 59.2%. The pore volume was 241.75 cm3. The soil columns and sterile deionized water to be used for mobility tests were cooled to 3.5±1.5°C, the temperature at which experiments were conducted, and the soil was then again compressed with the compacting plate to compensate for any disturbance of the soil column in the period between packing and inoculation. The soils were moistened from below by connecting a bottle of water to the stem of the Buchner funnel. Sterile deionized water was added at a rate of approximately 10 cm h"1 until the water level was approximately 2 cm above the soil surface. The water was then drained to the surface of the soil column, and a circular mound with a radius of 1 cm of dry sterile soil was placed in the center of that mound. A 1.0 mL inoculum of 1.0 x 108 cells was placed in the center of that mound. An additional 1-cm layer of sterile soil was added to the top of the column, thereby covering the inoculated mound and making the column surface approximately level to prevent horizontal movement of the inoculum. Deionized water was added to the soil surface. Physical disturbance of the surface of the soil column was avoided by pouring the water onto A1 strips placed above the soil surface. A total of four pore volumes of effluent was collected in the flask. The rate of water flow through the column was maintained at approximately 0.8 pore volumes per h. To determine the extent of lateral movement of the microorganisms and the possible transport of bacteria along the walls of the column, the inoculum was introduced at the center of the soil surface in a circle of approximately 1-cm diameter. After the column was leached with four pore volumes and allowed to drain, five vertical cores (5 cm in length) were taken from the soil column using a 5-mL syringe (1.4 cm i.d., 1.7 cm. o.d.) from which the luer end was cut. A core taken from the center of the column was divided into sections taken from depths of 0.0 to 1.7, 1.7 to 3.4, and 3.4 to 5.0 cm. Cores were also taken at distances of 2 cm and 4 cm between the core center and the column center. Two cores 5 ------- were taken at each such distance at diametrically opposed points per column. Tests of lateral movement were conducted with duplicate columns. The total number of bacteria in each core and core section was then determined. Determinations were made of the percentage of the cells added to the soil that appeared in the effluent (percentage transported) and the percentage that could be recovered from both the effluent and the soil column at the end of the test period (percentage recovery). Triplicate columns were inoculated with 1 mL of bacterial suspension. The bacteria in the effluent were counted after passage of four pore volumes of deionized water, and the number of bacteria remaining in the soil of two of the three inoculated columns was determined after the effluent was collected. For this purpose, all of the soil was removed from the column, the soil was shaken with deionized water for 5 min on a rotary shaker operating at 12 0 rpm, and counts were made after the larger particles were allowed to settle for 5 min. Adsorption of bacteria to soil was determined by a procedure based on the difference in gravity sedimentation rates of the bacterial cells and sand, silt, and clay size fractions of the soil. At the experimental temperature of 3°C, the time required for the size fraction >2 jL/m of the soil to settle below an 8-mm depth is predicted by Stokes' law (Jackson, 1974) to be 65 min. To determine the amount of water-dispersible clay (smaller than 2 jL/m) that remained suspended with bacteria at the sampling depth, a 2 0-g portion of Kendaia loam was shaken for 1 h at 3.5±1.5°C with 100 mL of deionized water. The suspension was then diluted to 1 L with deionized water and allowed to settle for 4.5 h (the settling time was calculated to collect particles <2 jL/m diameter) . A 25-mL portion was then taken from the 5-cm depth of a 1-L graduated cylinder and dried overnight at 105°C. The water-dispersible clay constituted 2.6% of the total soil, or 12% of the total clay content. To measure bacterial adsorption at 3°C, 2 0-g samples of Kendaia loam and approximately 10® cells were shaken for 1 h with 100 mL of deionized water. The suspension was allowed to settle for 65 min in a sterile 250-mL graduated cylinder. Determinations were made of the number of bacteria at the 8-mm depth of the soil suspension (S8) and of a control (C8) to which no soil was added. The soil and control suspensions were again thoroughly mixed, and the numbers of bacteria in the soil suspension (St) and the control (Cf) were determined from a depth of approximately 1 to 2 cm. The drop plate method (Reed and Reed, 1948) was used for bacterial counts. The data were expressed as adsorption coefficients (Kd) where Kd = F/D (Ct - F)"1 6 ------- and F is the number of bacteria adsorbed, and D is the concentration of soil in the suspension. The value of D is 0.2 (20 g/100 mL). The adsorption coefficients are expressed as the lower or higher bound of bacterial adsorption. The lower-bound adsorption value assumes that bacteria retained by clay particles of equal or smaller size are not considered to be sorbed, and the higher-bound adsorption value considers all bacteria-clay interactions as adsorption. To determine F, the equation takes into account bacterial settling and the interaction between bacteria and water-dispersible clay. The lower-bound of adsorption is expressed by the following equation, which does not consider the interaction between bacteria and water-dispersible clay in the top 8-mm sampling depth, Flow — ~ (^8 ^ ^/Cg) * The higher-bound of adsorption is expressed by the following equation, which considers the interaction between bacteria and water-dispersible clay in the top 8-mm sampling depth, Fhigh = St - (S8 Ct/C8) d - Wd X Ct/C8)"1 where Wd is the water-dispersible fraction of the clay (0.12). CELL PROPERTIES All assays described below were conducted at 3°C, which was the temperature used in tests of bacterial transport. Hydrophobicity was determined by measuring bacterial adherence to hydrocarbons (BATH) and hydrophobic-interaction chromatography (HIC) . For the BATH procedure, a modification of the method of Rosenberg et al. (1980) was used with octane as the assay hydrocarbon. Bacteria grown in Trypticase-soy broth for 24 h at 3 0°C were washed and resuspended in a buffer-salts solution at pH 7.1 containing 22.2 g of K2HPO^ -3H20, 7.3 g of KH2P04, 1.8 g of urea, and 0.2 g of MgS04-7H20 per liter to an optical density of 0.2 at 550 nm. A mixture of 4.0 ml of washed cells (108/ntl) and 0.00, 0.25, 0.5, 0.75, or 1.0 ml of octane was added to test tubes and mixed with a vortex mixer for 60 sec. The contents of each test tube were placed in a 5-ml syringe with a luer-lock to avoid possible mixing of the organic layer with the aqueous phase. The phases were allowed to separate for 30 min, and then the light absorbance at 400 nm of the aqueous phase was measured with a Hitachi U-2 000 spectrophotometer. The percentage of adherence to octane is expressed as 100 times the ratio of (absorbance of the octane-free bacterial suspension minus that of the aqueous layer) to the absorbance of the octane-free suspension. Hydrophobic-interaction chromatography, which measures the amount of bacteria retained by a hydrophobic gel (Dillon et al., 7 ------- 1986; Mozes and Rouxhet, 1987), was also used to determine cell hydrophobicity. Octyl-sepharose gel (Pharmacia, Uppsala, Sweden) was washed with 4 M NaCl prior to being packed in duplicate wool- plugged Pasteur pipettes to a final bed volume of 2 ml. Bacteria grown in Trypticase-soy broth for 24 h at 3 0°C were washed twice with 4 M NaCl and resuspended in 4 M NaCl to a density of approximately 108 cells per ml. The bacterial suspension (1 ml) was then applied to the gel bed and eluted with 5 ml of 4 M NaCl. Another column was eluted with deionized water to assess the extent of filtration of the cells. The percentage of hydrophobicity is expressed as 100 times the ratio of absorbance of the original cell suspension at 400 nm minus that of the eluate (nonretained bacteria) to the absorbency of the original suspension. The data indicate the percentage of cells retained by the hydrophobic gel. Net surface electrostatic charge was determined by electrostatic-interaction chromatography (ESIC) and by measuring zeta potential. ESIC is a measure of the bacterial affinity for cation- or anion-exchange resins (Pedersen, 1981; Stenstrom, 1989). Pasteur pipettes plugged with glass wool were filled with either CM-sepharose CL-6B anion exchanger or DEAE-sepharose CL-6B cation exchanger (Pharmacia) to a final bed volume of 2 ml. Duplicate columns of the two resins were washed with 0.9% NaCl prior to column packing. Bacteria grown in Trypticase-soy broth for 24 h at 30°C were washed twice with 0.9% NaCl and resuspended in 0.9% to a density of 108 cells/ml. The bacterial suspension (1.0 ml) was added to the columns, and the cells were eluted with 5 ml of 0.9% NaCl. The percentage of cells retained is expressed in the same way as for the BATH procedure. The electrophoretic mobility of the bacteria was measured with a Lazer Zee meter model 501 (Mozes et al., 1987; van der Mei et al., 1987). This instrument measures the zeta potential by determining the rate of bacterial movement in a known electric field. The potential difference between the electrodes was 150 V. The cells were suspended in deionized water (pH 7) at a concentration of 10® cells per ml before determination of electrophoretic mobility at 20°C. The measurements of zeta potential were corrected to 3°C by multiplying the measured zeta potential by (1-0.02(20-3). TRANSPORT THROUGH AQUIFER SOLIDS The bacterial strain used in the experiments was isolated by Gannon et al. (1991) from samples of Kendaia loam obtained in Aurora, NY. This strain (Pseudomonas sp. strain KL2) is amphitrichously flagellated and is characterized by an average cell diameter of 0.4 /im and an average cell length of 1.8 /im. From the 19 strains tested by Gannon et al. (1991), this strain was selected because it showed higher than average mobility in homogeneous columns of disturbed Kendaia loam soil. The bacteria were grown at 30°C for 24 h (200 mL of medium in a 250-mL flask) in Trypticase- 8 ------- soy broth on a rotary shaker (100 rpm). After being collected by centrifugation and washed twice with deionized water with a resistivity of 18.3 Mohms-cm, the bacteria were resuspended in either deionized water or a 0.01 M NaCl solution to achieve concentrations of 1.0 X 10s and 1.0 X 109 cells per mL. The pH of these bacterial suspensions was 7.0. Ten minutes prior to the breakthrough experiments, the extent of cell aggregation was assessed by examining wet mounts under a light microscope and by comparing differences in turbidity of the bacterial suspension at 0 and 5 min using a Spectronic 20 spectrophotometer at a wavelength of 550 nm, following procedures described by Calleja (1984) . No bacterial aggregation was observed in either deionized water or NaCl solution. The sandy aquifer material used originated from Eastport, NY. The Suffolk County Department of Health provided auger drillers to obtain aquifer material from a depth of 11 m. Special precautions were taken not to mix the aquifer material with surface soil. After being air-dried and passed through a 2-mm sieve, this aquifer material was found to contain 0.07% clay, 1.88% silt, 4.55% fine sand, 3.1% medium sand and 90.4% coarse sand. Breakthrough experiments were conducted using 0.3-m long plexiglass columns (Soil Measurement Systems, Tucson, AZ) with an internal diameter of 0.05 m. Bottles containing 0.01 M NaCl, deionized water, or the bacterial suspension were connected to the column via a peristaltic pump used to maintain a constant flow velocity. The sand, which was constantly stirred to avoid particle segregation while it was poured into the column, was packed uniformly to a bulk density of 1.750±0.012 Mg M"3 by gently tapping the wall of the columns. One pore volume (PV) was equal to 204 cm3. All the tubing, glassware and valves used in the experiments were autoclaved to avoid contamination. After being sterilized by irradiation with 60Co (2.5 Mrad) , the columns were set up vertically and saturated with sterile deionized water. Upon saturation, a constant upward flow velocity was established using a peristaltic pump. Two velocities were selected: 1.0 X 10"4 m s"1 (3.5 PV h"1) and 2.0 X 10"4 m s"1 (6.9 PV h"1) . Two types of boundary conditions could in principle be adopted in column breakthrough experiments: (a) a step increase in concentration or (b) a pulse-type boundary condition. The latter was chosen, largely to limit the risk of clogging at the inlet end of the column. To obtain a pulse-type boundary condition, the bacterial suspensions were substituted for a period of 1 h for the sterile deionized water that previously percolated through the column. For the experiments in which bacteria were suspended in a NaCl solution, the bacterial pulse was followed by a sterile 0.01 M NaCl solution. Otherwise, the flow of deionized water was resumed. In an experiment designed to determine the effect of a sudden reduction in the ionic strength of the solution, a pulse of bacteria suspended in the NaCl solution was followed by 0.01 M NaCl 9 ------- solution for 1 h and then by deionized water for 2 h. In each experiment, the effluent from the column was collected using a fraction collector. The bacterial concentration in each fraction was determined by the drop plate method (Reed and Reed, 1948) on Trypticase-soy agar using triplicate samples for each dilution. All the breakthrough experiments were carried out in duplicate at 3±1.5°C to limit bacterial growth and death. Numerical integration of the untransformed data, using the trapezoidal method, yielded the percentage of bacteria recovered in the effluent after a given period of time. The breakthrough of a tracer (chloride) was determined separately under the same conditions by applying a 1-h pulse of 0.01 M NaCl followed by deionized water. The chloride concentration in successive fractions of the effluent was measured with a chloridometer (Haake Buchler Instruments, Saddlebrook, NJ) . In all cases, perfectly symmetric breakthrough curves showed that chloride behaved as a tracer and that preferential pathways were absent from the column. STATISTICAL ANALYSIS OF DATA The data were subjected to regression analysis using the Data Desk software package (Odestra Corp., Northbrook, IL) . Significance was determined by the F-ratio, which is the ratio of the mean square for regression to the mean square residual. RESULTS TRANSPORT THROUGH SOIL Bacteria were obtained from Kendaia loam that were able to grow using benzene, chlorobenzene, or toluene as sole source of C and energy. They were identified by standard bacteriological techniques as strains of Pseudomonas. Achromobacter. Bacillus, and Enterobacter. The isolates able to degrade benzene were designated Pseudomonas Benl and Achromobacter Ben2, those degrading chlorobenzene were designated Pseudomonas CB1, Bacillus CB2, and Bacillus CB3, and the bacteria growing on toluene were designated Enterobacter Toll, Enterobacter Tol2, Enterobacter Tol3, and Pseudomonas Tol4. The bacteria obtained from diesel fuel- contaminated soil were designated Achromobacter DF1, Pseudomonas DF2, and Flavobacterium DF3, those from the sewage treatment plant were designated Enterobacter strains IS1 and IS2, and those from Kendaia loam as Enterobacter KL1 and Pseudomonas KL2 and KL3. Pseudomonas KL2 was used to study the direction of movement of bacteria through the soil. The distribution of bacteria in the column showed that the direction of bacterial flow was chiefly downwards in both soil columns tested (TABLE 1) . The presaturation of the soil columns resulted in vertical water flow,- and bacteria 10 ------- TABLE 1. Distribution of Pseudomonas KL2 in columns of soil after passage of four pore volumes of water Sample Sample Cell no. X 10^ sen g Location depth (cm) Column 1 Column 2 Center 0.0-1.7 980 2,200 1.7-3.4 1,400 730 3.4-5.0 720 690 Left, 0.4-2.0 cmf 0-5.0 79 21 Right, 0.4-2.0 cmt 0-5.0 14 0.75 Left, 2.4-4.0 cmf 0-5.0 0.75 0.01 Right, 2.4-4.0 cmt 0-5.0 0.90 <0.01 tLateral distance and left or right of center core. 11 ------- thus moved with the water. Because little horizontal movement was evident and essentially no bacteria reached the walls, movement of cells along the column wall did not appear to contribute to measurements of the vertical mobility of bacteria. In the studies of bacterial transport, the agreement between the numbers of colony-forming units on replicate plates from triplicate columns of soil was good (TABLE 2), except for Bacillus CU4519 and Bacillus CB3, for which fewer than 105 of the 10s cells added to the top of the soil were transported and the agreement among replicate counts was poor. In tests of the numbers of added bacteria that were recovered, replicate counts of the number of colony-forming units agreed with a standard deviation of ±2 0%. Marked differences in the fraction of the added bacteria that moved through the soil were evident among the various species (TABLE 2). For Enterobacter IS2, 15% of the cells were transported, whereas only 0.01% of the added cells of Bacillus CB3 were found in the effluent as viable bacteria. No consistent pattern in mobility was evident among the strains of a genus; thus, more than 5% of the cells of two strains of Enterobacter and three strains of Pseudomonas were transported as compared to <1% of three other strains of the same two genera. The benzene degraders (strains Benl and Ben2) moved to a greater extent than the isolates able to use chlorobenzene (strains CB1, CB2, and CB3) or toluene (Toll, Tol2, Tol3, and Tol4). Of the eight isolates selected for their presumed greater mobility, more than 2% of the cells of six strains (Enterobacter IS2, Enterobacter IS1, Pseudomonas KL2, Pseudomonas DF2, Achromobacter DF1, and Enterobacter KL1) were transported through the soil, whereas the percentages were lower for Pseudomonas KL3 and Flavobacterium DF3. For the bacterium originally obtained from a groundwater aquifer (Arthrobacter Lula D), 7.7% of the cells moved through the soil. The percentage of the added cells that were recovered in the soil and the effluent varied from essentially 100% for Achromobacter Ben2 and Bacillus CB2 to 4.3% for Bacillus CB3 (TABLE 2). Although some strains of a genus showed high recoveries, far lower recoveries were found among other strains of the same genus. The fact that the recoveries of only 5 of the 19 strains were below 25% indicates that high percentages of the cells of many strains did not lose viability and were not irreversibly sorbed to soil particles. A comparison of recovery percentages and the transport percentages indicates that many of the viable cells were retained in the soil column. The data show that the recoveries exceeded 50% for 7 of the 10 isolates for which >2% of the cells were transported, but such high recoveries were found for only 1 of the 9 isolates for which <1% of the cells were transported. Regression analysis indicated that transport was correlated with recovery (F ratio = 5.08, significant at P = 0.05). 12 ------- TABLE 2. The numbers and percentages of bacteria transported and recovered Bacterium Bacteria transportedf (%) Bacteria recoveredf (%) Enterobacter IS2 15±2 61±6 Enterobacter IS1 13±3 60±13 Pseudomonas KL2 8 . 2±1.2 4 6±6 Arthrobacter Lula D 7.7±1.9 39±4 Pseudomonas DF2 6.9±0.4 53±12 Achromobacter Ben2 6.8±0.8 107±13 Pseudomonas Benl 5.9±0.4 71±4 Bacillus CB2 4.1±1.2 104±20 Achromobacter DF1 3.9±0.8 39±4 Enterobacter KL1 2.2±0.4 86±7 Pseudomonas KL3 0.9±0.3 48±3 Enterobacter Tol2 0.9±0.1 14±1 Enterobacter Tol3 0.9±0.1 64±1 Pseudomonas Tol4 0 . 3±0.1 6.5±1.5 Enterobacter Toll 0.2±0.1 34±1 Pseudomonas CB1 0.2±0.1 25±5 Bacillus CU4519 0.1±0.1 9 . 4±1.3 Flavobacterium DF3 0.1±0.01 5 . 4±1.5 Bacillus DB3 0.01±0.00 4 . 3±0.4 fMean ± standard deviation 13 ------- The values for adsorption coefficient (Kd) of the bacteria ranged from 0.0 (i.e., a lower-bound Kd = no bacteria adsorbed) for Enterobacter IS1 to infinity (i.e., a higher bound Kd = all bacterial were adsorbed) for Pseudomonas Tol4 (TABLE 3) . A comparison of the mean values of the lower- and higher-bound Kd values with the transport percentages shows that 8 of 10 bacteria for which >2% of the cells were transported had Kd values <10.0, whereas 8 of 9 bacteria for which <1% of the cells were transported had Kd values >10.0. Thus, a high percentage of cells of strains with low Kd values moved relatively freely through the soil, whereas a low percentage of cells of strains with high Kd values were transported at a significant rate under identical conditions. The relationship between the percentage of the cells transported and the lower- but not the upper-bound Kd values was statistically significant. The F values for the regressions were 7.40 and 3.26, respectively. Regressions indicated that recoveries were related to the Kd values (P = 0.05). When the lower-bound Kd values exceeded 20 or the upper-bound Kd values exceeded 100 (Enterobacter Tol2, Pseudomonas Tol4 and CB1, and Bacillus CU4519) , 25% or fewer of the cells were recovered. This probably reflects the inability to detect a significant fraction of the sorbed cells by the counting procedure. CELL PROPERTIES The hydrophobicities of the bacteria varied markedly. Measurements of hydrophobicity based on bacterial adherence to octane (the BATH method) revealed that less than 10% of the cells of Enterobacter sp. IS2, Pseudomonas sp. KL 2 and Bacillus strains CU4 519 and CB3 adhered to the hydrocarbon, whereas more than 70% of the cells of Bacillus sp. CB2 and Pseudomonas sp. Tol 4 were retained (TABLE 4) . No relationship is evident between the results of the BATH assay and the percentage of cells transported or the classification of the isolates. For most of the test bacteria, more cells were retained by the hydropic gel in the determinations by hydrophobic-interaction chromatography (HIC) than by octane. Except for Achromobacter sp. Ben2, more than 25% of the cells adhered to the gel (TABLE 4). No relation is evident between the results of the HIC assay and the 14 ------- TABLE 3. Adsorption coefficients of bacteria Bacterium Adsorution Lower bound coefficient (Kd) Higher bound Enterobacter IS2 4.5 5.5 Enterobacter IS1 0.0 0.0 Pseudomonas KL2 5.5 8 . 5 Arthrobacter Lula D 5.5 8.0 Pseudomonas DF2 0.85 0.9 Achromobacter Ben2 13 25 Pseudomonas Benl 6.0 8.5 Bacillus CB2 18 36 Achromobacter DFl 7.5 11 Enterobacter KL1 3.5 4.5 Pseudomonas KL3 9.0 24 Enterobacter Tol2 29 145 Enterobacter Tol3 6.5 8.5 Pseudomonas Tol4 45 00 Enterobacter Toll 9.5 16 Pseudomonas CB1 26 150 Bacillus CU4519 30 410 Flavobacterium DF3 13 17 Bacillus CB3 12 24 15 ------- TABLE 4. Relationship between characteristics of bacteria and their transport through soil Cells trans- Cells retained m Zeta Cell ported BATH HIC Anion Cation potential length Cap Bacteria (%) assay assay exchange exchange (-mV) (f/m) sul< Enterobacter IS2 15 8 29 98 14 24 0.7 + Enterobacter IS1 13 21 29 98 29 24 0.7 + Pseudomonas KL2 8.2 6 28 97 9 21 1.4 + Arthrobacter Lula D 7.7 13 91 98 7 31 0.8 - Pseudomonas DF2 6.9 17 29 99 5 19 0.8 + Achromobacter Ben2 6.8 31 7 98 23 18 1.4 - Pseudomonas Benl 5.9 25 45 100 5 26 2.1 - Bacillus CB2 4.1 85 58 98 21 8 0.9 + Achromobacter DR1 3.9 61 66 98 10 13 1.9 + Enterobacter KL1 2.2 17 28 99 25 21 1.8 - Pseudomonas KL3 0.9 21 32 98 7 32 1.8 - Enterobacter Tol2 0.9 19 39 99 47 21 2.6 - Enterobacter Tol3 0.9 11 41 98 15 19 2.2 - Pseudomonas Tol4 0.3 75 50 96 10 21 1.7 + Enterobacter Tolll 0.2 69 50 99 9 11 1.0 - Pseudomonas CB1 0.2 44 64 97 18 21 2.5 - Bacillus CU4519 0.1 5 77 100 20 36 1.6 - Flavobacterium DF3 0.1 29 59 98 39 13 1.3 - Bacillus CB3 0. 01 5 61 98 7 15 2.1 + ------- percentage of cells transported or the genera in which the bacteria are classified. Moreover, as indicated by the F value, regression analysis showed that the correlation between the results of the BATH and HIC assays was poor. Elution of the columns with deionized water rather than 4 M NaCl led to recovery of 100% of the added cells of all strains tested, indicating that the cells were not removed in the assay by filtration. Measurements were made of the net surface charge by determinations involving ESIC and zeta potentials. In the chromatographic procedure, anion- and cation-exchange resins were used. Most cells of the 19 strains were retained by the anion- exchange resins, and 97 to 100% of the cells were retained by this method (TABLE 4). On the other hand, appreciable differences were noted in the percentage of cells retained by the cation-exchange resins. More than 30% of the cells of Enterobacter sp. Tol2 and Flavobacterium sp. DF3 but less than 10% of the cells of Pseudomonas strains KL2, DF2, Benl, and KL3, Arthrobacter sp. Lula D, Enterobacter Tol4, and Bacillus sp. CB3 were retained. The zeta potentials of the bacteria also differed, ranging from values of -8 mV for Bacillus sp. CB2 to -36 mV for Bacillus sp. CU4519. A relation was not evident between the surface charge of the 19 bacteria determined by these means and their mobilities through soil or generic placement. The cell sizes and the presence of capsules were determined for each of the 19 organisms. The width of the cells did not differ appreciably, ranging from 0.5 to 0.7 jJm. In contrast, the lengths varied from 0.7 to 2.6 fJm (TABLE 4). Transport of the bacteria was statistically related to length of cells (F = 11.8, significant at P = 0.05) as indicated by regression analysis. Eight of the strains produced capsules and ten formed none. However, nonencapsulated and capsulated cells were evident among the strains showing both good (>1% transport) and poor mobilities (<1% transport). Only 10 of the 19 bacteria were tested for the presence of flagella. Eight strains were flagellated. Enterobacter strains IS2 and IS1, Pseudomonas strains DF2 and Benl, and Bacillus sp. CU4 519 had peritrichous flagella, Pseudomonas sp. KL2 and Bacillus strains CB2, CU4519, and CB3 bore lophotrichous flagella Pseudomonas sp. KL2 and Bacillus sp. CB2 were amphitrichous, and Pseudomonas sp. DF2 and Bacillus strains CU4519 and CB3 were monotrichous. Flagella were thus present on cells of strains that exhibited poor (<1% transport) and good (>1% transport) mobilities. TRANSPORT THROUGH AQUIFER SOLIDS In the experiments involving low flow rate (1.0 X 10~Sii s"1) and low bacterial concentration (1.0 X 108 cells mL"1), a distinct effect of the ionic strength of the carrying liquid phase on the 17 ------- extent of bacterial breakthrough was evident (Fig. 2). When the bacteria were suspended in 0.01 M NaCl solution, their concentration (C) in the effluent never reached more than 2.2% of the input concentration, (Co). In contrast, the relative concentration C/Co approached unity after approximately 70 min when the bacteria were suspended in deionized water. After 2 h of flow through the columns, 100% of the chloride tracer and 60% of the bacteria suspended in deionized water were transported through the column vs. only 1.5% of the cells suspended in 0.01 M NaCl solution. Compared with the breakthrough of chloride, the breakthrough of bacteria suspended in deionized water was significantly retarded (Fig. 3). The time for first measurable appearance of cells in the effluent was not greatly different in both cases; i.e., approximately 12 min for chloride and 15 min for bacteria. However, whereas the relative concentration of chloride in the effluent increased rapidly thereafter, reaching a value of 1.0 only 30 min after application of the pulse, the ratio C/Co for the bacteria increased more gradually. The tail ends of the breakthrough curves for chloride and bacteria suspended in deionized water appeared to coincide, a sharp drop occurring in both cases at about 75 min. A similar drop in C/Co at approximately 7 5 min was also apparent for bacteria suspended in 0.01 M NaCl solution. However, it is necessary to bear in mind the experimental error involved in. the evaluation of the bacterial concentration as illustrated by the scatter. The bacterial concentration in the effluent after 100 min became negligibly small both in the systems with deionized water and 0.01 M NaCl solution. In the experiments involving a higher flow rate (2.0 X 10"4m s"1) , the breakthrough was similar to that described above with a few differences. When bacteria were suspended in 0.01 M NaCl solution, more cells (3.9% vs. 1.5%) were transported through the column, and their relative concentration reached 7.3% (vs. 2.2% observed earlier) (Fig. 4). Nevertheless, reduction of the ionic strength of the liquid phase resulted in a significantly increased rate of transport (77%) and in a peak relative concentration reaching 1.0. The higher flow rate produced a shortened time to the breakthrough of chloride; after only 5 min, chloride appeared in the effluent, and its relative concentration then increased sharply to reach 1.0 in less than 10 min. Essentially all (99%) of the chloride passed through the column. The bacteria suspended in deionized water followed the same pattern (Fig. 5) with, however, less apparent delay than in the first experiment. The time for bacterial breakthrough was between 5 and 10 min, and the relative cell concentration reached 1.0 approximately 30 min after application of the pulse. Thereafter, the behavior of the bacteria suspended in deionized water coincided with that of chloride. In particular, the tail ends of their breakthrough curves were congruent, precisely the same drop in C/Co being observed in both case between 65 and 70 min. A similar decrease of C/C0 was 18 ------- 0.080 0.060 0.040 0.020 Hours Figure 2. Breakthrough of Pseudomonas sp. KL2 with 0.01 M NaCl (flow rate 1.0 X 10"4 in s"1, equivalent to 3.5 pore volume h"1). 19 ------- 1.0 0.8 0.6 0.4 0.2 Deionized M. °o° cPO O 000 o cp°^o°1^ o w o • • • • 3 • • • %. • • • 0.5 1.0 Hours o chloride • bacteria ftabt I t»t • 1.5 Figure 3. Breakthrough of Pseudomonas sp. KL2 with deionized water (flow rate 1.0 X 10"4 m s"1, equivalent to 3.5 pore volumes h"1) . 20 ------- 1 CJ CJ B •H 4-> cd U 4-> C •H Pi 0.080 0.060 0.040 0.020 0.01 M NaCl • • _L * • • f 0.5 1.0 Hours 1.5 Figure 4. Breakthrough of Pseudomonas sp. KL2 with 0.01 M NaCl solution and flow rate of 2.0 X 10"4 m s"1, equivalent to 6.9 pore volumes h"1. 21 ------- 1 u u 8 •H cd 4-i & 03 CJ S u > •H 4J CO '—I Si Pd 1.0 0.8 0.6 0.4 0.2 peionized H^O O o° o " o_ ^ % CP o chloride • bacteria 0.5 1.0 Hours 1.5 Figure 5. Breakthrough of Pseudomonas sp. KL2 with deionized water at a flow rate of 2.0 X 10"4 m s"1, equivalent to 6.9 pore volumes h"1. 22 ------- observed within the same time period when the bacteria were suspended in 0.01 M NaCl solution. When a cell density of 1.0 X 109 cells mL"1 was used, the breakthrough curves for bacteria in 0.01 M NaCl solution and deionized water were almost identical except that the relative concentration of bacteria at the peak approached 1.0 at the low ionic strength, whereas C/CQ was 0.7 at the higher ionic strength (Fig. 6 and 7) . A precipitous drop in relative concentration occurred in both cases between 70 and 8 0 min. This time range corresponded closely to the tail ends of the chloride and bacterial breakthrough curves that were previously noted under similar flow conditions. The percentages of bacteria transported through the column were 44% with 0.1 M NaCl and 57% with deionized water. The effect of reducing the ionic strength (replacing 0.01 M NaCl solution with a pulse of deionized water) of the inflowing solution on the breakthrough of bacteria is shown in Fig. 8. A control was conducted in which 0.01 M NaCl was used for the entire test period. When only NaCl solution was used, a single peak (0.022) of bacterial breakthrough followed by a tail was observed. The continuous application of NaCl solution did not displace bacteria already retained in the column. However, when the NaCl solution was replaced with deionized water, a second peak of bacterial breakthrough was observed. The relative bacterial concentration at the second peak reached 0.12, which was 4 times higher than the first peak. The decrease in ionic strength lowered the retention of bacteria in the column. DISCUSSION Macropore flow may be a major mechanism of bacterial transport in soils. Therefore, the use of undisturbed soil columns might have provided data on bacterial transport that would have particular relevance to circumstances prevalent in the field (Smith et al., 1985). However, columns of homogeneous soil were used to avoid uncontrolled, preferential movement of bacteria through macropores and thus to permit a definition of the factors that control the movement of bacteria through the soil matrix itself. For the development of the procedure for determinations of mobility, a loamy soil was selected to avoid the extremes of limited bacterial sorption and nearly free movement in sandy soils on the one hand and the restricted movement resulting from mechanical filtration and increased sorption in fine structured soils on the other hand. Had these more extreme conditions been imposed, the ability of the procedure to detect small differences in bacterial mobilities might not have been evaluated. Saturated soil with a constant head of water was used to mimic bacterial movement under conditions of saturated flow. This permitted the occurrence of mass transport of the cells in the sufficiently large pores of the homogeneous soil. Bitton et al. 23 ------- CJ> CJ> c o •H 4-1 tfl 5-1 4-1 G 0) U C o CJ 0) > •H 4-1 to r-H O) ctj Hours Figure 6. Breakthrough of Pseudomonas sp. KL2 with 0.01 M NaCl at 1.0 X 10V cells mL"1 (flow rate 1.0 X 10'* m s"1 equivalent to 3.5 pore volumes h"1). 24 ------- Figure 7. Breakthrough of Pseudomonas sp. KL2 with deionized water at 1.0 X 10v cells mL"1 (flow rate equivalent to 3.5 pore volumes h"1) . 1.0 X 10" m s-1 25 ------- 0.12 0.09 0.06 0.03 B ~ ei . *B nfel 1—«- 2 3 Hours M A Figure 8. Breakthrough of Pseudomonas sp. KL2 at 1.0 X 109 cells mL~1 using 0.01 M NaCl for 1 h followed by deionized water (flow rate 1.0 X 10"4 m s"1 equivalent to 3.5 pore volumes h"1) . 26 ------- (1974) showed that movement of bacteria through soil columns stopped when the water content was at or below field capacity, and Madsen and Alexander (1982) demonstrated that movement of bacteria through soil was not detectable in the absence of a transporting agent such as water. The procedure described here has several advantages for testing bacterial mobility in the soil matrix. Spurious data on mobility resulting from bacteria moving at the interface between the soil and the column wall are avoided through the use of a relatively wide column and the coating of the column walls with petrolatum to bind soil particles to the walls. Channels through which bacteria could move preferentially were eliminated by grinding and sieving the soil prior to preparing the column and, to ensure reproducible measurements of transport, by uniform packing to a fixed bulk density. Bacterial death from predation or parasitism was avoided because such predators and parasites were killed by irradiating the soil. Increases in cell numbers arising from growth and decreases associated with starvation were prevented by performing the tests of transport at 2 to 5°C. Furthermore, the marked differences in mobility among the isolates suggest that the proposed procedure does indeed distinguish among bacteria with different capacities for movement. For inoculation of the soil surface with bacteria that can degrade organic pollutants at some underground site, some of the added cells must move through the soil to the below ground zone of contamination. Evidence exists, however, that introduced organisms may fail because they are not transported to sites containing the chemical (Goldstein et al., 1985). In this context, it is worth noting that many of the carefully controlled experiments in which inoculation resulted in biodegradation required transport to soil depths of only 10 cm (Barles et al., 1979; Edgehill and Finn, 1983; McClure, 1972). Measurements such as those described in the present study will enable extrapolation of the potential penetrability of the bacteria to considerably greater depths. The capacity to degrade a chemical in culture is a necessary but not sufficient requisite for successful biodegradation in the field because, in addition to other traits, the inoculum strain must possess the traits that enable it to move through soil, subsoil, or aquifer materials to reach the area of chemical contamination. The ability of bacteria to be transported to subsurface contaminated zones may be evaluated from bacterial properties such as cell size and their susceptibility to adsorption. The correlation between these properties and mobility can be determined by the procedure here proposed. Such a correlation may enable the classification of bacteria according to their potential mobility. The susceptibility of a bacterium to predation or parasitism in the matrix through which it must pass must also be determined before its ability to reach the zone of contamination can be predicted. 27 ------- The data show a significant inverse correlation between the lower-bound Kd values and the fraction of cells transported through the column. The values for recovered cells transported varied from nearly zero to 25% at Kdl <2, whereas the values never exceeded 8% at Kdl >2. Adsorption, when sufficiently strong, can effectively retard the bacteria. If adsorption is weak, the transport of cells through the soil matrix may be controlled by mechanical filtration. The correlation between Kdl and Kdh was good enough to make their use in many cases interchangeable. The dimensions of the bacterial cells affected their mobility. As the cell length increased, the highest value for recovered cells transported among bacteria of a given length decreased. Thus, the longer the cell, the less its likelihood of passing through the soil matrix. Cell length was not significantly correlated with the recovery, Kdh, or Kdl. Cell hydrophobicity, net surface electric charge, and the presence of capsular polysaccharides were evaluated because they are properties of bacterial cells that appear to be involved in adsorption of bacteria to solid surfaces (Fattom and Shilo, 1984; Rosenberg and Kjelleberg, 1986). The sizes of the cells (Corapcioglu and Haridas, 1984; Pekdeger and Matthess, 1983) were tested because larger cells may be more readily removed by filtration than smaller cells. The presence of flagella might also impede movement, so their occurrence on the test strains was investigated. Varying degrees of hydrophobicity were observed among the bacteria, but the results of the BATH and HIC assays did not agree. The nonuniformity of the bacterial surface may cause a bacterium to be hydrophilic in one assay and hydrophobic in another (Stenstrom, 1989) . Moreover, hydrophobicity is not a definitive characteristic but varies with the hydrocarbon used for the assay (Marshall and Cruickshank, 1973; Stotzky, 1985). Measurements of zeta potential revealed that all of the test bacteria had net negative surface charges. However, bacteria with positive charges on nonuniform cell surfaces may adhere to negatively charged particles of soil (Marshall, 197 6). No pattern was observed between the extent of transport and bacterial surface charges. Adherence of bacteria to solid surfaces may result from their having extracellular polysaccharides (Costerton et al. , 1978 ; Sutherland, 1983). Of the strains for which more than 1.0% of the cells passed through the soil, six had capsules, indicating that capsules do not necessarily hinder transport. Measurements were made of cell size because large bacteria presumably are less likely to pass through soil pores than small cells, and a statistical relationship between size and transport of bacteria through soil was evident. Although cell appendages could impede mobility, a relationship was not observed between flagellation and transport. 28 ------- Motility was not considered important in the present study because transport was tested at low temperatures. The present findings suggest that it should be possible to obtain bacteria that have both the capacity to biodegrade unwanted organic compounds and the ability to move through earth materials to sites containing these chemicals, as indicated by the observation that high percentages of two benzene degraders and one chlorobenzene-utilizing bacterium moved through soil in appreciable numbers. The movement of bacteria through homogeneous sand increased when the inflowing solution had a low ionic strength. When deionized water was used, the bacteria moved readily through the sand column after an initial period of retention. The high mobility of bacteria may result from the relatively inert surfaces of the aquifer materials at low ionic strength, thus limiting adsorption, and large pores and pore necks which allowed bacteria to pass through with minimal filtration. The breakthrough of bacteria and chloride differed in that bacterial breakthrough was slower, transport of bacteria was less than chloride, and the relative concentration of bacteria increased gradually and tailed slightly. These differences indicated that the cells were briefly retained in the porous medium. The retention of bacteria is generally attributed to filtration in the contact zones of adjacent pores, sedimentation in the pores and/or adsorption (Corapcioglu and Haridas, 1984) . Gerba et al. (197 5) noted that removal of bacteria appears to occur largely at or near the soil surface. Filtration at constrictions in drainage channels and in nontransmissing pores can limit bacterial movement through porous media (Vinten and Nye, 1985) . Bacterial adsorption may result from van der Waals forces, electrostatic interactions, hydrophobicity and polymer bridging (Marshall et al. , 1971). If the aquifer material has low adsorption capacity, the potential adsorption sites will be filled by the bacteria that arrive first. The bacteria that follow can either travel freely or replace those cells that are adsorbed. Thus, bacterial breakthrough would be retarded only until the adsorption sites were filled. In addition, the adsorption of bacteria may reduce the diameter of pore necks and enhance filtration to some extent. The salt solution dramatically reduced bacterial breakthrough. The total bacterial breakthrough was low because the cells were removed from solution by interactions with the sand matrix. Cations such as Ca, Mg, and Na increase bacterial adsorption by neutralizing the negative surface charge of the bacteria (Yates and Yates, 1988). The enhanced retardation of bacterial transport in the presence of NaCl may result from a further compression of the electrical double-layer arising from the increase in ionic strength. The diffuse double-layer may be more compressed and the interparticle distances reduced, thus creating more opportunities for adsorption (Marshall, 1975). When deionized 29 ------- water was used, nearly all the bacterial cells moved through the system after the initial stage of retention, suggesting that the pores and pore necks were not restricting bacterial passage. It is unlikely that NaCl increased the number of terminal pores or changed the pore size distribution of the aquifer material because of the low clay content of the aquifer material. The replacement of the NaCl solution with deionized water resulted in a second peak of bacterial breakthrough. The retention caused by the adsorption mechanisms appeared to be reversible when ionic strength was changed. Filtration would not likely be reversible unless the porous medium was disturbed. Differences in the transport of bacteria noted in experiments with 0.01 M NaCl and deionized water were more marked at 1.0 X 108 than at 1.0 X 109 cells per mL. This may be linked with available filtration and adsorption sites. At low cell concentrations, the retention sites presumably were not filled so that most of the bacteria were adsorbed, whereas most of the retention sites presumably were filled at a higher cell concentration, so that the additional bacteria traveled freely or replaced bacteria that were retained. However, tests with deionized water failed to reveal appreciable differences between the two cell concentrations, possibly a result of the reduction in ionic strength that decreased the adsorption capacity of the sand. An increase in flow velocity somewhat reduced bacterial retention, a decrease that may be related to the reduction in bacterial residence or "reaction" time at higher flow velocity. The experimental results suggest that adsorption significantly contributed to the retention of bacteria and that bacterial movement through aquifer sand was enhanced by reducing the ionic strength of the inflowing solution. Cell density and flow velocity also influenced bacterial movement. For bioremediation of contaminated sandy aquifers, manipulation of ionic strength may thus be a means to facilitate movement of bacteria to the site of organic contamination. 30 ------- REFERENCES Barles, R. W. , C. G. Daughton, and D. P. H. Hsieh. 1979. Accelerated parathion degradation in soil inoculated with acclimated bacteria under field conditions. Arch. Environ. Contain. Toxicol. 8:647-660. Baveye, P., and A. Valocchi. 1989. An evaluation of mathematical models of the transport of biologically reacting solutes in saturated soils and aquifers. Water Resour. Res. 25:1413- 1421. Bell, R. G., and J. B. Bole. 1978. Elimination of fecal coliform bacteria from soil irrigated with municipal sewage lagoon effluent. J. Environ. Qual. 7:193-196. Bitton, G., J. M. Davidson, and S. R. Farrah. 1979. On the value of soil columns for assessing the transport pattern of viruses through soils: a critical outlook. Water Air Soil Pollut. 12:449-457. Bitton, G. and C. P. Gerba. 1984. Groundwater pollution microbiology: The emerging issue. p. 1-7. In G. Bitton and C. P. Gerba (eds) Goundwater pollution microbiology. J. Wiley, New York. Bitton, G., N. Lahav, and Y. Henis. 1974. Movement and retention of Klebsiella aeroaenes in soil columns. Plant Soil 40:373- 380. Borden, R. C. , and P. B. Bedient. 1986. Transport of dissolved hydrocarbons influenced by oxygen-limited biodegradation. 1. Theoretical development. Water Resour. Res. 22:197 3-1982. Brown, K. W., H. W. Wolf, K. C. Donnelly, and J. F. Slowey. 1979. The movement of fecal coliforms and coliphages below septic lines. J. Environ. Qual. 8:121-125. Calleja, G. C. 1984. Identifying the problems: Theoretical and experimental approaches, p. 21-91. In Microbial aggregation. CRC Press, Boca Raton, Florida. Corapcioglu, M. Y., and A. Haridas. 1984. Transport and fate of microorganisms in porous media: a theoretical investigation. J. Hydrol. 72:149-169. Corapcioglu, M. Y., and A. Haridas. 1985. Microbial transport in soils and groundwater. A numerical model. Adv. Water Resour. 8:188-200. Costerton, J. W., G. G. Geesey, and K.-J. Cheng. 1978. How bacteria stick. Sci. Am. 238:86-95. 31 ------- Dazzo, F., P- Smith, and D. Hubbell. 1973. Vertical dispersal of fecal coliforms in Scranton fine sand. Proc. Soil Crop Sci. Soc. Fla. 32:99-102. Dillon, J. k. , J. A. Fuerst, A. C. Hayward, and G. H. G. Davis. 1986. a comparison of five methods for assaying bacterial hydrophobicity. j. Microbiol. Methods 6:13-19. Duboise, S. M. , B. E. Moore, and B. P. Sagik. 1976. Poliovirus survival and movement in a sandy forest soil. Appl. Environ. Microbiol. 31:536-543. Edgehill, R. u., and R. K. Finn. 1983. Microbial treatment of soil to remove pentachlorophenol. Appl. Environ. Microbiol. 45:1122-1125. Fattom, A., and M. Shilo. 1984. Hydrophobicity as an adhesion mechanism of benthic cyanobacteria. Appl. Environ. Microbiol. 47:135-143. Gannon, J. T. , V- B. Manilal, and M. Alexander. 1991. Relationship between cell properties and the transport of bacteria through soil. Appl. Environ. Microbiol. 57:190-193. Gerba, C. P-, and J. C. Lance. 1978. Poliovirus removal from primary and secondary sewage effluent by soil filtration. Appl. Environ. Microbiol. 36: 247-251. Gerba, C. P., C. Wallis, and J. L. Melnick. 1975. Fate of wastewater bacteria and viruses in soil. J. Irrig. Drain. Div., Proc. Am. Soc. Civ. Eng. 101(IR3):157-174. Germann, P. F., M. S. Smith, and G. W. Thomas. 1987. Kinematic wave approximation to the transport of Escherichia coli in the vadose zone. Water Resour. Res. 23:1281-1287. Goldshmid, J., D. Zohar, Y. Argaman, and Y. Kott. 1973. Effect of dissolved salts on the filtration of coliform bacteria in sand dunes, p. 147-157. In S. H. Jenkins (ed.) Advances in water pollution research. Pergamon Press, New York. Goldstein, R. M., L. M. Mallory, and M. Alexander. 1985. Reasons for possible failure of inoculation to enhance biodegradation. Appl. Environ. Microbiol. 50:977-983. Hagedorn, C., E. L. McCoy, and T. M. Rahe. 1981. The potential for groundwater contamination from septic effluents. J. Environ. Qual. 10:1-8. Harvey, R. W., L. H. George, R. L. Smith, and D. R. LeBlanc. 1989. Transport of microspheres and indigenous bacteria through a 32 ------- sandy aquifer: Results of natural- and forced-gradient tracer experiments. Environ. Sci. Technol. 23:51-56. Hattori, T. and R. Hattori. 197 6. The physical environment in soil microbiology: An attempt to extend principles of microbiology to soil microorganisms. CRC Crit. Rev. Microbiol. 4:423-461. Jackson, M. L. 1974. Soil chemical analysis - Advanced course, p. 110-114. Published by author, Dept. of Soil Sci., Univ. of Wisconsin, Madison, Wis. Krone, R. B., G. T. Orlob, and C. Hodgkinson. 1958. Movement of coliform bacteria through porous media. Sew. Indus. Wastes 30:1-13. Madsen, E. L., and M. Alexander. 1982. Transport of Rhizobium and Pseudomonas through soil. Soil Sci. Soc. Am. J. 46:557-560. Marshall, K. C. 1975. Clay mineralogy in relation to survival of soil bacteria. Annu. Rev. Phytopathol. 13:357-373. Marshall, K. C. 197 6. Interfaces in microbial ecology. Harvard University Press, Cambridge, MA. Marshall, K. C. 1980. Adsorption of microorganisms to soils and sediments. p. 317-329. In G. Bitton and K. C. Marshall (eds.) Adsorption of microorganisms to surfaces. Wiley, New York. Marshall, K. C., and R. H. Cruickshank. 1973. Cell surface hydrophobicity and the orientation of certain bacteria at interfaces. Arch. Mikrobiol. 91:29-40. McCarty, P. L., B. E. Rittmann, and E. J. Bouwer. 1984. Microbiological processes affecting chemical transformations in groundwater. p. 89-115. In G. Bitton, and C. P. Gerba (ed.) Groundwater pollution microbiology. Wiley, New York. McClure, G. W. 1972. Degradation of phenylcarbamates in soil by mixed suspension of IPC-adapted microorganisms. J. Environ. Qual. 1:177-180. Molz, F. J., M. A. Widdowson, and L. D. Benefield. 1986. Simulation of microbial growth dynamics coupled to nutrient and oxygen transport in porous media. Water Resour. Res. 22:1207-1216. Mozes, N., F. Marchal, M. P. Hermesse, J. L. Van Haecht, L. Reuliaux, A. J. Leonard, and P- G. Rouxhet. 1987. Immobilization of microorganisms by adhesion: Interplay of 33 ------- electrostatic and non-electrostatic interactions. Biotech. Bioeng. 30:439-450. Mozes, N. , and P. G. Rouxhet. 1987. Methods for measuring hydrophobicity of microorganisms. J. Microbiol. Methods 6:99- 112 . Pedersen, K. 1981. Electrostatic interaction chromatography, a method for assaying the relative surface charges of bacteria. FEMS Microbiol. Lett. 12:365-367. Peele, T. C. 193 6. Adsorption of bacteria by soils. Cornell Univ- Agric. Sta. Memoir 197, p. 3-18. Pekdeger, A., and G. Matthess. 1983. Factors of bacteria and virus transport in groundwater. Environ. Geol. 5:49-52. Rahe, T. M. , C. Hagerdon, E. L. Mc Coy, and G. F. Kling. 1978. Transport of antibiotic-resistant Escherichia coli through western Oregon hillslope soils under conditions of saturated flow. J. Environ. Qual. 7:487-494. Reed, R. W. , and G. B. Reed. 1948. Drop plate method of counting viable bacteria. Can. J. Res. (Sec. E), 26:317-326. Rittmann, B. E. , P. L. McCarty, and P. V. Roberts. 1980. Trace- organics biodegradation in aquifer recharge. Ground Water 18:236-243 . Rosenberg, M. , D. Gutnick, and E. Rosenberg. 1980. Adherence of bacteria to hydrocarbons: A simple method for measuring cell- surface hydrophobicity. FEMS Microbiol. Lett. 9:29-33. Rosenberg, M., and S. Kjelleberg. 1986. Hydrophobic interactions: Role in bacterial adhesion. Adv. Microb. Ecol. 9:353-393. Schaub, S. A., and C. A. Sorber. 1977. Virus and bacteria removal from wastewater by rapid infiltration through soil. Appl. Environ. Microbiol. 33:609-619. Smith, M. S., G. W. Thomas, R. E. White, and D. Ritonga. 1985. Transport of Escherichia coli through intact and disturbed soil columns. J. Environ. Qual. 14:87-91. Sosbey, M. D., C. H. Dean, M. E. Knuckles, and R. A. Wagner. 1980. Interactions and survival of enteric viruses in soil materials. Appl. Environ. Microbiol. 40:92-101. Stenstrom, T. A. 1989. Bacterial hydrophobicity, an overall parameter for the measurement of adhesion potential to soil particles. Appl. Environ. Microbiol. 55:142-147. 34 ------- Stotzky, G. 1985. Mechanisms of adhesion to clays with reference to soil systems, p. 195-253. In D. C. Savage and M. Fletcher (ed.), Bacterial adhesion: mechanisms and physiological significance. Plenum Publishing Corp., New York. Sutherland, I. W. 1983. Microbial exopolysaccharides - Their role in microbial adhesion in aqueous systems. CRC Crit. Rev. Microbiol. 10:173-201. Tan, Y. 1989. Transport of bacteria in porous media. Ph.D. thesis. Australian National Univ., Canberra, Australia. Taylor, S. W. , and P. R. Jaffe. 1990. Substrate and biomass transport in a porous medium. Water Resour. Res. 26:2181- 2194 . van der Mei, H. C., A. H. Weerkamp, and H. J. Busscher. 1987. A comparison of various methods to determine hydrophobic properties of streptococcal cell surfaces. J. Microbiol. Methods 6:277-287. Vinten, A. J. A., and P- H. Nye. 1985. Transport and deposition of dilute colloidal suspensions in soil. J. Soil Sci. 36:531- 541. Viraraghavan, T. 1978. Travel of microorganisms from a septic tile. Water Air Soil Pollut. 9:355-362. Wilson, J. T. , J. F. McNabb, D. L. Balkwill, and W. C. Ghiorse. 1983. Enumeration and characterization of bacteria indigenous to a shallow water-table aquifer. Ground Water 21:134-142. Wollum, A. G. , II., and D. K. Cassel. 1978. Transport of microorganisms in sand columns. Soil Sci. Soc. Am. J. 42:72- 76. 35 ------- |