Enhancing bacterial transport for bioaugmentation of aquifers using low ionic strength solutions and surfactants

PII: S0043-1354(98)00291-7

Wat. Res. Vol. 33, No. 4, pp. 1090±1100, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/99/$ - see front matter

ENHANCING BACTERIAL TRANSPORT FOR BIOAUGMENTATION OF AQUIFERS USING LOW IONIC STRENGTH SOLUTIONS AND SURFACTANTS M QUN LI1 and BRUCE E. LOGAN2**

Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ 85721, U.S.A. and 2Department of Civil and Environmental Engineering, Penn State University, University Park, PA 16802, U.S.A.

1

(First received August 1997; accepted in revised form June 1998) AbstractÐThe transport of bacteria in contaminated aquifers over a distance of only one meter can require tens to thousands of unsuccessful collisions of bacteria with soil grains. Previous work has shown that low ionic strength (IS) solutions and the nonionic surfactant Tween 20 can reduce bacterial adhesion to ultraclean surfaces such as glass and quartz porous media. In this study, we examined whether these results could be generalized to soils and to other surfactants, by measuring the retention of two species of radiolabeled microbes over short (1 cm) distances in soil minicolumns. Calculations were also made, using the clean-bed ®ltration theory, to evaluate if bacterial transport distances are sucient for bioagumentation to occur over a large region of the subsurface. Collision eciencies were expressed using the ®ltration model in terms of the sticking coecient, a, de®ned as the fraction of collisions that are successful. In glass bead columns, a's for monoclonal populations were reduced from a = 0.19 (Alcaligenes paradoxus) and a = 0.01 (CD1), to a < 0.008 for Tween 80-phosphate bu€er solutions and a < 0.0054 for low ionic strength (0.01 mM) solutions (Darcy velocity, U = 10ÿ3 m sÿ1; Hammaker constant = 10ÿ20 J; and ¯uid properties of water at 228C). Low a's were also obtained using other nonionic surfactants (Tween 80, Triton 100 and 705, POE-10, Brij + 35) and an anionic biosurfactant, all added at concentrations above their critical micelle concentration (CMC). Although sticking coecients were also reduced by an order-of-magnitude for natural soils, sticking coecients remained too high to permit wide dispersal of cells over distances of >1 m. For A. paradoxus, a was reduced using a low ionic strength solution from 0.72 to 0.083 for the Arizona soil and from 1.7 to 0.2 for the Ringold soil; for CD1, a was reduced from 0.57 to 0.09 for the Ringold soil. Based on the soil grain diameters of these soils (127 mm, Arizona soil; 224 mm, Ringold soil), a's in this range will permit transport distances (de®ned as 99.9% reduction in cell concentration) of 01 m (U = 10 m/d, 108C) which may be sucient for creating small, bioactive zones. However, in order to increase bacterial transport over distances >1 m, methods other than simple solution chemistry changes will be needed to enhance aquifer bioaugmentation operations. # 1999 Elsevier Science Ltd. All rights reserved Key wordsÐAlcaligenes paradoxus bacteria, bioremediation, column tests, pollutant, soil, subsurface

INTRODUCTION

Bacteria can be used for subsurface remediation to enhance the degradation of speci®c pollutants either by adding nutrients to stimulate the growth of existing subsurface populations (biostimulation) or by directly injecting microbial cultures into the aquifer (bioaugmentation). A major impediment to bioaugmentation is that the soil acts as an ecient ®lter and reduces the concentration of suspended bacteria by several orders-of-magnitude within 10 to 100 cm of the well. The retention and growth of these microbes over such a short distance can lead to well clogging and failure of a bioremediation process. The rapid removal of bacteria within the aquifer results from a combination of high collision fre*Author to whom all correspondence should be addressed. [Tel.: +1-814-863-7908; Fax: +1-814-863-7304; Email: [email protected]].

quencies and high attachment probabilities between the bacteria and soil grains. It can be shown using a standard ®ltration model to describe bacterial transport (Martin et al., 1996) that a nonattaching bacterium would have to undergo >500 collisions to be successfully transported a distance of only 1 meter from a well under typical bacteria and groundwater conditions (cell diameter of 1 mm, super®cial velocity of 1 m/d, soil grain diameter of 120 mm, porosity of 0.33 and 108C). Collision frequencies can be decreased slightly by increasing groundwater velocities. Bacterial attachment probabilities, however, vary widely and can be substantially altered through changes in solution chemistry (Fletcher, 1980; Gordon and Millero, 1984; van Loosdrecht et al., 1987; Gross and Logan, 1995). Bacterial sticking coecients, de®ned as the rate bacteria stick to a soil grain to the rate they collide (a), measured in the laboratory are typically 0.1±1 for laboratory grown cells suspended in water at

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ionic strengths typical of groundwater (2 mM) or higher, but a values may be much lower for indigenous microbes (Harvey and Garabedian, 1991; Martin et al., 1992; Gross and Logan, 1995; Jewett et al., 1995; Johnson et al., 1996). Harvey and Garabedian (1991) calculated sticking coecients of 0.0054 < a < 0.0097 for DAPI-stained indigenous microbes used in ®eld tests. Bacterial a's are dicult to measure in the laboratory, using a traditional approach of calculating a from steady state breakthrough concentrations in column tests. In instances where a's are high, it is usually necessary to use high cell concentrations in the column in¯uent to obtain measurable concentrations of cells in the column e‚uent. However, high colloid concentrations can ®ll the soil grain surface and invalidate the assumptions of the cleanbed ®ltration theory used to calculate a (Yao et al., 1971; Liu et al., 1995). Column media can become ®lled, or ``jammed'', with colloids with as little as 3% of the soil grain surface area covered, preventing other suspended particles from interacting with soil surfaces (Rijnaarts et al., 1996). When bacteria have extremely low a's, it is dicult to obtain accurate estimates of a from column breakthrough concentrations, unless very long columns are used, since e‚uent concentrations will nearly equal in¯uent concentrations. For example, Jewett et al. (1993) calculated that a column r60 m long would be necessary to accurately (95% con®dence interval) measure a low a (0.0021) for column conditions used by Martin et al. (1992). Precise estimates of a can be obtained over several orders-of-magnitude in a single column set up, when particle ®ltration rates are based on total mass retention, instead of breakthrough concentrations. Sticking coecients as low as 3  10ÿ5 can be measured over a transport distance of only 1 cm in minicolumns packed with 40 mm glass beads using the microbe and radiolabel kinesis (MARK) procedure (Gross et al., 1995). Using the MARK test, a variety of chemicals (Tween 20, sodium forms of dodecyl sulfate, PPi and periodate, lysozyme and protease K) known to a€ect bacterial attachment to glass beads were tested (Gross and Logan, 1995). All of these chemicals, except the nonionic surfactant Tween 20, either increased cell sticking coecients, or reduced them by less than an order-of-magnitude. However, a was calculated to decrease from a = 0.38 (IS = 70 mM) to a = 0.0016 when a monoclonal population of Alcaligenes paradoxus was suspended in either low IS water (IS = 0.01 mM) or water containing a high concentration of Tween 20 (0.1 vol% in a phosphate bu€er, IS = 70 mM). Bacterial sticking coecients for laboratorygrown bacteria will need to be reduced by one to two orders-of-magnitude, or in the range of 10ÿ2
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augmentation (Johnson et al., 1996). Numerous studies have shown that low ionic strength (IS) solutions and surfactants can substantially reduce bacterial adhesion to ultraclean glass and quartz surfaces (Fontes et al., 1991; Gannon et al., 1991; Martin et al., 1992; Gross and Logan, 1995; Rijnaarts et al., 1996). Despite this work, however, it is not well known to what extent these laboratory results, using cm-long columns, can be applied to soils over larger distances necessary for bioaugmentation schemes. For the present study, two strains of microorganisms (A. paradoxus; and CD1, a groundwater isolate) were used in the MARK procedure to determine whether large reductions in a observed for glass beads could also be obtained for soils. We examined the e€ectiveness of low ionic strength solutions, several nonionic surfactants and a biodegradable anionic surfactant recently shown to reduce cell attachment in sand columns (Bai et al., 1997), to enhance the transport of cells in columns packed with two soils, cleaned glass beads and cleaned quartz media. Our results indicated that both low ionic strength and surfactant solutions substantially increase bacterial transport, but that larger decreases in sticking coecients for soils are required to disperse cells over distances sucient to make bioaugmentation an e€ective remediation technology. MATERIALS

Cultures A. paradoxus is a gram-negative, nonmotile rod isolated from a contaminated soil that is able to degrade aromatic compounds including 2,4-dichlorophenoxyacetic acid (2,4D) (Wu et al., 1993). Cultures were stored frozen in a mineral salts medium (MSM), revived in tryptic soy broth, washed and incubated in MSM plus yeast extract and 2,4D and grown to a ®nal concentration of 02  108 mlÿ1 as described in Gross and Logan (1995). Cells were radiolabeled by adding 3 ml of the cell suspension to a sterile test tube containing 30 ml of 3 H-leucine (ICN, 1 Ci/ml). The tube was rotated for 0.5 h at room temperature before 1 ml of this solution was diluted with 100 ml of the solution used in the column tests to produce a ®nal cell concentration of 0106 mlÿ1. CD1 is a gram-negative, nonmotile, subsurface isolate from a Department of Energy ®eld site in Oyster, VA. This microbe was provided by A. Mills (University of Virginia). Frozen cells were thawed, incubated in a 10% solution of a PTYG solution until stationary growth (24± 35 h) transferred (1:100) again to 10% PTYG and incubated to stationary growth (Martin et al., 1995). Leucine uptake was much slower for CD1 than A. paradoxus. Therefore, the cell suspension was diluted 1:100 with the carrying solution used in the column experiments (®nal concentration 0106 mlÿ1) and 100 ml of this solution was incubated with 3 H-leucine (40 ml) for 18 h on a shaker table. All chemical solutions were made using ultra pure water (Milli-Q, MQ; Millipore). Mineral salts media consisted

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Qun Li and Bruce E. Logan

(per liter) of: 1.5 g KH2PO4, 1.982 g Na2HPO4, 0.5 g NH4Cl, 54.6 mg MgSO4, 14 mg CaCl2, 5 mg ZnSO4
7H2O, 2.5 g Na2MoO4
2H2O and 0.22 mg FeCl3
6H2O. PTYG contained (per liter): 0.5 g peptone, 0.5 g tryptone, 1 g yeast extract, 1 g glucose, 0.6 g MgSO4
7H2O and 0.07 g CaCl2
2H2O. Solutes used in column experiments Radiolabeled cells were suspended in three di€erent solutions; a high ionic-strength (IS = 70 mM) phosphate buffer, arti®cial groundwater (AGW, IS = 2 mM) and a low IS solution consisting of MQ water (IS = 0.01 mM). Cell suspensions (106 mlÿ1) were prepared in these solutions as described above, except in low IS experiments, where the ionic strength was reduced by washing cells using ®ltration or centrifugation techniques; cells were then resuspended in solutions appropriate for di€erent experiments. In experiments using CD1, cell suspensions were washed by syringe-®ltering cells onto a 0.2 mm pore-diameter (25 mm) ®lter (Supor Acrodisc, Gelman Scienti®c) and resuspending the cells by backwashing the ®lter with 50 ml of MQ water. In experiments using A. paradoxus, cells were washed three times with MQ water by centrifuging the cells for 5 min at 6000  g and resuspending the pellet in MQ water. The phosphate bu€er contained (per liter): 0.51 g KH2PO4, 0.52 g K2HPO4, 0.147 g CaCl2; NaCl was added to produce an IS = 70 mM. AGW contained: 0.019 g NaCl, 0.02 g KCl, 0.017 g CaSO4, 0.048 CaCO3, 0.03 g Ca(NO3)2, 0.013 KNO3, 0.001 K2HPO4, 0.065 MgSO4. Prior to an experiment all solutions were sterilized by ®ltration through a 0.2 mm syringe ®lter. Surfactants Six nonionic surfactants were used at concentrations above their critical micelle concentration: Tween 20 (0.11% w/v, Calbiochem), Tween 80 (0.1% w/v, Calbiochem), Triton X-100 (0.1% vol%; New England Nuclear), Triton X-705 (0.7% w/v, Sigma), Brij + 35 (0.3% w/v Sigma) and POE-20 (60 mg/l; Sigma). Monorhamnolipids (MRLs) have recently been shown to reduce cell attachment in pumped sand columns 5 cm long (Bai et al., 1997) and were used at a concentration (250 mg/l) above the CMC. Surfactants were added to radiolabeled bacterial suspensions and rinse solutions at the desired ®nal concentration and the suspension was shaken for 45 min prior to an experiment. Porous media Four media were used to pack the columns: two cleaned media (glass beads and quartz) and two soils (Arizona and Ringold). The 40 mm diameter glass beads and quartz (158 mm mean diameter) were prepared using a wet sedimentation technique and cleaned as previously described (Martin et al., 1996). The Arizona soil was collected from the North Fallow Field at the University of Arizona farm from a depth of 3±6 feet below the surface (in the unsaturated zone) and passed through a 500 mm mesh. This soil is on average 90% sand, 7% silt, 3% clay and 0.1% organic carbon (pers. comm., Michael Young, Department of Soil, Water and Environmental Sciences, University of Arizona). The soil was rinsed several times to remove ®nes by hand shaking the soil (0250 g) in a ¯ask (1000 ml) with MQ water until the supernatant was clear and dried overnight at 658C. An average grain diameter of 127 mm was

measured from projected areas based on a number distribution (Martin et al., 1996). The Ringold soil was obtained from near-surface sediments of the Ringold formation (McCaulou et al., 1995; Martin et al., 1996), rinsed and dried as described above. The Ringold soil (grain diameter of 224 mm) is 1% silt and clay and contains 1.1% organic carbon (dry weight basis) (McCaulou et al., 1995).

Column experiments Bacterial sticking coecients were measured for the four di€erent porous media by measuring bacterial retention in minicolumns using the MARK test as previously described in detail (Gross and Logan, 1995; Gross et al., 1995). In this test syringe tubes (0.8 cm inner diameter, 3 cm3) are wet packed with 1.5 g of the porous medium supported by a GF/F ®lter (Whatman) in the bottom of the tube and mounted to a multiple ori®ce vacuum box, equipped with LuerLok connectors. All solutions are added to the top face of the media using a pipetteman. The vacuum was set to produce a super®cial velocity of 010ÿ3 m/s. The protocol for the bacterial injection experiment consisted of rinsing the media in the column with 2 ml of the bacteria-free carrying solution, adding 2 ml of the radiolabeled bacterial suspension, rinsing with another 4 ml of the bacteria-free solution and then maintaining the vacuum until the column appeared to be dry. Based on a typical column porosity of 0.4, each 2 ml of applied solution was equivalent to 10 pore volumes for the top 1 cm of the column. The column was then cut at the bottom and sliced into 1±2 mm slices while it was extruded (using the syringe plunger) through the top of the tube. The length of the slice was calculated from its weight. Bacterial retention in each slice was calculated from the sample radioactivity using scintillation counting. The fraction of bacteria retained in each slice, Ri, was calculated from the total radioactivity of the cells added to the column as Ri ˆ

N0 ÿ

Ni X

Niÿ1

,

…1†

where N0 is the concentration of bacteria added to the sample (dpm), Ni is the concentration of bacteria in the slice and Ni ÿ 1 is the total number of bacteria retained in previous slices. All samples were run in triplicate with controls to account for any unassimilated radiolabeled compounds or radiolabeled compounds released by the cells into solution (Gross et al., 1995; Martin et al., 1996).

Cell surface charge and hydrophobicity The cell z-potentials were measured, using a capillary electrophoresis device, as reported by Glynn (1998). Cell hydrophobicities were measured using the bacterial adhesion to hydrocarbons (BATH) test (Rosenberg, 1984). For hydrophobicity tests cells (5 ml) were added in triplicate to glass test tubes (16  155 mm) containing 1 ml of n-hexadecane (Sigma), vortexed for 1 min and left to stand undisturbed for >30 min in order to allow the phases to separate. Cell concentrations in the water phase were then measured using the acridine orange direct count (AODC) method (Hobbie et al., 1977). Controls were run to account for cell adsorption to glass walls as previously described (Gross and Logan, 1995).

Bacterial transport for bioaugmentation

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Table 1. Porous media parameters and average values used in ®ltration model calculationsa Porous medium Parameter

Symbol

Arizona

Ringold

Quartz

Glass

dc y Z l

127 0.40 0.0052 37

224 0.40 0.0029 12

158 0.36 0.0047 29

40 0.40 0.024 538

Soil grain diameter (mm) Soil porosity Collector eciency Filter coecient (mÿ1)

a Conditions varied slightly between experiments, but average values used were: water velocity (super®cial) of 10ÿ3 m sÿ1, cell density of 1.07 g cmÿ3, bacterial equivalent radius of 1 mm, Hammaker constant of 10ÿ20 J, and ¯uid properties of water at 228C.

Theory The e€ect of the di€erent media grain sizes on colloid ®ltration eciencies can be compared using the steady state clean-bed ®ltration equation (Yao et al., 1971) C ˆ exp…ÿalL†, C0

…2†

where C0 and C are the concentrations of bacteria entering and leaving the column of length L, l = 3(1 ÿ y)Z/2dc is the ®lter coecient, y the bed porosity, Z the collector eciency calculated using the Rajagopalan and Tien (RT) model (Rajagopalan and Tien, 1976; Logan et al., 1995), dc the soil grain diameter and a the sticking coecient de®ned as the fraction of particle collisions that result in successful attachment. The number of collisions a completely nonattaching cell would experience during transport over a distance L is lL (Martin et al., 1996). Typical values used in ®ltration calculations are summarized in Table 1. For the given parameters, clean-bed conditions are expected. Even if all bacteria injected into the column attached to the media grains, only 0.08% of the surface area would be covered. This is less than that reported to produce clogging or blocking in porous media (Liu et al., 1995; Rijnaarts et al., 1996). The fraction of bacteria removed in the column, R, is related to the volume-averaged concentration of bacteria in the column in¯uent (C0) and e‚uent (C), by R = 1 ÿ C/C0 (Gross et al., 1995). Therefore, the sticking coecient for bacteria in each slice, ai, can be calculated as a function of the thickness of each slice, Li using ai ˆ ÿ

ln…1 ÿ Ri † lLi

…3†

and the overall sticking coecient,  a, for the whole column was calculated (Martin et al., 1996) as ln…1 ÿ  aˆÿ

n X iˆ1

lL

Ri † :

RESULTS

Ionic strength The resuspension of laboratory grown cultures of A. paradoxus in low ionic strength (IS = 0.01 mM) water reduced their adhesion to all four types of media (glass, quartz and two soils) compared to experiments with higher ionic strength solutions. Sticking coecients decreased from ar 1 in AGW to a < 0.2 in the low IS water (Fig. 1). Although a

is assumed to be constant in ®ltration calculations, in general ai decreased with column length in all column experiments using AGW independent of the type of porous medium. Decreases in ai with transport distance have previously been noted using cleaned glass (Albinger et al., 1994) and quartz (Johnson et al., 1995; Martin et al., 1996) media. In some experiments (primarily those using low IS water) there were slight increases in ai over the length of the column. These intracolumn changes in ai may be important since the magnitude of the change in ai within a column is larger than di€erences between replicate columns (Fig. 1). The decrease in the overall column sticking coecient,  a, with ionic strength is signi®cant (p < 0.001) based on average values obtained in the high (70 mM) and low IS (0.01 mM) experiments (Fig. 2). However, there were no signi®cant changes in a as a result of reducing the ionic strength by an order-of-magnitude from values used in laboratory cultures (70 mM) to values typical of groundwater (2 mM). In a 70 mM solution some of the  a's were greater than unity (1.66 and 2.48 on the Ringold soil and quartz media). Values greater than unity have been previously observed for highly destabilized particles and re¯ect inaccuracies in either collision frequencies predicted using the RT model, or incorrect assignment of a single particle diameter (used to calculate the collector eciency) for a heterogeneous medium containing a large size distribution of collectors (Hornberger et al., 1992; Logan et al., 1995; Martin et al., 1996). The lowest sticking coecients were always obtained in columns packed with glass beads, but the relative ordering of  a for the other three media indicated that sticking coecients were strain and media speci®c (Fig. 2). The relative ranking of sticking coecients in AGW for A. paradoxus was glass < Arizona < Ringold 1 quartz. This order was slightly di€erent for CD1 with the Ringold soil having the highest a, resulting in the order glass beads < quartz < Ringold soil. Comparison of microbial properties and sticking eciencies The average sticking coecients of monoclonal populations of A. paradoxus were consistently higher than those measured for CD1, except for the

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Qun Li and Bruce E. Logan

Fig. 1. Sticking coecients (ai) of Alcaligenes paradoxus vs transport distance in columns packed with four di€erent porous media as a function of solution ionic strength (IS). Symbols are averages of individual values (ÿ) for experiments run in triplicate.

low IS glass bead experiments (Fig. 2). When using arti®cal groundwater (IS = 2 mM) in experiments with the Ringold soil, for example, the sticking coecient of A. paradoxus ( a=1.7) was three times larger than that of CD1 ( a=0.57). In low IS water, there was a slightly smaller di€erence in a's between the two cell types:  a=0.2 for A. paradoxus was larger by a factor of 2.2 than the value of  a=0.09 measured for CD1. The two bacteria had nearly identical sticking coecients on glass beads in low IS solutions, despite large di€erences in  a (a factor of 21) in the high IS solutions. The order-of-magnitude reduction in  a produced by a low IS solution on glass surfaces may therefore be species-speci®c. The reduction of

IS by four orders-of-magnitude reduced the sticking coecients of A. paradoxus by one order-of-magnitude (a factor of 54) and has been previously observed to substantially reduce the attachment of Pseudomonas ¯uorescens P17 to silica beads (Jewett et al., 1995) and P. aeruginosa to a rotating glass disk (Martin et al., 1992). However, the  a of CD1 was reduced by only a factor of 2.4 as a result of the same 4-log reduction in ionic strength. Part of the di€erences in sticking coecients between the two bacteria observed here may be a result of the hydrophobic nature of their surfaces. van Loosdrecht et al. (1990) demonstrated that bacterial attachment to glass surfaces increased with cell hydrophobicity, as indicated by contact angle

Bacterial transport for bioaugmentation

Fig. 2. Average sticking coecients for Alcaligenes paradoxus (solid lines, ®lled symbols) and CD1 (dashed lines, open symbols) in four di€erent porous media as a function of ionic strength.

measurements. A. paradoxus was more hydrophobic than CD1, as indicated by a higher partitioning of A. paradoxus into hexadecane (79.32 3.3% vs 59.7 2 2.3% for CD1; Table 2). However, bacterial adhesion to glass surfaces is also a function of the surface charge of the cell. A. paradoxus had a zpotential (ÿ33 mV) that was, on average, slightly less negative than that of CD1 (ÿ31 and ÿ41 mV) although the bimodal charge distribution of CD1 makes this comparison uncertain (Table 2). A larger a for A. paradoxus than CD1, however, is at least consistent with that found for other bacteria, based on their relative hydrophobicities and electrostatic charges (van Loosdrecht et al., 1990; Gross and Logan, 1995). E€ect of surfactants The addition of the nonionic surfactant, Tween 20, to solutions containing a phosphate bu€er reduced the  a's of both A. paradoxus and CD1 (Table 3) but was less e€ective at reducing a than the use of a low IS solution alone. The addition of a surfactant reduced the  a's to about half those measured in just AGW. The ai's, measured as a function of column distance after the addition of the surfactant, generally

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paralleled those, measured using only AGW, but the ai's produced by the addition of a surfactant did not resemble those produced using just low IS water (Figs 3 and 4). The di€erent patterns in ai's with column length for the surfactant and low IS experiments suggest that the mechanism of achieving lower ai's, using a nonionic surfactant, may not the be the same as that produced by low IS water. The presence of phosphate ions in surfactant solutions is important in reducing sticking coecients, although the reason for this e€ect is not clear. The addition of Tween 20 to phosphate-bu€ered solutions, even at high ionic strengths (70 mM) reduced  a's below those measured for otherwise identical surfactant solutions made up in the lower ionic strength AGW (IS = 2 mM) as shown in Table 3. For example, when AGW (2 mM ionic strength) was replaced with phosphate bu€er (70 mM ionic strength)  a was reduced from 0.015 2 0.001 to 0.0061 2 0.001. We hypothesize that the phosphate ion aids in binding the surfactant to the porous medium, although we have not further tested this hypothesis. Other nonionic surfactants and one anionic surfactant were tested using the phosphate bu€er (IS = 70 mM) to see if larger reductions in  a could be achieved than those measured for Tween 20. The  a's for the other surfactants varied, but Tween 20 was the most e€ective surfactant tested for reducing the sticking coecient (Fig. 5) because this surfactant produced the greatest reduction in  a. The anionic monorhamnolipid (MRL) was the least e€ective surfactant, reducing  a by only 10%. Other surfactants commonly used for groundwater remediation studies, including Tween 80, Triton 100 and 750, Brij + 35 and POE-10, reduced  a by 73 to 92%. Separate experiments were conducted on di€erent days using Tween 20. We have found that the MARK method is sensitive enough to measure slight changes in sticking coecients on di€erent days (Johnson and Logan, 1996). The range of  a's measured using Tween 20 with the same strain of A. paradoxus on di€erent days over a period of four months is within the range of  a's measured for the di€erent surfactants (except for the rhamnolipid). Thus, we conclude that nonionic surfactants

Table 2. Particle surface charge measurements of cells in AGW and relative hydrophobicities of A. paradoxus and CD1 based on the extent of cell partitioning into hexadecane Cell concentration in AGW in hexadecane test (106mlÿ1) Bacteria A. paradoxus CD1

z-Potential (mV) ÿ33 ÿ31, ÿ41

Electorphoretic mobility (mm cm/V s) ÿ2.6 ÿ2.5, ÿ3.3

Tween 20 (vol%) basis

P-bu€er

P-bu€er + hexadecane

Cells in hexadecane (%)

0 0.1 0 0.1

2.07 20.55 2.04 20.23 1.33 20.15 1.19 20.12

0.43 20.045 1.96 20.07 0.54 20.03 1.19 20.10

792 3 3.9 2 7.2 602 2 0.0 2 1.3

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Qun Li and Bruce E. Logan

Table 3. Summary of sticking coecients for two bacteria suspended in di€erent solutions, in four types of porous media. Solution ionic strengths were: AGW, 2 mM; P-bu€er, 70 mM; low IS, 0.01 mM Average sticking coecient,  a Alcaligenes paradoxus Porous medium Arizona Ringold Quartz Glass

AGW 0.722 0.04 1.70 2 0.07 1.58 2 0.01 0.192 0.01

AGW + Tween 20 P-bu€er + Tween 20 0.442 0.03 1.462 0.05 0.592 0.02 0.015 20.001

nm 0.4320.03 0.3820.11 0.00612 0.001

CD1 Low IS

AGW

0.083 20.005 nm 0.202 0.01 0.57 20.06 0.082 0.00 0.28 20.02 0.0050 20.0001 0.010 2 0.000

AGW + Tween 20

Low IS

nm 0.33 20.005 0.15 20.01 0.008 2 0.006

nm 0.090 2 0.007 0.044 2 0.005 0.0054 20.0008

Fig. 3. E€ect of a nonionic surfactant (Tween 20) added to an arti®cial groundwater (AGW; IS = 2 mM) on the sticking coecients (ai) of Alcaligenes paradoxus, measured at di€erent distances in columns packed with four di€erent porous media. Data for sticking coecients in low ionic strength (IS = 0.01 mM) water are taken from Fig. 1. Symbols are averages of individual values (ÿ) for experiments run in triplicate.

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Fig. 4. Sticking coecients (ai) of CD1, measured at di€erent distances in columns packed with four di€erent porous media in di€erent chemical solutions. Symbols are averages of individual values (ÿ) for experiments run in triplicate.

all have the same general e€ects on the sticking coecients of bacteria, although the exact value of  a is a function of the bacterial strain, porous medium and even daily variations in culture conditions. While the mechanisms by which surfactants lower  a are not well understood, it is clear that adding a surfactant reduced the partitioning of the two bacteria into hexadecane (Table 2) suggesting that sur-

factants act to reduce cell hydrophobicity. However, it is also just as likely that the adsorption of surfactants onto the cell surface created steric barriers to cell adhesion to the mineral surfaces. The use of Tween molecules with greater lengths did not correlate with lower  a's, however, so we were unable to further separate the e€ects of cell hydrophobicity and steric interferences on  a.

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Fig. 5. Average sticking coecients ( a) of Alcaligenes paradoxus, suspended in a phosphate bu€er (IS = 70 mM) before and after the addition of various surfactants. Some experiments were repeated to examine the variability in  a over time. The results for Tween 20 span a period of four months (MRL = monorhamnolipid).

DISCUSSION

Bacterial transport in porous media is limited by high collision frequencies and large a's between the cell and the porous medium particles. Nonionic surfactants have previously been shown to decrease cell attachment to a variety of surfaces (Zierdt, 1979; Humphries et al., 1987; Bai et al., 1997), but the e€ect of surfactants on bacterial transport in soil columns has not been suciently quanti®ed and scaled up to larger systems. Minicolumn experiments reported here, quantify the extent of enhanced bacterial transport in porous media, by direct measurement of cell deposition in the soil column. When this data was interpreted using ®ltration theory it was calculated that Tween 20 and several nonionic surfactants could reduce bacterial a's by an order-of-magnitude. Similarly, low IS solutions also substantially decreased overall bacterial attachment and reduced cell sticking coecients. Based on the results for the two species examined here, low IS solutions produce lower values of a and therefore, would be preferable to surfactants for bioaugmentation of aquifers. Is this reduction in a sucient for bioaugmentation purposes? Well-to-well distances for bioremediation are usually several to tens of meters and therefore it seems reasonable to assume that bacteria should be transportable over at least half of these distances in appreciable numbers. In order to predict the extent of cell transport over these larger distances, the sticking coecients measured for the Arizona soil were used in the ®ltration equation to scale up cell transport over distances larger than the 1 cm intervals measured in the mini-

columns. Assuming a groundwater velocity of 10 m dÿ1 during cell injection for the Arizona soil with low IS water (a = 0.083), it was calculated using equation 2 that there would be a 99.9% reduction in cell concentrations within 0.92 m of the well (Fig. 6). While this is a substantial increase in transport distance, compared to a distance of 0.11 m calculated for cells suspended in arti®cial groundwater (a = 0.72), lower sticking coecients are required for ®eld use. If a sticking coecient of 0.01 could be obtained, cells would be transported 7.6 m before a 3-log reduction in cell concentration, while for a = 0.001, this distance could be increased to 76 m. For the groundwater ¯ow conditions assumed here (U = 10 m dÿ1; 108C; Arizona soil), a nonattaching cell 1 mm in diameter would have to undergo 870 collisions to be transported 10 m. At a super®cial velocity of 1 m dÿ1, 3540 collisions would be necessary. The use of surfactants and low ionic strength solutions can substantially decrease collision eciencies, but experimental results presented here indicate that further reductions in a are necessary to transport cells over these large distances. There was relatively little di€erence between a's measured for highly cleaned quartz and the Arizona soil (0.1% organic matter) suggesting that the presence of organic matter is a less important issue than media sphericity. Sticking coecients were typically an order-of-magnitude higher for columns packed with quartz particles than those for spherical glass beads, suggesting that irregular particle shapes contributed to high collision eciencies. Unfortunately, little can be done to increase particle

Bacterial transport for bioaugmentation

Fig. 6. Comparison of bacterial transport distances in soil, assuming di€erent sticking coecients, soil properties of dc=127 mm and y = 0.41 for Arizona soil, groundwater ¯ow of U = 10 m dÿ1 at 108C and bacteria with a diameter of 1 mm and density of 1.07 g cmÿ3.

sphericity of natural soils in the ground to obtain lower a's. Despite the lack of transport of bacteria over large distances, bioaugmentation is proceeding in ®eld tests (Duba et al., 1996). Instead of trying to disperse bacteria over large distances, investigators have chosen to create smaller, bioactive zones of <1 m (McCarty et al., 1998). It is feasible to distribute cells over these smaller distances and the injection of nutrients into smaller bioactive zones can be more carefully controlled than more widespread nutrient additions and therefore are more likely to support the injected microbes than the indigenous community. There is evidence that cells can have surface properties that allow them to be transported over great distances. Sticking coecients of indigenous bacteria measured in ®eld tests, for example, have been reported to be <0.01 (Harvey and Garabedian, 1991). The factors that contribute to these low sticking coecients need to be elucidated in order to design more ecient schemes for subsurface injection of laboratory-derived microoganisms for bioaugmentation purposes. AcknowledgementsÐWe thank T. Camesano and J. Glynn for helping obtain bacterial surface charge data and three anonymous reviewers for their comments. Monorhamnolipids were kindly provided by Dr Raina Miller, Department of Soil and Water Science, University of Arizona. This research was supported by grant number ES-04940 from the National Institute of Environmental Health Sciences, NIEHS. Its contents are solely the responsibility of the authors and do not necessarily represent ocial views of the funding agency. REFERENCES

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