Proceedings of the Fifth International Symposium of In-situ and On-site Bioremediation, April 19-22, San Diego,CA.

ENHANCING BIOCOLLOID TRANSPORT TO IMPROVESUBSURFACE REMEDIATION

Bruce E. Logan and Terri A. Camesano (Penn State University, University Park,PA), Brock Rogers (URS Greiner Woodward Clyde, Phoenix, AZ)

Yan Fang (University of Arizona, Tucson, Arizona)

ABSTRACT: Bioaugmentation can be an effective method of subsurface remediationbut well clogging must be minimized by reducing bacterial adhesion to soil particlesin the vicinity of the well. In this paper, we review recent work performed in ourlaboratory to determine the most effective methods for increasing bacterial transportdistances in groundwater aquifers. The presence of a non-aqueous phase liquid(NAPL) increased bacterial transport despite data that indicated bacteria favorablypartitioned into hydrophobic NAPL phases. Gas sparging also increased bacterialtransport distances compared to those obtained when bacteria were suspended in anartificial groundwater, but gas sparging was not as effective as using low ionicstrength (IS) water or surfactants. Filtration models predict reduced bacterialtransport at low versus high flow velocities, but the opposite effect was observedwhen bacteria were motile. For efficient bioaugmentation, we recommend that motilebacteria be suspended in low ionic strength (~10-5 M) water, and that they be injectedat low pumping velocities (~1 m/d) to minimize bacterial attachment.

INTRODUCTIONSubsurface remediation can be enhanced by injecting bacteria into the aquifer

via a procedure known as bioaugmentation, but bacteria-sized (~1 :m) colloidalparticles readily adhere to soil particles and may not be transported more than onemeter in soils leading to well clogging. In order to efficiently treat large volumes ofsoils by injecting bacteria from wells, bacterial transport distances of 10's of metersmust be obtained (Gross and Logan, 1995). Measuring factors that could sufficientlyreduce bacterial attachment to achieve these large transport distances would requirelaboratory columns 1 to 10 m long using a conventional approach of measuring cellconcentrations in column breakthrough tests (Jewett et al., 1993), so smaller-scalemethods were developed to screen bacterial attachment properties.

Using the MARK method (Gross et al., 1995) we have previously summarizedthe effects of solution IS, dissolved and sorbed organic matter, and various chemicaladditives on bacterial transport (Johnson et al., 1996). Attachment was quantifiedusing filtration theory in terms of the collision efficiency, ", defined as the ratio of therate that bacteria stick to a soil grain to the rate that they strike it. Of the chemicalsexamined, surfactants such as Tween-20 have been found to be the most effective,reducing " by 2.5 orders of magnitude on glass surfaces, while dissolved naturalorganic matter has little effect. The most consistent method of increasing bacterialtransport in a variety of soils and porous media is decreased IS. Decreasing the ISfrom that of growth media (10-1 M) to that of ultrapure water (10-5 M) decreased "

Proceedings of the Fifth International Symposium of In-situ and On-site Bioremediation, April 19-22, San Diego,CA.

(1)

(2)

of Pseudomonas fluorescens P17 from 0.18 to 0.026 (Johnson et al., 1996). We summarize here the results of more recent research in our laboratory to

determine the effects of other factors, such as fluid velocity, cell motility and gassparging. Because non-aqueous phase liquids (NAPLs) may be present at manyheavily contaminated sites, we also examined the effect of NAPLs on bacterialtransport.

METHODSColumn experiments were performed by adapting the MARK procedure,

which consists of measuring the retention of radiolabeled cells in short columns(Gross et al., 1995), to transport experiments using longer columns (7 to 15 cm). Thebacterium used for all experiments was Pseudomonas fluorescens strain P17, a Gramnegative motile rod. Cells were radiolabeled by incubating a cell suspension with 3Hleucine. In MARK tests cells are pulled by vacuum through an open-ended shortcolumn. For the longer column experiments reported here we pumped bacterialsuspensions through capped glass or stainless steel columns packed with a sandy soilcollected from the North Fallow Field at the University of Arizona farm, or cleanedquartz (average grain diameters of 127 :m and 200 :m, respectively).

After the cells were passed through a column, the column was rinsed andsliced into sections. The fraction of bacteria retained in each slice, Ri, was

where N0 is the concentration of bacteria added to the sample (dpm), Ni is theconcentration of bacteria in the slice, and Ni-1 is the total number of bacteria retainedin previous slices. To account for incomplete radiolabel recovery, samples werespiked with additional radiolabeled cells as previously described (Camesano andLogan, 1998). The extent of bacterial attachment was evaluated using a steady stateclean bed filtration equation

where C0 and C are the concentrations of bacteria entering and leaving the column oflength L, is the filter coefficient, 2=0.40 the bed porosity, 0 thecollector efficiency calculated using the RT model (Logan et al. 1996), dc the soilgrain diameter, and " the collision efficiency.

The fraction of bacteria removed in the column, R, is related to theconcentration of bacteria in the column effluent, C/C0, by R=1-C/C0. Therefore, thecollision efficiency for bacteria in each slice, "i, can be calculated as a function of thethickness of each slice, Li using . The overall collision efficiencyfor the whole column was calculated from the total removal of cells in the columncalculated by adding up the removal from all slices.

Proceedings of the Fifth International Symposium of In-situ and On-site Bioremediation, April 19-22, San Diego,CA.

RESULTS AND DISCUSSION

Average Flow Velocity. According to filtration theory, the fraction of bacteriaretained should decrease as velocity increases (producing a constant "). Ourexperiments found the opposite to be true (Table 1). The collision efficienciesincreased with velocity for velocities ranging from <1 to several hundred m/d. At avelocity of 0.56 m/d, the overall " was 0.003 for a 7-cm column. Under typicalgroundwater conditions, this would result in a one-log reduction in cell concentrationafter the bacteria had traveled 7 m. At >110 m/d, " was high enough to cause ~70%of the bacteria to be retained in just a 7-cm column, conditions that would renderbioaugmentation difficult as continued cell injection at this high cell retention wouldeventually clog the well.

Cell Motility. Cell motility was likely the reason for the observed changes in cellretention with fluid velocity (Camesano and Logan 1998). Bacteria swim at typicalvelocities of 3.5 m/d (Mercer et al., 1993). Thus, at low bulk fluid velocities,bacterial motility could allow bacteria to avoid collisions with soil grains. However,at velocities much larger than swimming speeds of the cells, we hypothesized that thebacteria would be trapped in the flow. We found that at high velocities " wasessentially constant, results that supported our hypothesis. To test our theory that cellmotility was responsible for extremely low collision efficiencies at low fluid velocities,we conducted experiments with bacteria made non-motile by staining with acridineorange (which also killed the cells). Collision efficiencies for non-motile bacteria hadessentially constant "’s over the range of fluid velocities (Table 1) supporting ourconclusions regarding cell motility at low flow velocities.

Ionic Strength (IS). P17 has a zeta potential (z) of -40.45 mV and is also relativelyhydrophobic. Due to its very negative surface charge, the attachment of P17 is likelygoverned by electrostatics. Because the soil and cells are both negatively charged theyrepel each other at the neutral pHs used in our experiments; this repulsive force isenhanced using low IS water. Low IS water (~10-5 M) reduced bacterial attachment

Proceedings of the Fifth International Symposium of In-situ and On-site Bioremediation, April 19-22, San Diego,CA.

Figure 1. Effect of NAPL (tetrachloroethylene) on the fraction ofbacteria retained in a soil column versus that predicted by filtration

theory. Adapted from Rogers (1997).

by approximately a factor of 20 compared to that in a higher IS artificial groundwater(0.004 M) at higher fluid velocities (Table 1). The reduction in attachment under lowIS conditions is consistent with the results obtained in previous studies using naturalsoils at high flow velocities in MARK tests (Johnson et al., 1996).

Effect of NAPLs on Transport. The presence of a NAPL residual (21.3 % of voidspaces filled) increased the overall transport of bacteria by 22% (Figure 1). Thecumulative fraction retained along the length of the column decreased from R=0.78("=0.025) to R=0.61 ("=0.016).

The reduction in bacterial attachment in the presence of a NAPL phase is aresult of the occupation of stagnant flow regions, or the micropore spaces, by theNAPL. This had the effect of blocking a portion of the flow paths in the column, thusincreasing the pore velocity of the bacterial suspension. Filtration theory predicts thatan increase in velocity will decrease the collector efficiency (0), which would have theeffect of reducing the fraction retained. An alternative explanation for the enhancedtransport could be that the NAPL is blocking favorable sites on the collector surface.If these sites are inaccessible to the bacteria, the probability of their attachment willdecrease thereby enhancing their transportability. However, sorbed PCE, in theabsence of a residual phase, did not affect PCE transport (Rogers, 1997). A decreasedattachment to the soil surfaces, due to hydrophobic repulsion between P17 and theNAPL was also considered as a possible explanation. However, hydrophobicity testsindicated that P17 favorably partitions into PCE. Therefore, the presence of ahydrophobic phase should have increased, not decreased bacterial retention in thecolumn as observed in our experiments.

Proceedings of the Fifth International Symposium of In-situ and On-site Bioremediation, April 19-22, San Diego,CA.

Figure 2. Gas sparging decreased the fraction of bacteria retained in a quartz-mediacolumn (data from Fang and Logan, 1999).

Gas Sparging. Bacterial transport can be enhanced for some bacterial species by gas(N2) sparging, but the enhancement due to the gas flow for P17 is less than thatobtained using surfactants (Figure 2). The fraction of bacteria retained in a 10-cmlong quartz-media column was reduced from R=0.38 to 0.15 by gas sparging whilewater was simultaneously being pumped through the column. Addition of a non-ionicsurfactant (0.1% v/v Tween 20) solution reduced the fraction of bacteria retained toR=0.06 (Fang and Logan, 1999).

The enhancement of bacteria transport by gas sparging is likely due to themobile gas-water interface. Because P17 is a relatively hydrophobic bacterium weexpect that cells will preferentially sorb to the gas-water interface. The sorption ofbacteria on the gas-water interface is essentially irreversible since capillary energyprovides a large attractive force to hold the bacteria on the gas-water interface. Thus,the movement of the gas-water interface during gas sparging results in the increasedforward transport of the cells in comparison to saturated water conditions. However,the increase in bacterial transport due to sparging is not as large as that possible usingsurfactants or low IS water.

CONCLUSIONSBacterial transport can be increased in natural soils by using low IS water

(~0.01 mM). For the motile species examined here cell attachment is minimized bypumping bacteria at low water velocities (~1 m/d) even though filtration modelspredict that attachment will be decreased with increased water velocity. Gas spargingwill not be as effective as low IS water in increasing bacterial penetration ingroundwater aquifers.

Proceedings of the Fifth International Symposium of In-situ and On-site Bioremediation, April 19-22, San Diego,CA.

ACKNOWLEDGEMENTSThis work was funded by grant ES-04940 from the National Institute of HealthSciences, NIEHS, to the University of Arizona. Its contents are solely theresponsibility of the authors and do not necessarily represent official views of thefunding agency. These studies were conducted while the authors were at theUniversity of Arizona, although current author affiliations are given above.

REFERENCES

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Fang, Y. and B.E. Logan. 1999. “Bacterial transport in gas sparged porous media.”J. Environ. Engng. In press.

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Gross, M.J. and B.E. Logan. 1995. “Influence of different chemical treatments ontransport of Alcaligenes paradoxus in porous media.” Appl. Environ. Microbiol.61(5):1750-1756.

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Li, Q. and B.E. Logan. 1999. Enhancing bacterial transport for bioaugmentation ofaquifers using low ionic strength solutions and surfactants. Wat. Res. 33(4):1090-1100.

Logan, B.E., D.G. Jewett, R.G. Arnold, E. Bouwer and C.R. O'Melia. 1995.“Clarification of clean-bed filtration models.” J. Environ. Eng. 121(12):869-873.

Mercer, J.R., R.M. Ford, J.L. Stitz, and C. Bradbeer. 1993. “Growth rate and effectson fundamental transport properties of bacterial populations.” Biotechnol. Bioengin.42:1277-1286.

Rogers, B. 1997. “Bacterial transport in NAPL-contaminated porous media.” M.S.Thesis, University of Arizona, Tucson.