United States Environmental Protection Agency Robert S. Kerr Environmental Research Laboratory Ada OK 74820 Research and Development EPA/800/S2-91/010 June 1991 *> EPA Project Summary Movement of Bacteria Through Soil and Aquifer Sand M. Alexander, R.J. Wagenet, P.O. Baveye, J.T. Gannon, U. Mingelgrin, and Y. Tan 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 bacte- ria in the soil matrix was developed. Nineteen bacterial strains were selected that differed In their ability to be trans- ported through soils. Measurements were made of sorptlon partition coeffi- cient, hydrophoblctty, net surface elec- trostatic charge, zeta potential, cell size, encapsulation, and flagellation of the cells. Only sorptlon and cell length were correlated with transport of the bacteria through soil. The breakthrough curves for Paeudomonaa sp. KL2 moving through a column packed with a sandy aquifer material were determined. Ionic strength of the Inflowing solution, bac- terial density, and velocity of water flow were found to have an effect on break- through. This Project Summary was deveh opad by EPA'a Robert S. Kan Environ- mental Research Laboratory, Ada, OK, to announce key findings of the research project that la Hilly documented In a separate report of the tame title (aee Project Report ordering Information at back). Introduction Approximately 50% of the United States population depends upon ground water as a source of drinking water. Consequently, the contamination of ground water by or- ganic chemicals is widely recognized as a critical environmental problem. The trans- port and mobility of bacteria in soil and subsurface materials has been a subject of interest for the past few decades be- cause of the environmental Importance of these organisms. The biorerrieolation 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 contamina- tion. Such inoculation is necessary if mi- croorganisms degrading the chemical contaminants are not present in the haz- ardous waste site or adjacent ground wa- ter. Many toxic organic chemicals persist at underground hazardous waste sites de- spite being readily biodegradable under laboratory conditions. When thfe occurs, introduced bacteria selected for their ca- pacity to degrade target contaminants, and capable of surviving and proliferating after injection in the aquifer, might be used to promote bbdegradation. The introduction of bacteria to degrade toxic wastes in sort and subsurface has generated renewed interest in the transport and mobity of bacteria. However, the mobility of the added bacteria may determine their effectiveness for in situ bioremediation. R bacteria ca- pable of degrading a contaminant 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 contami- nant plume. Considerable attention has been given to the mobility of bacteria and other micro- organisms in soil and subsurface materi- als. These studies were conducted primarily because of concern with the dissemination Printed on Recycled Paper ------- of pathogens from land spreading opera- tions, ground-water recharge, or the dis- posal of manure or municipal sludge. Several studies have shown poor mobility of the investigated species of bacteria through soil. However, considerable move- ment of some bacteria was observed in field studies. 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. It is, however, generally acknowledged that the movement of bacteria in a homoge- neous porous medium is poor because of both adsorption and mechanical filtration of bacterial cells, which have been sug- gested as mechanisms for their retention in soils. In a study by various investigators, 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 wa- ter, 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 gradi- ent, the relative breakthrough of cells into a sampling well located 1.7 m from the injection well was less than 1%. Further studies found nearly a 16-fold greater breakthrough of Escherichia coli through intact cores compared to disturbed cores of a sandy loam. It was concluded that soil structure and the velocity of water flow are critical in determining movement of bacte- ria through soil. Scientists reviewed a number of math- ematical models describing the concurrent growth of bacteria and transport of biode- gradable substrates in saturated porous media; these models assume that bacteria form either continuous biofilms or discrete microcolonies on surfaces of solid par- ticles and hence are immobile. However, many environmental factors greatly affect bacterial transport. Ionic strength is par- ticularly important. The literature 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. On the basis of the electri- cal double layer developing in the vicinity of charged surfaces, it was concluded that the number of bacteria attracted to sur- faces lessened with decreased electrolyte concentrations, whereas adsorption of cells to surfaces increased with higher electro- lyte concentrations. Researchers reported that the application of rain water or distilled water will result in the desorption of viruses from soil particles. Additional studies re- ported that complete blockage of pores due to soil dispersion does not occur in sodic soils leached with water of low elec- trolyte 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. The effect of ionic strength on move- ment of bacteria through subsurface earth materials seems to have been largely ig- nored 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 mobility of bacteria in soil by avoid- ing uncontrolled variations in bacterial be- havior 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 addi- tion, a study was conducted to determine the influence of certain properties of bacte- rial 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 chlo- ride tracer. The effect of variations in the ionic strength of an inflowing solution, bac- terial cell density, and flow velocity on the transport of bacteria through the earth ma- terials was evaluated. Discussion Macropore flow may be a major mecha- nism of bacterial transport in soils. There- fore, the use of undisturbed soil columns might have provided data on bacterial trans- port that would have particular relevance to circumstances prevalent in the field. However, columns of homogeneous soil were used to avoid uncontrolled, preferen- tial 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 de- terminations of mobility, a loamy soil was selected to avoid the extremes of limited bacterial sorption and nearly free move- ment in sandy soils on the one hand and the restricted movement resulting from me- chanical 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 mobili- ties might not have been evaluated. Saturated soil with a constant head of water was used to mimic bacterial move- ment under conditions of saturated flow. This permitted the occurrence of mass transport of the cells in the sufficiently large pores of the homogeneous soil. Re- searchers showed that movement of bac- teria through soil columns stopped when the water content was at or below field capacity, and other investigators demon- strated that movement of bacteria through soil was not detectable in the absence of a transporting agent such as water. The procedure described here has sev- eral advantages for testing bacterial mobil- ity in the soil matrix. Spurious data on mobility resulting from bacteria moving at the interface between the soil and the col- umn wall are avoided through the use of a relatively wide column and the coating ol 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 ol transport, by uniform packing to a fixed bulk density. Bacterial death from preda tion or parasitism was avoided because such predators and parasites were killec by irradiating the soil. Increases in eel numbers arising from growth and decrease: associated with starvation were preventec by performing the tests of transport at 2 tc 5°C. Furthermore, the marked difference; in mobility among the isolates suggest tha the proposed procedure does indeed dis tinguish among bacteria with different ca parities for movement. For inoculation of the soil surface with bacteria that can degrade organic pollut ants at some underground site, some o the added cells must move through the soi to the zone of contamination at depth Evidence exists, however, that introducec organisms may fail because they are no transported to sites containing the chemi cal. In this context, it is worth noting tha many of the carefully controlled expert ments in which inoculation resulted in bio degradation required transport to soi depths of only 10 cm. Measurements suet as those described in the present stud] will enable extrapolation of the potentia penetrability of the bacteria to consider ably greater depths. The capacity to degrade a chemical ii culture is a necessary but not sufficien requisite for successful biodegradation ii the field because, in addition to other traits the inoculum strain must possess the trait: ------- that enable it to move through soil, subsoil, or aquifer materials to reach the area of chemical contamination. The ability of bac- teria to be transported to subsurface con- taminated zones may be evaluated from bacterial properties such as cell size and their susceptibility to adsorption. The cor- relation between these properties and mo- bility can be determined by the procedure here proposed. Such a correlation may enable the classification of bacteria ac- cording to their potential mobility. The sus- ceptibility of a bacterium to predatbn or parasitism in the matrix through which it must pass must also be determined before its ability to reach the zone of contamina- tion can be predicted. It was possible to estimate an upper and lower boundary on the soil to water distribution coefficient of the bacteria (KJ. The data show a significant inverse corre- lation between the lower-bound K,, values and the fraction of cells transported through the column. The values for recovered cells transported varied from nearly zero to 25% at lower-bound K,, <2, whereas the values never exceeded 8% at lower-bound K,, >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 mechani- cal filtration, the correlation between KJ and Kjn 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 bnger the cell, the less its likelihood of passing through the soil matrix. Cell length was not significantly correlated with the recovery, higher-bound K,, or lower-bound K,,. Cell hydrophobicity, net surface elec- tric charge, and the presence of capsular polysaccharkdes were evaluated because they are properties of bacterial cells that appear to be involved in adsorption of bacteria to solid surfaces. The sizes of the cells were tested because larger cells may be more readily removed by filtration than smaller cells. The presence of f lagella might also impede movement, so their occur- rence on the test strains was investigated. Varying degrees of hydrophobicity were observed among the bacteria, but the re- sults of two different assays for hydropho- bicity did not agree. The nonuniformity of the bacterial surface may cause a bacte- rium to be hydrophilic in one assay and hydrophobic in another. Moreover, hydro- phobicity is not a definitive characteristic but varies with the hydrocarbon used for the assay. Measurements of zeta potential re- vealed that all of the test bacteria had net negative surface charges. However, bac- teria with positive charges on nonuniform cell surfaces may adhere to negatively charged particles of soil. 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 potysaccharides. 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 trans- port. Measurements were made of cell size because large bacteria presumably are less likely to pass through soil pores than small cells, and a statistical relation- ship between size and transport of bacte- ria through soil was evident. Although cell appendages could impede mobility, a rela- tionship was not observed between flagel- lation and transport. Motility was not considered important in the present study because transport was tested at low tem- peratures. The present findings suggest that it should be possible to obtain bacteria that have both the capacity to biodegrade un- wanted organic compounds and the ability to move through earth materials to sites containing these chemicals, as indicated by the observation that two benzene de- graders and one chlorobenzene-utilizing bacterium moved through soil in appre- ciable numbers. The movement of bacteria through ho- mogeneous sand increased when the inflowing solution had a bw ionic strength. When debnized water was used, the bac- teria moved readily through the sand col- umn after an initial period of retention. A 0.01 M solution of NaCI dramatically reduced bacterial breakthrough. The total bacterial breakthrough was bw because the cells were removed from solution by interactbns with the sand matrix. The re- placement of the NaCI solution with debn- ized water resulted in a second peak of bacterial breakthrough. The retention caused by the adsorption mechanisms ap- peared to be reversible when bnic strength was changed. Differences in the transport of bacteria noted in experiments with 0.01 M NaCI and debnized water were more marked at 1.0 X 10* than 1.0 X 10* cells per ml. An increase in f bw velocity somewhat reduced bacterial retention, a decrease that may be related to the reduction in bacterial resi- dence or "reaction" time at higher fbw velocity. Conclusions The experimental results suggest that adsorption significantly contributed to the retention of bacteria and that bacterial movement through aquifer sand was en- hanced by reducing the bnb strength of the inflowing solution. Cell density and fbw velocity also influenced bacterial move- ment. For bbremediatbn of contaminated sandy aquifers, manipulation of ionic strength may thus be a means to facilitate movement of bacteria to the site of organb contamination. •&U. S. GOVERNMENT PRINTING OFFICE: 1991/548-028/20224 ------- M. Alexander, R.J. Wagenet, P.C. Baveye, J.T. Gannon, U. Mingelgrin, and Y. Tan are with Cornell University, Ithaca, NY 14853. John T. Wilson is the EPA Project Officer (see below). The complete report, entitled "Movement of Bacteria Through Soil and Aquifer Sand,* (Order No. PB91-164277/AS; Cost: $15.00, subject to change) will bo available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Robert S. Kerr Environmental Research Laboratory U.S. Environmental Protection Agency Ada, OK 74820 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati, OH 45268 BULK RATE POSTAGE & FEES PAID EPA PERMIT NO. G-35 Official Business Penalty for Private Use $300 EPA/600/S2-91/010 ------- |