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
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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:
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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
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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
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