Soil Biul. Biuthtm. Vol. 2). No. 12. pp. 1155-1160. 1991
Primed in Great Britain. All rights reserved
Copyright (IJ "*l Pergamon Press pK
EPA/600/J-92/076
BACTERIAL TRANSPORT THROUGH
HOMOGENEOUS SOIL
J. T. GANNON, U. MINCELGRIN.' M. ALEXANDER and R. J. WAGENET
Department of Soil. Crop and Atmospheric Sciences, Cornell University. Ithaca. NY 14853, U.S.A.
(Antpted 28 Junr 1991)
Summary—The transport of microorganisms in soils is of major importance for bioremediation of
subsurface polluted zones and for pollution of groundwater with pathogens. 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 lest period were minimal because temperatures of 2-5*C were maintained and
predators and parasites were eliminated by "Co 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 and 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. ^< •—-.
INTROIHCTION
The bioremedialion 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 in-
oculation is necessary if microorganisms degrading
the chemical contaminants are not present in the
hazardous-waste site or adjacent groundwaters.
Should the introduced bacteria fail to survive or
move through the unsaturiucd zone or aquifer, bio-
remediation will not occur. It has thus been reported
(hat a /7-nitrophcnol-dcgrading bacterium added to
the soil surface failed to mineralize much of the nitro
compound unless it was mixed into the soil
(Goldstein ti al.. 1985).
Considerable attention has been given to the
mobility of bacteria and other microorganisms in
•.oil and subsurface materials. These studies were
conducted primarily because of concern with the
I dissemination of pathogens from land spreading
operations, groundwater recharge or the disposal of
manure or municipal sludge (Gerba el al., 1975;
Brown <•/ al.. 1979; Bell and Bole. 1978). Several
studies have shown poor mobility of the investigated
species of bacteria through soil (Bitton el al.. 1974;
Wollum and Cassel. 1978: Madsen and Alexander.
1982). However, considerable movement of some
bacteria was observed in Held studies (Schaub and
Sorbcr, 1977; Viraraghavan. 1978), and rainfall or
artificial additions of water enhance the transport of
viruses through soil (Duboisc ti al., 1976; Gerba and
Lance, 1978; Sobscy ft al., 1980). It is unclear
whether the movement of bacteria that has been
observed occurred through the soil matrix or through
•Permanent adJreit: Institute of Suili and Water, Volcani
Center. Bet Dagan. Israel.
tAuthor for correipondence.
the macropores or channels that afford the organisms
a relatively unhindered passage (Hagcdorn ft al..
1981; Rahe tt al.. 1978). Both adsorption (Hattori
and Hattori. 1976; Marshall. 1980) and mechanical
filtration (Pekdcger and Matthess. 1983; Smith vi al..
1985) of bacterial cells have been suggested as mech-
anisms for their retention in soils. Soil structure and
the velocity of water flow appear to be major determi-
nants of the movement of bacteria (Smith ci al., 1985;
Harvey el al.. 1989). The use of soil columns for
studies of bacterial transport has been suggested to
give rise to misleading results (Bitton n al.. 1979).
Our objectives were to develop a reproducible
procedure that would yield consistent measurements
of relative mobility of bacteria in soil by avoiding
uncontrolled variations in bacterial behavior and to
relate transport to efficiency of recovery and adsorp-
tion of the cells, in the procedure that was developed.
flow through macropores did not occur.
MATERIALS AND METHODS
Bacteria able to degrade benzene, chlorobcnzenc or
toluene were isolated by enrichment culture in sol-
utions containing 100 mg of the organic compound.
l.6g K,HPO4.0.4 g KH,PO4.0.5 g (NH4),SO4. 25 mg
CaSO, :H;O. 0.2 g MgS'o, 7H:O and 2.3 mg FeSO,
7H:O I"' dtionized 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 Biodcgradaiion was
determined by spcctrophotometric 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 toil, Kendaib loam and liquid from the
primary settling tank of the Ithaca. N.Y. sewage
treatment plant were placed on top of the soil column
1155
-------
1156
J. T. GANNON ti al.
described below. The soil column was then leached
with 4 pore vol of water. 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
Anhrobacier sp. Luta D from an aquifer was pro-
vided by J. L. Sinclair, both from Cornell University.
The bacteria were grown in Trypticase-soy broth at
30 C for 24-48 h. the cells were harvested by centrifu-
gation and washed twice, and the organisms were
suspended and diluted in 0.9% NaCI solution. The
cell suspensions were cooled to 3.5± I.5:C for In
prior to their addition to columns of soil. Bacterial
counts were made on Trypticase-soy agar using trip-
licate samples from the effluent of each soil column
and triplicate plates per dilution. The plates were kept
at.30 C for 24-48 h.
Kendaia loam (37.1% sand. 40.4% silt.
22.5% clay. pH 6.4. cation-exchange capacity
26.5cmolkg~') was air-dried, ground, .sieved
(<2mm) and then sterilized by "'Co irradiation
(2.5 Mrad). Aseptic conditions were maintained
during the experiments. The primary clay species in
this soil is vermiculitic. but small amounts of mica
and chlorite are also present. The soil was packed in
600-ml Buchncr funnels. 10 cm dia. each containing
a fritt' i-glass disc with pore sizes of 40-60 pm. Tests
had indicated that the pore sizes in the disc permitted
passage of essentially only bacteria and water-
dispcrsiblc clay, and the rate of flow during the
experiments remained constant for the 4 pore vol of
water passed through the columns, indicating that
clogging did not occur. The funnel was attached to a
500-ml Erlcnmcycr flask lilted with a sidesrm con-
taining Hg in a bulb to control the flow rate. The
walls of the funnel were coated with a thin layer of
sterile petrolatum added in liquid form at >60 C.
The petrolatum scaled the interface between the
funnel walls and the soil. The soil was pressed down
with a fitted brass compacting plate to a depth of
5cm to give a bulk density of 1.080±0.062gem'1
and a porosity of 59.2%.
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.
The columns were moistened from below by connect-
ing a bottle of water.to the stem of the Buchner
funnel. Sterile deionized water was added at a rate of
ca I Ocm h ' until the water level was ca 2 *m above
the soil surface. The water was then drained to the
surface of ihe soil column, and a circular mound .ith
a radius of I cm of dry sterile soil was applied to the
center of the soil surface. A I.O-ml inoculum of
I x 10* cells was placed in the center of that mound.
An additional I-cm layer of sterile toil was added to
the lop of Ihe column, thereby covering the inocu-
lated mound and making the column surface approxi-
mately level. Deionized water was added to the soil
surface after inoculation. Physical disturbance of the
surface of Ihe toil column was minimized by pouring
the water onto Al strips placed above Ihe soil surface.
A total of 4 pore vol of effluent WAS collected.
The rale of water flow through the column was
maintained at ca 0.8 pore vol h"1.
The distribution of microorganisms in the column
•t the end of the test and possible transport of
bacteria along the walls of the column were deter-
mined as follows. After the column was leached with
4 pore vol and allowed to drain, five vertical cores
(5cm in lengih) were taken from the soil column
using a 5-ml syringe (1.4cm i.d., 1.7cm 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 and
4cm between the core center and the column center.
Two cores were taken at each such distance at
diametrically opposed points per column. Determi-
nations of bacterial distribution were conducted wiih
duplicate columns. The total number of bacteria in
each core and core section was then determined.
Determinations were made of the proportion of the
cells added to the soil that appeared in the effluent
(percentage transported) and the proportion 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. The
bacteria in the effluents and those remaining in (he
soil of two of the three inoculated columns, were
counted after passage of 4 pore vol of deionized
water. For this purpose, the soil removed from the
column was shaken with deionized water (1:2.5) for
5 min on a rotary shaker operating at 120 rev mm '.
and bacterial 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 sedi-
mentation rates of the bacterial cells and sand, silt
and clay size fractions of the soil. At an experimental
temperature of 3 C. the time required for the size
fraction >2/
(I)
F it the number of bacteria adsorbed ml*'. D is the
concentration of toil in the tuipension, and the value
-------
Bacterial transport
1157
of D is 0.2 (20 g 100 ml"'). Lower and upper bounds
for KA were calculated. The lower-bound adsorption
value assumes that bacteria retained by clay panicles
of equal or smaller size are not considered to be
sorbed, and the upper-bound adsorption value con-
siders 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.
Tible 1. Diitribution of Pieudomonas KL2 in columns of soil after
pauage of 4 pore vol ol water
b. - S, - (S, x C,/C,)
(2)
The upper-bound of adsorption is expressed by the*
following equation.
F»p - [S, - (S, C,/C,] (I - W< x QC.) -' (3)
where Wd is the water-dispersible fraction of the clay
RESULTS
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, Achromobacier, Bacillus and
Enierobacier. The isolates able to degrade benzene
were designated Pseudomonas Benl and Achro-
mobacier Bcn2, those degrading chlorobenzene were
designated Pseudomonas CBI. Bacillus CB2 and
Bacillus CB3 and the bacteria growing on toluene
were designated Enierobacier Toll. Enierobacter
Tol2. Enierobacter Tol3 and Pseudomonas Tol4. The
bacteria obtained from diesel fuel-contaminated soil
were designated Achromobacier DFI. Pseudomonas
DF2 and Flai-ohacierium DF3. those from the sewage
treatment plant were designated Enierobacier strains
IS I and IS2 and those from Kendaia loam as Enter-
obacier KLI and Pseudomonas KL2 and KL3. The
last three isolates were not selected because of their
ability to metabolize aromatic compounds.
Pseudomonas KL2 was used 10 study Ihe direction
of movement of bacteria through the soil column.
Sample
location
Center of column
Left. 1.3-2.7 cm'
Right, 1.3-2.7 cm'
Left, 3.3-«.7cm'
Right. 3.3-4.7 cm'
Sample
depth
0.0-1.7
1.7-3.4
3.4-5.0
0-5.0
0-5.0
0-5.0
0-5.0
Cell no.
Column 1
980
1400
720
79
14
0.75
0.09
Column 2
2200
730
690
21
0.75
0.01
5% of the cells of two strains of Enterohacier
and three strains of Pseudomonas were transported as
compared to < I % of three other strains of each of
T»bk 2 The number* and percentage* of bacteria transported and recovered
Bacteria Bacteria Bacteria
transported transported' recovered*
(I0*cful (%) (•/.)
Bacterium
Enirtubacirr IS2
Enittiihacitt ISI
/'irw/nmiwHu KL2
Anhrtibticiri Lula D
PmJimimui DF2
Aehromahatitr Ben2
Pvuilimonai Benl
AII-I//UI CB2
Aih'iimotvtit' DFI
F.ntrriihaiiri KLI
PiHulimonat KL3
Knir'ittvt lr' Tol2
KnttrHhtirirr Tol3
PiruJiim/mat T»I4
fjutrobtiflrr Toll
Pimlimimiai C'BI
Aid//*. CU45I9
Flat ntaf/rrwm DFI
Borillia CB3 •
25-31
26-50
22-32
14-26
13-24
6.4-8.5
5.0 5.8
14-30
2.4-4.0
1.6^2.6
2.3-4.4
2.03.0
1.3-1.5
0.49-1.1
0.60-1.5
0.15-0.48
0-0.29
0.12-0.22
0.0053-0.016
IJ±2
13 3
8.2 1
7.7 1.9
6.9 0.4
6.8 0.8
5.9 0.4
4.1 1.2
3.9 O.K
2.2 0.4
0.9 O.J
0.9 O.I
0.9 O.I
0.3 0.)
0.2 ±0.1
0.2 ±0.1
O.I ±0.1
O.I ±0.01
0.01 1 0.00
61 6
60 I.'
46 6
39 4
53 12
107 IJ
71 4
104 20
39 4
»6 ^
48 3
14
64
6.5 .J
34
25
94 I
5.4 .5
4.3 0.4
'Mean 1 standard deviation
-------
1158
J. T. GANNON el al.
the same two genera. The benzene degraders (strains
Ben I and Ben2) moved to a greater extent than the
isolates able to use chloroben/jne (strains CB1, CB2
and CB3) or toluene (Toll. Tol2, Tol3 and Tol4). Of
the eight isolates selected Tor their presumed greater
mobility, most were relatively mobile. More than 2%
of the cells of six strains (Enterobacter IS2, Enter-
obatttr ISl. Pseudomonas KL2, Pseudomonas DF2,
Achromobacier DPI and Enterobacter (KLI) were
transported through the soil, whereas the percentages
were lower Tor Pseudomonas KL3 and Flavobacterium
DF3. For the bacterium originally obtained from an
aquifer (Arthrobacier Lula D), 7.7% of the cells
moved through the soil.
The proportions of the added cells that were
recovered in the soil and the effluent varied from
essentially 100% for Achromobacier Ben2 and Bacil-
lus 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 a
high percentage of the cells of many strains did not
lose viability and were not strongly sorbed to soil
particles. A comparison of recovery percentages and
the transport percentages indicates that many of the
viable cells were retainer in the soil column. A
regression of percentage of cells transported on per-
centage of bacteria recovered gave an F value of 5.08
(significant at P - 0.05). 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. This is
not surprising since, if it is assumed that low recovery
results from sorbed cells not re-entering the stream of
flowing water or their loss of viability, strains with
many cells strongly sorbed or inactivated are not
likely to have many viable cells transported.
The A'j values of the bacteria ranged from 0.0 for
Enterobacter ISl to infinity (upper bound of Ka) for
Pseudomonas Tol4 (Table 3). The value of infinity
reflects adsorption of all the cells. A comparison of
the mean values between the lower- and higher-
3. Advorpnon coefficient of bacteria
Kt value (ml g ')
Bacterium
Enirrnha. irr \S2
Enltrtthai-ttr IS)
Psrudumona* KL2
Arlhrohaclri Lula D
PuuJoiMonai DF2
Achromohaeitt Ben2
Pimlomuniu Benl
Bacillus CB2
Aelirttmohaeltr DPI
Enifrohacirr KLI
Pitudamaniu KLJ
£>irrr0rW/rrTol2
Enurohaeitr Toll
PiruJamonaj ToM
Enirrotmtlrr Toll
Pmnlamanai CBI
AK-///U CIM5I9
FlMi'hiult'Him Of)
BacUlia CB)
Loiter
bound
4.5
0.0
5.5
5.5
0.85
1)
6.0
IS
7.5
3.5
9.0
29
6.5
43
».5
:»
JO
13
12
Upper
bound
5.5
0.0
8.5
8.0
0.*
:s •
8.5
36
II
4.5
24
145
k.S
x .
16
150
410
17
24
bounds of Kd values with the transport percentages
shows that 8 of 10 bacteria for which >2% of the
cells were transported had mean Kd values 10.0. Thus, a high
percentage of cells of strains with low K^ values
moved relatively freely through the soil, whereas a
low percentage of cells of strains with high A.'0 values
were transported at a significant rate under identical
conditions.
The relationship between Ka and transport is es-
pecially striking if recovery is considered. Thus, for
strains for which <50% of the cells were recovered.
<1% of the cells were transported for all the 8
isolates having mean Kt values of > 10.0. Similarly.
>2% of the cells were transported for 8 of the 9
species with recoveries >35% and mean Kd values
< 10.0. Mobility is thus strongly dependent on bolh
adsorption and viability.
The relationship between the percentage of the cells
transported and the lower- but not the upper-hound
Kt values was statistically significant. The I'values for
the regressions were 7.40 ,md 3.26. respectively.
Regressions indicated that recoveries were related
to the A^ values (P =0.05). When the lower-bound
Kt values exceeded 20 or the upper-bound A'd values
exceeded 100 (Enterobacter Tol2. Pseudomtmas Tol4
and CBI and Bacillus CU45I9). 25% or less of the
cells were recovered. This probably reflects the in-
ability to detect a significant fraction of the sorbed
cells in the procedure used for counting.
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 el at.. 1985). However, because the geometry
of macropores in the field may change frequently due
to wetting, drying, freezing, thawing or the burrowing
of invertebrate animals, movement of bacteria
through the soil matrix may be necessary for
biodegradative microorganisms to reach much of the
chemical that is well dispersed in the soil. Columns of
homogeneous soil were used to minimize uncon-
trolled, preferential movement of bacteria through
macropores and thus to permit a definition of such
factors as mechanical filtration and adsorption th.ii
control the movement of bacteria through the soil
matrix itself. In the development of the procedure for
determination of mobility, a loamy soil was selected
to avoid the extremes of limited bacterial sorption
and relatively free movement in sandy soils on the
one hand and the restricted movement resulting from
extensive mechanical filtration, as well as extensive
sorption. in fine-textured soils on the other hand.
Had a sandy or a fine-textured soil been used, the
ability of the procedure to detect small differences in
bacterial mobilities might have been reduced. Fur-
thermore, species that were found to be extensively
transported in a disturbed soil lacking a network of
macropores would likely move even more readily in
a natural, non-disturbed soil with significant macro-
porosity.
-------
Bacterial transport
1159
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. (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 here described has several advan-
tages 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 rela-
tively wide column. Macro pores through which bac-
teria could move preferentially were eliminated by
grinding, sieving the soil prior to preparing the
column and uniform packing to a fixed bulk density.
The slow wetting of the column from the bottom
prevented the formation of preferential paths during
saturation of the column with water. Bacterial death
from predation or parasitism was avoided because
sterile soil was used. Increases in cell numbers arising
from growth and decreases associated with starvation
were prevented by performing the tests of transport
at 2-5 C. Furthermore, the marked differences in
mobility among the isolates suggest that the proposed
procedure docs indeed distinguish among bacteria
with different capacities for movement.
For inoculation of the surface of the soil with
bacteria that can degrade organic pollutants to lead
to the destruction of (hose compounds at some
underground site, some of the added cells must move
through ihc soil to the below ground zone of con-
tamination. Evidence exists, however, that intro-
duced organisms may fail because they are not
transported to the sites containing the pollutant
(Goldstein ft al.. 1985). In this context, it is worth
noting that many of the carefully controlled exper-
iments in which inoculation resulted in biodegrada-
tion required transport to soil depths of only 10 cm
(Barles ct al.. 1979: Edgchill and Finn. 198?: Me-
Clure. 1972). Measurements such as those made in
our study will enable extrapolation of the potential
penetrability of the bacteria to considerably greater
depths. The data show a significant inverse corre-
lation between the mobility of the bacteria and the
lower bound A'd values. There is good reason why the
lower bound of Kd correlates better with transport
than the higher bound. Adsorption on clay panicles
equal in size to or smaller than the cells (this adsorp-
tion being included in the upper bound but not in the
lower bound) may not affect mobility as much as
adsorption on bigger particles. Bacteria adsorbed on
particles smaller than the cells may move to some
extent together with the particles-on which they are
adsorbed. Such adsorption, at times, may even en-
hance the transport because it retards adsorption on
bigger particles. Thus, the lower bound, which in-
cludes only adsorption on particles larger than the
cells, is likely to correlate (inversely) better than the
upper bound with mobility. Mechanical filtration and
adsorption can effectively retard bacterial transport.
If adsorption it weak, the transport of cells through
the soil matrix may be controlled by other factors,
such as mechanical filtration. We have found that
bacterial transport was strongly correlated with cell
size (P = 0.01) (Gannon et al., 1991), suggesting the
importance of mechanical filtration. When Ka is high
(e.g. >IO), mobility should be low even for small
bacteria provided that mechanical filtration is limited
by large pore sizes. Yet for bacteria with low Ka
values (e.g. < '.0), cell size should be critical. Adsorp-
tion and recovery were also inversely related. It has
also been found that a significant correlation did not
exist between mobility and hydrophobicity. net sur-
face charge and capsule formation of these bacteria
(Gannon et al., 1991).
The present findings suggest that it should be
possible to obtain bacteria that have both the ca-
pacity to biodegrade unwanted organic compounds
and the ability to move through earth materials to
sites containing such compounds. By selecting more
mobile bacteria, the physical constraint to bioremedi-
ation with inoculated bacteria may be overcome, and
species able to destroy pollutants may move to the
sites of pollution.
Acknowledgements—This work was sur ported by a cooper-
ative agreement with the USEPA Robert S. Kerr Environ-
mental Research Laboratory. Ada. Oklahoma and h> a
grant from the Mellon Foundation. We thank J. T. Wilson
Tor helpful suggestions. This report has not been subjected
to peer and administrative review by the USEPA and
therefore may not necessarily reflect the views of the
Agency, and no official endorsement should be inferred
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