WATER RESOURCES RESEARCH, VOL. 49, 2424–2436, doi:10.1002/wrcr.20174, 2013

Transport and fate of microorganisms in soils with preferential flow
under different solution chemistry conditions
 ° nek1
Yusong Wang,1 Scott A. Bradford,2 and Jirı Simu
Received 25 June 2012; revised 14 February 2013; accepted 27 February 2013; published 17 May 2013.

[1] Laboratory and numerical studies were conducted to investigate the transport and fate ofEscherichia coli D21g and coliphage ’X174 in saturated soils with preferential ﬂow under
different solution ionic strength (IS ¼ 1, 5, 20, and 100 mM) conditions. Preferential ﬂow
systems were created by embedding a coarse-sand lens (710 mm) into a ﬁner matrix sand(120 mm). Complementary transport experiments were conducted in homogeneous sandcolumns to identify controlling transport and retention processes, and to independently
determine model parameters for numerical simulations in the heterogeneous experiments.
Results from homogeneous and heterogeneous transport experiments demonstrate that
retention of E. coli D21g and ’X174 increased with IS, while the effect on E. coli D21g in
ﬁner sand was much greater than in coarse sand. This microbe transport behavior was welldescribed by numerical simulations. The importance of preferential ﬂow on microbetransport was found to be enhanced at higher IS, even though the overall transport
decreased. However, the contribution of preferential ﬂow was much higher for E. coli D21g
than ’X174. Deposition proﬁles revealed signiﬁcant cell retention at the interface of thecoarse-sand lens and the ﬁne-sand matrix as a result of mass transfer. Cell release from thepreferential ﬂow system with a reduction of solution IS exhibited multipulse breakthroughbehavior that was strongly dependent on the initial amount of cell retention, especially at
the lens-matrix interface.
 ° nek (2013), Transport and fate of microorganisms in soils with preferential flow
Citation: Wang, Y., S. A. Bradford, and J. Simu
under different solution chemistry conditions, Water Resour. Res., 49, 2424–2436, doi:10.1002/wrcr.20174.

1.

Introduction[2] Waterborne disease outbreaks associated with drinking water in the United States during 1971–2002 are known
to have resulted in 575,457 cases of illness and 79 deaths,
with 14% caused by bacteria, 19% by protozoa, 8% by viralpathogens, and 47% by unknown acute gastrointestinal
illness [Reynolds et al., 2008]. Wastes from humans,
domesticated and wild animals, birds, and insects
frequently contain high concentrations of pathogenic microorganisms [U.S. Department of Agriculture, 1992; U.S.
Environmental Protection Agency, 1998; Gerba and Smith,2005] that serve as pathogen sources in agricultural settings.
Pathogenic microorganisms can be transported to streams
by surface water runoff and to ground water by recharge
through the vadose zone. These contaminated surface andground water supplies may eventually serve as drinking
water and/or irrigation water for fresh produce. If these contaminated water supplies are not adequately treated before

1
Department of Environmental Sciences, University of California,
Riverside, California, USA.
2
U.S. Salinity Laboratory, USDA, ARS, Riverside, California, USA.

Corresponding author: Y. Wang, Department of Environmental
Sciences, University of California, Riverside, CA 92521, USA.
(Ywang032@ucr.edu)©2013. American Geophysical Union. All Rights Reserved.
0043-1397/13/10.1002/wrcr.20174

use, ground water typically receives minimal or no treatment, then they can put the public’s health at risk.
[3] Much research has examined physical (size of the
microbe and the porous medium, microbe concentration,water velocity, water content, and surface roughness) andchemical (surface chemistry of the microbe and soil, andaqueous solution pH, ionic strength (IS), and chemical composition) factors that inﬂuence the retention of microorganisms in homogeneous porous media under relatively uniform
ﬂow [Mills et al., 1994; Mccaulou et al., 1995; Hendry etal., 1999; Yee et al., 2000; Dong et al., 2002; Bradford etal., 2006b; Chen and Walker, 2007]. Mechanisms of
microbe retention have been inferred from repacked column
breakthrough curves (BTCs) and retention proﬁles (RPs),
batch experiments, and complimentary micromodel studies
that allowed for direct microscopic observation [Ochiai etal., 2006]. The mechanisms governing transport and retention of microbes in porous media have been described byseveral review articles [Stevik et al., 2004; Sen and Khilar,
2006; Bradford and Torkzaban, 2008].
[4] Field, lysimeter, and undisturbed soil column
experiments have frequently revealed that microorganisms,
colloids, and other contaminants can travel much deeper andfaster than would be predicted based on results from laboratory studies in homogeneous porous media [Bales et al.,
1989; Abu-Ashour et al., 1994; Pivets and Steenhuis, 1995;
Pivetz et al., 1996; Jiang et al., 2010]. This phenomenon
has been ascribed to transport in preferential ﬂow pathways
created by plant roots, burrowing earthworms [Beven and2424

WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

Germann, 1982; Madsen and Alexander, 1982; Unc andGoss, 2003; Cey et al., 2009], and/or natural structural heterogeneities [Wollum and Cassel, 1978]. Preferential ﬂow canalso occur in soils and aquifers with strong contrasts inhydraulic conductivity between sediment layers [Harvey etal., 1993]. Considerable research has indicated that colloids
and microorganisms can be transported in preferential ﬂow
pathways [McGechan and Lewis, 2002; Jarvis, 2007; Panget al., 2008; Cey et al., 2009; Cey and Rudolph, 2009; Passmore et al., 2010]. Preferential ﬂow has also been implicatedin the rapid transport of bacteria to ﬁeld tile drains [Evans
and Owens, 1972; Dean and Foran, 1992; Guzman et al.,2009]. However, most of this research is qualitative in nature
because of difﬁculty in quantifying the physical and chemical complexities of the soil matrix and macropore systemwith regard to microbe transport. This gap in information
currently presents a great obstacle to predicting thefate of microbes in natural environments [McCarthy and
McKay, 2004].
[5] The exchange rate of water between the macropore
and the matrix, or between layers with contrasting hydraulicconductivities, will likely be a critical factor in determining
the rate of microbe migration in preferential ﬂow systems[Harvey et al., 1993; Morley et al., 1998; Allaire-Leung etal., 2000a, 2000b; Allaire et al., 2002a, 2002b] because of
the potential for greater retention of microbes in the matrix
as a result of enhanced chemical interactions, lower hydrodynamic forces, and smaller pore spaces. It is also possiblethat microbes may be physically excluded from the matrix,
and in this case transport of microbes would exclusively
occur in macropores. Optimum conditions for microbe transport in preferential ﬂow systems will therefore likely depend
on a wide variety of chemical and physical factors of boththe matrix and macropore domains, but little quantitative
research has addressed this issue, and the relative importanceof these factors has not yet been fully determined [Fontes
et al., 1991].
[6] It is important to be able to describe water ﬂow inthe preferential ﬂow domain in order to quantitatively
simulate the transport of microorganisms in the ﬁeld.
Various conceptual models of preferential ﬂow exist thatare dependent on a great number of parameters (e.g., macropore hydraulic properties, mass transfer terms, and mac ° nek and van Genuchten, 2008]. It isropore geometry) [Simudifﬁcult, if not impossible, to independently determine allof the required model parameters under natural conditions
 ° nek et al., 2003]. Thus, some assumptions have to be[Simu
made to simplify the preferential ﬂow path for simulation
purposes. Artiﬁcial macropores can be systematicallycreated by leaving small cylindrical openings in repacked
columns [Pivetz and Steenhuis, 1995; Castiglione et al.,2003] or by packing different sized sands to generate layers
and/or lens with contrasting hydraulic conductivities[Fontes et al., 1991; Saiers et al., 1994; Morley et al.,
1998; Bradford et al., 2004]. The study of preferential ﬂow
and transport through artiﬁcial macropores provides anopportunity to overcome many modeling challenges,
because the macropore geometry and hydraulic properties
can be well deﬁned and controlled in a repeatable manner[Pivetz and Steenhuis, 1995; Castiglione et al., 2003;
Guzman et al., 2009; Arora et al., 2011, 2012]. Hence, it ispossible to isolate and identify factors that have the greatest

inﬂuence on preferential ﬂow and transport in such systems. Furthermore, controlled preferential ﬂow and transport studies provide valuable information onparameterization that is needed to help adapt numericalmodels to complex natural systems.
[7] Most studies of colloid and/or microbe transport
through artiﬁcial macropores have focused on understanding the inﬂuence of physical heterogeneity on BTCs
[Fontes et al., 1991; Saiers et al., 1994; Morley et al.,1998] and RPs [Bradford et al., 2004]. Numerical simulations of collected data demonstrate the importance of transverse mixing at the interface between the coarse-sand lensand the ﬁne-sand matrix [Saiers et al., 1994; Morley et al.,1998] and the spatial distribution of retained colloids[Bradford et al., 2004]. Relatively little research has investigated the inﬂuence of microbe size and solution chemistry
in preferential ﬂow systems. Fontes et al. 1991 studied bacteria transport under two different IS conditions in a column with a coarse-sand lens imbedded in a ﬁner soil matrix
and demonstrated decreasing transport potential with anincrease in IS. However, bacteria RPs were not determinedin this study, and the data were not modeled. Furthermore,no published research has examined the release behavior of
microbes with a reduction of solution IS in systems with
artiﬁcial macropores. Consequently, our understanding of
the effects of solution chemistry on microbe transport inpreferential ﬂow systems is still incomplete.
[8] The objective of this research was to study the
coupled inﬂuence of physical and chemical factors on thetransport of microorganisms in preferential ﬂow systems.
Transport experiments were conducted for two different
sized microorganisms (Escherichia coli D21g and coliphage ’X174) in packed columns with an artiﬁcial macropore at various IS conditions. Complementary transport
experiments were also conducted with these microorganisms in homogeneously packed columns to independently
determine many of the model parameters. RPs and therelease of retained microorganisms after altering the IS of
the soil solution were additionally studied for heterogeneous columns. Numerical simulation of the ﬂow and transport behavior of the preferential ﬂow system with
HYDRUS (2D/3D) facilitated the quantiﬁcation of ﬂow
and transport processes, especially the mass transfer andrelease behavior at the matrix/macropore interface.

2.

Materials and Methods

2.1. Sands and Electrolyte Solutions[9] Two sizes of Ottawa (quartz) sand were used in thecolumn experiments described later. The median grain size(d50) of these sands were 120 and 710 mm (referred to as
ﬁne sand and coarse sand later), and their corresponding
coefﬁcients of uniformity were equal to 1.6 and 1.3, respectively. A salt cleaning procedure was employed to remove
kaolinite clay from the sand surfaces [Bradford and Kim,
2010] before use in the experiments to eliminate any background interference from clay particles.
[10] Electrolyte solutions that were used in column
experiments consisted of autoclaved, deionized (DI) waterwith its pH 5.8 and the IS level adjusted to 0, 1, 5, 20, and100 mM using NaCl or NaBr. These IS levels were selected
to create a range of adhesive conditions between the2425

WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW
Table 1. Measured Zeta Potentials and Calculated Mean Interaction Energy Barrier Height and Depth of the Secondary Minima for E.
coli D21g and ’X174 on Approach to the Quartz Sanda
Zeta Potential (mV)

Energy Barrier Height (kT)E. coli D21g

Secondary Minima (kT)’X174

E. coli D21g

’X174

IS
(mM)

Fine
Sand

CoarseSand

E. coli
D21g

’X174

Fine
Sand

Coarse
Sand

Fine
Sand

Coarse
Sand

Fine
Sand

Coarse
Sand

Fine
Sand

Coarse
Sand

1
5
20
100

80
81
80
40

83
75
63
30

60
52
43
13

20
15
8.5
6.4

5520
4317
2920
59

5657
4149
2502
15.7

11.2
6.5
1.6
NB

11.2
6.4
1.6
NB

N/A
0.5
2.6
23.4

N/A
0.5
2.7
26.4

N/A
0.01
0.03
NB

N/A
0.01
0.03
NB

a

NB, no energy barrier; N/A, not applicable.

microorganisms and sand. Bromide served as a conservative tracer in the column experiments. The efﬂuent concentration of bromide was determined using a bromideselective electrode (Thermo Scientiﬁc Orion bromide electrode ionplusV Sure-FlowV).
R

R

2.2. Microbes[11] E. coli D21g and coliphage ’X174 were selected as
representative microorganisms for column transport experiments. E. coli D21g is a Gram-negative, nonmotile bacterial
strain, which has minimal lipopolysaccharides, and negligible amounts of extracellular polymeric substances [Walker
et al., 2004]. The effective diameter of E. coli D21g is 1.84mm [Walker et al., 2004]. Coliphage ’X174 is considered to
be a conservative indicator for human virus transport. It is aspherical, single-stranded DNA coliphage with a 27 nm
diameter and low hydrophobicity [Dowd et al., 1998].
[12] E. coli D21g was cultured in Luria-Bertani broth(LB broth, Fisher Scientiﬁc, Fair Lawn, NJ) containing
0.03 mg/L gentamycin (Sigma, St. Louis, MO) on a rotary
shaker overnight (12–18 h) at 37 C. Then, 2 mL of liquid
culture was transferred onto a LB media plate containing
0.03 mg/L gentamycin, and the plates were cultured overnight (12–18 h) at 37 C. Sterile water was placed on theplates, and the colonies were gently harvested using a sterile glass rod to release the colonies into solution. The bacteria suspension was then centrifuged to separate whole cells
from the solution. The supernatant was decanted, and thebacteria were resuspended. These processes were repeated
two times before diluting the concentrated suspension into
the desired electrolyte solution to ensure that all traces of
the growth medium were removed. A fresh cell suspension
was prepared right before the start of each experiment. The
concentrations of E. coli D21g in inﬂuent, efﬂuent, and soil
solution were determined using a spectrophotometer(Unico UV-200, United Products & Instruments, Dayton,
NJ) at 600 nm [Torkzaban et al., 2008] and by the spread
plating method [Clesceri et al., 1989] when necessary (e.g.,
low concentration).
[13] Coliphage ’X174 was propagated at 37 C overnighton bacterial host E. coli CN-13 (ATCC 700609) at mid-exponential growth phase in Typtic soy broth supplemented
with 1% nalixidic acid. The lysate was centrifuged (5000 
g for 20 min) and ﬁltered (0.45 mm ﬁlter) to remove the celldebris and to recover the coliphage. The concentration ofcoliphage ’X174 in aqueous solutions was determinedusing the double agar overlay method 1601 [U.S. Environ-mental Protection Agency, 2001] with bacterial host E. coli
CN-13. The number of plaque forming units (pfu) was
determined by counting the plaque density. All coliphage
assays were run in duplicate and diluted as necessary.
[14] E. coli D21g and coliphage ’X174 were added to
the NaBr solutions to achieve inﬂuent concentrations (C0)of approximately 1.0  108 cells/mL and approximately
1.0  107 pfu/mL, respectively. The suspension was continuously mixed during the column experiment using amagnetic stirrer. The values of C0 for both microbes were
measured three times during the course of a transport
experiment to assess the reproducibility of the measurements and microbial survival. The spectrophotometer readings for E. coli D21g were within 1% of C0, and thestandard deviation of the spread plating method was 14.3%
of C0. The standard deviation of repeated measurements of’X174 was 13.1% of C0. No systematic decrease in C0 was
observed over the duration of the column experiments, andthis indicates that little inactivation occurred during thisinterval.
2.3. DLVO Calculations[15] The zeta potential of coliphage ’X174, E. coli
D21g, and Ottawa sand crushed to a powder in various solution chemistries (Table 1) was calculated from experimentally measured electrophoretic mobilities using a
ZetaPALS instrument (Brookhaven Instruments Corporation, Holtsville, NY) and the Smoluchowski equation. The
total interaction energy of the ’X174 and E. coli D21g
upon approach to the Ottawa sand under the various solution chemistries was subsequently calculated (Table 1)
using Derjaguin-Landau-Verwey-Overbeek (DLVO) theory
and a sphere-plate assumption [Verwey and Overbeek,
1948; Derjaguin, 1954]. Electrostatic double-layer interactions were quantiﬁed using the expression of Hogg et al.
1966 using zeta potentials in place of surface potentials.
The retarded London-van der Waals attractive interaction
force was determined from the expression of Gregory
1981. Values of the Hamaker constant used in these calculations were 4  1021 J [Penrod et al., 1996] and 6.5 
1021 J [Rijnaarts et al., 1995] for ’X174 and E. coli
D21g, respectively.
2.4. Homogeneous Column Experiments[17] Glass chromatography columns (15 cm long and 4.8
cm inside diameter) were used in homogeneous transport
experiments. A schematic of the experimental setup is
given by Bradford et al. 2002. The columns were wet

2426

WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW
Table 2. Experimental (Pore Water Velocity v) and Fitted Model(Bromide Dispersivity , Microbe Attachment Rate Coefﬁcientkatt, and Maximum Solid Phase Concentration of Microbes, Smax)
Parameters From the Homogeneous Fine- and Coarse-Sand
Column Experiments With E. coli D21g and ’X174 at Different
Solution IS Conditionsa
E. coli D21g
ISv
(mM) (cm/min) (cm)

katt

Fine
sand

1
5
20
100

0.33
0.31
0.34
0.37

0.10
0.10
0.13
0.14

Coarse
sand

20
100

10.3
10.3

0.52 0.001
0.59 0.005

Smax

0.0004 0.01
0.066 1.1
0.2
3.5
0.48
4.8
0.1
0.4

’X174

R

2

katt

Smax

R2

0.99 0.33 0.17 0.98
0.99 ND
ND ND
0.95 ND
ND ND
0.99b 0.68c 0.16c 0.96d0.034 0.54d
1.00 ND
ND ND
0.97 ND
ND NDa
The goodness of the model ﬁt is quantiﬁed by the coefﬁcient of linear
regression (R2) on breakthrough data unless otherwise noted. ND, notdetermined.
b
Determined using RP information.
c
Parameters for site 1.
dParameters for site 2

packed with a given size of Ottawa sand. The porosity ofthe packed columns was between 0.36 and 0.38, and thelength of the columns was about 13 cm. Aqueous solutions
were pumped upward through the vertically oriented columns at a steady ﬂow rate using a peristaltic pump. The
pore water velocities in the homogeneous column experiments (Table 2) were selected to be consistent with thosemeasured in the heterogeneous column experiments
described later.
[22] Two pore volumes (PVs) of a selected NaCl solution were ﬂushed through a column, and the sand wasallowed to equilibrate with this solution (phase 0) before
initiating a microbial transport experiment. Microbial transport experiments were carried out in two phases. First, several PVs of microbe suspension and NaBr were introduced
into the column at a constant rate and IS (phase 1). Second,
NaCl solution was ﬂushed through the column at the same
ﬂow rate and IS as in phase 1 until the efﬂuent microbe
concentration returned to a baseline level (phase 2). Efﬂuent samples were continuously collected during the transport experiment at selected intervals using a fraction
collector. The efﬂuent samples were then analyzed for Br
Table 3. Mass Balance Information (Efﬂuent, Sand, and Total)for Homogeneous Fine- and Coarse-Sand Column Experiments
With E. coli D21g and ’X174 at Different Solution IS
Conditionsa
Recovery (%)
E. coli D21g

Fine sandCoarse
sand
a

’X174

IS
(mM)

Effluent

Sand

Total

Effluent

Sand

Total

1
5
20
100
20
100

103
44
12
2
98
92

ND
58
83
85
3
12

103
102
95
87
101
104

69
ND
ND
36
ND
ND

1
ND
ND
1
ND
ND

70
ND
ND
37
ND
ND

ND, not determined.

Figure 1. A representative picture of (left) heterogeneouscolumn with lens in the center and (right) simulated ﬂow
ﬁeld.

and microbe concentrations as described earlier. Replicate
transport experiments were conducted for the different solution chemistry conditions.
[23] The microbial RPs in the sand of some columns
were determined following completion of phases 1 and 2.
The saturated sand was carefully excavated into tubes containing excess DI water. The tubes were shaken for 15 min
to liberate any reversibly retained microbes, and the concentrations of E. coli D21g and coliphage ’X174 in theexcess solution were determined as described earlier. The
volume of solution and mass of dry sand was determinedfrom mass balance. The microbe mass recovery (%) was
subsequently determined during phases 1 and 2 and for theRPs from mass balance and the injected amount (Table 3).
2.5. Heterogeneous Column Experiments[25] Heterogeneous column experiments were conductedin a plexiglass (acrylic) column, 22 cm long and 13.2 cm
inside diameter. A polyester membrane (Saatiﬁl PES 18/13)
with an 18 mm nominal pore size was placed at the bottomof the column and connected to a hanging water column(tube) to control the bottom boundary pressure. Preferential
ﬂow systems were created by packing the ﬁne (120 mm) andcoarse (710 mm) sands into the column as follows: (i) thecolumn was ﬁlled with autoclaved DI water to about one
third of the column height, and a 30 cm long plastic tube
with outside diameter of 1.14 cm was held in the center ofthe column; (ii) the ﬁne sand was incrementally wet packed
into the matrix portion of the column (outside the plastictube) to a height of 20 cm; (iii) excess water in the plastic
tube was drained from the bottom; (iv) the tube was carefully pulled out from the column without disturbing the surrounding ﬁne matrix sand and leaving a 1.14 cm diameterhole in the center of the column; (v) the hole was then ﬁlled
to a height of 20 cm with the coarse sand using a funnel tocreate a preferential ﬂow lens; and (vi) the column was thensaturated with water from the bottom. The porosity of thecolumns ranged from 0.34 to 0.36. Figure 1 shows an example of the artiﬁcial preferential ﬂow path.
[26] Solutions were delivered onto the surface of the heterogeneous column at a steady ﬂow rate using a rain

2427

WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

Figure 2.

A schematic of the setup for transport experiments in heterogeneous columns.

simulator connected to a peristaltic pump. The watervelocity was selected in order to just maintain saturated
conditions (several millimeters of ponding at the surface)in the column through the experiments. Figure 2 shows aschematic of the experimental setup used for the preferential bromide and microbial transport experiments. Similar
procedures were carried out to collect and analyze efﬂuent
samples in the homogeneous and heterogeneous column
experiments. Furthermore, phases 0, 1, and 2 were conducted in an analogous fashion. After recovery of the
BTCs, the distribution of retained microbes in the heterogeneous column was quantiﬁed as follows. Sand samples
were taken at six equally spaced depths and three locations,
namely, the lens, the matrix in the vicinity of the lens, andthe bulk matrix. The determination of the amount ofretained microbes in the sand was then determined in a similar manner to the homogeneous column experiments.
Phases 1 and 2 of the heterogeneous column experiments
were replicated for most solution chemistry conditions.
[27] Following recovery of the BTC for the microbes
and bromide, some of the columns underwent an additional
experimental phase 3 to determine the release of retained
microbes with a reduction in solution IS. In this case, columns were ﬂushed with autoclaved DI water at the same
velocity as during phases 1 and 2 until the released microbe
concentration in the efﬂuent returned to a baseline level.
The efﬂuent samples were collected and analyzed using thesame protocols as during phases 1 and 2.
[28] The saturated hydraulic conductivity (Ks) for theﬁne and coarse sands was directly measured to be 0.3 and11 cm/min, respectively. However, the value of Ks in thematrix and lens was observed to vary somewhat for eachheterogeneous column experiment due to small differencesin packing and porosity. A more representative value of Ks
for the matrix and lens was determined as follows. The
value of Ks for the matrix was determined from thebreakthrough time of the Br tracer from the matrix.
The value of Ks for the lens was subsequently determinedas the difference in the measured total ﬂow rate and thematrix ﬂow rate.

3.

Numerical Modeling[29] The HYDRUS-1D and HYDRUS (2D/3D)
 ° nek et al., 2008] codes were used to simulate the[Simutransport of bromide and microbes in the homogeneous andheterogeneous column experiments, respectively. The
HYDRUS codes simulate water ﬂow using the Richards
equation. Bromide transport was simulated using theadvection-dispersion equation (ADE). Microbe transportand retention was simulated using the ADE with ﬁrst-orderterms for kinetic retention and release as shown later forthe 2-D case:@ ðC Þ
@
@C
@qi C

Dij
  katt C þ kdet S
¼
@t
@xi
@xj
@xi

(1)

@ ðS Þ
¼  katt C  kdet S;@t(2)

where subscripts i and j denote coordinate directions, C[NcL3 ; L and Nc denote the units of length and number ofmicrobes, respectively] is the microbe concentration in the
aqueous phase, S [NcM1 ; M denotes units of mass of soil]
is the microbe concentration on the solid phase, Dij [L2T1]
is the hydrodynamic dispersion coefﬁcient, qi [LT1] is theDarcy water velocity in i direction, kdet [T1] is the microbe
detachment rate coefﬁcient, katt [T1] is the microbe attachment rate coefﬁcient,  is the water content, and  [ML3]is the bulk density. The parameter accounts for time- andconcentration-dependent blocking using a Langmuirian
approach as [Adamczyk et al., 1994]:
¼1

S
;
Smax

(3)

where Smax [NcM1] is the maximum solid phase concentration of microbes. In the case of ’X174 at solution
IS ¼ 100 mM, a similar two-site kinetic retention was
employed.

2428

WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

4.

Figure 3. Representative plots of observed and simulated(model) normalized Br concentrations (C/C0) in (top)homogeneous ﬁne-sand and coarse-sand columns as afunction of PV and (bottom) the heterogeneous column as afunction of time. Parameter values are given in Tables 2
and 4.
[30] The homogeneous column transport experiments
were simulated using a third-type boundary condition at the
inlet and a zero concentration gradient at the outlet. The
HYDRUS codes include a nonlinear least squares optimization routine to inversely estimate model parameters byﬁtting to experimental data. This option was used to determine Br and microbe transport parameters by ﬁtting the experimental data from the homogeneous column
experiments. In particular, the dispersivity was determinedfrom the Br BTC, and the transport parameters (Smax, katt,kdet) for microbes at given solution chemistry conditions
were determined from measured microbe BTCs and/or RPs(Table 2).
[31] Bromide and microbe transport parameters obtained
from the homogeneous column experiments were subsequently used in HYDRUS simulations of the heterogeneouscolumn experiments with preferential ﬂow. In this case, a
2-D axisymmetrical geometry was employed, and themodel domain was 20 cm  6.6 cm. This domain was
subdivided into two vertical regions, with the left region
representing the coarse lens in the center of the column
with a radius of 0.57 cm and the remaining region representing the ﬁne matrix sand. Domain discretization was
designed to reduce simulation times while minimizing
mass balance errors. A constant water ﬂux boundary condition was employed at the soil surface, and a constant head
was applied at the bottom boundary. A third-type boundary
was chosen for the solute transport boundary condition at
the inlet, and a zero concentration gradient was selected at
the column outlet.

Results and Discussion

4.1. Bromide[32] Representative plots of observed and simulated
BTCs (C/C0 versus PV) for the Br tracer in the homogeneous ﬁne-sand and coarse-sand columns are shown in Figure
3. Bromide acted as a good conservative tracer, with goodmass recovery and breakthrough at one PV. The 1-D ADE
provided an excellent description of the homogeneous Br
BTCs (Figure 3) when the dispersivity was inversely optimized to the data (R2 > 0.98). Dispersivities were around0.12 and 0.55 cm (Table 2) for ﬁne- and coarse-sand columns, respectively.
[33] Figure 3 also provides representative observed andsimulated BTCs for Br in a heterogeneous column thatexhibited preferential ﬂow. The very early breakthrough ofBr (labeled as T1) demonstrated the extremely fast movement of water and solute through the artiﬁcial macropore.
The peak value of C/C0 in this ﬁrst pulse was around 0.20–
0.27, indicating that 20%–27% of the total water ﬂux
occurred through the macropore (Table 4). A second,higher (C/C0 ¼ 0.83–0.90) Br pulse (labeled as T2)
occurred after about 22 min due to breakthrough of Br
from the matrix material. The value of C/C0 does not reachone for the matrix pulse because of the difference in arrival/ending times of the matrix and lens pulses; for example, the matrix pulse is diluted by NaCl solution from thelens. As mentioned earlier, the measured Br breakthroughtimes for the lens and matrix regions were used to reﬁneestimates of the hydraulic properties in the heterogeneouscolumn experiments. Table 2 provides a summary of thesolute transport parameters that were used in subsequentHYDRUS simulations for each column. The values of thelens and matrix Ks (Table 4) and water ﬂuxes (Table 4)
were very consistent for the different column experiments,
and this indicates that the artiﬁcial macropore was highly
duplicable and had limited variation. Furthermore, theagreement between observed and simulated Br BTCs wasvery good (R2 > 0.92 in Table 4). Simulations therefore
provided an excellent description of water ﬂow and Br
transport in the heterogeneous systems.
4.2. E. coli D21g
[34] Figure 4 (top) presents observed and simulated
BTCs for E. coli D21g in homogeneous ﬁne sand when thesolution IS equaled 1, 5, 20, and 100 mM. Figure 4 (bottom) gives similar information for the coarse sand when the
Table 4. Experimental Values of the Local (Lens and Matrix)
Saturated Hydraulic Conductivity (Ks), the Percentage of Local
Flux to Total Flux, and the Percentage of Local Transport to Total
Transport in the Heterogeneous Column Experiments for E. coli
D21g at Different Solution IS Conditions
Lens

MatrixIS(mM)

Ks(cm/min)

Flux(%)

Transport(%)

Ks(cm/min)

Flux(%)

Transport
(%)

1
5
20
100

11.2
11.8
10.5
10.6

25
27
27
20

26
38
68
99

0.31
0.31
0.31
0.33

75
73
73
80

74
62
32
1

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WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

Similar to experimental observations, the values of katt andSmax increased with IS and were larger in the ﬁner than thecoarser sand (Table 2). The value of kdet was low in allcases; thus kdet was set as 0 for all simulations.
[37] A partial explanation for differences in the E. coli
D21g transport behavior in coarse and ﬁne sands isobtained from the ﬁltration theory [Yao et al., 1971], which
predicts that E. coli D21g mass transfer to the sand surface
is greater for the ﬁner than the coarser sand. Table 1
presents the calculated mean interaction energy height anddepth of the secondary minimum for E. coli D21g on
approach to the quartz sand. Unfavorable attachment conditions (i.e., the presence of a signiﬁcant energy barrier in Table 1) are predicted to occur for all the solution IS.
However, the depth of the secondary energy minimum
increased with IS (Table 1) and followed a qualitatively
similar trend to the amount of cell retention (Table 3).
[38] It should be mentioned that mean interaction energies do not reﬂect the potentially signiﬁcant inﬂuence of
microscopic heterogeneities on cell retention [Duffadar
and Davis, 2008]. Evidence for the potential role of microscopic heterogeneities is obtained from the mass balance
information (Table 3) and from ﬁtted model parameters(Table 2). In particular, values of Smax increased with IS
and in the ﬁner sand (Table 2). Furthermore, the total mass
Figure 4. Observed and simulated (model) normalized
efﬂuent concentrations (C/C0) of E. coli D21g as a functionof PV in homogeneous (top) ﬁne-sand and (bottom) coarsesand columns at selected solution IS conditions. Parametervalues are given in Table 2.
IS was 20 and 100 mM. Corresponding RPs and mass balance information for these systems are presented in Figure
5 and Table 3, respectively. RPs are plotted in Figure 5 as
the normalized retained concentration of cells in the sand
(S/C0) as a function of the distance from the column inlet.
Retention parameters (katt, kdet, and Smax) were estimatedby nonlinear least squares optimization to the experimental
data (Table 2).
[35] Less breakthrough was observed for both ﬁne sandand coarse sand as the IS increased (Figure 4). The efﬂuent
recovery rate for E. coli D21g decreased from 103% to 2%
in the ﬁne sand as the IS increased from 1 to 100 mM (Table 3). A similar trend occurred in the coarse sand, butchanges in IS had a much less signiﬁcant inﬂuence on thetransport of E. coli D21g in coarse than in ﬁne sand (Table2). For example, when the IS ¼ 100 mM, the recovery in
the efﬂuent was equal to 2% and 92% in the ﬁne and coarse
sands, respectively. Similar E. coli D21g transport experiments in the coarse sand were not conducted at IS ¼ 1 and5 mM because almost complete breakthrough occurred at
IS ¼ 20 and 100 mM. The RPs for E. coli D21g in the ﬁne
and coarse sand (Figure 5) followed complimentary trends
to the BTCs shown in Figure 4, with increasing cell retention with IS and in the ﬁner sand. The RPs tended todecrease exponentially with depth as predicted by the ﬁltration theory [Yao et al., 1971].
[36] Simulations provided a good description of the E.
coli D21g BTC and RP data (R2 > 0.95 in Table 2), including the blocking behavior at intermediate IS conditions.

Figure 5. Observed and simulated (model) normalized
solid phase concentrations (S/C0) of E. coli D21g as a function of depth in homogeneous (top) ﬁne-sand and (bottom)
coarse-sand columns at selected solution IS conditions.
Parameter values are given in Table 2.

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WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

Figure 6. Observed and simulated (model) normalized
efﬂuent concentrations (C/C0) of E. coli D21g as a functionof time in heterogeneous columns at selected solution IS
conditions. Parameter values are given in Tables 2 and 4.
recovery in the ﬁne sand decreased from around 100% at
IS ¼ 1 mM to around 87% at IS ¼ 100 mM (Table 3). Thisobservation suggests that a small amount (<13%) of E. coli
D21g was irreversibly retained (primary minimum) in theﬁne sand at IS ¼ 100 mM. Conversely, mass balance information indicates that E. coli D21g reversibly interacted in asecondary minimum (>95%) at lower IS and in the coarse
sand.
[39] Figure 6 presents observed and simulated BTCs forE. coli D21g in the heterogeneous columns when the solution IS equaled 1, 5, 20, and 100 mM. Corresponding massbalance information for these systems is presented in Table
5. Recall that the ﬁrst pulse in the BTC is associated withtransport through the lens, whereas the second pulse is controlled by the matrix. When the IS ¼ 1 mM the efﬂuent recovery of E. coli D21g was 96% (Table 5), and the BTCs
for E. coli D21g (Figure 6) and Br (Figure 3) were very
similar. However, the matrix breakthrough time for E. coli
D21g was slightly earlier than for Br (21.5 compared to23.0 min). This suggests that size exclusion may increases
the transport velocity of E. coli D21g by constraining cells
to faster ﬂow domains and larger pore networks than Br[Fontes et al., 1991; Ryan and Elimelech, 1996; Morley etal., 1998; Ginn, 2002].

[41] Similar to homogeneous columns (Figure 4), the recovery of E. coli D21g in the efﬂuent of the heterogeneouscolumns decreased from 96% to 13% (Table 5) as the solution IS increased from 1 to 100 mM (Figure 6). This trend
is consistent with data presented by Fontes et al. 1991. The
peak value of C/C0 in the ﬁrst pulse of the BTC for the lensdecreased over a narrow range from 25% to 13% as the IS
increased from 1 to 100 mM. In contrast, the peak value of
C/C0 in the second pulse of the BTC for the matrix
decreased over a larger range from 77% to almost 0% as
the IS increased from 1 to 100 mM. The contribution ofpreferential ﬂow for the overall transport of E. coli D21g
increased from 26% to 99% as IS changed from 1 to 100
mM (Table 4). This shows an interesting phenomenon thathas not been reported by other researchers. Increasing thesolution IS decreased the overall transport but increased therelative importance of preferential ﬂow.
[42] Figure 7 shows the RPs for E. coli D21g in the lens,in the matrix adjacent to the lens, and in the bulk matrix
sand when the IS ¼ 100 mM. Similar to the homogeneouscolumn experiments, greater amounts of E. coli D21g
retention occurred in the matrix than the lens. Furthermore,
the RPs in the lens and bulk matrix sands were quite similar
to those observed in the corresponding homogeneouscoarse- and ﬁne-sand column experiments (Figure 5),
respectively. The RPs of the matrix in the vicinity of lensprovided strong evidence for the interaction between thelens and matrix sands. Simulation results presented byMorley et al. 1998 are consistent with this ﬁnding. In particular, retained concentrations of E. coli D21g in the matrix were higher next to the lens than in the bulk matrix at
all depths. Deviation in the amount of E. coli D21g retention in the matrix adjacent to the lens and the bulk matrix
was especially signiﬁcant at depths greater than 6 cm.
These observations demonstrate signiﬁcant amounts of celltransport from the lens to the surrounding matrix andenhanced cell retention in the matrix than the lens.
[43] The transport and retention of E. coli D21g in theheterogeneous columns were again simulated using HYDRUS. Parameters values used in these simulations wereobtained from corresponding Br data and E. coli D21g datain the homogeneous columns (Table 2). Longitudinal dispersivities of coarse and ﬁne sands were set as the averagevalues obtained from homogeneous experiments (0.55 cm
and 0.12 cm) for all columns. Transverse dispersivity was
set to be one tenth of the longitudinal dispersivity. The
simulated BTCs (Figure 6) matched with the observed

Table 5. Experimental Mass Balance Information (Efﬂuent During Phases 1 and 2, With DI Flush During Phase 3, and Total) for theHeterogeneous Column Experiments With E. coli D21g and ’X174 Under Different Solution IS Conditionsa
Recovery (%)
R2

E. coli D21g

’X174

IS
(mM)

Br

E. coli
D21g

’X174

Effluent

DI

Total

Effluent

DI

Total

1
5
20
100

0.92
0.98
0.96
0.94

0.98
0.71
0.9
0.88

0.85
ND
ND
0.95

96
61
32
13

ND
32
61
68

96
93
93
81

98
ND
ND
50

ND
ND
ND
2

98
ND
ND
52

a

The goodness of the model ﬁt is also quantiﬁed by the coefﬁcient of linear regression (R2) on breakthrough data using model parameters in Tables 2
and 4. ND, not determined.

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WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

Figure 8. Semilog plots of observed normalized efﬂuent
concentrations (C/C0) of E. coli D21g as a function of timein heterogeneous columns at selected solution IS conditions
including phase 3.

Figure 7. Observed and simulated (model) normalized
solid phase concentrations (S/C0) of E. coli D21g as (top) afunction of depth in heterogeneous column at three different locations (lens, matrix in the vicinity of lens, and matrix) and (bottom) simulated spatial distribution of E. coli
D21g in HYDRUS at solution IS ¼ 100 mM. [Color ﬁgure
can be viewed in the online issue, which is available atwileyonlinelibrary.com.]
BTCs reasonably well for all IS conditions (R2 > 0.71 in
Table 5). The simulations also provided a reasonable
description of the measured RPs in the heterogeneous column (Figure 7). No signiﬁcant water ﬂow was observed at
the interface of the lens and matrix in the simulation. Masstransfer at the lens-matrix interface was not very sensitive
to the longitudinal dispersivity but was controlled by thetransverse dispersivity and especially the concentration
gradient.
[44] Additional experiments were conducted to better
understand the signiﬁcance of cell retention at the lensmatrix interface. In particular, after completion of phases 1
and 2 the heterogeneous columns were eluted with DI
water (phase 3) to study cell release when the secondary
minimum was eliminated. The release process was not
simulated because of the coupling between cell retention
parameters and variable IS. Figure 8 presents the release
curves for E. coli D21g during phase 3. Similar to Figure 6,
early breakthrough from the lens was observed just afterswitching to DI water. Release curves in homogeneouscolumn experiments typically produce a sharp spike[Bradford et al., 2007; Torkzaban et al., 2010a]. In contrast, the ﬁrst pulse of the release curve in the heterogeneous columns was more gradual and of a longer duration.
This observation supports the above conclusion about theinteraction and mass transfer at the lens-matrix interface. A
second sharp spike with extremely high cell concentrations
came out from the matrix at about 22 min (consistent withthe breakthrough time for the matrix). Greater release wasobtained by ﬂushing with DI water in systems with higher
IS and greater cell retention during phases 1 and 2. The
overall recovery rates of E. coli D21g (breakthrough andrelease) are provide in Table 5. Similar to the homogeneouscolumns, the overall recovery was lower than 100% anddecreased with increasing IS.
4.3. Coliphage uX174[45] Figure 9 presents observed and simulated BTCs for’X174 in homogeneous ﬁne sand when the IS equaled 1
and 100 mM. In contrast to E. coli D21g, the BTCs for’X174 were slightly retarded, and the amount of retardation increased with IS. Similar behavior has been observedfor other nanoparticles [Torkzaban et al., 2010b]. The
recovered ’X174 in the column efﬂuent was equal to 69%
and 36% when the IS was 1 and 100 mM, respectively. The
two-site kinetic model was selected for ’X174 when
IS ¼ 100 mM for better performance. The transport simulation provided a good description of this data (R2 > 0.96).

Figure 9. Observed and simulated (model) normalized
efﬂuent concentrations (C/C0) of ’X174 as a function of
PV in homogeneous ﬁne-sand columns at solution IS ¼ 1
and 100 mM. Parameter values are given in Table 2.

2432

WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

Figure 10. Observed and simulated (model) normalized
efﬂuent concentrations (C/C0) of ’X174 as a function oftime in heterogeneous columns at solution IS ¼ 1 and 100
mM. Parameter values are given in Tables 2 and 4. For the
IS ¼ 100 mM experiment the values of Smax were optimized
to 0.04 and 0.15 for the two sites.
The parameter kdet was not needed to describe this data on
a nonlog scale, suggesting mainly irreversible retention.
Similarly, very small amounts of ’X174 were recoveredfrom the sand (<2%). The RPs for ’X174 could therefore
not be accurately determined. The effect of microscopic
heterogeneities on colloid retention has been reported toincrease, as the colloid size decreases [Duffadar and Davis,
2008]. These observations suggest that ’X174 was subject
to irreversible, primary minimum attachment to a larger
extent than E. coli D21g.
[46] Retention of ’X174 was less than E. coli D21g
when the IS ¼ 100 mM, but greater than E. coli D21g whenthe IS ¼ 1 mM. These differences in the retention dependon the values of katt and Smax given in Table 2. Filtration
theory [Yao et al., 1971] predicts that katt is proportional tothe product of the colloid collector efﬁciency () and the
sticking efﬁciency (). The value of  was predicted[Tufenkji and Elimelech, 2003] to be much larger for’X174 ( ¼ 0.28) than E. coli D21g ( ¼ 0.02). Conversely,  and Smax depend in a complex manner on thedepth of the secondary minimum and hydrodynamic forces[Shen et al., 2010], surface macromolecules [Kim et al.,2009], and physical and chemical heterogeneities [Bendersky and Davis, 2011]. Furthermore, the inﬂuence of the secondary minimum and chemical heterogeneity changes with
IS and colloid size.
[47] Transport experiments for ’X174 in the ﬁne sandwhen the IS ¼ 5 and 20 mM were not conducted because
the expected differences in the BTCs (intermediate to 1 and100 mM data shown in Figure 9) were within the analytic
error. Similarly, ’X174 retention in the coarse sand at IS
equal to 1 and 100 mM was initially assumed to be zero
because the E. coli D21g data (Figure 4) exhibited muchless retention in the coarse than the ﬁne sand for given IS
conditions (Table 3). The validity of this assumption will
be veriﬁed in the heterogeneous column experiments discussed later.
[48] Figure 10 presents BTCs for ’X174 in heterogeneous columns when the IS equaled 1 and 100 mM. Chemistry had a very similar inﬂuence on the transport of ’X174
as for E. coli D21g (Figure 6). In particular, the overall

transport decreased with an increase in the IS, but the contribution of preferential ﬂow increased. The recovery
changed from 98% to 50% when the IS was 1 and 100 mM,
respectively. The peak value of C/C0 in the lens (ﬁrst) andmatrix (second) pulses changed from 28% to 18% and 99%
to 58% when the IS was 1 and 100 mM, respectively. In
contrast to E. coli D21g, higher values of C/C0 wereobserved for ’X174 at comparable IS conditions, and thecontribution of preferential ﬂow was much lower (26%–
34% with an increase in IS). This observation suggests that
’X174 is less inﬂuenced by physical heterogeneity than E.
coli D21g and thereby behaves more similar to Br becauseof its smaller size.
[49] The simulation could not provide a good descriptionof the ’X174 data in the heterogeneous column when usingparameter values from the homogeneous ﬁne-sand column
experiments. Large variations in C0 and the degree of sandcleaning could cause signiﬁcant difference in Smax for theﬁne sand in the homogeneous and heterogeneous column
experiments. Therefore, we used the same values of kattfrom the homogeneous ﬁne-sand column and optimized
separate values of Smax to the heterogeneous matrix data.
Values of R2 were greater than 0.85 with the optimized
Smax.
[50] In contrast to E. coli D21g (Figure 8), only small
amounts of ’X174 were released from the lens and matrix
during phase 3 when the IS was reduced from 100 mM to
DI water. Similar to the minimal recovery of ’X174 fromthe sand, this observation reﬂects that ’X174 was mainly
irreversibly retained in a primary minimum.
4.4. Limitations of the Mathematical Model[51] It needs to be emphasized that the mathematicalmodel used here to describe the transport and fate of microorganisms has its limitations with respect to the complexityof processes involved in the transport of microorganisms insoils, as well as with respect to modeling preferential transport. The model considers attachment/detachment as ﬁrstorder processes and neglects other processes such as straining and/or size exclusion. Additionally, only Langmuiriantype blocking was considered, while other blocking models
could have been used as well. Microbes were treated ashaving a uniform size distribution, and aggregation andother biological activities were not considered in themodel. Note that the analysis of the effects of these various
processes can be found elsewhere (e.g., Bradford et al.
2003 for straining and size exclusion; Bradford et al.
2006a for different blocking mechanisms; Bradford et al.
2005 for ripening behavior; Bradford et al. 2006b for cellaggregation ; Gargiulo et al. 2006 for cell growth; andBradford et al. 2006c for virus inactivation).
[52] It should also be stressed that application of our 2-D
model for preferential ﬂow in natural systems is difﬁcult
because it requires detailed information on the spatial distribution of soil hydraulic properties which produce preferential ﬂow paths. To overcome this limitation, ﬂow andtransport in preferential pathways are sometimes describedusing a dual permeability model that divides ﬂow into fast
 ° nek and vanand slow regimes [e.g., Jarvis, 2007; Simu
Genuchten, 2008]. However, this approach is more common for variably saturated conditions than for fully saturated conditions.

2433

WANG ET AL.: TRANSPORT OF MICROORGANISMS WITH PREFERENTIAL FLOW

5.

Summary and Conclusions[53] DLVO calculations indicate that the depth of thesecondary minimum increased and that the height of theenergy barrier deceased with an increase in IS. Consequently, retention of E. coli D21g and ’X174 wasenhanced as the solution IS increased in both homogeneousand heterogeneous sand columns. This effect of IS was
found to be especially signiﬁcant for E. coli D21g in theﬁner sand. In general, E. coli D21g was mainly reversibly
retained in the sand as a result of interactions in a secondary minima, whereas ’X174 was largely irreversible
retained as a result of primary minima interactions. HYDRUS proved to be a powerful tool to simulate the transportand deposition behavior of E. coli D21g and ’X174 in bothhomogenous and preferential ﬂow systems under the different IS conditions.
[54] The relative amount of preferential transport of
microbes through the lens increased with IS. This indicatesthat preferential transport of microbes became more important under conditions of higher overall retention. Cell RPs
demonstrated signiﬁcant amounts of mass transfer at theinterface between the lens and matrix. Cell release with areduction in solution IS exhibited multipeaked breakthrough in the preferential ﬂow systems. The amount ofcell release was sensitive to the initial distribution ofretained cells, especially at the lens-matrix interface. Accurate predictions of microorganism transport and fate inﬁelds with preferential ﬂow and transients in solution
chemistry require information on BTCs, RPs, and release
behavior.
[55] The artiﬁcial macropore method proved to be a reliable and repeatable way to study transport processes in systems with preferential ﬂow. Further study will be done toinvestigate the inﬂuence of macropore geometry with theartiﬁcial macropore method, which can help improve ourunderstanding of natural environments. Numerical models
still need to be improved to better characterize mass transfer at the matrix-lens interface and the release of microorganisms with transients in solution IS. Furthermore,alternative mathematical models are warranted to predict
transport behavior in more complicated systems and atlarger scales in order to connect the studies in idealized
artiﬁcial macropore system with the real processes innatural systems.
[56] Acknowledgments. We would like to thank Lorena Altamirano
for her help in conducting some of the experimental research presented
herein. This research was supported in part by the USDA, ARS, NP 214,
and Multistate Research Funded project W-2188. The USDA is an equal
opportunity provider and employer.

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