Article
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Release of E. coli D21g with Transients in Water Content
Yusong Wang,*,† Scott A. Bradford,‡ and Jiri Simunek††

Department of Environmental Sciences, University of California, Riverside, California 92521, United States
U.S. Salinity Laboratory, USDA, ARS, Riverside, California 92521, United States‡

S Supporting Information*

ABSTRACT: Transients in water content are well-known tomobilize microorganisms that are retained in the vadose zone.
However, there is no consensus on the relative importance ofdrainage and imbibition events on microorganism release. To
overcome this limitation, we have systematically studied therelease of Escherichia coli D21g during cycles of drainage andimbibition under various solution chemistry and initialconditions. Results from these column studies revealed theinﬂuence of imbibition and drainage on D21g release. In
particular, imbibition eﬃciently released cells from the air−water interface (AWI) that were initially retained under steadystate unsaturated conditions by expansion of water ﬁlms and destruction of the AWI. Conversely, signiﬁcant release and transportof cells during drainage only occurred below a critical water saturation (water ﬁlm thickness). In this case, a fraction of the cells
that were initially retained on the solid−water interface (SWI) partitioned into the mobile aqueous phase and the AWI as thereceding water ﬁlm thickness decreased during drainage. The eﬃciency of cell release from the SWI during drainage was muchless than for the AWI during imbibition. Cycles of drainage and imbibition removed cells from the SWI and the AWI,
respectively. However, the peak concentration and amount of cells that were released increased with the number of retained cells
and the amount of drainage and imbibition, and decreased with the number of drainage and imbibition cycles. Release of cells
during drainage and imbibition was found to be more pronounced in the presence of a weak secondary minimum when the ionicstrength (IS) was 5 mM NaCl. Increases in the solution IS decreased the inﬂuence of water transients on release, especiallyduring drainage. Complete recovery of the retained cells could be achieved using both IS reduction and cycles of drainage andimbibition, even when the cells were retained under favorable attachment conditions. In general, cell release was more
pronounced with transients in water content than transients in IS when the IS ≥ 5 mM.

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controlled process.4,40−44 Conversely, transients in watercontent and solution chemistry can rapidly mobilize retained
microorganisms and/or colloids.2−8,12−15,34,40,45−49
Transients in water content can promote the release ofretained microorganisms by several incompletely understoodmechanisms.10,11,13,40,48−55 The AWI increases and the waterﬁlm thickness decreases with a decrease in the water saturation
during drainage.33,56−58 Microbes on the SWI may partition
into the AWI when the water ﬁlm thickness is smaller than the
microbe radius.9,33,56 A moving AWI can potentially liberate
microbes from the SWI during drainage.50,59 The AWI is
destroyed, water ﬁlms expand, and the pore water velocity
increases during water imbibition.34,40 Microbes associated with
the AWI, AWS contact line, and in thin water ﬁlms may bereleased by these processes during imbibition.10,11,13,34,40,49,50,55
Larger microbes are expected to be more eﬃciently released bydrainage and imbibition than smaller microbes.52,60,61

INTRODUCTION
The vadose zone is an important barrier to protect groundwaterfrom pathogenic microorganisms and other colloids that canpose a threat to the public health.1 Transients in water content
and solution chemistry triggered by inﬁltration and drainage
events, evapotranspiration, and water table ﬂuctuations are very
common in the vadose zone. Much evidence exist thattransients in water content and solution chemistry may
mobilize retained microorganisms and can promote theirtransport through the vadose zone.2−15 Consequently, an
understanding of the release of microorganisms with transientsin water content and solution chemistry is necessary to protect
water resources and public health.
Microorganisms may be attached to the solid−water interface(SWI) or strained in small pores spaces that occur near grain−
grain contacts and larger-scale surface roughness locations
under saturated conditions.16−27 In addition, microorganisms
may also be attached to the air−water interface (AWI) or
strained at the air−water solid (AWS) contact line and in thinwater ﬁlms under unsaturated conditions.28−40 Release ofretained microorganisms is typically low under steady water
ﬂow, water saturation, and solution chemistry conditions.11,40
In this case, release is considered to be a slow, diﬀusion© 2014 American Chemical Society

Received: April 23, 2014
Revised: July 18, 2014
Accepted: July 20, 2014
Published: July 21, 2014
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Articlebackground interference from clay particles, the sand wastreated by a salt cleaning procedure described by Bradford andKim.14 Electrolyte solutions for the column experiments
consisted of autoclaved deionized (DI) water (pH 5.8) with
the IS adjusted to 0, 1, 2.5, 5, 10, 20, and 100 mM using NaCl
to create a range of adhesive conditions between the bacteria
and sand.
Escherichia coli D21g. Escherichia coli D21g was employedin the transport experiments discussed below. It is a Gramnegative, nonmotile bacterial strain that produces minimal
amounts of lipopolysaccharides and extra-cellular polymeric
substances.67 The surface properties of D21g have been wellcharacterized in terms of surface charge, acidity, and hydrophobicity.68,69 We selected D21g for our studies because it has
relatively simple surface properties that helped us to isolate theinﬂuence of water transients on release.
The culture and harvest procedures used in this study havebeen described by Wang et al.69 In brief, D21g was cultured
overnight (12−18 h) at 37 °C in Luria−Bertani broth (LBBroth, Fisher Scientiﬁc, Fair Lawn, NJ) containing 30 mg/L
gentamycin (Sigma, St. Louis, MO), transferred onto a LB
media plate containing 30 mg/L gentamycin, and the plates
were cultured overnight (12−18h) at 37 °C. The colonies were
harvested into sterile water, and then the bacteria suspension
was centrifuged and resuspended two times to remove all tracesof the growth medium. A fresh cell suspension at the desired
electrolyte solution was prepared right before the start of eachexperiment. The concentrations of D21g in inﬂuent (Co ≈ 108
cells mL−1) and eﬄuent samples were determined using a linear
correlation between spectrophotometer (Unico UV-200,
United Products & Instruments, Dayton, NJ) readings at 600
nm and known cell concentration. The detection limit wasapproximately 1% of Co when using the spectrophotometer,whereas the standard deviation of the conventional spread
plating method was 14.3% of Co.69
The Supporting Information (SI) contains details pertaining
to the calculation of interaction energies between D21g and the
SWI for the various IS conditions (SI S1). Results from thesecalculations are summarized in SI Table S1. Information is alsopresented in SI S1 about the expected interaction of D21g with
the AWI.
Column Experiments. An illustration of the experimental
setup that was used in the transport and release experiments isgiven in SI Figure S1. Column experiments were conducted in aplexiglass (acrylic) column that was 22 cm long and had aninside diameter of 13.2 cm. E. coli D21g suspensions or eluting
solution, which were mixed continuously with a magnetic
stirrer, were delivered to the top of the column at a steady ﬂow
rate using a rain simulator connected to a peristaltic pump
(Barnant Company, Barrington, Illinois). The capillary pressure
head in the column was measured with three miniature stainlesssteel tensiometers (Chemiquip Products Co.,West New York,NJ) inserted at 5, 10, and 15 cm from the upper sand surface.
Data from the tensiometers were registered using a CR7
datalogger (Campbell Scientiﬁc, Logan, UT). Stainless steel
tubes were located on the opposite side of the column to thetensiometers to facilitate air entry and exit. These tubes werecapped during saturated conditions. A polyester membrane(Saatiﬁl PES 18/13) with an 18 μm nominal pore size wasplaced at the bottom of the column and connected to a hanging
water column (tube) to control the bottom boundary pressure.
The membrane has a bubbling pressure head of about 130 cmH2O. Neither the column body nor the nylon membrane

There presently is no consensus on the relative importanceof drainage and imbibition events on colloid or microorganism
release.11,13,50,51,62 Some studies have attributed release only to
imbibition,49 whereas others have found that release occurred
during both drainage and imbibition.11,13,40,50 In the latter case,the amount of release is sometimes observed to be more
pronounced during drainage11 and other times imbibition.13
These conﬂicting results suggest a strong sensitivity of
microorganism release to the initial conditions and saturation
dynamics that has not yet been fully resolved. For example, theaccessibility of microorganisms retained on the SWI to the AWI
is expected to increase with decreasing water saturation.33
Hence, the amount of release during imbibition may therefore
depend on how much of the pore space was previously drained.
If retained microbes on the SWI are preferentially found in thesmallest regions of the pore space, then little interaction orrelease of microbes with the AWI is expected at higher water
saturations. As noted in the preceding paragraph, release of
microbes retained on the AWI, AWS contact line, and in thinwater ﬁlms under unsaturated conditions is expected to be very
diﬀerent from microbes attached or strained on the SWI. The
eﬃciency of release from the SWI and AWI is therefore
expected to be very diﬀerent, but still is not qualiﬁed.
Several researchers have reported that release duringdrainage and imbibition is sensitive to the chemistry of theaqueous and solid phases,4,11,13,55,59,62−64 as well as the surface
properties of the colloids.64,65 These chemical properties
inﬂuence the strength of the adhesive interaction between the
microbe and the SWI and/or AWI. Enhanced microbe
retention and release is expected when the adhesive interaction
is stronger and weaker, respectively. For example, larger
amounts of colloid release have been observed during transientwater content conditions when the solution ionic strength (IS)and the magnitude of the secondary minimum werelower.11,55,59,62−64 However, the combined inﬂuence of initialconditions (e.g., amount of retained microbes) and interaction
strength in determining release during drainage and imbibition
has not yet been determined. In addition, chemical
perturbations, such as a reduction in IS, can induce releasefrom the SWI and AWI by shifting the net interaction forcesfrom attractive to repulsive.3,4,8,15,66 The relative importance oftransients in water content and solution chemistry on microberelease still has not been quantiﬁed.
The overall objective of this research is to improve ourunderstanding of microbial release during transient watercontent conditions. In particular, we systematically study therelease of Escherichia coli D21g from the AWI during
imbibition, from the SWI during drainage, and from the SWI
and AWI during cycles of drainage and imbibition undervarious solution chemistry conditions. Other experiments wereconducted to investigate the eﬃciency of cell release from the
SWI and AWI for various amounts of drainage and imbibition,and the combined inﬂuence of transients in saturation andsolution IS on release. Results demonstrate that considerationof saturation dynamics, initial conditions, and release eﬃciency
can explain many of the reported discrepancies in colloid
release behavior during drainage and imbibition in theliterature.

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MATERIALS AND METHODS
Porous Media and Electrolyte Solutions. Ottawa(quartz) sand with a median grain size (d50) of 120 μm wasused in the column experiments. In order to eliminate any
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Articleretained the cells.69 Eﬄuent samples were collected from thehanging water column using a fraction collector and monitored
for E. coli D21g concentrations using a spectrophotometer as
described above. An electronic balance (Sartorius Master Series,
LP Models, Germany) was used to determine the total waterbalance in the column over time. The average water saturation
was calculated from the initial balance reading under saturated
conditions and the change in water storage.
Ottawa sand was wet packed into the column to a height of
20 cm. The porosity of the column was around 0.34, and thepore volume was around 930 mL under saturated conditions.
Around 2 pore volumes of a selected NaCl solution was ﬂushed
through the column to achieve steady state ﬂow (saturated/unsaturated), and to allow the sand to equilibrate with thesolution. Saturated and unsaturated transport experiments were
initiated by injecting about two pore volumes of cell suspension(Phase 1) and then eluting NaCl solution with the same IS(Phase 2) to the column top at a steady-state ﬂow rate (SITable S2). The column was ﬂushed with eluting NaCl solution
until no signiﬁcant concentration of bacteria was detected.
Saturated conditions were maintained in the column by keeping
a ﬁne layer of ponding at the top boundary and 0 pressure headat the bottom boundary. Unsaturated, steady-state water ﬂow
conditions at selected water saturations were achieved by
reducing the inﬂow rate of water at the column top and by
simultaneously increasing the suction at the bottom of thecolumn. The latter was done by lowering the hanging watercolumn until the readings of the tip tensiometers showed thesame values, that is, a unit hydraulic head gradient wasachieved.
Various drainage and/or imbibition conditions were
implemented in the column (Phase 3) to study the release
behavior of E. coli D21g (SI Table S2). Drainage was conductedwith no ﬂow at the top boundary while maintaining a negative
pressure at the bottom. By adjusting the height of the hanging
water column, the sand column was drained to diﬀerent levels.
Imbibition was initiated by increasing the ﬂow rate at the top of
the column and/or by elevating the bottom boundary pressure.
The imbibition process was considered ﬁnished when steadystate ﬂow was reached and the concentration in the eﬄuent
went down back to background levels.
Additional details about the column packing (SI S2),
replication of transport and release experiments (SI S3, Table
S2, and Figure S2−S3), and a summary of experimental
conditions and mass balance information (Table S2) are givenin the SI.

Figure 1. An example of cell release from the AWI during imbibition
when the IS = 5 mM NaCl (Phases 1, 2, and 3). Initial deposition
occurred under unsaturated conditions (around 0.64 water saturation),and then the sand was saturated using two imbibition sequences(denoted by I1 and I2). Also included in the ﬁgure is a breakthroughcurve for D21g under saturated conditions. See SI Table S2 forexperimental details and mass balance information. Legends: blue
diamond as breakthrough and release concentrations for unsaturated
conditions, red triangle as breakthrough concentration for saturated
conditions, blue line as average saturation for unsaturated conditions,
red line as average saturation for saturated conditions.

previously been attributed to destruction of the AWI and
expansion of water ﬁlms which liberate cells that were retainedat the AWI, triple point, or in thin water ﬁlms. Interestingly, theamount of cell release during the two imbibition steps (21.9%)
was similar to the enhancement of cell retention caused byunsaturated (in comparison with saturated) conditions duringphases 1 and 2 (22.7%). This suggests that the extra cellretention under unsaturated conditions can be released by
imbibition, and that this cell release was very eﬃcient.
Cell Release From the SWI During Drainage. Figure 2
presents an example of D21g release from the SWI duringdrainage when the IS=5 mM NaCl. In this case, phases 1 and 2
occurred under saturated conditions, and then the sand was
drained during phase 3 by lowering the bottom boundary
condition to −120 cm. No cell release was observed when the■

RESULTS AND DISCUSSION
Cell Release From the AWI. Figure 1 shows breakthroughand release curves for D21g as a function of eﬄuent volume
when the solution IS = 5 mM NaCl. Breakthrough curves(BTCs) are shown for both saturated (Sw = 1) and unsaturated(Sw = 0.64) conditions. Enhanced retention was observed whenretention occurred under unsaturated (80.7%) than saturated(58%) conditions (SI Table S2). This ﬁnding is consistent withthe reported literature and has primarily been attributed toattachment to the AWI, ﬁlm straining, or retention at the air−water−solid triple point. 28−39,64 Cell release from theunsaturated sand was initiated during phase 3 using two
imbibition steps that are shown in the ﬁgure. Step increases inwater saturation each produced a cell release pulse with similar
peak concentrations that were equal to the maximum value ofthe unsaturated BTC. Cell release during imbibition has

Figure 2. An example of D21g release from the SWI during drainage
when the IS = 5 mM NaCl (Phases 1, 2, and 3). Phases 1 and 2 wereconducted under saturated conditions, and then the sand was drained
during phase 3 by lowering the bottom boundary condition. See SI
Table S2 for experimental details and mass balance information.
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Articlewater saturation was greater than 0.6. Conversely, signiﬁcantamounts of cell release occurred when the water saturation wasless than 0.6. This observation suggests that release duringdrainage was triggered when a critical saturation was reached.
This behavior was further conﬁrmed by replicate information in
the SI (Table S2 and Figure S3). The critical saturation concept
may help explain the divergence of ﬁndings in the literature on
the reported eﬀects of drainage on colloid release.11,13,51 It
should be noted that a critical water saturation is likely to be
media speciﬁc, but it is theoretically related to capillary pressure
and/or water ﬁlm thickness terms that are more general.34,51
However, it is possible that the critical water saturation will also
depend on the solution chemistry and properties of the
microbe, and additional studies are needed to fully resolve thisissue.
In addition, a drainage release pulse was observed in Figure 2
that initially increases and then decreases with increasing
amounts of drained water. The above observations are notconsistent with a constant diﬀusion controlled release process,which predicts that the eﬄuent concentration would
continuously increase with decreasing water saturation becauseof the smaller water volume and lower ﬂow rates. Rather, theresults suggest that drainage below a critical saturation level (orﬁlm thickness) can remove a fraction of the cells from the SWI,
and transport these cells with the aqueous phase. Microbes on
the SWI experience diﬀerent forces and torques as the waterﬁlm thickness decreases during drainage.70,71 For example,
microbes will be subjected to strong capillary forces and can bedirectly incorporated into the AWI when the water ﬁlm
thickness is smaller than the microbe radius.9,34,50 Eventually,
the microbes will begin to experience interaction energies
arising from both the SWI and AWI, which will alter the force
and torque balance at a particular location, and may mobilize
some of the microbes from the SWI to the aqueous phase and/or cause a fraction of the cells to partition into the AWI. The
rate of transport of a released cell pulse during drainage will bevery slow because the water velocity rapidly decreases withdecreasing water content.72
Cell Release by Drainage and Imbibition. Figure 3
presents examples of D21g release during a drainage andimbibition cycle for diﬀerent amounts of initial cell retention
when the IS = 5 mM NaCl. Short and long input pulse
durations (1.5 and 3.1 PVs) were employed during phase 1 toobtain diﬀerent initial conditions. Phases 1 and 2 were again
conducted under saturated conditions, and then the sand was
drained and imbibed during phase 3 by adjusting the boundary
conditions at the bottom (constant −90 cm) and top (waterﬂux was changed from 0 to 23.5 mL min−1) of the column. Allof the retained cells were initially on the SWI. Release of cells
from the SWI was initiated by water drainage. As noted in thesection above, this occurred due to the presence of thinreceding water ﬁlms that partition some of the cells from the
SWI to the aqueous phase and/or the AWI. The relative
importance of the drainage and imbibition release pulses
depended on the initial conditions during phases 1 and 2. In
particular, the release pulse became more pronounced withlarger input pulse durations because greater amounts of cells
were initially retained on the SWI. Cells that are directly
associated with the AWI will be released during imbibition
when the AWI is destroyed (Figure 1). Imbibition will also
produce an expansion of water ﬁlms (and ﬂow rates) that will
enhance the mobility of cells that were released from the SWI
during drainage. Hence, imbibition may complete the release

Figure 3. An example of D21g release during a drainage andimbibition cycle for diﬀerent initial conditions when the IS = 5 mM
NaCl (Phases 1, 2, and 3). Short and long input pulse durations (1.5
and 3.1 PVs) were employed during phase 1 to obtain diﬀerent initialconditions. Phases 1 and 2 were conducted under saturated conditions,and then the sand was drainage and imbibed during phase 3 byadjusting the boundary conditions at the bottom and top of thecolumn. See SI Table S2 for experimental details and mass balance
information. Legends: blue diamond as release concentration for longdeposition, red triangle as release concentration for short deposition,
blue line as average saturation for long deposition, red line as average
saturation for short deposition.

pulse initiated during drainage. Higher release pulses during
imbibition than drainage reﬂect diﬀerences in the eﬃciency of
release from the SWI and AWI that will be discussed below.
Figure 4 presents an example of D21g release with repeatedcycles of similar amounts of water drainage and imbibition

Figure 4. An example of D21g release with repeated cycles of similar
amounts of water drainage and imbibition when the solution IS = 5mM NaCl (Phases 1, 2, and 3). Phases 1 and 2 were conducted undersaturated conditions, and then the sand was repeatedly drained andimbibed during phase 3 by adjusting the boundary conditions at thebottom and top of the column. D# and I# denote the drainage andimbibition number (#), respectively. See SI Table S2 for experimental
details and mass balance information.

when the solution IS=5 mM NaCl. Phases 1 and 2 wereconducted under saturated conditions, and then the sand was
repeatedly drained and imbibed during phase 3 by adjusting theboundary conditions at the bottom (−90 cm) and top (waterﬂux was changed from 0 to 23.5, and then 41.2 mL min−1
during each cycle) of the column. In contrast to Figure 3, two
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Article(Figure 4). Apparently the amount of cell release was a functionof the lowest water saturation during drainage, with drainage tolower water saturations promoting greater amounts of cellrelease from the SWI. The water ﬁlm thickness decreases andthe air−water interfacial area increases as the water saturation
decreases.33,56−58 Both of these factors suggest that greater
amounts of cells will be incorporated into the AWI at lower
water saturations during drainage. Imbibition will subsequently
release these cells to the water phase because of destruction of
the AWI and expansion of water ﬁlms.
Cell Release For Diﬀerent IS Conditions. Cell releasewith drainage and imbibition was discussed above for a solution
IS of 5 mM NaCl. Solution IS eﬀects the height of the energy
barrier and the depth of the secondary minimum between
D21g and the SWI. In particular, the energy barrier decreases
and the depth of the secondary minimum increases for an
increase in solution IS (SI Table S1). It is therefore notsurprising that the solution IS has also been reported toinﬂuence the amount of colloid release from the SWI duringdrainage and imbibition.4,11,59,63,64 Repulsive electrostatic andvan der Waals interactions are expected between D21g and the
AWI for the various IS conditions (SI S1). Conversely,
microbes that partition into the AWI are subjected to strong
capillary forces.9,34,50 Additional studies were conducted tobetter understand the coupled eﬀects of IS on cell release fromthe SWI during drainage and imbibition.
Figure 6 presents examples of D21g release during drainage
and imbibition when the solution IS = 100, 10, 5, and 2.5 mM
NaCl during phases 1, 2, and 3. Phases 1 and 2 were conductedunder saturated conditions, and during phase 3 the sand wasﬁrst drained by lowering the bottom boundary condition to−90 cm, and then imbibed by changing the top ﬂux from 0 to
23.5 mL min−1. The amount of cell retention during phases 1
and 2 increased with the solution IS (99%, 97%, 62%, and 46%
when the IS equals 100, 10, 5, and 2.5 mM NaCl, respectively)
because of its eﬀect on the interaction energy (SI Table S1).
Conversely, the amount of cell release with drainage andimbibition did not simply increase with a decrease in IS. Rather
cell release during drainage and imbibition depended on boththe initial amount of cell retention during phases 1 and 2, andthe solution IS. When the IS = 100 mM, no signiﬁcant release
was observed for either drainage or imbibition due to the strong
secondary minimum interaction between the cells and the sandsurface (SI Table S1). At an IS = 10 mM NaCl, cell release was
not observed during drainage, but a small release pulse was seen
during imbibition. Conversely, signiﬁcant amounts of cellrelease were observed with both drainage and imbibition whenthe IS = 2.5 and 5 mM NaCl. This observation indicates thatdrainage and imbibition is eﬀective at releasing cells from the
SWI when they are interacting in a shallow secondary
minimum.11,55,59,62−64 The largest amount of cell releaseoccurred when the IS = 5 mM NaCl because greater amountsof cell retention initially occurred in 5 than 2.5 mM NaClduring phases 1 and 2. Similar to Figure 3, greater amounts ofcell release tended to occur during imbibition than drainage.
Diﬀerences in the arrival time of the release pulses during
imbibition likely reﬂect the inﬂuence of the initial distributionof retained cells and/or the strength of interaction with the
SWI (SI Table S1).
Figure 7 present an example of D21g release with transientsin solution IS and water content. Phases 1 and 2 wereconducted in 100 mM NaCl. Phase 3 was conducted bysuccessively lowering the solution IS from 100, to 20, to 5, to 0

imbibition steps were used to release the retained cells. Each
imbibition step produced a corresponding pulse of released
cells, but the second pulse was much smaller than the ﬁrst. The
ﬁrst and second imbibition steps both released cells bydestruction of the AWI and water ﬁlm expansion. However,cell release is expected to be more pronounced at lower water
saturations because the rate of change in the air−waterinterfacial area is greater56 and small amounts of water ﬁlm
expansion may be suﬃcient to release cells that were pinned by
the AWI.34 The total amount of colloid release has beenreported to increase with the amount of imbibition.48
It is interesting to note that one drainage and imbibition
cycle in Figure 4 did not remove all of the retained cells. Eachdrainage and imbibition cycle released additional cells.
However, the peak concentration and the amount of released
cells diminished as the number of drainage and imbibition
cycles increased. Similar to information presented in Figure 3,
this observation suggests that the eﬃciency of cell release fromthe SWI is low in comparison to the AWI (Figure 1). This
ineﬃciency is likely due to spatial variability in the adhesive
force and water ﬁlm thickness. In particular, the adhesive force
can vary as a result of chemical heterogeneity, surface
roughness, and diﬀerences in the strength of primary andsecondary minimum interactions.73 The water ﬁlm thickness
will also vary with the water saturation, the surface roughness,the pore-scale geometry, and the solid phase wettability.33,74
Figures 3 and 4 demonstrate that diﬀerences in initialconditions and release eﬃciencies from solid−water and air−water interfaces can help explain the wide variety of reported
transient release behavior under unsaturated conditions.11,13,34,40,48,51,59
Cell Release with Diﬀerent Amounts of Drainage.
Figure 5 presents an example of D21g release during three

Figure 5. Examples of D21g release (IS = 5 mM during phases 1, 2,
and 3) during three continuous drainage and imbibition cycles when
the column was successively drained to lower water saturations of 0.57,
0.36, and 0.27 by lowering the bottom boundary pressure. D# and I#
denote the drainage and imbibition number (#), respectively. See SI
Table S2 for experimental details and mass balance information.

continuous drainage and imbibition cycles when the column
was successively drained to lower water saturations of 0.57,
0.36, and 0.27 by lowering the bottom boundary pressure.
Similar peak concentrations and release amount were observedfor each drainage and imbibition cycle. In contrast, decreasing
amounts of cell release were observed for each drainage andimbibition cycle when draining to the same water saturation
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ArticleFigure 6. An example of D21g release during drainage (left) and imbibition (right) when the solution IS = 100, 10, 5, and 2.5 mM NaCl duringphases 1, 2, and 3. Phases 1 and 2 were conducted under saturated conditions, and then the sand was drained during phase 3 by lowering the bottom
boundary condition to −90 cm. The water ﬂux at the top of the column was 20.5 to 24.3 mL min−1 for imbibition events, and the ﬁnal saturation
ranged from 0.70 to 0.75. See SI Table S2 for experimental details and mass balance information.

Figure 7. An example of D21g release with transients in solution IS and water saturation. Phases 1and 2 were conducted in 100 mM NaCl. Phase 3
was conducted by successively lowering the solution IS from 100, to 20, to 5, to 0 mM NaCl and initiating drainage and imbibition cycle(s) as
indicated in the ﬁgure. D#, I#, and IS# denote the drainage, imbibition, and IS number (#), respectively. See SI Table S2 for experimental details andmass balance information.

as an eﬀective barrier to minimize the risks of microbial
contamination of groundwater. However, this belief does notconsider the confounding eﬀects of transients in water content
and solution chemistry that are ubiquitous in the vadose zone
as a result of inﬁltration, drainage, evapotranspiration, watertable ﬂuctuations, and/or changes in surface water levels. Data
presented in this manuscript demonstrate that D21g transportand release will be highly sensitive to transient conditions. In
particular, drainage and imbibition may both mobilize large
numbers of D21g, especially at low solution IS. The amount of
release strongly depends on the initial conditions (number andlocation of retained cells, the solution chemistry, and the
distribution of cells on the SWI and/or AWI) and thesaturation dynamics (the initial water saturation when the cells
were retained, and the amount of drainage and imbibition).
Release of cells from the AWI was much more eﬃcient thanfrom SWI. Consequently, greater amounts of microbe releaseand transport are expected during water imbibition whenmicrobes are added to the soil under unsaturated conditions. In
addition, drainage of soil to lower water contents will facilitate
the release of more microbes from the SWI during imbibition.
Both of these factors will increase the risks of microorganism

mM NaCl and initiating drainage and imbibition cycle(s) ateach IS as indicated in the ﬁgure. Cell release was observed
directly after switch to IS = 5 and 0 mM due to reduction and/or elimination of the secondary minimum (SI Table S1).
Release during drainage and imbibition for each step IS level
was very similar to that shown in Figure 6. In general, cellrelease by drainage and imbibition was greater than by areduction in solution IS when the IS ≥ 5 mM. After switching
to DI water, an additional 10% of the retained cell mass could
be recovered by drainage and imbibition. The total recovered
cell mass balance was close to 100%. Collectively, the results
indicate that step reductions in solution IS and/or multiple
drainage and imbibition cycles can mobilize all of the retained
cells, even when the initial conditions produced a deep
secondary minimum that was favorable for cell retention (SITables S1 and S2).
Environmental Implications. Most microbial transport
studies have been conducted under steady-state water content
and solution chemistry conditions. Results typically suggest
limited mobility of micoorganisms in the subsurface because ofretention to the SWI and/or AWI, especially under unsaturated
conditions. Consequently, the vadose zone is commonly viewed
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transport through the vadose zone to contaminate groundwater
resources. Additional studies and model development areneeded to extend the ﬁndings of this study to othermicroorganisms, soil types, and transient conditions that
occur in natural environments.

■

ASSOCIATED CONTENT

S Supporting Information*

This Supporting Information section contains details pertaining
to (i) calculation of interaction energies between D21g and the
SWI, and D21g and the AWI for the various IS conditions (S1);(ii) a summary of interaction energy results for D21g and theSWI (Table S1); (iii) column packing (S2) and an illustrationof the experimental setup (Figure S1); (iv) replication oftransport and release experiments (S3 and Figures S2, S3); and(v) a summary of experimental conditions and mass balance
information (Table S2). This material is available free of charge
via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author*Phone:951-823-9431; e-mail: ywang032@ucr.edu.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
This research was supported by the 214 Manure and ByproductUtilization Project of the USDA-ARS. Mention of trade names
and company names in this manuscript does not imply any
endorsement or preferential treatment by the USDA. We also
acknowledge the help of Teresa Clapp in conducting thesetransport and release experiments.

■

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