Environ. Sci. Technol. 2007, 41, 1265-1271

Transport and Deposition of
Metabolically Active and Stationary
Phase Deinococcus radiodurans
in Unsaturated Porous MediaG . G A R G I U L O , * ,† S . A . B R A D F O R D , ‡J . Š I M Ů N E K , § P . U S T O H A L , †H. VEREECKEN,† AND E. KLUMPP†
Agrosphere (ICG-IV), Institute of Chemistry and Dynamics ofthe Geosphere (ICG), Forschungszentrum Jülich GmbHD-52425, Jülich, Germany, Salinity Laboratory, USDA-ARS,
450 West Big Springs Road, Riverside, California 92507-4617,
and Department of Environmental Sciences, University ofCalifornia, Riverside, California 92521

Bioremediation is a cost-efficient cleanup technique thatinvolves the use of metabolically active bacteria to degrade
recalcitrant pollutants. To further develop this technique
it is important to understand the migration and deposition
behavior of metabolically active bacteria in unsaturated
soils. Unsaturated transport experiments were therefore
performed using Deinococcus radiodurans cells that were
harvested during the log phase and continuously suppliedwith nutrients during the experiments. Additional experiments
were conducted using this bacterium in the stationary
phase. Different water saturations were considered in thesestudies, namely 100 (only stationary phase), 80, and 40%.
Results from this study clearly indicated that the physiologicalstate of the bacteria influenced its transport and depositionin sands. Metabolically active bacteria were morehydrophobic and exhibited greater deposition than bacteria
in the stationary phase, especially at a water saturationof 40%. The breakthrough curves for active bacteria also had
low concentration tailing as a result of cell growth ofretained bacteria that were released into the liquid phase.
Collected breakthrough curves and deposition profiles
were described using a model that simultaneously considers
both chemical attachment and physical straining. New
concepts and hypotheses were formulated in this model
to include biological aspects associated with bacteria growth
inside the porous media.

Introduction
Various contaminants have been improperly disposed of or
inadvertently released into the vadose zone. Infiltrating water,which serves to recharge to groundwater supplies, may
subsequently pass through these contaminated regions andleach contaminants to drinking water supplies. Bioremediation strategies such as bioaugmentation, biostimulation,and natural attenuation have been proposed as cost-effectivemeans to clean up these contaminated sites (1, 2). These
techniques rely on the use of metabolically active bacteria* Corresponding author phone: 0049(2461)618783; fax: 0049(2461)618100; e-mail: g.gargiulo@fz-juelich.de.
† Institute of Chemistry and Dynamics of the Geosphere.
‡ USDA-ARS.
§ University of California.
10.1021/es062854a CCC: $37.00
Published on Web 01/20/2007

 2007 American Chemical Society

to degrade or immobilize recalcitrant pollutants (3-8).
Bioaugmentation involves the introduction of cultured
microorganisms that are able to metabolize and grow on thecompounds of interest. In biostimulation the subsurface
environment is modified by adding limiting nutrients to“stimulate” native bacteria that are capable of degrading
contaminants. Natural attenuation uses the existing microbialcommunity and subsurface conditions to contain the spreadof contamination and to reduce concentrations.
Bioremediation has many advantages compared withother cleanup methods. For example, since the contamination can be treated in situ, the technology does not incur
removal and disposal costs and thus post-cleanup costs canbe substantially reduced. Although bioremediation holds
great promise for dealing with intractable environmental
problems, it is important to recognize that much of thispromise has yet to be realized. This is due in part to our lackof understanding of microorganism interactions in differenthydrologic environments and our inability to efficiently
deliver microorganisms from an injection point to a contaminant plume or source zone. The design of efficientbioremediation strategies can be improved by a better
understanding of the complex interplay of chemical, biological, and physical factors affecting bacteria transport anddeposition.
One of the unresolved problems in bioaugmentation
operations is our inability to predict the distribution of the
inoculated bacteria throughout the soil profile. This information is needed to ensure adequate contact between
specialized bacteria and target compounds. Early adsorptionof bacteria can be detrimental to a successful remediation
process. In particular, very little is known about transportand deposition of bacteria that are metabolically active. The
majority of transport studies have been performed using
bacteria cells that were unable to grow and reproduce, orwere harvested in the late stationary phase of growth (4, 7,
9, 10).
In practical bioremediation applications, the bacteria cells
must be metabolically active and be able to reproduce. In
this case, the bacteria must be supplied with nutrients or beable to use components of the liquid or solid phases as anutrient source. The status of bacteria needs to be consideredsince the transport behavior of metabolically active andnonactive bacteria cells may exhibit significant differences.
Microbiological effects associated with the activity of thecells have typically been neglected in transport studies, but
may prove to be significant for many practical bioremediation
applications.
Differences in transport and deposition behavior can becaused by bacteria surface properties (6, 8, 11) that will
influence interactions with the surfaces of solid soil particles(8). The nature of bacteria cell surfaces is a function of their
physiological state (3, 12). It has been reported in the literaturethat the growth phase influences the cell surface hydrophobicity (13-15) and electrophoretic mobility (16). The hydrophobicity of bacteria surfaces has an important influenceon their attachment to a solid matrix (3, 11, 13). Sincehydrophobicity also plays a role in the initial adhesion ofbacteria to the air-water interface (4, 17, 18), its influenceon bacteria transport under unsaturated conditions can also
be expected.
Incorporation of microbial processes into transportmodels has typically proceeded along two separate lines ofinvestigation (19): bacteria transport has commonly been
considered to be similar to that of inert colloids (4, 10); or
the biodegradation of dissolved contaminants has been
VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 1265

modeled in the presence of a stationary bacteria that responds
to various concentrations of substrates and electron acceptors(20-22). Since changes in metabolic activity may significantly
alter the deposition rates of bacteria to solid surfaces, wehave investigated how the physiological state of the bacteria
impacts their movement through unsaturated systems andhow these processes and factors can be mathematicallymodeled. An existing transport model that simultaneously
accounts for attachment/detachment and time- and depthdependent straining processes (23) has been expanded toconsider possible changes due to cellular division and to
take into account the more dynamic situations resulting fromthe metabolic activity of bacteria.

Materials and Methods
Bacteria. Deinococcus radiodurans (DSMZ 20539) is a Grampositive, nonmotile, non-spore-forming, spherical (diameter1.0-1.5 µm), obligate aerobic bacterium. Bacteria were grownon the agar plates consisting of ATCC Medium 220: CASOAGAR (Merck 105458), peptone from casein (15.0 g), peptone
from soy meal (5.0 g), NaCl (5.0 g), agar (15.0 g), and distilledwater (1000.0 mL). For column experiments the cells were
cultivated at 30 °C in a nutrient broth that was agitated at
130 rpm using a thermostated shaker (Certomax HK BRAUN). The nutrient broth consisted of casein peptone, tryptic digest(10.0 g), yeast extract (5.0 g), glucose (5.0 g), NaCl (5.0 g), anddistilled water (1000.0 mL). The bacteria cells were harvested
from the nutrient broth during the log phase by gentle
centrifugation (10 min, 7100g, 25 °C) and resuspended in
fresh media.
More detailed information on the surface characteristics
and morphology of Deinococcus radiodurans has beenpreviously reported (11). The low degree of hydrophobicityof this bacterium was quantified using the Microbial Adhesion
to Hydrocarbon (MATH) approach (24).
Sand. Sterile fused silica sand (Teco-Sil, C-E Minerals
Greenville, USA) was used as the porous medium in thecolumn experiments. The median grain size and the coefficient of uniformity of this sand were determined to be 567µm and 1.87, respectively. The van Genuchten (25) water
retention curve and hydraulic conductivity function parameters were determined from multistep outflow data andinverse modeling (26). According to this approach, theporosity was 0.428, the saturated hydraulic conductivity was
0.576 cm min-1, and the empirical parameters R, n, and lwere 0.0234 cm-1, 12.13, and 3.617, respectively (11).
Column Experiments. Only stationary phase bacteria
could be used in transport experiments conducted at 100%water saturation because these bacteria are obligate aerobes.
Both stationary-phase and log-phase bacteria were used inunsaturated transport experiments (80 and 40% water
saturation). Description of the setup used in the packed
column experiments has been previously reported (11). Onlyan abbreviated discussion is provided below.
The experimental column was constructed from Plexiglas
and was 8 cm in diameter and 22.5 cm long. The air phasein the column was maintained at atmospheric pressure bymeans of two aeration openings. The column was equippedwith tensiometers and pressure transducers near the top, inthe middle, and at the bottom of the column to measure thematrix potential and to verify the uniformity of water
saturation within the column under steady-state unit gradient(uniform matrix potential with depth) conditions. At the topof the column a sprinkling plate equipped with 80 stainlesssteel needles was used to evenly distribute the influent overthe sand surface at a rate of 165-170 mL/h. The water andthe solution were supplied to the sprinkling plate using apiston pump. The bottom was formed by a porous stainlesssteel plate covered with a hydrophilic membrane (polyester
fabric, mesh size 15 µm). The suction at the bottom of the1266 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 4, 2007

column was adjusted to achieve unit gradient by changing
the drip point elevation of a hanging water column. Steadystate water flow conditions were kept constant for theremainder of the transport experiment. The total column
water content was continuously monitored gravimetricallyusing a digital balance. The column bottom was connectedsequentially with PVC tubes to a conductivity probe (tracer),
spectrophometer, and UV detector (bacteria). Both thebacteria and the tracer outflow concentrations were monitored online.
For full water saturation (100%), the column was completely evacuated using a vacuum pump and subsequently
flushed from the top with degassed buffer solution. In
experiments conducted at 80% water saturation, water was
introduced into the column by slowly pumping upward at
a constant rate until a water saturation of approximately
80% was achieved. In contrast, water saturation of 40% was
obtained by gradually reducing the inflow rate at the top
while at the same time increasing the suction pressure at thebottom until unit gradient conditions were achieved (11).
Before initiating the transport experiment with stationary
phase cells, the sand pack was equilibrated by flushing itwith several pore volumes of phosphate buffer solution (pH) 7 and 10-4 M). The solution used for the experiment withmetabolically active bacteria was composed of peptone fromcasein (15.0 g), peptone from soy meal (5.0 g), NaCl (5.0 g),
and distilled water (1000.0 mL) diluted 1000 times.
The final spatial distribution of retained bacteria wasmeasured following each transport experiment according to
the procedure described elsewhere (11). The deposition
profile of retained cells per gram of dry sand was determinedfrom measured values of the total organic carbon content(TOC) in the sand. Experimental mass balance was calculated
independently for the cells in the effluent and retained cells
and normalized by the total number injected into the column.
Theoretical Basis. The HYDRUS-1D code (27) was used
to characterize the experimental data collected in this study.
The bacteria transport was modeled using a modified formof the convection-dispersion equation, in which 2 kinetic
adsorption sites were considered, and first-order kinetic
growth was assumed on the solid and liquid phases. The
mass balance equation is defined as follows:

∂s1
∂s2
∂θC
∂C
∂qC
∂
θD
+ Fb
+ Fb
)
+ µwθC + µsFb
∂t
∂t
∂t
∂x
∂x
∂x
(s1 + s2) (2)()

where θ is the volumetric water content; Fb [ML-3; where M
and L denote units of mass and length, respectively] is thesoil bulk density; t [T; T denotes units of time] is the time;q [LT-1] is the flow rate; x [L] is the spatial coordinate; D[L2T-1] is the dispersion coefficient; C [NcL-3; where Nc is thenumber of bacteria cells] is the bacteria concentration in the
aqueous phase; s1 [NcM-1] and s2 [NcM-1] are the solid-phase
concentrations associated with sites 1 and 2, respectively;
and µw [T-1] and µs [T-1] are the liquid- and solid-phase growthcoefficients, respectively.
Several studies (23, 28) divided sorption sites into two
fractions (s ) s1 + s2) and assumed different rates or processes
occurring at each sorption site. Bradford et al. (23) assumed
that the first fraction of sorption sites, s1, represents processesof attachment/detachment and the second fraction, s2,
represents irreversible straining. The mass balance equation
for the solid phase is given as follows when growth on thesolid phase is also considered:F∂(s1 + s2)∂s)F) θkaC - kdFbs1 +∂t∂t
θkstrψxC + µsFb(s1 + s2) (3)TABLE 1. Percentage of Bacteria in the Outflow and Retainedin the Column
log phase

FIGURE 1. Deinococcus radiodurans growth curve (left) andhydrophobicity % (right) measured as a function of time.
where ka [T-1] is the attachment coefficient; kd [T-1] is thedetachment coefficient; kstr [T-1] is the straining coefficient;and ψt and ψx are dimensionless colloid retention functions
that account for time- and depth-dependent deposition onsite 2, respectively. In this analysis, attachment to the solid
phase and the air-water interface can be lumped in the ka
term. Similarly, straining in pores and water films is also
considered to be lumped using the kstr term.
Bradford et al. (23) hypothesized that the influence of
depth-dependent straining processes on colloid retention
can be described using the following coefficient:

ψx )

(

)

dc + x - x0 -β
dc(4)

where dc [L] is the median diameter of the sand grains, x0 [L]is the coordinate of the location where the straining process
starts (in this case the surface of the soil profile), and β is anempirical factor controlling the shape of the spatial distribution.
The code allowed us to simultaneously fit the bacteriatransport parameters (ka, kd, kstr, β, µl and µs) while consideringboth the breakthrough curve and the retention profile in the
nonlinear least-squares optimization routine. To minimize
the potential for non-unique parameter fits, we used available
experimental information to limit the number of parametersthat were considered in these optimizations. Our selection
procedure for parameter optimization will be discussed below
in the Model Applications section. The mean pore watervelocity and dispersivity that were used in simulations
reported herein were obtained by fitting the solution of theconvective dispersion equation for the tracer breakthroughcurve.

Results and Discussion
Growth Curve and Hydrophobicity. Figure 1 shows thegrowth curve of Deinococcus radiodurans obtained bymonitoring the cells suspension optical density (OD) andthe bacteria surface hydrophobicity (measured using the
MATH test) over the course of the bacterial growth curve.
The first increase of the OD was detected between 6 and 7h, with its value increasing from 0.06 to 0.08, indicating theend of the lag phase and the beginning of the log phase(bacteria binary division). After 28-29 h the OD stopped
changing. This observation was clear evidence that the log
phase was completed and the cells’ metabolic activity ended.
After about 30 h the bacteria culture entered the stationary
phase; i.e., only weak metabolic activity and no increase in
the number of bacteria.

stationary phasesaturation

outflow

tailretained

outflow

retained

80%
40%

75%
39%

6%
8%

45%
87%

88%
47%

14%
53%

Inspection of Figure 1 demonstrates that the growth
conditions influence the cell surface hydrophobicity of
Deinococcus radiodurans. The initial hydrophobicity percentage was zero and nearly constant, with values scattered
between 0 and 3%, during the lag phase. The hydrophobicity
then increased during logarithmic growth, reaching a maximum value of almost 10% at the beginning of the stationary
phase. As the stationary phase advanced, the hydrophobicity
started to decrease and reached the initial value of 0% after
about 100 h. In summary, during a period of high growth
rates the bacteria were observed to become more hydrophobic, reaching a maximum value at the end of the log
phase and the beginning of stationary phase, whereas lower
hydrophobicity was measured during the subsequent phaseof nutrient starvation, until it returned to its initial value.
These results are in agreement with other observations
reported in the literature that indicate starvation decreased
bacteria hydrophobicity and their adhesion to solid surfaces(13-15, 29, 30). Such changes in bacteria hydrophobicity
have been attributed to changes in the surface molecularcomposition during the different life phases of the bacteria.
Proteins and polysaccharides are molecules involved ininteractions of bacteria cells with the solid interface (31).
Since proteins and amino acids are hydrophobic components
of extracellular polysaccharide, increased numbers of thesemolecules cause an increase in the surface bacteria hydrophobicity (8). Moreover, it was observed that bacteria
starvation caused a decrease in the concentration of surface
proteins (32). The bacteria hydrophobicity observed during
the growth stage may be attributed to an initial increase in
the amount of proteins on the bacteria surface during thelog phase. A subsequent decrease in hydrophobicity may
thus be caused by the decrease in the surface proteins during
cell starvation.
Gargiulo et al. (11) reported on differences in shape andmorphology of the log and stationary phase Deinococcus
radiodurans. Log phase Deinococcus radiodurans tend toform pairs and tetracoccus-shaped aggregates of around 2-4µm in diameter when grown in nutrient both. Conversely,
stationary phase Deinococcus radiodurans occurred primarily
as single cells. Differences in the size of log and stationary
phase Deinococcus radiodurans likely occurred as a result ofdifferences in cell surface hydrophobicity (Figure 1). Colloid
stability and aggregation is reported to be sensitive to thesurface hydrophobicity (33, 34); i.e., hydrophobic colloids
are less stable than hydrophilic colloids.
Decreasing hydrophobicity of soil bacteria during starvation has been recognized to be a result of a survival strategy(14). Under optimal growth (nutrient) conditions, it isbeneficial for soil bacteria cells to be more hydrophobic andto firmly attach to solid surfaces and to each other. On theother hand, under unfavorable nutrient conditions, thehydrophilic microorganisms are smaller (less aggregation)and may more easily detach themselves from solid surfaces
and be transported in the liquid phase to a new location in
the environment that is richer in nutrients.
Transport and Deposition. Table 1 provides mass balance
information for the bacteria transport experiments. Very goodmass balance was obtained for the stationary phase bacteria(100-102%), and provides a high degree of confidence inour experimental procedures. Conversely, it was not possible
VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 1267

FIGURE 2. Comparison of breakthrough curves for Deinococcus
radiodurans at different saturations (100, 80, and 40%) and in different
growth phases (stationary and log phases). Only stationary-phasebacteria could be used in transport experiments conducted at 100%water saturation because the bacteria are obligate aerobes. Herethe relative concentration, C/Co where Co is the influent bacteria
concentration, is plotted as a function of pore volume.
to close the mass balance for bacteria with cells in the log
phase because of binary division that occurred during theexperiment. The total number of cells recovered after theexperiment exceeded by about 30% the total number of cells
injected in the soil column.
Figure 2 compares the breakthrough curves (here relative
concentrations, C/Co, are plotted as a function of porevolume) for Deinococcus radiodurans in the stationary and/or growing (log) phases at saturations of 100, 80, and 40%.
Only stationary phase bacteria could be used in transport
experiments conducted at 100% water saturation because
these bacteria are obligate aerobes. The breakthrough curves
show significant differences in the maximum relative concentrations, in the curve shapes, and in the concentration
trailing between bacteria in the stationary and metabolically
active phases at the various saturations.
For a given saturation (80 and 40%), lower effluent
concentrations were observed for the metabolically active
bacteria than for the stationary phase cells (Figure 2).

Although breakthrough curves at 80% saturation reached aplateau for bacteria in both stationary and growth phases,
their maximum relative concentrations were different. The
maximum relative concentration for the metabolically active
cells in the log phase (C/Co ) 0.63) was lower than for cells
in the stationary phase (C/Co ) 0.83). At 40% saturation, themaximum relative concentrations in the outflow were alsosignificantly different for the stationary (C/Co ) 0.5) and logphase (C/Co ) 0.3) bacteria. This enhanced retention for the
metabolically active bacteria may be attributed to more cellcell interactions as a result of differences in hydrophobicity(Figure 1) and larger cell/aggregate (1.0 compared to 2-4µm for stationary and log-phase cells, respectively) sizes.
Both attachment and straining are predicted to increase withincreasing cell/aggregate size (23, 35).
For a given growth phase (stationary or growth), lower
effluent concentrations were also observed for decreasingwater saturation (Figure 2). The maximum relative concentration for the stationary phase cells at 100, 80, and 40%
water saturation was 0.92, 0.83, and 0.5, respectively. The
maximum relative concentration for active cells at 80 and
40% water saturation was 0.63 and 0.3, respectively. Increased
retention with decreasing water saturation has been observed
by other researchers (4, 9-11, 18, 36), and has been attributed
to attachment at the air-water interface (4, 9, 10), film
straining (18), and more recently to straining (11, 36).
The overall shape of breakthrough curves shown in Figure
2 for bacteria in different phases was different as well. Bacteria
in the resting phase at 40% water saturation clearly exhibita decreasing rate of deposition with time. In contrast, in themetabolically active phase the deposition of successive cells
on each other is not prevented. Metabolically active bacteria
cells that are deposited on the sand surface apparently do
not inhibit the retention of additional bacteria because theactive cells have stronger cell-cell interactions. The enhanced
cell-cell interactions for metabolically active bacteria could
be explained with the increased cell hydrophobicity during
the experiment (Figure 1) (29) and the higher number andextension of surface macromolecules compared to bacteria
in the resting mode (30, 32). This could have resulted inenhanced polymer bridging.
Contrary to the stationary state, the breakthrough curveof the log-phase bacteria showed pronounced tailing, withthe outflow concentrations not reaching zero values evenafter 15 pore volumes (Figures 2, 3A, and 4A). This phe-

TABLE 2. Model Parameters for Deinococcus radiodurans in Different Growth Phases and at Different Saturations
saturation
100%
value

ka (min-1)
kd (min-1)
kstr (min-1)
βR2
R 2 (btca)
R 2 (rpa)

4.54 × 10-5
2.27 × 10-3
5.12 × 10-3
0.432
0.994
0.994
0.980

ka (min-1)
kd (min-1)
kstr (min-1)µs (min-1)
βR2
R 2 (btca)
R 2 (rpa)

N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A

80%
S.E.coeff.

40%
value

S.E.coeff.

Deinococcus radiodurans (stationary phase)
1.69 × 10-4
3.38 × 10-3
5.27 × 10-4
2.13 × 10-2
2.78 × 10-2
5.33 × 10-3
1.27 × 10-3
1.73 × 10-2
4.72 × 10-4
0.432
0.996
0.998
0.954

9.77 × 10-2
7.70 × 10-2
7.77 × 10-2
0.432
0.928
0.931
0.896

2.15 × 10-2
1.79 × 10-2
4.34 × 10-3

Deinococcus radiodurans (log phase)
4.29 × 10-3
4.39 × 10-4
6.84 × 10-4
1.57 × 10-4
1.72 × 10-2
2.73 × 10-3
4.87 × 10-4
5.20 × 10-5
0.432
0.957
0.957
0.959

1.08 × 10-2
1.64 × 10-3
9.33 × 10-2
1.45 × 10-4
0.432
0.956
0.865
0.984

1.35 × 10-3
3.81 × 10-4
6.30 × 10-3
3.05 × 10-5

N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A

value

a btc ) breakthrough curve; rp ) retention profile.

1268 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 4, 2007

S.E.coeff.

FIGURE 3. Measured and fitted breakthrough curves (A) and retention
profiles (B) for Deinococcus radiodurans for cells in the stationary(100 and 80% water saturation) and log (80% water saturation)phases. In A the relative effluent concentration is plotted as afunction of time. In B the normalized concentration (number ofbacteria recovered in the sand, Nc, is divided by the number in aunit volume of the input bacterial suspension, Ni) per gram of dry
sand and these are plotted as a function of the distance from thecolumn inlet.
nomenon was attributed to cell growth. We hypothesize thatthe pronounced tailing was due to continuous bacteria
multiplication in the solution and due to the mechanism
called “cell division mediated transport” (37, 38). During cell
division mediated transport the mother cells deposit on themineral surface, grow, divide, and finally release the daughter
cells into the aqueous phase.
The retention profiles at all saturations and for both active
and nonactive cells were not exponential with depth aspredicted by classical filtration theory (see values of β in
Table 2). The deposition profiles exhibited invariably greater
retention in the section adjacent to the column inlet (Figures
3Band 4B). This incongruence with CFT has been reported
by other researchers under both saturated (5, 23, 39-44) andunsaturated (10, 11, 36) conditions. The log-phase bacteria
showed a more pronounced presence of bacteria at thecolumn inlet than stationary bacteria at both saturations (80and 40%). The log phase bacteria likely had enhanced cellcell interactions and straining at the column inlet presumably
due to the presence of more extensive protein molecules on
the bacteria surface that caused “polymer bridging” and dueto their larger cell/aggregate sizes. The relative number ofbacteria present throughout the soil column is also higher
in the case of log-phase cells compared with the stationaryphase cells. This observation can be attributed to enhanced

FIGURE 4. Measured and fitted breakthrough curves (A) and retention
profiles (B) for Deinococcus radiodurans at 40% saturation for cells
in stationary and log phases. In A the relative effluent concentrationis plotted as a function of time. In B the normalized concentrations
per gram of dry sand are plotted as a function of the distance fromthe column inlet.
attachment due to different surface characteristics of themetabolically active bacteria compared with the resting modecells. In particular, proteins on the surface of active bacteria
cells enhance the hydrophobicity and attachment along thesoil packing.
Model Application. For stationary phase transport experiments the values of µw and µs were considered equal tozero because no growth occurred, and attachment (ka andkd) and straining (kstr) model parameters were fit to breakthrough curves and deposition profiles. Model fits were more
complicated for log phase transport experiments because
growth also occurred in the liquid and solid phases. In thiscase, attachment (ka and kd), straining (kstr), and growth (µw
and µs) model parameters were fit to breakthrough curves
and deposition profiles. To minimize the number of fitting
parameters that were optimized, the value of β in eq 4 was
set equal 0.432 based on previously published findings (23)when significant straining occurred. According to thismodeling approach the influence of attachment and straining
processes dominate different regions of the deposition profile(23); i.e., straining dominates near the sand surface andattachment dominates at greater transport distance.
Very good agreement was obtained between the observed
and simulated data for the metabolically active and resting
phases of the bacteria at both saturations (Figures 3A and B,
4A and B). Values of optimized parameters are summarizedin Table 2. The attachment, ka, detachment, kd, and straining,
kstr, coefficients increased as the saturation decreased (Table
VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 1269

2) for both the resting mode cells and the log-phase bacteria.
The attachment coefficient, which lumps attachment to thesolid phase and the air-water interface, slightly increased
as saturation decreased, probably due to the enhanced
attachment to the air-water interface as the air-waterinterfacial area increased (11). Increasing values of kd withdecreasing saturation, suggests that attachment to the airwater interface is approaching linear equilibrium conditions(45). Straining has been hypothesized to increase withdecreasing water saturation because more of the water flux
flows through small straining sites at lower saturations (11).
For a given water saturation, the value of kstr was very
similar for stationary- and log-phase cells. This finding
suggests that straining is not much affected by the growthstage for these bacteria. In contrast, parameter values for ka
and kd were very dependent on the growth stage of the cells.
In particular, the values of kd were significantly lower for
metabolically active cells. This observation indicates thatstronger cell-cell interactions occurred for attached bacteria
under active growth conditions. It is well-known that many
bacteria are capable of forming a biofilm. Under certain
conditions the bacteria cells communicate with each otherby means of a process called quorum sensing and detect
when they are assembled in large numbers as opposed towhen they are essentially alone (46). According to this theory,
bacteria in favorable growing conditions secrete and sense
small signalling molecules to communicate and to promotebiofilm formation. The metabolically active cells are not likely
to reject each other and the deposition of one cell should notinhibit the deposition of the next cell. The cells try to get as
close as possible to each other to form a more resistant
structure. This effect may be further enhanced by increasedhydrophobicity of metabolically active bacteria and theassociated higher number and extension of proteins on thesurface of these bacteria.
Mass balance information after recovery of the concentration plateau region of the breakthrough curve (before
significant detachment occurred) also supports this enhanced
cell-cell interaction hypothesis. Attachment for active andstationary cells accounted for 59.1 and 14.9% of the retainedcell mass at a water saturation of 80%, respectively. Similarly,
attachment for active and stationary cells accounted for 35.2
and 16.6% of the retained cell mass at a water saturation of
40%, respectively. Hence, increased deposition of log-phasecompared to stationary-phase bacteria are attributed primarily to attachment. Straining was always the dominantmechanism of retention (>83.4%) for the stationary-phasebacteria. For log-phase cells, however, straining accounted
for 40.9 and 64.8% of the retained cells at a water saturationof 80 and 40%, respectively.
The plateau region of the breakthrough curves shown in
Figure 2 did not change much with time for the log-phasebacteria transport experiments, and suggests that µl was low
and only accounted for a small fraction of Co. Preliminarymodel fits to this data also indicated that the value of µl wasvery low (<0.46 × 10-4 min-1), and that the value of µs was
significantly higher (1 order of magnitude) than that of µl.
This result indicates that the deposited cells have a higher
division rate compared with the cell in the bulk solution. To
minimize the potential for non-unique parameter fits, wetherefore set the value of µl to zero in subsequent simulations
with log-phase cells. Table 2 provides values of µs when µl
was set equal to zero. The tailing of the bacteria outflow
concentrations could be well described by this model
formation at both saturations (80% and 40%) (Figures 3A
and 4A). These observations support our hypotheses that
deposited bacteria are particularly active in biofilm formation
under favorable growth conditions and that the observed
tailing in the breakthrough curves was primarily due to releaseof the daughter cells into the aqueous solution from attached
1270 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 4, 2007

mother cells (cell-division mediated transport).
Results from this study clearly indicate that the physiological state of the bacteria will influence its transport anddeposition in soils producing a more dynamic system andresulting in different effluent breakthrough curves anddeposition profiles for metabolically active bacteria. Additional studies are warranted to better understand bioremediation processes and to design more effective technologies. For example, further transport studies with different
bacteria strains during their active states, and to better deducemutual bacterial interactions, are desirable. The coupledinfluence of various pollutants, nutrients, substrates, andelectron acceptors on the physiological state of the bacteria
and their transport potential are topics of future studies.

Acknowledgments
G.G. thanks the Forschungzentrum Juelich, Germany, forthe financial support of this project.

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Received for review December 1, 2006. Accepted December 12,
2006.
ES062854A

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