Adhesion and Deadhesion of Ureolytic Bacteria on

Sand under Variable Pore Fluid Chemistry

Surabhi Jain 1 and Dali Naidu Arnepalli, Ph.D., M.ASCE 2
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.

Abstract: It is essential to understand the phenomena of adhesion, deadhesion, and transport of microorganisms in porous media to scale up
bioengineering processes. In this study, the adhesion and deadhesion of two ureolytic microorganisms were investigated in loose sand using a
set of flow-through column experiments by varying the pore fluid chemistry. An increase in the ionic strength altered the surface-charge
properties of the microbes and the selected geomaterial, which in turn reduced the energy barrier, leading to significant adhesion of microbes
on the sand surface. After microbial adhesion, permeation of a lower-ionic-strength solution exhibited considerable bacterial deadhesion from
the sand column, indicating the reversible nature of the interaction between bacteria and the sand surface. The physicochemical adhesion and
deadhesion mechanisms are elucidated in terms of extended Derjaguin-Landau-Verwey-Overbeek theory. The variation of the energy barrier
between the microbe and sand, the appearance of primary and secondary energy minima attributed to the change in pore fluid chemistry, and
its influence on microbial adhesion and deadhesion on to the sand were also studied. DOI: 10.1061/(ASCE)EE.1943-7870.0001708. © 2020
American Society of Civil Engineers.
Author keywords: Ureolytic bacteria; Adhesion; Deadhesion; Ionic strength; pH; Derjaguin-Landau-Verwey-Overbeek (DLVO).

Introduction properties of microbes and sorbent, which in turn determines the

Comprehending the mechanism of microbial adhesion, deadhesion,
and its transport in saturated porous media is essential in biogrout-
ing, bioaugmentation, bioremediation, and wastewater treatment
(Ginn et al. 2002; Kim et al. 2008). Bioaugmentation and biogrout-
ing techniques involve the injection of solutions containing mi-
crobes with an adequate amount of nutrients to the targeted
zone of the subsurface for improving its engineering properties
(Fauriel and Laloui 2012; Chu et al. 2014). In this process, the mi-
crobes could be detached and flushed away from the targeted zone,
in turn making the process inefficient. Previous researchers have
investigated the effect of physical, chemical, and biological factors
on the collision, attachment, rate of deposition, and detachment of
microbes onto the solid surface (Choi et al. 2017; Roy et al. 2019).
The governing factors considered in the microbe–solid surface in-
teraction studies include bacterial characteristics, solution chemis-
try, and porous media properties (Li et al. 2006; Torkzaban et al.
2008).
Various strategies have been proposed to control the rate of bac-
terial adhesion and transport in porous media for the successful im-
plementation of bioengineering processes in the field (Mills et al.
1994; Harkes et al. 2010). Among the many strategies reported in
the literature, control of pore fluid chemistry, particularly the ionic
strength, has been found to be efficacious (Redman et al. 2004).
The ionic strength of the pore fluid governs the surface-charge
1 deadhesion, and transport in porous media have been explained
Research Scholar, Dept. of Civil Engineering, Indian Institute of Tech-
nology Madras, Chennai, Tamil Nadu 600036, India. ORCID: https://orcid
.org/0000-0002-8838-420X. Email: surabhi.28.09.91@gmail.com
2
Associate Professor, Dept. of Civil Engineering, Indian Institute of
Technology Madras, Chennai, Tamil Nadu 600036, India (corresponding
author). ORCID: https://orcid.org/0000-0002-7972-6116. Email: arnepalli@
iitm.ac.in
Note. This manuscript was submitted on June 26, 2019; approved on
December 3, 2019; published online on March 30, 2020. Discussion period
open until August 30, 2020; separate discussions must be submitted for
individual papers. This paper is part of the Journal of Environmental En-
gineering, © ASCE, ISSN 0733-9372.
rate of adhesion of bacteria and their transport through the porous
media (Torkzaban et al. 2008). Few studies concerned biocemen-
tation, and a weakly sorbed ureolytic Sporosarcina pasteurii has
been immobilized on the sand surface by injecting a 50-mM ionic
strength pore fluid to achieve homogeneous cementation (Whiffin
et al. 2007; Harkes et al. 2010). However, the majority of the stud-
ies failed to explain the mechanism of microbial adhesion, its
reversibility, and the effect of change in ionic strength on adhesion
and deadhesion. Besides, the pH of the pore solution and geoma-
terial varies widely based on their origin, mineralogical variation,
and environmental condition, among others. Although pH influen-
ces the microbial adhesion and transport through porous media, its
effect has not been established (Cheng et al. 2014).
Given this, the present study aimed to examine the physical dep-
osition, physiochemical adhesion, and deadhesion of two ureolytic
microorganisms on to the sand surface by varying the ionic strength
and pH of pore fluid by conducting flow-through column experi-
ments. Attempts were also made to obtain the parameters represent-
ing the microbial adhesion under different pore fluid chemistries by
fitting the experimental data of flow-through column experiments
using theoretical models.
The surface properties of microbes are comparable to those
of colloidal particles due to their similarity in size and surface-
charge distribution. Consequently, microbes obey the principles
of colloid–surface interaction in terms of adhesion and deadhesion
on solid surfaces (Choi et al. 2017). Given this, microbial adhesion,
based on classical Derjaguin-Landau-Verwey-Overbeek (DLVO)
theory, where the overall interaction energy is the sum of van der
Waals force and electrostatic double-layer interaction or repulsion
energy between the microbes and collector surface (Loosdrecht
et al. 1989; Redman et al. 2004; Zhang and Olson 2012). However,
the classical theory does not consider short-range repulsion Born
potential, which creates a very small yet significant primary/
secondary minima, steric interaction, and hydrophobic interactions,
among others (Redman et al. 2004). Further, few studies have
modified the DLVO theory by incorporating the Born potential

© ASCE 04020038-1 J. Environ. Eng.

J. Environ. Eng., 2020, 146(6): 04020038

 when estimating the total interaction energy profile for understand-
ing the physical deposition, non-DLVO interaction, and appearance
of primary/secondary minima in a short separation range in satu-
rated porous media (Hahn et al. 2004; Choi et al. 2017). Given this,
the present study attempts to explain the mechanisms of adhesion
and deadhesion of microbes by emphasizing the surface charge of
geomaterial and microbes, as well as their electrostatic double-layer
interaction, based on extended DLVO theory.
Materials and Methods
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.
Microorganism and Culture Preparation
Two types of ureolytic bacteria used for the study were S. pasteurii
and Proteus species because these organisms have been success-
fully employed for the biocementation and microbially induced car-
bonate precipitation (MICP) processes (Nemati et al. 2005; Zhao
et al. 2014). Alkaliphilic S. pasteurii (ATCC 11859) was procured
from the American type culture collection, Manassas, Virginia. The
other ureolytic Proteus species chosen for the study was isolated
from garden soil of Indian Institute of Technology Madras campus,
Chennai, India. For obtaining the pure culture of selected microbes,
the streak plate method was employed using a nutrient agar Petri
plate at a temperature of 30°C. Nutrient agar was prepared by steri-
lizing 13 g of nutrient broth (Himedia, Mumbai, India) with 1.5%
agar in 1 L of deionized water. The composition of the nutrient
broth was maintained as 1.5 g of beef and yeast extract, 5 g of pep-
tone, and 5 g of NaCl in 1 L of sterile deionized water at a pH of
7.4
0.2. Ureolytic activity was confirmed by streaking the mi-
crobes in urea agar base plates. The change in color of the urease
agar from yellowish orange to pink is attributed to the increase in
pH due to the urease enzyme activity of the microbes (Achal and
Pan 2011). Colony morphology, Gram staining, and scanning elec-
tron microscopic (SEM) image analyses were performed to identify
the morphological characteristics of the organisms (Jain and
Arnepalli 2019). Glycerol stock of the chosen microbes was pre-
pared in 50% glycerol [Volume/Volume (v=v)] and stored at −80°C
(Jain and Arnepalli 2019).
The glycerol stock culture was revived by growing it in 100 mL
of nutrient broth urea (NBU) in 250-mL Erlenmeyer flasks for
12–16 h. After 12–16 h of incubation, 1 mL of culture from the
flask was transferred into fresh 100 mL NBU and incubated in
an orbital shaker by agitating at 120 revolutions per minute
(rpm) and by maintaining the temperature at 30°C. The NBU media
was prepared by adding 8 g of nutrient broth and 20 g of urea in 1 L
of deionized water. Microbial cells were harvested from the late
exponential growth phase and centrifuged at 8,000 rpm at 4°C
to obtain microbial pellets. Further, these pellets were rinsed twice
using sterile water and resuspended in solutions with different ionic
strengths and pH. The ionic strength of the solution was adjusted by
adding CaCl2 · 2H2 O, whereas the pH of the pore fluid was con-
trolled in the range of 59 by adding diluted NaOH or HCl. The pore
fluid chemistry considered in this study enables the survival of the
chosen bacteria (Anbu et al. 2016).
The microbial culture prepared under variable pore fluid
chemistry was analyzed for surface-charge properties in terms
of their zeta potential using a ZetaPals instrument (Brookhaven
Instruments, Holtsville, New York). The average zeta potential
value of three independent measurements was taken for each mi-
crobial cell suspension having ionic strength of 0.1, 1, 10, 30, and
100 mM, and pH ranging from 5 to 9. The bacterial concentration
(referred as bacterial density) in the solution was determined by
measuring its optical density at 600-nm wavelength, OD600 , using
© ASCE 04020038-2
J. Environ. Eng., 2020, 146(6): 04020038
a UV-visible spectrophotometer (Agilent Carry 100, Santa Clara,
California). Further, a cell counting chamber known as a hemocy-
tometer was used for measuring the cell population with the help of
an optical microscope (Nikon Instruments, Melville, New York).
Selection of Geomaterial
Indian standard Ennore fine sand was chosen for investigating the
organism’s attachment on it using a flow-through column setup.
Because the selected geomaterial is highly crystalline nonreactive
quartz, the effect of chemical and mineralogical heterogeneities
imposed by it on the microbial interaction is minimized. For this
purpose, sand samples from Tamil Nadu Minerals, Chennai, India,
were procured. The sand sample was rinsed several times using
deionized water for removing impurities that may cause an error
in optical density measurement. Before the commencement of
the experiment, oven-dried sand was sterilized for 20 min in an
autoclave by maintaining pressure of 104 kPa (15 psi) and a tem-
perature of 121°C to avoid the influence of native microbes. The
specific gravity and particle-size distribution characteristics of the
selected geomaterial were determined with the help of a helium gas
pycnometer (Quatachrome Instruments, Boynton Beach, Florida)
as per ASTM D5550 (ASTM 2006b) and by conducting mechani-
cal wet sieve analysis according to ASTM D422 (ASTM 2007),
respectively.
Further, the pH of the soil sample was measured by using a pH
electrode connected to a water quality analyzer (Elico P138,
Hyderabad, India). X-ray diffraction analysis was carried out to
quantify the mineralogical composition of sand. The surface-charge
characteristics of sand in different pore fluid chemistries were de-
termined based on the electro-osmosis theory using the following
relationship:
εp ζ
vavg ¼ E ð1Þ
μ z
where vavg = average electro-osmotic velocity; εp = permittivity of
the pore fluid; ζ = surface charge of the sample, which was obtained
by considering the average value of three independent experiments
conducted; μ = solution viscosity; and EZ = applied electric field
strength.
Flow-Through Column Setup
Fig. 1 depicts the schematic view of the flow-through column
setup. The pictorial view of the experimental setup is incorporated
as an insert in Fig. 1 for enhancing the clarity. The setup is made of
plexiglass having an internal diameter of 30 mm and a length of
70 mm. A set of Whatman filter papers is placed on either side
of the sand sample, having a height of 60 mm to retain the sand
particles in position. A layer of scouring pad was placed next to
the filter paper to create drainage layers on either side of the sand
layer. The porosity of the soil specimen was maintained around
43% to simulate the loose-sand condition that may prevail in
the field. It can be observed from the reported literature that bio-
cementation is an effective process in treating the loose-sand hav-
ing the soil porosity range of 35%–40% (Whiffin et al. 2007). To
achieve the desired porosity, the dry unit weight of the sand column
was maintained as 15.1 kN=m3 by preparing a specimen using the
free-fall technique. A flow regulator was connected between the
storage tank and the inlet of the soil column to maintain a predeter-
mined flow rate.
Before the experimentation, glassware and chemicals were steri-
lized to avoid cross-contamination of other microorganisms present
in them. Before experimenting, the sand column was saturated by
J. Environ. Eng.

Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.
Fig. 1. Schematic view of flow-through column experimental setup.
permeating the deionized water from top to bottom until a steady
flow condition has reached. Upon ensuring the complete saturation
of the sand column, a series of breakthrough experiments were per-
formed for investigating the adhesion and deadhesion mechanisms
of microbes on to the sand. Initially, a known pore volume of sterile
water having a predetermined ionic strength and pH was injected
into the saturated sand column by maintaining a constant flow rate
of 180 mL=h to ensure a sufficient contact period for bacterial ad-
sorption (Harkes et al. 2010). The passing of approximately 5 pore
volumes of the ionic solution was enough to ascertain the chemical
equilibrium between the geomaterial and the pore fluid in the sand
column. However, flushing of the soil column was continued until
the chemical equilibrium was attained, i.e., ionic strength and pH of
effluent reaches the influent fluid chemistry.
Consequently, a bacterial solution of nearly 4 pore volumes
(≈4.5 × 18 ¼ 81 mL) having identical pore solution chemistry
with that of the previous step was introduced into the soil column
under similar flow conditions. For assessing the physical deposition
of microbes on the sand, experiments were conducted by permeat-
ing the bacterial solution in sterile deionized water. This process
was continued until the bacterial concentration in the effluent at-
tained a constant value. Subsequently, the deionized water having
similar solution chemistry was introduced to assess the deadhesion
of the microbe. During the entire adsorption and desorption pro-
cess, the maximum retention time available for bacteria is about
12 min for any pore fluid chemistry, which in turn eliminates
the possible growth of microbes within the porous media.
Approximately 4 mL of the influent and effluent (i.e., one-fourth
of a pore volume) was sampled at a regular interval into a sterile test
tube, and the bacterial concentration present was determined by
measuring the optical density, OD600 . The normalized effluent con-
centration (i.e., ratio of the bacterial density of effluent, Ct , and
© ASCE 04020038-3
J. Environ. Eng., 2020, 146(6): 04020038
influent bacterial density, C0 ) was monitored throughout the ex-
periment. Experiments were conducted in triplicate over a range
of ionic strengths and pH values, and the mean value was reported.
The microbial transport through the soil column can be represented
by the following relationship [Eq. (2)] (Freeze and Cherry 1979):


∂C Dl ∂ 2 C V f ∂C
¼ − ð2Þ
∂t R ∂x2 R ∂x
where C = concentration of microbial suspension; t = time;
Dl = longitudinal hydrodynamic dispersion coefficient; R =
retardation coefficient; vf = seepage velocity (≈18 × 10−4 m=s);
and x = linear length of soil column (≈0.06 m).
For assessing the unknown parameters (Dl and R) in the preced-
ing governing equation, a numerical tool called Studio of
Analytical Models (STANMOD) was employed by considering
the nonlinear sorption behavior (Toride et al. 1999). In the analysis
of STANMOD, a flux-averaged concentration mode, pulse input
boundary condition with zero initial concentration, and zero pro-
duction was chosen. Further, the distribution coefficient, K p was
determined based on the following relationship [Eq. (3)]:
ρ × Kp
R¼1þ ð3Þ
η
where ρ = density; and η = porosity of the soil sample.
Sterile water (without bacteria in it) having an ionic strength one
order magnitude less than that used for the adsorption process was
injected, to assess the deadhesion mechanism. For instance, if a
sand column was saturated with a bacterial solution of known con-
centration and ionic strength of 100 mM, the deadhesion studies
were performed with sterile water having 10-mM ionic concentra-
tion. In these experiments, the effluent optical density and micro-
bial concentration were determined to assess the cumulative
fraction of desorbed bacteria from the sand column. The initial bac-
terial population in the sand column was obtained by considering
the total organisms adhered to the sand column per unit volume of
the sample. The adhesion and deadhesion studies using flow-
through column experiments were performed under the isothermal
condition of 25°C.
Principles of Extended DLVO Theory
The adhesion and deadhesion of the microbes on the sand surface
depend on the total interaction energy between the collector surface
and colloidal bacteria. For this purpose, the present study employed
an extended DLVO theory to quantify the net attraction and repul-
sion energy between the bacteria and sand surface. As per the
extended DLVO theory, the total interaction energy (ETot ) is the
summation of electrostatic double-layer repulsion (EEDL ), van
der Waals interaction energy (EVDW ), and Born interaction energy
(EBorn ). The overall interaction energy of the microbial–sand sys-
tem was modeled as a sphere–plate interaction, and the same is
presented as follows (Hogg et al. 1966):


1 þ expð−khÞ
EEDL ¼ πε0 εr ap 2ζ b ζ s ln
1 − expð−khÞ

2 2
 ð−2khÞ

þ ðζ b þ ζ s Þ ln 1 − exp ð4Þ
where ε0 = vacuum permittivity (8.85 × 10−12 F=m); εr = relative
dielectric permittivity of pore fluid; ap = equivalent radius of the
bacteria obtained as 1.03 and 0.76 μm for S. pasteurii and Proteus
species, respectively, from SEM analysis (Fig. S1); ζ b and ζ s = zeta
potential values of bacteria and sand grains, respectively, obtained
over a range of ionic strengths and pH; and k = inverse of
J. Environ. Eng.
 Debye length (1=m), which can be obtained using the following
relationship:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2 × 103 e2 N A I
k¼
ϵ0 ϵr KT
where e = electron charge (1.602 × 10−19 C); N A = Avogadro’s
number (6.02 × 1023 molecules/mol); I = ionic strength (mol=L);
K = Boltzmann’s constant (1.381 × 10−23 J=K); and T = absolute
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.
temperature in Kelvin.
The van der Waals interaction energy was calculated by using
the following relationship:


−Aap 14h −1
EVDW ¼ 1þ
6h λ
where A = Hamaker constant (6.5 × 10−21 J for quartz); λ = char-
acteristic wavelength of the dielectric (100 nm); and h = separation
distance between the microbes and sand surface. The Born inter-
action energy, EBorn is presented in Eq. (7)


−Aσ6B 8ap þ h 6ap − h
EBorn ¼ þ
7,560 ð2ap þ hÞ7 h7
where σB = born collision diameter, which equals to 0.5 nm
(Hahn et al. 2004). With this, the total energy was calculated as
per the Eq. (8)
ETot ¼ EEDL þ EVDW þ EBorn
Description of Experimental Results
Microorganism and Phenotypic Characterization
The pure culture of S. pasteurii and Proteus species were cultured
in a nutrient agar Petri plate after 24 h of incubation at 30°C. The
colony morphology of S. pasteurii showed an irregular form with a
wavy or undulated edge. It formed a dull creamy color opaque
colony of size nearly 1–3 mm. The microorganism was motile
and viscid in nature with slightly raised elevation in its side view.
In contrast, the colonies of Proteus species were circular in geom-
etry with an entire margin and convex elevation. This organism
formed tan-colored punctiform colonies and moved out in waves
across the plate.
The Gram-staining images were obtained with the help of an
optical microscope of both the bacterial smear. Gram-staining re-
sults showed that S. pasteurii was a rod-shaped, Gram-positive mi-
crobe that retained a crystal violet color after staining. In contrast,
the cell wall of Proteus species exhibited pink color and proved to
be a Gram-negative, rod-shaped microbe. Both organisms were
able to hydrolyze urea due to their urease enzyme activity, which
was identified by the change in color of urea agar upon culturing
the microbes in urea agar base (Jain and Arnepalli 2019). Scanning
electron microscope images showed that S. pasteurii cells had a
length of 2–2.5 μm and diameter of 0.5–0.7 μm, and the cells of
isolated Proteus species were 1–1.2 μm in length and 0.4–0.5 μm
in diameter. Further, an empirical expression for determining the
cell density was developed, which is reported in Eqs. (9) and
(10) for S. pasteurii and Proteus species, respectively
© ASCE
J. Environ. Eng., 2020, 146(6): 04020038
ð5Þ
ð6Þ
Fig. 2. Variation of zeta potential of the selected species and sand with
ionic strength.
Cell concentrationS:pasteurii ×107
cells
¼ 8.6 × OD1.36 ð9Þ
ð7Þ 600
mL


7 cells
Cell concentrationProteus ×10 ¼ 80 × OD1.95
600 ð10Þ
mL
ð8Þ
The surface-charge characteristics of the selected organisms
were assessed in terms of zeta potential over the range of ionic
strength of 0.1–100 mM. Fig. 2 indicates that that the chosen cells
of S. pasteurii and Proteus species were negatively charged in the
neutral and low-ionic-strength solutions. With an increase in ionic
strength, the surface negativity decreased and became positive at
ionic strengths of 50 and 90 mM for Proteus and S. pasteurii, re-
spectively. The surface-charge behavior of the selected microbes
is analogous to charged colloidal particle, where the increase in
electrolyte concentration prevents the straying away of counterions
from the charged surface. As a result, the diffused electrical double
layer gets compressed, eventually resulting in a lower value of zeta
potential (Hsu and Huang 2002; Nikhil John and Arnepalli 2019;
Kollannur and Arnepalli 2019).
Further, to delineate the effect of pH on the surface-charge prop-
erties of the selected microbes, the zeta measurements were carried
out over a pH range of 5–9 (Fig. S2). Unlike in the case of ionic
strength, the pH variation has not imparted a significant impact on
the measured surface-charge properties, and the values were scat-
tered in a short range of −37 to −50 mV for the organisms con-
sidered in this study. The obtained variation can be treated as
nominal owing to the fact that the zeta potential of S. pasteurii
and Proteus species was found to be −49 and −45 mV, respec-
tively, when measured using deionized water. Similar observations
on pH buffering were reported by Hsu and Huang (2002) and
Ohmori and Glatz (1999). This could be due to the inherent nature
of the organisms to undergo ion exchange or specific adsorption
in order to preserve a constant chemical milieu surrounding the
cell wall. It is worth mentioning that the Gram-positive and
Gram-negative organisms exhibited a neutral charge (referred as
isoelectric point) when they were incubated at pH 2–3 and pH
4–5, respectively (Mitchell and Santamarina 2005).
04020038-4 J. Environ. Eng.
 Table 1. Physicochemicomineralogical properties of fine sand
Property Value
Specific gravity 2.65
Particle-size distribution characteristics (fraction by weight)
Gravel size (i.e., >4.75 mm) 0
Coarse sand size (i.e., ≤4.75 and >2 mm) 0
Medium sand size(i.e., ≤2 and >0.425 mm) 47
Fine sand size (i.e., ≤0.425 and >0.075 mm) 53
Fines (i.e., ≤0.075 mm) 0
D10 (mm) 0.2
D30 (mm) 0.3
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.

D60 (mm) 0.4

Uniformity coefficient, Cu 2.1
Coefficient of curvature, Cc 1.0
USCS soil classification (ASTM D2487) SP
Maximum void ratio, emax 0.82
Minimum void ratio, emin 0.63
pH 7.4
Major minerals present Quartz (89%) and
kyanite (11%) Fig. 3. Variation of normalized effluent concentration with pore
volume for S. pasteurii during adhesion and deadhesion of microbes
over a range of ionic strengths.
Geomaterials Characterization
Table 1 presents the grain-size distribution characteristics of the displacement of existing pore fluid by uniform and undispersed
selected fine sand. Based on the particle-size distribution character- flow of the bacterial solution. Once the influent bacterial solution
istics, the selected geomaterial can be classified as poorly graded filled within the pore space, the subsequent measurement in the
sand as per the Unified Soil Classification System [ASTM D2487 effluent showed a sudden rise in bacterial concentration. For exam-
(ASTM 2006a)]. The quantitative phase analysis of X-ray diffrac- ple, in the case of the bacterial solution with zero ionic strength
tion patterns of the fine sand revealed that the selected geomaterial (neutral), the normalized effluent concentration reached 0.8
has 89% quartz and 11% aluminosilicates (i.e., kyanite). The sur- immediately after 1 pore volume of flow for both S. pasteurii and
face charge of sand when equilibrated over a range of ionic strength Proteus species. A similar trend was observed in the case of a
and pH values is shown in Figs. 2 and S2. Unlike ionic strength, the lower-ionic-strength value (0.1 mM) where nearly 90% of influent
effect of pore solution pH on surface-charge properties of sand is bacterial concentration has exited the sand column. This reflects a
found to be negligible. At neutral pH, the zeta potential value of plug or piston flow with insignificant physical deposition and
sand was found to have an electronegativity value of −45 mV. physicochemical sorption of microbes on sand. The minute fraction
of bacteria retained by the sand may be due to the physical straining
and/or surface characteristics attributed to angularity, the roughness
Discussion of Experimental Findings
of the collector surface.
With the further increase in ionic strength, there was a notice-
Effect of Ionic Strength on Adhesion and Deadhesion able decrease in the effluent concentration. For instance, when
of Bacteria tested using 30-mM ionic strength, the retention of bacteria in-
The adhesion, deadhesion, and transport mechanisms of S. pas- creased, and hence Ct =C0 declined to the range of 0.3–0.35 for both
teurii and Proteus species were studied using a saturated sand col- organisms. The microbial retention reached its maximum in the
umn. Based on the measured bacterial density, OD600 in the influent
and effluent, the normalized effluent concentration, Ct =C0 , was ob-
tained using Eqs. (9) and (10). The variation of normalized effluent Table 2. Transport parameters for different species over range of ionic
bacterial concentration with pore volume over a range of ionic strengths
strengths for both the species was obtained. The breakthrough Transport parameter
Ionic
curves (BTCs) revealed the effect of ionic strength on the adhesion
strength Species Dl
mechanism of microbes on the sand surface. Further, the experi- (mM) name (×10−6 m2 =s) R K p (L=kg) R2
mentally obtained BTCs were modeled using STANMOD to obtain
the bacterial transport parameters of saturated sand. 0 S. pasteurii 0.0025 1.14 27.9 0.99
For enhancing the confidence, the BTCs obtained using the 0.1 0.0025 1.15 27.9 0.99
1 1 1.20 35.91 0.99
analytical model were compared with that of experimentally ob-
10 1.76 32.9 5,789 0.94
tained BTCs for S. pasteurii as shown in Fig. 3. For the sake of 30 1.48 109 19,715 0.97
completeness, the BTCs pertaining to Proteus are reported in 50 2.23 2,336 423,540 0.99
the Supplemental Data (Fig. S3). It can be observed that the adhe- 100 128 16,070 2,991,475 0.83
sion and deadhesion values (rising and waning portion of the break- 0 Proteus 0.003 1.14 35.4 0.97
through curve) coincide with the fitted values. Table 2 depicts the 0.1 0.008 1.17 35.5 0.98
bacterial transport parameters such as D1 , R, and K p along with the 1 2.84 1.25 46 0.99
coefficient of determination R2 over a range of ionic strengths. 10 8 26.5 4,623 0.95
It is evident from the figures that the normalized bacterial 30 3.08 83.9 15,037 0.99
concentration in the effluent is insignificant up to 1 pore volume 50 128 16,070 2,991,475 0.83
100 128 16,070 2,991,475 0.83
irrespective of ionic strength of influent. This is due to the

© ASCE 04020038-5 J. Environ. Eng.

J. Environ. Eng., 2020, 146(6): 04020038

 cases of high ionic strength (50 and 100 mM), consequently strug-
gling to achieve microbial equilibrium between the influent and
effluent, which in turn resulted in negligible microbial density
in the effluent. This strong retardation of the species explains
the absence of a tailed flow as was evident in the rest of the ionic
strengths considered in this study. The behavior of an increase in
species retardation with ionic strength was clearly evident from the
obtained retardation coefficient values (Table 2). Fig. 3 also indi-
cates that beyond 1 pore volume, the effluent concentration was
increased steadily and reached a constant value of nearly 2–3 pore
volumes. The delay in arriving at the equilibrium concentration is
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.

attributed to physical deposition and/or physicochemical sorption

of the bacteria.
The microbial retardation can be due to the adhesion/sorption of
species. It was evident from the zeta potential analysis that the sur-
face negativity of the microbes and fine sand decrease with an in-
crease in ionic strength. This could be the reason for the increased
rate of microbial adhesion onto the sand surface. Incidentally, sim-
ilar adhesion behavior has been reported in the literature for various
bacterial species, including Pseudomonas putida, Escherichia coli, Fig. 4. Deadhesion of bacteria from sand column upon change of dead-
and Pseudomonas fluorescence, among others (Choi et al. 2017; hesion pore fluid chemistry.
Torkzaban et al. 2008).
After the bacterial adhesion (i.e., beyond 5 pore volumes),
desorption with deionized water having a similar solution chemis-
try (i.e., without bacteria in it) showed the deadhesion of the mi-
crobe under stable physicochemical conditions. In Figs. 3 and S3
for S. pasteurii and Proteus species, the tailing slope, which indi-
cates the bacterial deadhesion, varied with the initial ionic strength
of the effluent. For example, in the case of 100-mM ionic strength,
no microbial concentration in the effluent could be observed during
the desorption stage, indicating irreversible microbial adhesion on
to the sand surface. Further, at intermediate to high ionic strengths
(10–50 mM), the breakthrough curve showed a sudden drop, and
thereafter no microbial concentration was evident in the effluent.
Whereas for the neutral to low-ionic-strength values, i.e., up to
1 mM, the tailing slope showed complete flushing of organisms
from the sand column. The reason for significant deadhesion of
microorganisms in the aforementioned cases may be the weak
physical adhesion onto the sand surface, which allows the microbes
to be easily knocked out under a small hydrodynamic force (Choi
et al. 2017).
To add further clarity toward the reversible nature of microbial
adhesion, the deadhesion experiments were repeated on the sand
column where the bacteria were deposited early up to 5 pore vol- Fig. 5. Variation of normalized effluent concentration with pore
umes. First, the number of microbes retained in the sand column volume over a range of pH values for S. pasteurii and Proteus.
was calculated after completion of the deadhesion process corre-
sponding to the injection of 8 pore volumes. Afterward, additional
deadhesion experiments were performed by flushing the bacteria-
loaded sand matrix with solutions of ionic strength one order less percentage of released bacteria was above 95%, where most of the
than that of the initial value. The amount of bacteria released was retained bacteria were flushed away with the 0.1 mM solution.
calculated in terms of the cumulative percentage of bacteria con-
centration eluted from the sand column against the number of pore
volumes of influent injection, and the same is reported in Fig. 4. Effect of pH on the Attachment of Bacteria on to the
In the first set of deadhesion experiment, 10 mM ionic strength Sand Surface
solution was injected in the column in which bacteria were already The adhesion of S. pasteurii and Proteus species was also evaluated
adhered using the fixation fluid of 100-mM ionic strength. The over a pH range of 5–9 using a saturated sand column. In Fig. 5, the
fraction of released amount of bacteria was 0.7 and 0.8 for normalized effluent concentration Ct =C0 is plotted against the
S. pasteurii and Proteus species, respectively, which shows nearly number of pore volume, which demonstrated the effect of pH on
30%–35% of bacteria were still retained in the sand column after the adhesion mechanism. The obtained experimental values were
deadhesion using 8 pore volumes of solution (Fig. 4). In the second fitted with the analytical solution obtained using STANMOD.
set of deadhesion experiments, 0.1 mM ionic strength solution was The effluent concentration was zero for approximately 1 pore
injected in the microbial-sand column, which was initially saturated volume due to the undispersed bacterial flow and uniform displace-
with a bacterial solution having 10-mM ionic strength. Contrary to ment of the solution that previously existed in the saturated sand
the previous observations, the results indicated that the cumulative column. After 1 pore volume, the bacterial concentration started

© ASCE 04020038-6 J. Environ. Eng.

J. Environ. Eng., 2020, 146(6): 04020038

 increasing and reached mostly 0.9 within 2 pore volumes of flow.
There was not much variation in the effluent concentration in spite
of altering the pH of the pore solution for microbes used in this
study. Over the range of pH values considered, the BTCs were sim-
ilar to a piston flow, where most of the bacteria were flushed out
from the sand column without any retardation. Some microbes
were adhered to the sand surface by physical deposition or due
to the surface features of both bacteria and collector surface.
The fitted parameters, i.e., retardation coefficient and partition
coefficient obtained from the observed BTCs, were nearly similar
for all pH values considered. Incidentally, these values are compa-
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.
rable with those obtained using a low-ionic-strength solution
(i.e., 0.1 and 1.0 mM), and hence the estimated bacterial transport
parameters are not presented. The coefficient of determination R2 is
close to 0.99 for both bacteria over a range of pH values considered
in this study. This behavior of bacterial attachment can be explained
by considering the variation of bacterial and sand zeta potential val-
ues with pH (Fig. S2). Both the microbes and sand surface have
shown a buffer region and are highly electronegative in the pH
range of 5–9, which leads to elution of bacteria from the sand col-
umn. After 5 pore volumes in the effluent, the tailing showed all the
physically sorbed microbes were flushed from the sand column
over a wide range of pH conditions considered.
Substantiation of Interaction Mechanism of Microbes
with Sand Using Extended DLVO Theory
To comprehend the interaction mechanism of microbes with sand,
the total interaction energy profile for various ionic strengths as a
function of separation distance was plotted as depicted in Figs. 6
and S4 for S. pasteurii and Proteus species, respectively. It has been
reported in the literature that the double layer repulsion, van der
Waals interaction energy, and Born potential energy diminish
within a few nanometers from the surface of the sand. However,
the highest energy barrier and primary/secondary minima of bac-
terial interaction with the saturated sand column can occur at a sep-
aration distance of few micrometers. Given this, the present study
evaluated variation of total energy over a separation distance of
0–100 nm.
Fig. 6. (a) Total interaction energy as a function of separation distance
for S. pasteurii in different ionic strengths; and (b) indications of the
primary and secondary energy minima.
© ASCE 04020038-7
J. Environ. Eng., 2020, 146(6): 04020038
From Figs. 6 and S4, it can be seen that the energy profile had
different curves, and for the produced energy barrier, primary or
secondary minima varied with different ionic strengths. In the
low-ionic-concentration region (i.e., 0.1 mM), the energy profile
did not have any primary or secondary minimum with a high en-
ergy barrier of 1,988 and 1,794 kT for S. pasteurii and Proteus
species, respectively. Hence, both the bacteria and sand surface
exhibited a high repulsive force, and bacteria could not adhere
to the sand surface (as indicated in Fig. 3). For 1-mM ionic
strength, the energy barrier was 1,175 and 600 kT without any
primary or secondary energy minima for S. pasteurii and Proteus
species, respectively.
The corresponding flow-through column experiments showed
nominal retardation of bacteria within the sand matrix. With further
increases in the ionic strength value to the intermediate range of
10–30 mM, the energy barrier reduced, and consecutively, a sec-
ondary minima was noticed in the interaction energy curve. In this
ionic strength range, at first, bacteria repelled from the sand surface
with the produced repulsive force but retained or adhered to the
collector surface in the secondary energy minimum. The aforemen-
tioned mechanism of the rate of adhesion, which increased with the
ionic strength value, was noticeably shown in the breakthrough
curve experiments (Figs. 3 and S3). In the case of high ionic con-
centrations of 50 and 100 mM, the energy barrier reduced to nil,
and the energy profile showed a high primary energy minimum.
Bacteria in this ionic strength value could easily attach to the sand
surface in the primary minima without any energy barrier. For this
reason, in the higher-ionic-strength range, most of the bacteria ad-
hered to the sand surface, and the bacterial concentration in the
effluent was found to be nearly zero.
By decreasing the ionic strength from 100 to 10 mM during the
deadhesion experiment, bacteria attached in the primary minima
experienced a sudden high energy barrier and moved away from
the sand surface. However, the released bacteria were captured
by the sand during occurrence of the secondary energy minimum.
In the second set of deadhesion experiments, when the ionic
strength decreased from 10 to 0.1 mM, bacteria encountered a
higher energy barrier without any secondary energy minima; as
a result, all the adhered bacteria flushed away from the sand
Fig. 7. Total interaction energy as a function of separation distance for
S. pasteurii over a range of pH values.
J. Environ. Eng.
 column. The BTCs reported in Fig. 4 show that decreasing the con-
centration from 100 to 10 mM and from 10 to 0.1 mM released 70%
and 100% of the bacteria that were retarded during adhesion pro-
cess, respectively. It can be summarized from the deadhesion ex-
periment and the energy profile that the irreversibility nature of
microbial adhesion depends on the ionic strength value of the pore
solution.
The interaction energy profiles for different pH as a function
of separation distance are plotted in Figs. 7 and S5. It can be
observed that the total interaction energy profile varies with
the pH value, exhibiting a higher energy barrier without any pri-
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.
findings of the study it is possible to achieve homogenous
biomineralization in the field by engineering the uniform
distribution of the microbes and by ensuring the availability
of nutrient and cementation reagents in the subsurface zone of
interest.
Data Availability Statement
All data used during the study appear in the published article. There
are no codes and models associated with this submission.

mary or secondary energy minima for both the microbes considered

in this study. In the neutral pH value, the energy profile was
having the highest energy barrier of 2,400 and 2,225 kT for S. pas- Notation
teurii and Proteus species, respectively. This can be corroborated
with the breakthrough response of microbial adhesion over a The following symbols are used in this paper:
range of pH. Because bacteria and sand repelled each other A = Hamaker constant;
with a high energy barrier/repulsive force, most of the bacteria ap = equivalent radius of the bacteria;
eluted and could not adhere to the sand surface, as shown in C = concentration;
Fig. 5. C0 = influent concentration;
Ct = effluent concentration at time t;
Ct =C0 = normalized effluent concentration;
Conclusions D1 = longitudinal hydrodynamic dispersion coefficient;
A set of flow-through column experiments were conducted by EBorn = Born interaction energy;
varying the pH and ionic strength of the pore solution to investigate EEDL = summation of electrostatic double layer repulsion;
the adhesion and deadhesion of ureolytic microbes in a porous ETot = total interaction energy;
medium. Based on the analysis, the following significant conclu- EVDW = van der Waals interaction energy;
sions can be drawn from this study: Ez = electrical field strength;
• The surface-charge characteristics of ureolytic bacteria and sand e = electron charge (1.602 × 10−19 C);
get altered significantly upon the change in ionic strength of the h = separation distance between the microbes and collector
pore fluid. The modification of surface charge is responsible for surface;
altering total interaction energy between the microbes and sand I = ionic strength;
surface. At a low ionic strength, adhesion was prevented by the K = Boltzmann’s constant;
significant energy barrier produced by the repulsive electrostatic K p = distribution coeffcicent;
interaction of highly negatively charged bacteria and sand. An k = inverse of Debye length (1=m);
increase of ionic strength caused the reduction of the repulsive
N A = Avogadro’s number;
force, and hence the bacteria could adhere to the sand surface in
the primary or secondary energy minima. OD600 = optical density at 600 nm;
• Microbial adhesion was found to be reversible upon the change R = retardation coefficient;
in ionic strength of the pore fluid. A decrease in ionic strength R2 = coefficient of determination;
enhances the negativity of the surface charge of microbes, de- T = absolute temperature in Kelvin;
veloped the energy barrier, and is responsible for the release of t = time;
the retained bacteria from the sand surface. vavg = average electro-osmosis velocity;
• The slight difference in the observed rate of adhesion and dead- vf = seepage velocity;
hesion of two different microorganisms can be attributed to the x = length of soil column;
minute variation in the surface charge and size of microbes. εp = permittivity of the pore fluid;
Other parameters such as surface characteristics, i.e., cell wall εr = relative dielectric permittivity;
characteristics, hydrophobicity, and surface features, may also ε0 = vacuum permittivity;
play a role in the adhesion and deadhesion mechanisms, which
η = porosity;
demands a systematic study.
λ = characteristic wavelength;
• The physical deposition of bacteria in the deionized water as
well as in low-ionic-strength solutions may be attributed to μ = viscosity;
the surface roughness and hydrophobicity of the sand and ζ = zeta potential;
microbes. ζ b = zeta potential value of bacteria;
• Microbial adhesion was not significantly affected by the varia- ζ s = zeta potential value of sand grains;
tion in pH of the pore solution. Because the surface charge of ρ = density; and
microbes and sand were highly negative in the pH range of 5–9, σB = Born collision diameter.
it encountered a strong repulsive electrostatic force and a high
energy barrier. As a result, the microbes failed to adhere to the
sand surface. Supplemental Data
• Overall, the study demonstrated the role of pore fluid chemistry
on adhesion, deadhesion, and transportation of ureolytic bacter- Figs. S1–S5 are available online in the ASCE Library (www
ia in the sand, which is of practical relevance. Based on the .ascelibrary.org).

© ASCE 04020038-8 J. Environ. Eng.

J. Environ. Eng., 2020, 146(6): 04020038

 References
Achal, V., and X. Pan. 2011. “Characterization of urease and carbonic
anhydrase producing bacteria and their role in calcite precipitation.”
Curr. Microbiol. 62 (3): 894–902. https://doi.org/10.1007/s00284
-010-9801-4.
Anbu, P., C. H. Kang, Y. J. Shin, and J. S. So. 2016. “Formations of
calcium carbonate minerals by bacteria and its multiple applications.”
SpringerPlus 5 (1): 1–26. https://doi.org/10.1186/s40064-016-1869-2.
ASTM. 2006a. Standard practice for classification of soils for engineering
purposes (Unified Soil Classification System). ASTM D2487. West
Conshohocken, PA: ASTM.
Downloaded from ascelibrary.org by "Indian Institute of Technology, Kanpur" on 02/20/22. Copyright ASCE. For personal use only; all rights reserved.
ASTM. 2006b. Standard test method for specific gravity of soil solids by
gas pycnometer. ASTM D5550. West Conshohocken, PA: ASTM.
ASTM. 2007. Standard test method for particle size analysis of soils.
ASTM D422. West Conshohocken, PA: ASTM.
Cheng, L., M. A. Shahin, R. Cord-Ruwisch, M. Addis, T. Haranto, and
C. Elms. 2014. “Soil stabilisation by microbial induced calcium carbon-
ate precipitation: Investigation of some important physical and environ-
mental aspects.” In Proc., 7th Int. Congress on Environmental
Geotechnics, 1–8. New York: Curran Associates.
Choi, N. C., J. W. Choi, K. S. Kwon, S. G. Lee, and S. Lee. 2017. “Quan-
tifying bacterial attachment and detachment using leaching solutions of
various ionic strengths after bacterial pulse.” AMB Express 7 (1): 38.
https://doi.org/10.1186/s13568-017-0340-2.
Chu, J., V. Ivanov, and M. Naeimi. 2014. “Optimization of calcium-
based bioclogging and biocementation of sand.” Acta Geotech. 9 (2):
277–285. https://doi.org/10.1007/s11440-013-0278-8.
Fauriel, S., and L. Laloui. 2012. “A bio-chemo-hydro-mechanical model
for microbially induced calcite precipitation in soils.” Comput. Geotech.
46 (Nov): 104–120. https://doi.org/10.1016/j.compgeo.2012.05.017.
Freeze, R. A., and J. N. Cherry. 1979. Ground water. Englewood Cliffs, NJ:
Prentice Hall.
Ginn, T. R., B. D. Wood, K. E. Nelson, T. D. Scheibe, E. M. Murphy, and
T. P. Clement. 2002. “Processes in microbial transport in the natural
subsurface.” Adv. Water. Res. 25 (8–12): 1017–1042. https://doi.org/10
.1016/S0309-1708(02)00046-5.
Hahn, M. W., D. Abadzic, and C. R. O’Melia. 2004. “Aquasols: On the role
of secondary minima.” Environ. Sci. Technol. 38 (22): 5915–5924.
https://doi.org/10.1021/es049746d.
Harkes, M. P., L. A. van Paassen, J. L. Booster, V. S. Whiffin, and M. C. M.
van Loosdrecht. 2010. “Fixation and distribution of bacterial activity in
sand to induce carbonate precipitation for ground reinforcement.” Ecol.
Eng. 36 (2): 112–117. https://doi.org/10.1016/j.ecoleng.2009.01.004.
Hogg, R., T. W. Healy, and D. W. Fuerstenau. 1966. “Mutual coagulation of
colloidal dispersions.” Trans. Faraday Soc. 62 (2): 1638–1651. https://
doi.org/10.1039/tf9666201638.
Hsu, B. M., and C. Huang. 2002. “Influence of ionic strength and pH on
hydrophobicity and zeta potential of Giardia and Cryptosporidium.”
Colloids Surf., A 201 (1–3): 201–206. https://doi.org/10.1016/S0927
-7757(01)01009-3.
Jain, S., and D. N. Arnepalli. 2019. “Biochemically induced carbonate pre-
cipitation in aerobic and anaerobic environments by Sporosarcina
pasteurii.” Geomicrobiol. J. 36 (5): 443–451. https://doi.org/10.1080
/01490451.2019.1569180.
Kim, S. B., S. J. Park, C. G. Lee, N. C. Choi, and D. J. Kim. 2008. “Bacteria
transport through goethite-coated sand: Effects of solution pH and
coated sand content.” Colloids Surf., B 63 (2): 236–242. https://doi
.org/10.1016/j.colsurfb.2007.12.003.
© ASCE 04020038-9
J. Environ. Eng., 2020, 146(6): 04020038
Kollannur, N. J., and D. N. Arnepalli. 2019. “Methodology for determining
point of zero salt effect of clays in terms of surface charge properties.”
J. Mater. Civ. Eng. 31 (12): 04019286. https://doi.org/10.1061/(ASCE)
MT.1943-5533.0002947.
Li, X., C. Lin, J. D. Miller, and W. P. Johnson. 2006. “Pore-scale
observation of microsphere deposition at grain-to-grain contacts over
assemblage-scale porous media domains using X-ray microtomogra-
phy.” Environ. Sci. Technol. 40 (12): 3762–3768. https://doi.org/10
.1021/es0525004.
Loosdrecht, M. C. M. V., J. Lyklema, W. Norde, and A. J. B. Zehnder.
1989. “Bacterial adhesion: A physiochemical approach.” Microb. Ecol.
17 (1): 1–15. https://doi.org/10.1007/BF02025589.
Mills, A. L., J. S. Herman, G. M. Hornberger, and T. H. Dejesust.
1994. “Effect of solution ionic strength and iron coatings on mineral
grains on the sorption of bacterial cells to quartz sand.” Appl. Environ.
Microbiol. 60 (9): 3300–3306. https://doi.org/10.1128/AEM.60.9.3300
-3306.1994.
Mitchell, J. K., and J. C. Santamarina. 2005. “Biological considerations in
geotechnical engineering.” J. Geotech. Geoevviron. 131 (10): 1222–
1233. https://doi.org/10.1061/(ASCE)1090-0241(2005)131:10(1222).
Nemati, M., E. A. Greene, and G. Voordouw. 2005. “Permeability profile
modification using bacterially formed calcium carbonate: Comparison
with enzymic option.” Process Biochem. 40 (2): 925–933. https://doi
.org/10.1016/j.procbio.2004.02.019.
Nikhil John, K., and D. N. Arnepalli. 2019. “Factors influencing zeta
potential of clayey soils.” In Geotechnical characterisation and geoen-
vironmental engineering, edited by V. Stalin and M. Muttharam, 171–
178. Dordrecht, Netherlands: Springer.
Ohmori, K., and C. E. Glatz. 1999. “Effects of pH and ionic strength on
microfiltration of C. glutamicum.” J. Membr. Sci. 153 (1): 23–32.
https://doi.org/10.1016/S0376-7388(98)00239-7.
Redman, J. A., S. L. Walker, and M. Elimelech. 2004. “Bacterial adhe-
sion and transport in porous media: Role of the secondary energy mini-
mum.” Environ. Sci. Technol. 38 (6): 1777–1785. https://doi.org/10
.1021/es034887l.
Roy, S., S. Ghosh, N. Bhowmick, and P. K. R. Choudhury. 2019. “Role
of physicochemical factors on bacterial attachment in textile fibrous
media.” Water Environ. J. 33 (1): 21–30. https://doi.org/10.1111/wej
.12365.
Toride, N., F. J. Leij, and M. T. van Genuchetn. 1999. The CXTFIT code for
estimating the transport parameters from laboratory or field tracer
experiments: Version 2.2. Research Rep. No.137. Riverside, CA:
United States Salinity Laboratory, Agricultural Research Service.
Torkzaban, S., S. S. Tazehkand, S. L. Walker, and S. A. Bradford. 2008.
“Transport and fate of bacteria in porous media: Coupled effects of
chemical conditions and pore space geometry.” Water Resour. Res.
44 (4): 1–12. https://doi.org/10.1029/2007WR006541.
Whiffin, V. S., L. A. van Paassen, and M. P. Harkes. 2007. “Microbial car-
bonate precipitation as a soil improvement technique.” Geomicrobiol. J.
24 (5): 417–423. https://doi.org/10.1080/01490450701436505.
Zhang, H., and M. S. Olson. 2012. “Effect of heavy metals on bacterial
attachment in soils.” J. Environ. Eng. 138 (11): 1106–1113. https://doi
.org/10.1061/(ASCE)EE.1943-7870.0000573.
Zhao, Q., L. Li, C. Li, M. Li, F. Amini, and H. Zhang. 2014. “Factors
affecting improvement of engineering properties of MICP-treated
soil catalyzed by bacteria and urease.” J. Mater. Civ. Eng. 26 (12):
04014094. https://doi.org/10.1061/(ASCE)MT.1943-5533.0001013.
J. Environ. Eng.