Engineered applications of ureolytic 
biomineralization: A review
Authors: Adrienne J. Phillips, Robin Gerlach, Ellen 
Lauchnor, Andrew C. Mitchell, Alfred B. Cunningham, 
& Lee Spangler
NOTICE: his is an Accepted Manuscript of an article published in Biofouling on July 2013, 
available online: http://www.tandfonline.com/10.1080/08927014.2013.796550. 
Phillips AJ, Gerlach R, Lauchnor E, Mitchell AC, Cunningham AB, Spangler L, "Engineered 
applications of ureolytic biomineralization: A review," Biofouling 2013 29(6):715-733.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Microbialy-induced calcium carbonate (CaCO3) precipitation (MICP) is a widely explored and promising technology foruse in various engineering applications. In this review, CaCO3precipitation inducedviaurea hydrolysis (ureolysis) isexamined for improving construction materials, cementing porous media, hydraulic control, and remediating environmen-
tal concerns. The control of MICP is explored through the manipulation of three factors: (1) the ureolytic activity
(of microorganisms), (2) the reaction and transport rates of substrates, and (3) the saturation conditions of carbonate
minerals. Many combinations of these factors have been researched to spatialy and temporaly control precipitation. This
review discusses how optimization of MICP is atempted for diferent engineering applications in an efort to highlight
the key research and development questions necessary to move MICP technologies toward commercial scale applica-
tions.
Keywords:calcium carbonate; urea hydrolysis; biofilm; MICP; mineral precipitation; mineralization
Introduction
Contrary to the commonly known detrimental efects of
biofilms in industrial and medical environments, biofilms
may be used for beneficial engineering applications. In
particular, ureolytic biofilms or microbes which induce
calcium carbonate (CaCO3) precipitation (MICP) have
been studied widely for beneficial use in construction
materials, cementation of porous media, hydraulic
control, and environmental remediation (Figure 1). A pri-
mary research focus has been controling MICP by
manipulating parameters that influence the saturation
state to achieve specific engineering goals. In many
cases, engineered applications depend on controling the
rate and distribution of CaCO3 precipitationin situ,
which is governed by the spatial and temporal variation
in saturation state.
Several reviews addressing MICP for use in engi-
neering, particularly, construction applications and
cementation of porous media have been prepared previ-
ously. De Muynck, De Belie, et al. (2010) elegantly
reviewed the role of MICP in enhancing and rehabilitat-
ing construction materials. Siddique and Chahal (2011)
also reviewed MICP for use in construction materials,
specificaly focusing on concrete. Separately, Ivanov and
Chu (2008) and DeJong et al. (2010, 2011) comprehen-
sively highlighted the role of the biogeochemical MICP
processes in soil and porous media systems. In addition,
Al-Thawadi (2011) reviewed MICP for strengthening
of sand. This review focuses on how the spatial and
temporal control of MICP has been explored to treat
construction materials, consolidate porous media, control
hydraulics and remediate environmental problems.
Microbialy-induced CaCO3precipitation
The involvement of microorganisms in mineral precipita-
tion occursviadiferent mechanisms (Benzerara et al.
2011; Northup & Lavoie 2001; Fouke 2011). Firstly, bio-
logicaly-controled mineralization describes celular
activities which specificaly direct the formation of the
mineral, for example, the cel mediated process of exo-
skeleton, bone or teeth formation, or the formation of
intracelular magnetite crystals by magnetotactic bacteria
(Decho 2010; Benzerara et al. 2011). Secondly, biologi-
caly-influenced mineralization is the process by which
passive mineral precipitation is caused through the pres-
ence of cel surfaces or organic mater such as extracelu-
lar polymeric substances (EPS) associated with biofilm
(Decho 2010; Benzerara et al. 2011). Thirdly, biologi-
caly-induced mineralization is the chemical alteration of
an environment by biological activity that generaly
results in supersaturation and precipitation of minerals
(Stocks-Fischer et al. 1999; De Muynck, De Belie, et al.
2010). Often combinations of the three diferent pro-
cesses are active at the same time in a system. For
Engineered applications of ureolytic biomineralization: a review
Adrienne J. Philipsa,b*, Robin Gerlacha,b*, Elen Lauchnora, Andrew C. Mitchelc, Alfred B. Cunninghama,d
and Lee Spanglere
aCenter for Biofilm Engineering, Montana State University, Bozeman, MT, USA; bChemical and Biological Engineering Department, Montana State University, Bozeman, MT, USA; cInstitute of Geography & Earth Sciences, Aberystwyth University, Wales, UK; dCivil 
Engineering Department, Montana State University, Bozeman, MT, USA; eEnergy Research Institute, Montana State University, Boze-
man, MT, USA
Figure 1. Proposed ureolysis-driven MICP engineering applications. White crystal hatch patern represents CaCO3. (a) Sealingsubsurface hydraulic fractures (eg during wel closure); (b) manipulating subsurfaceflow paths to improve oil recovery; (c)
strengthening earthen dams or consolidating porous materials; (d) minimizing dust dispersal from surfaces; (e) sealing or remediating
concrete fractures; (f) coating PCB-oil contaminated concrete resulting from leaking equipment; (g) treating or coating limestone or
concrete to minimize acid erosion; (h) sealing ponds or reservoirs; (i) forming subsurface bariers to control salt water or
contaminated groundwater intrusion; (j) remediating the subsurface contaminated with radionuclides or toxic metals (represented by
radioactivity symbols); (k) treating fractures (in cap rocks, wel bore cements, or casing/cement/formation interfaces) to mitigate
leakage from geologicaly sequestered CO2injection sites.
instance, in the case of microbialy-induced calcium car-
bonate precipitation or mineralization (MICP), where the
celular activity influences chemical conditions (satura-
tion state) to promote mineralization, it is possible that
biologicaly-influenced mineralization is also occuring
since the cels themselves or their exudates may act as
nucleation sites for CaCO3crystal formation (Stocks-
Fischer et al. 1999).
MICP can occur as a byproduct of urea hydrolysis,
photosynthesis, sulfate reduction, nitrate reduction, or
any other metabolic activity that leads to an increase in
the saturation state of calcium carbonate (DeJong et al.
2010; Benzerara et al. 2011). This review focuses on
urea hydrolysis (ureolysis) to promote CaCO3precipita-
tion. In ureolysis-driven MICP, the celular or urease
enzyme activity influences chemical conditions (the satu-
ration state) to promote mineralization through four fac-
tors: (1) dissolved inorganic carbon (DIC) concentration,
(2) pH, (3) calcium concentration, and (4) potential
nucleation sites (Hammes & Verstraete 2002). Thefirst
three factors determine the saturation state, because DIC
and pH influence the carbonate ion concentration or
activity {CO32}. The fourth factor impacts the critical
saturation state (Scrit), which is the saturation state at
which nucleation (ie precipitation) occurs under the
given conditions. Additionaly, the species and the con-
centration of microbe(s), their ureolytic activity, the form
of microbial growth (ie biofilm or planktonic), tempera-
ture, salinity, injection strategy (ieflow rate, treatment
times), and reactant concentration (or activity) may
impact the saturation conditions and the efficiency and
extent of CaCO3 precipitation (Harkes et al. 2010;
Okwadha & Li 2010; Mortensen et al. 2011; Cuthbert
et al. 2012). Carefuly manipulating: (1) the ureolytic
activity of microorganisms, (2) the reaction and transport
rates of substrates, and (3) the saturation state may
greatly influence treatment efficacy.
Ureolytic activity of microorganisms
The urease enzyme can be found in a wide variety of
microorganisms (Mobley & Hausinger 1989; Hammes
et al. 2003) and contributes to the ability of the cel to
utilize urea as a nitrogen source (Feris et al. 2003;
Burbank et al. 2012). While urease production is quite
common across a wide range of soil organisms and
found in other natural environments, in the laboratory,
many researchers have examined ureolytic MICP using
the common soil organismSporosarcina pasteuriATCC
11859, formerlyBacilus pasteuri (Yoon et al. 2001).
S. pasteuriis non-pathogenic, does not readily aggregate
under most growth conditions, and produces large
quantities of active intracelular urease (Feris et al.
1996; Stocks-Fischer et al. 1999; DeJong et al. 2006).
S. pasteurihas been isolated from soil, water, sewage,
and urinal incrustations (De Muynck, De Belie, et al.
2010).
One disadvantage of studying laboratory strains is the
microbial complexity of real world environments. In the
context of soil stabilization, it was noted that injection of
these organisms may result in non-homogeneous distribu-
tion of the microbes, or the organisms may face chalenges
of competition or predation from native organisms (van
Veen et al. 1997; van Elsas et al. 2007; Burbank et al.
2011). As such, to maintain ureolytic populations in sub-
surface applications, it may be advantageous to stimulate
native atached (biofilm) ureolytic populations rather than
augmenting the environment with laboratory strains not
adapted to the treatment environment (Fujita et al. 2010;
DeJong et al. 2011; Tobler et al. 2011; Burbank et al.
2012). Also, when considering the augmentation of the
subsurface with certain organisms, particularlyS. paste-
uri,described as a facultative anaerobe (Feris et al. 1996;
Tobler et al. 2011) and more recently as an obligate aerobe
(Martin et al. 2012), it is important to consider the impact
of electron acceptors (for example, oxygen in the case of
S. pasteuri) on microbial growth. Although ureolytic
activity itself does not depend upon oxygen (Mortensen
et al. 2011), microbial growth and urease production could
be limited by the availability of electron acceptor. It has
been demonstrated thatS. pasteuricannot anaerobicaly
synthesizede novourease; therefore the active urease may
be limited to the existing enzyme injected with the aerobi-
caly grown inoculum (Martin et al. 2012). To overcome
chalenges associated with growth-coupled urease produc-
tion, stimulation of native populations, injection of elec-
tron-acceptor rich growth medium, or the injection of
urease enzyme might be considered.
Additionaly, mineral precipitation around cels can
influence ureolytic activity by either causing cel inactiva-
tion through membrane disruption or by limiting nutrient
transport to the cel (Stocks-Fischer et al. 1999; Parks
2009; Cuthbert et al. 2012). Zamareńo et al. (2009) sug-
gest that precipitation and entombment might be a passive
process, which the organisms cannot help but be involved
in. Alternatively, they suggest that the precipitation pro-
tects cels for a short period of time from detrimental cal-
cium concentrations. In an engineering application, it is
important to consider that entombment may lead to
reduced ureolysis and potentialy limit further precipita-
tion. To overcome inactivation and promote additional
CaCO3precipitation, resuscitation or reinjection of organ-
isms as wel as additional treatments may be required to
maintain an active ureolytic population and maximize pre-
cipitation (Tobler et al. 2011; Ebigbo et al. 2012).
Reaction and transport
Chemical reactions.During ureolysis-driven MICP, ure-
ase catalyzes the hydrolysis of one mole of urea to form
one mole of ammonia and one mole of carbamic acid
(Equation 1), which spontaneously hydrolyses to
carbonic acid and another mole of ammonia (Equation
2). Under circum-neutral conditions, the two moles of
ammonia become protonated by deprotonating water to
form two moles of ammonium (NH4+) and two moles of
hydroxide ions (Equation 3). The generated hydroxide
ions shift the equilibrium of DIC species towards bicar-
bonate (HCO3) and carbonate (CO32) (Equations 4
and 5) (Stocks-Fischer et al. 1999; Dick et al. 2006;
Mitchel et al. 2010).
COðNH2Þ2þH2O !UreaseNH2COOHþNH3 ð1Þ
NH2COOHþH2O !SpontaneousNH3þH2CO3 ð2Þ
2NH3þ2H2O !2NHþ4þ2OH ð3Þ
H2CO3 !HCO3þHþ ð4Þ
HCO3þHþþ2OH !CO23 þ2H2O ð5Þ
In the presence of sufficient calcium ion activity, sat-
uration conditions become favorable for CaCO3precipi-
tation (Equation 6).
Ca2þþCO23 !CaCO3ðsÞ ð6Þ
Kinetics of reactions.Although urease increases urea ure-
olysis rates 1014times over uncatalyzed rates (Mitchel
& Feris 2005), ureolysis is the rate-limiting step in
MICP. The concentration of bacteria, temperature, pH,
saturation conditions, and salinity have been shown to
influence ureolysis kinetics (Feris et al. 2003; Dupraz,
Parmentier, et al. 2009; Tobler et al. 2011). In general, a
higher concentration of cels producing urease has been
shown to positively impact the rate of ureolysis, as have
elevated (20 °Cvs10 °C) temperatures (Feris et al.
2003; Mitchel & Feris 2005; Tobler et al. 2011).
Several models to predict rates of ureolysis can be
considered. In conditions of excess urea, a zero order
model might be appropriate, where the rate of ureolysis,
rurea, is equal to the rate constant and not influenced by
the urea concentration [urea] (Equation 7):
rurea¼½ureatime¼ kurea ð7Þ
Most commonly,first order rate models are presented
(Equation 8) (Feris et al. 2003; Mitchel & Feris 2005;
Tobler et al. 2011; Cuthbert et al. 2012), where the ureol-
ysis rate is dependent on the urea concentration:
rurea¼ kurea½urea ð8Þ
Ureolysis rates have also been modeled using
Michaelis–Menten type expressions that include a term
accounting for non-competitive inhibition by ammonium
(Equation 9) (Fidaleo & Lavecchia 2003; Ebigbo et al.
2012). Herevmaxis the maximum rate of ureolysis,Km
is the half saturation coefficient, [P] is the concentration
of ammonium, andKP is an inhibition constant for
ammonium:
rurea¼ vmax½ureaðKmþ½ureaÞ1þ½PKP
ð9Þ
The rates of ureolysis are dependent on a wide range
of factors and have been extensively studied in MICP
systems, particularly in laboratory batch systems. Simple
batch studies with planktonic cels produce valuable
parameters to be used in MICP models, recognizing that
the same parameters may not be fuly transferable when
considering values associated with biofilm. Models can
help develop understanding of more complex environ-
ments not easily studied in the laboratory.
Transport.Influid systems relevant to MICP, both
advective and difusive transport occurs, and dominance
of one or the other depends on the system. Advection
refers to movement of a species withfluidflow. Difu-
sion refers to the movement of species independent from
the bulkfluid movement and driven by concentration or
electrostatic potential gradients. Thefluidflow conditions
(such as whether theflow is laminar or turbulent, axial
or radial) and thefluid properties (density and viscosity)
influence the advective and difusive properties of the
species transport. In MICP application, transport condi-
tions may be complex, particularly in the case of radial
flow where thefluid velocity changes with distance.
Damköhler (Da) number.The dimensionless Da number,
which describes the ratio of reaction rate to transport
rate, may serve as an important design tool in MICP
application. In biogeochemical processes such as MICP,
the reactions (particularly ureolysis) are coupled to the
transport of the reactive species. In general terms, Da
relates the reaction rate of a species to the advective or
difusive mass transport rate of that species (Equation
10) (Berkowitz & Zhou 1996; Dijk & Berkowitz 1998;
Domenico & Schwartz 1998).
Da¼Reaction rateTransport rate ð10Þ
More specificaly, Da depends on the kinetics of the
reaction and the transport through a specific reactor (or
natural) system. For example, in a plugflow system
where advective transport dominates, Da represents a
ratio of the reaction rate to the advective mass transport
rate of the species. When Da < 1, it does not indicate that
reaction is not occuring; it does, however, imply that
not al the supplied substrate is utilized and may be
transported from the reaction zone. Da values >1 indicate
that the reaction is limited by the transport rate for a
given length scale.
In a pulsedflow system or within stagnant pore
spaces, where difusive transport is likely to dominate,
Da is the ratio of reaction rate to the efective difusion
rate of the reactive species. In difusion dominated
cases, a Da < 1 indicates the reaction rate is limited by
reaction kinetics rather than difusion; however, given
enough time, the reaction may proceed to completion.
Alternatively, Da >1 indicates the reaction rate drives
the establishment of concentration gradients of reactive
species.
Da incorporates many of the factors related to reac-
tion and transport into a single unitless number, for ease
of comparison and design. The systematic analysis of Da
may reveal a functional design tool (for example, pre-
dictingflow rates or pulsed treatment times) for MICP
not previously explored.
Saturation conditions
CaCO3precipitation is ultimately governed by the satura-
tion state (SorΩ) of calcium carbonate where {Ca2+} and
{CO32} represent the activities of Ca2+and CO32 ions,
which are approximately equal to concentration for low
ionic strength conditions, andKsois the temperature-
dependent equilibrium solubility constant (Equation 11):
SorX¼fCa
2þgfCO23g
Kso ð11Þ
AtS= 1, the solution is considered in equilibrium
with the solid phase. IfS> 1, the solution is considered
supersaturated with respect to CaCO3and CaCO3precip-
itation is thermodynamicaly favored. IfS< 1, the solu-
tion is considered undersaturated and dissolution of solid
phase CaCO3, if present, is thermodynamicaly favorable
(Figure 2) (Stumm & Morgan 1996). The saturation
index (SI) is represented as the log10of the saturation
state (Equation 12). When SI is positive, then the solu-
tion is supersaturated andvice versa. Further detailed
calculations can be found in several publications of
potential interest to the reader (Feris et al. 2003; Dup-
raz, Parmentier, et al. 2009; Tobler et al. 2011).
SI¼log10ðSÞ ð12Þ
While the Sor SI predicts whether precipitation is
thermodynamicaly favored, it does not necessarily pre-
dict the saturation state at which precipitation begins
(Scrit).Scritor SIcritare empirical values which reflect
how highly supersaturated a solution must become
before precipitation is observed. This critical supersatu-
ration is related to overcoming the nucleation activation
free energy barier (Feris et al. 2003) and is likely
impacted by a variety of system parameters influencing
the activity of Ca2+and CO32 ions. Saturation values
in the literature for batch systems have been reported in
the range ofS= 12–436 (Feris et al. 2003; Mitchel &
Feris 2005, 2006a; Dupraz, Parmentier, et al. 2009;
Tobler et al. 2011).Scritmay depend on many factors,
including the kinetics of ureolysis, the initial cel den-
sity, the presence of nucleation points, and the presence
of organics.
Nucleation.As outlined above, it is quite possible that
combinations of diferent biomineralization processes are
active at the same time in a system. For instance, while
ureolysis can increase the saturation state of the bulk
environment (biologicaly-induced mineralization) the
precipitation process itself might be initiated by the
microbes serving as nucleation sites (biologicaly-influ-
enced mineralization) (Figure 3a and b) (Stocks-Fischer
et al. 1999; De Muynck, De Belie, et al. 2010). Once
Figure 2. Influence of saturation on precipitation in a cross
section of a groundwater aquifer (precipitates are represented
by white crystal hatch patern). Saturation states >1 (S > 1) and
saturation indices >0 (SI > 0) indicate that precipitation is
thermodynamicaly favored; saturation states <1 (S < 1) and
saturation indices <0 (SI < 0) indicate that dissolution is favored
if the mineral form is present. The saturation state can vary
spatialy and temporaly due to reaction and transport rates
which create concentration gradients (Zhang et al. 2010).
precipitation has commenced, ureolysis may maintain a
high SI and cels as wel as newly precipitated minerals
likely act as additional templates or nucleation sites to
facilitate crystal growth (Stocks-Fischer et al. 1999;
Hammes 2002). While the influence of cel surfaces as
nucleation sites has been widely discussed (Douglas &
Beveridge 1998), Mitchel and Feris (2006b) observed
an equalScritin solutions with and without bacterial cels
separated by dialysis membranes that alowed for trans-
port of solutes between the two solutions. In addition,
CaCO3nucleation has been noted in a variety of systems
to be influenced by the presence of certain proteins,
microbial biomolecules, EPS, other available passive
substrates, heterogeneous nucleation on botle wals or
be solely occuring homogeneously in solution (Mitchel
& Feris 2006b; Dupraz, Parmentier, et al. 2009; Fouke
2011).
Mineralogy.Three primary polymorphs of CaCO3exist:
calcite, vaterite, and aragonite. It is wel known that sur-
face-atached communities of microorganisms, or bio-
films, secrete EPS rich in polysaccharides and other
organic macromolecules. EPS and organic mater have
been linked to the formation of vaterite which may be
stabilized in the presence of certain organics (Braissant
et al. 2003; Rodriguez-Navaro et al. 2007). Vaterite has
been found as a minor, meta-stable, or transitional phase
in the formation of calcite (Tourney & Ngwenya 2009).
The maturation of CaCO3from vaterite to calcite may be
described by the Ostwald Step Rule where metastable
forms nucleate and then are replaced with more stable
forms, a sequential formation in time also known as
paragenesis (Morse & Casey 1988). The mechanisms of
initial nucleation, which may be influenced by the micro-
bial growth conditions, the presence of certain organics,
such as EPS, or the saturation conditions of thefluid, as
wel as subsequent maturation are not completely under-
stood (Morse & Casey 1988; Jiménez-López et al. 2001;
Braissant et al. 2003; Zamareńo et al. 2009). Crystal
size may be a factor in the efficacy of an MICP technol-
ogy. CaCO3 crystals precipitatedviaureolysis-driven
MICP have been observed to be generaly larger and less
soluble than those precipitated under the same abiotic
bulk solution conditions (Mitchel & Feris 2006b;
Mitchel et al. 2013).
To summarize, CaCO3precipitationviaureolysis-dri-
ven MICP is initiated by creating conditions oversaturat-
ed with respect to CaCO3, likely combined with the
increased abundance of cel surfaces as nucleation points
at the point of critical saturation andfinaly crystal
growth on nuclei (Feris et al. 2003).
Engineering applications
Construction materials
Biodeposition
Biodeposition refers to the deposition of MICP to protect
the surface of porous materials (such as limestone, con-
crete, or bricks) from water intrusion. MICP treatment
can decrease the ability of a material to absorb water,
restore the surface, and reduce further potential weather-
ing (Figure 1g) (Dick et al. 2006; De Muynck, De Belie,
et al. 2010). For example, in reinforced concrete, pores
Figure 3. Images of cels associated with minerals. (a) SEM image of tube-like calcium-containing minerals (with similar diameters
to bacterial cels) possibly entombingS. pasteurishaped cels. Other researchers have noted similarfindingsviaSEM analysis where
rod shaped bacteria-like structures were observed inside and adjacent to CaCO3crystals or as rod shaped impressions in the CaCO3crystal (Stocks-Fischer et al. 1999; Fujita et al. 2004; Mitchel & Feris 2005; De Muynck et al. 2008; Dupraz, Parmentier, et al.
2009); (b) CLSM image of bacterial cels (red and green) closely associated with CaCO3precipitates (grey). © Cambridge UniversityPress. Reprinted with permission. From Schultz et al. (2011).
might alow penetration of water and ions, particularly
chloride or acids, leading to deleterious corosive efects
to the embedded reinforcing steel (Dick et al. 2006;
Achal et al. 2011a; De Muynck et al. 2011). In a MICP
treated surface, CaCO3 can clog pores and decrease
water penetration through a protective calcite layer. Since
De Muynck, De Belie, et al. (2010) provided a very
comprehensive review of this topic, this review wil dis-
cuss how the experimental conditions, particularly the
promotion of ureolytic activity and application of sub-
strates, influence treatment efficacy.
First, ureolytic Bacilus sphaericus isolates from
calcareous sludge were found to be efective at CaCO3
precipitation on limestone cubes (Dick et al. 2006). The
cubes were immersed in liquid bacterial cultures to
promote biofilms and then immersed in urea and calcium
chloride treatments to promote CaCO3formation. It was
concluded, that isolates with a highly negative zeta
(ζ)-potential, an indication of electrical surface potential of
cels, would more successfuly colonize positiveζ-poten-
tial limestone. It was also concluded, that the high initial
urea degradation rate and the high surface covering with
CaCO3on the biofilm produce the most homogeneous and
coherent CaCO3coating to provide protection of limestone
from water intrusion (Dick et al. 2006).
De Muynck et al. (2008) performed similar biodepo-
sition tests on concrete cubes treated with urea and cal-
cium chloride or calcium acetate (an alternative to
corosive chloride) treatment solutions. Their study found
no diference between calcium sources when examining
B. sphaericusureolysis-driven MICP in terms of weight
gain of the samples due to precipitation or chloride pene-
tration resistance. Additionaly, they concluded that the
biofilm may act as a template or primer for initial depo-
sition of CaCO3(De Muynck et al. 2008). Secondly, De
Muynck, Verbeken, et al. (2010) examined the influence
of urea and calcium concentrations on MICP coating of
limestone. It was reported that increasing urea and cal-
cium concentrations and repeated treatment improved the
resistance of the limestone to water absorption due to
CaCO3precipitation. It was nevertheless concluded that
the benefits of increased urea and calcium chloride con-
centration should be balanced with the detrimental
impacts such as unwanted ammonium by-product forma-
tion or stone discoloration (De Muynck, Verbeken, et al.
2010). Finaly, De Muynck et al. (2011) investigated the
pore structure of French limestone base materials to
determine the impact on the penetration depth and pro-
tective performance ofB. sphaericusureolysis-driven
MICP deposits. More successful bacterial penetration of
larger pores resulted in more deposition in stones with
higher porosity.
Chunxiang et al. (2009) usedS. pasteuri-facilitated
MICP to coat cement with CaCO3biodeposits to study
corosion resistance. By altering the order of addition of
calcium and urea, the researchers increased the efective-
ness of the MICP deposits against water absorption and
acid corosion of the cement. They concluded that add-
ing calcium before urea to a stationary phase bacterial
culture produced a more compact CaCO3 deposit
because calcium influenced ureolysis activity and rates
which may impact the adhesion and thickness of the
CaCO3layer (Chunxiang et al. 2009). Whiffin (2004)
suggested that high calcium nitrate (Ca(NO3)2) concen-
trations may inhibit urease activity, although mixed
efects on activity were observed among environmental
isolates or a microbial consortium (Hammes et al. 2003;
Burbank et al. 2011). Therefore, depending on the organ-
isms’tolerance for calcium concentrations, a balance
might need to be struck between high Ca2+concentra-
tions which may inhibit ureolysis and low Ca2+concen-
tration which may not alow for the formation of
sufficiently protective deposits.
Biocement
Concrete is one of the most commonly used construction
materials, but it is prone to weathering and cracking.
Cracks form in concrete due to aging and/or freeze thaw
cycles which lead to pathways for corosivefluid intru-
sion (Bang et al. 2010; Jonkers et al. 2010; Achal et al.
2011b; Wiktor & Jonkers 2011). Healing of fractures in
concrete with MICP (Figure 1e) would be advantageous
since other sealants may degrade over time or are envi-
ronmentaly toxic, whereas CaCO3 may be a more
benign treatment (Siddique & Chahal 2011). Here, bioce-
ment refers to the use of MICP to produce binder materi-
als to seal fractures or improve strength and durability of
cementitious materials (such as adding microbes to
cement mixtures). Since this topic has been extensively
reviewed by others (De Muynck, De Belie, et al. 2010;
Siddique & Chahal 2011), this section wil focus on
investigations related to the control of MICP treatment
for both concrete fracture sealing and improvement of
cementitious material.
Bacteria in or applied to concrete may face chal-
lenges to their activity including smal pore sizes as con-
crete cures, which may damage or inhibit the penetration
of organisms, and the high pH, which may inhibit bio-
logical activity. Cement, or rather the water associated
with cement, can have a pH of 11–13 even after it is
completely cured (Bang et al. 2001; Jonkers et al. 2010).
Alkaliphilic spores embedded in concrete were observed
to retain culturability for <4 months presumably due to
cel damage as the cement cured and pore size decreased
(Jonkers et al. 2010). Given smal pore sizes and high
pH conditions, research has focused on the use of alkali-
philic organisms and/or methods to protect the organisms
in order to maintain viability and ureolytic activity dur-
ing treatment.
To protect microbial urease activity from high pH in
cement,S. pasteuricels were immobilized in polyure-
thane (PU) foam in cement fractures and treated with
urea/calcium solutions. Researchers found urease activity
was maintained and hypothesized that enzyme activity
might be stabilized for longer periods of time when
embedded in a matrix such as PU foam (Bang et al.
2001). Instead of immobilizing cels, Bachmeier et al.
(2002) investigated the use of urease immobilized in PU
foam, since this treatment methodology does not depend
upon maintaining cel viability for ureolysis. Immobi-
lized enzyme treatments showed decreased CaCO3pre-
cipitation rates, possibly due to difusion limitation of
either calcium or carbonate. However, increased enzyme
stability was observed at elevated temperature compared
to the free enzyme (Bachmeier et al. 2002). More
recently, Bang et al. (2010) immobilized varying concen-
trations ofS. pasteuricels on SiranTMglass beads to
fil into concrete cracks for crack remediation. Once
again immobilization was speculated to have stabilized
cel and urease activity from the adverse efects of the
high pH of the concrete (Bang et al. 2010).
Van Titelboom et al. (2010) studied the efficacy of
silica gel supplemented withB. sphaericuscels injected
into concrete fractures and treated with calcium chloride,
calcium acetate, calcium nitrate, and urea solutions. The
calcium source did not change the reduction in water
absorption (al sources worked to produce deposits in
fractures) indicating the possibility of using alternative
calcium sources. The necessity for some protection of
cels from the high pH in concrete was suggested as bac-
teria injected without gel failed to precipitate CaCO3,
although it is also possible that cels injected in the frac-
ture without silica gel may have not atached wel and
thus resulted in reduced treatment efficacy (Van Titel-
boom et al. 2010). Another approach to concrete fracture
remediation is self-healing, where healing agents are
released or activated when fractures form (Wiktor &
Jonkers 2011; Wang et al. 2012). In one unique study,
carying agents including PU or silica-gel,B. sphaericus,
and urea/calcium nitrate treatments were loaded into
separate glass capilaries and embedded in mortar, which
upon cracking fractured the glass capilaries alowing the
carying agents, cels, and treatment solutions to mix.
The bacteria retained ureolytic and CaCO3precipitating
activity after immobilization in both PU and silica, but a
more homogeneous distribution of CaCO3crystals was
observed in the silica gelvsthe PU foam which was
atributed to the ability of bacteria to distribute more
homogeneously through the less viscous silica sol
(before gelation) than PU pre-polymer (Wang et al.
2012).
Ureolytic MICP can potentialy improve the strength
of cement by incorporating cels into the cement mixture,
although high concentrations of cels may reduce the
compressive strength due to interference by the biomass
with the integrity of the mortar (Ramachandran 2001).
When certain cel concentrations of Bacilussp.,isolated
from commercialy available cement, were mixed into a
water, cement, sand mixture and cured in urea/CaCl2
treatment, the microbial cement was found to resist water
uptake beter and showed improved compressive strength
compared to the control cement (Achal et al. 2011b).
The compressive strength of fly ash or silica fume
amended concrete was also found to be improved by
MICP induced by B. megaterium(Achal, Pan, et al.
2011) andS. pasteuri(Chahal et al. 2012a, 2012b).
In summary, construction materials may be improved
by MICP. It has been shown that increasing the number
of treatment applications, changing the calcium source to
avoid deleterious impacts from chloride, applying treat-
ment to higher porosity materials, promoting biofilm
growth before calcium treatment, and varying the order
in which the constituents are applied (calcium before
urea) can yield improvements in protective CaCO3coat-
ingsviathe MICP process. Also, some promise was
found in using ureolytic MICP to improve the strength
of concrete and remediate concrete fractures, but immo-
bilization of cels or urease enzyme in gels or PU was
required to provide protection from high pH activity
inhibition or damage to cels during cement curing.
Immobilization in turn may lead to difusion limitations
and potentialy reduced precipitation. These studies dem-
onstrate the importance of protecting the urease activity
by either promoting cels to atach to the surface or
immobilizing them.
Cementation of porous media
Ureolysis-driven MICP to alter or improve the mechani-
cal properties of unconsolidated porous media has been
extensively investigated. This method has been proposed
to suppress dust (Figure 1d), reduce permeability in
granular media, improve soils, stabilize slopes (Fig-
ure 1c), and strengthen liquefiable soils (Golapudi et al.
1995; Feris et al. 1996; Whiffin et al. 2007; Bang et al.
2011; Burbank et al. 2011). CaCO3crystals precipitated
during MICP can bridge gaps between the grains in por-
ous media to bind them together; precipitation can also
reduce the pore throat size, porosity, and permeability,
and increase the stifness and strength of the porous
media matrix (DeJong et al. 2010). Much of the work to
date has been performed to improve the efficiency of
precipitation, maximize the extent of the treatments, and
balance chemical use to reduce costs forfield applica-
tion. In engineering applications such as sand consolida-
tion or soil strengthening, it is preferable to precipitate
CaCO3homogeneously over distance and use as litle
reactant volume as possible for economic reasons
(Harkes et al. 2010; van Paassen et al. 2010). While
preferential plugging may be efective in some engineer-
ing applications, non-homogeneous bacterial distribution
and non-homogeneous precipitation may have the disad-
vantage of near-injection-point plugging where substrates
are abundant, limiting the spatial extent of the treatment
(Cunningham et al. 2007; Gerlach & Cunningham 2011;
Mortensen et al. 2011). Proposed strategies for control-
ling precipitation include promoting the spatial distribu-
tion of ureolytic activity of cels or biofilm, manipulating
the transport and reaction rates of the reactive species
and promoting favorable saturation conditions in specific
regions.
Sand consolidation
Whiffin et al. (2007) described a sand stabilization treat-
ment method (BioGrout), which folowed S. pasteuri
inoculation with a calcium chloride solution to increase
bacterial adhesion to the sand before MICP treatment.
This treatment sequence achieved significant strength
improvement and porosity reduction in sand packed
columns. Although non-uniform precipitation was
observed along the length of the column, it was reasoned
that a more homogeneous distribution could be achieved
by shifting the balance of supply and conversion (ie Da)
by increasingflow rates or lowering conversion rates to
achieve higher reactant infiltration (Whiffin et al.
2007).
Folowing this initial Biogrout work, Harkes et al.
(2010) altered the ionic strength andflow rates, again
influencing the reaction kinetics and transport rates
related to Da, to study the impact on ureolytic bacterial
distribution in sand to prevent near-injection-point clog-
ging. Bacterial atachment was found to be positively
influenced by increased salinity or ionic strength of the
transportingfluids, which could be due to a decrease in
the electrostatic repulsion forces between the cels and
the porous media surfaces (Schol et al. 1990; Foppen
& Schijven 2006). However, the increase in ionic
strength might also promote atachment of cels near the
injection point and limit the spatial extent of the treat-
ment. So, by altering the transport rate (increasingflow)
of low ionic strength solutions Harkes et al. (2010)
observed a more homogeneous distribution of bacteria,
but cautioned against the loss of atachment and activity
when low ionic strength solutions are used. Transport of
bacteria through the matrix of a porous medium is a
complex function of the size and surface properties of
the cel, electrical interactions, theflow rate and the
chemistry of the transportfluid as wel as the pore size
distribution of the porous medium (Jenneman et al.
1985; Schol et al. 1990; Bouwer et al. 2000; Mitchel
& Santamarina 2005; Harkes et al. 2010). A balance
between ionic strength and transport could help promote
more homogeneous cel and ultimately ureolytic activity
distribution.
Much of the work presented has been performed on
a smaler scale in a laboratory-controled environment,
yet in 2010 van Paassen et al. embarked on a scaled-up
demonstration of MICP in 100 m3of sand to determine
the ground improvement abilities and the extent of pre-
cipitation. Similar to the injection strategies developed
by Whiffin et al. (2007) and Harkes et al. (2010), the
sand was inoculated withS. pasteuricels, cementation
solution folowed to promote bacterial cel adhesion and
then urea and calcium solutions were injected 10 times
over 16 days. As much as 40 m3of the 100 m3sand reac-
tor were cementedviaMICP with a visible wedge shape
between the injection and extraction wels (Figure 4)
(van Paassen et al. 2010).
Liquefiable soils
Other researchers have also examined scaled-up ureoly-
sis-driven MICP. Burbank et al. (2011) studiedfield-scale
ureolysis-driven MICP to strengthen liquefiable soils.
Liquefiable soils are loose granular soil deposits gener-
aly found in saturated conditions, which may undergo a
decrease in shear strength when subject to seismic waves
and contribute to man-made structure failure during
earthquakes (Burbank et al. 2011). Soils on the shore of
the Snake River (USA) were subjected to ureolytic bio-
mineralization treatments, which yielded soils cemented
with 1% by weight CaCO3in the near surface and
1.8–2.4% calcite below 90 cm (Burbank et al. 2011).
This was less precipitation than observed in laboratory
enriched samples, which was atributed to the lower
technical quality of the calcium source in thefield study.
Theirfindings also suggested higher concentrations of
CaCO3formed away from the injection point rather than
closer to the injection point. Researchers atributed this
to either (1) eluviation wherefine-grained materials or
Figure 4. Image of a cemented sand body from a large scale
Biogrout experiment. Reprinted from van Passen et al. (2010),
with permission from ASCE.
CaCO3particles may have been transported downward
with the infiltrating water, or (2) increased ureolysis and
possibly delayed subsequent precipitation occuring in
the deeper soil profile.
Subsurface barriers
In certain coastal areas, salt water intrusion into freshwa-
ter aquifers during groundwater extraction has become a
major problem. The problem is often addressed by creat-
ing underground dams or increasing artificial recharge of
fresh water to prevent migration of salt-laden water into
freshwater aquifers. Subsurface MICP bariers may be an
alternative to these methods (Figure 1i) (Rusu et al.
2011). Due to salt water intrusion into ground water,
MICP must be able to occur in saline conditions to be
applied in these environments. Mortensen et al. (2011)
assessed the influence of various environmental factors
on ureolysis-driven MICP to determine suitablein situ
environments. First, they observed that short term ureo-
lytic activity did not appear to be inhibited by anaerobic
conditions after cels were cultured aerobicaly, which
agrees withfindings by Parks (2009), Tobler et al.
(2011), and Martin et al. (2012). Secondly, they found
ful and half-strength seawater enhanced CaCO3precipi-
tation rates, possibly due to increased alkalinity and
cation availability (Mortensen et al. 2011). Finaly, the
authors note that manipulating the reaction and transport
rates by inhibiting precipitation with increased ammo-
nium concentrations or by controlingflow rates is
important in achieving homogeneous distribution of
MICP. These results demonstrate the potential of ureoly-
sis-driven MICP for developing subsurface bariers to
prevent salt water intrusion.
Aquaculture: impermeable crusts
One promising engineering application of ureolysis-dri-
ven MICP is the preparation of crusts to control seepage
from aquaculture ponds or reservoirs into underlying
soils or sands (Figure 1h). Stabnikov et al. (2011) used
the halotolerant, alkaliphilicBacilussp. VS1 isolate to
seal a sand-lined model pond. Successive percolation
treatments with high concentrations of urea and calcium
solutions resulted in a nearly impermeable crust on the
surface of the sand, which markedly reduced the seepage
rate, taking sand to the same permeability range as wel
compacted clay (Figure 5).
Dust suppression
Bang et al. (2011), showed the potential for using ureol-
ysis-driven MICP to suppress dust (Figure 1d). Dust
poses problems to human health and is traditionaly sup-
pressed by means of chemical application or watering
down which may be difficult to maintain or may use
environmentaly problematic chemicals. Ureolysis-driven
MICP is proposed as an alternative to consolidate dust
particles.S. pasteuri cels or urease and urea/calcium
chloride treatment solutions were sprayed over sand sam-
ples, which were then subjected to wind erosion tests.
Bang et al. (2011) found MICP dust control to be very
efective, but its efficiency was subject to the soil type
and grain size distribution, as wel as environmental con-
ditions such as humidity and temperature.
In summary, ureolysis-driven MICP has been
explored for several engineered applications involving
porous media, including consolidating sand or soils, cre-
ating subsurface bariers, sealing aquaculture ponds and
suppressing dust. These applications are often controled
by manipulating the transport and reaction rates to either
promote homogeneous deposition or controled deposi-
tion in selective areas. In MICP application to porous
media, a complex set of factors, including environmental
conditions may greatly influence the results of the treat-
ment.
Hydraulic control and environmental remediation
Radionuclide and metal remediation
Radionuclide remediation. The US Department of
Energy faces environmental remediation chalenges such
as the long-term management of the Hanford site in
Washington, USA, where groundwater is contaminated
with radionuclides (Waren et al. 2001; Fujita et al.
2004, 2008, 2010; Wu et al. 2011). Traditional treatment
methods such as pump and treat have been found inef-
fective at the site to remediate or prevent migration of
mobile radionuclide groundwater contaminants (Fujita
et al. 2008). Therefore, years of research have evolved
methods to stimulate ureolytic subsurface organisms to
Figure 5. Photograph of an 1 mm thick crust of calcite on a
sand surface. Reprinted from Stabnikov et al. (2011), with
permission from Elsevier.
promote CaCO3precipitation which in turn promotes co-
precipitation and solid phase capture of some of these
contaminants, in particular strontium-90, a uraniumfis-
sion by-product (Figure 1j). In subsurface environments
saturated with respect to CaCO3minerals, the co-precipi-
tation forms a long-term immobilization mechanism
while the90Sr decays (Mitchel & Feris 2006a; Fujita
et al. 2010; Wu et al. 2011).
Control of strontium co-precipitation in the subsur-
face has been widely researched by studying the rates of
ureolysis and precipitation. Waren et al. (2001) demon-
strated that 95% of total strontium was captured in the
solid phase during batch ureolysis-driven MICP experi-
ments. Further studies demonstrated thatS. pasteuriin
artificial groundwater media exhibited higher rates of
ureolysis at slightly elevated temperatures, strontium co-
precipitation increased with increasing CaCO3precipita-
tion rates, and higher ureolysis rates could reduce the
time to reach critical saturation (Scrit) which is important
since the greatest CaCO3 precipitation rates were
observed nearScrit(Feris et al. 2003; Fujita et al. 2004;
Mitchel & Feris 2005).
Since augmentation of subsurface environments with
microbes may not be ideal or feasible, Fujita et al.
(2008) investigated the potential of enriching native ure-
olytic organismsin situin the Eastern Snake River Plain
Aquifer (Idaho, USA) for the purpose of remediating
groundwater by co-precipitating strontium. The authors
suggest that multiple treatments with low concentrations
of a carbon source (molasses) to stimulate the subsurface
community folowed by the injection of urea can pro-
mote ureolytic subsurface populations (Fujita et al.
2008). Another microbial enrichment test was performed
with groundwater and sediment samples from wels at
the Hanford site in Washington, USA (Fujita et al.
2010). Urea stimulated sediment samples showed spe-
cific ureolytic activity 2–4 orders of magnitude higher
compared to groundwater samples, leading researchers to
hypothesize that greater activity was associated with
atached (or biofilm) communities compared to plank-
tonic cels (Fujita et al. 2010).
Metal remediation.Toxic metal (eg copper, arsenic, and
chromium) contamination in soil or groundwater has
been atributed to mining and smelting as wel as other
industrial activities. Toxic metal contamination is linked
to human health problems and curent remediation eforts
can be costly and relatively inefective. Traditional reme-
diation eforts include phytoremediation, removing, or
covering the soils with clean soil, on-site chemical leach-
ing of contaminants or bioremediation with toxic metal-
tolerant bacterial species (Achal et al. 2012). However,
these treatment methods may not be long-term solutions.
For example, in bioremediation many bacterial species
can decrease the solubility and thus immobilize metals
by changing their redox state. However, future changes
in oxidation-reduction potential could lead to remobiliza-
tion; therefore, an alternate remediation method is
CaCO3-based co-precipitation.
It was previously shown that chromate can be associ-
ated with CaCO3in co-precipitated form (Hua et al.
2007), also Achal et al. (2012) isolatedSporosarcina
ginsengisoli CR5, an arsenic-tolerant, urease-positive
bacterium and researched its MICP potential to remediate
arsenic contaminated soils. Although growth of the
organism was slowed in the presence of arsenic, signifi-
cant arsenic was removed from aqueous solution during
ureolytic MICP (Achal et al. 2012). Another study
focused on remediation of copperviathe MICP process
by the copper-tolerant, ureolytic organism,Kocuriaflava
CR1. Copper bioremediation studies were performed with
K.flavain urea and calcium containing batch with copper
concentrations up to 1000 mg l1 (Achal, Pan, et al.
2011). The authors reported a positive corelation
between higher urease production and higher copper
removal from aqueous solutions (Achal, Pan, et al. 2011).
In elevated concentrations, metals may be toxic
to organisms involved in remediation. Kurmaç (2009)
evaluated the impact of varying concentrations of lead, cad-
mium, chromium, zinc, copper, and nickel to ureolysis-dri-
ven MICP treatment technology in synthetic wastewater
amended with urea and calcium chloride. They found the
impact of metal toxicity on microbial substrate degradation,
as measured by the reduction in biochemical oxygen
demand (BOD), increased in the folowing order: Cd (I)
> Cu(I) > Pb(I) > Cr(VI) > Ni(I) > Zn(I) (Kurmaҫ2009).
In the application of MICP, metal toxicity may be a limiting
factor in treatment efficacy, but isolation of metal-tolerant
ureolytic organisms from contaminated environments may
improve the treatment potential.
Polychlorinated biphenyl containment
Additional recalcitrant contaminants threatening environ-
mental and human health are polychlorinated biphenyls
(PCBs), which can contaminate concrete surfaces when
PCB-containing oil leaks from equipment. Methods of
removing PCB-contaminated oil include solvent washing,
hydroblasting, or sandblasting folowed by encapsulation
in epoxy coating. Epoxy coating may be inefective due
to resurfacing of the oil over time (Okwadha & Li
2011). An alternative to epoxy coating is the use of
ureolysis-driven MICP to produce a coating to seal
PCB-contaminated concrete (Figure 1f). By applying
S. pasteuri cultures and urea/calcium treatment to the
surface of PCB-coated cement cylinders, surficial PCB-
containing oils were encapsulated. No leaching through
the MICP coating was observed and permeability was
reduced by 1–5 orders of magnitude (Okwadha & Li
2011).
Tab
le 
1. 
Su
mm
ary
 of
 co
ntr
ol 
par
am
ete
rs 
and
 ra
nge
s u
sed
 to
 pr
om
ote
 mi
cro
bial
ly 
ind
uce
d c
alci
um
 ca
rbo
nat
e p
rec
ipi
tati
on 
(M
IC
P).
Con
tro
l v
ari
abl
e 
Ran
ge
Gen
era
l a
sse
ss
men
t o
f s
ucc
ess
Rel
eva
nt r
efe
ren
ces
Ure
oly
tic
 ac
tiv
ity
 of
 or
ga
nis
ms
Ino
cul
ati
on
con
cen
tra
tio
n
0.0
3–
2.8
8 O
D 60
0
105
–10
9 cf
u m
l
1
Gre
ate
r b
act
eri
al 
con
cen
tra
tio
ns 
= fa
ste
r r
ate
s o
f
ure
oly
sis
 & 
pro
duc
e l
arg
er 
and
 le
ss 
sol
ubl
e c
rys
tal
s
Mitc
hell
 an
d F
erri
s (
20
06
b),
 H
ark
es 
et 
al. 
(2
01
0),
Ok
wad
ha 
an
d L
i (
20
10)
, T
obl
er 
et 
al. 
(2
011
)
Mic
roo
rga
nis
m
S. 
pas
teu
rii,
 B.
 le
ntu
s,
B. 
meg
ate
riu
m,
B.
sph
aer
icu
s,S
. g
ins
eng
iso
li,
K.
flav
a,
B.
pse
udo
fir
mus
,B.
 co
hni
i,
B. 
alk
ali
nitr
ilic
us,
nati
ve 
org
ani
sm
s o
r e
nzy
me
Bio
fil
m c
om
mun
itie
s 
may
 ha
ve 
hig
her
 ac
tiv
ity
;
sti
mul
ati
on 
of 
nati
ve 
ure
oly
tic
 or
gan
is
ms 
des
ira
ble;
str
ain
 sh
oul
d h
ave
 st
ron
g u
rea
se 
pro
duc
tio
n,
sho
uld
 no
t b
e p
ath
oge
nic
 or
 ge
neti
call
y 
mod
ifie
d if
bio
aug
men
tati
on 
is 
nec
ess
ary
Bac
hm
eie
r e
t a
l. (
20
02)
, 
Ha
mm
es 
et 
al. 
(2
00
3),
Whi
f fi
n (
20
04)
, 
Dic
k e
t a
l. (
20
06)
, F
ujit
a e
t a
l. (
20
08,
201
0),
 A
cha
l, 
Pan
, e
t a
l. (
20
11)
, B
urb
an
k e
t a
l. (
20
11)
,
Tob
ler
 et
 al.
 (2
011
), 
Wik
tor
 an
d J
on
ker
s (
20
11)
, 
Ach
al
et 
al. 
(2
01
2),
 C
uth
ber
t e
t a
l. (
20
12)
Car
bon
 so
urc
e 
BH
I, 
NB,
 Ye
ast 
ext
rac
t, 
Try
ptic
 so
y b
rot
h,
Pep
ton
e, 
Ace
tat
e, 
Lac
tos
e 
mot
her
 or
 C
orn
sta
rch
 li
quo
r, 
Mol
ass
es
Alte
rna
te 
car
bon
 so
urc
es 
may
 i
mpr
ove
 ec
ono
mic
fea
sib
ilit
y f
orfi
eld
 ov
er l
ab 
gra
de 
rea
gen
ts;
inj
ecti
on 
of 
a c
arb
on 
sou
rce
 pr
ior
 to
 ur
ea 
may
sti
mul
ate 
sub
sur
fac
e a
tta
che
d c
om
mun
itie
s
Sto
cks
-Fi
sch
er 
et 
al. 
(1
99
9), 
Fuj
ita
 et
 al.
 (2
00
8),
 A
cha
l
et 
al. 
(2
00
9, 
20
11a
), 
Mitc
hell
 et
 al.
 (2
01
0), 
van
Paa
sse
n e
t a
l. (
20
10)
, 
Bur
ban
k e
t a
l. (
20
11)
,
Mor
ten
sen
 et
 al.
 (2
011
), 
Tob
ler
 et
 al.
 (2
011
)
Rea
cti
on 
an
d t
ra
nsp
ort
Flo
w c
ond
iti
ons
 
Con
sta
nt
flo
w
Exa
mpl
es:
 0.
7 p
ore
 vo
lu
mes
 pe
r d
ay;
 0.
35
–
12 l
 h
1
Con
sta
nt l
ow
flo
w r
ate
s 
may
 le
ad 
to 
non
-
ho
mog
ene
ous
 C
aC
O 3
dist
rib
uti
on/
inj
ecti
on 
poi
nt
plu
ggi
ng;
 hi
ghe
r i
nje
cti
on 
rat
es 
= m
ore
 ev
en
dist
rib
uti
on 
of 
bac
ter
ia,
 ho
mog
eno
us 
Ca
CO
3
dist
rib
uti
on,
 mi
ni
miz
e i
nje
cti
on 
poi
nt 
ce
men
tati
on
(ie
 D
a
1)
Whi
ffi
n e
t a
l. (
20
07)
, 
Har
kes
 et
 al.
 (2
01
0),
Cun
nin
gha
m e
t a
l. (
20
11)
, 
Mor
ten
sen
 et
 al.
 (2
011
),
Sch
ultz
 et
 al.
 (2
011
), 
Tob
ler
 et
 al.
 (2
01
2)
Pul
se 
or 
per
col
ati
on
flo
w
Exa
mpl
e: s
tati
c i
nte
rva
ls 
bet
wee
n tr
eat
men
ts
Gro
wth
 tr
eat
men
ts 
may
 be
 us
ed 
to 
ove
rco
me
mor
tali
ty 
due
 to
 Ca
CO
3
ent
om
bm
ent
 & 
sup
ply
ele
ctr
on 
acc
ept
ors;
 pu
lse
dfl
ow
 m
ay 
giv
e 
mor
e
ho
mog
ene
ous
 C
aC
O 3
pre
cip
itat
ion
DeJ
on
g e
t a
l. (
20
06)
, 
De 
Mu
ync
k, 
Ver
bek
en,
 et
 al.
(20
10)
, v
an 
Paa
sse
n e
t a
l. (
20
10)
, 
Bur
ban
k e
t a
l.
(20
11)
, C
un
nin
gha
m e
t a
l. (
20
11)
, 
Mor
ten
sen
 et
 al.
(20
11)
, S
tab
nik
ov 
et 
al. 
(2
011
), 
Al 
Qab
an
y e
t a
l.
(20
12)
, E
big
bo 
et 
al. 
(2
01
2), 
Tob
ler
 et
 al.
 (2
01
2),
Lau
ch
nor
 et
 al.
 (2
01
3), 
Phi
lli
ps 
et 
al. 
(2
01
3)
Vis
cos
ity
Lo
w (i
e c
los
e t
o 
wat
er) 
to 
hig
h (
PU
) 
Lo
w v
isc
osit
yfl
uid
s c
an 
pen
etr
ate 
sm
alle
r fr
act
ure
s/
tre
at
men
t a
rea
s 
wit
h l
ess
 pu
mpi
ng 
pre
ssu
re;
bac
ter
ial
 di
stri
but
ion
 m
ay 
be 
mor
e h
om
oge
nou
s i
n
les
s v
isc
ous
 so
lut
ion
s
Cun
nin
gha
m e
t a
l. (
20
11)
, 
Wan
g e
t a
l. (
20
12)
Te
mpe
rat
ure
 
10
–60
 °C
Inc
rea
sed
 ur
eol
ysi
s r
ate
s o
bse
rve
d a
t h
igh
er
te
mpe
rat
ure
; u
rea
se 
enz
ym
e c
an 
wit
hst
and
 ev
en
hig
her 
te
mpe
rat
ure
s t
han
 m
eso
phi
lic
 ur
eol
ytic
all
y
acti
ve 
cell
s, 
par
tic
ula
rly
 if 
im
mob
iliz
ed
Bac
hm
eie
r e
t a
l. (
20
02)
, F
erri
s e
t a
l. (
20
03)
, 
Mitc
hell
and
 Fe
rris
 (2
00
5, 
20
06a
), 
Du
pra
z, 
Par
men
tie
r, e
t a
l.
(20
09)
, T
obl
er 
et 
al. 
(2
011
), 
Ban
g e
t a
l. (
20
11)
Sali
nit
y
0.3
6–
100
 g l
1
Inc
rea
sed
 sa
lin
ity
 m
ay 
inc
rea
se 
alk
ali
nit
y, 
ure
oly
sis
rat
es, 
and
 ba
cte
rial
 ad
sor
pti
on;
 in
cre
ase
d s
ali
nit
y
may
 in
cre
ase
 ti
me 
del
ay 
to
S cri
t; 
pho
sph
ate
s 
may
dec
rea
se 
pre
cip
itat
ion
 ra
tes
Fer
ris 
et 
al. 
(2
00
3),
 D
upr
az 
, 
Men
ez,
 et
 al.
 (2
00
9),
Dup
raz
, P
ar
men
tie
r, e
t a
l. (
20
09)
, 
Har
kes
 et
 al.
 (2
01
0),
Mor
ten
sen
 et
 al.
 (2
011
), 
Rus
u e
t a
l. (
20
11)
, S
tab
nik
ov
et 
al. 
(2
011
)
(Co
nti
nue
d)
Carbon dioxide sequestration
With atmospheric carbon dioxide (CO2) concentrations
on the increase, mitigation strategies are being explored
widely. One proposed mechanism for reducing emissions
is the capture and storage of CO2in deep geologic reser-
voirs, such as deep saline aquifers. The efficacy of this
mitigation method depends on preventing potential CO2
leakage either back to the surface or into overlying aqui-
fers. Possible reasons for leakage may include: (1)
decreased wel bore integrity, possibly due to the coro-
sive efect of supercritical CO2, also known as carbon-
ation, or fractures in those wel cements; or (2) areas of
increased cap rock permeability (Huerta et al. 2008; Bar-
let-Gouédard et al. 2009; Wigand et al. 2009; Carey
et al. 2010). Traditional wel repair methods include the
use of cements (such asfine cement); however, these
may be of higher viscosity than the aqueous solutions
used to promote MICP. Higher viscosityfluids may not
adequately penetrate smal pore spaces and potentialy
not seal microfractures where low viscosity supercritical
CO2couldfind leakage pathways. As such, MICP may
be an efective tool to seal fractures or high permeability
leakage zones in the context of CO2sequestration (Fig-
ure 1k), and may also be efective in helping to reliably
abandon wels after fossil fuel extraction (Figure 1a).
Three proposed methods to which ureolysis-driven
MICP have been suggested to contribute to in situCO2
leakage mitigation are formation trapping, solubility trap-
ping, and mineral trapping (Dupraz, Menez, et al. 2009;
Mitchel et al. 2010). MICP may reduce permeability to
mitigate leakage potential (formation trapping). Also, the
storage of CO2might be enhanced by ureolysis-driven
MICP by increasing the dissolved CO2(as carbonate or
bicarbonate) in the subsurface formation water (solubility
trapping). Finaly, ureolysis-driven MICP might enhance
the precipitation of dissolved CO2in carbonate minerals
(mineral trapping) (Mitchel et al. 2010).
Formation trapping.Engineered MICP has been pro-
posed to protect wel cements from supercritical CO2,
plug microfractures in the near wel environment and
reduce permeability in cap rock (Mitchel et al. 2010;
Philips et al. 2013). In these applications, the spatial
extent and temporal efficiency of precipitation must be
controled. Experiments under atmospheric conditions
have led to evolved injection strategies to promote more
uniform spatial distribution of CaCO3. Pulseflow, with
brieffluid injection folowed by batch biomineralization
periods, rather than continuousflow injections precipi-
tated less CaCO3near the influent in sand column reac-
tors. Additionaly, reducing the SI near the injection
point during periods of active biomineralization reduced
near-injection-point plugging (Cunningham et al. 2009,
2011; Schultz et al. 2011; Ebigbo et al. 2012). Recently,
Tab
le 
1. 
(C
ont
inu
ed
)
Con
tro
l v
ari
abl
e 
Ran
ge
Gen
era
l a
sse
ss
men
t o
f s
ucc
ess
Rel
eva
nt r
efe
ren
ces
Sat
ura
tio
n c
on
diti
ons
pH
4.5
–13
Pea
k e
nzy
me 
acti
vit
y =
 p
H 8
.0;
 op
ti
mal
S. 
pas
teu
rii
gro
wth
 = 
pH
 8.
5; 
ext
re
me 
(lo
w a
nd 
hig
h) 
pH
env
iro
nm
ent
s 
may
 be
 de
tri
men
tal 
to 
acti
vit
y; 
onc
e
for
med
 C
aC
O 3
is 
res
ilie
nt t
o a
cid
 att
ack
 w
hen
pH
 > 
1.5
 (a
t c
ert
ain
 ti
me 
sca
les
); 
pH 
inc
rea
ses
 af
ter
ure
oly
sis 
are
 fo
llo
wed
 by
 p
H d
ecr
eas
e d
ue 
to
Ca
CO
3
pre
cip
itat
ion
Gol
lap
udi
 et
 al.
 (1
99
5), 
Sto
cks
-Fi
sch
er 
et 
al. 
(1
99
9),
Ban
g e
t a
l. (
20
01)
, 
Ach
al 
et 
al. 
(2
00
9),
 C
hu
nxi
an
g
et 
al. 
(2
00
9),
 D
upr
az 
, 
Men
ez,
 et
 al.
 (2
00
9),
 D
upr
az,
Par
men
tie
r, e
t a
l. (
20
09)
, 
Ban
g e
t a
l. 
(2
01
0),
 O
kw
ad
ha
and
 Li
 (2
01
0), 
Tob
ler
 et
 al.
 (2
011
)
Ure
a/c
alci
um
con
cen
tra
tio
n
Ure
a: 
6 m
M–
1.5
 M
Equ
im
ola
r u
rea
/C
a2
+
rati
o 
may
 be
 op
ti
mal 
sin
ce
gre
ate
r r
eac
tan
t c
onc
ent
rati
on 
= hi
ghe
r k
ine
tic 
rat
es
(bu
t o
nly
 to
 a 
cer
tai
n p
oin
t) 
& t
o b
ala
nce
 re
age
nts
for
 re
acti
on 
but
 to
 no
t a
dd 
ext
ra 
un
wan
ted
che
mic
als 
to 
env
iro
nm
ent
; c
alci
um
 ni
tra
te 
or 
ace
tat
e
alte
rna
tiv
es t
o c
alci
um
 ch
lor
ide
War
ren
 et
 al.
 (2
00
1), 
Fer
ris 
et 
al. 
(2
00
3),
 D
e 
Mu
ync
k
et 
al. 
(2
00
8),
 C
hu
nxi
an
g e
t a
l. (
20
09)
, 
Du
pra
z,
Par
men
tie
r, e
t a
l. (
20
09)
, 
De 
Mu
ync
k, 
Ver
bek
en,
 et
 al.
(20
10)
, 
Ok
wad
ha 
an
d L
i (
20
10)
, 
Van
 Ti
ttel
bo
om
 et
 al.
(20
10)
, B
urb
an
k e
t a
l. (
20
11)
, 
Cu
nni
ng
ha
m e
t a
l.
(20
11)
, 
Mor
ten
sen
 et
 al.
 (2
011
), 
Sta
bni
ko
v e
t a
l.
(20
11)
, T
obl
er 
et 
al. 
(2
011
), 
Lau
ch
nor
 et
 al.
 (2
01
3)
Cal
ciu
m: 
25
μM
–1.
25
 M
Sat
ura
tio
n st
ate 
(Ω
,
So
rS
crit
)
12–
436
Sat
ura
tio
n st
ate
 an
d c
riti
cal 
sat
ura
tio
n st
ate
infl
uen
ce 
spa
tial
 & 
te
mpo
ral
 pr
eci
pita
tio
n o
f 
Ca
CO
3
Fer
ris 
et 
al. 
(2
00
3),
 Mi
tch
ell 
an
d F
erri
s (
20
05,
 20
06
b),
Dup
raz
, P
ar
men
tie
r, e
t a
l. (
20
09)
, T
obl
er 
et 
al. 
(2
011
)
these injection strategies have been used to seal hydrau-
lic fractures in 70 cm diameter sandstone cores under
ambient (Philips et al. 2013) and high pressure (Philips
et al. personal communication) conditions.
Solubility and mineral trapping.Spore and biofilm-form-
ingBacilusspecies are resistant to high pressures and
supercritical CO2 (Mitchel et al. 2008, 2009).
Accordingly, Mitchel et al. (2010) studied S. pasteuri,
for ureolysis-driven CaCO3precipitation with a range of
initial13C–CO2head pressures and urea concentration in
artificial groundwater. Precipitated CaCO3was heavily
enriched in13C–CO2 and the fraction of13C–CO2
increased with increasing headspace pressure and urea
concentrations, suggesting that ureolysis enhanced the
amount of carbonate in the CaCO3 derived from
headspace CO2(g) (mineral trapping). Dupraz, Menez,
et al. (2009) also studiedS. pasteuri in artificial
groundwater to determine the transformation of CO2into
a solid carbonate phase (mineral trapping) under diferent
temperature and salinity conditions (relevant to subsur-
face saline aquifer conditions) with diferent partial pres-
sures of CO2. While no temperature dependence of
CaCO3precipitation rates was found in their studies, it
was observed that increased salinities increased alkinal-
ization and ureolysis rates, but created a delay in time
before CaCO3precipitation began (Dupraz, Menez, et al.
2009). Finaly, Mitchel et al. (2010) also demonstrated
that as pH increases, the DIC increases and headspace
CO2(g) decreases (solubility trapping). It was concluded
that ureolysis-driven MICP in the subsurface can poten-
tialy increase the security of long-term CO2storage. On-
going research suggests ureolysis-driven MICP also
occurs at high pressures (>73 bar) and those derived
minerals are relatively stable under the time scales tested
when subjected to supercritical CO2exposure (Mitchel
et al. 2013).
In summary, control of ureolysis-driven MICP for
remediating subsurface environments of strontium con-
taminated groundwater, toxic metal contaminated soils
and groundwater, PCB-contaminated concrete or improv-
ing security of geologicaly sequestered CO2has been
widely explored. Research has focused on methods to
maintain ureolytic activity and understand the transport
and reaction rates of urea and calcium, which influence
CaCO3saturation conditions (Table 1). Ureolysis-driven
MICP may efectively treat a wide variety of engineering
chalenges, but care should be taken to consider the
maintenance of ureolytic activity (viability of organisms)
under adverse contaminant exposure.
Summary
Much of the literature surounding ureolysis-driven
MICP focuses on controling the wide-range of parame-
ters that influence precipitation. The range of variables
and the optimum values determined for specific MICP
applications indicate that there is not one‘recipe’for
controling MICP in engineered applications. The suc-
cess of MICP treatment depends on the ability to precipi-
tate CaCO3at appropriate locations and times. Ureolysis-
driven MICP is controled by three main parameters, (1)
the ureolytic activity (of microorganisms), (2) the reac-
tion and transport rates of the substrates, and (3) the sat-
uration conditions of carbonate minerals (Table 1).
First, organisms or enzyme are either injected or
stimulated to provide the catalyst for ureolysis and cels
may act as nucleation sites for precipitation to occur.
Several chalenges suround maintaining the ureolytic
activity of microorganisms, such as adverse environmen-
tal conditions (eg high pH or toxic metals), electron
acceptor (eg oxygen) limitations, entombment in calcium
carbonate, and nutrient difusion limitations causing cel
inactivation after entombment.
Second, the reaction rates and the transport rates of
reactants are manipulated, for example, by changing the
flow conditions (eg velocity) or reagent concentrations.
Factors such asfluid salinity and temperature can influ-
ence the rates of ureolysis and mineral precipitation.
Flow rate andfluid viscosities can influence the transport
conditions. Exploring the dimensionless Da, which is the
ratio of reaction rate to transport rate, as a tool in MICP
design under various conditions may provide valuable
insight for controling ureolysis and precipitation and
ultimately the success of MICP engineered applications.
Finaly, whether calcium carbonate has the thermody-
namic propensity to precipitate is governed by the satura-
tion conditions, and the location and timing of
precipitation can be influenced by the presence of nucle-
ation sites. TheSor SI is determined by the activity of
Ca2+and CO32 andScritor SIcritare empirical values
which reflect how highly supersaturated a solution must
become before precipitation is observed.Scritcan be
influenced by a variety of factors including but not lim-
ited to ureolysis kinetics and the availability of nucle-
ation sites.
A wide range of factors can impact the saturation
state to promote precipitation of CaCO3in engineered
MICP technologies (Table 1). Since controling satura-
tion conditions and precipitation in time and space is a
multi-factored reactive transport chalenge, modeling has
become an essential tool to optimize injection and treat-
ment strategies. Curent models, carefuly interpreted and
calibrated, explore promotion of favorable saturation
state and predict treatment efficacy while decreasing the
need for labor-intensive laboratory experiments (Ebigbo
et al. 2010, 2012; Zhang & Klapper 2010; Barkouki
et al. 2011; Fauriel & Laloui 2011).
Improving the economic and environmental feasibil-
ity of ureolysis-driven MICP treatment must be consid-
ered in the transition from laboratory tofield-relevant
scale engineered MICP technologies. There is an eco-
nomic limitation to the use of laboratory grade nutrient
sources infield applications and alternate nutrient
sources such as inexpensive industrial wastewater, lac-
tose mother liquor (dairy industry), and corn steep liquor
(starch industry) may ofer a possibility of cheaper nutri-
ent sources (Achal et al. 2009, 2011a; Mitchel et al.
2010). Additionaly, large volumes of reactant and the
production of bacterial cultures for injection (if neces-
sary) may make certain engineered applications of MICP
economicaly chalenging compared to traditional treat-
ments. Optimizing treatment strategies may reduce cost
by minimizing unnecessary injection or the excessive use
of amendments. Unwanted by-products from ureolysis
such as NH4+, have to be considered and controled at
least in certain prospective applications. NH4+is undesir-
able, since groundwater aquifer health may be harmed,
stone discolored, or subsurface communities changed by
metabolic competition (eg outcompeting bioaugmented
organisms) due to NH4+salts or conversion products (De
Muynck, De Belie, et al. 2010; Tobler et al. 2011).
While promising and efective treatment strategies using
MICP have been demonstrated, additional research is
necessary in order to improve economic feasibility,
define optimal treatment strategies and reduce unwanted
by-products.
Outlook
With the wide variety of ureolysis-driven MICP applica-
tions being researched and developed around the world,
there remain a number of technology development chal-
lenges and thus research opportunities. In order to
improve the potential for successful MICP application,
additional strategies have to be developed through fur-
ther research including, but not limited to: (1) investigat-
ing the potential of biofilm-based MICP approaches
compared to suspended cel-based approaches, specifi-
caly diferences in ureolysis and mineral precipitation
kinetics, mineralogy, mineral reactivity and stability
between atached and planktonic cultures; (2) determin-
ing the optimal substrate balance (eg urea and calcium)
for various MICP applications with the goal of optimiz-
ing CaCO3precipitation efficiency, which may increase
economic feasibility and reduce production of unwanted
byproducts; (3) investigating nano- to micro-scale
mineral nucleation processes and determining the efects
on subsequent mineral growth, morphology, and stability
at larger scales; (4) improving mathematical models
describing MICP processes in porous media by develop-
ing quantitative descriptions of fundamental processes at
the micro- and macro-scale (eg ureolysis and growth
kinetics, precipitation kinetics, crystal growth, and
microbe-mineral interactions) as wel as integrating these
process descriptions into Darcy-scale models for large-
scale application design; (5) experimenting at larger
scales, which, together with the developed models, wil
alow for the evaluation of the importance of transport
processes in controling MICP for engineeredfield appli-
cation; (6) developingin situmonitoring technologies
(such as geophysical methods) that alow assessment
of success infield applications; and (7) evaluating
long-term stability of MICP treatments compared to
conventional (eg cement-based) technologies.
It is evident that the implementation of MICP-based
technologies on afield scale requires the expertise of
many disciplines, and multi-disciplinary research and
development teams wil be necessary. This review sum-
marizes the research results across many proposed engi-
neered applications in an efort to inspire researchers to
address the key research and development questions nec-
essary to move MICP technologies toward commercial
scale applications.
In conclusion, ureolysis-driven MICP has been
suggested for a wide variety of engineered treatments
including modification of construction materials, cement-
ing porous media, hydraulic control, and remediating
environmental contaminants (Figure 1). A majority of the
literature focuses on promoting ureolytic activity, under-
standing the reaction and transport conditions, and ulti-
mately manipulating the saturation state to achieve the
desired timing and location of CaCO3precipitation. Many
potential applications of ureolysis-driven MICP exist,
including those discussed in this review and other appli-
cations such as stabilizing building foundations or slopes;
minimizing erosion, stabilizing grounds prior to tunnel-
ing; sealing tunnel seepage; strengthening earthen dams
and dikes; strengthening dunes to protect shorelines or
prevent desertification; as wel as removing calcium from
waste streams (Hammes 2002; DeJong et al. 2010, 2011).
A diverse, multi-disciplinary research efort including
field demonstrations, modeling, and elucidation of the
fundamental mechanisms of ureolysis-driven MICP has
and wil continue to aid in the efort of transitioning
MICP-based technologies from the laboratory to thefield.
Acknowledgments
Funding was provided from the US Department of Energy
(DOE) under NETL No. DE-FE0004478 and DE-FE0009599
and Zero Emissions Research Technology Center (ZERT),
Award No. DE-FC26-04NT42262, DOE EPSCoR Award
No. DE-FG02-08ER46527 and Subsurface Biogeochemical
Research (SBR) Program, contract No. DE-FG02-09ER64758.
Additional funding was provided through National Science
Foundation Award No. DMS-0934696 and European Union
Marie Curie Reintegration Grant, No. 277005. Any opinions,
findings, conclusions, or recommendations expressed herein are
those of the authors and do not necessarily reflect the views of
the DOE. Special thanks to Peg Dirckx for the artwork. The
authors also wish to thank James Connoly and the anonymous
reviewers for their valuable suggestions to improve the
manuscript.
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