Construction and Building Materials 25 (2011) 3791–3801

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Construction and Building Materials

journal homepage: www.elsevier.com/locate/conbuildmat

Review

Effect of ureolytic bacteria on concrete properties

Rafat Siddique a, Navneet Kaur Chahal b,⇑
a
Department of Civil Engineering, Thapar University, Patiala, Punjab, India
b
Department of Biotechnology and Environmental Sciences, Thapar University, Patiala, Punjab, India

a r t i c l e i n f o a b s t r a c t

Article history: Bacteria are incredibly diverse. Numerous diverse bacterial species participate in the precipitation of
Received 28 December 2010 mineral carbonates in various natural environments, including soils, geological formations, freshwater
Received in revised form 4 March 2011 biofilms, oceans and saline lakes.
Accepted 11 April 2011
Bacteria are believed to affect carbonate precipitation both through affecting local geochemical condi-
Available online 25 May 2011
tions and by serving as potential, nucleation sites for mineral formation. A novel technique for the reme-
diation of damaged structural formations has been developed by employing a selective microbial
Keywords:
plugging process, in which metabolic activities promote precipitation of calcium carbonate in the form
Bacteria
Concrete
of calcite. Recently, microbial mineral precipitation resulting from metabolic activities of some specific
Bioremediation microorganisms in concrete to improve the overall behavior of concrete has become an important area
Microbial mineral precipitation of research. It has been hypothesized that almost all bacteria are capable of CaCO3 production because
Concrete properties precipitation occurs as a byproduct of common metabolic processes such as photosynthesis, sulfate
reduction, and urea hydrolysis.
In this review paper, an overview of bacteria, their types based on the classification has been studied.
Even the effect of bacteria on various parameters in concrete proves to be beneficial development. Based
on the studied properties like compressive strength, permeability, water absorption, chloride ingression,
the microbial mineral precipitation appears to be a promising technique at this state of development.
Ó 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3792
1.1. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3792
1.2. Morphology of bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3792
1.3. Growth and reproduction of bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3792
2. Ureolytic activity and carbonate biomineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3793
3. Bioremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3794
3.1. Role of bacteria in concrete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3794
3.2. Applications of bacteria in concrete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3796
3.2.1. Microbial concrete as an alternative surface treatment for concrete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3796
3.2.2. Microbial concrete as concrete crack remediation/healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3797
3.2.3. Microbial concrete as water purifier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3797
4. Effect of bacteria on various parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3798
4.1. Compressive strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3798
4.2. Permeability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3799
4.3. Water absorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3799
4.4. Chloride ingression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3800
5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3800
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3800

⇑ Corresponding author.
E-mail address: navneetkchahal@gmail.com (N.K. Chahal).

0950-0618/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.conbuildmat.2011.04.010
3792 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801
1. Introduction
Microbial mineral precipitation resulting from metabolic activ-
ities of some specific microorganisms in concrete to improve the
overall behavior of concrete has become a considerable area of
research. These bacteria are able to influence the precipitation of
calcium carbonate by the production of urease enzyme. Precipita-
tion of calcium carbonate crystals occurs by heterogeneous
nucleation on bacterial cell walls once supersaturation is achieved.
The application of concrete is rapidly increasing worldwide and
therefore the development of bacterial mediated concrete is
urgently needed for environmental reasons. As presently, about
8% of atmospheric carbon dioxide emission is due to cement pro-
duction, mechanisms that would contribute to longer service life
of concrete structures would make the material not only more
durable but also self repair, i.e., the autonomous healing of cracks
in concrete. The potential of bacteria to act as self healing agent in
concrete has proven to be a promising future. This field appears to
be more beneficial as bacterial concrete appears to produce more
substantially more crack plugging minerals than control specimens
(without bacteria).
A promising sustainable repair methodology is currently being
investigated and developed in several laboratories, i.e., a technique
based on the application of mineral producing bacteria. The appli-
cation for ecological engineering purposes is becoming increas-
ingly popular as is reflected by recent studies where bacteria
were applied for removal of chemicals from waste water streams
[1], for bioremediation of contaminated soils [2] and removal of
green house gases from landfills [3]. The applicability of specifi-
cally mineral producing bacteria for sand consolidation and lime-
stone monument repair [4–8] and filling of pores and cracks in
concrete have been recently investigated [9–12]. In all these stud-
ies so far, bacteria or derived ureolytic enzymes were externally
applied on cracked concrete structures or test specimens, i.e., as
surface treatment or repair system. An integrated healing agent
would save manual inspection and repair and moreover increase
structure durability. Addition of such an agent to the concrete mix-
ture would thus save both money and the environment as less
maintenance and use of environmental friendly repair material is
needed.
Microbial carbonate precipitation (biodeposition) decreases the
permeation properties of concrete. Hence, a deposition of a layer of
calcium carbonate on the surface of concrete resulted in a decrease
of water absorption and porosity.
1.1. Bacteria
Bacteria (singular: bacterium) are relatively simple, single-celled
(unicellular) organisms. Their genetic material is not enclosed in a
special nuclear membrane, bacterial cells are called prokaryotes.
Typically a few micrometers in length, bacteria have a wide range
of shapes, ranging from spheres to rods and spirals. Bacteria are
ubiquitous in every habitat on Earth, growing in soil, acidic hot
springs, radioactive waste, water, and deep in the Earth’s crust, as
well as in organic matter and the live bodies of plants and animals.
There are typically 40 million bacterial cells in a gram of soil and a
million bacterial cells in a milliliter of fresh water; in all, there are
approximately five nonillion (5  1030) bacteria on Earth, forming
much of the world’s biomass. Bacteria are vital in recycling
nutrients, with many steps in nutrient cycles depending on these
organisms, such as the fixation of nitrogen from the atmosphere
and putrefaction. However, most bacteria have not been character-
ized, and only about half of the phyla of bacteria have species that
can be grown in the laboratory. The study of bacteria is known as
bacteriology, a branch of microbiology.
1.2. Morphology of bacteria
Bacteria display a wide diversity of shapes and sizes, called mor-
phologies. Bacterial cells are about one tenth the size of eukaryotic
cells and are typically 0.5–5.0 lm in length. However, a few species
– for example Thiomargarita namibiensis and Epulopiscium fishelsoni
are up to half a millimetre long and are visible to the unaided eye.
Among the smallest bacteria are members of the genus Mycoplasma
which measure only 0.3 micrometres, as small as the largest
viruses. Some bacteria may be even smaller, but these ultramicro-
bacteria are not well-studied.
Most bacterial species are either spherical, called cocci
(sing. coccus, from Greek kókkos, grain, and seed) or rod-shaped,
called bacilli (sing. bacillus, from Latin baculus, stick). Some rod-
shaped bacteria, called vibrio, are slightly curved or comma-
shaped; others, can be spiral-shaped, called spirilla, or tightly
coiled, called spirochaetes. A small number of species even have
tetrahedral or cuboidal shapes. More recently, bacteria were dis-
covered deep under the Earth’s crusts that grow as long rods with
a star-shaped cross-section. The large surface area to volume ratio
of this morphology may give these bacteria an advantage in nutri-
ent-poor environments. This wide variety of shapes is determined
by the bacterial cell wall and cytoskeleton, and is important be-
cause it can influence the ability of bacteria to acquire nutrients,
attach to surfaces, swim through liquids and escape predators.
1.3. Growth and reproduction of bacteria
Unlike multicellular organisms, increases in the size of bacteria
(cell growth) and their reproduction by cell division are tightly
linked in unicellular organisms. Bacteria grow to a fixed size and
then reproduce through binary fission, a form of asexual reproduc-
tion. Under optimal conditions, bacteria can grow and divide extre-
mely rapidly, and bacterial populations can double as quickly as
every 9.8 min. In cell division, two identical clone daughter cells
are produced. Some bacteria, while still reproducing asexually,
form more complex reproductive structures that help disperse
the newly-formed daughter cells. Examples include fruiting body
formation by Myxobacteria and aerial hypae formation by Strepto-
myces or budding. Budding involves a cell forming a protrusion
that breaks away and produces a daughter cell.
In the laboratory, bacteria are usually grown using solid or
liquid media. Solid growth media such as agar plates are used to
isolate pure cultures of a bacterial strain. However, liquid growth
media are used when measurement of growth or large volumes
of cells are required. Growth in stirred liquid media occurs as an
even cell suspension, making the cultures easy to divide and trans-
fer, although isolating single bacteria from liquid media is difficult.
The use of selective media (media with specific nutrients added or
deficient or with antibiotics added) can help identify specific
organisms.
Most laboratory techniques for growing bacteria use high levels
of nutrients to produce large amounts of cells cheaply and quickly.
However, in natural environments nutrients are limited, meaning
that bacteria cannot continue to reproduce indefinitely. This nutri-
ent limitation has led the evolution of different growth strategies.
Some organisms can grow extremely rapidly when nutrients
become available, such as the formation of algal (and cyanobacte-
rial) blooms that often occur in lakes during the summer. Other
organisms have adaptations to harsh environments, such as the
production of multiple antibiotics by Streptomyces that inhibit
the growth of competing microorganisms. In nature, many organ-
isms live in communities (e.g. biofilms) which may allow for
increased supply of nutrients and protection from environmental
stresses. These relationships can be essential for growth of a partic-
ular organism or group of organisms (syntrophy).
 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801
Bacterial growth follows three phases. When a population of
bacteria first enters a high-nutrient environment that allows
growth, the cells need to adapt to their new environment. The first
phase of growth is the lag phase, a period of slow growth when the
cells are adapting to the high-nutrient environment and preparing
for fast growth. The lag phase has high biosynthesis rates, as pro-
teins necessary for rapid growth are produced. The second phase
of growth is the logarithmic phase (log phase), also known as the
exponential phase. The log phase is marked by rapid exponential
phase. The rate at which cells grow during this phase is known
as the growth rate (k), and the time it takes the cells to double is
known as the generation time (g). During log phase, nutrients are
metabolized at maximum speed until one of the nutrients is
depleted and starts limiting growth. The final phase of growth is
the stationary phase and is caused by depleted nutrients. The cells
reduce their metabolic activity and consume non-essential cellular
proteins. The stationary phase is a transition from rapid growth to
a stress response state and there is increased expression of genes
involved in DNA repair, antioxidant metabolism and nutrient
transport.
2. Ureolytic activity and carbonate biomineralization
Biomineralization is defined as a biologically induced precipita-
tion in which an organism creates a local micro-environment with
conditions that allow optimal extracellular chemical precipitation
of mineral phases [13]. Numerous diverse microbial species partic-
ipate in the precipitation of mineral carbonates in various natural
environments including soils, geological formations, fresh water
bio films, oceans and saline lakes. The precise role of the microbes
in the carbonate precipitation process is still not clear. Almost all
bacteria are capable of calcium carbonate precipitation [14]. A
novel technique for the remediation of damaged structural forma-
tions has been developed by employing a selective microbial plug-
ging process, in which metabolic activities promote precipitation
of calcium carbonate in the form of calcite. Biomineralization of
calcium carbonate is one of the strategies to remediate cracks in
building materials.
Recently, microbial mineral precipitation resulting from meta-
bolic activities of some specific microorganisms in concrete to
improve the overall behavior of concrete has become an important
area of research. It has been hypothesized that almost all bacteria
are capable of CaCO3 production because precipitation occurs as a
byproduct of common metabolic processes such as photosynthesis,
sulfate reduction, and urea hydrolysis [15].
The hydrolysis of urea by the widely distributed enzyme urease is
special in that it is one of the few biologically occurring reactions
that can generate carbonate ions without an associated production
of protons. When this hydrolysis occurs in a calcium-rich
environment, calcite (calcium carbonate) precipitates from solution
forming a solid-crystalline material. The binding strength of the
precipitated crystals is highly dependent on the rate of carbonate
formation and under suitable conditions it is possible to control
the reaction to generate hard binding calcite cement (or biocement).
The urease enzyme (e.g. urea amidohydrolase; EC 3.5.1.5) is
common in many microorganisms and ureolysis can be induced
in a lab setting by adding urea [3]. One mol of urea is hydrolyzed
intracellularly to 1 mol of ammonia and 1 mol of carbamate (Eq.
(1)), which spontaneously hydrolyzes to form an additional 1 mol
of ammonia and carbonic acid (Eq. (2)). These products subse-
quently equilibrate in water to form bicarbonate, 2 mol of ammo-
nium, and 2 mol of hydroxide ions Eqs. (3) and (4). The latter give
rise to a pH increase, which in turn can shift the bicarbonate equi-
librium, resulting in the formation of carbonate ions (Eq. (5)),
which in the presence of soluble calcium ions precipitate as CaCO3
3793
Eqs. (6) and (8). Eq. (7) is an overall reaction for the system, show-
ing that urea and calcium are added to the system, and ammonium
and calcium carbonate are products.
COðNH2 Þ2 þ H2 O ! NH2 COOH þ NH3 ð1Þ
NH2 COOH þ H2 O ! NH3 þ H2 CO3 ð2Þ
H2 CO3 ! HCO3 þ Hþ ð3Þ
2NH3 þ 2H2 O ! 2NH4þ þ 2OH ðpH increaseÞ ð4Þ
HCO3 þ Hþ þ 2OH ! CO2
3 þ 2H2 O ð5Þ
CO2
3 þ Ca
2þ
! CaCO3 ðcarbonate precipitationÞ ð6Þ
COðNH2 Þ2 þ 2H2 O þ Ca2þ ! 2NH4þ
þ CaCO3 ðoverall reactionÞ ð7Þ
Calcium carbonate is an appropriate mineral to use for the
reduction of porosity of underground formations for many reasons.
Ca2+ is one of the most abundant cations and carbonate ions
(HCO3 and CO2 3 ) are some of the most abundant anions in most
subsurface waters [16]. In order to produce the most mineral mass,
utilizing elements already present in the subsurface is a more effi-
cient method than adding another chemical. Injection of supercrit-
ical CO2 into the underground formations will also make more
carbonate ions by the dissolution and disassociation of CO2, which
in turn will be used to precipitate more mineral.
Bacterial calcium carbonate precipitation results from both pas-
sive and active nucleation. Passive carbonate nucleation occurs
from metabolically driven changes in the bulk fluid environment
surrounding the bacterial cells. This increases the mineral satura-
tion and induces nucleation [17]. In the ureolysis driven system,
this occurs from an increase in pH due to ammonification [5].
Active carbonate nucleation occurs when the bacterial cell surface
is utilized as the nucleation site. The cell clusters exhibit a net elec-
tronegative charge which favors the adsorption of Ca2+ ions. The
Ca2+ ions attract CO23 and HCO
3
ions, which will eventually form
calcium carbonate precipitates [15,18]. Although it is known that
there are many different types of bacteria capable of calcium car-
bonate precipitation, it has been hypothesized that there are spe-
cific attributes of certain bacteria that promote and affect CaCO3
precipitation more than others [15]. It has already been noted that
cell walls have an inherent electronegative charge that affect the
binding of certain ions [19], but the extracellular polymeric sub-
stance (EPS) associated with biofilms may also be involved. Biofilm
cells are contained in the EPS matrix and may use it as an attach-
ment device, for structural support, and/or protection [20]. The EPS
matrix is composed primarily of polysaccharides and, depending
on the side chains attached to the polysaccharides (e.g. carboxyl
groups, pyruvate, phosphate, or sulfate), the matrix can exhibit
an overall negative charge. This negative charge is important in
trapping metal ions within the EPS matrix [21].
One of the primary applications of biomineralization is the
plugging of porous media with applications leaning toward biore-
mediation [22]. Because plugging of porous media can occur in
many different environmental locations and involve many differ-
ent factors, such as soil alkalinity, temperature, and pressure, it is
important to monitor the effectiveness of the bacteria’s ability to
precipitate out calcium carbonate in each different environmental
situation. Research done by [23] showed that the hydrolysis of urea
by Bacillus pasteurii (now reclassified as Sporosarcina pasteurii [24]
is temperature dependent and that the highest calcite precipitation
rates occurred near the point of critical saturation [25]. It also high-
lighted the fact that calcite precipitation is kinetically dependent
3794 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801
on saturation state and independent of temperature. This research
by [23] emphasized the impact of environmental conditions on cal-
cite precipitation that were previously noticed by Nadson, as re-
corded by [25]. Members of the genus Bacillus are Gram-positive,
rod-shaped, endosporeforming bacteria commonly found in soil
[26]. B. pasteurii, a member of this genus, converts urea to ammo-
nium carbonate more actively than any other known bacterium.
Therefore, B. pasteurii and other members of the Bacillus genus
are pincorporated into studies to determine their influence on cal-
cium carbonate precipitation in various environments. Experi-
ments performed indicated that urease activity at high pH in
B. pasteurii favored calcium carbonate precipitation [5]. Upon
examination of the sand grains from columns used in the experi-
ment, bacterial cells were shown encased in calcite crystals, which
indicated that the bacteria acted as a nucleation site for the miner-
alization process, an example of active nucleation.
Another study conducted by [15] looked at strain specific
CaCO3 precipitation. Isolates collected from various soil locations
in Belgium yielded some crystal growth and urease activity and,
when sequenced, showed that all the isolates were closely related
to one another and the group Bacillus sphaericus. Other close rela-
tives of the group are B. pasteurii, Bacillus psychrophilus, Bacillus
globisporus, Planococcus okeanokoites, and Filibacter limicola.
Microbiologically induced (also called ‘‘bacteriogenic’’) calcium
carbonate precipitation is comprised of a series of complex bio-
chemical reactions, including concomitant participations of a bac-
terium like B. pasteurii, urease (urea amidohydrolase; EC 3.5.1.5),
and high pH. In this process, an alkalophilic soil microorganism,
like B. pasteurii, plays a key role by producing urease that hydro-
lyzes urea to ammonia and carbon dioxide. The ammonia increases
the pH in surroundings, which in turn induces precipitation of
CaCO3, mainly as a form of calcite.
In aqueous environments, the overall chemical equilibrium
reaction of calcite precipitation can be described as [27]:
Ca2þ þ CO2
3 ! CaCO3 #
The solubility of CaCO3 is a function of pH and affected by ionic
strength in the aqueous medium. Generally in a medium, say pro-
vided with urea–CaCl2 medium that supports microbial growth,
additional ions including NHþ  +  +
4 , Cl , Na , OH , and H , may affect
chemically induced CaCO3 precipitation at different pHs. Microbio-
logically induced calcium carbonate precipitation occurs via more
complicated processes than chemically induced precipitation.
The bacterial cell surface with a variety of ions can non-
specifically induce mineral deposition by providing a nucleation
site. Ca2+ is not likely utilized by microbial metabolic processes;
rather it accumulates outside the cell. In medium, it is possible that
individual microorganisms produce ammonia as a result of enzy-
matic urea hydrolysis to create an alkaline micro-environment
around the cell. The high pH of these localized areas, without an
initial increase in pH in the entire medium, commences the growth
of CaCO3 crystals around the cell. Possible biochemical reactions in
urea–CaCl2 medium to precipitate CaCO3 at the cell surface can be
summarized as follows [5]:
2þ
Ca2þ þ Cell ! Cell-Ca

Cl þ HCO3 þ NH3 ! NH4 Cl þ CO2
3 cium carbonate polymorphs [30]. Bacterial deposition of a layer
2þ
Cell-Ca þ CO2
3 ! Cell-CaCO3 #
3. Bioremediation
The Bacterial cell surface with variety of ions can non-
specifically induce mineral deposition by providing the nucleation
site. Bacteria have the largest surface area to volume ratio of any
life form [17] and have a net electronegative charge [19]. The
combination of the large surface area and net negative charge
facilitates the binding of dissolved metal ions on the surface of
the Bacteria. In order to determine the maximum amount of cal-
cium carbonate that can be precipitated under extreme conditions,
the effect of common contaminants on the kinetics of the process
have to be considered.
Certain bacteria are capable of surviving in very harsh environ-
ments, and some prefer an extreme pH and higher temperatures.
The normally slow production of toxic chemicals at mining sites
can be accelerated by the presence of chemolithotrophs which
oxidize Fe2+ to Fe3+ and sulfur to SO24 A specific chemolithotroph,
Thiobacillus ferrooxidans, oxidizes iron and sulfur, but is also
capable of immobilizing the portion of metal leachate it comes in
contact with, eventually encapsulating itself in a mineral phase
of mainly iron oxides [17]. By showing that mineral formation
can occur under extreme or toxic conditions, biomineralization
can be viewed as a possible mechanism for cleaning up hazardous
environments.
When used in bioremediation work, bacteria are often in the
presence of toxic substances, and it is important to know if these
toxic substances inhibit the effectiveness of the bacteria. Ureolytic
bacteria capable of precipitation inherent in the contaminated
aquifers [23]. The strategy is to accelerate the precipitation process
by adding urea, thus slowing the transport of harmful chemicals in
the subsurface. The addition of ureolytic bacteria could be an easier
and more efficient method for removing the excess calcium in the
water; ureolysis would be induced with the addition of urea, and
calcium carbonate would precipitate.
An experiment performed [15], showed that up to 85% of cal-
cium was removed from industrial wastewater using this method.
The use of bacteria in bioremediation processes has potential to be
more environmentally friendly, efficient, and cost effective. The use
of inherent bacteria eliminates the need for the addition of bacteria
and costly reagents.
A concrete product prepared by mixing a cement paste contain-
ing microbial cells in particular ratio is known as microbial con-
crete. It has diverse and wonderful properties, due to its major
property of crack remediation or self healing it is also known as self
healing concrete.
3.1. Role of bacteria in concrete
Microorganisms are incredibly diverse and include bacteria,
fungi, archaea and some microscopic plants and animals such as
plankton. Use of bacteria in concrete is recent thirst now a day in
research. However a new approach that a microbial mineral depos-
it constantly occurs in natural environments. The primary role of
bacteria in the precipitation process has been ascribed to their abil-
ity to create an alkaline environment through various physiological
activities [28].
Certain Negatively charged nature and specific functional
groups of microbial cell walls favors the binding of divalent cations
(Ca2+ and Mg2+), thereby making microorganisms ideal crystal
nucleation site [29]. Specific proteins present in biological extracel-
lular polymeric substances cause the formation of different cal-
of calcite on the surface of the specimens resulted in a decrease
of capillary water uptake and permeability towards gas. The t
ype of bacterial culture and medium composition had a profound
impact on calcium carbonate crystal morphology.
Microbial mineral precipitation (biodeposition) involves various
microorganisms, pathways and environments. Considerable re-
search on carbonate precipitaton by bacteria has been done by
using ureolytic bacteria. These bacteria are able to influence the
 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801
precipitaton of calcium carbonate by the production of urease
enzyme. This enzyme catalyzes the hydrolysis of urea to CO2 and
ammonia, resulting an increase of the pH and carbonate concentra-
tion in the bacterial environment [5].
Immobilization technique for remediation of cracks in concrete,
where microbial cells are encapsulated in polymers has been
adapted to enclose Calcium Carbonate precipitation in the gap to
enhance the strength for selective concentration [9]. Microbial
calcite precipitation (MCP) occurs as a byproduct of common
microbial metabolic process, such as urea hydrolysis, photosynthe-
sis, sulfate reduction. These different metabolic processes increase
the alkalinity (pH and dissolved inorganic carbon) and thereby
favoring the calcium carbonate precipitation [31]. Fig. 11 depicts
the formation of Calcium carbonate on the cell wall of bacteria.
Calcium carbonate precipitation is a general process in the
bacterial world under appropriate conditions [14]. Some bacteria
and fungi can induce precipitation of calcium carbonate extracellu-
larly through a number of processes that include photosynthesis,
ammonification, denitrification, sulfate reduction and anaerobic
sulphide oxidation [32,33].
B. pasteurii produces intracellular urease constitutes close to 1%
of the cell dry weight [6]. Bacillus pasteruii, a common soil bacte-
rium can induce the precipitation of calcite. As a microbial sealant,
CaCO3 exhibited its positive potential in selectively consolidating
Fig. 1. A new calcite layer (surface II) formed over the concrete surface (surface I).
Fig. 2. Magnified image of calcite crystals found on the concrete surface.
3795
simulated fractures and surface fissures in granites and in the
consolidation of sand. Besides this, a durability study on concrete
beams treated with bacteria, exposed to alkaline, sulfate and
freeze–thaw environments were also studied. The durability per-
formance increased with increase in the concentration of bacteria.
Microbial calcite precipitation was quantified by X-ray diffraction
(XRD) analysis and visualized by SEM [10].
Biodeposition of a calcium carbonate layer on degraded lime
stone by five different strains of the B. sphaericus group and one
strain of Bacillus lentus [7]. They found that Bacillus strains were
capable of depositing calcium carbonate, but different in amount.
Further to obtain restoring and protective calcite layer on degraded
limestone, five different strains of the B. sphaericus group and one
strain of B. lentus were tested for their ureolytic driven calcium car-
bonate precipitation. Although all the Bacillus strains were capable
of depositing calcium carbonate, differences occurred in the
amount of precipitated calcium carbonate on agar plate colonies.
Seven parameters involved in the process were examined: calcite
deposition on limestone cubes, pH increase, urea degrading capac-
ity, extracellular polymeric substances (EPS)-production, biofilm
Fig. 3. Developing calcite crystals at higher magnification. It can be seen that rod-
shaped objects, consistent with the dimensions of B. pasteurii are spread around the
crystals.
Fig. 4. Rod shaped impressions consistent with the dimensions of B. Pasteurii,
spread around the calcite crystals.
3796 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801

formation, f-potential and deposition of dense crystal layers. The
best calcite precipitating strains were characterized by high ureo-
lytic efficiency, homogeneous calcite deposition on limestone
cubes and a very negative f-potential.
Biomineralization as biologically induced precipitation in which
an organism creates a local environment with conditions that
allow optimal extracellular chemical precipitation of mineral
phases [13]. Bio-mediated production of calcite crystals by carcin-
ogenic bacteria which has great applicable value for the restoration
of deteriorated calcareous monuments, because of its high purity
and coherency [34]. Weathered concrete samples made with
Portland cement or with blast furnace slag cement and fouled by
lichens were treated with Thiobacillus bacteria and an appropriate
nutrient, by submersion or sprinkling. SEM and XRD analysis
revealed a dense layer of calcite and vaterite crystals [35].
Biomineralization of calcium carbonate is one of the strategies
to remediate cracks in building materials because cracks not only
influence the service durability on concrete structure, but also
harmful for the structure safety [36].
Bacterial deposition of a layer of calcite on the surface of the
specimens resulted in a decrease of capillary water uptake and
gas permeability [37]. The effects of bacterial carbonate precipita-
tion (biodeposition) on the durability of mortar specimens with
different porosity. The surface deposition of calcium carbonate
crystals decreased the water absorption with 65–90% depending
on the porosity of the specimens. As a consequence, the carbon-
ation rate and chloride migration decreased by about 25–30%
and 10–40% respectively. An increased resistance towards freezing
and thawing was also noticed. The results obtained with the biode-
position treatment were similar as those obtained with conven-
tional surface treatments [38].
Microbial calcite precipitation was quantified by X-ray diffrac-
tion (XRD) analysis and visualized by SEM [38]. The specimens
with bacteria did not develop any micro cracks, as they did
not expand much unlike control specimens when subjected to
alkali aggregate reactivity, sulfate attack, drying shrinkage and
freeze–thaw. Fig. 1 shows that a new layer (surface II) was
formed over the surface of the cement mortar beam (surface I).
The elemental composition of surface I was found to be charac-
teristic of cement material, and the elemental composition of
surface II, was found to be predominantly calcite material, which
formed an impermeable layer and increased the durability
performance.
Magnified image of calcite crystals found on the surface II of the
bacterial specimen, subjected to freeze thaw, is shown in Fig. 2.
Bacteria were found in intimate contact with the calcite crystals
(Fig. 3). Rod-shaped impressions, consistent with the dimensions
of B. pasteurii were found in the calcite crystals, which formed on
the surface of the specimens in Fig. 4.
3.2. Applications of bacteria in concrete
3.2.1. Microbial concrete as an alternative surface treatment for
concrete
An important measure to protect concrete against damage is
diminishing the uptake of water [39]. Many of the physical and
chemical deterioration mechanisms of concrete are related to
aggressive substances present in aqueous solution.
Surface treatments play an important role in limiting the
infiltration of water and consequently of detrimental components
– into concrete. Broad arrays of organic and inorganic products
are available in the market for the protection of concrete surfaces,
such as a variety of coatings, water repellents and pore blockers.
But these means of protection beside their favorable influences
even show disadvantageous aspects such as:

 Degradation over time.

 Need for constant maintenance.
 Different thermal expansion coefficient of the treated layers.
 Use of certain solvents contributes to environmental pollution
as well.

Bacterial induced carbonate mineralization is a novel and eco-

friendly strategy for the protection and remediation of stone and
mortar [40].

Table 1
Compressive strength values of 7 and 28 days tests with Portland cement mortar
cubes mixed with biomass [10].

Bacillus pasteurii, 7-days compressive 28-days compressive

cells (cm3) strength (MPa) strength (MPa)
Average Standard Average Standard
deviation deviation
Control 0 47 1.38 55 1.27
Live 3.8  103 55 2.39 62 3.23
3.8  10s 54 1.55 63 1.97
3.8  107 57 1.27 65 0.87
Killed 3.8  103 53 3.73 56 2.36
3.8  105 59 3.46 55 2.42
3.8  107 60 1.81 61 0.92
Live 7.6  103 46 2.08 65 0.81
7.6  105 50 2.73 55 2.33
7.6  107 55 1.28 55 0.44
Killed 7.6  103 57 1.47 59 3.19
7.6  10s 61 2.88 56 2.57
Fig. 5. (Above) SEM Micrograph of mortar without any microorganisms [41]. 7.6  107 66 1.41 58 0.79
(Below) SEM micrograph of mortar with anaerobic microorganisms of 105/ml cell
concentration [41]. Average and standard deviation values obtained from triplicate samples.
 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801 3797

Table 2
Effect of the anaerobic microorganisms addition on mortar strength [41].

Cell conc./ml Average mortar compressive strength (MPa)

of water
3 days 7 days 14 days 28 days
Strength ± S.D % Increase relative Strength ± S.D % Increase relative Strength ± S.D % Increase relative Strength ± S.D % Increase relative
to control to control to control to control
Nil 8.67 ± 0.28 – 12.60 ± 0.47 – 16.00 ± 0. 81 – 23.13 ± 0. 23 –
10 8.68 ± 0.44 0 12.74 ± 0.89 1.11 16.21 ± 0.22 1.31 24.21 ± 0. 43 4.66
102 8.76 ± 0.47 1.04 12.87 ± 0.46 2.14 16.44 ± 0. 38 2.75 25.00 ± 0. 88 8.08
103 8.80 ± 0.69 1.49 12.98 ± 0.81 3.01 16.87 ± 0. 64 5.43 25.40 ± 0. 84 9.81
104 8.89 ± 0.87 2.55 13.4 ± 0. 53 6.34 17.10 ± 0.37 6.87 25.44 ± 0. 97 9.98
105 9.34 ± 0.81 7.73 14.70 ± 0.74 16.67 19.50 ± 0. 42 21.87 28.98 ± 0. 86 25.29
106 9.20 ± 0.28 6.11 13.80 ± 0.58 9.52 17.50 ± 0. 81 9.38 26.52 ± 0. 27 14.65
107 8.86 ± 1.01 2.19 13.00 ± 0.23 3.17 17.00 ± 0. 45 6.25 25.69 ± 0.74 11.06

These findings are very important with respect to concrete crack remediation.

3.2.2. Microbial concrete as concrete crack remediation/healing 1. Bacillus subtilis

When cracks appear in the concrete, the possibility for corro- 2. Bacillus thuringiensis
sion of the embedded steel arises which could eventually ruin 3. B. sphaericus.
the integrity of the structure. Without immediate attention, the
cracks can expand and cause extensive damage. Current forms of These cells can be readily extracted from soil and cultured.
concrete crack remediation are structural epoxy, resins, epoxy (Ratio: 0.1–50)
mortar, and other synthetic filler agents. Cement paste may comprise (but not limited to) traditional
These synthetic solutions often need to be applied more than cement (normal Portland cement or blast furnace slag cement).
once as the cracks expand. Clearly there is a need for an effective, These microbial cells have sufficient resistance against strong
long-term, environmentally safe method to repair cracks in con- alkalinity even after they are mixed in the cement paste and
crete structures. Several research groups have investigated the against high temperature during production process.
possibility of biomineralization as an effective method to remedi-
ate cracks and fissures in concrete structures.
Cracks filled with a mixture of B. pasteurii (now reclassified as
Table 4
S. pasteurii) and sand showed a significant increase in compressive Various microbes used in concrete.
strength and stiffness, compared to cracks without cells. Micros-
copy confirmed the presence of calcite crystals and cells near the Applications Types of bacteria

surface of the cracks [10]. Microbial concrete as crack healer Bacillus pasteurii
Other groups have noted that biomineralization can be used in Deleya halophila
Halomonas eurihalina
the conservation of ornamental limestone statues or carvings,
Myxococcus xanthus
similar to its use in concrete remediation. Myxococcus xanthus is Bacillus megaterium
capable of precipitating calcium carbonate. The CaCO3 cements Microbial concrete as surface treatment Bacillus sphaericus
pre-existing calcite grains on the pore walls of the limestone Microbial concrete as water purifier Bacillus subitilis
Bacillus sphaericus
without completely plugging the pore. The resulting crystals are
Thiobacillus
strongly attached and more resistant to stress than were the
pre-existing calcite grains [8].

16
Increase of Compressive Strength (Mpa)

3.2.3. Microbial concrete as water purifier

Concrete and steel are arguably the most widely used construc-
14
tion materials in the world today. Steel bars are embedded in con-
crete to produce stronger building structures and the concrete
provides the added benefit of protecting the steel bars from the 12
elements.
Microbes which have been used to create excellent water puri- 10
fication effect comprises
8

Table 3
6
Effect of E. coli microorganism additions on mortar strength [41].

Cell concentration/ Average compressive strength (Mpa) 4

ml of water
3 days 7 days 14 days 28 days
2
Nil 8.72 ± 0.53 12.58 ± 0.23 16.32 ± 0.26 23.13 ± 0.61
10 8.68 ± 0.47 12.60 ± 0.28 16.33 ± 0.13 23.00 ± 0.31
102 8.74 ± 0.38 12.44 ± 0.25 16.28 ± 0.47 23.11 ± 0.27 0
103 8.44 ± 0.28 12.53 ± 0.42 16.23 ± 0.37 23.14 ± 0.27 12.7 19.5 25.4
104 8.58 ± 0.69 12.61 ± 0.45 16.00 ± 0.42 23.10 ± 0.11
105 8.71 ± 0.44 12.56 ± 0.23 16.34 ± 0.38 23.12 ± 0.31 Crack Depth (mm)
106 8.18 ± 0.64 12.49 ± 0.14 16.29 ± 0.34 23.60 ± 0.24
107 8.73 ± 0.68 12.66 ± 0.16 16.41 ± 0.22 22.50 ± 0.36 Fig. 6. Increase of compressive strength of Portland cement mortar cubes with
different depths in presence of Bacillus pasteurii [10].
3798 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801

Concrete plate (microbial conc.) purifies water impurities after
some time (2 weeks), black muddy colour is changed to clean
colour in 2 months. It also eliminates smell of water. B.O.D. of
1400 can be reduced to B.O.D. of 1.4.
Microbial concrete as water purifier has the following advantages:
 Useable as water purifier tank walls.
 Floor lining of a water purifying facility in homes, industrial
plants.
 The cement containing microbial cells can be effectively used
for purifying water such as river water or lake water and in par-
ticular can be effectively used at a location where water flows at
a low rate with stagnation.
SEM examination done by [41] shows that a mortar made with
a cell concentration of 105/ml concentration, the pores are almost
completely filled with narrow strands of filler as shown in (Fig. 5).
Effect of the anaerobic microorganisms addition on mortar
strength is best explained in Tables 2 and 3. Various applications
of microbes in concrete is explained in Table 4.
4. Effect of bacteria on various parameters
4.1. Compressive strength
The effects of microbes on compressive strength of cement mor-
tar cubes at age of 7 and 28 days was studied [10]. They found that
inclusion of microbial biomass (B. pasteurii) enhanced the compres-
sive strength (Table 1).
They used live and killed cells of different concentrations of
B. pasteurii and found that the live cells, at lower concentrations,
increase the compressive strength of cement mortar with a longer
incubation period. Their findings have been summarized in Fig. 6.
The effects of addition of anaerobic microorganism, Shewanella
species on compressive strength of cement sand mortar [41]. They
found that the strength of mortar cubes increased at all levels of
anaerobic microbe addition. They reported 25% increase in 28 days
compressive strength of cement mortar was achieved with the
addition of about 100,000 cells/ml of mixing water.
Compressive strength of concrete with B. pasteurii as microbes,
had marginal (5–10%) increase in strength (in case of BU and

Fig. 7. Carbonation rate constants, K, for different grades of mortar applied with different types of surface treatments after 6 weeks of accelerated carbonation tests [37].

Fig. 8. Sorptivity coefficient K. for different grades of mortar applied with different types of surface treatments [37].
 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801 3799

6 Except for the water repellents, similar tendencies were observed

water absorbed (kg /sq.m)

between the gas permeability and carbonation rate results.

5 The rate of carbonation and the performance of the surface
treatment were correlated to the water–cement ratio. Carbonation
4 was shown to be related to the nature and connectivity of the
pores, with larger pores giving rise to higher carbonation depths.
3 Significant differences in carbonation depth between treated and
untreated specimens were already noticeable after 2 weeks of
2 accelerated carbonation.
The protective effect of the biodeposition treatment towards
1 carbonation could be improved by additional treatments with bac-
teria and a calcium source or an increased concentration of calcium
0 ions.
10 20 40 60 80 100 120 140 160 180 Film forming coatings and sealants to be effective against car-
Time (hours) bonation, the thickness of the treatment should be at least
200 lm [39]. The mean thickness of the carbonate layer was about
Bac. Sph CaCl2 CaCl2 30–50 lm, nevertheless an improved resistance towards carbon-
Bac Sph CaAc ation was already noticed in Fig. 7.
Bac.Sph NaCa
Reference 4.3. Water absorption

Fig. 9. The influence of surface treatment on the rate of water absorption versus
time for w/c 0.6 mortar cubes [37].
BP bacterial suspended in urea–CaCl2, Bacteria suspended in
phosphate buffer when conc. with BW (Bacteria suspended in
water) had marginal decrease in strength (10%) when compared
to controlled concrete samples [10].
4.2. Permeability
Permeability of concrete is believed to be the most important
characteristic of concrete that affects its durability. The principal
result of the intrusion of chloride (i.e., salt-water) into concrete is
the corrosion of the reinforcing steel. Once this occurs, the struc-
ture will no longer maintain its structural integrity; the lifespan
is reduced, and the general safety of the public is severely
degraded. It is increasingly apparent that for many concrete mem-
bers, the ability of the concrete to resist chloride penetration is an
essential factor in determining its successful performance over an
extended period.
The decrease in gas permeability due to the biodeposition treat-
ments resulted in an increased resistance towards carbonation.
To determine the increase in resistance towards water penetra-
tion a sorptivity test was carried out [37]. Sorpitivity coefficient K
(cm 5 1/2) was obtained by using following expression.
Q pffiffi
¼K t
A
when Q is the amount of water absorbed, A is cross-section of
specimen that was in contact with water (cm2), ‘t’ is the time
(Fig. 8). Sorptivity coefficient K for different grades of mortar
applied with different types of surface treatments helped to
determine the increase in resistance towards the water penetration
which is best depicted (Fig. 8). So, absorption of bacteria and
precipitation of carbonate crystals resulted in weight increase of
the mortar specimens.
So, absorption of bacteria and precipitation of carbonate
crystals resulted in weight increase of the mortar specimens.
The most pronounced reduction in water absorption compared
to untreated samples was reached for the most porous mixture w/c
(0.7) and where urea, nutrient broth and external calcium source
were provided : the most of water absorbed by the mortar samples
after 2 h was the decreased by factor 5 (Fig. 9).

Fig. 10. Chloride migration coefficients, Dnssm, for different grades of applied with different types of surface treatments [37].
3800 R. Siddique, N.K. Chahal / Construction and Building Materials 25 (2011) 3791–3801
Fig. 11. Calcium carbonate formation on cell wall of bacteria (Wikipaedia).
4.4. Chloride ingression
Resistance towards chloride penetration of biodeposition
treated samples was measured with the use of an accelerated
migration test, chloride migration coefficient [37]. The increased
resistance towards the migration of chlorides of cubes treated with
biodeposition was similar to that of the acrylic coating and the
water repellent silanes and silicones and larger than in the case
of the silanes/siloxanes mixture, which were all reported to be
effective in decreasing the rate of reinforcement corrosion (Fig. 10).
5. Conclusions
Based on the studied properties, the conventional methods to
protect concrete from degradation proves to be the advantageous
property. Microbial mineral precipitation appears to be a promis-
ing technique. The type of bacterial culture and medium composi-
tion had a profound impact on calcium carbonate crystal
morphology. The use of pure culture resulted in a more pro-
nounced effects. Metabolic activities of some specific microorgan-
isms in concrete are responsible to improve the overall behavior of
concrete. It has been hypothesized that almost all bacteria are
capable of CaCO3 production because precipitation occurs as a
byproduct of common metabolic processes such as photosynthesis,
sulfate reduction, and urea hydrolysis. Even the effect of bacteria
on various parameters in concrete proves to be beneficial develop-
ment. Based on the studied properties like compressive strength,
permeability, water absorption, chloride ingression, the microbial
mineral precipitation appears to be a promising technique at this
state of development.
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