Ecological Engineering 37 (2011) 554–559

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Improved strength and durability of fly ash-amended concrete by microbial

calcite precipitation
Varenyam Achal a,∗ , Xiangliang Pan a , Nilüfer Özyurt b
a
Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology & Geography, Chinese Academy of Sciences, 818 Beijing Road, Urumqi 830 011, China
b
Department of Civil Engineering, Bogazici University, Istanbul 343 42, Turkey

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

Article history: Fly ash acts as a partial replacement material for both Portland cement and fine aggregate. An innova-
Received 13 August 2010 tive approach of microbial calcite precipitation in fly ash-amended concrete has been investigated. This
Received in revised form is the first report to discuss the role of microbial calcite precipitation in enhancing the durability of fly
21 September 2010
ash-amended concrete. The present study investigated the effects of Bacillus megaterium ATCC 14581 on
Accepted 7 November 2010
compressive strength, water absorption and water impermeability of fly ash-amended mortar and con-
Available online 26 January 2011
crete. Mortar specimens were used for compressive strength and water absorption tests, while concrete
specimens were used for water impermeability tests. At the fly ash concentrations of 10%, 20% and 40%
Keywords:
Bacillus megaterium
in mortars, bacterial cell enhanced mortar compressive strength by 19%, 14% and 10%, respectively, com-
Fly ash pared to control specimens. Treated mortar cubes absorbed more than three times less water than control
Concrete cubes as a result of microbial calcite deposition. Microbial deposition of a layer of calcite on the surface of
Calcite the concrete specimens resulted in substantial decrease of water uptake and permeability compared to
Durability control specimens without bacteria. Microbial cells also prevented ingress of water effectively in different
Environmentally friendly concentrations of fly ash-amended concrete. Scanning Electron Micrography (SEM) analyses evidenced
the direct involvement of bacteria in calcite precipitation. The approach of the present study gives us dual
environment friendly advantages. First, use of fly ash-a recovered resource reduces depletion of natural
resources and also reduces the energy-intensive manufacturing of other concrete ingredients, leading
to savings in both energy usage and emissions of greenhouse gases. And second, use of bacterial cells
to improve strength and durability of fly ash-amended concrete further provides greener and economic
options.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction In the past, use of fly ash in concrete was restricted to specific

Addition of fly ash to concrete has become a common practice
in recent years. Fly ash is a finely divided residue resulting from
powdered coal combustion and acts as a pozzolanic material, i.e.,
the particles react with water and lime to produce cementitious
products (Fraay et al., 1989). Reasons for fly ash addition include
economy and enhancement of certain properties such as worka-
bility and pumpability of fresh concrete and of hardened concrete.
Studies have been published concerning the effect of fly ash on con-
crete porosity and resistivity, pore solution chemistry, oxygen and
chloride ion diffusivity, carbonation rates and passivation (Mangat
and Gurusamy, 1987; Thomas and Matthews, 1992; Montemor
et al., 2000).
∗ Corresponding author. Tel.: +86 151 99137383; fax: +86 991 7885300.
E-mail address: varenyam@gmail.com (V. Achal).
amounts by related codes, since decrease in strength and conse-
quently durability of concrete materials were reported (Khatib,
2005). However, in the new era higher and more effective use of
end products/by products have become more important, since nat-
ural resources are limited and use of by-products is advantageous
environmentally and economically. Therefore, the efforts of many
researchers studying the use of high amounts of fly ash in concrete
show that if necessary precautions are taken, higher amounts of
fly ash can be used in concrete without sacrificing either strength
or durability (Berryman et al., 2005). It is well known that the
deterioration of concrete structures usually involves movement of
aggressive gases and/or liquids from the surrounding environment
into the concrete followed by physical and/or chemical reac-
tions within its internal structure, possibly leading to irreversible
damage (Claisse et al., 1997). Therefore, transport properties and
mechanical properties (compressive strength) are the important
factors for concrete durability. There are a variety of ways to reduce
ingress of external agents in concrete, mainly by use of chemi-

0925-8574/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecoleng.2010.11.009
 V. Achal et al. / Ecological Engineering 37 (2011) 554–559
cal, mineral admixtures and external coatings. But these are not
permanent solutions and also they are not eco-friendly. A novel
strategy to enhance durability of cement based materials is biomin-
eralization of calcium carbonate. Biomineralization is defined as
a biologically induced precipitation in which an organism cre-
ates a local micro-environment, with conditions that allow optimal
extracellular chemical precipitation of mineral phases (Hamilton,
2003). These technologies have been successfully used for consol-
idation of sand columns, for repair of limestone monuments and
for remediation of cracks in concrete (De Muynck et al., 2010).
Biomineralization occurs through chemical reactions mediated by
metabolic activities of an organism in certain environmental con-
ditions. Species of the Bacillus group are able to precipitate calcite
on their cell constituents and in their micro-environment by con-
version of urea into ammonia and carbon dioxide (Hammes et al.,
2003; Achal et al., 2009a,b). The bacterial degradation of urea
locally increases the pH and promotes the microbial deposition of
carbon dioxide as calcium carbonate in a calcium rich environment
(Warren et al., 2001). The basic reaction of the calcocarbonic system
is:
CO3 2− + Ca2+ ↔ CaCO3 (Ksp = 3.8 × 109 ) (1)
where Ksp is the solubility product.
The driving force for precipitation of CaCO3 is the supersatura-
tion level S, defined by the ratio of the ionic product:
S = (Ca2+ ) × (CO3 2− )/Ksp (2)
During microbial urease activity, 1 mole of urea is hydrolysed intra-
cellularly to 1 mole of ammonia and 1 mole of carbamate (Eq. (3)),
which spontaneously hydrolyses to form an additional 1 mole of
ammonia and carbonic acid (Eq. (4)) (Burne and Marquis, 2000).
CO(NH2 )2 + H2 O → NH2 COOH + NH3 (3)
NH2 COOH + H2 O → NH3 + H2 CO3 (4)
These products subsequently equilibrate in water to form bicar-
bonate and 2 moles of ammonium and hydroxide ions (Eqs. (5) and
(6)).
H2 CO3 ↔ HCO3 − + H+ (5)
2NH3 + 2H2 O ↔ 2NH4 + + 2OH− (6)
These two reactions (Eqs. (5) and (6)) give rise to a pH increase,
which in turn shifts the bicarbonate equilibrium, resulting in the
formation of carbonate ions (Eq. (7)). This pH increase takes place
initially in the local micro-environment around the bacterial cell,
and propagates in the bulk solution of the bacterial cell suspension.
HCO3 − + H+ + 2NH4 + + 2OH− ↔ CO3 2− + 2NH4 + + 2H2 O (7)
Thus, the carbonate concentration will increase, inducing an
increase in S (according to Eq. (2)) and resulting in CaCO3 precipi-
tation around the cell, in the presence of soluble calcium ions.
The present study discusses the role of calcite precipitation
induced by Bacillus megaterium ATCC 14581 to enhance the dura-
bility of fly ash-amended cement mortar and concrete. Mortar and
concrete specimens incorporating bacterial cells were prepared by
replacing cement with fly ash at the concentrations of 10%, 20%
and 40%. Further compressive strength, water absorption and water
impermeability tests were carried out and results were compared
with control specimens.
555
Table 1
Chemical analysis of cement and fly ash (%).
SiO2 CaO Al2 O3 MgO Fe2 O3 K2 O Na2 O SO3 TiO2
Cement 21.04 62.11 5.02 2.44 3.12 0.71 0.33 3.12 –
Fly ash 51.47 3.82 33.72 0.67 3.16 1.56 0.65 0.51 1.32
2. Materials and methods
2.1. Materials
Ordinary Portland cement conforming to IS 12269-1987 was
used. Locally available clean, well-graded, natural river sand hav-
ing fineness modulus of 2.89 conforming to IS 383-1970 was used
as fine aggregate. The coarse aggregate was a crushed limestone,
with a maximum size of 29.5 mm. The fly ash was obtained locally
from power plant. The fineness of the fly ash (retained on a 45-␮m
sieve) was 28.8%. The chemical analyses of fly ash and cement are
presented in Table 1.
2.2. Microorganism
B. megaterium ATCC 14581 was used throughout this study. The
culture was maintained at nutrient agar medium (pH 7.5). To pre-
pare mortars and concrete specimens for compressive strength,
water absorption and water impermeability tests, bacterial cells
were grown in nutrient broth containing urea (NBU) media as
described by Achal et al. (2009a). Bacterial culture was grown at
37 ◦ C under shaking condition (130 rpm).
2.3. Viability test of bacteria in fly ash amended mortar/concrete
The survival rate of incorporated B. megaterium ATCC 14581 in
mortar and concrete specimens was determined at different ages
(3-, 7- and 28-days). The number of viable bacterial cells as cfu/ml
was calculated by serial dilution technique. The specimens at differ-
ent ages were crushed to powder in a hand-held mortar, suspended
in aliquots of NBU medium, homogenized by repeated mixing on
a vortex mixer and ultrasonic treatment in a water bath, and sub-
sequently serially diluted and plated on nutrient agar plate and
incubated at 37 ◦ C overnight.
2.4. Compressive strength test
To study the compressive strength test of cement mortar, B.
megaterium ATCC 14581 was grown in NBU media. The cement
to sand ratio was 1:3 (by weight), and the water to cement ratio
was 0.47. The bacterial culture to cement ratio was also maintained
at 0.47. Fly ash was added by replacing the amount of cement at
the concentrations of 10%, 20% and 40%. A cube mould of 70.6 mm
was used, as per IS 4031-1988. Sand and cement were thoroughly
mixed, adding along with grown culture of B. megaterium ATCC
14581 correspondence to 5 × 107 cfu/ml. Cubes were cast and com-
pacted in a vibration machine. After de-molding, all specimens
were cured in NBU medium at room temperature until compression
testing at the intervals of 3, 7 and 28 days. Control specimens were
also prepared in a similar way without adding bacterial cells. Com-
pression testing was performed using an automatic compression
testing machine.
2.5. Water absorption test
To determine the increase in resistance towards water penetra-
tion, a sorptivity test, based on the RILEM 25 PEM (II-6), was carried
out on mortars. The mortars were prepared exactly the same way as
556 V. Achal et al. / Ecological Engineering 37 (2011) 554–559
for compressive strength tests. The mortar specimens were cured
in media for 28 days. After curing, the specimens were dried at
45 ◦ C in a ventilated oven, establishing a mass equilibrium of less
than 0.1% between two measurements at 24 h intervals. The speci-
mens were then exposed to 10 ± 1 mm of water (water level about
2 mm above the base of the specimen). This was done in an atmo-
sphere of 20 ◦ C and R.H. of 60%. At regular time intervals (15 min,
30 min; 1 h, 1.5 h, 3 h, 5 h, 8 h, 24 h, 72 h, 96 h, 120 h, 144 h and
168 h) the specimens were removed from the water and weighed,
after drying the surface with a wet towel. Immediately after the
measurement the test specimens were submerged again. The sorp-
tivity coefficient, k [cm s−1/2 ], was obtained by using the following
expression:
Q √
=k t
A
where Q is the amount of water absorbed [cm3 ]; A is the cross sec-
tion of the specimen that was in contact with water [cm2 ]; t is the
time [s], Q/A was plotted against the square root of time, then k
was calculated from the slope of the linear relation between the
former.
2.6. Water impermeability test
For conducting the water impermeability test, concrete cubes of
dimension 150 mm (M20 grade) were prepared with grown culture
of B. megaterium ATCC 14581 grown in NBU media correspondence
to 5 × 107 cfu/ml and all specimens were cured in the same media
at room temperature for 28 days. Ordinary Portland cement, fine
aggregate (medium-sized natural river sand) and crushed stone
coarse aggregate with maximum size of 29.5 mm was used. The
ratio of cement:sand:coarse aggregate was 1:1.54:2.86. The water,
cement ratio was 0.47. The bacterial culture to cement ratio was
also maintained at 0.47. Fly ash was added by replacing the amount
of cement at the concentrations of 10%, 20% and 40%. Control sam-
ples (without bacterial cells) were also prepared in the similar
manner. After air drying for 24 h, specimens were firmly secured
in position in the impermeability test apparatus and analysed as
per DIN 1048. Atmospheric pressures of 1, 3 and 7 bars were
applied sequentially 24 h each. At the end of the test, each spec-
imen was removed and split into two halves for determining the
water penetration depth. Resistance to penetration is a measure of
impermeability of concrete.
2.7. SEM analysis
To study the role of microbially induced calcite precipitation,
fly ash amended mortar and concrete specimens were observed
under scanning electron microscope (SEM). After the compressive
strength test, the broken specimens were collected and dried at
60 ◦ C for 24 h. The samples were gold-coated with a sputter coating
machine (Emitech K575) and examined using a JEOL JSM-5410LV
(JEOL, United Kingdom) SEM at accelerating voltages ranging from
15 to 35 kV.
2.8. Statistical analysis
All the experiments were performed in triplicate. The data was
analysed by Analysis of Variance (ANOVA) and the means were
compared using Tukey’s test. All the analyses were performed using
GraphPad Prism (5.01) software.
(8)
Fig. 1. Survival count of B. megaterium ATCC 14581 obtained from different con-
centrations of fly ash amended mortar (M) and concrete (C) specimens at different
ages.
3. Results and discussion
3.1. Survivability of bacteria in fly ash amended mortar/concrete
Serial dilution technique allowed estimation of the number
of viable bacterial cells (cfu/ml) present in mortar and concrete
specimens at different ages. To count bacteria, it must be free
from the cement matrix and ideally brought in a single cell
(non-clumped) suspension (Jonkers et al., 2010). The initial bac-
terial dose, while preparing mortar and concrete specimens, was
5 × 107 cfu/ml. It is clearly indicated from Fig. 1 that number
of viable bacterial cells substantially decreased with increasing
specimen age but number of bacteria were sufficient enough to
carry out their job. At 10% fly ash concentration, the numbers of
viable cells in mortar and concrete specimens were recorded as
7.3 × 106 cfu/ml and 6.1 × 106 cfu/ml, respectively, at the age of 3
days. The number of viable cells at 3 days was maximum in 40%
fly ash amended concrete (8.3 × 106 cfu/ml) followed by similar
concentration in mortar (8.1 × 106 cfu/ml) while these specimens
contained 8.3 × 104 cfu/ml and 9.0 × 104 cfu/ml cells in mortar and
concrete samples, respectively, at 28 days. Mortar specimen (with-
out fly ash) contained 3.5 × 106 cfu/ml cells while 4.7 × 106 cfu/ml
cells were recorded in case of concrete specimen (without fly ash)
at the age of 3 days. At the age of 28 days, these specimens contained
3.2 × 104 cfu/ml and 5.2 × 104 cfu/ml cells in mortar and concrete
samples, respectively. Also bacterial count was more in case of fly
ash amended mortar or concrete specimens irrespective of their
ages and higher the percentage of fly ash, more was survival rate.
So it can be concluded that due to more number of pores in fly ash
amended specimens (especially at higher concentrations), bacte-
ria might get better aeration for better growth. Control specimens
which were prepared without added bacteria did not yield viable
bacterial cells.
3.2. Compressive strength test
Compressive strengths of cement mortar cubes amended with
fly ash were tested to determine the efficiency of improvement by
bacterial cells i.e., B. megaterium ATCC 14581. Fig. 2 summarizes
the 3, 7 and 28 day compressive strength of cement mortar cubes
containing microbial cells. Higher the concentration of fly ash,
lower was compressive strength with all the mortars with or with-
out bacterial cells. But, the compressive strength had significantly
 V. Achal et al. / Ecological Engineering 37 (2011) 554–559
Fig. 2. Effect of B. megaterium ATCC 14581 on the compressive strength of cement
mortar cubes amended with different concentrations of fly ash at 3, 7 and 28 days.
Error bars show standard deviation (n = 3) (BM – Bacillus megaterium).
increased for the mortar cubes that contained microbial cells
irrespective of the fly ash concentration used to make it. Mixing of
bacterial cells in mortar cubes (without containing fly ash) showed
around 21% improvement in compressive strength at 28 days
(28 MPa) with respect to control specimen (23.2 MPa), whereas
improvement in the compressive strength of mortars (containing
10% fly ash) by bacterial cells was 19% (27.6 MPa) compared to
control specimen (23.1 MPa). At the 20% fly ash concentration in
mortars, the bacterial cell enhanced its compressive strength by
14% compared to control specimen. There was also good improve-
ment in the compressive strength of mortars containing 40% fly ash
with bacterial cells, as it lead to 10% improvement in the strength
compared to control specimen. Therefore, it can be concluded that
B. megaterium ATCC 14581 significantly improved the strength of
mortars amended with even higher concentrations of fly ash at 28
days. This improvement in compressive strength is probably due to
deposition of CaCO3 on the microorganism cell surfaces and within
the pores of cement–sand matrix, which plug the pores within the
mortar (Ramakrishnan et al., 1998; Ramachandran et al., 2001).
The overall trend of an increase in compressive strength up to 28
days might be attributed to the behavior of microbial cells within
the cement mortar matrix. During the initial curing period, micro-
bial cells obtained good nourishment, because the cement mortar
was still porous; but growth was not proper as there was com-
pletely new environment for microbes (Achal et al., 2009a). There
was a measurable increase in compressive strength of cement
mortar cubes prepared with bacterial cells, supported by previ-
ous studies (Ramachandran et al., 2001; Bang et al., 2001; Ghosh
et al., 2005; Achal et al., 2009a). Further, it was concluded that the
increase in compressive strengths is mainly due to consolidation
of calcium carbonate that is induced by bacteria in the pores of the
cement mortar cubes.
3.3. Water absorption test
Fig. 3 shows the influence of the microbially induced calcite
precipitation on the water absorption rate in mortar cubes. Over
a period of 168 h (7 days), the cubes amended with fly ash (0%,
10% and 20%) with bacterial cells absorbed nearly 3.5 times less
water (Fig. 3b) than the control cubes (Fig. 3a) while in case of cubes
containing 40% fly ash, mortars absorbed two times less water com-
pared to control. The presence of bacteria resulted in a significant
decrease of the water uptake compared to control specimens. Sim-
557
Fig. 3. The influence of the (a) control and (b) microbial treatment on the rate of
water absorption versus time for mortar cubes amended with different concentra-
tions. Error bars show standard deviation (n = 3).
ilar observations were made by previous reports (De Muynck et al.,
2008a,b; Achal et al., 2010a).
The deposition of a layer of calcium carbonate on the surface and
inside pores of the mortar specimens resulted in a decrease of water
absorption and permeability. Once the pores are filled with inert
material like calcium carbonate, continuous channels for passage
of water, air and pollutants are sealed and reduction in ingress of
water and chlorides is observed. This deposition by microbial action
can seal the voids, pores, and cracks of microscopic size where
other sealants cannot go. As a consequence, the ingress of harmful
substances may be limited. Nemati and Voordouw (2003) noticed
a decrease in the permeability of sandstone cores after injecting
CaCO3 forming reactants. From this experiment, it is clear that the
presence of a layer of carbonate crystals on the surface by bacterial
cells has the potential to improve the resistance of cementitious
materials towards degradation process.
3.4. Water impermeability test
The water impermeability results of concrete are presented in
Table 2. The top surface of concrete specimens was more imper-
meable to water compared to side surface. The top surface water
penetration was 9.3 mm in bioremediated specimens compared to
33.1 mm (in control) in case of concrete specimens without fly ash.
At 10% fly ash concentration, top surface water penetration was
also found to very low in bioremediated specimens (9.7 mm) com-
pared to 36.5 mm in control, while at the fly ash concentration of
40% top surface water penetration was 20.8 mm in bioremediated
specimens compared to 41.5 mm in control concrete. These results
558 V. Achal et al. / Ecological Engineering 37 (2011) 554–559
Fig. 4. Scanning electron micrographs of (a) fly ash-amended mortar, (b) close view of fly ash-amended mortar surface and (c) fly ash-amended concrete; showing microbially
induced calcite precipitation (Cc – calcite crystals).
Table 2
Water penetration in fly ash-amended concrete prepared with and without bacterial
cells.
Fly ash (%) Top penetration (mm) Side penetration (mm)
Control Biotreated Control Biotreated
0 33.14 ± 2.2c * 9.32 ± 1.5d * 41.44 ± 2.7d * 18.67 ± 2.5cd *
10 36.52 ± 2.5b * 9.73 ± 2.1c * 43.18 ± 2.3c * 19.78 ± 1.5c *
20 39.32 ± 1.7ab * 10.48 ± 1.2b * 46.72 ± 2.4b * 21.72 ± 2.1b *
40 41.56 ± 2.3a * 20.88 ± 1.6a * 57.81 ± 1.4a * 33.72 ± 2.0a *
*
Values bearing different letter in the same column are significant at P < 0.05. All
values are mean ± SD (n = 3).
indicated that the permeability of the concrete cubes with bacterial
cells was lower than that of the control specimens irrespective of
concentrations of fly ash amended in it. Bacterial cells were highly
effective in preventing the ingress of water especially for specimens
prepared by using lower concentration of fly ash. Bacterial cells
also prevented ingress of water effectively in 40% fly ash-amended
concrete compared to control specimens. The lower permeability
of the bioremediated cubes was probably due to a denser interfa-
cial zone formed due to calcite precipitation between the aggregate
and the concrete matrix when compared with that of the control
specimens.
The resistance to water is measured as penetration depth at the
top and the sides of the concrete cubes. The penetration at the sides
is higher than that at the top. The authors believe that the reason for
having higher values of water penetration at the top surfaces are
due to better compaction and closing of pores at the top which was
also supported by previous study (Achal et al., 2010a). Understand-
ably, calcite precipitates better on the top surface due to gravity.
Bacterial intervention has dramatically reduced the depth of pene-
tration regardless of the media. This demonstrates the profound
effect of the proposed process on the permeability of concrete.
This can be extremely beneficial in remediating porous or dete-
riorated concrete. Research has indicated that a concrete which
is low in permeation properties lasts longer without exhibiting
signs of distress and deterioration (Nolan et al., 1995). The bac-
terial intervention was far more effective on top than on the sides.
The precipitated calcite crystals help in lowering the permeability
of water and other harmful substances thus increasing the dura-
bility of concrete (Van Tittelboom et al., 2010). Consistent with
compressive strength and water absorption tests, biocalcification
also showed improved water impermeability results.
3.5. SEM analysis
Calcite precipitation in mortar and concrete specimens by B.
megaterium ATCC 14581 was visualized by SEM analysis. A dense
growth of calcite crystals embedded with bacterial cells was
observed in the specimens (Fig. 4). Bacteria were found in inti-
mate contact with the calcite crystals. On closer observations,
rod-shaped bacteria associated with calcite crystals were found.
This deposition serves as a barrier to harmful substances from
entering the sample, and thus improves its impermeability. The
presence of crystalline calcite associated with bacteria indicates
that it served as a nucleation site during the mineralization pro-
cess (Stocks-Fischer et al., 1999; Achal et al., 2010b). Based on these
results it can be concluded that biocalcification by B. megaterium
plays an important role in enhancing the durability of any building
materials or structures.
4. Conclusion
Microbially induced calcite precipitation has been demon-
strated as a new potential method to enhance the durability of
fly ash-amended concrete. The present work deals with the mea-
surement of compressive strength, water absorption and water
impermeability (which are the most important parameters influ-
encing the durability of concrete) for bacterially treated mortars
and concrete specimens and finally its performance. The technique
of microbial calcite precipitation significantly improved strength
of fly ash-amended concrete and enhanced its water imperme-
ability. Further SEM analysis confirmed the presence of calcite
induced by bacteria. The use of this biological technique is highly
desirable because the calcite precipitation induced as a result of
microbial activities is pollution free and natural. The current work
demonstrates that this approach has great promise as a novel
biological metabolic product that can be utilized to improve the
durability of fly ash amended concrete and other such building
materials.
Acknowledgements
This work was supported by Knowledge Innovation Program
of Chinese Academy of Sciences (KZCX2-YW-335 and KZCX2-YW-
135), Program of 100 Distinguished Young Scientists of the Chinese
Academy of Sciences and National Natural Science Foundation of
China (40673070, 40872169 and 41072195).
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