jmb

J. Microbiol. Biotechnol. (2015), 25(8), 1328–1338

http://dx.doi.org/10.4014/jmb.1411.11037 Research Article

Review

Effect of Microorganism Sporosarcina pasteurii on the Hydration of

Cement Paste
Jun Cheol Lee1, Chang Joon Lee2, Woo Young Chun3, Wha Jung Kim3, and Chul-Woo Chung4*
1
School of Architecture and Civil Engineering, Kyungpook National University, Daegu 702-701, Republic of Korea
2
Department of Architectural Engineering, Chungbuk National University, Cheongju 362-763, Republic of Korea
3
School of Architectural, Civil, Environmental, and Energy Engineering, Kyungpook National University, Daegu 702-701, Republic of Korea
4
Department of Architectural Engineering, Pukyong National University, Busan 608-739, Republic of Korea

Received: November 14, 2014

Revised: April 13, 2015
Accepted: April 14, 2015
First published online
April 15, 2015
*Corresponding author
Phone: +82-51-629-6084;
Fax: +82-51-629-6081;
E-mail: cwchung@pknu.ac.kr
pISSN 1017-7825, eISSN 1738-8872
Years of research have shown that the application of microorganisms increases the
compressive strength of cement-based material when it is cured in a culture medium. Because
the compressive strength is strongly affected by the hydration of cement paste, this research
aimed to investigate the role of the microorganism Sporosarcina pasteurii in hydration of
cement paste. The microorganism’s role was investigated with and without the presence of a
urea-CaCl2 culture medium (i.e., without curing the specimens in the culture medium). The
results showed that S. pasteurii accelerated the early hydration of cement paste. The addition
of the urea-CaCl2 culture medium also increased the speed of hydration. However, no clear
evidence of microbially induced calcite precipitation appeared when the microorganisms were
directly mixed with cement paste.

Copyright © 2015 by Keywords: Compressive strength, hydration, characterization, microorganism, Sporosarcina

The Korean Society for Microbiology pasteurii
and Biotechnology

Introduction nucleus on the bacterial cell [18]. This becomes a nucleation

Concrete has been widely used as a construction and
building material. During the service life of concrete, it
experiences weathering, including abrasion, shrinkage, and
expansion cracking associated with freeze–thaw cycles,
sulfate attack, alkali silica reaction, biological degradation,
and so on. To remediate the integrity of concrete, a broad
range of organic and inorganic products have been proposed
as self-healing materials [8, 9].
Microbially induced calcite (CaCO3) precipitation (MICP)
is one of the processes that drives the self-healing of
concrete. The concept is to use the urease activity of
microorganisms [9]. When microorganisms are in contact
with urea, they hydrolyze urea into CO2 and ammonia. The
alkalinity increases as a result. Because the negatively
charged bacterial cells favor binding of divalent cations
such as Ca2+, the HCO3- (CO2 that is produced during
urease activity and dissolved in solution) reacts with Ca2+
in the solution and forms a heterogeneous calcite (CaCO3)
site for a continuous MICP process [5-8] that repairs
damaged concrete.
MICP has also been shown to increase the compressive
strength of cementitious materials [1, 4, 7, 13, 18, 19]. The
increase in the compressive strength was associated with a
reduction of the porosity by filling of the pore space with
calcite when the cementitious specimens were cured in a
culture medium. MICP has been used to coat the surface of
cement-based materials to increase their durability [16, 17].
It was clearly shown that MICP can be successfully induced
in the open pore spaces to remediate damage when it is
applied after cracking [9].
Note that it is cumbersome to apply a urea-CaCl2 culture
medium in the curing of cementitious materials. The
medium is known to drive the increase in the compressive
strength, but it is economically infeasible to use urea in
mortar or concrete because the same curing condition is
unlikely to be applied for real concrete structures. Therefore,
research has been conducted to improve the strength and

J. Microbiol. Biotechnol.

durability of mortar or concrete using microorganisms without
a urea-CaCl2 culture medium. Ghosh et al. [5] reported that
the compressive strength of cement mortar was positively
affected, although no clear evidence of calcite precipitation
was observed. Note that the available literature is still
limited with respect to the role of microorganisms on
hydration of cement paste with and without a urea-CaCl2
culture medium, so further investigation is necessary.
In this study, the role of the microorganism Sporosarcina
pasteurii (ATCC 11859) in hydration of cement paste with
and without the presence of a culture medium was
investigated. S. pasteurii was chosen because it has been
known to survive in a cementitious environment (high-pH,
calcium-rich environment) and was also shown to increase
the compressive strength in the presence of a culture
medium [9, 7, 13].
Materials and Methods
Preparation of Microorganisms
As mentioned, S. pasteurii (ATCC 11859) was used for this study.
The strain was purchased from the Korean Biological Resource
Center (Daejon, Korea). Tryptic soy broth (TSB) culture medium
was used to grow the microorganisms. The TSB culture medium
was stored in autoclave conditions for 15 min at 121oC to eliminate
other microorganisms that may be present. The microorganisms
were inoculated in the TSB culture medium, and the medium with
the microorganisms was shaken at 170 rpm for 24 h at 30oC under
microaerobic conditions to facilitate rapid growth [14]. Then the
TSB culture medium with the microorganisms was centrifuged at
8,000 rpm, washed twice with distilled water, and diluted in
distilled water to obtain a mixing water with an optical density of
1.0 at 600 nm (approximately 107 cells/ml). This diluted solution
of 107 cells/ml was used as an original source. Mixing water with
cell concentrations of 105, 103, and 101 cells/ml was obtained by
diluting the original source.
Observation of S. pasteurii
Before the cement paste samples were prepared, the presence
and activity of the microorganisms that were grown in TSB culture
were verified using a urea-CaCl2 culture medium. The medium
was prepared using 3 g/l nutrient broth, 20 g/l urea, 2.12 g/l
NaHCO3, 10 g/l NH4Cl, and 3.7 g/l CaCl2·2H2O [6, 14]. The pH of
the urea-CaCl2 medium was adjusted to 6.0 using 6 N HCl
solution [14]. The urea-CaCl2 culture medium was used to facilitate
MICP after the microorganisms were grown. The MICP process
Table 1. Chemical composition of type I Portland cement.
Compound name CaO SiO2 SO3 Fe2O3 Al2O3
Conc. (%) 66.681 19.051 3.808 3.797 2.888
Effect of Microorganism on Cement Hydration 1329
caused by the microorganisms under atmospheric conditions was
observed using a light transmission optical microscope (biological
microscope model NSB-50T/B; Samwon Ltd., Korea) and a field
emission scanning electron microscope (FE-SEM model SU8200;
Hitachi Ltd., Japan).
Semi-Adiabatic Calorimetry
Distilled water containing no microorganisms was used to
make a plain cement paste sample. Note that no culture medium
solution was used to mix the cement paste samples with the
microorganisms. Two cell concentrations, 103 and 107 cells/ml,
were added directly to the cement paste to investigate the effect of
the cell concentration on the hydration of cement paste without
the urea-CaCl2 culture medium.
To make the specimens, 2,000 g of type I Portland cement that
conforms to the American Society for Testing and Materials
(ASTM) C 150 specification and 700 ml of distilled water (w/c =
0.35) containing the targeted cell concentration were used. The
chemical composition of type I cement is shown in Table 1. The
materials were mixed by following the American Society for
Testing and Materials (ASTM) C 305 specification.
After a cement paste sample of w/c 0.35 was prepared, it was
immediately poured into a container having a diameter of 60 mm
and a height of 72 mm encapsulated within polystyrene foam. A
thermocouple was immersed inside each cement paste sample to
measure the temperature rise of the cement paste during the early
hydration period. The temperature rise of specimens with and
without the microorganisms can be used to understand their
effect on early hydration of cement paste.
Hydration Study
To investigate the role of the microorganisms in the hydration
kinetics of cement paste, 10 g of type I Portland cement and 10 ml
of distilled water (or urea-CaCl2 culture medium) containing
107 cells/ml (w/c = 1) were poured into a 50 ml plastic tube. The
cap of the tube was sealed, and the tube was vigorously shaken to
mix the cement, water, and microorganisms. A water-to-cement
ratio yielding a loose concentration was chosen to improve the
effectiveness of the evaluation by reducing the effect of
microorganisms being captured within the pores and becoming
inactive. Only one concentration level, 107 cells/ml, was chosen
for this experiment. Cement paste mixed with distilled water
containing no microorganisms was also prepared to provide a
reference guideline. After shaking, the lid of the tube was opened
again, and the plastic tube was filled with N2 gas to limit further
ingress of ambient air into the specimen.
To understand the reaction kinetics of cement paste with
the microorganisms, quantitative X-ray diffraction (XRD) and
MgO K2O TiO2 ZnO SrO P2O5
1.893 1.17 0.357 0.15 0.126 0.079
August 2015 ⎪ Vol. 25 ⎪ No. 8
1330 Lee et al.

differential scanning calorimetry/thermogravimetric analysis (DSC/

TGA) were used at sample ages of 1, 3, 7, and 28 days. Because it
was difficult to directly measure the amount of C-S-H during
hydration, the amount of calcium hydroxide was measured to
estimate the calcium silicate hydration.

X-Ray Diffraction
The crystalline structure of w/c = 1 cement paste samples with
and without the microorganisms was examined by XRD, using a
Rigaku D/Max-2500 instrument (Rigaku, Tokyo, Japan). For
quantitative Rietveld analysis, 10% of TiO2 (rutile) was added to
the specimens as an internal standard, and the specimens were
gently ground to equally disperse the rutile into the cement paste.
The scanning angle 2θ was varied from 5o to 90o with a step size of
0.02o and a dwell time of 1.5 sec. The working voltage was 40 kV,
and the electric current was 200 mA. The scanned data at the ages
of 1, 3, 7, and 28 days were first analyzed using the EVA software.
The data were compared with the Inorganic Crystal Structure
Database (ICSD) to obtain the phase analysis. A quantitative
Rietveld analysis was conducted using TOPAS 4.2 to determine
the amount of calcium hydroxide in the cement paste samples.
The following ICSD entries were used: 1841, tricalcium aluminate;
1956, anhydrite; 9197, brownmillerite (tetracalcium aluminoferrite);
27039, ettringite; 31330, rutile; 59327, monocarbonate; 62363,
Friedel's salt; 63250, hydrocalumite; 64759, hatrurite (tricalcium
silicate); 79550, larnite (dicalcium silicate); 79674, calcite; 100138,
monosulfate; 29210, quartz; 9863, periclase; and 202220, portlandite
Fig. 1. Light transmission optical microscopy images of
(calcium hydroxide). Note that no ICSD entry was available for
samples.
hemicarbonate, so the amount of hemicarbonate could not be
(A) Without inoculation of S. pasteurii into urea-CaCl2 culture medium,
investigated.
and (B) S. pasteurii being inoculated into urea-CaCl2 culture medium.

Differential Scanning Calorimetry/Thermogravimetric Analysis

To verify the amount of calcium hydroxide in the w/c = 1
cement paste samples with the microorganisms, the samples were
analyzed at the ages of 1, 3, 7, and 28 days using DSC/TGA
equipment (SDT Q600; TA Instrument, Japan). For the analysis,
the temperature was raised from 25oC to 1,000oC at a heating rate
of 10oC/min. The weight loss and endothermic DSC peak at about
450oC were used to calculate the amount of calcium hydroxide in
the samples. The amount of calcite in the specimens was also
verified using DSC/TGA data.
Results
Observation of S. pasteurii
Fig. 1 shows optical microscopy images of the culture
medium before and during inoculation. Before inoculation
with the microorganisms (Fig. 1A), the urea-CaCl2 culture
medium showed no evidence of MICP. However, as the
microorganisms were inoculated into the medium, dark
spherical material clearly appeared (Fig. 1B). The dark
color was associated with a lack of light transmission,
indicating that the observed material was solid. A round
shape appears in Fig. 1B because the microorganisms tend
to gather and form spheres when they are active. Note that
each sphere cannot be directly related to an individual
calcite crystal. In fact, it is a group of calcite crystals being
precipitated onto the shell of the microorganisms.
The observed SEM images are presented in Fig. 2. At
1,000× (Fig. 2A), the surface of the sphere was rough with
some open spaces. The SEM image at 6,000× (Fig. 2B)
shows that the crust of the sphere consisted of many small
crystals. The microscopy observations clearly showed that
the microorganisms used in this experimental work
actively formed calcite under ambient conditions when the
urea-CaCl2 culture medium was available.
Semi-Adiabatic Calorimetry
The temperature rise of the specimens containing 103 and
107 cells/ml is presented in Fig. 3. Although the differences
in the temperature rise were minimal, the addition of the

J. Microbiol. Biotechnol.

Fig. 2. Scanning electron microscopy images of the shell
of S. pasteurii at (A) 1,000× magnification and (B) 6,000×
magnification.
microorganisms clearly increased the early temperature
rise of the cement paste samples. The temperature rise of
the samples also increased when the cell concentration was
increased. The results indicate that the incorporation of the
microorganisms into cement paste accelerated hydration of
the cement paste.
Hydration Study
X-ray diffraction. The XRD patterns of hydrated cement
paste (w/c = 1) with the microorganisms are presented in
Fig. 4. The XRD pattern of unhydrated cement powder is
also shown in Fig. 4A. The observed phases in the hydrated
cement paste samples and their amounts identified by
Rietveld quantitative analysis are shown in Tables 2–5.
Although the XRD scan was performed from 5° to 90° for
quantitative Rietveld analysis, Fig. 4 shows only the XRD
patterns from 5° to 35° to facilitate the identification of
phases associated with calcium silicate and aluminate
hydration.
According to Fig. 4A, the XRD patterns of all the 1-day-
Effect of Microorganism on Cement Hydration 1331
Fig. 3. Temperature rise of cement paste with and without
S. pasteurii.
Gray line: plain cement paste; black line: cement paste with 103 cell/ml
of S. pasteurii; black dotted line: cement paste with 107 cell/ml of
S. pasteurii.
old specimens showed hydrated phases of ettringite (at
9.1o) and calcium hydroxide (portlandite, at 18.1o). In the
cement paste mixed with urea-CaCl2 or urea-CaCl2 and the
microorganisms, a clear indication of Friedel’s salt (AFm
structure that incorporates a Cl- ion in the crystal structure)
was observed. Because Friedel’s salt was not observed in
the absence of urea-CaCl2, the results indicate that the
available Cl- ions in the urea-CaCl2 culture medium entered
the AFm structure. Gypsum (at 11.6o) in the unreacted
cement disappeared when the cement was hydrated. Some
unreacted ferrite (brownmillerite: C4AF), C3S, C2S, and C3A
was also observed (at 2θ angles above 30o) in all the
samples after 1 day. However, the presence of C3A was
slightly unclear. The peak at 29.4o (after 1 day) was calcite
that originated from unreacted cement powder. The peak
at 27.4o was the rutile internal standard used for quantitative
Rietveld analysis. Quartz was observed in all the samples
at 26.5o at all ages. A very small peak of monosulfate was
identified at 9.8o, except in the cement paste mixed with
urea-CaCl2 and the microorganisms. Although it was not
shown in Fig. 4A, periclase (MgO) was also identified in all
the samples at all ages.
After 3 days (Fig. 4B), cement paste and cement paste
with the microorganisms started to develop hemicarbonate
(at 10.6o). Monosulfate completely disappeared in all the
samples. Ettringite and portlandite were still observed, and
the peak intensity of portlandite increased after 3 days.
However, the cement paste with urea-CaCl2 and urea-
August 2015 ⎪ Vol. 25 ⎪ No. 8
1332 Lee et al.

Fig. 4. XRD patterns of hydrated cement paste with S. pasteurii after (A) 1 day of hydration, (B) 3 days of hydration, (C) 7 days of
hydration, and (D) 28 days of hydration.
(E) Same as (D) but focused on 5 to 15o. □ : cement paste, ◇ : cement paste with S. pasteurii, △ : cement paste with urea-CaCl2 culture medium,
○ : cement paste with S. pasteurii and urea-CaCl2 culture medium, ☆ : unhydrated cement paste

CaCl2 plus the microorganisms still showed Friedel’s salt as
the dominant form of AFm. No hemicarbonate was observed
in these samples. Unreacted C3S, C2S, and ferrite were still
observed after 3 days. After 7 days (Fig. 4C), the XRD
patterns were similar to those of the 3-day-old specimens.
The peak intensity of hemicarbonate and Friedel’s salt
continued to increase, and ettringite still existed. The peak
intensity of portlandite also increased after 7 days. The
calcium silicate peaks (C3S and C2S) clearly showed a
reduction in XRD intensity. The peak intensity of ferrite
decreased after 7 days.
After 28 days (Fig. 4D), the peak intensity of portlandite
dominated that of all the other peaks, so Fig. 4E shows the
patterns redrawn for easy identification of the hydrated
calcium aluminate phases. According to Fig. 4E, the XRD
peak pattern was similar to that of the 7-day-old specimens,
but the development of monocarbonate (at 11.62o) was
observed in the plain cement paste and cement paste with
the microorganisms. In the samples with urea-CaCl2 and
urea-CaCl2 plus the microorganisms, the dominant form of
the AFm phase was still Friedel’s salt.
Differential scanning calorimetry/thermogravimetric
analysis. Fig. 5 shows the thermal behavior of the unreacted
cement powder, hydrated cement paste with and without
the microorganisms, and hydrated cement paste with urea-
CaCl2 and urea-CaCl2 plus the microorganisms. The
unreacted cement (Fig. 5A) showed a very small amount of
thermal activity at about 110oC and 380oC. This is most
likely related to the thermal transition of calcium sulfate
phases (gypsum to hemihydrate and to anhydrite). A small
exothermic reaction was observed at about 680oC. This can
be related to the decomposition of calcite. The measured
total weight loss of calcite was about 1.1%.
After 1 day (Fig. 5B), thermal activity appeared in the DSC
curves at around 60–110oC in all the cement paste samples.
The thermal activity can be associated with the decomposition
of ettringite and also possibly with desorption of water
from the C-S-H phases. The data show a noticeable

J. Microbiol. Biotechnol.
 Effect of Microorganism on Cement Hydration 1333

Table 2. Amounts of phases after 1 day of hydration (weight Table 4. Amounts of phases after 7 days of hydration (weight
percent). percent).
C CS CU CSU C CS CU CSU
C3S 13.897 13.724 7.987 10.448 C3S 4.378 2.186 1.259 0.625
C2S 8.473 8.438 10.667 9.13 C2S 9.655 11.002 9.857 11.836
C3A 0.582 0.597 0.738 0.712 C3A - - - -
C4AF 7.289 7.142 4.131 5.46 C4AF 3.922 4.725 3.51 3.447
Periclase (MgO) 1.09 1.09 1.122 1.102 Periclase (MgO) 0.748 0.718 1.136 1.005
Anhydrite (CaSO4) 0.578 0.576 0.956 0.755 Anhydrite (CaSO4) 0.564 0.637 0.645 0.954
Quartz (SiO2) 0.457 0.449 0.952 0.144 Quartz (SiO2) 0.163 0.17 0.35 0.616
Calcite (CaCO3) 5.385 5.338 12.714 6.28 Calcite (CaCO3) 11.126 11.191 12.087 10.643
Ettringite 6.239 6.182 6.002 5.551 Ettringite 3.864 4.378 3.766 4.917
Monosulfate 0.946 0.92 0.55 - Monosulfate - - - -
Hemicarbonate - - - - Hemicarbonate - - - -
Monocarbonate - - - - Monocarbonate - - - -
Friedel’s salt or hydrocaluminate - - 2.684 1.977 Friedel’s salt or hydrocaluminate - - 9.595 10.262
Portlandite (CH) 7.327 7.201 5.62 4.962 Portlandite (CH) 9.356 10.924 8.821 13.034
Amorphous 47.737 48.343 45.877 53.479 Amorphous 56.224 54.069 48.974 42.661
Total 100 100 100 100 Total 100 100 100 100
C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste
with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii. with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii.

Table 3. Amounts of phases after 3 days of hydration (weight Table 5. Amounts of phases after 28 days of hydration (weight
percent). percent).
C CS CU CSU C CS CU CSU

C3S 5.181 5.241 2.751 2.359 C3S 1.195 1.389 1.097 0

C2S 13.385 13.214 10.589 9.919 C2S 5.377 5.779 6.715 7.634
C3A - - - - C3A - - - -
C4AF 6.275 6.271 3.891 3.974 C4AF 0.836 1.094 0.797 -
Periclase (MgO) 1.039 1.063 1.753 0.885 Periclase (MgO) 0.676 0.883 0.674 1.012
Anhydrite (CaSO4) 0.633 0.617 0.642 0.774 Anhydrite (CaSO4) 0.535 0.463 0.982 0.614
Quartz (SiO2) 0.203 0.204 0.137 0.295 Quartz (SiO2) 0.24 0.232 0.412 0.132
Calcite (CaCO3) 12.524 12.886 10.664 9.848 Calcite (CaCO3) 10.361 11.448 10.791 8.734
Ettringite 5.777 5.809 4.089 4.109 Ettringite 2.818 2.4 2 2.178
Monosulfate - - - - Monosulfate - - - -
Hemicarbonate - - - - Hemicarbonate - - - -
Monocarbonate - - - - Monocarbonate 7.395 7.192 - -
Friedel’s salt or hydrocaluminate - - 5.991 5.694 Friedel’s salt or hydrocaluminate - - 7.195 6.449
Portlandite (CH) 9.188 9.766 6.318 9.752 Portlandite (CH) 10.874 9.712 9.803 8.521
Amorphous 45.795 44.929 53.175 52.391 Amorphous 59.693 59.408 59.534 64.726
Total 100 100 100 100 Total 100 100 100 100
C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste C = Plain cement paste; CS = cement paste with S. pasteurii; CU = cement paste
with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii. with urea-CaCl2; and CSU = cement paste with urea-CaCl2 and S. pasteurii.

August 2015 ⎪ Vol. 25 ⎪ No. 8

1334 Lee et al.

Fig. 5. DSC/TGA data from cement pastes with S. pasteurii after (A) no hydration, (B) 1 day of hydration, (C) 3 days of hydration,
(D) 7 days of hydration, and (E) 28 days of hydration.
Red line: heat flow; black line: weight loss; solid line: plain cement paste; dotted line: cement paste with S. pasteurii; bold dotted line: cement paste
with urea-CaCl2 culture medium; double line: cement paste with S. pasteurii and urea-CaCl2 culture medium.

endothermic DSC peak at 440oC with accompanying weight observed. This can be associated with calcite decomposition.
loss in the TGA curve. This thermal activity is known to However, the weight loss in the TGA curve was not clearly
indicate the decomposition of portlandite (calcium hydroxide) accompanied by thermal activity in the DSC curve. After
[15]. At about 680oC, some amount of weight loss was 3 days (Fig. 5C), 7 days (Fig. 5D), and 28 days (Fig. 5E), the

J. Microbiol. Biotechnol.

thermal behaviors of the cement paste samples were
similar to those of the 1-day-old specimens. The thermal
analysis data were further used to derive the amount of
calcium hydroxide in the hydrated cement paste samples in
order to support the results of the quantitative XRD
analysis.
Quantitative analysis. The amounts of the phases
observed in hydrated cement pastes with and without the
microorganisms are shown in Tables 2–5. The amount of
C3S clearly decreased as hydration progressed, and C3A
seemed to react immediately during the first day of
hydration, but the amount of C2S did not show a clear
trend. Therefore, C2S did not seem to react significantly
during the 28-day hydration period with or without the
presence of microorganisms. The amount of C4AF showed
an overall decrease as hydration progressed.
In all the cement paste samples, the amount of ettringite
generally decreased as hydration proceeded. In the plain
cement paste and plain cement paste with the microorganisms,
the amount of monosulfate was about 0.9% after 1 day,
and then it disappeared. As mentioned, the amount of
hemicarbonate could not be characterized because of
its absence in the ICSD. After 28 days, the amount of
monocarbonate in plain cement paste with and without the
microorganisms was about 7.2–7.4%. In the cement paste
with urea-CaCl2, the amount of Friedel’s salt increased from
2.7% after 1 day to 9.6% after 7 days and then decreased to
7.2% after 28 days. In the cement paste with urea-CaCl2 and
the microorganisms, the amount of Friedel’s salt also
increased from 2.0% after 1 day to 10.3% after 7 days and
decreased to 6.5% after 28 days.
Figs. 6A-6D show the amount of calcium hydroxide
(obtained by Rietveld quantitative analysis) in the cement
paste with and without the microorganisms. The amount of
calcium hydroxide generally increased as hydration
progressed. The specimens with the microorganisms had
more calcium hydroxide after 3 and 7 days, but the amount
of calcium hydroxide decreased between 7 and 28 days.
The amount of calcium hydroxide in the cement paste
samples was also verified using DSC/TGA (Figs. 6E-6H).
The weight loss at around 400-450oC was quantified
because calcium hydroxide is known to decompose in this
temperature range [15]. From the TGA results shown in
Figs. 6E-6H, the cement paste with the microorganisms
always showed a higher amount of calcium hydroxide for
the first 7 days. After 28 days, although the plain cement
paste with the microorganisms showed slightly more
calcium hydroxide than the plain cement paste, the cement
paste with urea-CaCl2 and the microorganisms showed
Effect of Microorganism on Cement Hydration 1335
slightly less calcium hydroxide than the cement paste with
only urea-CaCl2. According to the quantitative phase analysis
of the cement paste with and without the microorganisms,
the presence of S. pasteurii affected the hydration of the
cement paste by reducing the amount of calcium hydroxide
after 28 days.
Discussion
The microorganism S. pasteurii was found to increase the
early hydration rate of cement paste (Fig. 3). This finding
can be related to an early increase in the amount of calcium
silicate hydration. Because it is difficult to characterize the
amount of C-S-H owing to its amorphous nature, the
amount of calcium silicate hydration was evaluated using
the amount of calcium hydroxide. When the microorganisms
were used, the amount of calcium hydroxide clearly
increased after 3 and 7 days regardless of whether the urea-
CaCl2 culture medium was used. This tendency seemed to
be maintained until 28 days, but the increase in the amount
of calcium hydroxide was not clearly observed after 28
days when urea-CaCl2 was present. The results of XRD and
DSC/TGA in this research do not clearly verify the
formation of calcite by MICP when the microorganisms
were directly incorporated during mixing. To remove the
problem of a lack of nutrition and to promote MICP, the
urea-CaCl2 culture medium was incorporated into the
cement paste during mixing (cement paste samples were
mixed with urea-CaCl2 culture medium at w/c = 1). This
was done to investigate whether the microorganisms can
be active when sufficient nutrition is available, even though
they were captured within the pore structure of the cement
paste with lack of oxygen. Note that the specimen was not
cured in the urea-CaCl2 culture medium, but the amount of
nutrition for MICP when hydration began was sufficient
considering the 1:1 ratio of water (in this case, urea-CaCl2
culture medium) to cement.
The XRD patterns of cement paste with 107 cells/ml
(Fig. 4) were similar to that of plain cement paste. The
decrease in the calcite peak and the peak widening at 29.4o
(angle 2θ) were observed in all the cement paste samples
after 3, 7, and 28 days. The peak intensity at 29.4o also
decreased as a function of the hydration time. This result
may indicate that small or poorly crystalline calcite was
formed, but amorphous calcite usually does not form
because calcite is very crystalline, with a strong preferred
orientation that yields a sharp XRD peak. Therefore, after
1 day, the observed XRD pattern at 29.4o was calcite, but
the calcite was consumed to form hemicarbonate after 3
August 2015 ⎪ Vol. 25 ⎪ No. 8
1336 Lee et al.

Fig. 6. Calcium hydroxide content of cement pastes with and without S. pasteurii.
(A) Calcium hydroxide content of plain cement paste obtained by Rietveld quantitative analysis, (B) calcium hydroxide content of cement paste
with S. pasteurii obtained by Rietveld quantitative analysis, (C) calcium hydroxide content of cement paste with urea-CaCl2 culture medium
obtained by Rietveld quantitative analysis, (D) calcium hydroxide content of cement paste with S. pasteurii and urea-CaCl2 culture medium
obtained by Rietveld quantitative analysis, (E) calcium hydroxide content of plain cement paste obtained by DSC/TGA, (F) calcium hydroxide
content of cement paste with S. pasteurii obtained by DSC/TGA, (G) calcium hydroxide content of cement paste with urea-CaCl2 culture medium
obtained by DSC/TGA, and (H) calcium hydroxide content of cement paste with S. pasteurii and urea-CaCl2 culture medium obtained by DSC/TGA.

J. Microbiol. Biotechnol.

and 7 days and later to form monocarbonate after 28 days.
The peak widening at 29.4o after 3, 7, and 28 days is
expected to be better correlated to the formation of C-S-H,
as indicated in other reports [2].
It is still possible to consider that the calcite produced
by MICP was consumed to form hemicarbonate and
monocarbonate. The transition from hemicarbonate to
monocarbonate after 28 days can be related to the results of
MICP. However, no clear differences between the XRD patterns
of cement paste with and without the microorganisms were
observed, and the thermal analysis (DSC/TGA) provided
no clear evidence for MICP. In fact, the TGA curve gave
some indication of calcite decomposition, but it was not
clearly associated with the DSC peak. In addition, the
amount of calcite in the samples with the microorganisms
did not differ from the amount in the plain cement paste
sample.
The main difference between the XRD patterns of plain
cement paste and that with the urea-CaCl2 culture medium
was the formation of Friedel’s salt. However, as mentioned
earlier, the formation of Friedel’s salt was associated with
the presence of CaCl2 in the urea-CaCl2 culture medium.
Other than that, no clear differences between cement paste
with or without the microorganisms and with or without
urea-CaCl2 were observed. Further research is necessary to
understand the role of the microorganisms in the hydration
kinetics of cement paste when they are directly incorporated
into the system.
According to the results, it can be concluded that calcite
formation (MICP) was not observed when the microorganisms
were directly incorporated into the cement paste during
mixing, regardless of whether the urea-CaCl2 culture
medium was used. It seems that the microorganisms were
active during the first day of hydration (as evidenced by
the calorimetry peak). After hardening, continuous MICP
did not seem to occur; thus, it is possible that the metabolism
of the microorganisms ceased after they were captured
within the pore structure of the cement paste. In other
words, the inactivity of the microorganisms might have
been associated with the lack of available oxygen and carbon
dioxide for continuous MICP (metabolism of S. pasteurii
microorganisms). In addition, the microorganisms might
have occupied the available pore space, which could
inhibit the growth of hydration products. It is not clear
whether the microorganisms can recover their activity
when they are open to the ambient conditions. It is also not
clear whether the produced calcites are incorporated into
the AFm structure as hemi- or monocarbonate. Further
research is necessary to answer these questions. However,
Effect of Microorganism on Cement Hydration 1337
it is certain from our microscopy observation that MICP
occurred under ambient air conditions (Figs. 1 and 2). The
results from other studies also indicate that MICP occurs
when the microorganisms are supplied from outside with
sufficient nutrition (urea-CaCl2 culture medium) [10, 12,
13]. We found that the microorganisms only accelerated the
early hydration of cement paste.
Acknowledgments
This research was supported by both a grant(14RDRP-
B076268) from Regional Development Research Program
funded by Ministry of Land, Infrastructure and Transport
of Korean government and a National Research Foundation
of Korea (NRF) grant funded by the Korean government
(MEST) (No. NRF-2010-0015142).
References
1. Bachmeier KL, Williams AE, Warmington JR, Bang SS. 2002.
Urease activity in microbiologically-induced calcite precipitation.
J. Biotechnol. 93: 171-181.
2. Cong X, Kirkpatrick RJ. 1996. 29Si MAS NMR study of the
structure of calcium silicate hydrate. Adv. Cem. Based Mater.
3: 144-156.
3. Douglas S, Beveridge TJ. 1998. Mineral formation by
bacteria in natural microbial communities. FEMS Microbiol.
Ecol. 26: 79-88.
4. Ghosh S, Biswas M, Chattopadhya BD, Mandal S. 2009.
Microbial activity on the microstructure of bacteria modified
mortar. Cem. Concr. Compos. 31: 93-98.
5. Ghosh S, Mandal S, Chattopadhyay BD, Pal S. 2005. Use of
microorganism to improve the strength of cement mortar.
Cem. Concr. Res. 35: 1980-1983.
6. Hammes F, Boon N, Villiers JD, Verstraete W, Siciliano SD.
2003. Strain-specific ureolytic microbial calcium carbonate
precipitation. Appl. Environ. Microbiol. 69: 4901-4909.
7. Jonkers HM, Thijssen A, Muyzer G, Copuroglu O,
Schlangen E. 2010. Application of bacteria as self-healing
agent for the development of sustainable concrete. Ecol. Eng.
36: 230-235.
8. Metayer-Levrel GL, Castanier S, Orial G, Loubiere JF,
Perthuisot JP. 1999. Applications of bacterial carbonatogenesis
to the protection and regeneration of limestones in
buildings and historic patrimony. Sediment. Geol. 126: 25-34.
9. Muynck WD, Belie ND, Verstraete W. 2010. Microbial
carbonate precipitation in construction materials: a review.
Ecol. Eng. 36: 118-136.
10. Muynck WD, Debrouwer D, Belie ND, Verstraete W. 2009.
Bacterial carbonate precipitation improves the durability of
cementitious materials. Cem. Concr. Res. 38: 1005-1014.
August 2015 ⎪ Vol. 25 ⎪ No. 8
1338 Lee et al.
11. Myszka K, Czaczyk K. 2009. Characterization of adhesive
exopolysaccharide (EPS) produced by Pseudomonas aeruginosa
under starvation conditions. Curr. Microbiol. 58: 541-546.
12. Park SJ, Park YM, Chun WY, Kim WJ, Ghim SY. 2010.
Calcite-forming bacteria for compressive strength improvement
in mortar. J. Microbiol. Biotechnol. 20: 782-788.
13. Ramachandran SK, Ramarkrishnan V, Bang SS. 2001.
Remediation of concrete using micro-organisms. ACI Mater.
J. 98: 3-9.
14. Stocks-Fischer S, Galinat JK, Bang SS. 1999. Microbiological
precipitation of CaCO3. Soil Biol. Biochem. 31: 1563-1571.
15. Taylor HFW. 1997. Cement Chemistry. Thomas Telford
J. Microbiol. Biotechnol.
Edition, Academic Press, London.
16. Tiano P, Biagiotti L, Mastromei G. 1999. Bacterial biomediated
calcite precipitation for monumental stones conservation:
methods of evaluation. J. Microbiol. Methods 36: 139-145.
17. Tiano P, Cantisani E, Sutherland I, Paget JM. 2006.
Biomediated reinforcement of weathered calcareous stones.
J. Cult. Herit. 7: 49-55.
18. Tittelboom KV, Belie ND, Muynck WD, Verstraete W. 2010.
Use of bacteria to repair cracks in concrete. Cem. Concr. Res.
40: 157-166.
19. Whiffin VS. 2004. Microbial CaCO3 precipitation for the
production of biocement. Dissertation, Murdoch University.