research article jmb reviethe concept is to use the urease activity of microorganisms [9]. when...

J. Microbiol. Biotechnol.

J. Microbiol. Biotechnol. (2015), 25(8), 1328–1338http://dx.doi.org/10.4014/jmb.1411.11037 Research Article jmbReviewEffect of Microorganism Sporosarcina pasteurii on the Hydration ofCement PasteJun Cheol Lee1, Chang Joon Lee2, Woo Young Chun3, Wha Jung Kim3, and Chul-Woo Chung4*

1School of Architecture and Civil Engineering, Kyungpook National University, Daegu 702-701, Republic of Korea2Department of Architectural Engineering, Chungbuk National University, Cheongju 362-763, Republic of Korea3School of Architectural, Civil, Environmental, and Energy Engineering, Kyungpook National University, Daegu 702-701, Republic of Korea4Department of Architectural Engineering, Pukyong National University, Busan 608-739, Republic of Korea

Introduction

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)

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

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

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: [email protected]

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2015 by

The Korean Society for Microbiology

and Biotechnology

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.

Keywords: Compressive strength, hydration, characterization, microorganism, Sporosarcina

pasteurii

Effect of Microorganism on Cement Hydration 1329

August 2015⎪Vol. 25⎪No. 8

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-CaCl2culture 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

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

Table 1. Chemical composition of type I Portland cement.

Compound name CaO SiO2 SO3 Fe2O3 Al2O3 MgO K2O TiO2 ZnO SrO P2O5

Conc. (%) 66.681 19.051 3.808 3.797 2.888 1.893 1.17 0.357 0.15 0.126 0.079

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

(calcium hydroxide). Note that no ICSD entry was available for

hemicarbonate, so the amount of hemicarbonate could not be

investigated.

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

Fig. 1. Light transmission optical microscopy images ofsamples.

(A) Without inoculation of S. pasteurii into urea-CaCl2 culture medium,

and (B) S. pasteurii being inoculated into urea-CaCl2 culture medium.



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-

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-

Fig. 3. Temperature rise of cement paste with and withoutS. 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.

Fig. 2. Scanning electron microscopy images of the shellof S. pasteurii at (A) 1,000× magnification and (B) 6,000×

magnification.

1332 Lee et al.


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

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 ofhydration, 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



Table 2. Amounts of phases after 1 day of hydration (weightpercent).

C CS CU CSU

C3S 13.897 13.724 7.987 10.448

C2S 8.473 8.438 10.667 9.13

C3A 0.582 0.597 0.738 0.712

C4AF 7.289 7.142 4.131 5.46

Periclase (MgO) 1.09 1.09 1.122 1.102

Anhydrite (CaSO4) 0.578 0.576 0.956 0.755

Quartz (SiO2) 0.457 0.449 0.952 0.144

Calcite (CaCO3) 5.385 5.338 12.714 6.28

Ettringite 6.239 6.182 6.002 5.551

Monosulfate 0.946 0.92 0.55 -

Hemicarbonate - - - -

Monocarbonate - - - -

Friedel’s salt or hydrocaluminate - - 2.684 1.977

Portlandite (CH) 7.327 7.201 5.62 4.962

Amorphous 47.737 48.343 45.877 53.479

Total 100 100 100 100

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.

Table 3. Amounts of phases after 3 days of hydration (weightpercent).

C CS CU CSU

C3S 5.181 5.241 2.751 2.359

C2S 13.385 13.214 10.589 9.919

C3A - - - -

C4AF 6.275 6.271 3.891 3.974

Periclase (MgO) 1.039 1.063 1.753 0.885

Anhydrite (CaSO4) 0.633 0.617 0.642 0.774

Quartz (SiO2) 0.203 0.204 0.137 0.295

Calcite (CaCO3) 12.524 12.886 10.664 9.848

Ettringite 5.777 5.809 4.089 4.109

Monosulfate - - - -




Portlandite (CH) 9.188 9.766 6.318 9.752

Amorphous 45.795 44.929 53.175 52.391

Total 100 100 100 100




C CS CU CSU

C3S 4.378 2.186 1.259 0.625

C2S 9.655 11.002 9.857 11.836

C3A - - - -

C4AF 3.922 4.725 3.51 3.447

Periclase (MgO) 0.748 0.718 1.136 1.005

Anhydrite (CaSO4) 0.564 0.637 0.645 0.954

Quartz (SiO2) 0.163 0.17 0.35 0.616

Calcite (CaCO3) 11.126 11.191 12.087 10.643

Ettringite 3.864 4.378 3.766 4.917

Monosulfate - - - -




Portlandite (CH) 9.356 10.924 8.821 13.034

Amorphous 56.224 54.069 48.974 42.661

Total 100 100 100 100




C CS CU CSU

C3S 1.195 1.389 1.097 0

C2S 5.377 5.779 6.715 7.634

C3A - - - -

C4AF 0.836 1.094 0.797 -

Periclase (MgO) 0.676 0.883 0.674 1.012

Anhydrite (CaSO4) 0.535 0.463 0.982 0.614

Quartz (SiO2) 0.24 0.232 0.412 0.132

Calcite (CaCO3) 10.361 11.448 10.791 8.734

Ettringite 2.818 2.4 2 2.178

Monosulfate - - - -


Monocarbonate 7.395 7.192 - -


Portlandite (CH) 10.874 9.712 9.803 8.521

Amorphous 59.693 59.408 59.534 64.726

Total 100 100 100 100



1334 Lee et al.


endothermic DSC peak at 440oC with accompanying weight

loss in the TGA curve. This thermal activity is known to

indicate the decomposition of portlandite (calcium hydroxide)

[15]. At about 680oC, some amount of weight loss was

observed. This can be associated with calcite decomposition.

However, the weight loss in the TGA curve was not clearly

accompanied by thermal activity in the DSC curve. After

3 days (Fig. 5C), 7 days (Fig. 5D), and 28 days (Fig. 5E), the

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.



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

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-CaCl2culture 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

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.



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,

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).

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