Construction and Building Materials 26 (2012) 532540

Contents lists available at ScienceDirect

Construction and Building Materials

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

Use of silica gel or polyurethane immobilized bacteria for self-healing concrete

Jianyun Wang a,b, Kim Van Tittelboom a, Nele De Belie a,, Willy Verstraete b
a
Magnel Laboratory for Concrete Research, Faculty of Engineering and Architecture, Ghent University, Technologiepark Zwijnarde 904, B-9052 Ghent, Belgium
b
Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

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

Article history: Cracks in concrete are the main reason for a decreased service life of concrete structures. It is therefore
Received 23 December 2010 more advisable and economical to restrict the development of early age small cracks the moment they
Received in revised form 14 May 2011 appear, than to repair them after they have developed to large cracks. A promising way is to pre-add heal-
Accepted 17 June 2011
ing agents to the concrete to heal early age cracks when they appear, i.e. the so-called self-healing
Available online 14 July 2011
approach. In addition to the more commonly studied polymeric healing materials, bacterial CaCO3 pre-
cipitation also has the potential to be used for self-healing. It is more compatible with the concrete matrix
Keywords:
and it is environment friendly. However, bacterial activity decreases a lot in the high pH (>12) environ-
Bacillus sphaericus
Immobilization
ment inside concrete. In this research, the possibility to use silica gel or polyurethane as the carrier for
Silica gel protecting the bacteria was investigated. Experimental results show that silica gel immobilized bacteria
Polyurethane exhibited a higher activity than polyurethane immobilized bacteria, and hence, more CaCO3 precipitated
CaCO3 in silica gel (25% by mass) than in polyurethane (11% by mass) based on thermogravimetric analysis.
Strength regain However, cracked mortar specimens healed by polyurethane immobilized bacteria had a higher strength
Coefcient of water permeability regain (60%) and lower water permeability coefcient (10101011 m/s), compared with specimens
healed by silica gel immobilized bacteria which showed a strength regain of only 5% and a water perme-
ability coefcient of 107109 m/s. The results indicated that polyurethane has more potential to be
used as a bacterial carrier for self-healing of concrete cracks.
2011 Elsevier Ltd. All rights reserved.

1. Introduction especially useful to repair deep-micro cracks and it can restrain

Concrete is the one of the most popular construction materials.
However, it is quite vulnerable to cracking because of its inherent
heterogeneity and the non-ideal service environments. Since cracks
provide an easy path for water and other aggressive substances like
Cl and SO24 to penetrate inside the concrete matrix, they should be
repaired in time to prolong the service life of concrete structures.
Generally, the normal repair methods follow the procedure of
monitoring, detecting and repairing. The repair work will be per-
formed after the cracks are discovered. Repair agents are applied
from the outside and penetrate into the cracks. This technology is
quite suitable for repairing large cracks. For small and deep cracks,
it will be difcult for healing agents to reach the inner part. There-
fore, an alternative repair method by means of a self-healing process
is being strived for. Healing agents are incorporated into the
concrete matrix during casting. When cracks appear, healing agents
will be released from within the concrete and ow into cracks to seal
the cracks from the inside to the outside. A self-healing method is
Corresponding author. Tel.: +32 9 264 55 22; fax: +32 9 264 58 45.
E-mail addresses: jianyun.wang@ugent.be (J. Wang), kim.vantittelboom@u
gent.be (K. Van Tittelboom), nele.debelie@ugent.be (N. De Belie), willy.verstrae
te@ugent.be (W. Verstraete).
early-age cracks to develop to large cracks.
Self-healing properties in concrete may be obtained by different
methodologies, such as secondary hydration of unhydrated
cement, addition of bers, and encapsulation of polymers [15].
Another alternative self-healing material is bacterially produced
calcium carbonate [69]. Compared with the healing agents like
expanded additives and polymers, the proposed bio-mineral
(CaCO3) is more compatible with the concrete matrix and more
environmentally friendly. Most bacteria are able to induce carbon-
ate precipitation under suitable conditions [1013]. In general,
there are three mechanisms associated with bio-carbonate precip-
itation. One is the dissimilatory sulfate reduction carried out by
sulfate reducing bacteria under anoxic conditions. The second is
the degradation of organic acids. Another pathway is related to
the nitrogen cycle, in particular the degradation of urea by ureolyt-
ic bacteria [14]. Among the three pathways to precipitate CaCO3,
decomposition of urea by ureolytic bacteria is easier to operate
and control [15,16].
In our previous research, Bacillus sphaericus was found to be able
to precipitate calcium carbonate (CaCO3) on its cell constituents and
in its micro-environment by decomposition of urea (CO(NH2)2) into
2
ammonium (NH 4 ) and carbonate (CO3 ). The latter subsequently
promotes the microbial deposition of CaCO3 in a calcium rich

0950-0618/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.conbuildmat.2011.06.054
 J. Wang et al. / Construction and Building Materials 26 (2012) 532540
environment. Through this process, the bacterial cell is coated with a
layer of CaCO3. The aim of this study is to use this bio-CaCO3 to heal
concrete cracks autonomously. The problem is that bacterial cells
cannot be added to cement specimens directly. On one hand, bacte-
rial activity decreases a lot in the high pH (>12) environment as pres-
ent in concrete. On the other hand, bacterial cells might be destroyed
during the process of hydration. Jonkers et al. indicated that bacteria
did not survive due to the decreasing of pore diameters during the
hydration of the cement materials [17]. Therefore, a suitable carrier
is necessary to immobilize bacteria and to protect them from the
harsh environment in concrete. In this work, silica gel and polyure-
thane were used as the carrier for the bacteria.
The term silica sol is derived from silicic acid sol. Silica sols are
colloidal dispersions of silicic acid in water. Silica gel is a popular car-
rier for microorganisms, like bacterial cells, yeast and algae, because
it has good properties of mechanical, thermal and photochemical
stability, biological inertness (not a food source for bacteria), and
suitable matrix porosity for the transmission of molecules and ions
[18,19]. In our previous work, silica gel immobilized bacteria were
used to manually heal cracks in concrete. The mixture made of silica
sol and bacterial suspension (containing bacterial cells and NaCl)
was injected into simulated cracks by a syringe. When gel formation
(caused by high concentrations of Na+ and Cl) began, the injection
was repeated several times until the crack was completely lled.
After silica sol became a gel, the specimens were immersed into
the medium consisting of urea and Ca2+ and then precipitation of
CaCO3 occurred. Water permeability of the specimens decreased
about 3 orders of magnitude after this treatment [20]. In contrast
to the previous work, in the current study the immobilized bacteria
together with nutrients and other agents, encapsulated in glass
tubes, were incorporated into the specimens during casting. When
cracking occurs, the glass tubes will break and healing agents will
ow out into the cracks. Silica gel forms in situ when silica sol meets
with Ca2+ from the concrete matrix and from the healing agent pre-
incorporated inside the specimens. At the same time, bacterial cells
are immobilized into the silica gel. When bacteria meet with urea
and Ca2+, CaCO3 precipitates.
Polyurethane (PU) is widely used as a waterproof material. PU
with immobilized bacteria has already been used to repair concrete
cracks [21]. In 2001 Bang et al. rst used PU foam to immobilize
bacteria for manual repairing of concrete cracks. The PU foam, con-
taining bacterial cells, was cut into equal-sized pieces. Afterwards,
PU foam strips were placed into simulated cracks of mortar speci-
mens. The specimens were then incubated in a urea-CaCl2 medium
at room temperature. As a result of CaCO3 precipitation, the 7d
compressive strength of the cracked specimens remediated by PU
immobilized bacteria was increased by 12% compared with the
ones only remediated with PU. Different from the method de-
scribed above, in which PU foam with immobilized bacteria was
applied externally (pre-formed and placed into the cracks manu-
ally), in this work bacteria and PU prepolymer were applied inter-
nally (to heal cracks from the inside). PU foam should form in the
crack automatically when cracking occurs and bacteria are incor-
porated inside the foam at the same time. The aim of this work
was to investigate the potential use of silica gel or polyurethane
immobilized bacteria to bring about self-healing concrete.
2. Materials and methods
2.1. Bacterial strain
The bacterial strain used in the experiments was B. sphaericus LMG 22,557
(Belgian coordinated collection of microorganisms, Ghent). This strain has a high
urease activity (40 mM urea hydrolyzed. OD1 h1), long survival time [22] and
can produce CaCO3 in a simple and controllable way [23].
The medium used to grow B. sphaericus consisted of yeast extract and urea. The
yeast extract medium was rst autoclaved for 20 min at 120 C and the urea solu-
tion was added which was sterilized by means of ltration through a sterile
533
0.22 lm Milipore lter (Millipore, USA). The nal concentrations of yeast extract
and urea were 20 g/L. Cultures were incubated at 28 C on a shaker at 100 rpm
for 24 h. Bacterial cells were harvested by centrifuging (7000 r/min, 7 min, Eppen-
dorf MiniSpin, Hamburg, Germany) the 24 h-old grown culture and the cells were
resuspended in saline solution (NaCl, 8.5 g/L). The concentration of bacterial cells
was 109 cells/mL.
2.2. Survival test of the bacteria
In this experiment it was tested how long the bacteria can remain viable and
sustain high urease activity. Batches of 2 mL bacterial solution (109 cells/mL, same
as in the Section 2.1) were added into a sterile vial (12.5 mm (diameter)  46 mm
(height), VWR). The vials were then closed tightly and put in the incubator at
28 C. At certain time intervals, three vials were taken out from the incubator. Bac-
teria of each vial were inoculated into 100 mL sterile deposition medium (yeast ex-
tract 20 g/L, urea 20 g/L and Ca(NO3)2.4H2O 79 g/L). The media were then put on the
shaker (28 C, 100 rpm) for three days. The amount of urea decomposed by bacteria
after three days was calculated based on the total ammonium nitrogen (TAN) mea-
sured in the deposition medium. Since one mole of urea (CO(NH2)2) produces 2 mol
of NH
4 , the amount of NH4 can thus indicate the amount of urea decomposed, and
hence the amount of CaCO3 precipitation. TAN concentrations were measured calo-
rimetrically by the method of Nessler [24].
2.3. Activity of bacteria after being immobilized into silica gel and polyurethane
2.3.1. Immobilization of bacteria
Immobilization of bacteria into silica gel: Levasil 200/30% sol, with a specic sur-
face area of 200 m2/g and a solid content of 30% was used to embed bacterial cells.
Two concentrations of saline solutions were used. The saline solution with 8.5 g/L
NaCl was used to re-suspend centrifuged bacteria. Another saline solution with
60 g/L NaCl (represented as HS) was used to make silica sol become silica gel. Silica
sol, bacterial suspension (BS, 109 cells/mL) and HS were mixed together with the
volume ratio 5:1:4. About 2 h later, the silica sol became gel and bacterial cells were
thus incorporated inside the gel.
Immobilization of bacteria into polyurethane: A two-component polyurethane
(MEYCO MP 355 1 K, BASF), represented as PU, was also used to encapsulate bacte-
rial cells. The volume ratio of component A of PU (polyurethane prepolymer, PU A),
component B of PU (accelerator, PU B) and bacterial suspension (BS, 109 cells/mL)
was 5:0.5:1. About 15 min after mixing of the three components, PU foam formed
and the bacterial cells were embedded inside the foam.
At the same time, silica sol and PU were also combined with dead bacteria (dead
bacteria were obtained by autoclaving living cells at 120 C for 20 min) and pre-
pared without bacteria as a control.
2.3.2. Bacterial activity after immobilization
The bacterial activity was evaluated by bacterial ureolytic activity (ability to
decompose urea) and carbonatogenesis activity (ability to precipitate CaCO3). The
bacterial ureolytic activity was expressed as the amount of the urea decomposed
by bacteria in the urea solution (20 g/L), which was determined by measuring the
conductivity of the urea solution. One mole of urea (CO(NH2)2) produces 2 mol of
2
NH 4 and 1 mol of CO3 . Therefore, the more urea is decomposed, the higher the
conductivity of the urea solution will be. The relationship between urea decom-
posed and conductivity is shown in the following equation [14]:
Urea decomposed mM conductivity ms cm1  9:6 1
The bacterial carbonatogenesis activity was determined by the decomposition
of urea in the deposition medium (DM) consisting of 20 g/L urea and 79 g/L
Ca(NO3)2.4H2O. The amount of CaCO3 precipitated by bacteria can be indicated by
the quantity of urea decomposed in the deposition medium. The more urea decom-
posed, the more CaCO3 formed. Since the deposition medium consisted of urea and
Ca(NO3)2, the increase of CO23 because of urea decomposition and the decrease of
Ca2+ and CO2 3 because of the formation of CaCO3 would make it difcult to relate
conductivity with the amount of urea decomposed. Therefore, the decomposed urea
was calculated also by measuring the TAN values in the deposition medium.
Living bacteria and dead bacteria, after being immobilized into silica gel and PU,
were immersed into 50 mL urea solution and 50 mL deposition medium, separately.
Besides, as the rst control, the same amount of free bacterial cells (un-immobi-
lized) was also added to the same urea solution and deposition medium. As the sec-
ond control, silica gel and PU without bacteria were also immersed into the same
media. The experiments were done in triplicate. The conductivity of the urea solu-
tion was measured every 24 h for 4 days. The TAN value was measured after the dif-
ferent series of silica gel and PU were immersed into the deposition medium for
3 days.
One week later, the original urea solutions, which were used to immerse SG and
PU immobilized bacteria, were poured out and 50 mL new urea solutions (also
20 g/L) were added to the same SG and PU immobilized bacteria. The conductivity
values of the new solutions were measured to investigate whether the immobilized
bacteria still had urease activity after being in silica gel and PU for one week.
534 J. Wang et al. / Construction and Building Materials 26 (2012) 532540
2.3.3. Thermogravimetric analysis (TGA) of CaCO3 precipitation
TGA was used to detect the formation of CaCO3 inside the silica gel and PU foam.
After being immersed into the deposition medium for three days, the different ser-
ies of silica gel and PU foam mentioned in Section 2.3.2 were dried in the oven at
50 C for 24 h. Samples of silica gel were ground into powders and samples of PU
foam were cut into small pieces to t for the pan (used for loading samples) of
the TGA instrument (SDT 2960 Simultaneous DSCTGA). The temperature increased
from room temperature to 1000 C at a speed of 10 C/min in argon atmosphere.
The weight loss of the samples during the process of heating was recorded and
shown in a weight-temperature graph.
2.3.4. Scanning electron microscope (SEM) analysis
The morphology of the calcium carbonate precipitated by immobilized bacteria
was studied in a FEI QUANTA 200F SEM at accelerating voltage of 20 kV. Secondary
electron imaging (SEI) was used for electron micrography. Samples were com-
pletely dried in the oven at 50 C for 2 days and were gold coated by a Baltec
SCD030 Sputter Coater before examination.
2.4. Self-healing by means of immobilized bacteria
2.4.1. Glass tubes with healing agents
To incorporate immobilized bacteria into mortar specimens, glass tubes with a
length of 40 mm and an inner diameter of 3 mm were used to carry the healing
agents (shown in Fig. 1). First, glass tubes were glued together and one of their ends
was sealed by an adhesive (Schnellklebstoff X 60, HBM). Then the healing agents
were injected into each tube from the other end, which was sealed afterwards.
For self-healing by using silica-gel immobilized bacteria, two glass tubes were
glued together. One tube was lled with bacterial suspension (BS, same as in Sec-
tion 2.3.1) and silica-sol. The other tube was lled with the same deposition med-
ium (DM) as described in Section 2.3.2. For self-healing by use of PU immobilized
bacteria, three glass tubes were glued together. One compartment of the tubes
was lled with PU A. The second one was lled with PU B together with deposition
medium. The third one was lled with BS. Since dead bacteria were obtained by
autoclaving living cells at 120 C for 20 min, BSA is used to represent dead bacteria
in the following gures and graphs.
2.4.2. Mortar specimens
The self-healing efciency of bacteria incorporated mortar specimens was eval-
uated by measuring the strength regain and the decrease of water permeability.
Therefore, two kinds of mortar specimens, prisms (40 mm  40 mm  160 mm)
for investigating the strength regain and cylinders (U = 80 mm, H = 22 mm) for
measuring water permeability, were made with a water to cement ratio of 0.5
and a sand to cement ratio of 3 by using ordinary Portland cement CEM I 52.5 N.
First, a 10 mm mortar layer was brought into the molds. After this layer was com-
pacted by means of vibration, two reinforcement bars with a diameter of 2 mm and
a length of 14 cm (for prisms) or two steel bers with a diameter of 1 mm for cyl-
inders were positioned on top of it (Fig. 2). Meanwhile, two or three sets of glass
tubes were also put on top of the layer. For the specimens with silica gel immobi-
lized bacteria, three sets of glass tubes were embedded (Fig. 2a) while for the spec-
imens with PU immobilized bacteria, two sets of tubes were incorporated (Fig. 2b)
to make sure that the total volume of the healing agents was the same. Afterwards,
the molds were completely lled with mortar and vibrated. After casting, all molds
were put in an air-conditioned room (curing room) with a temperature of 20 C and
a relative humidity of more than 90% for 24 h. After demolding, the specimens were
placed in the same room for two weeks.
Silica sol
SG DM
Silica-sol + dead bacteria
SG-BSA DM
Silica-sol + living bacteria
SG-BS DM
PU A
PU
PU B+ DM
H2 O
PU A
PU-BSA PU B+ DM
Dead bacteria
PU A
PU-BS PU B+ DM
Living bacteria
Fig. 1. Glass tubes with different healing agents.
For the prisms, ve different series were made: four series containing glass
tubes with healing agents SGBS, SGBSA, PUBS and PUBSA; one reference series
without glass tubes or healing agents. Each series had three replicates. For the cyl-
inders, also ve different series were made: four series containing glass tubes with
healing agents SG, SGBS, PU and PUBS; one reference series without glass tubes
or healing agents. Each series consisted of six replicates.
Additionally, extra sets of glass tubes, which were the same as the ones used in
mortar specimens, were also stored for two weeks under identical conditions in the
curing room. Three sets of glass tubes were kept for each series of specimens. After
two weeks, they were broken manually and the healing agents owed into a beaker.
In the beaker, silica gel or PU foam formed with or without bacteria, urea and Ca2+
incorporated inside. The beakers were put into a tightly closed jar with a high rel-
ative humidity (more than 90%) inside. One week later, samples from different
beakers were taken out for TGA analysis to detect the formation of CaCO3.
2.4.3. Crack formation
Two weeks later, the prisms were taken out from the curing room and realistic
cracks were created in the prisms by means of a crack width controlled three-point
bending test, as shown in Fig. 3a. A metal plate was glued at the bottom of the
prisms (in the middle part). Crack width was measured by a linear variable differ-
ential transformer (LVDT), which was also attached at the bottom of the specimens
[25]. The crack width was increased with a speed of 0.0005 mm/s until a crack
width of 0.5 mm was obtained, followed by unloading. After unloading, the remain-
ing crack width was about 0.35 mm.
The cylinders were also taken out of the curing room after two weeks and were
subjected to a splitting test to make cracks, as shown in Fig. 4. The same kind of me-
tal plates and plastic blocks were glued at both sides of the cylinder. The crack
width was controlled by the average value of the crack opening measured by two
LVDTs attached at both sides of the cylinders. At rst, the average crack width
was also developing with a velocity of 0.0005 mm/s until a crack width of
0.5 mm was obtained. However, it was found that some of the cylinders (with steel
bers) were easily broken when the crack width reached 0.5 mm. Therefore, the
load was stopped when the crack width reached 0.4 mm. After de-loading, the
remaining crack width was about 0.25 mm.
2.5. Evaluation of self-healing
2.5.1. Strength regain
After performing the bending test, all prisms were placed again into the air-
conditioned room. One week later, strength regain was measured by reloading
the prisms. The self-healing efciency was evaluated by comparing the peak load
obtained during the rst loading cycle and the reloading cycle (L2/L1, in Fig. 3b).
2.5.2. Water permeability test
The healing efciency was also investigated by measuring the water permeabil-
ity after crack healing. The method used is modied from the low pressure water
permeability test used by Wang et al. [26]. After the splitting test, three specimens
in each series were used for the water permeability test since some specimens had
broken completely during the splitting test.
During the splitting test, it was noticed that some uid (silica sol or polyure-
thane) came out of the tubes and migrated into the cracks. So the cracked cylinders
were put in the curing room again to wait till the sol became gel and the polyure-
thane foam formed. Since the healing agents were gradually owing out because of
the capillary force, it took more time in real specimens for the sol to become gel
(about 34 h) and for polyurethane foam forming (about 24 h) than in beakers.
Then, the cylinders were immersed into water for 3 days. After that, specimens
were taken out from water and the surfaces were dried under room temperature.
The surface dry cylinders were then mounted inside the PVC ring. Subsequently,
they were vacuum saturated as described in NBN B 24-213 [27]. The vacuum satu-
ration allows to establish a steady state ow condition in a specimen during the
water permeability test. The specimens were rst vacuumed in a vacuum chamber
for 23 h and then de-mineralized water was added into the chamber while keep-
ing the vacuum condition. When the cylinders were completely immersed into
water, the vacuum was stopped. The cylinders were kept immersed in water for an-
other 24 h. Then the cylinders were taken out from the chamber and put into the
setup of the water permeability test. The PVC ring which was used to mount cylin-
ders was accurately t with the rubber seals of the setup for measuring the water
permeability to avoid leakage (Fig. 5).
The whole setup was kept watertight to make sure that the specimens were in a
water saturated situation before every measurement. No water evaporation oc-
curred from the specimens during the whole test period. The time for the decrease
of the water level from h0 till hf in the glass tube was measured at regular time
intervals during 30 days of testing. This water ow rate was related with the water
permeability of the cracked specimens. Based on Darcys law, the coefcient of
water permeability of the specimen can be calculated by the following equation:
 
aT h0
k ln 2
At hf
 J. Wang et al. / Construction and Building Materials 26 (2012) 532540 535

(a) Silica sol as carrier (b) PU as carrier

Fig. 2. Cylinders with the glass tubes which were lled with healing agents.

(a) (b)

Fig. 3. Setup of the bending test and the obtained loading curves.

Fig. 4. Layout of the splitting test.

in which k is the coefcient of water permeability (m/s); a is the cross-section area of

the glass tube (m2); A is the cross-section area of the cylinder (m2); T is the thickness
of the cylinder (m); t is the time of water falling from h0 to hf (s); h0 and hf are the
initial and nal water levels (cm).

Fig. 5. Setup of the water permeability test.

3. Results and discussion

In the following curves and graphs, the average values are decomposed compared the values at the 4th week and the 6th week.
shown together with the standard deviation which is represented This was due to the adjustment of the pH to 7. The pH of the original
by means of error bars. media was about 6.5. This indicates that a neutral pH is more prot-
able for bacterial activity. Because of this long term viability, the bac-
3.1. Survival of the free bacteria teria have great potential to be used in self-healing concrete.

It can be seen from Fig. 6 that bacterial ureolytic activity did not 3.2. Activity of bacteria immobilized in silica gel and polyurethane
decrease at all after 24 weeks. The amount of urea decomposed by
bacteria was almost the same from the beginning (Time 0) till the From Fig. 7a, it can be seen that the presence of dead bacteria
end (24 weeks). There was a slight increase in the amount of urea did not result in an increase of the conductivity of the urea
536
20
in sterile deposition media
Urea decomposed (g/L)
15
10
0
Time 0 2 weeks
solutions. For the urea solutions to which SG or SG + BSA was
added, the increase of conductivity in the rst 24 h was due to
the release of ions (Na+ or Cl), which were used to transform silica
sol into a gel. Since the quantity of the released ions was limited,
the conductivity after 24 h reached a stable state. The conductivity
in the urea solution with the addition of free bacterial cells
increased rapidly within 24 h and then decreased slightly until it
became stable afterwards. The reason for the decrease is the
volatilization of NH3 produced from NH 4 . After the equilibrium be-
tween NH3 and NH 4 was reached (after 2 days), the conductivity
stabilized. The conductivity of the urea solution with silica gel
immobilized bacteria increased at a slower rate compared with
the one with free cells. Three days later, the conductivity reached
a stable value. The slower decomposition rate of silica gel immobi-
lized bacteria was due to the decrease of bacterial activity after
being immobilized into silica gel. Therefore, it took more time to
decompose the same amount of urea by immobilized bacteria than
by non-immobilized bacteria. The slower increasing rate of con-
ductivity of the urea solution with immobilized bacteria might also
be attributed to the retarding effect of gel on the diffusion of urea.
Therefore, bacterial cells inside silica gel gradually came in contact
with urea and then decomposed it. The produced NH 4 and CO3
diffused through silica gel into the solution and caused the increase
of the conductivity. The whole process took more time compared
with when the reaction happened directly in the urea solution. It
was also noticed that the nal conductivity of the urea solution
with SG + BS was even slightly higher than the one with free cells.
The reason could be that some ammonia was trapped inside the gel
and couldnt escape anymore.
The situation with PU immobilized bacteria was to some extent
similar, as shown in Fig. 7b. The urea solutions, with only PU foam
or PU foam together with dead bacteria, showed a limited increase
in conductivity which indicated that no urea was decomposed. The
urea solutions with PU immobilized living bacteria displayed an
obvious increase of the conductivity as time went on, but at a slower
speed compared with the ones with non-immobilized bacteria. This
was also due to the inuence of the carrier (polyurethane foam).
The nal value was about one-third of the peak value of the free cells.
This indicated that bacteria still had activity to decompose urea after
immobilization into PU foam. Yet the overall activity decreased con-
siderably, which means that less CaCO3 might be produced than ini-
tially expected.
In Fig. 7a and b, the data for free BS were the same. The amount
of free BS was the same as that in SG and PU. So the activity of un-
immobilized bacteria, SG immobilized bacteria and PU immobi-
lized bacteria can be compared (Fig. 7c). Based on the conductivity
measurements, the amount of urea decomposed by bacteria (non-
immobilized or immobilized) can be calculated. As shown in
Fig. 7c, free cells decomposed about 70% of the urea and immobi-
lized bacteria decomposed 50% (silica gel immobilized bacteria)
or 30% (polyurethane immobilized bacteria). After being immobi-
J. Wang et al. / Construction and Building Materials 26 (2012) 532540
4 weeks 6 weeks 8 weeks 10 weeks 12 weeks 14 weeks 18 weeks 24 weeks
Fig. 6. Ureolytic activity of the bacteria at different storage time.
lized in the carriers, bacterial ureolytic activity can still be kept
(from 30% to 70%). Bacteria immobilized into silica gel had higher
ureolytic activity than those immobilized into polyurethane.
It can be seen from Fig. 7d that the immobilized bacteria both in
SG and PU can still decompose urea after one week. The amount of
urea decomposed was almost the same as that in the rst week
which means the ureolytic activity of immobilized bacteria did
not decrease. Although the bacteria did not decompose all the urea
in the solution in the rst week before the conductivity reached a
stable value, they started to decompose urea again after removing
the old urea solution and adding the new urea solution. That is be-
cause the bio-decomposition of urea was governed by bacterial
urease activity. Urea is the inducer or the substrate of this enzyme
reaction. At rst, the amount of urea was large enough to trigger
the urease activity, so initially the speed of urea decomposition
was fast. During the process of reaction, the amount of urea grad-
ually decreased followed by a slowing of the reaction speed, and
the amount of ammonium gradually increased. The ongoing reac-
tion was also affected by the accumulation of ammonium and
ammonia in the solution, which could inhibit bacterial activity
[28]. Indeed, when the quantity of urea decreased to a limited
2
value and the ammonium and ammonia accumulated to a certain
degree, the reaction was inhibited completely, resulting in some
residual urea. However, when the urea solution was refreshed,
the conductivity of the solution increased again. Hence, after
removing the inhibitory end-products (ammonium and ammonia),
and adding new urea, the bacterial urease activity can completely
recover.
3.3. CaCO3 precipitation in silica gel and PU foam
No urea was decomposed in the deposition medium with only
silica gel, or only PU foam, or silica gel with dead bacteria or PU
foam with dead bacteria. Dead bacteria did not decompose urea,
which corresponded with the result stated in Section 3.1. There
was about 14 g/L (70%) and 6 g/L (30%) urea decomposed in the
deposition medium by SG and PU immobilized bacteria, respec-
tively, which indicated that about 23 g/L and 10 g/L CaCO3 was
formed.
CaCO3 normally decomposes into CaO and CO2 in the tempera-
ture range of 650800 C [29]. Therefore, due to the release of CO2,
there should be a weight loss during that temperature range if
CaCO3 exists in the samples. As shown in Fig. 8a and b, there was
an obvious weight decrease at the temperature range from
650 C to 800 C (gray lines). This decrease was due to the decom-
position of CaCO3 into CO2 and CaO, which corroborated the exis-
tence of CaCO3 precipitated by SG and PU immobilized bacteria.
Based on the graphs, it can be seen that CaCO3 amounted to about
25% and 11% (by mass) of the silica gel and polyurethane samples,
respectively. There was more CaCO3 precipitation in the silica gel
than in the PU foam. The results correspond with the fact that
 J. Wang et al. / Construction and Building Materials 26 (2012) 532540 537

(a) It can be seen from the described results that the bacteria, after
being immobilized into silica gel or polyurethane, still have ureolyt-
ic activity and carbonatogenesis activity, and can precipitate CaCO3.
However, for a real self-healing process, it is not practical to supply
urea and Ca2+ by immersing concrete structures into the deposition
medium or by spraying the deposition medium afterwards. Each
necessary component should be incorporated inside concrete at
one time. To mimic a real self-healing process, specimens were
incorporated with healing agents lled in glass tubes during casting
(see Section 2.4.2). During the rst loading cycle to make cracks, the
glass tubes were broken and healing agents ew out into cracks and
mixed together. It is assumed that bacteria could produce CaCO3 in
this self-healing process. Therefore, some silica gel and polyure-
(b) thane samples were taken for TGA analysis to detect the formation
of CaCO3 after breaking the glass tubes manually. The TGA results
(see Fig. 9) proved the existence of CaCO3 precipitation. In Fig. 9,
comparing (a and b) with (c), and (d and e) with (f), it can be seen
that there is an obvious weight decrease in the temperature range
from 650 C to 750 C, similar with Fig. 8. This was due to the
decomposition of CaCO3. No DTG peak in this temperature range
was observed in the series without living bacteria. The weight per-
centage of CaCO3 precipitation in silica gel and polyurethane was
about 12% and 10% by mass, respectively. The amount of precipita-
tion decreased when the bacteria were subjected to a mimic self-
healing process. This was due to the limited supply of the deposi-
16
(c) tion medium. Bacterial activity and the amount of deposition
14 medium containing urea and Ca2+ are the two most important fac-
Urea decomposed in
urea solution (g/L)

12 tors for CaCO3 precipitation by immobilized bacteria. When more

10 deposition medium was supplied, like in Section 2.3.2 (by immers-
8 ing into 50 mL DM), more precipitation occurred. In the mimic self-
healing process, the deposition medium was lled in glass tubes.
6
The amount was limited by the volume and the number of the glass
4
tubes used. The total volume was less than 50 mL. Therefore, the
2 quantity of CaCO3 precipitation in a mimic self-healing process
0 was less than that was obtained from the immersion method.
Free cells SG+BS PU+BS
Fig. 10 shows the scanning electron micrograph of CaCO3 pre-
cipitation (indicated by the black arrow) in silica gel and PU foam.
(d) It can be seen that the CaCO3 particles from silica gel immobilized
bacteria had a regular cubic shape, and the size was about 100 lm.
CaCO3 in PU foam showed an irregular block shape with a similar
particle size (also about 100 lm). It can be seen from Fig. 10a
and c that CaCO3 particles were distributed more homogenously
in silica gel than in PU foam. The reason is that polyurethane pre-
polymer is more viscous than silica sol (a state before gel). So it is
easier for bacteria to distribute homogeneously in silica sol than in
polyurethane before silica sol became gel or polyurethane foam
formed.

3.4. Self-healing efciency in cracked mortar specimens

3.4.1. Strength regain

Fig. 7. Ureolytic activity of un-immobilized and immobilized (silica gel or polyure-
thane) bacteria, as indicated by the increase of the conductivity: (a) the conductivity of
the urea solutions with different kinds of SG additions ( added with un-
immobilized bacteria (free cells), as a comparison with immobilized bacteria,
added with silica gel, added with silica gel immobilized dead bacteria, added
with silica gel immobilized living bacteria), (b) The conductivity of the urea solutions
with different kinds of PU foam additions ( added with un-immobilized bacteria
(free cells), as a comparison with immobilized bacteria, same as the one in (a),
added with PU foam, added with PU immobilized dead bacteria, added with
PU immobilized living bacteria), (c) amount of urea decomposed (calculated from the
conductivity values in (a) and (b)) by un-immobilized bacteria, SG immobilized
bacteria and PU immobilized bacteria after three days, and (d) The conductivity of the
new urea solutions with the original SG and PU immobilized bacteria (The old solutions
were poured out and the new solutions were added at the 7th day, therefore the time to
measure conductivity in the graphs was started at the 168th hour).
bacteria had a lower ureolytic activity after being immobilized in
PU foam than in silica gel.
The amount of strength regain of cracked specimens of different
series is shown in Fig. 11b. There was no strength regain in the
specimens which had no healing agents incorporated (reference).
One example of the loading curves of a reference specimen can
be seen in Fig. 11a. The strength regain in the series of SG + BSA
were due to silica gel itself since dead bacteria cannot precipitate
CaCO3. Higher strength regain was obtained in the series of SG + BS
because of the bacterial CaCO3 precipitation inside silica gel. Yet,
the levels of strength regain of both series were quite limited, i.e.
less than 5%. Specimens with PU immobilized bacteria had more
strength regain, about 50% to 80%. No obvious difference of
strength regain was observed in the specimens between the use
of living bacteria and dead bacteria. It can be concluded that the
strength regain was mainly due to polyurethane since the amount
of CaCO3 precipitated inside PU foam was quite limited. Although
538 J. Wang et al. / Construction and Building Materials 26 (2012) 532540

100 0.4 100 0.4

)
)
0.3 80 0.3
90

Deriv.Weight (%/
Deriv.Weight (%/

Weight (%)
Weight (%)

0.2 60
80 0.2
0.1 40

70 0.1
0 20

60 -0.1 0 0
0 200 400 600 800 1000 0 200 400 600 800 1000
Temperature ( ) Temperature ( )
(a) SG+BS (b) PU+BS
Fig. 8. TGA graphs of samples from SG + BS and PU + BS (The gray and black lines were the weight (percentage) changes and the derivation of the weight (percentage)
changes as a function of temperature, respectively).

(a) SG (b) SG+BSA (c) SG+BS

(d) PU (e) PU+BSA (f) PU+BS

Fig. 9. TGA graphs (the gray and black lines were the weight (percentage) changes and the derivation of the weight (percentage) changes as a function of temperature,
respectively) of the silica gel and polyurethane samples taken from the reaction products from the healing agents (lled in the glass tubes) after breaking the glass tubes
(series of (af) were shown in Fig. 1).

bacteria can precipitate more CaCO3 in silica gel than in PU foam,
silica gel itself has limited strength to contribute to the strength
regain of the specimens. PU foam is an organic polymer. It has a
higher strength and there will be a stronger bond between polymer
and crack wall. Hence higher strength regain can be obtained when
using PU.
3.4.2. Water permeability
During the 30 days of testing, the water permeability coefcient k
of the specimens gradually reached a stable value. The nal k values
of each series are shown in Fig. 12. The k values of the reference spec-
imens (without healing agents incorporated) ranged from 4  106
to 7  106 m/s. The situation of the specimens with only silica gel
was similar to the reference specimens and the nal k value was
about 106 m/s which indicated that silica gel in the crack had lim-
ited capacity to decrease the water permeability. One specimen of
this series had higher water permeability than the untreated ones.
The reason was because this specimen had a larger crack width
(the initial crack width was 0.5 mm and decreased to 0.35 after
de-loading; the other specimens had an initial crack width of
0.4 mm). This demonstrates that crack width has a profound inu-
ence on water permeability. The water permeability of the
specimens with silica gel immobilized living bacteria (SG + BS)
decreased about two orders of magnitude compared with the refer-
ence ones. The nal k values of this series varied from 109 to
107 m/s. Adding living bacteria to silica gel decreased the water
permeability because of the lling effect of CaCO3 produced by bac-
teria. The specimens with polyurethane (with or without bacteria)
showed lower water permeability compared to the ones with silica
gel. For the series of PU, the nal k value was about 6  1011 to
7  1011 m/s. Due to the un-complete lling of the crack by poly-
urethane foam in one replicate, this specimen had an extremely high
water permeability (with a k value about 106 m/s) compared with
the other two replicates. For the series of PU + BS, the nal k value
was around 3  1011 to 6  1011 m/s, slightly lower than that of
PU without living bacteria, because some pores of polyurethane
foam were lled by bacterial CaCO3 precipitation, and hence helped
to decrease the water permeability.
Bacterial CaCO3 had a positive effect on decreasing the water
permeability. This positive effect depended on the amount of
CaCO3 precipitation. When using silica gel as the carrier, the bacte-
ria had a higher ureolytic activity and carbonatogenisis activity,
and thus can produce more CaCO3 precipitation inside silica gel.
Therefore, there was an obvious difference in water permeability
coefcient of the specimens between the series of SG and SG + BS.
Polyurethane is normally used as a waterproong material. So the
 J. Wang et al. / Construction and Building Materials 26 (2012) 532540 539

(a) CaCO3 in silica gel (100x) (b) CaCO3 in silica gel (1000x)

(c) CaCO3 in PU foam (100x) (d) CaCO3 in PU foam (1000x)

Fig. 10. Scanning electron micrographs of CaCO3 precipitation in the silica gel and PU foam.

3
(a)
2.5
Load (kN)

1.5

0.5

0
0 200 400 600 800
Crack width (m)

100
(b)
Fig. 12. Water permeability of the cracked cylinders after being repaired by the
80
Strength regain (%)

different incorporated healing agents.

60

40 After this proof of concept step in real mortar specimens, it can

20
0 tant, or for structures that are difcult to reach for inspection
Reference SG+BSA SG+BS PU+BSA
Fig. 11. Strength regain of cracked specimens of different series: (a) loading curves
of a reference specimen (Black line: First loading curve; Gray line: Second loading
curve), and (b) overall strength regain of cracked specimens.
water permeability of the specimens can be decreased a lot when
only using polyurethane. Since bacteria had a lower carbonatogen-
esis activity after being immobilized in PU foam, less CaCO3 precip-
itation occurred inside PU foam than in silica gel. Therefore, the
positive effect of the bacterial CaCO3 in the polyurethane series
was not as obvious as in the silica gel series.
be seen that the self-healing system in the study has a potential to
be used in practical application. Self-healing has most benet for
structures where good durability and long service life are impor-
PU+BS
and maintenance. More efforts will be done on getting more CaCO3
precipitation and searching for a more suitable capsule for the
healing agents because the glass tubes may be easily broken during
mixing.
The extra costs resulting from incorporating the self-healing
healing system in concrete is difcult to estimate accurately for
the moment since it is still in the state of proof of concept.
Based on the case of our current research, the cost for a concrete
(80 /m3) with self-healing property would be increased by
728% (using PU + BS) and 521% (using SG + BS), depending on
the amount of healing agents added. However, the cost for the
later maintenance and repair work would be greatly decreased.
540 J. Wang et al. / Construction and Building Materials 26 (2012) 532540
Therefore, applying the self-healing system in concrete structures
is a potential solution for the sustainable development of con-
crete structures.
4. Conclusions
Bacteria can still conserve ureolytic activity and carbonatogenesis
activity after being immobilized into silica gel and polyurethane
foam. TGA results proved the formation of the bacterial CaCO3
precipitation in a mimic self-healing process which indicated the
great potential use of immobilized bacteria in self-healing concrete
cracks. More self-healing efciency (higher strength regain and more
pronounced decrease of water permeability) was obtained from the
specimens with incorporated polyurethane immobilized bacteria.
Therefore it is promising to use polyurethane immobilized bacteria
to self-heal early concrete micro cracks. Further research is needed
to increase the amount of bio-CaCO3 to obtain more self-healing
efciency.
Acknowledgements
The authors appreciate the nancial support from the Research
Foundation Flanders (FWO-Vlaanderen) for this study (Project No.
G.0157.08). The authors express their thanks to the Department of
Inorganic Chemistry and the Department of Physics for providing
TGA and SEM analysis.
References
[1] He H, Guo ZQ, Stroeven P, Stroeven M, Johannes Sluys L. Self-healing capacity
of concrete-computer simulation study of unhydrated cement structure. Image
Anal Stereol 2007;26:13743.
[2] Yang YZ, Lepech MD, Yang EH, Li VC. Autogeneous healing of engineered
cementitious composites under wet-dry cycles. Cem Concr Res 2009;39:
38290.
[3] Sahmaran M, Li VC. Durability properties of micro-cracked ECC containing high
volumes y ash. Cem Concr Res 2009;39:103343.
[4] Dry C. Matrix cracking repair and lling using active and passive modes for
smart timed release of chemicals from bers into cement matrices. Smart
Mater Struct 1994;3:11823.
[5] Dry C, McMillan W. Three-part methylmethacrylate adhesive system as an
internal delivery system for smart responsive concrete. Smart Mater Struct
1996;5:297300.
[6] Ramachandran SK, Ramakrishnan V, Bang SS. Remediation of concrete using
micro-organisms. ACI Mater J 2001;98:39.
[7] De Muynck W, Debrouwer D, De Belie N, Verstraete W. Bacterial carbonate
precipitation improves the durability of cementitious materials. Cem Concr
Res 2008;38:100514.
[8] Jonkers HM, Schlangen E. Self-healing of cracked concrete: a bacterial
approach. In: Proceedings of FRACOS6: fracture mechanics of concrete and
concrete structures. Italy: Catania; 2007. p. 182126.
[9] Ramakrishnan V. Performance characteristics of bacterial concrete a smart
biomaterial. In: Proceedings of the rst international conference on recent
advances in concrete technology. America: Washington DC; 2007. p. 6778.
[10] Stocks-Fischer S, Galinat JK, Bang SS. Microbiological precipitation of CaCO3.
Soil Biol Biochem 1999;126(14):2534.
[11] Rodriguez-Navarro C, Rodriguez-Gallego M, Ben Chekroun K, Gonzalez-Munoz
MT. Conservation of ornamental stone by Myxococcus xanthus-induced
carbonate biomineralization. Appl Environ Microbiol 2003;69(4):218293.
[12] Castanier S, Le Me0 tayer-Levrel G, Perthuisot J. Carbonates precipitation and
limestone genesisthe microbiogeologist point of view. Sediment Geol
1999;126:923.
[13] Hammes F, Boon N, de Villiers J, Verstraete W, Siciliano SD. Strain-specic ureolytic
microbial carbonate precipitation. Appl Environ Microbiol 2003;69(8):49019.
[14] De Muynck W. Microbial interactions with mineral building materials.
Doctoral thesis. Magnel Laboratory for Concrete Research and Laboratory for
Microbial Ecology and Technology. Gent University; 2009.
[15] De Muynck W, Verbeken K, De Belie N, Verstraete W. Inuence of urea and
calcium dosage on the effectiveness of bacterially induced carbonate
precipitation on limestone. Ecol Eng 2010;36(2):99111.
[16] Qian CX, Wang JY, Wang RX, Cheng L. Corrosion protection of cement-based
building materials by surface deposition of CaCO3 by Bacillus pasteurii. Mater
Sci Eng C 2009;29(4):127380.
[17] Jonkers HM, Thijssen A. Bacteria mediated remediation of concrete structures.
In: Proceedings of the second international symposium on service life design
for infrastructures. The Netherlands: Delft; 2010. p. 83340.
[18] Soltmann U, Raff J, Selenska-Pobell S. Biosorption of heavy metals by solgel
immobilized Bacillus sphaericus cells, spores and s-layers. J SolGel Sci Technol
2003;26:120912.
[19] Soltmann U, Bottcher H. Utilization of solgel ceramics for the immobilization
of living microorganisms. J SolGel Sci Technol 2008;48:6672.
[20] Van Tittelboom K, De Belie N, De Muynck W, Verstraete W. Use of bacteria to
repair cracks in concrete. Cem Concr Res 2010;40:15766.
[21] Bang SS, Galinat JK, Ramakrisshnan V. Calcite precipitation induced by
polyurethane-immobilized Bacillus pasteurii. Enzyme Microb Technol 2001;28:
4049.
[22] Wang JY, Van Tittelboom K, De Belie N, Verstraete W. Potential of applying
bacteria to heal cracks in concrete. In: Proceedings of the second international
conference on sustainable construction materials and technologies. Italy:
Ancona; 2010. p. 180718.
[23] Dick J, De Windt W, De Graef B, Saveyn H, Van Der Meeren P, De Belie N, et al.
Bio-deposition of a calcium carbonate layer on degraded limestone by Bacillus
species. Biodegradation 2006;17:35767.
[24] Ivanov VM, Figurovskaya VN, Barbalat Yu A, Ershova NI. Chromaticity
characteristics of NH2Hg2I3 and I2: molecular Iodine as a test form alternative
to Nesslers reagent. J Anal Chem 2005;60(7):70710.
[25] Van Tittelboom K, De Belie N. Self-healing concrete by means of encapsulated
polymers. In: Proceedings of the 13th international conference on polymers in
concrete (ICPIC). Portugal: Madeira; 2010. p. 68188.
[26] Wang K, Jansen DC, Shah SP, Karr AF. Permeability study of cracked concrete.
Cem Concr Res 1997;27(3):38193.
[27] NBN B 24-213. Belgische norm: Proeven op metselstenenWateropslorping
onder vacuum; 1976.
[28] Whifn VS. Microbial CaCO3 precipitation for the production of biocement.
School of biological sciences and biotechnology. Murdoch University, Perth;
2004. p. 20.
[29] Oniyama E, Wahlbeck PG. Application of transpiration theory on TGA data:
calcium carbonate and zinc chloride. Thermochimica Acta 1995;250:4153.