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 Smart Materials and Structures

Smart Mater. Struct. 28 (2019) 075041 (12pp) https://doi.org/10.1088/1361-665X/ab2012

Non-ureolytic microbial self-repairing

concrete for low temperature environment
Yilin Su1,2,3 , Jianhang Feng1,2,3, Qiwei Zhan1,2,3, Yi Zhang1,2,3 and
Chunxiang Qian1,2,3
1
School of Materials Science and Engineering, Southeast University, Nanjing 211189, People’s Republic
of China
2
Jiangsu Key Laboratory for Construction Materials, Southeast University, Nanjing 211189, People’s
Republic of China
3
Research Institute of Green Construction Materials, Southeast University, Nanjing 211189, People’s
Republic of China

E-mail: seusyl@163.com

Received 24 March 2019, revised 26 April 2019

Accepted for publication 8 May 2019
Published 18 June 2019

Abstract
To improve the self-healing efficiency of bacterial concrete, one type of bacteria which could
survive, germinate and induce calcium carbonate precipitation at 7 °C was investigated and
incorporated into concrete directly. The results showed that after healing for 14 d, cracks with
400–500 μm width could be sealed, and higher water resistance was obtained for cracked
specimens with bacteria, compared with plain cracked concrete. Therefore, it could be concluded
that the bacteria has the potential of promoting concrete self-healed at low temperature.
Keywords: concrete, bacteria, self-healing, low temperature, calcium carbonate

(Some figures may appear in colour only in the online journal)

1. Introduction such as superabsorbent polymers, could improve self-healing

Cement-based materials are widely used material in con-
struction. With the high demand for longer service life of
building and low carbon emissions, durability of cement-
based materials is an important research direction nowadays.
However, due to its brittleness and harsh application
environment, cement-based materials are inevitable to crack.
If the cracks in materials are not repaired timely, hostile
species, such as chloride and sulfate ions, will ingress and
accelerate the deterioration of cement-based materials [1–3].
Therefore, repairing cracks of cement-based materials is an
effective method to extend their usage time. Manual repair
methods are commonly used [4], but they are hardly to heal
inaccessible cracks [5] and cost much [6]. Thus, exploring
cement-based materials with self-healing performance is
highly desirable.
Incorporating mineral admixtures [7] or capsules [8]
containing calcium hydroxide is one method to promote
further hydration of unhydrated cementitious materials [9–12]
and carbonation [13], forming C–S–H gel and calcium car-
bonate to trigger crack healing. In addition, expansion agents,
efficiency of cement-based materials by swelling after which
water penetrates into cracks [14, 15]. Although both of the
aforementioned methods are easy to operate and eco-friendly,
the crack width that could be totally repaired is no more than
200 μm. Therefore, self-healing technology, which could
repair cracks above 200 μm, is needed.
Application of microorganisms in cement-based materi-
als is a promising strategy to improve the self-healing effect.
The maximum repaired crack width is 970 μm until now [16],
by using microbial induced calcium carbonate precipitation
technology in cement-based materials. Moreover, incorpora-
tion bacteria could regain the strength [17, 18] and improve
the durability [19] of cracked cement-based materials. How-
ever, to maintain good bacterial activity, curing temperature
during bacterial self-healing process is always 20 °C–30 °C
[20–28], which causes doubt on whether this method can be
used in low-temperature service environment of concrete.
Hence, it is especially important for the development of low
temperature resistance bacterial self-healing agent, which
could be used to repair cracks of concrete in underground
parking garage and canals, as suggested in the previous report

0964-1726/19/075041+12$33.00 1 © 2019 IOP Publishing Ltd Printed in the UK

Smart Mater. Struct. 28 (2019) 075041 Y Su et al

Table 1. Composition of culture medium.

Nutrient substance Mass (g) Main function

Peptone 10.0 Nitrogen and carbon sources
Beef extract 3.0 Nitrogen and carbon sources,
NaCl 5.0 Inorganic salt
FeCl2 0.1 Inorganic salt
Deionized water 1000 Water

[29]. Compared with traditional methods, such as temperature

gradient domestication for bacteria, selection and cultivation
of low temperature resistant bacteria is a more effective way.
Lysinibacillus sp. has been proved to be a alkali-resistant
microorganism, which could induce calcium carbonate pre-
cipitation by binding positively charged calcium ions on net
negative charged cell walls and forming extracellular poly-
meric substances [30–32]. Therefore, in this paper, a type of
Lysinibacilus sp. was selected and its mineralization product
in low-temperature alkaline environment was investigated. Figure 1. Sketch of reactor and design.

Self-healing performance of cement-based materials incor-

porated the bacteria was verified as well. bacterial sample was placed in an oven at 35 °C for 1 d before
observation.

2. Materials and methods 2.2. Spore germination in alkaline environments

It is noted that the pH inside the concrete matrix can reach

2.1. Bacterial strain
A type Lysinibacillus sp. was purchased from China Center of
Industrial Culture Collection (CICC), which showed a low
temperatures resistant. They are gram-positive, aerobic and
spore-forming bacteria with elliptical or columnar shape.
Bacteria strain after selection was stored in ultra-low temp-
erature refrigerator (Thermo 902-ULTS, America) at −78 °C.
After autoclaved at 121 °C for 25 min, 3 ml bacteria spores
was inoculated in 200 ml liquid medium, and proportion of
the liquid medium was shown in table 1. Then the medium
was incubated at (7±3) °C on a shaker at 170 r min−1 with
air flow for 72 h.
Spore germination in the liquid medium was determined
by a qualitative procedure involving the changes in OD600
(optical density measurement at 600 nm) of spore suspensions
during germination [33]. Sample solutions were obtained every
4 h up to 72 h and the optical density and pH values of them
were measured by using multifunctional microplate detector
(Bio-Tek, America) and pH meter (Hengxin AZ8601, Taiwan),
respectively. Moreover, the microscopic morphology of the
bacteria was observed by FE-SEM (field emission scanning
electron microscopy, FEI-Sirion). The preparation steps for
SEM samples were as follows: (1) Collecting the cells: cen-
trifuging the culture solution at 8000 rpm for 3–5 min, dis-
carding the supernatant and pouring the 2.5% glutaraldehyde
fixative for fixation for 2–4 h; (2) Washing and dehydrating:
after being washed 3 times by deionized water, dehydration
was carried out by using the gradients were 30%, 50%, 95%,
100% ethanol, 15–20 min for each operation. After that, the
12∼13, especially in freshly cast concrete. While in the
crack zone, the pH can be lower in the range of 9–11 caused
by dilution of water flow and carbonization. Therefore, the
spore germination in alkaline environments was investi-
gated. Bacterial spores was inoculated into different liquid
medium with the initial pH of 9, 10, 11, 12 and 13, adjusted
by NaOH solution. The composition of the medium and
other growth conditions except alkalinity were the same as
the method mentioned in the section 2.1. Those mediums
were incubated at (7±3) °C on a shaker at 170 r min−1
with air flow for 3 d.
In addition, the viability of bacteria in fresh cement
paste was also studied, bacteria solution with concentration
of 1.36×108 cells ml−1 that was measured by flow cyt-
ometer (Guava easyCyte 5) were incorporated into cement
paste with the w/c ratio of 0.5 directly to replace water.
After 3 d, 7 d and 14 d, the samples were removed from the
standard curing room (20 ±1 °C, RH 95%) and then
ground into powders using a sterile mortar and pestle [34]
until the all the particle sizes were below 2.36 mm. After
which about 50 g of the powdered sample were suspended
in 250 ml of fresh liquid medium as mentioned before and
the resulting suspension was sonicated in a water bath for
10 min at a low frequency. Those fresh liquid medium were
incubated at (7±3) °C on a shaker at 170 r min−1 with air
flow for 3 d after being filtrated through sterile gauze and
adjusted pH to 12.
The optical density (OD) of the culture was measured
and recorded every 4 h at a wavelength of 600 nm for 72 h.
The germination of spores at different pH values and viable

2
Smart Mater. Struct. 28 (2019) 075041
Table 2. Mixture design for verifying mineralization capacity at low temperature.
Group Bacteria liquid/(ml) Calcium solution/(ml)
1 50 200
2 50 200
Table 3. Mixing proportion of cement-based sample.
Group Sand/(g) Cement/(g) Bacteria liquid/(ml)
1 1200 600 0 6
2 1200 600 50
3 1200 600 100
cell retention in cement pastes cured for different time were
therefore obtained.
2.3. Mineralization products in low temperature
Currently, there are two main non-ureolytic mechanisms
applied to microbial self-repairing concrete, which are
involved hydration of carbon dioxide promoted by enzyme
named carbonic anhydrase and organic carbon decomposed
by bacteria [35, 36]. To explore the mechanism of miner-
alization in low temperature, reaction of microbial induced
calcium carbonate was conducted at constant temperature of
(7±3) °C for 24 h. 50 ml bacterial medium at the end of
exponential growth phase were incorporated into 200 ml
solution with a concentration of 1.0 mol l−1 calcium source.
The proportion design was shown in table 2 and the reactor
was illustrated in figure 1. In order to verify whether the
bacteria could deposit calcium carbonate by secreting carbo-
nic anhydrase at low temperature, calcium nitrate was used as
a calcium source, and carbon dioxide was produced by
reacting sodium bicarbonate with acetic acid, as shown as
mode 1 in figure 1. This carbon dioxide generator was
replaced every 6 h. Moreover, in order to explore whether
low-temperature mineralization performance comes from
microbial decomposing organic carbon, calcium lactate was
used as a calcium source, and the reactor without the carbon
dioxide generating device was introduced air continuously, as
shown as mode 2 in figure 1. After 3 d of mineralization, the
samples were transferred to a vacuum drier to oven-dried at
60 °C until the mass change in 24 h was less than 0.1%.
Subsequently, the products in the mineralized samples were
investigated by using DTA-TG (NETZSCH STA449F3
simultaneous thermal analysis meter). The temperature
increased from room temperature to 1000 °C with a rate of
20 °C min−1. In addition, for analyzing the crystal structure
and micro-morphology of biogenic calcium carbonate, partial
precipitates were washed by deionized water and absolute
ethanol for three times respectively before oven-dried, and
then x-ray diffraction (XRD, Bruker D8-Discover, Germany)
and FE-SEM (FEI-Sirion) was used for characterization.
Y Su et al
Calcium source/(1 mol l−1) Bacteria in solution/(cells ml−1) pH
7
calcium nitrate 2.7×10 10
calcium lactate 2.7×107 10
Calcium source/(g) Water/(g) Bacteria in mortars/(cells m−3)
270 0
6 220 1012
6 170 1013
2.4. Self-healing properties of mortar in low temperature
2.4.1. Preparation of mortar specimens. Mortar specimens
with the size of 40 mm×40 mm×160 mm were prepared
by mixing sand, tap water, P·II 52.5 cement and microbial
self-healing agent, which contained bacteria liquid with
108 cells ml−1 and calcium sources. The mix design is
shown in table 3 and the mixing process was according to
Chinese standard GBT17671-1999. After cuing for 24 h,
specimens with dimensions of 40×40×160 mm were
demoulded and kept in standard curing room (20 ±1 °C,
RH 95%) for 7 d.
2.4.2. Cracks creation and repairing incubation process.
Different methods were used in pre-cracking for different
cracking widths. For crack width below 200 μm, cracks
were obtained by three-point bending at a low loading speed
until the first crack appears. For cracks with a larger crack
width, Cracks were introduced according to Luo et al [37].
The broken prismatic specimens was reassembled and
embedded with nails of different diameters for controlling
crack width. All sides of specimens except the cracking
surface were wrapped with epoxy resin after reassembled.
The cracked specimens were immersed in deionized water in
an plastic container which was open to the atmosphere. In
addition, air was passed into the water constantly by using a
tube and a pump. The system was put in a refrigerator with
temperature of (7±3) °C. For observing the self-healing
efficiency of cracks, the specimens were taken out after
14 d.
2.5. Characterization methods for self-healing efficiency
2.5.1. Visual observation of cracking area.Specimens were
removed from water and stored at 25±3 °C and 60%±
10% RH for three hours, after which the changes of
cracking area were observed and recorded at the same
magnification by using stereo microscope (SMZ745, Nikon,
Japan). To quantitatively characterize self-healing effects,
the software named ‘Image-Pro plus’ was used for counting
the number of pixel dots occupied by cracking area before
3
Smart Mater. Struct. 28 (2019) 075041 Y Su et al

Figure 2. Curing conditions for capillary absorption test.

and after self-healing. The area repair ratio was expressed

by the equation (1)

Area repair rate % = (A 0 - At ) A 0 ´ 100%, (1 )

where, A0 is the number of pixel dots corresponding to

cracking area before repairing and At is those of cracking
area after repairing of t days.

2.5.2. Capillary absorption. Capillary water absorption tests

were carried out to quantitatively assess the water resistance
of the samples before cracking, after cracking, and after
healing. In order to show the obvious difference on the water
resistance of repaired specimens with bacteria or not, all
cracks were introduced by embedded with nails and crack
width was controlled between 0.4 and 0.5 mm and the content
of bacteria in microbial samples was 1013 cells m−3. Two
groups of specimens, the reference group and the microbial
group, were prepared, and the mixture ratios were referred
to group 1 and group 3 in table 3, respectively. Three types
of each group were set up for investigating the change of
water absorption, which were un-cracked samples, cracked
samples and repaired samples. Moreover, all specimens were
subjected to the same maintenance procedure for eliminating
the influence of hydration age, as shown in figure 2. Before
testing the absorption, the samples were oven-dried at 40 °C
until the mass change was below 0.1%, after which the
samples were sealed by epoxy except for cracking surface.
The process of sorptivity was conducted based on ASTM C
1585 [38] considering the inner surfaces of cracks.
Subsequently the rate of water absorption (mm/s1/2) was
obtained through the slope of the curve that was absorption I
(mm) against the square root of time (s1/2). The specimens
were fully submerged in water with the water level higher
than top surfaces and the mass change of the specimens was
recorded in successive immersion time. A wet towel was used
to wipe the water on the surface before weighing and
specimens were re-submerged immediately after measuring.
Absorption (mm) as s function of time was expressed by
Dm t
I= = S · t1 2 + b,
Ar r 0
Figure 3. Monitoring of growth and pH changes in cultivation
process.
(g mm−3), S is the rate of absorption (mm/s1/2), b is the
initial value of I at the start time.
2.5.3. Permeability. Permeability of specimens was
characterized by evaluating of water flow passing through
the specimen during a certain period [39, 40]. The test method
was according to the method reported by Chen [41]. The
cylinder specimens with f 100 mm×50 mm was connected
to the polyvinyl chloride (PVC) pipe. The crack width of
specimens was 400–500 μm and the dosage of bacteria in
microbial samples was 1013 cells m−3. Mix designs of
reference samples and bacterial samples were the same as
that of group 1 and 3 in table 3, respectively. The water in the
PVC pipe was kept a fixed height of 100 cm. The test lasted
5 min and the amount of water penetrating through the mortar
was measured by an electronic scale. The water permeability
test was conducted twice, before and after crack healing. The
cracked specimens after curing for 7 d were fully immersed in
water for 24 h to be saturated. After which the initial water
permeability was measured. Samples after being repaired for
14 d was tested again. The permeability coefficient could be
(2 )
calculated according to (3).

where Δmt is the mass change at time t, Ar is the area of

samples exposed to water (mm2) and ρ0 is the density of water k = Q ´ L A ´ Dh, (3 )

4
Smart Mater. Struct. 28 (2019) 075041
Figure 4. (a) Scanning electron microscopy and (b) Energy dispersive spectrometry of bacteria after incubation for 48 h.
where k is permeability coefficient, m s−1; Q is amount of
water flow, m3 s−1; L is height of specimen, m; A is area of
section, m2; Δh is head difference, m.
The recovery of water permeability (RWP) for each set of
the three healed specimens was calculated as follows:
RWP = (k i - k h ) k i ´ 100%,
where ki is the initial permeability coefficient, kh is the
permeability coefficient of healed specimens.
2.5.4. Characterization of product in cracks. After 14 d
repairing incubation, a set of sample repaired by bacteria in
cracking area was chosen to observe morphology of repairing
products. The sample was coated by platinum with thickness of
Y Su et al
20 nm for SEM observation (Sirion, FEI, America). Meanwhile,
the components of precipitation were probe d by the energy
dispersive spectrometer (GENESIS 60 S) connected with SEM.
3. Results and discussion
(4 )
3.1. Bacteria germination and growth in low temperature
The optical density of the microbial culture medium at low
temperature was shown in figure 3. This bacteria grow rapidly
between 24 h and 36 h and entered the stationary phase at
44 h, in which the maximum cell density was 1.36×
108 cells ml−1. At the same time, the pH of the culture
decreased slightly during the initial stage, after which
5
Smart Mater. Struct. 28 (2019) 075041 Y Su et al

Figure 5. Spores germination under alkaline conditions.

pH gradually rose and stabilized. After 48 h incubation, the

bacterial culture was weakly alkaline before blended with
cement-based material. The bacteria are long rod shaped with
size of 1.1–1.7×0.5–0.7 μm (figure 4), which was observed
by scanning electron microscope combined with phosphorus
and sulfur shown in the energy spectrum.
The growth of cells and germination of spores in dif-
ferent pH environments was shown in figure 5. Microbial
growth and germination could proceed when the pH of the
growth medium was 12. It could be seen that the lag phase
during bacterial growth was prolonged as pH increased,
which indicated that this type of bacteria could be resistant
to alkaline environment. However, the growth rate sig- Figure 6. Thermal analysis of mineralized products.
nificantly decreased at the pH of 13 and the final optical
density was 0.1 approximately, which meant that bacterial
spores could not germinate in the environment at pH 13.
Moreover, bacteria could survive in cement-based materials
during the first 14 d, but the problem of extension of lag
phase could be observed as well. It could be attributed that
the germination of bacterial spores needed relatively long
time in high alkali cement-based materials. In addition,
concentration of viable cell retention decreased with the
curing time increased. It could be related to the process of
refinement of pore structure during the hydration, reducing
the living space of microorganisms. All in all, in the cracks
of which pH value was in the range of 9–11, this type of
bacteria has potential to survive of cement-based materials
in low temperature.

3.2. Mineralization product synthesized in solution

Figure 6 shows the DSC, TG and corresponding DTG curves

of the mineralization precipitation under two different
mineralizing modes, in which carbonates are derived from
hydration of carbon dioxide accelerated by carbonic anhy-
drase and consumption of organic matter respectively. As
shown in figure 6(a), there are three main thermal decom-
position peaks occurred at the temperatures of 166.38, 607.39
Figure 7. XRD diffraction spectrum of the mineralized precipitate.
and 660.39 °C, which involve the loss of crystal water, the
decomposition of calcium nitrate and calcium carbonate.
Meanwhile, the mass loss of mineralized products during the

6
Smart Mater. Struct. 28 (2019) 075041
Figure 8. SEM image of biogenic calcium carbonate.
heating shows that the content of less, which is about 16.05%
of sediment. Similarly, there are two main thermal decom-
position peaks in figure 6(b). The exothermic phenomenon at
313.9 °C is attributed to the combustion of calcium lactate.
Conversely, the endothermic peak corresponding to 702.64 °C
is the thermal decomposition endothermic peak of calcium
carbonate, which accounted for 49.09% of sediment. The
ability of this kind of microorganism to deposit carbonate at
low temperature is mainly derived from the decomposition of
small molecular organic matter.
Meanwhile, the XRD diffraction spectrum of the miner-
alized precipitate is shown in figure 7. The crystals were
mainly calcium carbonate. Compared with the chemically
synthesized calcium carbonate which was bought from
Macklin (Purity: 99.9%), calcium carbonate induced at low
temperature (T=7 °C) by Lysinibacillus sp. in both modes
was calcite. Figure 8 was the SEM image of biogenic calcium
carbonate. Calcium carbonate produced in the way of mode 1
at low temperature was irregular ellipsoid shape, as shown in
figure 8(a), and particle size of that calcite was in the range of
5–50 μm. To the contrary, it could be observed that the
morphology of calcium carbonate obtained by using mode 2
was mainly cube, of which the particle sizes is around 30 μm
as shown in figure 8(b). The difference in morphology of
7
Y Su et al
calcium carbonate in two ways may be attributed to different
crystal nucleation methods. In mode 1, the production of
calcium carbonate mainly relies on the carbonic anhydrase
produced by microorganisms to accelerate the dissolution of
carbon dioxide. But at low temperature, this extracellular
enzyme activity could be affected, more of bacteria played the
role of nucleating sites, and the size of calcium carbonate
formed varies. On the contrary, in the mode 2, the inorganic
carbonate produced through the process of the bacteria
decomposing organic matter is widely distributed in the
solution, so that the crystal size formed was relatively
uniform.
3.3. Self-healing effect of mortar at low-temperature
3.3.1. Visual quantification of cracks repairing. Optical
microscopy was used for capturing images and analyzing.
Figure 9 shows the self-repairing ability under low
temperature conditions, when this bacteria dosage was
1013 cell m−3. The crack with size of 400–500 μm was not
only almost filled completely, but also the surface of the
specimens was covered by white precipitates. Moreover,
when optically magnifying the repaired crack region, it can be
Smart Mater. Struct. 28 (2019) 075041
Figure 9. Surface repairing effect of specimen with the bacteria dosage is 1013 cell m−3.
seen that the white transparent crystal particles are closely
packed and filled in the crack.
By using image analysis software Image J, the statistical
analysis of repair rates of different cracking widths results are
shown in figure 10. Reference specimens showed very limited
crack-healing potential for cracks of width below 100 μm,
which could be attributed to the further hydration of cement
particles. But for the crack with a larger crack width, the
healing effect was not effective. The specimens with the
bacteria dosage of 1012 cells m−3 showed a huge improve-
ment in self-healing ability, which could be found sealing
effect for cracks with a width less than 300 μm. In addition,
8
Y Su et al
more bacterial dosage further enhances repairing perfor-
mance. After 14 d incubation, satisfactory closure results for
cracks with widths below 500 μm were achieved, and cracks
within 300 μm can be completely repaired. The visual
observation of the repair effect indicates that this bacteria
has profoundly improved the capability of crack-healing
under low temperature condition.
3.3.2. Improvement of water resistance. The change of water
resistance was characterized by capillary water absorption and
water permeability. As shown in figure 11, the un-cracked
cement-based specimen showed low water sorptivity, however,
Smart Mater. Struct. 28 (2019) 075041
Table 4. Permeability coefficients and RWP values of bacterial and control samples.
Group Initial permeability coefficient/(×10−5 m s−1)
Reference mortar 1.72
Bacterial mortar 1.80
Figure 10. Area repair rate of specimens with different cracking
width after healing 14 d.
Figure 11. The change of water absorption in different specimens.
the cracking has a huge damage to this capability. After
incubation for 14 d in water at 7 °C, both the references and the
specimens with microbe showed a reduction of water absorption.
While the rate of absorption was calculated based on ASTM C
1585, the obvious trend about the initial rate of absorption were:
Cracked-reference samples>Cracked-microbial samples>
Repaired-reference samples>Repaired-microbial samples>
un-cracked-reference samples>un-cracked-microbial samples.
In general, the cracked specimens with bacteria healing showed a
notable reduction of the amount of water absorbed and was
closing to un-cracked sample, which means the resistance to
water was rebuilt by the biological precipitates. The decrease of
9
Y Su et al
Permeability coefficient after healing/(×10−5 m s−1) RWP/(%)
1.54 10.47
0.29 83.89
water absorption in the references could be related to the
further hydration of unhydrated particles in the repairing
incubation. At the same time, it was found that the specimens
with microbe showed lower water absorption than the
references by comparing the water absorption under same
cracking condition, which might be attributed optimiz-
ation of the pore structure of the surface by microbial
mineralization [35].
The water permeability coefficient and RWP of samples
are shown in table 4. It could be found that after healing for
14 d in water at 7 °C, bacterial specimens showed a
significant decrease on water permeability coefficient, while
the coefficient of control samples after incubating for 14 d
was not reduced obviously. The RWP values of microbial
mortars and reference mortars were 83.89% and 10.47%,
respectively. Therefore, it could be concluded that microbial
induced calcium carbonate precipitation in cracks could be
resistant to water flow, thus improving the impermeability of
cracked bacterial samples. Although no healing effect could
be observed in control samples with 0.4–0.5 mm crack width,
as shown in figure 10, the impermeability of cracked control
samples was slightly improved, which could be inferred that
further hydration product might be generated inner of cracks.
3.4. Morphology and component of crack fillers
Figure 12 shows the SEM images of crack fillers at different
depths from the cracking surface. It could be observed that
crystallized product is densely packed at the depth of
0–800 μm from the cracking surface. After being analyzed by
energy spectrum, crystallized product was calcium carbonate,
the sizes of which were between 40 and 100 μm. In particular,
bacteria could also be seen on the product, as shown in
figures 12(e) and (f). As the depth deepened, the dis-
continuous calcium carbonate communities distributed
between 800 and 1600 μm from the cracking surface and
these calcium carbonates had a smaller particle size than those
from the shallow layers, which might be related to the lower
content of calcium and carbonate ion in deep of the crack than
the shallow. At a deeper distance from the cracking surface,
over than 1600 μm, the elemental composition of these
regions was consistent with the normal cement matrix ana-
lyzed by the energy spectrum.
4. Conclusions
This research revealed the bacteria Lysinibacillus sp could be
used as a bacterial based self-healing agent to reach the aim of
autogenous healing of concrete under the low-temperature
condition. The resistance to alkaline environments and the
Smart Mater. Struct. 28 (2019) 075041 Y Su et al

Figure 12. Micromorphology and energy spectrum analysis of precipitates along the depth of crack after 14 d incubation, (a) and
(b) distribution of precipitates in cracks; (c) in the depth direction of the crack, 0 to 800 μm from the crack surface; (d) magnification
observation of external deposits (0 to 400 μm from the crack surface); (e) magnification observation of internal deposits (400 to 800 μm from
the crack surface); (f) bacteria on the internal deposits; (g) and (h) magnification observation of internal deposits (800 to 1600 μm from the
crack surface); (i) and (j) magnification observation of cement matrix (over 1600 μm from the crack surface), ‘ ’ shows the magnified area,
‘ ’ shows the points for EDS analysis.

10
Smart Mater. Struct. 28 (2019) 075041 Y Su et al

Figure 12. (Continued.)

11
Smart Mater. Struct. 28 (2019) 075041
capability of inducing calcium carbonate were analyzed at
low temperature. Moreover, self-healing effect of bacterial
mortars at low temperature was investigated. The results
showed that the early age cracks with 0.3–0.4 mm width
could be completely repaired when the 1013 cells m−3 bacteria
were incorporated into mortars, and the crack fillers were
mainly calcium carbonate with different sizes. Furthermore,
calcium carbonate production induced by bacteria at low-
temperature condition could be attributed to the consumption
of small molecules of organic matter. In the future, as organic
compounds used in bacterial liquid might negatively affect
concrete properties [42], the components of the bacterial
liquid will be further optimized.
ORCID iDs
Yilin Su https://orcid.org/0000-0001-6280-2584
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