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Effect and Mechanism of Encapsulation-Based Spores on Self-

Healing Concrete at Different Curing Ages
Tianwen Zheng, Yilin Su, Xuan Zhang, Hengyi Zhou, and Chunxiang Qian*

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ABSTRACT: It has become an intelligent and environmental protection method to repair concrete cracks based on microbial-
induced calcium carbonate precipitation (MICP). However, due to the high-alkali environment in concrete, even the microbial
spores with strong alkali resistance find it difficult to survive for a long time, which affects the long-term self-healing effect of concrete
cracks. In this paper, low-alkali sulfo-aluminate cement (SC) was used as a carrier to encapsulate spores, and the effects of the spore
group and microbial group on the basic performances of concrete were studied. Then, the area repair ratio, water permeability, the
repair ratio of anti-chloride ion penetration, and ultrasonic velocity were used to evaluate the self-healing efficiency of cracks, and the
self-healing effects of two kinds of microbial self-healing agents on concrete cracks with different curing ages were further studied.
Moreover, the growth, enzyme activity, and microbial morphologies of spores with and without encapsulation immersed in the
simulated pore solution of cement-based materials at different times were studied to discuss the protective effect of the carrier on
spores. Compared with the reference group, the results showed that the addition of two microbial self-healing agents would slightly
affect the basic performances of concrete, but both were within the control range of concrete materials. For the early-age cracks, the
two kinds of microbial self-healing agents could achieve a good self-healing effect, but for the later-age cracks, the concrete cracks of
the microbial group could still be repaired well, while the self-healing effect of the spore group was greatly reduced. Moreover, the
white precipitates generated at the crack mouth were all calcite CaCO3. In addition, the self-healing mechanism of different microbial
self-healing agents on concrete cracks was discussed carefully. This study provides a new idea and method for the engineering
application of microbial self-healing concrete.
KEYWORDS: carrier, self-healing efficiency, curing ages, protective, self-healing concrete

1. INTRODUCTION of concrete structures to decrease and shorten their service

Due to the advantages of convenient use, low price, high
strength, and good durability, concrete has become the most
widely used building material in the world, which can be used in
modern civil engineering construction fields such as housing
engineering, bridge engineering, ports, and so on.1−3 However,
the brittleness, low tensile strength, and large shrinkage
deformation of concrete materials4,5 lead to the inevitable
production of different types of microcracks on the concrete
surface in the long-term service process, which greatly affects the
esthetics and functionality of buildings. At the same time, the
appearance of cracks will affect the waterproofing ability, bearing
capacity, and safety of concrete structures. Meanwhile, cracks
will accelerate the corrosion of reinforcement, alkali aggregate
reaction, and carbonation speed, which can cause the durability
life.6−8 Therefore, timely and efficient repair of concrete cracks
can not only improve the service life of concrete structures but
also save maintenance costs and reduce economic losses.
In recent years, with the development of microbial
mineralization technology and concrete materials, using the
microbial-induced calcium carbonate precipitation (MICP)
technology to repair concrete cracks has become a research
Received: September 10, 2020
Accepted: November 4, 2020

© XXXX American Chemical Society https://dx.doi.org/10.1021/acsami.0c16343

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hotspot at home and abroad. This method mainly uses the
mineralization of microorganisms to generate calcium carbonate
with good compatibility with cement-based materials, leading to
the cracks being effectively repaired and the self-healing purpose
of concrete cracks being achieved.9−12 At the same time,
microbial self-healing technology has the advantages of being
environmentally friendly, timely, and efficient and leads to
multiple repairs of cracks, providing a new idea and method for
the research of self-healing concrete and the realization of
intelligent civil engineering materials.
Gollapudi et al.13 used the principle of microbial mineraliza-
tion and demonstrated the feasibility of microorganisms for self-
healing of concrete cracks. Ramachandran et al.14 adopted the
MICP technology for the repair of concrete cracks, and the
results showed that bacteria could induce calcium carbonate
precipitation to repair cracks. In addition, the strength and
stiffness of concrete could be restored to a certain extent.
Meanwhile, from the application of MICP technology in the
passive repair of concrete cracks to the realization of self-healing
concrete, scholars have done a lot of research studies. However,
due to the influence of the high-alkali environment in concrete
and the increasingly dense pore structure, it is difficult for
microorganisms to survive in concrete for a long time, which
significantly affects the self-healing efficiency of concrete cracks
in the later stage. Jonkers et al.15 directly added a kind of
Alkalophilic bacillus into concrete, and the bacterial viability in
concrete was studied. The results showed that the survival
number of bacterial spores decreased by more than 90% within
one month. He pointed out that the survival time of
microorganisms could be prolonged by carrier technology.
Therefore, a variety of materials are used as carriers to
encapsulate microorganisms, such as polyurethane,3,16,17 hydro-
gel,18−20 microcapsule,21−23 and other organic polymer
materials. Moreover, graphite nanoplatelets (GNPs),3 iron
oxide nanosized particles (IONPs),24 silica gel,17,25 expanded
clay particles,5,26,27 expanded perlite (EP) particles,28,29
ceramsite,30 calcium sulfo-aluminate cement,31 recycled aggre-
gate,32 aerated concrete granules,33 lightweight aggregates
(LWAs),3,34,35 diatomaceous earth (DE),36 and many other
inorganic materials are used as carriers. These carriers can
protect microorganisms from the complex environment inside
the concrete to a certain extent, prolong the survival time of
microorganisms, and improve the self-healing efficiency of
concrete cracks in later ages.
However, the above studies only verify that the carrier
technology can effectively achieve the self-healing purpose of
concrete cracks, but the self-healing effect of concrete cracks at
different curing ages has been rarely studied. In addition, there
are few studies on the reasons and mechanisms of the different
self-healing effects of concrete cracks by microorganisms with
and without encapsulation. Therefore, it is urgent to carry out
research on the self-healing effect of cracks at different curing
ages. Meanwhile, the enzymatic activity, quantity, and
morphology changes of microorganisms at different curing
ages should be clarified, which can provide a theoretical basis
and experimental support for the application of microbial self-
healing concrete.
Generally, the microorganism that could induce mineraliza-
tion to generate calcium carbonate was adopted in this article,
and low-alkali sulfo-aluminate cement (SC) was used as the
carrier material. Furthermore, a kind of microbial self-healing
agent containing spores was prepared by the sugar-coated
method. The protective effect of the carrier on spores was
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studied in a simulated pore solution of cement-based materials.
Meanwhile, the experiment of spores without encapsulation was
set for comparison. The enzyme activity, bacterial number, and
morphology of spores with and without encapsulation under
different soaking times were tested to reflect the protective effect
of the carrier on spores. Moreover, the two kinds of microbial
self-healing agents were added into concrete, and the effects of
the spore group and microbial group on the workability of
concrete were studied. Then, the self-healing efficiencies of
cracks were characterized by the area repair ratio, water
permeability, the repair ratio of anti-chloride ion penetration,
and ultrasonic velocity. Moreover, the self-healing effects of
concrete cracks at different curing ages were compared. Finally,
the self-healing mechanism of concrete cracks with different
kinds of microbial self-healing agents was analyzed and
discussed in detail.
2. MATERIALS AND METHODS
2.1. Microorganism and Culture Medium. A kind of Bacillus
mucilaginous L3 producing carbonic anhydrase (CA) was used in this
study, which was purchased from the China Center of Industrial
Culture Collection (CICC).37−40 The bacteria were first cultured in a
liquid medium containing 10.0 g of sucrose, 3.0 g of yeast extract, and
2.5 g of potassium hydrogen phosphate per liter of distilled water (pH =
7.0). Then, the culture medium was packed in an Erlenmeyer flask and
autoclaved at 121 °C for 25 min. After inoculation, the bacteria were
incubated in a constant-temperature shaking incubator (HZQ-X160,
Huamei biological instrument, China) at 25 °C and 170 rpm for 96 h.
During the cultivation process, the bacterial growth and changes of pH
value in the medium solution were tested every 4 h, and the pH value
was determined by a pH meter (PHS-3C, LEICI, China). Then, 50 mL
of microbial vegetative cells (1% v/v) grown in the stationary phase was
taken to inoculate again in a sterilized 5 L small microbial fermentation
tank (MC-JGF-5L, Beijing Mancang Technology Co., Ltd., China)
containing a medium (the ingredients are shown in Table 1) for 64 h. In
Table 1. Composition of the Culture Medium for B.
Mucilaginous L3
nutrient substance mass (g) main function
sucrose 50.0 carbon sources
yeast extract 15.0 nitrogen sources
potassium hydrogen phosphate 12.5 inorganic salt
manganese sulfate 0.025 spore-transforming agent
deionized water 5000 water
addition, MnSO4·H2O was added in the culture medium to promote
the transformation of spores.31,41 Then, spray-drying was performed to
transform microbial liquid spores into powdery spores; the drying
temperature and peristaltic pump speed were 180 °C and 8 rpm in the
spray-drying process, respectively. Finally, the powdery spores obtained
were conserved in a refrigerator at 4 °C.
2.2. Testing the Number of Microbial Vegetative Cells,
Spores, and Morphology. In the process of microbial culture, the
optical density (OD) in the medium solution was measured to reflect
the bacterial growth, where OD600 is the optical density of microbial
vegetative cells at the wavelength of 600 nm. The higher the OD600
value was, the more cells there were. OD600 was tested by a
multifunctional enzyme labeling instrument (Varioskan LUX, Thermo
Fisher). In addition, the cell concentration and the number of powdery
spores per gram after spray-drying were determined by a flow cytometer
(FACS Calibur, Becton Dickinson). Moreover, the morphologies of
microbial spores and vegetative cells were observed by a field emission
scanning electron microscope (FE-SEM, FEI-Sirion, Holland). The
specific morphology observation steps were as follows. (1) Washing:
The microbial vegetative cells and spores were washed with deionized
water three times to remove the inorganic salts and then with 25%
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Table 2. Oxide Percentages (%) of the Used Cement

chemical compositions CaO SiO2 Al2O3 Fe2O3 SO3 MgO K2O P2O5
ordinary Portland cement 56.03 20.72 6.3 2.68 2.31 2.54 0.92
sulfo-aluminate cement 44.12 7.49 35.01 1.25 7.47 1.32 0.2 0.27

Figure 1. Schematic diagram of the synthesizing microbial self-healing agent.

Figure 2. Images of the prepared microbial self-healing agent.

ethanol three times to remove the organic substances. (2) Drying: The encapsulation (Figure 3) were immersed in the simulated pore solution
microbial vegetative cells and spores after washing were diluted 1000 of cement-based materials (as shown in Table 3). After soaking for 7,
times with deionized water after sterilization, dropped on a conductive
adhesive, and then placed into a vacuum constant-temperature drying
oven at 60 °C for 24 h before the test.
2.3. Preparation of Microbial Self-Healing Agent. Sulfo-
aluminate cement (SC) has the characteristics of early and high
strength, good water, frost, and corrosion resistance, and long-term
stability. In addition, the alkalinity of sulfo-aluminate cement pore
solution is about 10.0, so low-alkaline SC is used as the protective
material for microbial spores. Table 2 shows the chemical composition
of ordinary Portland cement (P·II 42.5) and sulfo-aluminate cement
(SC). The microbial self-healing agent encapsulated with spores was
prepared by the sugar-coated method. By controlling the rotation speed
of the sugar-coating machine, the deionized water containing the
culture medium (10.0 g/L sucrose, 3.0 g/L yeast extract, and 2.5 g/L
potassium hydrogen phosphate) was continuously sprayed to roll the
powdery spores into microspheres with the size of 1.18−2.36 mm,
which was the first sugar-coating. Moreover, low-alkaline SC was added,
and the second sugar-coating was carried out by spraying deionized
water continuously. Meanwhile, the ratio of water and sulfo-aluminate
cement for the second sugar-coating was controlled at about 0.1 (w/c).
Finally, the core−shell microbial self-healing agent with the particle size
of 3.35−4.0 mm was obtained. The schematic diagram of the Figure 3. Spores and spores with encapsulation immersed in the
preparation is shown in Figure 1. Moreover, the inner core was simulated pore solution of cement-based materials. (a) Spores and (b)
powdery spores, and the outer shell was low-alkaline SC as the spores with encapsulation.
protective material. The microbial self-healing agent prepared is shown
in Figure 2. In addition, the microbial self-healing agent was put into a
standard curing room (RH = 90 ± 5%; T = 20 ± 3 °C) for curing for 3 Table 3. Simulated Pore Solution of Cement-Based Materials
days to ensure that the microbial self-healing agent would not be broken
during the concrete preparation process. substance Ca(OH)2 NaOH KOH K2SO4 pH
2.4. Research on Protective Performance of the Carrier. To
study the protective effect of SC on spores, the spores with and without content (mol/L) 0.002 0.25 0.54 0.003 13.90

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Table 4. Mix Design of Concrete (in Mass)
cement fly ash mineral powder water water reducer
group (kg/m3) (kg/m3) (kg/m3) (kg/m3) (kg/m3)
reference 261 56 56 164
spores 261 56 56 164
microbial 261 56 56 164
Figure 4. Specific schematic diagram of crack creation and self-healing incubation.
14, 28, and 56 days, the spores with encapsulation were taken out and
crushed to obtain the spores inside the core−shell granules under
aseptic conditions. Then, an equal quantity of spores with and without
encapsulation was cultivated in the alkaline environment of cement-
based materials. The protection effect of the carrier on the spores was
reflected by testing the germination growth of spores, enzyme activity
change, and morphology of the microorganism under different soaking
times. Moreover, the growth of spores was represented by an OD600
value. The enzyme activity change could be tested by a colorimetric
method; for the specific test method, one could refer to previous
research,42 which was mainly based on the different catalytic efficiencies
of different enzyme activities. The enzyme activity was indirectly
reflected by the amount of p-nitrophenol. The more the p-nitrophenol
that was produced, the greater was the optical density, and the higher
was the carbonic anhydrase activity. Changes in the morphology of
microorganisms during the growth process were observed by a
fluorescent inverted microscope (DS-Fi3, Nikon, Japan). The micro-
scope was equipped with “NIS-Elments D” software connected to a
computer. In addition, the images were taken using phase-field
illumination and a 60× inverted objective.
2.5. Performance of Concrete. 2.5.1. Preparation of Concrete
Specimens. Concrete specimens with diameter of 100 mm and height
of 50 mm were prepared by mixing cementitious materials, sand, stone,
tap water, water reducer, calcium source, and microbial self-healing
agents. Meanwhile, concrete specimens with the size of 150 mm × 150
mm × 150 mm were prepared to test the compressive strength. The
mixing process of concrete was according to the Chinese standard GB/
T2419-2005, and the mix design is shown in Table 4. After curing for 24
h in a standard curing room (RH = 90 ± 5%, T = 20 ± 3 °C), specimens
were demolded and put back again for further curing.
2.5.2. Concrete Slump Test. The concrete slump mainly refers to the
plasticizing performance and pumpability of concrete and reflects the
flow characteristics of fresh concrete. The workability represented
whether the concrete is easy to operate and construct, including water
retention, fluidity, and cohesiveness of concrete. It is an important
condition to ensure normal construction of concrete, which can be
determined by the slump test. The workability of concrete has an
important influence on the strength, compatibility, and durability of
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sand stone calcium nitrate microbial self-healing
(kg/m3) (kg/m3) (kg/m3) agent (kg/m3)
5.22 776 1072
5.22 776 1072 5.0 2.5
5.22 768.5 1072 5.0 7.5
concrete. In addition, after a period of time, concrete will experience
slump loss, which is the concrete slump loss over time. So a 1 h slump
loss over time can also reflect the quality of fresh concrete. The concrete
mixture slump and 1 h slump loss over time are tested by the Chinese
national standard GB/T50080-2016 “Standard for test method of
performance on ordinary fresh concrete”.
2.5.3. Concrete Apparent Density. Density refers to the mass of
matter per unit volume. Generally, the matter is continuous without
gaps. Therefore, density is equal to the ratio between its mass and its
volume, which is the density of the material. However, for concrete
materials, pores are generated due to the difference in particle size and
shape of various raw materials. In this case, the density obtained by
dividing the mass of the pile by the unit volume is not the same as the
density of the material itself, which is called the apparent density. As an
important part of the concrete design process, the apparent density of
concrete can be classified according to the different apparent densities.
If the apparent density of concrete is more than or equal to 2800 kg/m3,
it is heavy concrete; if the apparent density of concrete is between 1950
and 2800 kg/m3, it is ordinary concrete; and if the apparent density of
concrete is less than 1950 kg/m3, it is light concrete.43 The test method
to determine the apparent density of a concrete mixture is carried out in
accordance with the Chinese national standard GB/T50080-2016.
2.5.4. Setting Time Test. The setting time of concrete can be divided
into the initial setting time and the final setting time. The initial setting
time is the time required from the mixing of cementitious materials and
aggregates with water until the concrete cement mortar body begins to
lose plasticity. However, the final setting time starts from the
cementitious material and aggregate mixing with water until the
concrete cement mortar completely loses its plasticity and begins to
produce strength. The setting time of concrete is greatly significant in
construction. To ensure that the concrete paste has enough time to
remain flowable during the engineering construction, the initial setting
time should not be too short. At the same time, for the concrete that has
formed the shape of the engineering structure to gain strength as soon
as possible, so that it can bear the load, the final setting time should not
be too long. The test method for the setting time of concrete should be
in accordance with the Chinese national standard GB/T50080-2016.
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2.5.5. Concrete Compressive Strength. The compressive strength of
concrete, as one of the main indicators to judge the quality of concrete,
is of great significance to the classification of concrete strength grades.
To test the mechanical properties of concrete and the development
over time, the compressive strength test of concrete with different mix
designs is conducted. The test ages are 3, 7, 14, and 28 days. The
concrete compressive strength test is carried out according to the
Chinese national standard GB/T 50081-2019 “Standard for the test
methods of concrete physical and mechanical properties”. In addition,
the test equipment is a TYA-2000 electrohydraulic pressure testing
machine (Wuxi Xinluda Instrument Equipment Co., Ltd, China).
2.6. Crack Creation and Self-Healing Incubation Conditions.
For the cylindrical concrete specimen, to obtain cracks that were close
to the real situation, the cylindrical specimen was made by splitting. The
specific creation steps were as follows. (1) The surface of the cylindrical
concrete specimen was cleaned and dried and then two circles of
adhesive tape were wrapped around the specimen to prevent it from
being completely damaged during the splitting process. (2) The
cylindrical concrete specimen wrapped with adhesive tape was placed
under the electrohydraulic pressure testing machine, and the loading
speed was maintained at 0.1−0.2 kN/s. Moreover, the specimen was
observed carefully, and when visible microcracks appeared on the
surface, loading was immediately stopped and the specimen was
removed. Finally, cracks with the width of 0.3−0.5 mm could be
obtained. Then, the concrete specimens with cracks underwent a dry−
wet cycle healing. In each dry−wet cycle, the specimens were placed in
water for 12 h and were then exposed to air for 12 h. In addition, the
healing temperature was maintained at 25 ± 2 °C, and the self-healing
efficiency of specimens was tested after 28 days of healing time. The
specific schematic diagram of crack creation and self-healing incubation
is shown in Figure 4.
2.7. Characterization Methods for Self-Healing Efficiency.
2.7.1. Image Visualization and Quantification. For concrete cracks, it
was the most intuitive method to obtain and analyze the crack images
directly. First of all, a high-power camera (D780, Nikon, Japan) was
used to vertically photograph the images of the specimen before and
after repair. The specific schematic diagram is shown in Figure 5. Then,
Figure 5. Schematic diagram of the image device.
“Image-J” software was used to binarize the crack images. The pixel
value could be determined by analyzing the gray value of the crack area.
The threshold segmentation of the crack area was achieved by setting
the gray threshold to 115 so as to obtain the images before and after
repair. Finally, the repair area of the crack was selected for pixel
statistics, and the area repair ratio could be calculated according to the
proportion of the crack area pixel to the initial crack pixels after 28 days
of healing time. The calculation equation is as follows:44−46
A 0 − A 28
area repair ratio = × 100%
A0 (1)
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where A0 is the number of crack area pixel dots before healing and A28 is
the number of crack area pixel dots after 28 days of healing time.
2.7.2. Permeability. The impermeability of concrete refers to the
amount of water permeated through the specimen per unit time. When
there are cracks in concrete, water can quickly pass through the
specimens through the cracks. If the cracks are repaired, it will take
more time for water per unit volume to penetrate through the
specimens. Therefore, water permeability can be used to characterize
the changes in impermeability before and after repair. The test method
for water permeability resistance was based on a previous report.40
During the test, the concrete specimens were connected to a poly(vinyl
chloride) (PVC) pipe with dimension φ 100 mm × 1200 mm, and the
water inside the PVC pipe was maintained at a constant height of 1000
mm (Figure 6). Before the test, the joint between the specimen and the
Figure 6. Schematic diagram of the water permeability test.
PVC pipe was sealed tightly with epoxy resin to avoid leakage. The
experiment lasted for 5 min, and the quantity of water that passed
through the specimens was measured by an electronic scale (MAX-
C6002, China). The test was conducted twice, before and after 28 days
of healing time. Then, the water permeability could be calculated by
Darcy’s law shown in eq 2
k = Q ·L /A ·Δh (2)
where k is the water permeability (m/s), Q is the amount of water flow
(m3/s), L is the height of specimens (m), A is the area of the section
(m2), and Δh is the head difference (m).In addition, the water
permeability would vary with the crack width. However, for the same
specimen, the crack width was the same, and the relative water
permeability could be better used to express the improvement of the
impermeability performance of the specimen before and after repair.
The relative water permeability could be calculated by the following eq
3
α = k 28/k 0 (3)
where α is the relative water permeability, k0 is the water permeability of
the specimen before healing (m/s), and k28 is the water permeability of
the specimen after 28 days of healing time. The larger the relative water
permeability was, the worse was the self-healing effect of concrete
cracks.
2.7.3. Rapid Chloride Permeability Test (RCPT). When the concrete
cracks are repaired, the material transport will be reduced to a certain
extent. Therefore, the rapid chloride permeability test (RCPT) can be
used to characterize the self-healing effect of cracks.7,47 The rapid
chloride permeability method was based on electric flux. Under the
action of DC voltage, the chloride ion in the solution could move to the
positive direction through the concrete specimen. By measuring the
electricity passing through the concrete specimen within a certain
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Figure 7. Rapid chloride permeability test device: (a) schematic diagram and (b) physical picture.
Figure 8. Schematic diagram of ultrasonic testing crack repair.
period of time, the resistance of the concrete specimen to chloride ion
permeability before and after repair could be reflected. The specific test
method should be implemented in accordance with the Chinese
national standard GB/T 50082-2009 “Standard for test methods of
long-term performance and durability of ordinary concrete”. A concrete
electric flux meter (SDL-6, Shanghai Torrent Equipment Co., Ltd.,
China) was used for the test instrument. The specific schematic
diagram of the rapid chloride permeability test is shown in Figure 7.
Before the test, the concrete specimens should be vacuum-saturated for
24 h, and then the specimens were installed in the test tank, and the
tightness between the specimens and the test tank was checked. Then,
3.0% NaCl solution and 0.3 mol/L NaOH solution were sequentially
injected into the test tank on both sides of the specimens. Moreover, the
copper mesh in the test tank injected with NaCl solution should be
connected with the negative pole of the power supply, and the copper
mesh in the test tank injected with NaOH solution should be connected
with the positive pole of the power supply. Then, the power supply was
switched on, a constant DC voltage of 60 ± 0.1 V was applied, and the
initial current reading I0 was recorded. During the test, the current was
recorded every 15 min for 2 h. According to the curve of the current
changing with the conduction time, the electric flux of the specimen for
2 h was calculated (eq 4) and used to evaluate the anti-chloride
permeability ability of concrete specimens before and after repair.
Q = 900 (I0 + 2I15 + 2I30 + 2I45 + 2It + ... + 2I90 + 2I105
+ 2I120) (4)
where Q is the total electric quantity passing through the specimen (C),
I0 is the initial current (A), and It is the current in time (min) t (A).
According to eq 5, the repair ratio of the anti-chloride ion penetration of
concrete specimens before and after repair was calculated.
Q 0 − Q 28
η= × 100%
Q0 (5)
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where η is the repair ratio of anti-chloride ion penetration, Q0 is the
electric flux 2 h before healing (C), and Q28 is the electric flux 2 h after
28 days of healing time (C).
2.7.4. Ultrasonic Velocity. Ultrasonics detected the self-healing
effect of concrete cracks, which mainly used the diffraction
phenomenon of sound waves. When using ultrasonics to carry out
the horizontal measurement method, a group of planar transducers
(transmitting transducer and receiving transducer) was placed on both
sides of the crack. When cracks appeared in the concrete specimen, part
of the sound wave emitted by the transmitting transducer was reflected,
and the other part of the sound wave could only pass through the crack
and propagate to the receiving transducer, which increased the
propagation distance and caused changes in the transmitting time.
However, when the crack was filled with mineralized products, a
bridging effect was generated on both sides of the crack, and the path of
the diffraction wave received by the receiving transducer would change,
which could cause the ultrasonic wave velocity obtained to be different.
It can be seen from Figure 8 that when the concrete crack was filled
with mineralized products, the coupling degree of the two crack surfaces
was different due to the different positions of the mineralized products.
Then, during ultrasonic testing, the wave velocity received by the
receiving transducer would be different. Therefore, the self-healing
effect of cracks could be reflected according to the wave velocity. Since
the propagation speed of the sound wave in the solid was larger than
that in the air, when the amount of mineralization was more, the
measured wave velocity would be greater, and the self-healing effect of
cracks along the depth direction would be better.
2.8. Characterization of Precipitates at the Crack Mouth of
Specimens. After 28 days of healing time, the morphologies and
element compositions of precipitates produced at the crack mouth of
specimens were collected and analyzed by a field emission scanning
electron microscope (FE-SEM, FEI-Sirion, Holland) equipped with an
energy-dispersive spectrometer (EDS), and the polymorphs of
precipitates were examined by X-ray diffraction (XRD, D8-Discover,
Germany) using a parabolic filter Cu Kα (λ = 1.54056 Å), employing a
step size of 0.02°/s with 2θ ranging from 10 to 80°.
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3. RESULTS AND DISCUSSION
3.1. Preparation of Microbial Spores. Figure 9 shows the
bacterial growth curve and the changes in the pH value in the
Figure 9. Bacterial growth curve and pH changes in the cultivation
process.
medium solution during the microbial culture process. It could
be seen that bacteria were in a lag phase within 0−12 h; bacteria
grew slowly and the growth curve was flat and stable. After 12 h,
bacteria proliferated rapidly, the number of bacterial vegetative
cells increased in geometric progression, and bacteria had
entered into the logarithmic phase. When the incubation time
was increased to 44 h, the optical density (OD600) in the culture
medium changed slowly, indicating that the bacteria had entered
into a stationary phase. In this phase, the bacterial reproduction
rate decreased and the relative number of dead bacteria began to
increase. Meanwhile, the number of newly added bacteria and
dead bacteria reached a dynamic balance. After 76 h, due to the
consumption of nutrients and the accumulation of harmful
metabolites, the number of bacteria proliferation decreased,
while the number of dead bacteria increased significantly, and
the bacteria entered the decline phase. In addition, in the process
of bacterial growth, the pH value in the medium solution
decreased first and then increased, and the lowest pH value
could reach 4.8 at 40 h. The reason might be because the bacteria
would produce CO2 in the metabolism process and then dissolve
in the culture medium. Moreover, the carbonic anhydrase (CA)
produced by the bacteria would accelerate the hydration of CO2
and then form HCO3− in the solution.48 Therefore, in the early
process of bacterial reproduction, the pH value in the medium
showed weak acidity, resulting in the decline of the pH value.
Moreover, bacteria could decompose yeast extract to produce
Figure 10. Flowchart of powdery spore preparation.
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ammonia in the metabolism process,49 which could dissolve in
the medium and form OH−. In addition, HCO3− and CO32−
could be generated in the medium under the catalytic effect of
carbonic anhydrase (CA). Thus, it eventually caused the rise of
the pH value in the medium.
To obtain alkali-resistant microbial spores, 50 mL of microbial
vegetative cells with the concentration of 109 cells/mL measured
by a flow cytometer in the stationary phase was also inoculated
in a 5 L small-scale microbial fermentation tank. After
incubation for 64 h, the microbial liquid spores were spray-
dried; at the same time, the peristaltic pump speed was
maintained at 8 rpm and the drying temperature was 180 °C. In
addition, the liquid spores should be swayed every once in a
while to avoid the phenomenon of uneven spraying. The spray-
drying process lasted for 8 h, and microbial powdery spores
could be obtained. The specific flowchart of the powdery spore
preparation is shown in Figure 10.
Figure 11 shows the optical microscope (OM) and SEM
images of the microbial vegetative cells and powdery spores. It
could be seen that the microbial vegetative cells were short rod-
shaped, with a length of about 2.0−3.0 μm, while the spores were
spherical with the size of about 0.5 μm. The number of powdery
spores per gram was measured by a flow cytometer. After
dilution by 1000 times, thiazole orange and propidium iodide
were used as coloring agents. Finally, the number of microbial
powdery spores was calculated as 1.0 × 1010 CFU/g.
3.2. Concrete Performance. 3.2.1. Slump Test. The results
of slump and 1 h slump loss over time of different kinds of fresh
concrete are shown in Figure 12. It could be seen that the slump
and 1 h slump loss over time of different concretes were,
respectively, 140−170 and 20−35 mm, which meets the basic
design requirements of concrete, indicating that the workability
of the spore group and microbial group fulfilled the require-
ments. However, compared with the reference group, the slumps
of the spore group and microbial group increased. However, the
1 h slump loss over time of the spore group was slightly lower
than that of the reference group, and the 1 h slump loss over time
of the microbial group increased slightly. It could be attributed
to the fact that the powdery spores had a slight air-entraining
effect, which increased the fluidity and workability of concrete
and then increased the slump while decreasing the 1 h slump loss
over time of fresh concrete. For the microbial group, the
addition of a microbial self-healing agent to replace part of the
fine aggregate might increase the sand ratio of concrete, and the
specific surface area of the fine aggregate also increased, which
could increase the friction between the particles, and then
decreased the fluidity and workability of concrete. Finally, the
slump and 1 h slump loss over time would be increased.
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Figure 11. OM and SEM micrographs of B. mucilaginous L3. (a) Vegetative cells, (b) spores.

Figure 12. Concrete slump test. (a) Slump and (b) 1 h slump loss over time.

3.2.2. Apparent Density of Fresh Concrete. Figure 13 shows

the apparent density of different kinds of fresh concrete. It could
be seen that the apparent density decreased slightly with the
addition of microbial self-healing agents. However, the apparent
density of the spore group was the lowest, which might be due to
the air-entraining effect of spores. Compared with the reference
group, the apparent density was lower; the reason could be
attributed to the fact that the specific gravity of the microbial
self-healing agent (2.40 g/cm3) was lower than that of the fine
aggregate (2.70 g/cm3).
3.2.3. Setting Time. The setting times of different kinds of
fresh concrete are shown in Figure 14. It can be seen that
compared with the reference group, the initial setting time and
final setting time of the spore group and the microbial group
were reduced. While the setting time of the microbial group was
the smallest, the initial setting time and final setting time were
235 and 490 min, respectively. After adding different kinds of Figure 13. Concrete apparent density measured.
microbial self-healing agents, the main reason for the reduction
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Figure 14. Concrete setting time.
of setting time might be the early strength effect of calcium
nitrate, which could accelerate cement hydration. Compared
with the microbial group, the spores without encapsulation
belonged to organic substances, which would have adverse
effects on the setting time of concrete. Therefore, the setting
time of concrete would be reduced to a certain extent after the
spores were encapsulated with sulfo-aluminate cement.
However, the setting time of the spore group and microbial
group conformed to the relevant provisions, which would not
affect the performance and application of concrete.
3.2.4. Compressive Strength. Figure 15 shows the
compressive strength of different kinds of concrete at different
Figure 15. Concrete compressive strength at different curing ages.
curing ages. Compared with the reference group, it could be seen
that the compressive strength of the spore group at different
curing ages was improved, which was consistent with the
previous reports.44,50−52 This might be due to the fact that
calcium nitrate had the effect of early strength, and microbial
spores could be filled in the pores of concrete to improve the
compactness of the concrete matrix, which caused the
improvement of compressive strength. However, for the
microbial group, the early-age compressive strength of concrete
was slightly higher than that of the reference group (3, 7 days),
but with the increase of curing age (14, 28 days), the later-age
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compressive strength of concrete was slightly lower than that of
the reference group, which was consistent with the refer-
ences.44,53 The reasons could be because the early strength of
the carrier was higher, and then with the increase of curing age,
the grain strength of the microbial self-healing agent was
insufficient to meet the development of the later-age strength of
concrete, finally causing the decrease of the later-age
compressive strength of concrete.
3.3. Self-Healing Efficiency of Concrete Cracks.
3.3.1. Area Repair Ratio. Figure 16 shows the images of
concrete cracks with the width of 0.3−0.5 mm at different curing
ages (7, 14, 28, and 56 days) before and after 28 days of healing
time. It could be seen that the cracks of the reference group were
not repaired after 28 days of healing time, and only a small
amount of white precipitates was filled in the cracks, which
might be caused by the carbonation reaction of CO2 in the crack
zone. In addition, the area repair ratio of cracks at different
curing ages was less than 10% (Figure 17). However, for the
spore group, with the increase of curing age, the self-healing
effect of cracks gradually decreased, and the production of white
precipitates at the crack mouth also gradually decreased, which
was consistent with the research of Jonkers.15 The main reason
might be that the spores without encapsulation were affected by
the high-alkali environment of concrete and the increasingly
dense pore structure of concrete, and with the increase of curing
age, the spores would be dead. Moreover, the longer the curing
age was, the lower the survival rate of spores would be.
Therefore, for the early-age cracks (7, 14 days), spores could
survive and induce the formation of mineralized products to
repair the cracks, and the area repair ratio could be as high as
93%. However, for the later-age cracks (28, 56 days), spores
gradually died, so the self-healing effect of cracks was greatly
reduced, and the area repair ratio was reduced to less than 30%.
For the microbial group, the cracks were well repaired with the
increase of curing age, and a large number of white precipitates
were generated into the cracks. The area repair ratio of cracks at
all curing ages was as high as 90%. In particular, because the
cracks were created by the splitting method, the width of all
cracks could not be guaranteed to be the same, but the width of
all cracks was 0.3−0.5 mm, and the self-healing effect of the
crack had little relationship with the specific crack pattern, which
was strongly influenced by microorganisms. Thus, the results
showed that using low-alkaline sulfo-aluminate cement to
encapsulate spores could well protect spores, prolong the
survival time of spores in concrete, and improve the self-healing
effect of cracks in the later age.
3.3.2. Water Permeability. The water permeability of
concrete specimens before and after 28 days of healing time
calculated according to Darcy’s law is shown in Table 5. It could
be seen that after 28 days of healing time, the water permeability
of specimens had been reduced to a certain extent. However, the
water permeability of the reference group at different curing
times had decreased less, and the relative water permeability
could reach more than 75%. The main reason could be
attributed to the secondary hydration of unhydrated cement
particles.46,54 For the spore group, the water permeability of the
concrete specimen with the early-age cracks repaired decreased
by about 2 orders of magnitude. However, with the increase of
curing age, the self-healing effect of cracks decreased greatly, and
the relative water permeability increased significantly, which
indicated that the self-healing effect of cracks was seriously
reduced, and the results were consistent with that of the area
repair ratio (Figure 17). The main reason should be assigned to
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Figure 16. Images of cracks with different curing ages after 28 days of healing time. (a) Reference, (b) spores, and (c) microbial.

the fact that the survival rate of spores decreased with the
increase of curing age, resulting in the poor self-healing effect of
cracks in the later age. However, for the microbial group, the
curing age had little effect on water permeability, and the relative
water permeability was less than 5%. This might be because the
surface of cracks was filled with white precipitates (Figure 16),
which effectively blocked the pore structure of the cracks. Thus,
the impermeability of the specimens after repair could be greatly
improved.
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3.3.3. Repair Ratio of Anti-Chloride Ion Penetration. The
rapid chloride permeability test (RCPT) measures the amount
of electric flux passing through the specimen before and after
repair per unit time, which represents the resistance ability of
concrete to chloride ion erosion. Figure 18 shows the current
changes of concrete cracks with different curing ages before and
after 28 days of healing time in the process of electric flux
measurement. It can be seen that the current increased
significantly when the cracks were generated and the current
decreased to a certain extent after the cracks were repaired. For
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3.3.4. Ultrasonic Velocity. The better the self-healing effect of

concrete cracks is, the higher the ultrasonic velocity will be.
Therefore, the ultrasonic detection method can indirectly reflect
the self-healing efficiency of concrete cracks. Figure 20 shows
the results of the ultrasonic velocity of concrete cracks with
different curing ages before and after 28 days of healing time. It
could be seen that the ultrasonic velocity of the reference group
before and after 28 days of healing time had little change, and the
ultrasonic velocity of cracks after repair was slightly higher than
that of cracks before repair, which might be the secondary
hydration of unhydrated cement particles. For the spore group,
the ultrasonic velocity of early-age cracks after repair was much
higher than that of cracks before repair, and then with the
increase of curing age, the ultrasonic velocity of specimens after
repair gradually decreased. In addition, when the curing age was
56 days, the ultrasonic velocity after repair was only 1.3 km/s,
which was far lower than the ultrasonic velocity of early-age
cracks after repair. However, for the microbial group, because
the surfaces of cracks at different curing ages were well repaired,
the ultrasonic velocity of the specimens at different curing ages
Figure 17. Area repair ratio of concrete cracks at different curing ages
after 28 days of healing time. after repair had been significantly improved, which could reach
more than 2.7 km/s. These results showed that the spores with
Table 5. Water Permeability of Specimens at Different encapsulation could also produce healing products to repair
Curing Ages after 28 Days of Healing Time concrete cracks at different curing ages, which was consistent
with the results of the area repair ratio, water permeability, and
water water the repair ratio of anti-chloride ion penetration.
cracking permeability permeability relative water
age before healing after healing permeability 3.4. Morphology and Component of Precipitates at
group (days) (m/s) (m/s) (%) the Crack Mouth. Figure 21 shows the SEM micrographs of
reference 7 1.28 × 10−4 9.93 × 10−5 77.6 white precipitates at the crack mouth of different concrete
14 1.03 × 10−4 8.26 × 10−5 80.2 specimens with different curing ages. For the reference group, it
28 1.16 × 10−4 9.23 × 10−5 79.6 could be seen that the white precipitates generated at the crack
56 9.84 × 10−5 8.09 × 10−5 82.2 mouth at different curing ages were composed of some irregular
spores 7 1.32 × 10−4 2.21 × 10−6 1.67 massive particles, and they were relatively dispersed. For the
14 9.51 × 10−5 4.83 × 10−6 5.08 spore group, the white precipitates at the crack mouth of early-
28 9.37 × 10−5 2.19 × 10−5 23.4 age cracks (7 and 14 days) were irregular large particles formed
56 1.31 × 10−4 8.69 × 10−5 71.8 by the close accumulation of small flake and spherical particles.
microbial 7 1.37 × 10−4 1.05 × 10−6 0.77 However, with the increase of curing age, the white precipitates
14 1.18 × 10−4 9.77 × 10−7 0.83 at the crack mouth were mainly composed of particles with an
28 1.52 × 10−4 2.32 × 10−6 1.53 uneven particle size and relatively dispersed, which were similar
56 1.40 × 10−4 5.92 × 10−6 4.23 to those of the reference group. For the microbial group, the
white precipitates generated at the crack mouth of concrete at
the reference group, the current decreased slightly after 28 days any curing age were irregular and massive particles formed by
of healing time, and the changes were not obvious. The repair agglomeration of some flake or spherical particles, and the
ratio of anti-chloride ion penetration was less than 5% (Figure accumulation degree was dense. The reason for the different
19). However, for the spore group, when the curing age was 7 morphologies of the white precipitates at the crack mouth was
and 14 days, the repair ratios of anti-chloride ion penetration because some organic substances generated during the
were 45.7 and 37.1%, respectively. Moreover, with the increase metabolism process of bacteria adhered the mineralized product
of curing age, the current value increased again, and the repair particles together, and then the densely agglomerated large
ratio of anti-chloride ion penetration decreased to less than 5%. particles could be formed.55−57 In addition, since most spores
The results showed that for the early-age cracks (7 and 14 days), died in the later-age cracks of the spore group (28 and 56 days),
the repair ratio of anti-chloride ion penetration could reach more the mineralization reaction could not produce mineralized
products. Therefore, the morphology of white precipitates
than 37%. However, for the later-age cracks, the self-healing
produced at the crack mouth was similar to that of the reference
effect of cracks was very poor, and the repair ratio of anti-
group. The XRD spectra of white precipitates generated at the
chloride ion penetration of the spore group differed little from crack mouth are shown in Figure 22. It could be seen that the
that of the reference group. Moreover, for the microbial group, precipitates of different kinds of concrete at different curing ages
whether it was early-age cracks or later-age cracks, the current were calcite CaCO3. Compared with the standard PDF #05-
decreased significantly after 28 days of healing time, and the 0586 card, the diffraction angle of 2θ = 29.40° mainly
repair ratio of anti-chloride ion penetration was more than 44%. corresponded to the (104) crystal plane of calcite. The intensity
In general, using carrier technology to encapsulate spores could of other impurity diffraction peaks was negligible except for the
improve the repair ratio of anti-chloride ion penetration at calcite diffraction peaks, demonstrating that the purity of
different curing ages. precipitates was high. In addition, the diffraction peaks were
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Figure 18. Current of concrete specimens before and after 28 days of healing time in the electric flux test. (a) Reference, (b) spores, and (c) microbial.
Figure 19. Repair ratio of anti-chloride ion permeation for concrete
cracks at different curing ages after 28 days of healing time.
sharp, which indicated that the precipitates had good
crystallinity.
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3.5. Protection Effect of Carrier on Spores. To study the
difference in the self-healing effects of the spore group and
microbial group on concrete cracks at different curing ages, the
spores with and without encapsulation soaked in the simulated
pore solution of cement-based materials (Figure 3) were
cultivated for 120 h under an alkali environment (pH =
11.0).46,58 Then, the growth of spores in the stationary phase (72
h) and the changes of enzyme activity under different soaking
times were tested to reflect the protection of the carrier on the
spores. In addition, the morphological changes of spore
germination and growth during the 120 h culture period were
studied.59 Figure 23 shows the growth and enzyme activity of
spores with and without encapsulation immersed in the
simulated pore solution of cement-based materials for different
times after culturing for 72 h. It could be seen that for the spores
without encapsulation, with the soaking time increased, the
growth of spores and enzyme activity would decrease. When the
soaking time reached 28 and 56 days, the spores hardly
germinated and grew, and there was no enzyme activity,
indicating that almost all spores died, which was consistent with
the study of Jonkers.15 However, for the spores with
encapsulation, the microbial growth and enzyme activity were
better when the immersion time was increased. The OD600 value
and the enzyme activity value of different immersion times did
not change much. The reason could be attributed to the
protective effect of the carrier, which prevented the spores from
being affected by the high-alkali environment and the
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Figure 20. Ultrasonic velocity of concrete cracks at different curing ages before and after 28 days of healing time. (a) Reference, (b) spores, and (c)
microbial.
continuously dense pore structure in concrete. Therefore, the
spores could grow well and have a high enzyme activity.
Figure 24 shows the evolution of microbial morphologies in
the process of spore growth after the spores with and without
encapsulation were soaked in the simulated pore solution of
cement-based materials for 7 and 28 days. It could be seen that
when the spores without encapsulation were soaked in the
simulated pore solution of cement-based materials for 7 days,
with the increase of culture time (Figure 24a), the spores
gradually germinated and grew. After 24 h, most of the spores
grew into microbial vegetative cells; when the culture time
reached 48 h, all of the spores were transformed into vegetative
cells. However, due to the consumption of nutrients, with the
culture time increasing to 96 h, some vegetative cells were
transformed into spores, and when the culture time reached 120
h, all of the vegetative cells were again transformed into spores.60
Meanwhile, when the spores were soaked for 28 days, it could be
seen from Figure 24b that with the increase of culture time, the
spores failed to germinate into vegetative cells, indicating that
the spores had died and lost the ability to revive. However, for
the spores encapsulated with sulfo-aluminate cement, whether
immersed in the simulated cement-based material pore solution
for 7 or 28 days, spores germinated into vegetative cells and grew
well. With the increase of culture time, the spores underwent the
microbial evolution process of “spores−vegetative cells−
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spores”. The results showed that spores with encapsulation
still had the potential activity; when the suitable growth
conditions were met, these dormant spores could still germinate
and grow into vegetative cells. However, when the nutrients in
the culture medium were consumed, the microbial vegetative
cells again transformed into spores with potential activity, which
showed that adding a certain amount of nutrients in concrete not
only could increase the germination and growth of spores but
also could improve the self-healing effect of concrete
cracks.46,61−64 However, the addition of nutrients could affect
the mechanical properties of concrete to a certain extent.65
Therefore, adding a certain amount of nutrients in the
preparation process of the microbial self-healing agent could
be considered, which still needs to be further studied.
3.6. Self-Healing Mechanism of Concrete Cracks.
Microbial self-healing concrete is to add the required microbial
self-healing agent into the concrete during the mixing process,
and then the self-healing purpose of concrete cracks through the
mineralization of microorganisms. In this study, the microbial
self-healing agents could be divided into unloaded spores and
calcium sources and loaded spores and calcium sources. When
cracks occurred, for the early-age cracks (7 and 14 days) of the
spore group, water and air entered the crack, which could reduce
the pH value of the crack zone.46,58 Meanwhile, the spores into
the concrete would germinate and grow into vegetative cells. In
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Figure 21. SEM micrographs of precipitates at the crack mouth of specimens at different curing ages after 28 days of healing time. (a−d) is the curing
age of the reference group at 7, 14, 28, and 56 days, respectively. (e−h) is the curing age of the spore group at 7, 14, 28, and 56 days, respectively. (i−l) is
the curing age of the microbial group at 7, 14, 28, and 56 days, respectively.
Figure 22. XRD spectra of precipitates at the crack mouth of specimens
after 28 days of healing time.
addition, during the rapid reproduction of vegetative cells, an
extracellular carbonic anhydrase (CA) could be produced,
which could greatly improve the hydration reaction of CO2 in
air, and the hydration reaction rate constant was about 107 times
than that of nature,66−68 so as to realize the rapid dissolution of
CO2 in the concrete crack zone.
Due to the large amount of OH− existing in the concrete crack
zone, CO2 could be converted into HCO3− and CO32− under
the catalysis of CA, which provided a necessary condition for the
formation of calcium carbonate. In addition, there was abundant
Ca2+ in the concrete crack zone, and the microbial vegetative
cells were negatively charged, which could attract positively
charged Ca2+ to accumulate on their surfaces, and provided
nucleation sites for the deposition of mineralization products.
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Under the action of CA, CO32− continued to be generated in the
crack zone; meanwhile, in the presence of Ca2+, white CaCO3
precipitates with good compatibility with the concrete matrix
formed at the crack mouth, which were filled in the cracks and
achieved the self-healing purpose of concrete cracks.
For the spores with encapsulation, when concrete cracks at
different curing ages were generated, the spherical microbial self-
healing agent would break and then release the spores. Due to
the better protection effect of the carrier on the spores, the
spores in the dormant state would germinate, grow, and
reproduce in the crack zone, and then CA could be produced in
the metabolism process, so as to reverse the mineralization
reaction to repair concrete cracks, which was similar to the above
mineralization mechanism.
Whether it was the spore group or the microbial group, in the
self-healing process of concrete cracks, the pH value of the
solution in the crack zone would gradually rise with the increase
of crack depth.69 The deeper the crack was, the higher the pH
value of solution in the crack zone would be, which was
unfavorable for the germination and growth of spores.
Meanwhile, when cracks were formed, the spores would
germinate and grow into vegetative cells on the surface solution
of the crack zone, and Ca2+ in the concrete matrix would
continue to diffuse to the crack zone,38,39 which provided a
necessary condition for the formation of CaCO3. Furthermore,
the vegetative cells could act as nucleation sites and promote the
formation of CaCO3 precipitates. With the increase of healing
time, the crack surface was fully filled with CaCO3, and it was
difficult for water and air to enter the crack zone. Meanwhile, the
depletion of air in the smaller crack widths did not facilitate the
respiration of active spores and CaCO3 precipitates were not
conducive to generate. Thus, the healing process of concrete
cracks was from the crack surface to the inside, and the deeper
the crack was, the less CaCO3 precipitates were generated.
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Figure 23. Growth and enzyme activities of microorganisms immersed in the simulated pore solution of cement-based materials for different times. (a)
Spores and (b) spores with encapsulation.

Figure 24. Optical microscope images of spores without and with encapsulation immersed in the simulated pore solution of cement-based materials at
different incubation times. (a) Spores immersed for 7 days, (b) spores immersed for 28 days, (c) spores with encapsulation immersed for 7 days, (d)
and spores with encapsulation immersed for 28 days.

The self-healing schematic diagram of concrete cracks is
shown in Figure 25, and the specific steps of mineralization
product deposition are as follows.42,70,71
(1) Under the catalysis of CA produced by the micro-
organism, the hydration reaction of CO2 was greatly
improved, and a great amount of H+ and HCO3− could be
produced in the crack zone solution:
CA
⎯ H+ + HCO3−
CO2 (aq) + H 2O ⎯→
(3) Since the microbial vegetative cells were negatively
charged, positively charged Ca2+ would be accumulated
on their surfaces
Cell + Ca 2 + ↔ Cell − Ca 2 + (8)
(4) The microbial vegetative cells acted as nucleation sites to
promote the deposition of mineralized products:
(6) CO32 − + Cell − Ca 2 + → Cell − CaCO3↓ (9)

(2) In the alkali solution of the concrete crack zone, HCO3−
reacted with OH− to form CO32− and H2O:
HCO3− + OH− ↔ CO3− + H 2O
4. CONCLUSIONS
To explore the survival time of spores with and without
encapsulation in concrete and the self-healing effect of concrete
(7) cracks at different curing ages, low-alkaline sulfo-aluminate
cement was used as the carrier material, and a kind of microbial
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Figure 25. Self-healing schematic diagram of concrete cracks. (a) Spore group and (b) microbial group.
self-healing agent encapsulated with spores was prepared by the
sugar-coated method. The growth, enzyme activity, and
microbial morphologies of spores with and without encapsula-
tion immersed in the simulated pore solution of cement-based
materials at different times were studied to characterize the
protection effect of the carrier on spores. In addition, the spores
with and without encapsulation were applied to the concrete,
and the effects of microbial self-healing agents on the basic
properties of concrete and the self-healing efficiency of cracks
were also studied. Moreover, the mechanism of different self-
healing effects was discussed in detail, which provided the
experimental support and theoretical basis for the engineering
application of microbial self-healing concrete. The results are as
follows.
(1) Compared with the reference group, the slump and
compressive strength of the spore group slightly
increased, while the 1 h slump loss over time, apparent
density, and setting time slightly reduced. For the
microbial group, the slump and 1 h slump loss over
time slightly increased, while the apparent density and
setting time decreased slightly. The early-age compressive
strength of concrete (3 and 7 days) was higher, but the
later-age compressive strength decreased to a certain
extent.
(2) When the spores without encapsulation were added in the
concrete, the early-age concrete cracks (7 and 14 days)
were repaired well, but for the later-age cracks, the self-
healing effect was greatly reduced. When the spores were
encapsulated with sulfo-aluminate cement, the cracks
with different curing ages were all well repaired. The
results showed that the two kinds of microbial self-healing
agents could be used for the early-age concrete cracks, but
for the later-age cracks, the carrier was needed to
encapsulate the spores, which ensured the self-healing
effect of concrete cracks.
(3) After soaking in the simulated pore solution of cement-
based materials for 28 days, the spores without
encapsulation almost died, while the spores with
encapsulation could still germinate and grow into
vegetative cells in the alkali environment. However,
when the nutrients were consumed, the vegetative cells
would turn into dormant spores, which indicated that
adding nutrients could prolong the survival time of
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microorganisms and increase the self-healing effect of
concrete cracks.
(4) The white precipitates generated at the crack mouth were
mainly calcite CaCO3. In addition, the mechanism of
concrete cracks with different microbial self-healing
agents was discussed carefully.
■ AUTHOR INFORMATION
Corresponding Author
Chunxiang Qian − School of Materials Science and Engineering,
Research Center of Green Building & Construction Materials,
and Jiangsu Key Laboratory for Construction Materials,
Southeast University, Nanjing, Jiangsu 211189, P. R. China;
orcid.org/0000-0002-2515-2233; Email: cxqian@
seu.edu.cn
Authors
Tianwen Zheng − School of Materials Science and Engineering,
Research Center of Green Building & Construction Materials,
and Jiangsu Key Laboratory for Construction Materials,
Southeast University, Nanjing, Jiangsu 211189, P. R. China
Yilin Su − School of Materials Science and Engineering, Research
Center of Green Building & Construction Materials, and
Jiangsu Key Laboratory for Construction Materials, Southeast
University, Nanjing, Jiangsu 211189, P. R. China
Xuan Zhang − School of Materials Science and Engineering,
Research Center of Green Building & Construction Materials,
and Jiangsu Key Laboratory for Construction Materials,
Southeast University, Nanjing, Jiangsu 211189, P. R. China
Hengyi Zhou − School of Materials Science and Engineering,
Research Center of Green Building & Construction Materials,
and Jiangsu Key Laboratory for Construction Materials,
Southeast University, Nanjing, Jiangsu 211189, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsami.0c16343
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This research was supported by the National Nature Science
Foundation of China (Grant No. 51738003).
ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
ACS Applied Materials & Interfaces
■
REFERENCES
(1) Aldea, C. M.; Shah, S. P.; Karr, A. Permeability of Cracked
Concrete. Mater. Struct. 1999, 32, 370−376.
(2) Huang, H. L.; Ye, G. Simulation of Self-Healing by Further
Hydration in Cementitious Materials. Cem. Concr. Compos. 2012, 34,
460−467.
(3) Khaliq, W.; Ehsan, M. B. Crack Healing in Concrete Using Various
Bio Influenced Self-Healing Techniques. Constr. Build. Mater. 2016,
102, 349−357.
(4) Algaifi, H. A.; Abu Bakar, S.; Sam, A. R. M.; Abidin, A. R. Z.;
Shahir, S.; AL-Towayti, W. A. H. Numerical Modeling for Crack Self-
Healing Concrete by Microbial Calcium Carbonate. Constr. Build.
Mater. 2018, 189, 816−824.
(5) Tziviloglou, E.; Wiktor, V.; Jonkers, H. M.; Schlangen, E. Bacteria-
Based Self-Healing Concrete to Increase Liquid Tightness of Cracks.
Constr. Build. Mater. 2016, 122, 118−125.
(6) Zhang, J. L.; Wu, R. S.; Li, Y. M.; Zhong, J. Y.; Deng, X.; Liu, B.;
Han, N. X.; Xing, F. Screening of Bacteria for Self-Healing of Concrete
Cracks and Optimization of the Microbial Calcium Precipitation
Process. Appl. Microbiol. Biotechnol. 2016, 100, 6661−6670.
(7) Achal, V.; Mukerjee, A.; Reddy, M. S. Biogenic Treatment
Improves the Durability and Remediates the Cracks of Concrete
Structures. Constr. Build. Mater. 2013, 48, 1−5.
(8) Nain, N.; Surabhi, R.; Yathish, N. V.; Krishnamurthy, V.; Deepa,
T.; Tharannum, S. Enhancement in Strength Parameters of Concrete
by Application of Bacillus Bacteria. Constr. Build. Mater. 2019, 202,
904−908.
(9) De Muynck, W.; De Belie, N.; Verstraete, W. Microbial Carbonate
Precipitation in Construction Materials: A Review. Ecol. Eng. 2010, 36,
118−136.
(10) Achal, V.; Mukherjee, A. A Review of Microbial Precipitation for
Sustainable Construction. Constr. Build. Mater. 2015, 93, 1224−1235.
(11) Joshi, S.; Goyal, S.; Mukherjee, A.; Reddy, M. S. Microbial
Healing of Cracks in Concrete: A Review. J. Ind. Microbiol. Biotechnol.
2017, 44, 1511−1525.
(12) Li, W. T.; Dong, B. Q.; Yang, Z. X.; Xu, J.; Chen, Q.; Li, H. X.;
Xing, F.; Jiang, Z. W. Recent Advances in Intrinsic Self-Healing
Cementitious Materials. Adv. Mater. 2018, 30, No. 1705679.
(13) Gollapudi, U. K.; Knutson, C. L.; Bang, S. S.; Islam, M. R. A New
Method for Controlling Leaching Through Permeable Channels.
Chemosphere 1995, 30, 695−705.
(14) Ramachandran, S. K.; Ramakrishnan, V.; Bang, S. S. Remediation
of Concrete Using Micro-Organisms. ACI Mater. J. 2001, 98, 3−9.
(15) Jonkers, H. M.; Thijssen, A.; Muyzer, G.; Copuroglu, O.;
Schlangen, E. Application of Bacteria as Self-Healing Agent for the
Development of Sustainable Concrete. Ecol. Eng. 2010, 36, 230−235.
(16) Bang, S. S.; Galinat, J. K.; Ramakrishnan, V. Calcite Precipitation
Induced by Polyurethane-Immobilized Bacillus pasteurii. Enzyme.
Microb. Technol. 2001, 28, 404−409.
(17) Wang, J. Y.; Tittelboom, K. V.; De Belie, N.; Verstraete, W. Use of
Silica Gel or Polyurethane Immobilized Bacteria for Self-Healing
Concrete. Constr. Build. Mater. 2012, 26, 532−540.
(18) Wang, J. Y.; Snoeck, D.; Van Vlierberghe, S.; Verstraete, W.; De
Belie, N. Application of Hydrogel Encapsulated Carbonate Precipitat-
ing Bacteria for Approaching a Realistic Self-Healing in Concrete.
Constr. Build. Mater. 2014, 68, 110−119.
(19) Palin, D.; Wiktor, V.; Jonkers, H. M. A Bacteria-Based Bead for
Possible Self-Healing Marine Concrete Applications. Smart Mater.
Struct. 2016, 25, No. 084008.
(20) Soysal, A.; Milla, J.; King, G. M.; Hassan, M.; Rupnow, T.
Evaluating the Self-Healing Efficiency of Hydrogel-Encapsulated
Bacteria in Concrete. Transp. Res. Rec. 2020, 2674, 113−123.
(21) Wang, J. Y.; Soens, H.; Verstraete, W.; De Belie, N. Self-Healing
Concrete by Use of Microencapsulated Bacterial Spores. Cem. Concr.
Res. 2014, 56, 139−152.
(22) Zamani, M.; Nikafshar, S.; Mousa, A.; Behnia, A. Bacteria
Encapsulation Using Synthesized Polyurea for Self-Healing of Cement
Paste. Constr. Build. Mater. 2020, 249, No. 118559.
Q https://dx.doi.org/10.1021/acsami.0c16343
www.acsami.org Research Article
(23) Litina, C.; Al-Tabbaa, A. First Generation Microcapsule-Based
Self-Healing Cementitious Construction Repair Materials. Constr.
Build. Mater. 2020, 255, No. 119389.
(24) Khushnood, R. A.; Din, S. U.; Shaheen, N.; Ahmad, S.; Zarrar, F.
Bio-Inspired Self-Healing Cementitious Mortar Using Bacillus subtilis
Immobilized on Nano-/Micro-Additives. J. Intell. Mater. Syst. Struct.
2019, 30, 3−15.
(25) Van Tittelboom, K.; De Belie, N.; Muynck, W. D.; Verstraete, W.
Use of Bacteria to Repair Cracks in Concrete. Cem. Concr. Res. 2010, 40,
157−166.
(26) Wiktor, V.; Jonkers, H. M. Quantification of Crack-Healing in
Novel Bacteria-Based Self-Healing Concrete. Cem. Concr. Compos.
2011, 33, 763−770.
(27) Sierra-Beltran, M. G.; Jonkers, H. M.; Schlangen, E. Character-
ization of Sustainable Bio-Based Mortar for Concrete Repair. Constr.
Build. Mater. 2014, 67, 344−352.
(28) Zhang, J.; Liu, Y.; Feng, T.; Zhou, M.; Zhao, L.; Zhou, A.; Li, Z.
Immobilizing Bacteria in Expanded Perlite for the Crack Self-healing in
Concrete. Constr. Build. Mater. 2017, 148, 610−617.
(29) Zhang, J. G.; Zhao, C.; Zhou, A. J.; Yang, C.; Zhao, L.; Li, Z.
Aragonite Formation Induced by Open Cultures of Microbial
Consortia to Heal Cracks in Concrete: Insights into Healing
Mechanisms and Crystal Polymorphs. Constr. Build. Mater. 2019,
224, 815−822.
(30) Xu, J.; Wang, X. Z.; Zuo, J. Q.; Liu, X. Y. Self-Healing of Concrete
Cracks by Ceramsite-Loaded Microorganisms. Adv. Mater. Sci. Eng.
2018, 2018, 1−8.
(31) Xu, J.; Wang, X. Z. Self-Healing of Concrete Cracks by Use of
Bacteria-Containing Low Alkali Cementitious Material. Constr. Build.
Mater. 2018, 167, 1−14.
(32) Liu, C.; Xu, X. Y.; Lv, Z. Y.; Lu, X. Self-Healing of Concrete
Cracks by Immobilizing Microorganisms in Recycled Aggregate. J. Adv.
Concr. Technol. 2020, 18, 168−178.
(33) Tan, L. Z.; Reeksting, B.; Ferrandiz-Mas, V.; Heath, A.; Gebhard,
S.; Paine, K. Effect of Carbonation on Bacteria-Based Self-Healing of
Cementitious Composites. Constr. Build. Mater. 2020, 257,
No. 119501.
(34) Chen, H. J.; Peng, C. F.; Tang, C. W.; Chen, Y. T. Self-Healing
Concrete by Biological Substrate. Materials. 2019, 12, No. 4099.
(35) González, Á .; Parraguez, A.; Corvalán, L.; Correa, N.; Castro, J.;
Stuckrath, C.; González, M. Evaluation of Portland and Pozzolanic
Cement on the Self-Healing of Mortars with Calcium Lactate and
Bacteria. Constr. Build. Mater. 2020, 257, No. 119558.
(36) Wang, J. Y.; De Belie, N.; Verstraete, W. Diatomaceous Earth as a
Protective Vehicle for Bacteria Applied for Self-Healing Concrete. J.
Ind. Microbiol. Biotechnol. 2012, 39, 567−577.
(37) Xue, B.; Qian, C. X. Mitigation of Efflorescence of Wallboard by
Means of Bio-Mineralization. Front. Microbiol. 2015, 6, No. 1155.
(38) Qian, C. X.; Chen, H. C.; Ren, L. F.; Luo, M. Self-Healing of Early
Age Cracks in Cement-Based Materials by Mineralization of Carbonic
Anhydrase Microorganism. Front. Microbiol. 2015, 6, No. 1225.
(39) Qian, C. X.; Ren, L. F.; Xue, B.; Cao, T. J. Bio-Mineralization on
Cement-Based Materials Consuming CO2 from Atmosphere. Constr.
Build. Mater. 2016, 106, 126−132.
(40) Chen, H. C.; Qian, C. X.; Huang, H. L. Self-Healing
Cementitious Materials Based on Bacteria and Nutrients Immobilized
Respectively. Constr. Build. Mater. 2016, 126, 297−303.
(41) Xu, J.; Wang, X. Z.; Wang, B. Biochemical Process of Ureolysis-
Based Microbial CaCO3 Precipitation and Its Application in Self-
Healing Concrete. Appl. Microbiol. Biotechnol. 2018, 102, 3121−3132.
(42) Zheng, T. W.; Qian, C. X. Influencing Factors and Formation
Mechanism of CaCO3 Precipitation Induced by Microbial Carbonic
Anhydrase. Process Biochem. 2020, 91, 271−281.
(43) Mohammed, H.; Ortoneda-Pedrola, M.; Nakouti, I.; Bras, A.
Experimental Characterisation of Non-Encapsulated Bio-Based Con-
crete with Self-Healing Capacity. Constr. Build. Mater. 2020, 256,
No. 119411.
(44) Zheng, T. W.; Su, Y. L.; Qian, C. X.; Zhou, H. Y. Low Alkali
Sulpho-aluminate Cement Encapsulated Microbial Spores for Self-
ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
ACS Applied Materials & Interfaces www.acsami.org Research Article

Healing Cement-Based Materials. Biochem. Eng. J. 2020, 163, (64) Jiang, L.; Jia, G. H.; Jiang, C.; Li, Z. Sugar-Coated Expanded
No. 107756. Perlite as a Bacterial Carrier for Crack-Healing Concrete Applications.
(45) Luo, M.; Qian, C. X. Performance of Two Bacteria-Based Constr. Build. Mater. 2020, 232, No. 117222.
Additives Used for Self-Healing Concrete. J. Mater. Civ. Eng. 2016, 28, (65) Chen, X.; Yuan, J.; Alazhari, M. Effect of Microbiological Growth
No. 04016151. Components for Bacteria-Based Self-Healing on the Properties of
(46) Zheng, T. W.; Qian, C. X. Self-Healing of Later-Age Cracks in Cement Mortar. Materials 2019, 12, No. 1303.
Cement-Based Materials by Encapsulation-Based Bacteria. J. Mater. (66) Kucukoglu, K.; Gul, H. I.; Taslimi, P.; Gulcin, I.; Supuran, C. T.
Civ. Eng. 2020, 32, No. 04020341. Investigation of Inhibitory Properties of Some Hydrazone Compounds
(47) Siddique, R.; Nanda, V.; Kunal; Kadri, E. H.; Khan, M. I.; Singh, on hCA I, hCA II and AChE Enzymes. Bioorg. Chem. 2019, 86, 316−
M.; Rajor, A. Influence of Bacteria on Compressive Strength and 321.
Permeation Properties of Concrete Made with Cement Baghouse Filter (67) Genç, H.; Ramazan, K.; Zeynep, K.; Nastaran, S.; Umit, K.;
Dust. Constr. Build. Mater. 2016, 106, 461−469. Mustafa, Z.; Ihami, G.; Hasan, Z. Discovery of Potent Carbonic
(48) Yi, H. H.; Qian, C. X. Synthesis and Characterization of Calcium Anhydrase and Acetylcholinesterase Inhibitors: 2-Aminoindan β-
Carbonate Nanoparticles via Bacterial Mineralization in Steel Slag Lactam Derivatives. Int. J. Mol. Sci. 2016, 17, No. 1736.
Comprising Cementitious Materials. Sci. Adv. Mater. 2020, 12, 760− (68) Mirjafari, P.; Asghari, K.; Mahinpey, N. Investigating the
768. Application of Enzyme Carbonic Anhydrase for CO2 Sequestration
(49) Zhuang, D. X.; Yan, H. X.; Tucker, M. E.; Zhao, H.; Han, Z. Z.; Purposes. Ind. Eng. Chem. Res. 2007, 46, 921−926.
Zhao, Y. H.; Sun, B.; Li, D.; Pan, J. T.; Zhao, Y. Y.; Meng, R. R.; Shan, G. (69) Zhang, Y.; Qian, C. X.; Zhang, X. Physicochemical characteristics
H.; Zhang, X. K.; Tang, R. Z. Calcite Precipitation Induced by Bacillus of crack mouth solution in microbial self-healing mortar. J. Southeast
cereus MRR2 Cultured at Different Ca2+ Concentrations: Further Univ. 2020, 50, 101−108.
Insights into Biotic and Abiotic Calcite. Chem. Geol. 2018, 500, 64−87. (70) Xiao, L. L.; Lian, B. Heterologously Expressed Carbonic
(50) Chahal, N.; Siddique, R.; Rajor, A. Influence of Bacteria on the Anhydrase from Bacillus mucilaginosus Promoting CaCO3 Formation
Compressive Strength, Water Absorption and Rapid Chloride by Capturing Atmospheric CO2. Carbonates Evaporites 2016, 31, 39−
Permeability of Concrete Incorporating Silica Fume. Constr. Build. 45.
Mater. 2012, 37, 645−651. (71) Bayindir, S.; Caglayan, C.; Karaman, M.; Gülcin, I.̇ The Green
(51) Siddique, R.; Singh, K.; Kunal; Singh, M.; Corinaldesi, V.; Rajor, Synthesis and Molecular Docking of Novel N-Substituted Rhodanines
A. Properties of bacterial rice husk ash concrete. Constr. Build. Mater. as Effective Inhibitors for Carbonic Anhydrase and Acetylcholinester-
2016, 121, 112−119. ase Enzymes. Bioorg. Chem. 2019, 90, No. 103096.
(52) Andalib, R.; Abd Majid, M. Z.; Hussin, M. W.; Ponraj, M.;
Keyvanfar, A.; Mirza, J.; Lee, H. S. Optimum Concentration of Bacillus
Megaterium for Strengthening Structural Concrete. Constr. Build.
Mater. 2016, 118, 180−193.
(53) Al-Kheetan, M. J.; Rahman, M. M.; Chamberlain, D. A. Optimum
Mix Design for Internally Integrated Concrete with Crystallizing
Protective Material. J. Mater. Civ. Eng. 2019, 31, No. 04019101.
(54) Luo, M.; Qian, C. X.; Li, R. Y. Factors Affecting Crack Repairing
Capacity of Bacteria-Based Self-Healing Concrete. Constr. Build. Mater.
2015, 87, 1−7.
(55) Han, Z. Z.; Wang, J. J.; Zhao, H.; Tucker, M. E.; Zhao, Y. H.; Wu,
G. Z.; Zhou, J. X.; Yin, J. X.; Zhang, H. C.; Zhang, X. K.; Yan, H. X.
Mechanism of Biomineralization Induced by Bacillus subtilis J2 and
Characteristics of the Biominerals. Minerals 2019, 9, No. 218.
(56) Xu, G. J.; Li, D. W.; Jiao, B. Q.; Li, D.; Yin, Y. J.; Lun, L. M.; Zhao,
Z. Q.; Li, S. Biomineralization of a Calcifying Ureolytic Bacterium
Microbacterium sp. GM-1. Electron. J. Biotechnol. 2017, 25, 21−27.
(57) Baskar, S.; Routh, J.; Baskar, R.; Kumar, A.; Miettinen, H. I.;
Merja, V. Evidences for Microbial Precipitation of Calcite in
Speleothems from Krem Syndai in Jaintia Hills, Meghalaya, India.
Geomicrobiol. J. 2016, 33, 906−933.
(58) Wang, J. Y.; Jonkers, H. M.; Boon, N.; De Belie, N. Bacillus
sphaericus LMG 22257 is Physiologically Suitable for Self-Healing
Concrete. Appl. Microbiol. Biotechnol. 2017, 101, 5101−5114.
(59) Wang, Y. Z.; Soga, K.; Dejong, J. T.; Kabla, A. J. Microscale
Visualization of Microbial-Induced Calcium Carbonate Precipitation
Processes. J. Geotech. Geoenviron. Eng. 2019, 145, No. 04019045.
(60) Zhan, Q.; Yu, X.; Zhang, S.; Xu, Y.; Pan, Z.; Qian, C. Study on
Improving the Consolidation Properties of Microbial Cementitious
Material by Promoting Spore Germination Ratio. Constr. Build. Mater.
2020, 252, No. 119036.
(61) Erşan, Y. Ç .; De Belie, N.; Boon, N. Microbially Induced CaCO3
Precipitation through Denitrification: An Optimization Study in
Minimal Nutrient Environment. Biochem. Eng. J. 2015, 101, 108−118.
(62) Jiang, L.; Jia, G. H.; Wang, Y. Z.; Li, Z. Optimization of
Sporulation and Germination Conditions of Functional Bacteria for
Concrete Crack-Healing and Evaluation of their Repair Capacity. ACS
Appl. Mater. Interfaces 2020, 12, 10938−10948.
(63) Ryu, Y.; Lee, K. E.; Cha, I. T.; Park, W. Optimization of Bacterial
Sporulation Using Economic Nutrient for Self-Healing Concrete. J.
Microbiol. 2020, 58, 288−296.

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