Cement and Concrete Composites 113 (2020) 103718

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Cement and Concrete Composites

journal homepage: http://www.elsevier.com/locate/cemconcomp

Application of bacterial spores coated by a green inorganic cementitious

material for the self-healing of concrete cracks
Mingyue Wu a, Xiangming Hu a, b, *, Qian Zhang a, Weimin Cheng a, **, Di Xue a, Yanyun Zhao a
a
Key Lab of Mine Disaster Prevention and Control, College of Safety and Environmental Engineering, Shandong University of Science and Technology, No. 579,
Qianwangang Road, Xin’an Street, Huangdao District, Qingdao, Shandong, China
b
Department of Chemical Engineering and Safety, Binzhou University, Yellow River 5 Road, Pengli Street, Bincheng District, Binzhou City, Shandong, China

A R T I C L E I N F O A B S T R A C T

Keywords: The Bio-based self-healing concrete requires a healing agent with excellent properties. This study used carbide
Biocapsule slag, fly ash, and desulfurized gypsum to prepare a green inorganic cementitious material, and further developed
Biomineralization a biocapsule by coating bacterial spores with the prepared cementitious material. Specifically, at first, the
Self-healing
properties of the biocapsule were analyzed and the effect of its dosage on of cement mortar properties was
Concrete cracks
evaluated. Then, mineralization activity was employed to characterize the ability of bacterial spores in the
biocapsule to achieve mineralization. Finally, crack-healing efficiency and water permeability were evaluated to
study the effectiveness of the biocapsule in crack self-healing. As per the results, addition of biocapsules at a
dosage of 5% of the cement mass led to full healing of 150–550 μm width cracks. After healing, the permeability
of specimens containing the biocapsule declined by about two orders of magnitude as compared with reference
specimens (R).

1. Introduction referred to as the microbially induced calcite precipitation (MICP)

Concrete is one of the most widely-used building materials. How­
ever, due to external load, alkali–aggregate reactions, drying shrinkage,
and other factors, it unavoidably suffers internal or surface microcracks
during preparation and service [1–3]. If these microcracks are not
promptly repaired, external moisture and other corrosive substances
(mainly Cl and SO24 ) enter the interior of the concrete through these
cracks [4–6], which accelerates the corrosion of reinforcement materials
and deterioration of the concrete and shortens the service life of the
concrete [7–9]. Thus, it is of great importance to repair these micro­
cracks in a timely and effective manner.
The carbonate precipitate produced by bacterially induced miner­
alization is an environmentally friendly and economical material [10].
Currently, this carbonate precipitate is used in several engineering
fields, such as, the cementing of sandy soil [11–13], the remediation of
heavy metal pollution and radionuclide contamination [14–17], the
treatment of soil [18–21], and the surface protection of building mate­
rials [22–26]. In most engineering applications, bacterially induced
mineralization produces calcium precipitate, and this intervention is
technique [27,28]. Recently, this technique has been employed by re­
searchers both at home and abroad to repair concrete cracks and
improve concrete durability [29–33].
The MICP technique offers a new way of repairing concrete cracks
and can extend the service life of concrete. However, according to a
study by Jonkers et al. [34], when bacteria and their nutrients are
directly added to concrete during mixing, the bacteria can only survive
for a maximum of four months in the concrete, which is detrimental to
the long-term and sustainable repair of concrete cracks. To better protect
these bacteria so that they can survive the hydration period of concrete
and remain active in the concrete for a long time afterwards, it is
necessary to provide a protective carrier for them. Scholars both at home
and abroad have made many attempts to develop protective carriers for
bacteria. For example, Wang et al. adopted diatomite [1], silica gel [4],
polyurethane [7], hydrogel [35], microcapsules [36], and modified so­
dium alginate [37] for immobilization or coating of bacterial spores, so
as to realize the self-healing of concrete cracks. Among the above car­
riers, microcapsules were found to have a relatively good healing effect;
when microcapsules were used, the self-healing efficiency of cracks

* Corresponding author. Key Lab of Mine Disaster Prevention and Control, College of Safety and Environmental Engineering, Shandong University of Science and
Technology, No. 579, Qianwangang Road, Xin’an Street, Huangdao District, Qingdao, Shandong, China.
** Corresponding author.
E-mail addresses: xiangming0727@163.com (X. Hu), wmcheng@sdust.edu.cn (W. Cheng).

https://doi.org/10.1016/j.cemconcomp.2020.103718
Received 13 August 2019; Received in revised form 22 May 2020; Accepted 17 June 2020
Available online 17 July 2020
0958-9465/© 2020 Elsevier Ltd. All rights reserved.
M. Wu et al.
(with a maximum width of 970 μm) reached 80%. However, when the
dosage of microcapsules was 1–4%, the compressive strength of the
concrete declined by 15–34%, which can seriously jeopardize the
workability of concrete. Other bacterial carriers, such as porous
expanded clay pellets [38], graphite nanosheets [39], lightmass aggre­
gate [39], expanded perlite [40], zeolite [41], and sulpho-aluminate
cement [42], have also proven to be effective for the self-healing of
concrete cracks. However, there are still certain issues with the above
microbial carriers. For instance, they are not capable of completely
isolating microbial cells from concrete, which results in a decline in
microbial mineralization activity over time, and the addition of carrier
materials exerts a negative influence on the mechanical properties of
concrete. When using bacteria to heal concrete cracks, the following
three aspects should be comprehensively evaluated: (i) the properties of
the carrier materials themselves and their influence on the activity of
bacterial spores; (ii) the influence of the addition of carriers on the
workability of concrete; and (iii) the effect of bacteria on concrete-crack
self-healing. The key to effective self-healing of concrete cracks through
the MICP technique appears to lie in the selection of suitable bacterial
carriers.
This paper employed cementitious materials produced by carbide
slag, fly ash, desulfurized gypsum, and sodium silicate hydration reac­
tion as wall materials and green and environmentally friendly poly­
saccharides (xanthan gum and microcrystalline cellulose), yeast
powder, and bacterial spore powder as core materials to prepare a
biocapsule. Then, the biocapsule was used for the self-healing of con­
crete cracks. The carbide slag used comprises CaO and Ca(OH)2; the
released OH ions can damage the hyaloid structure on the surface of fly
ash, release Al2O3 and SiO2 from inside fly ash, and further lead to a
hydration reaction that generates cementitious materials [43]. The
cementitious materials not only have a certain strength but are also
highly compatible with concrete. The bacterial spore powder used was a
mineralized Bacillus cereus CS1 isolated in calcium carbide slag. The
carbide slag that is not involved in hydration can provide a primitive
environment for bacterial spores, further protecting bacterial spores
against the severe environment in concrete and extending the service life
of the concrete, which guarantees a continuous repair of concrete cracks.
After biocapsule preparation, this study proceeds to investigate its
properties, its effects on both the workability of concrete and the
mineralization activity of bacteria, and its effect on the self-healing of
concrete cracks. Hence, this work lays a theoretical and experimental
foundation for truly realizing effective microbially induced self-healing
of concrete cracks.
2. Materials and methods
2.1. Bacterial strain
The bacteria used in this study were Bacillus cereus CS1 (separated
from carbide slag), which is Gram-positive [44]. The MICP process is
realized by the production of urease, i.e., urea in the environment is
decomposed into NHþ 4 by urease, hence, increasing the alkalinity. A high
alkalinity tends to reduce the saturation index; thereby, shifting the
equilibrium of bicarbonates, leading to the production of CO23 ions. The
presence of bacterial cells and Ca2þ in their surrounding environment
would allow the CO23 ions to bond with Ca2þ ions and precipitate as
calcium carbonates [45]. The Bacillus cereus CS1 was cultured in a liquid
medium composed of yeast extract powder (5 g/L), peptone (10 g/L),
and NaCl (10 g/L). The above nutrients were purchased from Beijing
Aoboxing Bio-Tech Co., Ltd., China. The liquid medium was autoclaved
at 121 � C for 30 min in a high-temperature autoclave (G180T, Shanghai
Shenan Medical Instrument Factory, China) before use. The isolated
bacteria were cultured to a stable period, and then, stored in a refrig­
erator at a temperature of 4 � C, and sub-cultured regularly (usually 7 d).
When the bacteria were used for the test, the bacteria stored in the
refrigerator were activated and cultured to a stable period, and then,
2
Cement and Concrete Composites 113 (2020) 103718
transferred as inoculums in to liquid medium. The inoculum amount was
1% of the medium volume. The cultures were incubated (28 � C, 180
rpm) for 3 d. Subsequently, they were subjected to pasteurization (80 � C
for 20 min, then 5 min in ice-cold water) to minimize the vegetative
cells. The culture was then centrifuged (4 � C, 7000 rpm) and the su­
pernatant was removed. The paste obtained was first vacuum-dried for
3 d at room temperature, and then ground to achieve bacterial spore
powder.
2.2. Capsulation of the bacterial spores
The biocapsule was prepared according to the following steps:
a. The substances needed for the core were weighed according to the
core-material proportioning provided in Table 1, and the bacterial
spore powder was added to a suitable quantity of water for homo­
geneous dispersion. The mixture of core-material substances
(excluding the bacterial spore powder) was then added to the ho­
mogeneously dispersed bacterial spore powder, and they were well
mixed to obtain a lumpy mixture that was further kneaded until it
was no longer sticky.
b. The lumpy mixture was then slowly added into a multi-purpose
pelletizer 50 g at a time, to perform pressing, extruding and pellet­
ing, followed by rolling, drying and sieving (25–30 � C) to prepare
biocapsule core-material pellets of a consistent size.
c. The substances needed for the wall material were weighed according
to proportion given in Table 1, and the powdered wall materials
(carbide slag, desulfurized gypsum, and fly ash) were well mixed.
d. The surfaces of the core-material pellets prepared in step b were first
uniformly sprayed with a layer of sodium silicate, and then, poured
into the well-mixed wall-material powder. The wall-material powder
containing the core-material pellets was then shaken at a constant
speed until it uniformly adhered to the surface of the core-material
pellets under the action of the sodium silicate. The wall materials
were rolled at 25 � C. 14 days after hydration, their surfaces were
again uniformly sprayed with a layer of sodium silicate. Finally, the
material was dried to obtain biocapsule pellets of a consistent size.
The resulting biocapsule had a diameter of about 2,000 μm and a
bacterial spore concentration of about 9.6 � 108 cells per gram of bio­
capsule (dry weight). The preparation process is illustrated in Fig. 1. The
cross section of the biocapsule is shown in Fig. 2.
2.3. Properties of the biocapsule
The fresh cement mortar is a water-rich environment. The ability of a
biocapsule added to cement mortar to maintain its integrity during
mixing and mortar hydration constitutes the key to its use for healing
mortar cracks. Moreover, the ability of the bacterial spores immobilized
in the biocapsule to survive and produce calcium carbonate through
mineralization is also critical to concrete-crack healing. Hence, the
following tests were performed.
Table 1
The composition of biocapsules.
core materials (g) wall materials (g)
bacterial spore 0.656 distilled 50.000 sodium 15.211
powder water silicate
yeast extract 4.371 peptone 1.093 carbide slag 16.901
powder
xanthan gum 1.093 urea 2.252 desulfurized 21.127
gypsum
microcrystalline 32.786 CaCl2 4.995 fly ash 6.761
cellulose
M. Wu et al.
Fig. 1. Biocapsules preparation process (a: granulation; b: pressed cake; c: core
particles; d: molding).
Fig. 2. Image of biocapsule cross section.
2.3.1. Waterproofing properties of biocapsule wall materials
The prepared biocapsule specimens (with an initial mass of 3 g in
each group) were placed in deionized water and taken out at 1 d, 3 d, 5
d, 10 d, and 15 d, respectively, and dried with absorbent paper. The mass
of the biocapsules in each group was then measured, and the increment
of the mass value was adopted as the index to evaluate the water­
proofing properties of the biocapsule wall materials.
2.3.2. Storage stability of the biocapsule
Microcapsule specimens with an initial mass of 50 g were soaked in
deionized water for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, 60 h,
and 72 h, respectively, and then, taken out and dried in an oven at a
temperature of 60 � C. The weight of the dried microcapsule specimens
was recorded. Notably, in this section, the soaked biocapsules were
dried in a drying oven. The purpose was to investigate if the biocapsule
wall material broke during the dry and wet cycles. In the waterproof test
of the biocapsule wall material, only the absorbent paper was used to dry
the moisture of the surface of the bio-capsule without drying it.
3
Cement and Concrete Composites 113 (2020) 103718
2.3.3. Mineralization activity of bacterial spores in biocapsule
As concrete is a high alkali and high calcium environment, in order to
study the effect of this environment on the mineralization activity of
bacteria in biocapsules. The 4 g biocapsules were soaked in Ca(OH)2
with a concentration of 1.65 g/L for 6 h in each test. And the pH value of
Ca(OH)2 solution needs to be adjusted to 13 by NaOH (0.1 mol/L) so­
lution.. After taking out and drying, the wall material was carefully
removed and the core material was ground into a powder, and then, it
was added to the 133.4 mL of sterilized medium (yeast extract powder:
5 g/L, peptone: 10 g/L, NaCl: 10 g/L, CaCl2:0.75 mol/L, urea: 0.75 mol/
L. Urea was added by filtration and sterilization.). A total of 7 groups of
tests were set up, and the tests were stopped at 24, 48, 72, 96, 120, 144,
168, 192, 216, and 240 h. The resulting precipitate was then centrifuged
and washed three times with anhydrous ethanol and distilled water,
respectively. After cleaning, the precipitate was dried in a drying oven at
60 � C. Finally, 1 mol/L HCl solution was added to the precipitate until no
bubbles were generated, and the precipitate after the reaction was dried
again. The mineralization activity was calculated according to its
weight. The above test was repeated (n ¼ 3).
2.4. Biocapsules in mortar specimens
2.4.1. Influence of the biocapsule on the mechanical properties of mortar
Different dosages of the biocapsule were added to mortar specimens
(40 mm � 40 mm � 160 mm) to study the influence of the biocapsule on
their capillary water absorption and mechanical properties. Ordinary
Portland cement (CEM I 42.5 N), sand and water were used to prepare
the cement mortar specimens with a water to cement ratio of 0.65 and a
sand–binder ratio of 3. The biocapsule dosages were set at 0%, 3%, 5%,
7%, 9%, 11%, and 13% of the cement mass; the specimens receiving
these doses were denoted by R, CS3%, CS5%, CS7%, CS9%, CS11%, and
CS13%, respectively. The first step was to mix the biocapsules with
water and prepare cement mortar according to GB/T 17671-1999 [46].
After pouring, all the molds were placed in a curing room (20 � C, N95%
RH). 48 h (24 h in the case of R) later, the specimens were demolded and
placed in the same curing room. 28 d later, their strength was measured
according to GB/T 17671-1999.
2.4.2. Compatibility between biocapsule and mortar specimen fracturing
Breakage of the biocapsules is a prerequisite for the self-healing of
concrete cracks. Herein, 5% biocapsule-dosed cement mortar specimens
that had been fractured as described in 2.4.1 were examined to identify
the biocapsule breaking on the fracture surface, with the purpose of
verifying the compatibility in the fracture behavior of the biocapsules
and mortar specimens.
2.4.3. Influence of biocapsule on setting time and consistency of cement
mortar
The influence of the addition of biocapsules on the setting time and
consistency of cement mortar was determined according to GB/T 1346-
2001 [47]. The dosages of the biocapsule were again set at 0%, 3%, 5%,
7%, 9%, 11%, and 13% of the cement mass, respectively.
2.4.4. Survival of the biocapsule during mortar mixing
Biocapsules were added to cement mortar at a dose of 5% of the
cement mass, and the mortar was then mixed in a cement mortar mixer.
At the end of mixing, a portion of the cement mortar was extracted and
placed in a culture dish to observe the survival and morphology of the
biocapsules.
2.4.5. Capillary water absorption of mortar specimens
28 day-cured specimens were placed in an oven at 40 � C, and their
mass was measured every 24 h until a mass change of less than 1% was
detected. For the water absorption test, the four sides adjacent to the
surfaces that were to be soaked in water were wrapped with tin foil
paper to avoid evaporation. The specimen was then immersed in water
M. Wu et al. Cement and Concrete Composites 113 (2020) 103718

with a water level of 10 � 1 mm and taken out at certain intervals for
drying and weighing. The water absorption test was conducted in a
curing room (20 � C, N60% RH). The water absorption coefficient k
(g⋅cm 2⋅h 1/2) was determined according to Equation (1) [1,36].
pffi
Q
¼k t (1)
S
Q - quantities water absorption at different intervals (g); S - contact
area between the specimen and water (cm2); t – interval (h).
2.5. Application of capsulated spores for self-healing cracks
2.5.1. Specimen preparation
Both cuboid (40 mm � 40 mm � 160 mm) and cylindrical (Φ ¼ 80
mm, H ¼ 22 mm) specimens were prepared. Table 2 lists the composi­
tion of the specimens in each group. Group R included specimens con­
taining no biocapsules; groups C3%, C5%, CS3%, and CS5% included
specimens containing biocapsules (specifically, the biocapsules in
groups C3% and C5% contained no bacterial spores; the biocapsules in
groups CS3% and CS5% contained bacterial spores). After pouring, all
the molds were placed in a curing room (20 � C, N95% RH). 48 h (24 h in
the case of R) later, the specimens were demolded and placed in the
same curing room until testing time.
2.5.2. Cracking and curing conditions
After 28 d of curing, the cuboid specimen produced multiple cracks
in a three-point bending manner. One side of the specimen was placed
on the support cylinder of the test machine. The long axis of the spec­
imen was perpendicular to the support cylinder and the loading speed
was 0.01 mm/s. The cylindrical specimens produced multiple cracks via
split tensile test. During the test, the specimens were placed at the center
of the lower plate of the testing machine. Arc-shaped pads were placed
between the upper and lower plate and the specimen. When approach­
ing the arc-shaped gasket, the ball seat was adjusted to form uniform
contact, and the loading speed was 0.02 MPa/s. Crack width was
measured by means of a linear variable differential transformer (LVDT)
that was attached to the side of the specimens. The related content can
be observed in the supplementary file. The final widths of the cuboid and
cylindrical specimens were 150–550 μm and 230–340 μm, respectively.
The cracked specimens were then placed under three curing condi­
tions: a. soaking in water; b. soaking in DM (The DM was composed of
0.75 mol/L urea and 0.75 mol/L CaCl2); c. wet–dry curing cycles. In one
wet–dry curing cycle, the specimen was soaked in DM for 16 h and then
exposed in air for 8 h. The curing environment was 20 � C at N60% RH.
Notably, when the cylindrical specimens were cured under the condition
of c, the environmental conditions when it is exposed to the air was 20
�
C, N95% RH. All specimens were cured for 28 d.
area. Initial images of cracks in the specimens were captured as soon as
they were formed, and the specimens were observed at intervals of 7
d during the healing process. The Image J 8.0 image analysis program
was used to determine the initial crack area and the healed crack area of
a specimen; a scanning electron microscope (SEM) and Energy Disper­
sive Spectrometer (EDS) were employed to analyze the micro-
morphology of mineralization products in the cracks.
2.5.3.2. Water permeability test. The cured specimen was immersed in
water for 3 d; then, the specimen was taken out in water and its surface
was dried at room temperature. The dry surface specimen was installed
in a PVC ring, and the specimen was evacuated. First, the specimen was
evacuated in a vacuum chamber for 2–3 h, and then deionized water was
added into the vacuum chamber while maintaining the vacuum condi­
tions. When the specimen was completely immersed in water, the vac­
uum was stopped. The specimen was soaked in water for 24 h, and then,
removed from the test chamber and tested for water permeability. The
entire device was maintained watertight to ensure that the specimen is
saturated before each measurement. During the entire test period, no
moisture evaporation occurred in the specimen. During the 14 d test
period, the falling time of the water level in the glass tube from h0 to hf
was measured regularly. The schematic diagram of the water perme­
ability test can be observed in the supplementary file. The water
permeability coefficient was calculated according to Equation (2) [4]:
� �
aT
k ¼ ln
h0
(2)
At hf
Where k is the coefficient of water permeability (m/s); a is the cross-
section area of the glass tube (m2); A is the cross-section area of the
cylinder (m2); T is the thickness of the cylinder (m); t is the time of water
falling from h0 to hf (s);h0 and hf are the initial and final water levels
(cm), respectively.
The above test was repeated (n ¼ 3), and the average value was
calculated (precise to 1.0 � 10 3 cm/s).
3. Results
3.1. Biocapsule properties
3.1.1. Waterproofness of the biocapsule
After soaking in distilled water for different periods of time, the
biocapsules were taken out, dried, and weighed to assess waterproof­
ness. Fig. 3 shows the change in biocapsule mass with soaking time.
After 1 d of soaking, the biocapsule mass displayed a clear increase from

2.5.3. Assessment of self-healing efficiency of mortar cracks

The biocapsules primarily promoted self-healing of concrete cracks
because the microbes generated mineralization products that can seal
the cracks. Hence, crack healing area and water permeability were
employed to directly reflect the crack-healing efficiency.

2.5.3.1. Crack filling. The filling efficiency was assessed by the healing
ratio (r), defined as the ratio of the healed crack area to the initial crack

Table 2
Composition of the specimens in each series.
Group cement sand water biocapsules bacterial spore
(g) (g) (g) (g) powder

R 325 975 211 0 N

C3% 325 975 211 9.8 N
C5% 325 975 211 16.3 N
CS3% 325 975 211 9.8 Y
CS5% 325 975 211 16.3 Y
Fig. 3. Variation in weight over time after soaking of biocapsules.

4
M. Wu et al. Cement and Concrete Composites 113 (2020) 103718

3 g to 3.77 g, an increase of 25.7%. 3 d of soaking increased the mass to

3.9 g, and there was no further mass increase with additional increase in
the soaking time. Although the quality of the biocapsules increased
during the test, it was found during the test that there were no clear
differences between the morphology of biocapsules soaked for 1 d, 3 d,
7 d, and 15 d in distilled water; the wall materials neither fell off nor
burst. These results indicate that the biocapsules can survive in a water-
rich environment. This can also be confirmed in Section 3.2.3.

3.1.2. Storage stability of the biocapsule

After soaking in distilled water for a certain period of time, the
biocapsule was taken out, dried, and weighed to assess its storage sta­
bility. Fig. 4 shows a plot of the relationship between the biocapsule
mass and soaking time during the storage stability test. In the first 4 h of
soaking in distilled water, the biocapsule did not experience any mass
loss. The biocapsule mass declined from 50 g to 49.2 g in 10 h, a decline
rate of 1.6%. The biocapsule mass declined further from 49.2 g to 48 g in
48 h, a decline of 4%, possibly because the soluble substances on the
biocapsule surface had dissolved in the distilled water. After the 48 h
point, the biocapsule saw a substantial mass loss. According to the test
findings, when a biocapsule soaked for more than 48 h was taken out
and dried, some of the wall materials of the biocapsule fell off, which can
be interpreted as the primary cause of the decline of biocapsule mass.
3.1.3. Mineralization activity of bacterial spores in the biocapsule
Mineralization was used to characterize the activity of the bacterial
spores in the biocapsule. A certain concentration of Ca2þ was added to
the culture medium, the core material of the biological capsule was
carefully taken out and ground into a powder, and then, it was added to
the sterilized culture medium. The mineralization activity is expressed
by measuring the amount of CaCO3 in the culture medium. As shown in
Fig. 5, no minerals were produced within 24 h and a small amount of
product was not formed until 48 h. This is because during this time, the
bacterial spores in the core material were in the recovery growth stage.
In addition, Fig. 5 shows that as time progressed, the content of
mineralized products gradually increased, and the increase in product
decreased at 216 h. During the trial, the final mineralized product was
5.37 g. This shows that the bacterial spores encapsulated in the bio­
capsules had a certain activity and could mineralize Ca2þ in the
environment.
Fig. 5. Mineralization activity of bacteria in biocapsules.
3.2. Biocapsules in mortar specimens
3.2.1. Effect of different dosages of biocapsule on mechanical properties of
mortar specimens
The additive dosage of the biocapsule had some influence on the
strength of mortar specimens. As shown in Fig. 6, when the biocapsule
dosage was 0–5%, the strength of the mortar specimens increased with
an increase in the dosage. When the dosage was 5%, the strength of the
mortar specimens reached a maximum, and their compressive strength
and flexural strength were raised by 2.50% and 8.86%, respectively,
compared to the case where no biocapsules were added. When the
biocapsule dosage exceeded 5%, the strength of the mortar specimens
declined with an increase in dosage. The compressive strength of the
specimens declined more significantly than the flexural strength, with
the compressive strength of 13%-dosage specimens declining by 53.35%
of that of those with a 5% dosage. Based on the above data, the following
conclusion can be reached. When the additive dosage of the biocapsule
is less than 5% of the cement mass, the strength of the mortar specimens
increases with an increase in biocapsule dosage. However, when the
dosage exceeds 5%, a negative influence is exerted on the strength of
mortar specimens.

Fig. 4. Variation in weight over time after soaking and drying biocapsules. Fig. 6. Variation of mortar sample strength with the addition of biocapsules.

5
M. Wu et al.
3.2.2. Influence of different biocapsule dosages on setting time and
consistency of mortar
The biocapsule had some influence on the setting time of mortar, but
this influence was not significant. As shown in Fig. 7a, when a bio­
capsule dosage of 0–5% was added, the setting time of the mortar
increased with increased dosage but the increment was extremely small
(initial setting and final setting time both increased by 2.86%). Between
the additive dosages of 3% and 5%, there was only a difference of 1 min
in the final setting time. When the biocapsule dosage exceeded 5%, the
setting time of the mortar declined to some extent. The initial setting and
final setting time of the mortar at the dosage of 13% declined by 25 min
and 27 min compared with those for a dosage of 5%, respectively.
Overall, addition of biocapsules to the mortar had a little influence on
the setting time.
Similarly, biocapsules also had some influence on the consistency of
the mortar. As shown in Fig. 7b, the consistency of mortar declined with
an increase in biocapsule dosage. Specifically, when the dosage was
below 5%, the consistency of the mortar had a small amplitude of
decline (about 6.02%), whereas when it exceeded 5%, the consistency of
the mortar declined more substantially. In particular, when the dosage
reached 13%, the consistency of the mortar declined by 43.61%, which
can seriously restrict the application of mortar in engineering practice.
3.2.3. Survival of biocapsule during mortar mixing and fracture
compatibility between biocapsule and mortar
The ability of the biocapsule to maintain its integrity when added
into and mixed with newly prepared mortar is the key to its healing of
mortar cracks. Herein, biocapsules were added into fresh mortar at a
dose of 5% of the cement mass and mixed for 5 min. A portion of the
mortar was then extracted and placed in a culture dish. Fig. 8a and b
show the mortar-mixed biocapsules in the culture dish. After taking the
biocapsules out, cleaning them, and examining their surfaces, it was
found that 96.15% of the biocapsules (50 out of 52) were unbroken.
The ability of the biocapsule to break as a mortar specimen fracture
also plays a vital role in the healing of concrete cracks. This is because
without breakage of the biocapsule, its internal healing agent cannot be
released, making it impossible for the agent to heal concrete cracks. As
shown in Fig. 8c and d, when the specimen was fractured on the tester
after 28 d of curing, the biocapsules located on the fracture surface of the
specimen broke as well and the internal healing agent was exposed. This
phenomenon revealed high compatibility in the fracturing behavior of
the biocapsule and concrete.
Fig. 7. Variation of mortar setting time and consistency with the addition of biocapsules (a: setting time; b: consistency).
6
Cement and Concrete Composites 113 (2020) 103718
3.3. Influence of biocapsule on the water absorption of mortar specimens
Fig. 9 shows the influence of different dosages of the biocapsule on
the water absorption of cement specimens. In the period 0–3 h, there
were relatively small differences among specimens that had received
different biocapsule dosages, but after 5 h, such differences gradually
became more obvious. In terms of water absorption, the differently
dosed specimens followed the order (from low to high): CS5% < CS7%
< CS3% < CS9% < CS11% < R < CS13%. In other words, when the
dosage of the biocapsule was 5%, the water absorption of the cement
specimens was the lowest. With the dosage further increased, the water
absorption of the cement specimens gradually increased, when it
reached 13%, the water absorption of the cement specimens even
exceeded that of specimens containing no biocapsules in group R. In
addition, the water absorption of cement specimens with a dosage of 3%
was higher than that of those at dosages of 5% and 7%.
As shown in the above results, the biocapsule dosage had a signifi­
cant influence on the water absorption of cement specimens. This may
be the result of a large number of tiny pores in cement specimens. That
is, when biocapsules were added, the effective ingredients in cement
(such as SiO2 and Al2O3) engaged in a hydration reaction with the gel
products on the biocapsule surface, further cementing with each other,
and consequently, reducing the porosity of the cement specimens and
the availability of capillary channels for water. This caused the water
absorption of the cement specimens to decline in comparison to that of
specimens containing no biocapsules (R) with an increase in the bio­
capsule dosage (3–11%). However, with a further increase in the bio­
capsule dosage, the large size of the biocapsule pellets caused more large
pores to occur around them. On the macroscopic level, this was man­
ifested as an increase in the number or size of pores in the cement
specimens. Thus, within the dosage range 5–11%, the amplitude of the
decline in the water absorption of cement specimens decreased with an
increase in the biocapsule dosage, until the water absorption of cement
specimens dosed with 13% of biocapsules even exceeded that of speci­
mens containing no biocapsules in group R. In addition, the water ab­
sorption of the cement specimens with a dosage of 3% was higher than
that of those with dosages of 5% and 7%. This was primarily because, at
a dosage of 3%, the hydration reaction between cement and the bio­
capsule was insufficient to fill the pores inside the specimens, but with
an increase in the dosage, the pores in the specimens gradually
decreased, as manifested by the decline in water absorption.
3.4. Water permeability of specimens after crack healing
The water permeability test was conducted on cracked cylindrical
M. Wu et al.
Fig. 8. Biocapsules in the mortar after agitation (a and b); biocapsules rupture with the rupture of the mortar specimens (c and d).
Fig. 9. Effect of different capsule dosages on water absorption of
cement specimens.
specimens after 28 d of curing under different conditions. Fig. 10 shows
the final k value of each group. The k values of the specimens in groups
R, C3%, and C5% fell within the range of 3.80 � 10 7–8.91 � 10 7 m/s.
Under the same curing conditions, the water permeability of specimens
in group CS5% was uniformly lower than that of those in group CS3%,
suggesting that the specimens in group CS5% exhibited better healing
7
Cement and Concrete Composites 113 (2020) 103718
Fig. 10. Water permeability of different specimens after curing.
than those in group CS3%. The specimens under wet–dry curing cycles
in DM saw a decline of two orders of magnitude in permeability
compared with that of those in group R. This is because the minerali­
zation products generated by bacteria filled up the cracks and obstructed
the transport of water. The final k value of the specimens under wet–dry
curing cycles in DM was lower than that of the specimens that remained
soaked in DM. This is because, in the former case, the specimens had
opportunities to be exposed in air, and the bacteria on them could come
into contact with more O2, which could strengthen bacterial growth and
M. Wu et al.
increase activity, generating more mineralization products for crack-
filling, and eventually, lowering the permeability of the specimens.
As indicated by the permeability results, the permeability of healed
specimens was two orders of magnitude lower than that of unhealed
specimens, which suggests that the cracks in healed specimens were
compactly sealed by mineralization products.
3.5. Healing of concrete cracks
When the biocapsules in the concrete broke along with specimen
fracturing, the moisture and oxygen in the air entered through the cracks
and activated the bacterial spores in the biocapsules. The germinated
bacterial spores then engaged in metabolic activity and generated ure­
ase. Under the action of urease, urea decomposed, generated minerali­
zation products, and thus, healed the concrete cracks. The mechanism of
self-healing is presented in Fig. 11.
This study explored the effectiveness of biocapsule dosage for pro­
moting crack-healing in cement specimens by considering the activity of
the bacterial spores in the biocapsule, the content of the biocapsule, and
the setting of different curing conditions. C3% and C5% crack widths of
the specimens were 450 μm and 512 μm, respectively. According to the
results, under the same biocapsule content and curing time, there was no
clear difference in crack-healing in specimens in groups C3% and C5%
and in specimens containing no biocapsules (R, the crack width was 150
μm), which suggests that the specimens in groups C3% and C5% had
extremely low healing rates or even no healing at all. The cracks of CS3%
and CS5% specimens were 550 μm and 504 μm, respectively, and their
cracks were able to heal, and their healing efficiency increased signifi­
cantly with an increase in curing time. In the three different curing
schemes, the specimens under wet–dry curing cycles in DM had the
highest crack-healing efficiency, followed by those cured in DM, while
those cured in water had the lowest healing efficiency. Fig. 12 shows the
healing effect of the specimens in groups CS3% and CS5% under wet–­
dry curing cycles in DM. There was no clear healing in groups CS3% and
CS5% in the first 7 d, but at day 14, mineralization products had filled up
the cracks and healed almost all of them. On day 21, the cracks in the
Fig. 11. Mechanism of biocapsule self-healing concrete cracks.
8
Cement and Concrete Composites 113 (2020) 103718
specimens were completely healed.
Considering the lack of clear healing in the specimens in groups R,
C3%, and C5%, this study only discusses the healing efficiencies of the
specimens in groups CS3% and CS5% under different curing conditions.
As shown in Fig. 13, under the same curing scheme, the healing effi­
ciency of the specimens increased with continued curing. After 21 d of
curing, the maximum healing efficiencies of the specimens cured in
water, the specimens cured in DM, and the specimens under wet–dry
curing cycles in DM in groups CS3%/CS5% were 22.68%/34.51%
(Fig. 13a), 83.24%/90.12% (Fig. 13b), and 96.67%/100% (Fig. 13c),
respectively. Unlike the results by Wang et al. [36], the crack healing
efficiency of the specimen during maintenance in water was lower than
in DM. The reason for the discrepancy is that in the study by Wang et al.
the nutrients, urea, and calcium sources required by the bacteria were
added directly to the mortar, and the microcapsules contained only
bacterial spores. In this study, bacterial spores and their nutrients, urea
and calcium source organisms were encapsulated in biocapsules. The
amount of urea and calcium source exposed after the rupture of the
biocapsule was much lower than when they were directly added to the
mortar. The higher healing efficiency of curing in DM may be due to the
supplementation of urea and calcium sources in the biocapsules by urea
and calcium sources in the DM. In the mortar specimens cured in water,
the nutrients and precipitation media (CaCl2 and urea) in the capsules
diffused into the water and caused the content of precipitation media
around the biocapsule to decline. As a result, bacterial spores were not
able to decompose enough urea to generate CO23 after activation, and
the quantity of mineralization products was insufficient to fill up cracks.
Further research is needed on the self-healing effect of adding bacterial
nutrients, urea, and calcium sources directly to the cracks in the mortar.
The healing efficiency of the specimens under wet–dry curing cycles in
DM was higher than that of the specimens that remained soaked in DM.
This is because, during dry curing in the former case, bacteria attached
to the specimen surface came into contact with sufficient O2, engaged in
increased metabolic activity, excreted more urease, and generated suf­
ficient mineralization products.
M. Wu et al.
Fig. 12. Healing of specimen cracks in cured by dry-wet cycling in DM.
4. Discussion
4.1. Biocapsule properties
The biocapsule had a certain waterproofness and high storage sta­
bility, and the bacterial spores sealed within it were bioactive.
The raw materials for the biocapsule walls were carbide slag, fly ash,
sodium silicate, and blast-furnace slag. The carbide slag stimulates the
production of calcium silicate hydrate (C–S–H) gel and ettringite (AFt)
crystals [48] from fly ash and blast-furnace slag through hydration. The
SEM images in Fig. 14 show the biocapsule walls to have a relatively
9
Cement and Concrete Composites 113 (2020) 103718
compact internal structure after hydration. Fig. 14a shows the
needle-like substance AFt, and Fig. 14b shows the flocculent substance
C–S–H gel. In addition, during biocapsule preparation, a protective film
of sodium silicate was added to the outside of the biocapsule, which
further enhanced its waterproofness.
In practical engineering applications, concrete generally does not
experience immediate cracking after pouring, but it is subsequently
inevitably subject to rain and other severe weather. The ability of bio­
capsules on the concrete surface to maintain their integrity under the
action of wet–dry curing cycles is thus essential for their ability to heal
concrete cracks. Herein, the biocapsule was first directly soaked in
M. Wu et al. Cement and Concrete Composites 113 (2020) 103718

Fig. 13. Cracks healing efficiency of CS3% and CS5% specimens under different curing conditions (a: curing in water; B: curing in DM; C: dry-wet cycle curing
in DM).

Fig. 14. SEM images of wall material hydration.

distilled water, and then, taken out and dried to test its storage stability.
After nine soaking–drying cycles, the biocapsule mass only declined by
4%. Notably, the laboratory test conditions for the storage stability of
the biocapsule were far more severe than those experienced by the
biocapsule in reality, as it was subjected to unavoidable artificial stresses
during the test.
According to the mineralization activity test, sealed bacterial spores
remained active. That is to say, in the case of concrete cracking and
biocapsule-breakage, bacterial spores can germinate and grow in a
certain environment and can then metabolize and mineralize calcium
carbonate precipitate. When the biocapsule was directly added into the
culture solution containing Ca2þ ions, the change in the Ca2þ
concentration shows that bacterial spores began to germinate and grow
after 60 h, which is consistent with the germination time of the bacterial
spores encapsulated by microcapsules prepared by Wang et al. [36] with
melamine. The above results indicate that the biocapsule prepared in
this study has suitable properties to be applicable for the self-healing of
concrete cracks.
4.2. Compatibility between biocapsule and mortar
Addition of the biocapsule into mortar affected the mechanical
properties of the concrete, as manifested by the fact that the mechanical
properties of the concrete were enhanced when the dosage of the

10
M. Wu et al.
biocapsule was less than 5%. However, when its dosage exceeded 5%, a
negative influence was exerted on the mechanical properties of the
concrete. This is because the sodium silicate on the biocapsule surface
engaged in a hydration reaction with cement mortar, and the hydration
products of the biocapsule and cement mortar blended with each other.
Because of the solid structure of the biocapsule, when its dosage was
low, it could fill up the pores in mortar specimens, making the mortar
specimens more compact, and increase their strength. However, when
its dosage was high, the gaps among biocapsules in the mortar far
exceeded the volume of pores filled through mortar hydration, resulting
in more and bigger pores. As a result, a negative influence was exerted
on the strength of mortar specimens.
In addition, as shown in Fig. 6, when the content of the biocapsule
exceeded 5%, the strength of the specimen decreased significantly,
while an increase in the water absorption of the specimen was relatively
small. Although the biocapsule wall material could participate in the
hydration of the mortar specimen and reduce its internal voids, the
mechanical properties of the specimen were increased within a certain
range of dosage. However, a large content of biocapsule had a great
influence on the strength of the specimen. This is because the strength of
the biocapsule core material was much lower than that of the hydration
product, and when its proportion in the mortar was large, it seriously
affected the strength of the specimen. When the content of the bio­
capsule was 5–11%, the water absorption of the specimen was still lower
than that of the test specimen R, and the water absorption rate did not
exceed the specimen R until the content of the biocapsule reached 13%.
Fig. 15. SEM images and EDS spectrums of mineralized products.
11
Cement and Concrete Composites 113 (2020) 103718
This is because although the internal porosity of the specimen increased
as the amount of biocapsules increased, the biocapsule wall material had
a certain degree of water resistance and could reduce the capillary water
absorption to a certain extent. Moreover, due to the random distribution
of the biocapsules in the specimen, when the content was less than 13%,
there were fewer continuous pores formed in the specimen, which
resulted in a smaller increase in water absorption. On the whole, in this
study, biocapsules have been prepared that can improve the mechanical
properties of mortar to a higher degree than those developed in previous
studies [35–37]. The results of the study of addition of biocapsule into
mortar to understand the self-healing of cracks suggest that the dosage
of the biocapsule should be controlled to within 5%.
When the dosage was lower than 5%, the biocapsule exerted little
influence on the setting time and consistency of mortar. When added
into fresh mortar and subjected to mechanical mixing, the biocapsule
had an extremely high integrity rate. Furthermore, after the failure of
28-d mortar specimens, the biocapsules broke at the sites of cracks. To
sum up, there was high compatibility between the biocapsule and
mortar.
4.3. Micro-morphology of mineralization products
The mineralization products of the specimens in group CS3% were
taken out and examined under SEM and EDS (Fig. 15). As shown in
Fig. 15a, the mineralization products were densely stacked, and they
compactly filled the concrete cracks. These products could also prevent
M. Wu et al.
external harmful substances from invading the concrete and negatively
impacting its durability and workability [8]. This also explains why the
water permeability of the specimens declined after healing. Fig. 15d is a
local enlargement of Fig. 15b. According to Fig. 15d and f, the miner­
alization product of the bacteria was calcite-type calcium carbonate, and
calcium carbonate crystals were linked up by incompletely formed
crystals. Figs. 15e and f present the EDS spectra at the green circle in
Fig. 15c and d, respectively. As can be seen from Fig. 15e, in addition to
the Ca, C, and O elements, there were Na, Cl, N, P, and S elements in the
spectrum. In Fig. 15f, there are only Ca, C, and O elements. Therefore,
we believe that the black spot (ie, the green circle) indicated by the
arrow in Fig. 15c refers to Bacillus cereus. They are believed to have been
present at these locations because the surfaces of the bacterial cells were
electronegative during mineralization, and the Ca2þ in the environment
adsorbed bacterial cells and used bacteria as nucleation sites for calcium
carbonate crystallization.
5. Conclusions
This work uses an inorganic cementitious material to prepare a
biocapsule, explores the properties of the biocapsule and its influence on
the properties of cement mortar, and investigates the effectiveness of
these biocapsules for self-healing of concrete cracks through consider­
ation of the healed crack area and water permeability. The primary
conclusions are as follows:
(1) The biocapsule has good waterproofness and high storage sta­
bility, and the bacterial spores coated by it also have adequate
mineralization activity.
(2) When the dosage of the biocapsule was less than 5%, the setting
time of concrete increased with an increase in dosage. However,
when the dosage exceeded 5%, the setting time declined with an
increase in dosage. The consistency of cement mortar declined
with increased biocapsule dosage.
(3) When the biocapsule dosage was less than 5%, the strength of
concrete specimens increased with an increase in dosage. How­
ever, when the dosage exceeded 5%, their strength declined with
an increase in dosage. That is, the optimum dosage of the bio­
capsule was 5%.
(4) During cement mortar mixing, the biocapsule satisfactorily
maintained its integrity; in the case of breakage of concrete
specimens, the biocapsule also broke.
(5) Specimens with a biocapsule dosage of 5% had the lowest water
absorption; the water absorption of specimens with dosages
exceeding 5% increased with an increase in dosage.
(6) Concrete with the biocapsule as an additive could self-heal cracks
150–550 μm in width; specimens cured in DM exhibited better
self-healing (healing efficiency ¼ 83.24–90.12%) than that of
specimens cured in water (healing efficiency ¼ 22.68–34.51%).
In particular, specimens under wet–dry curing cycles in DM
exhibited best self-healing (healing efficiency ¼ 96.67–100%).
Compared with reference specimens (R), the permeability of
specimens containing the biocapsule declined by about two or­
ders of magnitude after healing.
Declaration of competing interest
The authors declared that they have no conflicts of interest to this
work.
Acknowledgement
This work was supported by the National Key R&D Program of China
[grant number: 2018YFC0807900]; the National Natural Science
Foundation of China [grant number: 51674038 and 51874193]; Taishan
Scholars Project Special Funding [grant number: TS20190935]; the
12
Cement and Concrete Composites 113 (2020) 103718
Shandong Province Natural Science Foundation [grant number:
ZR2018JL019 and ZR2017PEE024]; Qingchuang Science and Technol­
ogy Program of Shandong Province University [grant number:
2019KJG008]; the Shandong Province Science and Technology Devel­
opment Plan [grant number: 2017GSF220003]; the Key Program of the
National Natural Science Foundation of China [grant number:
51934004]; Scientific Research Foundation of Shandong University of
Science and Technology for Recruited Talents [grant number:
2017RCJJ010 and 2017RCJJ037]; Shandong Province First Class Sub­
ject Funding Project [grant number: 01AQ05202]; the SDUST Research
Fund [grant 2018TDJH102]; Taishan Scholar Talent Team Support Plan
for Advantaged & Unique Discipline Areas.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cemconcomp.2020.103718.
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