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ICSSD 2024 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

Mechanical Properties of Self-Healing Concrete (Bio-Concrete)

Modified with Limestone Powder

O.G. Mark 1, A.K Kamale1, J.K. Jolayemi1

1
Department of Civil Engineering, College of Engineering, Covenant University, km 10, Idiroko
Road, Sango, Ota, Ogun State, PMB 1023, Nigeria.

[email protected]
[email protected]

*
Corresponding email: [email protected]

Abstract. Many advantages over conventional concrete have been demonstrated by the use of bio-concrete, which is created by
adding microorganisms to conventional concrete. Research on the durability and strength of bio-concrete that uses limestone
powder as a filler instead of regular Portland cement is lacking. The aim of this study is to examine the mechanical properties of
bio-concrete made with bacillus subtils as the healing agent and limestone powder as a supplementary cementitious material
(SCM) at percentage replacement levels of 0%, 5%, 10%, and 15%. The materials used in this study were bacillus subtils as the
bacteria, regular Portland cement and limestone powder as the binders, fine and coarse aggregates. The mechanical tests carried
out were sulphate attack, alkaline attack, splitting tensile strength, compressive strength and flexural strength tests. The samples
were subjected to water ponding for three days to induce an initial crack width of no more than 0.3 mm. The samples'
microstructure were characterized using XRF, SEM-EDS, and XRD as well. Based on the data collected, the weight loss, strength,
and crack closure indices for each sample were calculated and compared to the control sample. In comparison to the control, it
was found that the bacteria's presence decreased the samples' crack width as the curing age rose. In addition, the strength,
durability, and microstructure of the self-healing concrete were enhanced by the bacteria and limestone powder, particularly when
the concrete contained 10% limestone powder. Based on this study, it can be inferred that bacillus subtils can cause concrete to
self-heal by forming spores that reduce or completely seal cracks in the concrete. In addition, including limestone powder can
result in concrete that heals more effectively.

Keywords: Bio-concrete, Limestone Powder, Supplementary Cementitious Material, Bacillus Subtils, Ordinary Portland
Cement.

1. Introduction
Globally, 14 billion cubic meters of concrete were used in 2020. By 2050, it is expected that there will
be a whopping 9.8 billion people on Earth, with 68% of them living in cities [1]. As a result, it is
inevitable that concrete use will rise over time, necessitating its sustainability. Although concrete is a
remarkable material for construction, its traditional binder, Portland cement has negative effects on the
surrounding environment because it releases carbon dioxide gas into the atmosphere during the
production process. Carbon dioxide is a greenhouse gas, which traps heat energy from the sun and causes
global warming. The production of cement contributes to greenhouse gas emissions, making up 7% to
8% of global emissions. Approximately one tonne of CO2 is released during the manufacture of one
tonne of cement. This CO2 is created during the burning of fuel and the calcination of raw materials.
Owing to this issue, using limestone powder as a supplementary cementitious material (SCM) to make

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ICSSD 2024 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

concrete in lieu of more cement shows promise. The term "mineral admixtures," or supplementary
cementitious materials (SCMs), refers to materials that are mixed with concrete to improve its
workability, permeability reduction, durability and pumpability. It also reduces the reactivity of alkalis
and improves the concrete's overall hardened properties through pozzolanic, hydraulic or both activities
[2]. Pozzolana is a siliceous or siliceous and aluminous substance that is produced when pozzolanic
activity of supplemental cementitious material reacts with calcium hydroxide (Ca(OH)2)) at room
temperature to produce a compound with binding capabilities. On its own, it has minimal to no cementing
properties.
Limestone powder is a byproduct of sedimentary rock and it is a pozzolanic supplementary cementitious
material that is primarily composed of calcium carbonate (CaCO3), found in the mineral calcite [3]. In
addition to being a common building material, limestone powder is essential to the production of cement
and lime. It is a great resource for many different uses because of its many attributes. It is a fairly soft
rock with a Mohs hardness of 3, making it easy to cut and shape for use in construction [4]. Additionally,
resistant to weathering and erosion, limestone powder is a durable material for floors, walls, and building
facades [5]. LP is an important resource for the production of cement and lime due to its high
concentration of calcium carbonate. When limestone is heated to high temperatures, it undergoes
calcination, converting into carbon dioxide (CO2) and lime (CaO). According to a research [6], the lime
produced can be used to make a number of products, including cement, plaster and mortar. In order to
improve concrete’s properties limestone powder can be added as a mineral during the manufacturing
process. This is a controversial claim because some studies have found the opposite to be true, despite
the majority of research showing that limestone powder can improve the strength and durability of
concrete.

According to a research [7], adding limestone powder to concrete can improve its tensile, flexural, and
compressive strength as well as reduce permeability and lengthen the life of concrete structures.
Additionally, a number of studies have shown that adding limestone powder to concrete strengthens its
properties. When compared to the control mix without limestone powder, the addition of 10% limestone
powder increased the compressive strength of the concrete by 16.45%, according to a study [8].
Similarly, another study [9] found that concrete's compressive strength increased by 7.9% when 10%
limestone powder was added. In addition to increasing concrete's compressive strength, limestone
powder can also increase concrete's flexural and splitting tensile strengths. The effects of limestone
powder on the flexural and splitting tensile strengths of concrete were investigated [10]. It was found
that adding 10% limestone powder increased the tensile strength by 20% and the flexural strength by
15%. Further research [11] stated that when the concentration of limestone powder rose, the compressive
strength decreased. Nevertheless, the negative impact of raising the concentration of limestone powder
was minimal up to 10%. Higher concentrations of limestone powder exhibited a more pronounced
negative impact on the compressive strength of concrete. The length change method is one way to find
out how well concrete specimens resist sodium sulfate attack. An increase in expansion strain indicates
a faster rate of degradation because gypsum and ettringite are being formed. According to a study [11],
for limestone (LS) cement concrete prepared using Type I, Type II, and Type V, respectively, they
showed the measured expansion strain with age up to 525 days.

One problem with conventional concrete, even with SCM added, is that it fractures readily under tensile
stress. Concrete structures deteriorate as a result of liquids entering the fissures on the surface, primarily
because the reinforcement bars corrode. The concrete's service life is shortened by these fissures, and
replacement costs rise accordingly. Various techniques are employed to treat fractures, even though it is
nearly impossible to prevent their formation. Certain methods of treating concrete that are currently in
use, like using chemicals and polymers, have been shown to have risks to human health and the
environment and to only yield temporary solutions. As a result, self-healing concrete fixes the problem
by filling in surface fissures and is a durable and environmentally friendly treatment option. This removes
the need for ongoing, challenging, costly, and time-consuming rehabilitation and restoration of concrete
structures. The strength and durability of the concrete will be improved by using self-healing concrete,
or "bio-concrete," mixed with limestone powder.
Something that can heal itself is said to be self-healing. There are two categories of self-healing
mechanisms in self-healing concrete [12]:
x Autogenous self-healing mechanism: Conventional concrete that uses only regular Portland
cement as a binder is typically batched so that some of the cement is anhydrous. This allows

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ICSSD 2024 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

moisture to seep into the concrete through surface cracks, hydrating the portion of the cement
that is not hydrated [12].
x Autonomous self-healing mechanism: Additionally, concrete can be purposefully made to heal
itself. which typically entails adding microorganisms that can prevent crack formation or
cementitious binding agents. When bio-concrete is cracked and moist, it can initiate the self-
healing process [12].

There are many potential applications for self-healing concrete, such as building new, durable structures
and repairing damaged ones. Self-healing concrete can be used to create maritime structures and other
infrastructure in harsh environments, as well as to repair concrete buildings, roads, and bridges [13].
Using bacteria of the genus bacillus, which can produce ammonia, carbon dioxide, bicarbonate, and
carbonate ions that will precipitate with the calcium ions in the concrete to form calcium carbonate
minerals, bacteria-based self-healing concrete, or "bio concrete," is a type of autonomous mechanism of
self-healing concrete. Calcium carbonate crystals further crystallize to fill in concrete fractures. [12].
The bacteria that are commonly used in bio concrete are bacillus pasteurii, bacillus cohni and bacillus
subtilis, which fill the voids and cracks in the concrete matrix by producing calcium carbonate as a
byproduct of their metabolism. The concrete can then be strengthened and repaired using this calcium
carbonate [12]. By doing this, one can significantly increase the concrete's lifespan and lessen the need
for expensive repairs [13]. According to a reseaarch [14], the microorganisms' production of calcium
carbonate can enhance the concrete's compressive strength by as much as 40%. Moreover, bio concrete
exhibits remarkable resistance to erosion from salt and acid. In general, bio concrete offers a more
environmentally friendly concrete than traditional concrete.

Several studies have looked into the self-healing properties of bacteria concrete, including the variables
influencing the healing process, possible uses for self-healing concrete and the environmental effects of
using materials derived from bacteria [15 -18]. The kind of bacteria or fungi used is one of the main
elements that affects the capacity of bacteria concrete to heal itself. Temperature, humidity, and the
availability of food are examples of environmental variables that have a big impact on how well
bacterium concrete cures itself [15]. Furthermore, adding organic materials like glucose and creating an
environment rich in nutrients can help bacteria in concrete heal themselves [16]. Concrete buildings can
self-heal microcracks up to 0.2 mm wide as a result of non-reacted cement particles in the concrete matrix
hydrating when they come into contact with incoming water [17]. The bacteria-based self-healing
mechanism has been shown to completely mend cracks up to 0.3 mm in width [12]. Calcium carbonate
will form on the concrete surface as a result of the following interaction between the CO2 present and
the calcium hydroxide found in the concrete matrix, as seen in Equation (1):
CO2+ Ca(OH)2 → CaCO3 + H2O (1)

There is a limited amount of CO2 available in this scenario, so the production of calcium carbonate is
limited. Since Ca(OH)2 is a soluble mineral, when water seeps into the fracture, it dissolves and diffuses
out through leaching. The self-healing process in bacteria-incorporated concrete is far more effective
because the bacteria actively convert calcium nutrients, as seen in Equation (2).
Ca(C3H5O2)2 + 7O2 → CaCO3 + 5CO2 + 5H2O (2)

In this case, calcium carbonate is directly produced by a microbial metabolic activity. This method results
in an efficient bacteria-based fracture sealing mechanism. In an alkaline environment, the ureolytic
bacteria bacillus subtilis JC3 can precipitate CaCO3 by converting urea into ammonium and carbonate
[18]. While the ammonia breakdown of urea raises the pH locally and promotes the microbiological
deposition of carbonate as calcite crystals in an environment rich in calcium, sealing the fissure, the pH
of the concrete remains constant.
Studies [17, 19] suggested that high-quality aggregates, low water-cement ratios, and pozzolanic
ingredients like fly ash or silica fume may help boost the strength and durability of bio-concrete. The
values of bacterial concrete's ultimate compressive strength, ultimate split tensile strength, and ultimate
flexural strength rise up to 10 milliliters of bacteria. After that, however, as the number of bacteria in the
concrete increased, the value of the ultimate compressive strength gradually decreased. The strongest
bacterial concrete at its maximum value was produced by 10 milliliters of bacteria [20]. According to a
research [21], the strength properties of bio concrete are enhanced by an increase in the volume of

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ICSSD 2024 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

bacteria present in the concrete. Given their direct influence on the longevity and functionality of the
concrete structure, bio-concrete's durability qualities are a crucial factor to take into account. Many
research works have examined the durability characteristics of bio-concrete, encompassing its ability to
withstand chemical attacks, freeze-thaw cycles, and corrosion. The enhanced corrosion resistance of bio-
concrete is one of its main benefits. Research has demonstrated that adding bacterial species like bacillus
subtilis and bacillus cohnii to the concrete mix raises the pH of the mixture, lowering the chance of
corrosion in the metal reinforcing bars (rebar) and other components of the concrete structure [22, 16].
Furthermore, bacterial species can help fill in gaps and fractures in the concrete matrix by forming
calcium carbonate and other minerals. This lowers the risk of corrosion by preventing the entry of water
and other corrosive agents [12].
Bio-concrete's durability has also been evaluated in freeze-thaw cycles. Research has demonstrated that
when compared to conventional concrete, bio-concrete containing bacterial species like bacillus cohnii
and sporosarcina pasteurii exhibits superior resistance to freeze-thaw cycles [17, 19]. This is because
certain bacterial species have the capacity to produce calcium carbonate, which can aid in filling in the
gaps and fissures that arise throughout the freeze-thaw cycle. One other crucial feature of durability is
bio-concrete's ability to withstand chemical attacks. Numerous investigations have looked into how
resistant bio-concrete is to chemical assaults, such as exposure to sulfates and acids. Findings have
demonstrated that, in comparison to conventional concrete, bio-concrete containing bacterial species like
bacillus subtilis and bacillus megaterium shows superior resistance to acid and sulfate attacks [22, 19].
Bacterial species can neutralize acidic compounds and stop sulfates from penetrating the concrete matrix
by forming calcium carbonate and other minerals. Apart from the type of bacteria, other elements like
the mix composition and curing conditions can also affect how durable bio-concrete is. Research has
indicated that the durability of bio-concrete can be enhanced by utilizing pozzolanic materials, premium
aggregates, and low water-to-cement ratios [17, 19].
Based on the percentage of compressive strength lost, a reasercher [16] examined the sulphate attack,
acid attack, and alkalinity attack characteristics of bio concrete that contained various bacteria at
particular ranges of bacteria volume. As the volume of the bacteria in the various bio concretes he studied
increased to 20 milliliters, he found that the durability properties significantly improved. There are still
unknowns regarding the use of limestone powder in bio-concrete, despite the fact that it has been the
subject of much research regarding its use as a filler in cement and concrete. One of the main concerns
of this research is the impact of limestone powder on the self-healing properties of bio-concrete. To
enhance the use of limestone powder in the production of bio-concrete and to comprehend the
fundamental mechanisms underlying these effects, more research is needed. In order to ensure that bio-
concrete containing limestone powder performs as intended over an extended period of time, its long-
term durability must also be evaluated.

This article concentrated on the experimental methods used to examine the durability and strength
characteristics of bio-concrete that substitutes regular Portland cement with powdered limestone. To do
this, bacillus subtilis—which can withstand the high alkalinity pH levels of the concrete it would be
placed in, was isolated from a soil sample near a cement manufacturing industry. It was cultured using
calcium lactate being prepared in a petri-dish at the required temperature and a slanted angled rather than
horizontal to accommodate more bacteria colonies on it and can be transferred to a liquid nutrient broth
shaken in orbital shaker to grow more colonies faster. In order to compare and contrast and determine
the best blend for strength and durability, limestone powder was added to ordinary Portland cement at
percentages of 0%, 5%, 10%, and 15% in the various concrete blends created from the aforementioned
combinations both with and without the bacteria. Tests for compressive strength, splitting tensile
strength, and flexural strength were used to examine the strength characteristics. Sulphate attack and
alkaline attack tests were used to examine the self-healing concrete's durability characteristics. Using
energy dispersive x-ray spectroscopy (EDXS), scanning electron microscopy (SEM), and x-ray
diffraction analysis (XRD) and x-ray fluorescence (XRF), the microstructure of the different concrete
blends was characterized.
By producing more sustainable and resilient concrete materials that can lower maintenance costs and
enhance the longevity of concrete structures, thereby improving urban safety and livability, this study
contributes to SDG 9 (Industry, Innovation, and Infrastructure) and SDG 11 (Sustainable Cities and
Communities).

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ICSSD 2024 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

2. Experimental Study
2.1. Materials

The materials utilized in this study were 42.5R Portland cement and limestone powder (LP) as the
binders, river sand as the fine aggregate and granite as the coarse aggregate. Tap water, potable enough
for drinking was used to mix the constituents appropriately. The bacteria (the self-healing agent) used in this
study was bacillus subtilis

2.2 Design of the Concrete Mix

Batching was carried out by weight in this study. A 1:2:4 prescribed mix and a 0.5 water/cement ratio
were applied. In addition, different concrete blends with and without bacteria were created by
substituting limestone powder for regular Portland cement at 0%, 5%, 10%, and 15%. The concrete
blends were then allowed to cure by water-ponding. The proportioning of the mix is as indicated in Table
1. For every regime (one sample containing bacteria and one sample not), three (3) samples were made,
and the average of the three samples' results was used. The sample without bacteria and without
limestone powder (conventional concrete) was the control sample. In total, eight (8) distinct types of
concrete combinations were produced. The concrete mixtures' names and descriptions are listed in Table
1. As an example, "NC0,100" denotes a normal concrete (without bacteria) that contains 0% limestone
powder and 100% Portland cement; "NC5,95" denotes a normal concrete (with bacteria) that contains
5% limestone powder and 95% Portland cement; "SHC0,100" denotes a self-healing concrete (with
bacteria) that contains 0% limestone powder and 100% Portland cement; and "SHC5,95" denotes a self-
healing concrete (with bacteria) that contains 5% limestone powder and 95% Portland cement.

Table 1: Design mix of the bio concrete.

Concrete Weight of Weight of Weight of Weight of Volume of
Type Cement (kg) Limestone fine Coarse bacillus
Powder (kg) Aggregate Aggregate subtilis (ml)
(kg) (kg)
NC0,100 7.2 0 14.4 28.8 0
NC5,95 6.84 0.36 14.4 28.8 0
NC10,90 6.48 0.72 14.4 28.8 0
NC15,85 6.12 1.08 14.4 28.8 0
SHC0,100 7.2 0 14.4 28.8 10
SHC5,95 6.84 0.36 14.4 28.8 10
SHC10,90 6.48 0.72 14.4 28.8 10
SHC15,85 6.12 1.08 14.4 28.8 10

2.3 Induction of Cracks on the Concrete Samples

The concrete constituents were cast into: steel molds of 100 mm x 100 mm x 100 mm cubes for the
compressive strength test and the durability tests; plastic molds of 100 mm diameter x 200 mm length
cylinders for the splitting tensile strength test and; wooden molds of 100 mm breadth x 100 mm depth x
500 mm length beams for the flexural strength test. The concrete samples in the molds were demolded
after 24 hours and cured for 3 days. An initial crack width of between 0.2 mm - 0.3 mm was induced in
each of the samples, according to a study [20], with the use of a compression machine. A well-calibrated
magnifying lens was used to measure the initial crack width. The pre-cracked concrete samples were
then returned into the water for curing, till the days of testing. At the days of testing and before crushing,
the crack closure index of each sample was determined as follows in Equation (3). Higher crack closure
index means that the concrete has a higher self-healing capacity, due to the presence of the bacteria.
Inferring that the cracks were completely or almost completely healed.
௜௡௜௧௜௔௟௖௥௔௖௞௪௜ௗ௧௛ሺ௠௠ሻ
Crack closure index = ௙௜௡௔௟௖௥௔௖௞௪௜ௗ௧௛ሺ௠௠ሻ
(3)

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IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

2.4 Preparation of the Hardened Pre-cracked Samples

Forty-eight (48) cube samples of the pre-cracked, cured normal concrete and forty-eight (48) cube
samples of the pre-cracked, cured self-healing concrete (as described in section 2.3) were brought out of
the curing tank at each curing age (7 days, 14 days, 21 days and 28 days of curing by water ponding),
for the compressive strength test, making a total of ninety-six (96) cube samples brought out for this test.
On each day of testing, triplicate samples of each concrete type were brought out of the curing tank, air-
dried and weighed. With the use of the magnifying lens again, the final crack width on each sample was
observed, measured and recorded. The crack closure index of each sample was determined as given in
Equation (3), while the average of the triplicate samples of each concrete type was taken as the average
crack closure index of each concrete type and at each curing age. The same procedure was done on the
samples for the splitting tensile strength, except that twelve (12) cylindrical samples of the pre-cracked,
cured normal concrete and twelve (12) cylindrical samples of the pre-cracked, cured self-healing
concrete (as described in section 2.3) were used and the curing age was at 28 days only. Similarly, for
the flexural strength test, twelve (12) reinforced concrete beams of the pre-cracked, cured normal
concrete and twelve (12) reinforced concrete beams of the pre-cracked, cured self-healing concrete (as
described in section 2.3) were used and the curing age was at 28 days only. The procedure was also
repeated for the durability tests (sulphate attack test and alkaline attack test). For each of the durability
tests, twenty-four (24) cube samples of the pre-cracked, cured normal concrete and twenty-four (24)
cube samples of the pre-cracked, cured self-healing concrete (as described in section 2.3) were used and
the curing ages were 28 days and 56 days.

2.5 Tests on the Strength Properties of the Hardened Bio Concrete

2.5.1 Compressive Strength Test
The compressive strength test was carried out according to standard code [23], using the compression
machine of Model YES-2000 with a 2000 kN maximum capacity. The cube sample was then placed
between the bearing surfaces of the compression machine. The metal plates of the machines or specimen
block were kept free of any loose grit or debris. The concrete cubes were positioned on bearing plates
with their centers of force precisely lined up with the resting machine plates. The force was applied
axially to the specimen without applying any shock. The specimen started to break in a specific area as
a consequence of the constant application of stress, until the specimen disintegrated completely. To
calculate the specimen's compressive strength, the highest force borne by the specimen was divided by
the cross-sectional area of the specimen cubes as seen in Equation (4). The average of the triplicate
samples of each concrete type was taken as the average compressive strength of each concrete type and
at each curing age. To further assess the self-healing capacity of the concrete samples, the crack closure
index was determined, using Equation (3). Higher crack closure index indicates a higher self-healing
capacity of the concrete.
ெ௔௫௜௠௨௠௟௢௔ௗ௔௧௙௔௜௟௨௥௘ሺேሻ
Compressive strength of concrete (N/mm2) = ஼௥௢௦௦ି௦௘௖௧௜௢௡௔௟௔௥௘௔௢௙௧௛௘௖௨௕௘௦௣௘௖௜௠௘௡ሺ௠௠మ ሻ (4)

2.5.2 Splitting Tensile Strength

The splitting tensile strength was carried out according to standard code [24], using the compression
machine described in section 2.5.1. The cylinders were placed in the compression machine with two
metal bars on either side, and the maximum applied load was recorded. The splitting tensile strength of
the concrete was determined using Equation (5). The average of the triplicate samples of each concrete
type was taken as the average splitting tensile strength of each concrete type and at each curing age. To
further assess the self-healing capacity of the concrete samples, the crack closure index was determined,
using Equation (3). Higher crack closure index indicates a higher self-healing capacity of the concrete.
ଶ୔
 ൌ (5)
஠୐ୈ

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IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

where,
T is the Splitting tensile strength (N/mm2),
P is the Maximum applied load (N),
D is the Diameter of the specimen (mm),
L is the Length of the specimen (mm)

2.5.3 Flexural Strength Test

The flexural strength of the reinforced concrete beam samples was tested via fully automated transverse
flexural testing machine, Model 2A8580, according to standard code [25]. Each sample was positioned
in the flexural testing machine on two roller supports, and the load was applied perpendicularly at the
middle of the beam till the sample failed. The maximum load at failure was noted and recorded. The
flexural strength was determined as given in Equation (6). The average of the triplicate samples of each
concrete type was taken as the average flexural strength of each concrete type and at each curing age. To
further assess the self-healing capacity of the concrete samples, the crack closure index was determined,
using Equation (3). Higher crack closure index indicates a higher self-healing capacity of the concrete.

Flexural Strength = (3PL)/(2bd2) (6)

where,
P is the maximum load,
L is the distance between the supports,
b is the width of the specimen,
d is the depth of the specimen

2.6 Tests on the Durability Properties of the Hardened Bio Concrete

2.6.1 Sulphate Attack Test

The test was carried out in accordance with standard code [26]. 5% sodium sulfate (Na2SO4) salt was
dissolved in water to create a sodium sulfate solution and stirred thoroughly in the water, until completely
dissolved, with a clear water as a confirmation. The initial weight of the triplicate pre-cracked concrete
samples set apart for the sulphate attack test was taken, recorded and averaged as W1 (kg). They were
then submerged in the sulphate solution for 28 and 56 days. On the day of testing, the triplicate pre-
cracked samples were removed from the sulphate solution, rinsed under a running tap water and allowed
to air-dry. The final crack width on each sample was taken. Likewise, the average final weight of the
sample was taken and recorded as W2 (kg). The average percentage weight loss was calculated as seen
in Equation (7). The samples were finally crushed, using the compression machine described in section
2.5.1, to determine their compressive strength after immersion, using Equation (4). The crack closure
index using Equation (3), the average compressive strength value using Equation (4) and the average
percentage weight loss using Equation (7) were determined, to assess the self-healing capacity of the
concrete produced in this research, when subjected to a harsh sulphate environment. The smaller the
average percentage weight loss, the higher the self-healing capacity of the concrete tested, in a harsh
sulphate environment.
ௐଵିௐଶ
Average percentage weight loss (%) = ௐଵ
ܺͳͲͲ (7)

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ICSSD 2024 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1492 (2025) 012026 doi:10.1088/1755-1315/1492/1/012026

where,
W1 is the average initial weight of the pre-cracked sample before immersion, in kg
W2 is the average final weight of the pre-cracked sample after immersion, in kg.

2.6.2 Alkaline Attack Test

The resistance of the bio concrete investigated in this research to alkaline environment was evaluated,
by carrying out alkaline attack test, according to standard code [27]. The methodology was similar to
that of the sulphate attack test, as described in section 2.6.1, except that the solution used in this test is
an alkaline solution whereby, 5% concentration of sodium hydroxide (NaOH) was mixed with water.

2.7 Microstructural Investigation

2.7.1 Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDXS)
The 28th day crushed samples of each concrete type were used to determine the morphology of the
concrete structure, via SEM. The elemental composition of the 28th day crushed sample of each concrete
type was analyzed using energy dispersive X-ray spectroscopy (EDXS).
2.7.2 X-Ray Diffraction (XRD)

This is one of the microstructural studies which was done on the concrete sample, mainly to determine
its mineralogy. It is crucial as the attributes of the concrete samples are all a consequence of their distinct
microstructural formations.

3. Results and Discussion

The physical and chemical characteristics of the binders used in this study are presented and discussed
in this section. Also, the results of the strength and durability properties, microstructure of the various
concrete types tested in this research are presented and discussed in this section.

3.1. Physical and Chemical Characteristics of the Binders

The physical and chemical properties of the binders (42.5R Portland cement and limestone powder) used
in this study are as seen in Table 2 and Table 3, respectively. Table 2 shows that the cement satisfied the
standard [28] with a specific gravity of 2.32, fineness of 5%, initial setting time of 50 minutes, final
setting time of 550 minutes, and standard consistency of 26%. According to standard code [29], the
primary oxide composition of the cement is Al2O3, SiO2, Fe2O3, and CaO, all of which were found in
Table 3. Its ignition loss was 0.08, meeting standard code’s maximum requirement of 10.0 [29].
According to Table 2, LP had a specific gravity of 2.44, which was higher than the cement used, a
fineness of 4.3%, a 90-minute initial setting time, a 580-minute final setting time, and a 30% consistency.
Similarly, its physical attributes were within the acceptable range for cement [28]. As a result, limestone
powder can be added to cement. It was determined by XRF analysis that its chemical properties, as listed
in Table 3, were within the acceptable range for binders [30] and that it could be used in concrete. It was
found that the limestone powder's Al2O3+SiO2+Fe2O3(%) was 78.64%, which was higher than 70% and
the nominal value specified in the standard [31] for pozzolanic materials. As per Ahmet's report, the
limestone powder satisfied the pozzolanic criteria because its MgO (%) was less than 5%, specifically
1.62%. The limestone powder's SO3(%) and Loss of Ignition (LOI) were, respectively, 0.33% and 9.97%.
As per Ahmet's report, the Limestone Powder's SO3(%) and LOI should be less than 3% and 10%,
respectively. Therefore, the Limestone Powder satisfied the requirements to be considered a pozzolanic
material that is suitable enough to be used in concrete.

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Table 2: Physical characteristics of the binders used.

Physical Characteristics 42.5R PC LP Standard [28]
[unit]
Consistency [%] 26 30 25 – 35
Initial setting time [minutes] 50 90 30 minimum
Final setting time [minutes] 550 580 600 maximum
Fineness [%] 5 4.3 10 maximum
Specific gravity 232 2.44 2.30 – 3.25

Table 3: Chemical composition of the binders used.

Oxide 42.5R PC [%] Standard [%] LP [%] Standard [%]
composition [29] [30]
Al2O3 4.98 4 – 12 11.10 10 – 17
SiO2 20.09 18 - 26 44.57 31 - 45
Fe2O3 1.80 1–6 22.97 10 - 40
Na2O 0.21 ≤ 1.0 0.44
CaO 64.19 58 – 66 5.52 4 - 20
K2O 0.53 ≤ 1.0 0.30 -
MnO - - 9.65 -
MgO 1.92 1–3 1.62 -
SO3 1.80 0.5 – 2.5 0.33 -
P2O3 - - 0.08 -
LOI 0.08 10 maximum 9.97 10 maximum

3.2. Strength Characteristics of the Hardened Bio-Concrete Samples

3.2.1. Compressive strength

The results of the average compressive strength and the average crack closure index of the various
concrete types, are presented in Fig. 1 and Fig. 2, respectively. From the average compressive strength
displayed in Fig. 1, an increase in compressive strength of the concrete from curing ages 7 days to 28
days was noted. This increment was due to the hydration of the cement and limestone powder (binder),
as well as the increased production of calcium carbonate by the bacteria during curing. The highest
strength was obtained after 28 days of curing and 10% replacement by limestone powder. These strengths
were 30.12 N/mm2 (with bacteria - SHC10,90) and 28.72 N/mm2 (without bacteria - NC10,90). As
explained by several researchers [32, 33, 34, 35], the higher strength results of NCs with LP and the
bacteria occurred due to the chemical and physical effects of the LP and the biological effects of the
bacteria. The small LP particles filled the voids between cement particles, which may have enhanced
packing density, decreased the essential void structure and therefore increased the compressive strength
at early ages [36]. LP can improve early strength by acting as nucleation sites for CH and C–S–H
products, leading to accelerated cement hydration [37]. According to a recent study [38], LP can also
react with cement’s C3A phase to create mono-carboaluminate hydrates, which increases early
compressive strength slightly. These findings suggest that the percentage replacement has a considerable
impact on crack index values. Larger crack index values, which are connected with larger replacement
percentages in Fig. 2, showed reduced final crack width. The ability of cementitious mortar to cure pre-
existing fractures is improved by the use of the bacillus subtilis bacteria immobilized on Nano- or Micro-
additives [39, 40]. This explains the outcomes presented in Fig. 2. Calcium carbonate may form as a
result of limestone generation inside the concrete matrix, aiding the material's capacity to self-heal [41],
which justifies the results shown in Fig. 1 and Fig. 2. There is evidence that cementitious composites'
capacity for self-healing can be improved by using bio immobilized limestone powder [42].

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35

Average Compressive Strength [N/mm2]

30

25

20

15

10

0
7 14 21 28

Curing Age [Days]

NC means Normal Concrete, SHC means Self-healing Concrete

NC0,100 NC5,95 NC10,90 NC15,85
SHC0,100 SHC5,95 SHC10,90 SHC15,85

Figure 1: Average compressive strength of the concrete types.

3.5
Average Crack Closure Index

2.5

1.5

0.5

0
7 14 21 28
Curing Age [Days]

NC means Normal Concrete, SHC means Self-healing Concrte

NC0,100 NC5,95 NC10,90 NC15,85

SHC0,100 SHC5,95 SHC10,90 SHC15,85

Figure 2: Average crack closure index of the concrete types.

3.2.2. Splitting Tensile Strength

The results of the average splitting tensile strength and the average crack closure index of the various
concrete types, are presented in Fig. 3 and Fig. 4, respectively. From the average splitting tensile
strengths displayed in Fig. 3, the concrete's splitting tensile strength initially declined with the rise in the
percentage replacement of limestone powder up until about 10% where it gained maximum strength
before declining at 15%. This increment in strength was due to the improvement of the cohesion and
binding properties in the concrete as well as the pozzolanic nature of the limestone powder. The highest
strength obtained after 28 days of curing was at 10% replacement were 2.51 N/mm2 (with bacteria -
SHC10,90) and 2.45 N/mm2 (without bacteria - NC10,90). The crack closure index tends to grow along

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with the limestone powder percentage replacement, as seen in Fig.4. This implies that increased
replacement rates may result in smaller cracks or less crack propagation, which would increase the crack
index values. The values from Fig. 4 shows that at 15% replacement with limestone powder for self-
healing concrete (bio concrete -SHC15,85), the crack index was higher, giving an 80%^rise over the
control (NC0,100) which shows a great reduction in the crack. This means that over time, the bacterial
self-healing concrete specimen’s crack will keep closing with a broader margin than conventional
concrete.

3
Average Splitting Tensile Strength [N/mm2]

2.5

1.5

0.5

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Curing Age

Figure 3: Average splitting tensile strength of the concrete types.

5
Average Crack Closure Index

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Curing Age

Figure 4: Average crack closure index of the concrete types.

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3.2.3. Flexural Strength

The results of the average flexural strength and the average crack closure index of the various concrete
types, are presented in Fig. 5 and Fig. 6, respectively. From the average flexural strengths displayed in
Fig. 5, the percentage substitution of limestone powder led to an increase in the concrete's flexural
strength. This increment was due to the improvement of the cohesion and binding properties in the
concrete as well as the pozzolanic nature of the limestone powder [30, 31]. The highest strength obtained
after 28 days of curing were 4.1 N/mm2 (samples with bacteria – SHC10,90) and 3.94 N/mm2 (samples
without bacteria – NC10,90), at 10% percentage replacement with limestone powder. A significant effect
of the percentage replacement of limestone powder as well as the curing conditions on the flexural
strengths of the concrete was noticed. This was noticed through the interaction effect plot of Fig. 5. This
showed that the increase in the percentage replacement of limestone powder from 0 to 15% resulted in
an increased flexural strength of the concrete [43]. Due to varying replacement percentages, the flexural
strength values for both the normal concrete and the self-healing concrete differed. As replacement
material percentage increased from 0% to 5%, flexural strength in both sets was seen to decline. Beyond
5%, a mixed pattern was seen, with either a rise in or a reduction in flexural strength, with 10% being
the maximum number. Self-healing concrete (bio concrete) and normal concrete are contrasted in Fig. 5,
with the former having superior strength for all replacement values. The findings are supported by
research [44,45] that found that after 28 days of curing, compressive strength (22%), split tensile strength
(16%), and flexural strength (11%) of bio-concrete mixes were superior to normal concrete. Larger
replacement percentages of LP (10% and 15%) and introduction of bacillus subtils may aid in reducing
the fracture in the flexural strength test, according to the information presented in Fig. 6, with SHC15,85
having the highest crack closure index of 71.4% rise over the control (NC0,100). This shows that adding
more limestone powder and introducing bacterial to concrete might increase its ability to withstand
flexural stress and stop cracks from spreading.

4.5

4
Average Flexural Strength [N/mm2]

3.5

2.5

1.5

0.5

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Curing Age

Figure 5: Average flexural strength of the concrete types.

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Average Crack Closure Index 3.5

2.5

1.5

0.5

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Curing Age

Figure 6: Average crack closure index of the concrete types.

3.3. Durability Characteristics of the Hardened Bio-Concrete Samples

3.3.1. Sulphate Attack

The results of the average compressive strength, the average weight loss index due to sulphate attack and
the average crack closure index of the various concrete types, after 28 and 56 days of immersion inside
sodium sulphate (Na2SO4), are presented in Fig. 7, Fig. 8 and Fig. 9, respectively. Based on the data from
Fig. 7, it can be concluded that the compressive strength is influenced by the proportion of material
replacement and the inclusion of bacillus subtils. The concrete with a 10% replacement in the regular
concrete sample (NC10,90) had the maximum compressive strength, giving a rise of 26.35% over the
control (NC0,100), at 56 days of immersion. This suggests that the replacement material increases the
concrete's resistance to sulfate attack. The results also show that a self-healing concrete (bio concrete)
with 10% replacement (SHC10,90) had the maximum compressive strength, giving a rise of 32.52%
over the control (NC0,100), at 56 days of immersion. According to a study [46], cement's resistance to
sulphate corrosion rises as its amount of limestone powder increases. Likewise, another study [16]
revealed that inclusion of bacteria in concrete can greatly improve the compressive strength of such
concrete. The results shown in Fig. 8 revealed that after being submerged in the Na2SO4 solution, the
weight of both the Self-Healing concrete (bio concrete) and the normal concrete samples decreased
significantly. In general, the higher the percentage replacement, the lower the weight loss. The
percentage drop varied from percentage to percentage. Inferring from the data presented in Fig. 8, the
reduction in weight varies as it shows there is better durability properties against the sodium sulfate
attack, exhibited by samples that contained more limestone powder as its binder compared to the
conventional concrete. A better durability property against the sodium sulfate attack was obtained with
SHC10,90, having 4.11% reduction in weight loss at 56 days of immersion in the sodium sulphate over
the control (NC0,100). This implies that both LP and bacillus subtils may help in controlling weight loss
in concrete, when exposed to harsh conditions like a sulphate environment. This finding, justified the
claims made by previous studies [7, 8], that adding limestone powder to concrete improves its resistance
to sulphate attack, likewise the claims made by previous studies [15 - 19] that introduction of bacteria
bacillus subtils and bacteria conhi improves the durability properties of concrete, especially its resistance
to sulphate attack. The weight index data, on the other hand, revealed that the weight loss was not
completely restored. The cracks induced as aforementioned exhibited more self-healing ability than the
samples without bacteria. Overall, the data in Fig. 9 shows that increasing the percentage replacement of
limestone powder in both self-healing (bio-concrete) and normal concrete samples may have resulted in
a reduction in crack widths and an increase in crack closure index values when subjected to a sulphate
attack, indicating the samples' high sulphate resistance.

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30
Compressive Strength due to Sulphate Attack

25

20
[N/mm2]

15

10

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Immersion 56 Days Immersion

Figure 7: Average compressive strength due to sulphate attack on the concrete types.

1.4
Average Weight Loss Index due to Sulphate

1.2

0.8
Attack

0.6

0.4

0.2

0
NC0,100 NC5.95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Immersion 56 Days Immersion

Figure 8: Average weight loss index due to sulphate attack on the concrete types.

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4.5

4
Average Crack Closure Index
3.5

2.5

1.5

0.5

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Immersion 56 Days Immersion

Figure 9: Average crack closure index of the concrete types due to sulphate attack.

3.3.2. Alkaline Attack

The results of the average compressive strength, the average weight loss index due to alkaline attack and
the average crack closure index of the various concrete types, after 28 and 56 days of immersion inside
sodium hydroxide (NaOH), are presented in Fig. 10, Fig. 11 and Fig. 12, respectively. Based on the data
from Fig. 10, it can be concluded that the compressive strength is influenced by the proportion of material
replacement and the inclusion of bacillus subtils. The concrete with a 10% replacement in the regular
concrete sample (NC10,90) had the maximum compressive strength, giving a rise of 16.86% over the
control (NC0,100), at 56 days of immersion. This suggests that the replacement material increases the
concrete's resistance to alkaline attack. The results also show that a self-healing concrete (bio concrete)
with 10% replacement (SHC10,90) had the maximum compressive strength, giving a rise of 22.37%
over the control (NC0,100), at 56 days of immersion. According to a study [46], cement's resistance to
alkaline attack rises as its amount of limestone powder increases. Likewise, another study [16] revealed
that inclusion of bacteria in concrete can greatly improve the compressive strength of such concrete.
Following the immersion in the NaOH solution, the weight of the Self-Healing (bio concrete) and the
normal concrete samples both significantly decreased, according to the data presented in Fig.11 and the
percentage decrease ranged from percentage to percentage replacement of LP and also upon the addition
of the bacteria. All samples lost weight as a result of the alkali attack, as evidenced by the decrease in
final weight from the original weight. The weight index readings that are just a little bit above or below
1 show a weight decrease. In general, the higher the percentage replacement with LP and also due to the
inclusion of the bacillus subtils, the less weight is lost. This demonstrates how the addition of limestone
powder and bacillus subtils improved the samples' resistance to an alkali attack. A better durability
property against the alkaline attack was obtained with SHC10,90, having 14.52% reduction in weight
loss at 28 days of immersion in the sodium sulphate over the control (NC0,100). This implies that both
LP and bacillus subtils may help in controlling weight loss in concrete, when exposed to harsh conditions
like an alkaline environment. This finding, justified the claims made by previous studies [7, 8], that
adding limestone powder to concrete improves its resistance to alkaline attack, likewise the claims made
by previous studies [15 - 19] that introduction of bacteria bacillus subtils and bacteria conhi improves
the durability properties of concrete, especially its resistance to alkaline attack. The weight index values,
however, indicate that not all of the weight was lost. The bacteria seemed to improve its durability
prowess for this test, the cracks induced as aforementioned exhibited more self-healing ability than the
samples without, as seen in Fig. 12. The self-healing property of the distinct concrete samples without
bacteria across various tests carried out seemed to be aided by the limestone powder added as an SCM,
where increments in limestone powder content resulted in a higher crack width index, this is obviously
due to the fineness of the powder and its hydrating properties. The best performing concrete type in this
context is NC15,85, having a higher crack closure index of 11% over the control (NC0,1100), at 56 days

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of immersion in the alkaline solution. In addition, in the samples containing bacteria, the limestone
healing properties were aided by the bacterial action covering up for the samples with lower limestone
content percentage. Among the concrete types tested for the alkaline attack test, SHC15,85 gave the
highest crack closure index, with 900% rise over the control (NC10,90), at 56 days of immersion.
Overall, Fig. 12 indicates that increasing the percentage replacement in both the self-healing concrete
(bio-concrete) and the regular concrete samples may have resulted in a decrease in crack widths and a
rise in crack index values when subjected to an alkali attack, indicating the samples' high alkali
resistance. This is in line with previous studies [47 - 49].

30
Average Compressive Strength due to Alkaline

25

20
Attack [N/mm2]

15

10

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Immersion 56 Days Immersion

Figure 10: Average compressive strength due to alkaline attack on the concrete types.

1.2
Average Weight Loss Index due to Alkaline

1.15

1.1
Attack

1.05

0.95

0.9
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC156,85
Concrete Type

28 Days Immersion 56 Days Immersion

Figure 11: Average weight loss index due to alkaline attack on the concrete types.

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12

Average Crack Closure Index due to Alkaline 10

8
Attack

0
NC0,100 NC5,95 NC10,90 NC15,85 SHC0,100 SHC5,95 SHC10,90 SHC15,85
Concrete Type

28 Days Immersion 56 Days Immersion

Figure 12: Average crack closure index of the concrete types due to alkaline attack.

3.4. Microstructural Analysis

The results of the scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDXS)
and the x-ray diffraction (XRD) for the control sample (NCO,100) and the optimum sample (SHC10,90),
after 28 days of crushing, are presented and discussed in this section. The self-healing concrete and
normal concrete samples’ Scanned Electron Microscopy (SEM) examination gave visible proofs of the
microstructural changes brought on by the changing parameters for example incorporating limestone
powder and adding bacillus subtils. Fig. 13 and Fig. 14 show how various phases, such as cementitious
matrix, aggregates, and healing agents, were distributed and connected to one another. The knowledge
of the healing mechanisms was aided by the use of the SEM images to link the microstructural findings
to the mechanical characteristics of the self-healing concrete. The elemental composition and their spatial
distribution were revealed by the self-healing concrete and the normal concrete samples’ Energy
dispersive X-ray spectroscopy (EDS) analysis. Calcium, silicon, aluminum, iron, and minor elements
added by the limestone powder and healing agents were among the numerous components contained in
the bio concrete that was identified and quantified using the spectroscopic data acquired from EDS. The
EDS revealed that the concrete contained calciumsilicatealuminoferrite (CSAF), one of the main
compounds found in cement; thus, adding limestone powder to conventional concrete improves its
binding capacity; the highest was found at 10% limestone content, which also justified the high strength
(compressive, splitting tensile, and flexural) obtained previously. SEM examination showed that sample
containing bacteria has a homogeneous surface texture, whereas that containing no bacteria has a
heterogeneous surface. Similarly, the interfacial transition zone (ITZ) in bacteria-containing sample
shows fewer gaps than in bacteria-free sample. The ITZ is the space between the binders and the
aggregate; sample containing bacteria has less ITZ and is less permeable to chemical assault; smaller
holes enhance durability. The distribution of the different mineralogical phases that were present was
shown by XRD examination of the self-healing concrete and the normal concrete sample. Different
crystalline phases, such as calcium silicate hydrates (C-S-H), portlandite, calcite (from limestone
powder), and other mineralogical components, were identified using the diffraction patterns acquired
from the XRD study. Ca(OH) level was high in normal concrete sample at 0%. Higher concentration of
Silicon oxide (‹ଶ ) was found at 10% The greater the CH, the faster the hydration process, which
promotes strength. The CS in standard concrete samples creates CSH, which speeds up the hydration
process and enhances strength and durability. The presence of Si in the self-healing concrete (bio
concrete) can enhance the interface strength and refine the pores, which can effectively reduce the water
permeability of concrete which is justified by previous studies [50, 51].

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(a) (b)

(c)
Figure 13: (a) SEMS (b) EDX (c) XRD of NC0,100.

(a) (b)

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(c)
Figure 14: (a) SEMS (b) EDX (c) XRD of SHC10,90.

4. Conclusion
The highest compressive strength was achieved when 10 ml of the bacteria was mixed into concrete
and also limestone powder was used as a supplementary cementitious material at 10% of the
concrete’s binder after 28 days of curing. This research provided that an optimum percentage of 10%
to 15% replacement of cement by limestone powder can be used to produce concrete with improved
durability and improved strength. Concrete containing limestone powder infused with bacteria will
provide concrete with higher mechanical strength properties, durability and a higher crack closure
index than conventional concrete without bacteria. Bio-concrete containing limestone powder and
exposed to water ponding curing condition (curing by complete immersion in water) will exhibit
better self-healing prowess than conventional concrete. The microstructural analysis shows that the
bacteria provided self-healing abilities to samples that contain them. This research concluded on the
notion that, bio-concrete containing limestone powder yields higher mechanical properties than
normal concrete containing limestone powder and conventional concrete. The optimum percentage
for maximum strength, durability and self-healing ability of bio-concrete containing limestone
powder of about 10% replacement of cement by limestone powder, 10 ml of bacillus subtils and
cured in a water-ponding condition can be recommended for use in the construction industry, for
self-heling concrete production.

Acknoledgements
The authors would like to express their gratitude to CUCRID and Covenant University administration
for their unwavering support.

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