5th Conference on Sustainability in Civil Engineering (CSCE’23)

Department of Civil Engineering

Capital University of Science and Technology, Islamabad Pakistan

“Bacterial Self-Healing for Sustainable Concrete: A

Comparative Study of Vegetative and Spore-Forming
Bacteria”

a
M Abdullah Akmal, b Syed Sohaib Aijaz, c Ali Raza*
a: Department of Civil Engineering, University of Engineering and Technology Taxila, Pakistan; Email:
[email protected]
b: Department of Civil Engineering, University of Engineering and Technology Lahore, Pakistan; Email:
[email protected]
c: Department of Civil Engineering, University of Engineering and Technology Taxila, Pakistan; Email:
[email protected]

Abstract- Affordability and availability of concrete is driving its use in building. Hence,
sustainable, and durable concrete is needed. Cracks from excessive water, creep, or
shrinkage cause concrete to fail. Crack creation reduces strength, and moisture alone or
with a toxic chemical like sulfur can induce steel corrosion and concrete degradation,
reducing the longevity of concrete. Hence, cracks must be filled, but mechanically
repairing cracks, especially micro and deep cracks, is laborious and expensive and cannot
be done in structural members that are not apparent. MICCP has been studied for concrete
durability. This research covers self-healing characteristics using bacterial species; the
effect of bio-concrete in prisms; and a comparative study using alkali-resistant spore-
forming bacteria and vegetative bacteria. For concrete to self-heal, Bacillus subtilis and
other alkali-resistant bacteria are added during mixing. This research indicates that Spore-
forming bacteria performed better than vegetative bacteria. The crack remediation due to
bacterial action was confirmed by performing SEM analysis which confirmed the self-
healing action of concrete.

Keywords- Self-Healing Concrete, Vegetative, Spore Forming, MICCP

1 Introduction
Concrete is inexpensive and commonly used in developing countries. As cracks form in concrete
over time due to shrinkage, temperature, or other factors, they reduce the material's strength and weaken it, resulting in
corrosion in steel-reinforced structures. When cracks exceed the allowed limit, they have a detrimental impact on the
concrete. Yet, concrete and steel bars begin to deteriorate when cracks surpass the 0.4-mm limit [1].

Various mechanical methods can be employed to fill cracks in concrete, such as manually filling the crack with epoxy
resin, which is expensive and cannot be used for small or deep cracks. In addition, regular oversight is required to monitor
the formation of cracks and fill them mechanically. Therefore, mechanical crack filling cannot be performed on non-visible
cracks developing in structural members [2]. An essential aspect of research is the improvement of the concrete industry
in terms of its durability and self-healing capabilities utilizing natural or artificial means.

The self-healing action of concrete can be achieved through two distinct methods: autogenous and autonomous healing
[3]. Autogenous healing of concrete refers to the concrete's inherent self-healing properties that can occur in the presence
of water. Calcium carbonate is formed due to the hydration of un-hydrated cement that is present in the concrete. Cement
hydration produces Ca (OH)2, which reacts with carbon dioxide in water to form CaCO3 crystals, allowing the concrete
to close itself. In general, we can say that there is no added substance or material for self-healing properties, and the entire

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 5th Conference on Sustainability in Civil Engineering (CSCE’23)
Department of Civil Engineering
Capital University of Science and Technology, Islamabad Pakistan

phenomenon is natural [4]. Several scholars have reported the development of autonomous self-healing. Wang Guiming
et al. [5] revealed that the self-healing process of cementitious materials by crystalline coating can be studied using SEM,
which reveals that it not only fills mortar fractures and seals pores but also repairs cracks. E. Schlangen [6] explained in
his investigations that experimental and numerical studies can explain how cracks that developed in early ages can be filled
or repaired by future hydration processes. It has been found that even after one day of hydration, the sample began to
regain its strength.

However, autonomous healing refers to the healing properties gained by introducing a healing substance into the cracks
[7]. It has been observed that bacterial self -healing is restricted to average crack width of 0.8 mm. At this point, a crack
self-heals, restoring its full strength and durability. Considering that intrinsic self-healing is limited to 0.4 mm, bacterial
self-healing can provide complete results with high efficiency [8]. However, Nguyen Ngoc Tri Huynh et al. created bio-
concrete to evaluate the compressive and flexural strength over largely induced artificial cracks. The cracks limit was 1-
18mm and was evaluate over period of 24 hours. Their research showed a increase in compressive and flexural strength
over small scale [9]. This research discusses the two most powerful healing phenomena: self-healing characteristics of
alkali-resistant spore-forming bacteria and alkali-resistant vegetative bacteria. The Bacterial bio-concrete refers to the
healing properties of concrete that are accomplished by employing a straightforward procedure to fill the cracks. The
fundamental mechanism is accomplished by creating a concrete mixture containing a precursor such as calcium lactate
(Ca(C3H5O2)2). When water enters the crack, bacteria start eating the Calcium lactate as food and start making calcium
carbonate, as a by-product, which accumulates in the crack to fill it. The microorganisms utilized in this type of concrete
are alkali-resistant bacteria from the genus Bacillus. Bacteria from this category are the most ideal since they produce
spores and can survive in harsh environments for more than 200 years. Because of its sustainable organic features, using
bacteria as a healing mechanism is one of the best techniques for producing this type of concrete. Also, some precursor
compounds result in an increase in concrete strength rather than a decrease in concrete strength. Microbially Induced
Calcium Carbonate Precipitation (MICCP) has sparked considerable interest as a viable, natural, and environmentally
friendly technology for autonomous concrete repair, resulting in greater concrete durability. A substantial study has been
undertaken on the use of MICCP to mend cracks in concrete [10].

2 Experimental Procedures

2.1 Materials.
To test the self-healing capabilities, concrete specimens with dimensions of 500x100x100 mm were
prepared. Specimens were cured for 24 hours at room temperature after molding. To evaluate the mechanical qualities,
150x300 mm cylinders were also prepared. All specimens were prepared in accordance with ASTM C31/C31M standards.
Tables 1-3 provide specifics on the concrete mix designs employed in this experiment. The compositions of the concrete
had a set parameter. The total time for stirring the concrete was set at 10 minutes.

Type Materials
Cement P.O 42.5 Ordinary Portland cement named
“Paidar Cement” (ASTM C-150 TYPE 1)
Water Domestic Water
Fine Aggregates River sand, Absorption Rate; 2.5060
Coarse Aggregates Crushed stone Aggregates, Absorption
Rate; 2.29
Precursor Compound Calcium Lactate with the specified
percentage of 1% of dry weight of cement
Autogenous Compound Bacillus Subtilus; alkali resistant bacteria
of genus Bacillus.

Table 1 Materials

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 5th Conference on Sustainability in Civil Engineering (CSCE’23)
Department of Civil Engineering
Capital University of Science and Technology, Islamabad Pakistan

Sr. # Wt. of water %Age Wt. of water Penetration

(W2) gm. mm

1 140 28 10

Table 2 Standard Consistency of Cement

Initial setting time Final setting time

218 mins (3.64 hours) 514 mins (8.55 hours)

Table 2.1 Settling time of Cement.

Cao SiO2 Al203 Fe2O3 MgO SO3 Na20 P2O5 MnO

63.5% 19% 5% 3% 2.25% 2.5% 0.4% 0.25% 0.4%

Table 2.2 Composition of Cement

Fine Fineness Specific Water Apparent Bulk Porosity

Aggregates Modulus gravity Absorption Density(kg/m3) Density(kg/m3)
%
(Apparent) %

2.5877 2.4903 2.5060 1345.01 1346.15 0.0651

%
Coarse Fineness Specific Water Apparent Bulk Porosity
Aggregates Modulus gravity Absorption Density(kg/m3) Density(kg/m3) %
(Apparent) %

6.5588 2.81 2.29 708.56 1349.32 0.17 %

Table 3 Properties of Aggregates

Moreover, Figure 1 and 2 shows each size level of fine and coarse aggregates.

Gradation curve for FA Gradation curve for CA

150
150
%age passing

%age passing

100 100

50 50

0 0
10 1 0.1 100 10 1
Sieve size mm Sieve size mm
ASTM Minimum ASTM Minimum
ASTM Maximum ASTM MAximum
%age passing of FA %age passing of CA

Figure 1 Gradation Curve for FA Figure 2 Gradation Curve for CA

Paper ID. 23-XXX Page 3 of 7

 5th Conference on Sustainability in Civil Engineering (CSCE’23)
Department of Civil Engineering
Capital University of Science and Technology, Islamabad Pakistan

2.2 Bacterial Cultures Protocol.

An Alkali Resistant Bacteria was selected considering the pH of concrete. As normal
pH of concrete ranges from 11 to 13 which is quite high for bacteria to survive, an alkali bacterium was needed for self-
healing of concrete. This research uses Bacillus Subtilus in both vegetative and spore forming culture for their comparative
study and to evaluate self-healing ability of concrete as shown in figure 3. The strains of Bacillus Subtilus were isolated
and the medium for their culture is generated. Then the culture was inoculated and incubated. After the incubation for the
required amount of time, culture was diluted with N-broth medium and then it was centrifuged in falcon tubes. The OD
was determined at 600nm by using spectrophotometer and pellets were dissolved in required amount of controlled distilled
water to obtain the bacterial water to be used in concrete. The strains of bacteria were cultured as per specification with
the concentration to be achieved as 106 cells/ml. The concentration at the time of synthesis was enhanced so that it can be
diluted at the time of use age.

Figure 3 Bacterial Solution

2.3 Preparation Of Micro-cracks.

After 24 hours of curing, concrete samples were pre-cracked using a three-point bending method. A smart crack gauge
with a 4-mm range was used to measure the width. SEM was used to bring in the images. A sample binarized image for
measuring the self-healing progress is shown in Fig. 4. The bacteria self-healing of the concrete samples was ultimately
characterized by measuring the overall healing area of the crack.

Cracks on
Upper layer
under
microscope.

Figure 4 SEM

Paper ID. 23-XXX Page 4 of 7

 5th Conference on Sustainability in Civil Engineering (CSCE’23)
Department of Civil Engineering
Capital University of Science and Technology, Islamabad Pakistan

3 Research Methodology
The methodology of this research involved the use of cement, sand, aggregates, precursor compound, and bacterial water
in a non-encapsulated way to produce bio-concrete. The selection of cement, sand and aggregates was made through ASTM
Codes involving ASTM C-150 TYPE 1, ASTM C144 and ASTM C33/C33M. A fixed mix design ratio of 1:1.5:3 with a
water-cement ratio of 0.49 was used. The precursor compound, calcium lactate was added at a specific percentage of 1%
of Cement Weight to the dry mixture. The selected bacterial strain was cultured in a nutrient-rich medium, and the bacterial
water was then harvested and added to the concrete mixture during the batching process. The precursor compound,
typically calcium lactate or calcium acetate, was also added to the mixture to provide a source of calcium ions for the
bacteria to convert into calcium carbonate. The mixture was then placed into molds and cured under optimal conditions to
allow the bacteria to consume the nutrients and produce calcium carbonate, which acted as a binding agent. The bio-
concrete was then tested for strength and durability using standard methods such as compressive strength, water absorption,
Modulus of Rupture and Self-healing Progress. Overall, this research aimed to investigate the feasibility of using non-
encapsulated bacterial water and precursor compounds in the production of bio-concrete, with the potential to develop
sustainable and durable construction materials.

To trace the progress of bio-concrete with respect to controlling specimens, slump cone tests, compression tests, modulus
of rupture tests, and scanning electronic microscopy tests were conducted.

A slump cone test was conducted to determine the workability of concrete. However, results indicated that concrete had
true slump, as the slump of the conventional concrete was recorded at 76 mm, while the slump for bio-concrete was 50mm,
as shown in Figure 5 below.

Figure 5 Slump Test

4 Results

4.1 Scanning Electron Microscopy.

Scanning Electron Microscopy (SEM) was used to analyze the morphology and minerology of the deposited calcium
carbonate caused by the induction of bacteria as a Self-healing agent.

White-Fillings
show Calcium
Carbonate
Precipitation in
Cracks.

Figure 6 SEM Results (Healed Microcracks)

Paper ID. 23-XXX Page 5 of 7

 5th Conference on Sustainability in Civil Engineering (CSCE’23)
Department of Civil Engineering
Capital University of Science and Technology, Islamabad Pakistan

White Crystals, showing in Figure 6, demonstrated the self-healing ability of bio-concrete. The specimens tested for
compressive strength were cut into little 37 × 37 mm cubes and then SEM examination was done on them. The Figure 6
depicts the outcome of the SEM study. This result from the SEM confirms the filling of a microcrack and justifies the
precipitation of calcium carbonate precipitation .

5 Conclusion
Following conclusions are drawn after deeply analyzing and studying the experimental results obtained by different test:

1 Locally developed Concrete incorporated bacteria can produce copious amount of minerals which can help in
filling of cracks.
2 Microbiological crack remediation is more efficient in shallow cracks than in deeper cracks primarily because the
microorganisms grow more actively in the presence of oxygen.
3 The results indicate that Spore-forming Bacteria shows better progress than Vegetative bacteria as it results in 97%
filling of cracks with calcium carbonate and shows increase in Compressive strength as compared to Vegetative
bacteria.
4 The results from the SEM Analysis further confirms that the increase in compressive strength is due to precipitation
of Calcium Carbonate and confirmed crack filling was observed.

6 Appendix
NO

Acknowledgment
Many people have contributed to this research and report, but the authors are grateful for the support, guidance & co-
operation of their Advisor Engr Syed Saqib without whom this research & report would not have been possible. His
continuous support & guidance helped us to work into & complete this research. We are honoured to acknowledge his
support.

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[8] M. Luo, C. Qian, and R. Li, “Factors affecting crack repairing capacity of bacteria-based self-healing concrete,” Constr. Build.
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[9] N. N. T. Huynh, K. Imamoto, and C. Kiyohara, “A Study on Biomineralization using Bacillus Subtilis Natto for Repeatability
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 5th Conference on Sustainability in Civil Engineering (CSCE’23)
Department of Civil Engineering
Capital University of Science and Technology, Islamabad Pakistan

[10] Y. L. Reddy and A. Pradesh, “Experimental Study on the Durability of Concrete,” Mukt Shabd J., vol. IX, no. VI, pp. 7785–
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