Materials Today: Proceedings xxx (xxxx) xxx

Contents lists available at ScienceDirect

Materials Today: Proceedings

journal homepage: www.elsevier.com/locate/matpr

An experimental investigation on self-healing concrete using

“Bacillus subtilis”
P.V. Premalatha a, *, M. Geethanjali b, S. Sundararaman c, C.S. Murali d
a
Department of Civil Engineering, M.I.E.T. Engineering College, Tiruchirapalli, Tamilnadu, India
b
Department of Civil Engineering, Surya College of Engineering, Tiruchirapalli, Tamilnadu, India
c
Department of Civil Engineering, Sri Manakula Vinayagar Engineering College, Puducherry, India
d
Department of Civil Engineering, CARE College of Engineering, Tiruchirapalli, India

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

Keywords: The current study attempts to measure the performance of concrete utilizing microbiologically generated bac­
Bacteria Concrete teria growth. This idea led to the creation of a highly unique concrete that employs bacteria to mend defects in
Bacillus Subtilis concrete. When cracks develop in concrete, they allow the ingress of water and various chemicals, thereby
Self-healing concrete
reducing its strength and durability and causing corrosion of the reinforcement. To solve this issue, bacterial
Cracks
Repair
concrete is developed which has an independent self-healing mechanism that assists in fracture repair by pro­
Durability ducing calcium carbonate crystals that fill micro-fissures and pores. In the present investigation, the bacterium
“Bacillus subtilis” was employed based on the literature study. This bacterium was cultured and utilized as it can
withstand harsh environmental conditions. The Bacterium “Bacillus subtilis” was added in volume of 10 ml, 20
ml, 30 ml, 40 ml and 50 ml in M25 concrete. The compressive strength of concrete was monitored at 7 days, 14
days and 28 days. It was observed that 30 cc of “Bacillus Subtilis” when cultures and added to the concrete gives
optimum compressive strength for a M25 grade concrete. Also, the inclusion of microorganisms improved
compressive strength of concrete significantly. This bacterium introduced to the concrete has successfully self-
healed the cracks thereby improving its durability.

1. Introduction absorptivity on concrete cubes due to the mixing of bacteria in concrete.

Most structures are built with concrete that cracks to allow
embedded steel reinforcing to take up the tensile loads. Crack formation
is mainly associated with the durability of concrete. Percolated fissures
can lead to leaks or the intrusion of toxic materials, causing the concrete
matrix to deteriorate or the reinforcement to corrode. In order to
enhance such concrete’s durability, water and other pollutants must be
kept out of reach which is practically not possible. Hence, self-healing
concept plays a major role in such circumstances. Self-healing refers
to its regaining ability on its own for a normal performance after heal­
ing. Bacteria transform the mineral precursor chemical into calcium
carbonate, which is more commonly known as limestone [1–4]. When a
crack occurs, ingress water activates this mechanism, and limestone
precipitation on the surface is observed. Bacteria-induced self-healing
concrete is expected to show increased durability, leakage prevention,
and service life extension. Many other researchers studied the effect on
the compressive strength, flexural strength, and water absorption and
The evaluated results indicate that concrete strength has increased
significantly. The present research mainly focuses on the comparison of
the strength of conventional and bio concrete cubes.
In Engineering, current material design is based on the concept of
damage prevention. Self-healing materials, as presented by Van der
Zwaag’s notion of damage management, are an alternative design
principle. Damage does not always result in problems if it is healed
through a self-healing process. These self-healing materials must fulfill
the expected number of functions and possess necessary self-healing
characteristics. Under normal circumstances, damages are detected,
followed by the delivery of a healing agent to the damage site, which
will induce the repairing process. In an ideal world, self-healing mate­
rials would be inexpensive and have qualities that are on par with or
better than currently available repairing materials, including the ability
to fix faults of any size, several times, and totally autonomously. When it
comes to concrete, the durability of the material is mostly examined for
the potential for damage.

* Corresponding author.
E-mail address: pvpremalatha@yahoo.co.in (P.V. Premalatha).

https://doi.org/10.1016/j.matpr.2023.08.118
Received 12 March 2023; Received in revised form 17 July 2023; Accepted 11 August 2023
2214-7853/Copyright © 2023 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the 2023 International
Conference on Advanced Technologies in Chemical, Construction and Mechanical Sciences.

Please cite this article as: P.V. Premalatha et al., Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2023.08.118
P.V. Premalatha et al. Materials Today: Proceedings xxx (xxxx) xxx

Healing by microbes is getting very popular as in this process; con­
crete already has a built-in healing mechanism due to its chemical,
physical, and mechanical reactions. Calcium carbonate precipitation is
the most important in this kind of self-curing. A crack width of 0.2 mm is
the average limit for which healing can still occur. Here the carbonation
reaction occurs in which dispersed carbon dioxide combines with the
calcium hydroxide deformed through the hydration process, and this
results in the formation of calcium carbonate. The precipitation of cal­
cium carbonate is also a principle of microbial healing [3–5]. Dormant
bacteria are reactivated by the ingress of water through the cracks.
Bacterial conversion of an ‘integrated mineral precursor chemical’
produces dense layers of calcium carbonate. When it comes in contact
with calcium lactate, bacteria merely serve as a catalyst.
Carbon dioxide is produced by the metabolic conversion of calcium
lactate, which combines with the calcium hydroxide in the concrete
matrix according to the chemical reaction in Equation (1) to form more
calcium carbonate. Massive creation of huge, over 100 m crystalline
calcium carbonate precipitates seals and blocks fissures, limiting further
infiltration of water and other chemicals that could damage the concrete
matrix or embedded reinforcement. Carbonate synthesis driven by
bacteria follows a variety of metabolic routes. Several researchers chose
urea hydrolysis as a viable bio cementation process [6,7].
The environment is alkalinized during ureolytic-driven carbonate
production, favoring the precipitation of calcium carbonate in the form
of calcite. One mole of urea is hydrolyzed into one mole of carbonate and
two moles of ammonium ions in the total reaction. Jonkers [5] pur­
posefully chose an organic calcium salts-based metabolic pathway over
urea hydrolysis to avoid potentially harmful effects on the concrete
matrix or embedded reinforcement when generated ammonia is subse­
quently converted to nitric acid by bacteria.
A healing agent is mostly made up of bacteria and a mineral pre­
cursor component. The first and most critical step is to select microor­
ganisms that were compatible with concrete. Bacteria should be able to
survive and thrive in such an alkaline environment. Because concrete
constructions are built to last at least 50 to 100 years, germs should be
able to survive for that long. As a result, a particular type of alkaliphilic
spore-forming bacteria was chosen. When life conditions grow less
favorable, bacteria create thick cell-walled spores. The use of appro­
priate bacteria in conjunction with calcium lactate as a mineral pre­
cursor compound resulted in the formation of calcium carbonate
precipitates in concrete cracks.
When calcium lactate and bacterial spores were given to the concrete
mixture in an unprotected state, mineral synthesis appeared to be
limited in time, most likely due to the precursor compound’s full inte­
gration in the matrix, limiting its access to bacteria. Direct addition also
appears to decrease the viability of bacterial spores to 2–4 months. This
is most likely due to further cement hydration reducing the pore size of
the cement paste. MIP (mercury intrusion porosimetry) indicates that
pore diameters decrease over time to less than 1 m, the average size of
Bacillus spores. When integrated bacterial spores are immobilized or
protected and the precursor molecule is kept available for bacterial
conversion, the possibility for long-term survival and activity increases.
Encapsulation of the two-compound healing agent in a protected
reservoir is the preferred solution.
(ii). Bacterial concrete is manufactured by inserting bacteria that can
continuously precipitate calcite in concrete. Microbiologically gener­
ated calcite precipitation is the scientific term for this process.
(iii). Bacillus subtilis, a bacterium that may effectively treat concrete
fractures. When the self-healing bacteria comes in contact with oxygen
and water, they will convert nutrients to calcium carbonate. Bacillus
subtilis creates calcite, which precipitates on an appropriate medium
supplied with calcium [see Eqn (1)]. This byproduct fills up the cracks
present in the concrete ensuring its durability.
The main focus of the research will be on how to create the correct
conditions for the bacteria to not only survive in the concrete but also to
manufacture enough calcite to mend cracks.
Ca(C3H5O2)2 + 7O2 → CaCO3 + 5CO 2 + 5H 2O (1)
CO 2 + Ca(OH) 2 → CaCO3 + H2 O
(Carbonation).
2.1. Preparation of sample- (Nutrient broth)
In a conical flask, 13 g of nutrient broth was added to 250 ml of
distilled water. Cotton is used to plug the conical flask. The soup is then
autoclaved for 15 min at 121 degrees Celsius.
• The flask was then removed and allowed to cool to room
temperature.
• To inoculate the broth without contaminating it, a laminar airflow
chamber was used.
• The laminar airflow chamber is cleaned with ethanol before using it.
• One loop of culture was taken and combined with the broth after it
had cooled.
• Then, for 24 h, the conical flask is maintained in the incubator shaker
at 37 degrees Celsius.
The pH study on the bacterial solution reveals that the culture of
Bacillus Subtilis is better in a solution of neutral to alkaline nature.
Hence care is taken that the pH of the solution is maintained at 7. The
above process is shown in Figs. 1, 2 and 3.
2.2. Maintenance of stock culture
Bacillus subtilis jc3 stock cultures were grown on nutrient agar
slants. The culture was streaked onto agar slants with an inoculating
loop and incubated at 37 degrees Celsius. Slant cultures were stored
under refrigeration (4 degrees Celsius) after 2–3 days of growth for a
total of 27 uses. Every 90 days, sub culturing was performed. Other
bacteria contamination was checked on nutrient agar plates on a regular

2. Materials and methods

The selection of bacteria in self-healing concrete dramatically affects

the mechanical property of the concrete. The germination of bacteria is
not possible in every environment. Selection of wrong bacteria results in
premature death and failure to achieve the desired outcome. In this
paper, Bacillus Subtilis is chosen based on a thorough literature study.
(i). Bacillus Subtilis is a bacillus found in soil and the human
gastrointestinal tract. Bacillus subtilis is a rod-shaped Bacillus species
that produces a strong, productive endospore that allows it to withstand
harsh environmental conditions. Fig. 1. Weighted medium.

2
P.V. Premalatha et al. Materials Today: Proceedings xxx (xxxx) xxx

Fig. 2. Nutrient broth in 250 ml medium.

Fig. 5. Maintenance of stock culture.

Fig. 3. Cultured Bacterial Sample.

basis (Figs. 4 and 5).(See Fig. 6).

3. Methodology Fig. 6. Mixing of Concrete.

3.1. Specimen casting
From the literature, guidance was obtained on casting specimens
with induced bacteria [8–13]. M25 grade concrete mix [14–17] was
prepared with ordinary Portland cement (OPC) and cubes of dimension
150 mm X 150 mm X 150 mm were cast. Specimens with bacillus subtilis
added from the total volume of concrete at 0 ml, 10 ml, 20 ml, 30 ml, 40
ml, and 50 ml have been cast. During the casting process, the cubes were
manually compacted into three layers. After 24 h, the specimens were
removed from the mould and water cured for 7, 14, and 28 days. After
curing, the specimens were tested for compressive strength with a
calibrated compression testing machine. Control specimens were cast of
concrete grade M25.
The slump test was done to check the workability of the concrete
after adding the bacteria. The results are presented in Fig. 7 and showed
a normal range.

Fig. 4. Petri-dish stock culture. Fig. 7. Slump test.

3
P.V. Premalatha et al. Materials Today: Proceedings xxx (xxxx) xxx

3.2. Specimen curing

Curing was done after the concrete has been placed in the mould and
hardened. Curing concrete properly results in increased strength and
lower permeability, as well as the avoidance of cracking where the
surface dries out prematurely. It is also important to avoid freezing or
overheating as a result of the exothermic setting of cement. Inadequate
curing can result in scaling, decreased strength, poor abrasion resis­
tance, and cracking. Fig. 8 shows the curing of Specimens.

3.3. Compressive strength test

The cubes were subjected to Compression test (Fig. 9) and the results
are presented in Fig. 10.

4. Results and discussion

4.1. Compressive strength results

Fig. 10 shows the compressive strength of bacteria added cubes. It is

observed that M25 grade bacterial concrete is having higher compres­
sive strength than the normal M25 grade concrete. When 30 ml of ba­
cillus subtilis bacteria is added, it gives the compressive strength of
33.32 N/mm2, which is the maximum. Hence, we can replace normal
M25 concrete by our M25 bacterial concrete thereby reducing the cost of
construction. Fig. 9. Cube testing.

4.2. Self-healing analysis

Calcium carbonate precipitating microorganisms is used to heal

cracks. After being immobilized on diatomaceous earth, these organisms
become embedded in the concrete matrix and begin the precipitation of
CaCO3. 20% of the ultimate load is applied to the concrete cube with 30
ml added bacteria specimen to induce the crack in concrete cubes. Once
there is a crack formation, this cube is removed from the compression
testing machine and left over for self-healing. In order to escalate the
self-healing process, water is sprayed over the specimen for 7 days. The
cracks present in the concrete allow the ingress of water resulting in the
chemical reaction with the water. The bacterial cell will be coated with a
layer of calcium carbonate, resulting in the microorganism’s death, but
the crack faces bonds together in the meantime. Fig. 11 shows the before
and after healing of cracked concrete cube.
Comparison of compressive strength of M25 to M25 Bacterial Con­ Fig. 10. Compressive strength of bacteria added cubes.
crete is shown in Fig. 12. However, the compressive strength of concrete
achieved after self–healing needs to be investigated, which may be taken
as a future scope of work.
From the above figure, it is observed that M25 grade bacterial con­
crete has higher compressive strength than the normal M25 grade

Fig. 11. Before and after healing of concrete cube.

concrete, so we can replace normal M25 concrete with our M25 bacterial
concrete. Hence the cost of construction is reduced.
However, one should not forget the limitations that bacterial con­
Fig. 8. Curing of Specimen. crete has, as mentioned below:

4
P.V. Premalatha et al.
Fig. 12. Compressive strength of m25 to m25 bacterial concrete.
• The cost of concrete is more and hence cannot be used for large-scale
construction
• The growth of bacteria is a challenging parameter, and a controlled
environment is essential.
• Standard codal procedures are not available for guidance.
Recommendations on usage of bacterial concrete:
Research on bacterial concrete is an emerging field and needs lots of
study to arrive at an optimum procedure. It is recommended that while
using the bacterial concrete, care should be taken that the culture of
bacteria is in a suitable medium and that the environment permits the
survival of bacteria and its performance.
5. Conclusion
The following conclusion is reached based on the current experi­
mental investigation.
• Bacillus subtilis can be produced safely and cheaply in the
laboratory.
• When 30 ml of bacillus subtilis bacteria is added, the compressive
strength reaches 33.32 MPa for a M25 grade concrete.
• M25 grade bacterial concrete has a higher compressive strength than
normal M25 grade concrete, and self-healing is achieved in bacterial
concrete.
• Because of its eco-friendliness and ease of use, bacterial concrete
technology has proven to be superior to many conventional tech­
nologies. This innovative concrete technology will soon serve as the
foundation for alternative and high-quality structures that are both
cost-effective and environmentally safe.
• The use of microbial concrete in construction could also simplify
some existing construction processes while revolutionizing new
construction methods.
Materials Today: Proceedings xxx (xxxx) xxx
CRediT authorship contribution statement
P.V. Premalatha: Formal analysis, Supervision, Writing - review &
editing. M. Geethanjali: Resources, Visualization, Writing – original
draft. S. Sundararaman: Formal analysis, Investigation. C.S. Murali:
Formal analysis, Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
References
[1] H. J. (2016). BIO CONCRETE AND BACTERIA BASED SELF HEALING CONCRETE.
International Journal of Research in Engineering and Technology, 05(05), 95–99.
doi:10.15623/ijret.2016.0505018.
[2] W. Khaliq, M.B. Ehsan, Crack healing in concrete using various bio influenced self-
healing techniques, Constr. Build. Mater. 102 (2016) 349–357, https://doi.org/
10.1016/j.conbuildmat.2015.11.006.
[3] J. Bashir, Bio concrete- The self-healing concrete, Indian J. Sci. Technol. 9 (1)
(2016) 1–5, https://doi.org/10.17485/ijst/2016/v9i47/105252.
[4] C. Kumar, New bacterial concrete to repair cracks through self healing technique,
Int. J. Eng. Res. V9 (06) (2020), https://doi.org/10.17577/ijertv9is060942.
[5] A.R.T. Jonkers, Discrete scale invariance connects geodynamo timescales,
Geophys. J. Int. 171 (2) (2007) 581–593, https://doi.org/10.1111/j.1365-
246x.2007.03551.x.
[6] DeMuynck W, deBelie N, Verstraete W (2007) Improvement of concrete durability
with the aid of bacteria.
[7] S.S. Bang, J.K. Galinat, V. Ramakrishnan, Calcite precipitation induced by
polyurethane-immobilized Bacillus pasteurii, Enzyme Microb. Technol. 28 (2001)
404–409.
[8] B. DeGraef, W. deWindt, J. Dick, W. Verstraete, N. deBelie, Cleaning of concrete
fouled by lichens with the aid of Thiobacilli, Mater. Struct. 38 (284) (2005)
875–888.
[9] E. Ezhilarasi, G. Geetha, K. Baskaran, Experimental study on flexural behaviour of
self healing concrete using Bacillus Substilis bacteria, Int. J. Eng. Res. V5 (12)
(2016), https://doi.org/10.17577/ijertv5is120160.
[10] Experimental Study of Behavior of Self Healing Concrete. (2017). International
Journal of Modern Trends in Engineering & Research, 4(8), 164–170. doi:
10.21884/ijmter.2017.4265.mcoz5.
[11] P, S., K, V., & S, V. (2019). Experimental Investigation on Bacterial Concrete Using
Bacillus Subtitles. International Research Journal of Multidisciplinary
Technovation, 207–219. doi:10.34256/irjmtcon27.
[12] Self-healing concrete repairs itself with bacteria, Ashley P. Taylor (2012).
[13] V. L. . (2016). EXPERIMENTAL INVESTIGATION ON SELF-HEALING BACTERIAL
CONCRETE. International Journal of Research in Engineering and Technology, 05
(06), 149–153. doi:10.15623/ijret.2016.0506029.
[14] IS : 10262 – 1982 “Recommended guidelines for concrete mix design”.
[15] IS 10262, “Recommended Guideline for Concrete Mix Design”.
[16] IS 456:2000, “Plain and Reinforced Concrete Code of Practice”.
[17] CONCRETE TECHNOLOGY” Theory and Practice by M.S. SHETTY.