Construction and Building Materials 202 (2019) 904–908
Contents lists available at ScienceDirect
Construction and Building Materials
journal homepage: www.elsevier.com/locate/conbuildmat
Enhancement in strength parameters of concrete by application of
Bacillus bacteria
Nidhi Nain a, R. Surabhi a, Yathish N.V. a,⇑, V. Krishnamurthy b, T. Deepa b, Seema Tharannum c
a
Department of Civil Engineering, PES Institute of Technology, Bangalore, India
Department of Civil Engineering, PES University, Bangalore, India
c
Department of Biotechnology, PES University, Bangalore, India
b
h i g h l i g h t s
The bacteria Bacillus megaterium, Bacillus subtilis and their consortia showed increased compressive strength by 22.5%, 14.3% and 15.8% respectively
when compared to that of conventional concrete.
Concrete being weak in tension is off the point of concern, the tensile strength of concrete improved by 18.49%, 25.3% and 19.58% upon using bacteria
Bacillus megaterium, Bacillus subtilis and their consortia respectively.
The SEM analysis clearly depicts the presence of rhombohedral calcite crystals and acetate ions were proved by spherical crystals in the SEM images as
shown in the journal.
The EDAX analysis gave a higher amount of CaO content in all the 3 treated concrete specimens.
Upon testing for the viability of bacteria present in concrete, by culturing them in the biotechnical laboratory. The colonies of bacteria added were found,
which proved the survival of Bacillus bacteria under harsh condition.
a r t i c l e
i n f o
Article history:
Received 20 July 2018
Received in revised form 8 January 2019
Accepted 10 January 2019
Available online 21 January 2019
Keywords:
Microbe induced micro-crack management
Compressive strength
Bacterial precipitation of calcite
Settlement
Shrinkage
Expansion
a b s t r a c t
The microbial-induced calcite precipitation (MICP) is a phenomenon of managing pre and post concrete
cracks. Species like Bacillus subtilis, Bacillus megaterium and a few consortia of species have been reported
to be useful to this self-healing of cracks that arise from shrinkage enhancement and settlement processes happening in concrete. This study aimed at addressing the issue of micro-crack management
and also to test whether the specific microbes would augment enhancement of compressive and tensile
strength of concrete. The pure cultures of the microbes along with the nutrients in requisite amounts
were added in water that was an ingredient to the concrete mix. Subsequently, the blocks were cast,
pond-cured for 7 and 28 days and tested for compressibility along with a test of tensile strength. The
results indicate that the compressive strength and split tensile strength of the species Bacillus subtilis,
Bacillus megaterium and consortia of both observed to be higher by 15% and more when compared with
conventional concrete with M30 concrete in 28 days. The study conclusively indicated that the microorganism demonstrated a positive role in not only enhancing the strength of concrete but also facilitating
self-healing of cracks. The bacterial application thus possesses feasibility to manage micro-cracks and
enhance the strength of concrete.
Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction
Concrete has proven to be useful since 6500 BCE [30] and has
now become the second most widely used element after water.
⇑ Corresponding author.
E-mail addresses: nidhinain17@gmail.com (N. Nain), surabhir8916@gmail.com
(R. Surabhi), yath.appi@gmail.com (N.V. Yathish), v.krishnamurthy@pes.edu
(V. Krishnamurthy), deepaoum1128@gmail.com (T. Deepa), seema@pes.edu
(S. Tharannum).
https://doi.org/10.1016/j.conbuildmat.2019.01.059
0950-0618/Ó 2019 Elsevier Ltd. All rights reserved.
On the contrary, concrete possesses lower tensile strength and less
ductility due to which it corroborates vulnerability towards the
generation of cracks. The production of cracks not only lowers
the strength of concrete but also gives access to deleterious substances. Besides this failure of concrete, micro-cracks are inherently present in concrete. Both the prior is well-known to result
in debilitation of structures.
Referring to a series of research carried out around the world,
various methodologies have been carried out sporadically with
the main motive to overcome deficiencies of cement concrete [1].
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N. Nain et al. / Construction and Building Materials 202 (2019) 904–908
The most common surface treatments use organic polymers
(epoxy, siloxane, acrylics and polyurethanes). These methods used
for managing micro-cracks, have a certain degree of toxicity and
also possesses certain drawbacks that make them not completely
efficient. A recent series of research shows a distinguish use of
microbial mineral precipitation resulting from metabolic activities
of favourable microorganisms in improving the overall behaviour
of concrete [3]. The process takes place near to or at any distance
away within the concrete. This process of bacterial application to
enhance concrete property is also termed as Biomineralization. In
the process, urease enzyme catalyzes the hydrolysis of urea into
ammonia and carbonate, this carbonate reacts with calcium and
thereby forming ‘calcium carbonate precipitate’ [14].
The past experimental journal shows the increase in the concrete property by use of various bacteria of genus Bacillus [8,19],
genus Sporosarcina, [16] and E. coli [21]. In coordination with the
prior researches, this journal proves experimentally the use of bacteria of genus Bacillus (Bacillus subtilis & Bacillus megaterium) in
enhancing concrete properties and also compares the suitability
of consortia of both the Bacterium for the same (Fig. 1.).
2. Materials and method
2.1. Materials
2.1.1. Bacteria
Bacillus subtilis and Bacillus megaterium are the two strains of gram-positive bacteria from genus ‘‘Bacillus” used for conducting the research. Bacillus subtilis is capable of forming highly resistant dormant endospores in response to nutrient
deprivation and other environmental stresses and are commonly found in gut commensal in humans and as well, are easily found in an upper layer of the soil. Bacillus
Fig. 1. Graphs showing the comparison of compressive strength of concrete
specimens.
megaterium is along with being a common soil bacterium, is also found in certain
food items including honey or on surfaces of various objects around us, for example,
clinical specimens, paper, stone etc (Fig. 2).
Bacillus subtilis was available in the Biotechnology department of PES University
and Bacillus megaterium was obtained from Microbial Type Culture Collection and
Gene Bank (MTCC) Chandigarh.
2.1.2. Cement
Ordinary Portland Cement 53 grade is used and tested for various properties as
per IS: 4031-1988 [37] and found to be confirmed to the various specification of IS:
12269-1987 [32] with a specific gravity of 3.10.
2.1.3. Fine aggregates
Locally available M-sand passing through 4.75 micro sieves is used as fine
aggregate. IS 2720 [33] is referred to obtain Specific Gravity. Fine aggregate works
as a filler in concrete to fill in the matrix and give a compacted structure bound by
cement. The specific gravity is found to be 2.70.
2.1.4. Coarse aggregates
A locally available aggregate of 20 mm downsize is used. IS 2386-1963 [34] is
referred to determine the Specific Gravity. The coarse aggregate occupies most of
the volume in concrete, it also adds on to the strength and resistance to abrasion.
The specific gravity is found to be 2.80.
2.1.5. Water
The quantity of water for the experiment is calculated as per mix design.
Whereas, 100 ml of water is replaced with 100 ml of bacterial solution for every
1000 ml of water.
2.2. Methods
2.2.1. Cultivation of bacteria
Prior to culturing of bacteria, the culture medium is prepared. A culture media
consists of water, carbon source, nitrogen source, trace elements and other growth
factors. For the media, the Nutrient broth is used with the compositions as mentioned in Table 1. The pure subculture of bacteria i.e. Bacillus subtilis and Bacillus
megaterium were prepared on nutrient agar medium on a petri dish. To avoid contamination by other species, all glassware and instruments used were previously
sterilized. The bacteria B. subtilis and B. megaterium forms irregular dry white colonies on the petri dish.
Using a platinum inoculating loop, few colonies, that is a loop full of required
bacteria is transferred into the prepared culture medium in a conical flask as part
of inoculation. This inoculated media is then kept for incubating at 37 °C temperature consisting of 150 rpm orbital shaker for overnight and a test of Optical Density
was then done to determine the concentration [19].
The bacterial concentration is kept at 108 cells/ml. The concentration of bacteria
is determined by finding ‘‘Optical density”, using a spectrophotometer. As visible
light passes through a cell suspension the light is scattered. The amount of light
scattered, measured in a spectrophotometer gives a value for Optical density. An
OD of 1 shows a concentration of 0.8 109 cells/ml.
2.2.2. Compression and split tensile test
The concrete cubes and cylinders were cast following M30 grade design according to IS 10262-2009 [35] with the addition of bacteria. The size of the cube is taken
as 150 mm 150 mm 150mm and cylinder of 150 mm diameter & 300 mm
height. The cubes were demolded after 24 h of casting and subsequently cured in
water for 28 days.
All the concrete specimens were prepared and tested as per IS 516-1959 [36].
2.2.3. SEM analysis
Scanning Electron Microscope (SEM) was used to analyse the morphology of the
concrete specimen and check for the calcite precipitation. After 28-days compressive strength test, a collective sample from three different levels that is from the
top, from 5 cm below and at the centre was taken for SEM analysis. This SEM analysis was done under gold plated samples with electron accelerating voltage of
10.0 kV and under magnification of 10 mm.
Table 1
Composition of nutrient broth. (13 g of nutrient
broth for every 1000 ml of water).
Fig. 2. Graph showing the split tensile strength of concrete specimens.
Composition
Content
Peptone
Sodium chloride
Yeast extracts
Meat extracts
5 g/l
5 g/l
2 g/l
1 g/l
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N. Nain et al. / Construction and Building Materials 202 (2019) 904–908
3. Results
3.1. Compressive strength
Upon conduction of compressive strength in Compression
Testing Machine, as mentioned in Table 2, the treated specimen
showed comparatively more strength than the conventional
3.2. Split tensile strength
Table 2
Tabulated result of compression test.
Concrete
Conventional
B. subtilis
B. megaterium
Consortia
Compressive Strength (N/mm2)
7th day
28th day
21.81 MPa
31.93 MPa
27.6 MPa
34.66 MPa
38.08 MPa
43.55 MPa
46.68 MPa
44.12 MPa
Table 3
Tabulated result of spit Tensile test.
Concrete
Conventional
B. subtilis
B. megaterium
Consortia
concrete for the same mix design. Conventional concrete shows
the strength of 38.08 MPa upon 28th day curing, whereas B. subtilis, B. megaterium and consortia show compressive strength of
43.55 MPa, 46.68 MPa and 44.12 MPa respectively i.e., the percentage increase in strength compared to conventional concrete by
14.36, 22.58 and 15.86% respectively.
Mechanism adopted was testing of specimens in Compression
Testing Machine and indirectly finding the tensile strength. The
results obtained showed a similar increase as that of compressive
strength when compared to the conventional concrete specimen.
As mentioned in Table 3, conventional concrete shows a tensile
strength of 3.04 MPa after 28th day of curing, whereas B. subtilis,
B. megaterium and consortia show a tensile strength of 4.11 MPa,
3.88 MPa and 3.92 MPa i.e., percentage increase compared to conventional concrete is 25.3, 18.29 and 19.51% respectively.
3.3. SEM (Scanning electron microscope)
2
Tensile Strength (N/mm )
7th day
28th day
1.86 MPa
2.23 MPa
3.64 MPa
3.49 MPa
3.28 MPa
4.11 MPa
3.88 MPa
3.92 MPa
Upon visually inspecting the specimens incubated with bacterial water, the presence of carbonate crystals was observed as
shown in Fig. 3. The visible rhombohedra crystals depict the presence of calcite ions, on the other hand, the acetate ions resulted in
spherical ions, which proves the success of the study based upon
previous investigations and findings [2,8,11].
Fig. 3. (a): The rectangular marked portion shows the presence of calcite in Bacillus subtilis Concrete. (b) Presence of Calcite precipitate in Bacillus megaterium Concrete. (c)
Presence of Calcite precipitate in Consortia Concrete.
N. Nain et al. / Construction and Building Materials 202 (2019) 904–908
Table 4
Results of Energy dispersive spectroscopy.
Concrete Specimen
Percentage of CaO in the specimen
by weight for 28 days curing
B. subtilis
B. megaterium
Consortia
79.08
80.65
79.90
3.4. EDAX (Energy dispersive spectroscopy)
To examine the amount of CaCO3 present in the treated sample,
an elemental constitutions examination of the sample was carried
out to determine the weight ratio and the results are as tabulated
in Table 4. Cement is composed of oxides of Ca, Si, Al, Fe and Sulphate. Compound ‘‘Calcium oxide” is one of the main factors which
accounts for strength in concrete, the composition of the same in
conventional concrete varies from 60 to 67%. After determining elemental constituents from EDAX, amount of CaO was calculated for
the treated and conventional concrete specimen and a very significant increase in the same was observed for the treated specimens
[9,29].
4. Discussion
The main motive of this experimental study was to determine
the change in concrete properties upon incubation of grampositive, calcite producing bacteria. This bacteria acts as a nutritive
medium which neither allows the loss of slump nor causes immediate setting as it provides workable time for concrete. The bacteria
were cultured and added in the water mix along with nutrient
broth. These bacteria acted as a site of nucleation that is bacterial
cell surface having negatively charged groups, attracts divalent
ions such as Ca2+, Mg2+ [4]. Urease enzyme then catalysis the
hydrolysis of urea into ammonia and carbonate, which then reacts
with the attracted divalent ion of Ca2+, thereby forming Calcium
carbonate [9]. This Calcium carbonate acts as a sealant and exhibits
positive potential to consolidate certain micro-cracks.
This production of Calcium carbonate causes better packing and
compaction of the concrete mix around them which gave the specimens much higher strength than controlled concrete specimen
[27]. Upon conduction of tests for strength, both compressive
and tensile, Bacillus megaterium showed a lower initial strength
i.e. after 7 days of curing when compared with Bacillus subtilis
and also when compared with consortia of both, which depicts that
B. megaterium has slower ability of obtaining the precipitation in
comparison with B. subtilis even after it’s proved viability after
7 days. Whereas after 28 days of curing (when according to IS
456: 2000) [38] a concrete specimen is supposed to attain 95% of
its life strength, all the 3 specimen that is with B. subtilis, B. megaterium and their consortia showed significantly high value of
strength compared to conventional concrete. This concluded that
the process of obtaining precipitation after consumption of ions
is significantly faster in B. subtilis when compared to that with B.
megaterium. The increased Calcium carbonate gives main focus
on crack healing efficiency directly strengthening the core. This
gives another proof of functioning of the bacteria.
The treated specimen and conventional specimen were both
tested and then compared. After comparison, Bacillus subtilis
showed a strength increase of 14.3%, Bacillus megaterium showed
a strength increase of 22.5% and the consortia of both showed a
percentage increase of 15.8% than conventional concrete. The test
results prove the functioning of microbiologically induced calcium
carbonate precipitation. The Calcium carbonate produced acts as a
catalyst for cement hydration and enhances hydrolyzation at faster
rater and hence increases the compressive strength of concrete and
also, there is an early gain in strength
907
A test for tensile strength, namely ‘‘Split Tensile Test” is conducted to determine the resistance of the concrete specimen to
withstand tension. Due to the brittle nature of concrete, it is very
weak in tension and is not expected to withstand high tensile load.
It is relatively difficult to conduct a direct tensile test, and thereby
either flexural test or split tensile test is conducted and tensile
strength is obtained based on formula mentioned in IS456: 2000
[38]. Like compressive strength, a similar increase in tensile
strength for all the 3 cases is observed compared to conventional
concrete. B. subtilis showed a percentage increase of 25.3%, B. megaterium showed a percentage increase of 18.29%, and also an
increase of 19.51% is seen in their consortia.
Scanned magnified images of the treated specimen is obtained
from SEM as shown in Fig. 3(a)–(c), showed the presence of Ettringite in the form of fibres and also white calcite precipitation which
is obtained as result of chemical reaction occurred due to the presence of bacteria. It also showed rhombohedra shaped crystals
which from the previous study is proved to be calcite ion [8,11].
EDAX analysis shows a higher content of CaO in treated concrete
compared to conventional concrete as mentioned in Table 4. In
comparison with B. subtilis and consortia of the concrete, B. megaterium exhibit an increased amount of CaO thus supporting the
higher value of compressive strength than other treated and conventional specimens. This higher CaO content is the main reason
for higher compressive strength in Bacillus megaterium when compared to other.
After introducing bacteria along with the nutrient, a sample
from specimen after 7 days of curing, is tested for bacterial viability using a simple plate count test on an agar plate. Bacterial colonies on the agar plate after an overnight of incubation proved that
bacteria were capable of withstanding the harsh alkaline environment in the interior of concrete. Hence, the bacteria survived in the
cement mixture and multiplied in early stages and produced Calcium carbonate as well adequately.
The Calcium carbonate deposition plugs the pores and certain
micro cracks in the concrete. It was also observed that once the
pores are plugged, the flow of nutrients and oxygen to bacteria
cells stopped, gradually cells died or form into endospores and acts
as organic fibre, thus increasing compressive strength. This study
concludes that bacteria from genus Bacillus is not only proved to
be efficient in crack healing ability [1,4,27] but also plays a great
role to increase the strength.
5. Conclusion
The following conclusions can be drawn based on the present
study The plate count test after curing proves the viability of bacteria
in concrete and its ability to withstand the harsh environment.
B. subtilis shows a percentage increase of 14.3% in compressive
strength and 25.3% increase in tensile strength compared to the
conventional specimen.
B. megaterium shows a percentage increase of 22.5% in compressive strength and 18.49% increase in tensile strength compared
to the conventional specimen.
Consortia of bacteria show a percentage increase of 15.8% in
compressive strength and 19.58% increase in tensile strength
compared to the conventional specimen.
The slower strength gains of B. megaterium depicts that B.
megaterium has a slower ability to obtain the precipitation in
comparison with B. subtilis even after it’s proved viability after
7 days.
Consortia show a percentage increase of 1.3% in compressive
strength compared to B. subtilis and a percentage decrease of
5.8% in same when compared to B. megaterium.
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N. Nain et al. / Construction and Building Materials 202 (2019) 904–908
Consortia show a percentage decrease of 4.8% in tensile strength
compared to B. subtilis and a percentage increase of 1.03% in
same when compared to B. megaterium.
SEM images and EDAX result analysis gives a strong proof of the
presence of calcite precipitation.
The study conclusively indicated that the microorganism
demonstrated a positive role in enhancing the strength of concrete
which is due to filling up of voids by calcite precipitation in the
concrete specimen. Hence, there is a possibility of self-healing of
microcracks for which further studies are required.
Conflict of interest
None.
Acknowledgements
This research was supported and financed by PES University,
Bangalore, India.
The authors show gratitude for technical assistance.
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