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Int. J. Life. Sci. Scienti. Res.

eISSN: 2455-1716
Jokyani and Chouhan, 2018
DOI:10.21276/ijlssr.2018.4.5.10

Research Article

Isolation, Characterization, and Application of Calcite Producing


Bacteria for Self-Healing Concrete Preparation
1 2
Diksha H. Jokyani , Dharmvir Chouhan *
1
Student, P. G Department of Microbiology, Dhote Bandhu Science College, Gondia, India
2
Assistant Professor cum HOD, P. G Department of Microbiology, Dhote Bandhu Science College, Gondia, India

*Address for Correspondence: Prof. D. A. Chouhan, Assistant Professor cum HOD, P.G Department of Microbiology,
Dhote Bandhu Science College, Kudwa Road Gondia, Maharashtra- 441614, India
Received: 18 Apr 2018/ Revised: 05 Jun 2018/ Accepted: 02 Aug 2018

ABSTRACT
Concrete being the foremost building material broadly used in the construction sector is subjected to crack formation due to low
tensile strength, durability, and ductility. So this issue is of great curiosity to the researchers in pursuit for the concrete production
with better properties. Micro-cracks are the main reason for structural failure occurs, when the load applied exceeds its limits.
This causes the seepage of water and other salts. In order to overcome this, the carbonate precipitating, non-pathogenic, spore-
forming, alkaline resistant strain of Bacillus subtilis has been explored as a bio-cementing material. Cracks in M20 grade concrete
4 5 6
blocks are injected by direct means with screened bacterial strain Bacillus subtilis (BH3) at the cell concentration of 10 , 10 , 10
CFU/ml with silica gel as an immobilizing agent and calcium lactate as a food source. The cracks were allowed to heal for
appropriate time duration at specific pH, temperature, and urea concentration. These findings suggest the potential of Bacillus
subtilis in an autogenously healing process.

Key-words: Bacillus subtilis, BH3, Carbonate precipitation, Calcite, Self healing and Bio-cementation

INTRODUCTION
A large amount of non-renewable resource is consumed in the cracking of concrete structures during its
by the construction engineering sector, most of which hardening and this ultimately leads to structural
contribute to the highest proportion of global CO2 deformation. Latter the ingress of some hazardous
emission at their production or application stage. The chemicals and moisture into the concrete may lead to
concrete production process is an energy-intensive decrement in its serviceability whereas; the penetration
process if in case of mining, transportation and of sulphates and chlorides in cracks causes the durability
processing are considered. Its production level lies at to be affected. Hence the more concern should be given
about 2.35 billion metric tonnes per year and contributes to this so as to prevent the expansion of cracks by a
an astonishing 10% of CO2 emission in the atmosphere [1]. sustainable means that might involve the natural
Another issue concerns is the huge maintenance costs microbial mechanism of bio-cementation which is
for structure built in past. About 10% of the bridges in promising.
the USA are considered structurally deficient and 10% of Autonomously healed concrete is nothing but the
it is considered functionally obsolete [2]. Apart from this biologically produced limestone by which the cracks can
the factors like freeze-thaw reactions, shrinkage, results be healed effectively. The screened bacterial strain of
genus “Bacillus” along with the calcium based nutrient
How to cite this article like calcium lactate and an immobilizing agent like silica
Jokyani DH, Chouhan D. Isolation, Characterization, and
Application of Calcite Producing Bacteria for Self-Healing Concrete gel is mixed in appropriate proportion and injected into
Preparation. Int. J. Life. Sci. Scienti. Res., 2018; 4(5): 2025-2036. the preformed cracks by direct means. In case if it is
added initially to the concrete mixture then the
Access this article online self-healing agent can lie in the dormant stage within the
www.ijlssr.com concrete for up to about 200 years. Due to any of the
damage the water starts to percolate deep into the
structure and the dormant bacterial form if get the

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Jokyani and Chouhan, 2018
DOI:10.21276/ijlssr.2018.4.5.10

growth favourable conditions to acquire its vegetative Microbiology, Dhote Bandhu Science College, Gondia,
form and starts to feed on calcium lactate consuming India from August 2017 to March 2018.
oxygen and thereby converting soluble calcium lactate to
Preparation and artificial cracking of concrete blocks-
insoluble calcium carbonate better called limestone.
The concrete blocks of M20 grade were first prepared
Here the consumption of oxygen by the bacterial strain is
with the mould size of 100 mm x 60 mm and left for 28
advantageous as oxygen is responsible for steel
days curing process. Further, the blocks were subjected
corrosion thereby enhancing the durability of steel
to indigenous cracking by plastic shrinkage method
structures [3], Since 1980’s several researchers had been
(plastic shrinkage cracks usually ranges between 1-2 mm
working on this concept and had used the different
bacterial strains such as Jonker et al. [4] used Bacillus depth) [8].
cohnii bacteria to precipitate CaCO3 while Santhosh et al. Determining Size of cracks- Size of the cracks in concrete
[5]
and Bang et al. [6] used Bacillus pasteurii. block was measured using scale [9].
As the bacterial cell wall is negatively charged, it will
draw the cations from the environment, Ca2+ and deposit Isolation of new ureolytic bacteria- Soil samples
it on their cell surface serving as a nucleation site [6]. pasteurized at 800C for 15 minutes then subjected to
tenfold dilution. The 0.2 ml of pasteurized diluted sample
Ca2+ + Cell Cell-Ca2+ 1 was then plated on sterile urea agar plates with 5% urea
concentration. Plates were incubated at 370C for 24 - 48
This bacterial genus is known to have specific urease
hrs and observed the isolated colonies. Pink color
activity that is utilized for the hydrolysis of urea into 1
colonies were selected and further screened based on
mole of ammonia and 1 mole of carbonic acid [7].
the capability to degrade urea in Christensen’s agar
H2N-CO-NH2 + H2O NH3 + H2N-CO-OH 2 medium and to tolerate high alkaline condition based on
pH optimization studies. The single selected potent
H2N-CO-OH + H2O NH3 + H2CO3 3
organism was in turn identified based on its
These two products was subsequently form 2 moles of
morphological and biochemical characteristics. Latter on
ammonium and hydroxide ion as,
the identified isolate was pure cultured on urea agar
H2CO3 HCO3- + H+ 4 slants and preserved at low temperature (40C) [10].

2NH3 + 2H2O 2NH4+ + 2OH- 5 Study on effect of environmental and nutritional


condition on urease production a capability of BH3
Reaction 4 and 5 turned in results in pH increase and strains- Bacillus subtilis strain with promising result was
shifts the equilibrium, resulting in the carbonate ion selected and the effect of environmental condition like
formation as, temperature, and nutritional condition such as urea
concentration were characterized. For temperature
HCO3- + H++2NH4+ +2OH- CO32- +2NH4+ + 2H2O 6
optimization, 1 ml of overnight growth culture was
The carbonate formed in reaction 6 was subsequently inoculated in 3 tubes containing urease broth except the
reacted with Ca2+ ions deposited on bacterial cell wall, control tube and incubated at 3 different temperature
leading to CaCO3 precipitation. ranges as 250C, 350C, and 450C then after 24 hours the
tubes were observed for change in color intensity and
Cell-Ca2+ + CO32- Cell- CaCO3 7 biomass was determined nephlometrically. For urea
MATERIALS AND METHODS concentration, optimization purpose 1 ml of overnight
Materials and Chemical used- Soil samples used for growth culture was inoculated in 3 tubes containing urea
isolation of potent strain were collected from 12 in concentration range of 2%, 5%, and 10% except the
different construction sites across the Gondia district, control tube and incubated at 350C. After 24 hours the
India. All the required chemicals in this work were tubes were observed for change in color intensity and
purchased from Hi-media laboratories, India. The biomass was determined nephlometrically. [11]
research work was carried out in the P.G Department of

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Jokyani and Chouhan, 2018
DOI:10.21276/ijlssr.2018.4.5.10
[14]
Growth Curve of Bacillus subtilis strains: The growth blocks .
curve of screened isolate was determined to set a
WSaturation – WOven Dried
growth comparison point at optimum condition. Three
ml of overnight growth culture was used to inoculate in
% water absorption = X 100
WOven Dried
30 ml of urea broth in 250 ml conical flask then the
Where,
culture was incubated for 27 hours at 350C, pH 8, and 5%
WSaturation = Weight of block after saturation
urea concentration. Inoculation time was considered as
WOven Dried= Weight of block after drying
zero time and the quantitative determination of growth
was carried out by spectrophotometer at 600 nm. RESULTS
Simultaneously the viable cell count was determined as a As the durability of concrete is affected by the cracks
colony forming units/ml (CFUs) [12]. The desired isolate leading to corrosion of reinforcing bars, the general
was purified and mixed with silica gel in 1: 100 dilution method of repairing is time consuming and expensive, so
factors with the cell count 104, 105, 106 cells so as to bio based calcite precipitation has been proposed as an
immobilize the bacterial cells and hence it can remain alternative and sustainable, environment friendly cracks
embedded for the long time in the concrete matrix. repair technique. The bio agent selected for this purpose
Along with it, calcium lactate is also added to it with was based on its tolerance to high pH, and continuous
molar mass 218 g/mole that serve as food source for formation of dense CaCO3 in the liquid medium.
bacterial cells. As it gets the growth favorable conditions According to analysis and study of blocks by the visual
it will soon start to precipitate CaCO3 and the bacterial examination of concrete core, the estimated size of
cells will be in turn coated with it resulting in the cracks varied from 0.3 to 0.5 mm [9]. Total 12 urease
autogenous healing [13]. positive Bacillus subtilis strains were isolated from soil
sample. Out of these 12 isolates Bacillus subtilis BH3
Water Permeability Test of Concrete- To determine the
strains were found to be most potent.
water absorption ability of bacterial concrete, this test
was carried out in a comparative manner. For this, the Optimization- The optimum temperature required for
control and bacteria treated concrete blocks were kept growth of Bacillus subtilis was 350C. Table 1 and Fig. 1
overnight saturated in the saline buffer and weighed. shows the optimization of temperature by using 3
Then both blocks were dried in the oven at 1000C for 24 different temperature ranges.
hours, cooled and again weighed. The obtained values
were then put into the formula given below to
determine percent water absorption by both the

Table 1: Measurement of bacterial growth at 3 different temperature ranges

Growth measure
Temperature range
S. No. Bacterial strains Ureolytic activity Interpretation
(0C) OD600
Color changes to The maximum
1. 25 1.51 ureolytic activity
mild pink
with effective
Color changes to
2. BH3 35 1.52 growth was
intense pink
observed at
Color changes to temperature range
3. 45 1.519
light pink of 350C
BH3= Bacillus subtilis

The specific urea concentration supporting the growth Table 2 and Fig. 2 show the effect of urea concentration
of selected ureolytic bacterial strains (BH3) was 5%. The on the bacterial growth and urea degrading ability of it.

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Jokyani and Chouhan, 2018
DOI:10.21276/ijlssr.2018.4.5.10

Effect of Temperature on growth of BH3


strain

Absorbance at 600 nm
1.525
1.52
1.515
1.51
1.505
25 35 45
Temperature range (0C)

Fig. 1: Effect of temperature on growth of Bacillus subtilis strains

Table 2: Measurement of bacterial growth at 3 different urea concentrations

Bacterial Urea Growth measure


S. No. Ureolytic activity Interpretation
strains concentration OD600

1. 2% 0.54 Color changes to light pink The maximum


ureolytic activity
Color changes to Intense with effective
2. BH3 5% 1.75
pink growth was
observed at 5% urea
3. 10% 1.69 Color changes to mild pink
concentration

Effect of urea concentration on growth of BH3


Absorbance at 600 nm

strain
2

1.5

0.5

0
2% 5% 10%
Urea concentration

Fig. 2: Effect of urea concentration on growth rate of Bacillus subtilis strains

Spectroscopic growth curve- The growth curve of referred as log phase. Nephlometric reading showed that
Bacillus subtilis was elucidated using the partially the cultures reached the stationary phase after 10 hours.
optimized condition. Table 3 and Fig. 3 indicate the After 24 hours the bacterial growth was inhibited due to
growth curve of B. subtilis isolates, which is considered media component depletion and the release of
as the selected isolate of this research paper. secondary metabolite that may be toxic to viable cells.
The maximum OD was seen between 6 to 10 hours and
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Jokyani and Chouhan, 2018
DOI:10.21276/ijlssr.2018.4.5.10

Table 3: Nephlometric absorbance values to construct growth curve of screened isolate of B. subtilis

Absorbance at 600 nm
Time in hours Trial 1 Trial 2 Trial 3 Average
1 0.162 0.164 0.180 0.168

2 0.372 0.343 0.417 0.377

3 0.651 0.621 0.682 0.651

4 0.911 1.001 1.073 0.995

6 1.400 1.545 1.521 1.488

8 1.618 1.647 1.690 1.651

9 1.671 1.677 1.700 1.682

10 1.720 1.677 1.710 1.702

11 1.723 1.694 1.721 1.712

12 1.731 1.717 1.729 1.725

25 1.019 1.724 0.940 1.227

26 0.911 0.853 0.848 0.870

27 0.908 0.847 0.769 0.841

Growth curve of strain BH3

2
OD at 600 nm

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1 2 3 4 6 8 9 10 11 12 25 26 27

Hours of incubation

Fig. 3: Growth curve of screened potent isolate of B. subtilis strains

Studies on concrete crack healing potential of BH3 of crack was reduced to 0.18 mm.
strains- In this investigation, the 3 concrete blocks, Block Similarly the Block 2 with crack size of 0.52 mm when
1, Block 2, and Block 3 were used for autogenous healing injected with 106 CFU/ml showed the efficient healing on
by the selected strains of Bacillus subtilis. Block 1 with 19th day of inoculation, whereas the Block 3 with crack
crack size of 0.45 mm width was injected with 105 CFU/ size 0.48 mm width, when injected with 104 CFU/ml,
ml along with silica gel and calcium lactate showed the showed the healing on 23rd day of inoculation with the
healing of cracks at 21st day of inoculation and the width reduction in crack size up to 0.25 mm.

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Jokyani and Chouhan, 2018
DOI:10.21276/ijlssr.2018.4.5.10
5
Table 4: Concrete crack healing potential of B. subtilis strains in block 1 (10 cells/ml)

S. No. Date Initial Characteristics Final characteristic (Width) Interpretation

1. 22nd January 0.45 mm

2. 23rd January 0.45mm


3. 24th January 0.44 mm
4. 25th January 0.43 mm
5. 26th January 0.41 mm
6. 27th January 0.4 mm
7. 28th January 0.4 mm
8. 29th January 0.38 mm
9. 30th January 0.37 mm
Block 1, inoculated
10. 31st January Grade: M20 0.37 mm
with the 105 cells/ml
11. 1st February Size: 100 X 60 mm 0.35mm of BH 3 strains,
Curing: 28 days showed the
12. 2nd February Crack developed by: 0.34 mm
effective healing
13. 3rd February plastic shrinkage type 0.31 mm within 21 days from
Width: 0.45 mm the day of
14. 4th February 0.28 mm
inoculation
15. 5th February 0.27 mm
16. 6th February 0.25 mm
17. 7th February 0.23 mm
18 8th February 0.2 mm
19. 9th February 0.19 mm
20. 10th February 0.19 mm
21. 11th February 0.18 mm

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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Jokyani and Chouhan, 2018
DOI:10.21276/ijlssr.2018.4.5.10

Fig. 4: Crack healing study in bacteria treated cracked concrete block (Block 1)

Table 5: Concrete crack healing potential of B. subtilis strains in block 2 (106 cells/ml)

Block characteristics Final characteristic


S. No. Date Interpretation
(Width)
1. 22nd January 0.52 mm
rd
2. 23 January 0.52 mm
th
3. 24 January 0.52 mm
th
4. 25 January 0.518mm
th Grade: M20 Block 2, inoculated with the
5. 26 January 0.516 mm
Size: 100 X 60 mm 106 cells/ml of BH 3 strains,
6. 27th January Curing: 28 days 0.514 mm showed the effective healing
th
7. 28 January Crack developed by: 0.51 mm within 19 days from the day of
th
8. 29 January plastic shrinkage type 0.48 mm inoculation
th Width: 0.52 mm
9. 30 January 0.47 mm

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10. 31st January 0.47 mm


st
11. 1 February 0.46 mm
nd
12. 2 February 0.44 mm
rd
13. 3 February 0.44 mm
th
14. 4 February 0.4 mm
th
15. 5 February 0.4 mm
th
16. 6 February 0.4 mm
th
17. 7 February 0.39 mm
th
18. 8 February 0.39 mm
th
19. 9 February 0.38 mm

Fig. 5: Crack healing study in bacteria treated cracked concrete block (Block 2)

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Jokyani and Chouhan, 2018
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4
Table 6: Concrete crack healing potential of B. subtilis strains in block 3 (10 cells/ml)

S. No. Date Block characteristics Final characteristic (Width) Interpretation

1. 22nd January 0.52 mm


2. 23rd January 0.52 mm
3. 24th January 0.52 mm
4. 25th January 0.518mm

5. 26th January 0.516 mm

6. 27th January 0.514 mm

7. 28th January 0.51 mm

8. 29th January 0.48 mm

9. 30th January 0.47 mm

10. 31st January Grade: M20 0.47 mm


Size: 100 X 60 mm Block 3, inoculated with the
11. 1st February 0.46 mm
Curing: 28 days 104 cells/ml of BH3 strains,
12. 2nd February Crack developed by: 0.44 mm showed the effective healing
plastic shrinkage within 23 days from the day
13. 3rd February 0.44 mm
type of inoculation
14. 4th February Width: 0.48 mm 0.4 mm

15. 5th February 0.4 mm

16. 6th February 0.4 mm


17. 7th February 0.39 mm
18. 8th February 0.39 mm
19. 9th February 0.38 mm
20. 10th February 0.26 mm
21. 11th February 0.26 mm
22. 12th February 0.25 mm
23. 13th February 0.25 mm

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Fig. 6: Crack healing study in bacteria treated cracked concrete block (Block 3)

Water absorption test by using saline buffer- The saline concrete block had shown the higher water absorption
buffer is used in this research work so as to determine as compared to the concrete block treated with potent
the increased resistance of concrete block towards the ureolytic bacterial BH3 strains. Table 7 numerically
water penetration and this test was conducted at a represents the bacterial influence on the water
laboratory level. According to this test, the normal permeability of blocks.

Table 7: Water saturation test results of bacteria healed cracked concrete

Blocks No. Weight (Saturation) Weight (Dried) Water absorption (%)

Block 1 192.73 183.70 4.91%

Block 2 188.05 174.80 7.58%


Block 3 152.30 140.10 8.70%
Control 131.1 119.4 9.79%

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DISCUSSION along with prevention of steel corrosion, the microbial


This study has revealed the BH3 strains of bacterial concrete technology had proved to be better than the
species (Bacillus subtilis) isolated from commercial conventional technologies. It may also be termed as
construction sites, have the high ureolytic activity and “Smart Bio Material”. The overall development of
can tolerate the pH range up to 12, which becomes the strength in bio-concrete is attributed to significant
primary factor for bio-cementation. Salmabanu L and reduction in water absorption capability by using potent
Suthar G [2] stated this genus of endolithic bacteria can bacterial strains of Bacillus subtilis immobilized by means
resist the pH range up to 13 and persist for the long of silica gel and supplemented with calcium lactate as
duration with efficient ureolytic activity. The isolated and food source. The efficient healing was shown with 105
screened potent isolate (BH3) was identified cells, in block 1 with the reduction in crack size from 0.43
phenotypically and biochemically same as done by Achal mm to 0.29 mm width at 21st day of inoculation.
et al. [11] and Cheng et al. [15] while the species level More exploratory works at large scale should be
characterization was achieved by 16s gene sequencing undertaken to determine the efficacy of bio cementation
technique. From the further optimization studies of BH3 for consolidations of building. To make the process
strains, it was clear that it shows the effective urea economical, microbial additives can repaired by
degrading activity at temperature 350C with 5% urea industrial growth of cells by employing the products as
concentration. Similar conclusion was given by Steubing lactose mother liquor and corn step liquor as nutrient
[12]
, who stated that change in temperature and source. Also, the durability of bacterial concrete should
nutritionally sound components can also affect the be studied under the various weathering conditions.
CaCO3 precipitation efficiency by ureolytic bacteria.
ACKNOWLEDGMENTS
Growth curve of this potent strains were determined
The author is grateful to P.G. Department of
nephlometrically that showed the log phase of these
Microbiology, Dhote Bandhu Science College, India for
strains during initial 6-10 hours and become stationary
their help and support in carrying out the present study.
after 10 hours further reduction in the growth rate after
Technical assistance and advice was provided by Dr. D. A.
24 hours. The cell count in the range 105 CFU/ml when
Chouhan so I would like to thank him for his
injected in block 1 with the crack width size of 0.45 mm
encouragement.
shows the significant reduction in crack size up to 0.18 in
21 days after inoculation which was more effective as CONTRIBUTION OF AUTHORS
compared to other 2 concrete block injected with 106 Research concept- Prof. D. A. Chouhan
and 104 CFU/ml cells. A similar finding was reported by Research design- Prof. D. A. Chouhan
Bai and Varghese [16] during an experimental Supervision- Prof. D. A. Chouhan
investigation on the strength properties of fly ash based Data collection- Diksha H. Jokyani
bacterial concrete. Data analysis and interpretation- Diksha H. Jokyani
The bacteria treated concrete block shows the significant Literature search- Diksha H. Jokyani
reduction in its water penetration ability compared to Writing article- Diksha H. Jokyani, Prof. D. A. Chouhan
normal concrete block due to the deposition of CaCO3 Critical review- Prof. D. A. Chouhan
matrix like structure around the bacterial cells thereby Article editing- Prof. D. A. Chouhan
blocking the pores and inhibiting the water percolation Final approval- Prof. D. A. Chouhan
through it. As with constant development in the field of
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DOI:10.21276/ijlssr.2018.4.5.10

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