Eco. Env. & Cons. 23 (September Suppl.) : 2017; pp.

(S57-S63)
Copyright@ EM International
ISSN 0971–765X

Influence of ureolytic bacteria in improving

performance characteristics of concrete
Prabhath Ranjan Kumar Soda1, V. Poornima2, R. Venkatasubramani3 and V. Sreevidya4

1,2
Department of Civil Engineering, Amrita University, Coimbatore, India.
3
Department of Civil Engineering, Dr. Mahalingam College of Engineering and Technology,
Coimbatore, India.
4
Department of Civil Engineering, Sri Krishna College of Technology, Coimbatore, India.

(Received 27 January, 2017; accepted 12 March, 2017)

ABSTRACT
A distinctive strength and durability associated phenomenon in numerous concrete structures are subtle
cracks and pores in the concrete. Principally, related cracks and pores result in increasing permeability of
cement concrete matrix. Entry of water, atmospheric pollutants and their byproducts can deteriorate the
matrix and diminish the strength and durability apparently. Also, it causes corrosion of the installed
reinforcement which in turn, enhances the maintenance cost. Previous research works have dealt with
conventional repair methods like epoxy injection, latex treatment and provision of further steel in design to
limit the crack width within permissible limit. On the other hand, bioconcrete is reliable in enhancing the
strength, durability and healing of cracks. In this research the bacterial strain, Bacillus licheniformis MTCC
3606, derived from soil is used for calcite precipitation and for achieving better strength and durability
properties in hardened concrete. Also, the bacteria was checked for compatibility with different type of
superplasticizers. It is inferred from the test results that the compressive and flexural strength got increased
significantly. It increased the resistance to acid attack and water absorption in concrete appreciably. SEM
analysis has shown the calcite precipitation inside the bacterial concrete. The results obtained from SEM
analysis were confirmed by XRD.

Key words : Bioconcrete, Bacillus Licheniformis MTCC 3606, Calcite precipitation, SEM, XRD.

Introduction time of structure. Even though the usage of several

The material which is being used for decades in the
field of construction is concrete, is good in compres-
sive strength but is deficient in durability properties
like resistance to water absorption, chloride attack,
acid attack, and the cracks formed due to drying
shrinkage. This leads to damage of the concrete by
which, harmful ingredients penetrate through the
cracks formed leading to corrosion of incorporated
steel in the RCC structures, thereby accelerating the
premature loss in structural integrity and the life
cementitious materials like fly ash, GGBS and silica
fume have checked the problems of porosity by in-
creasing its density and the problems due to CO2
emissions while manufacturing the cement up to
certain limit, the problems of micro cracks are still in
existence and the cost involved in repair works like
injecting with epoxy, treating with latex emulsion
being carried out to avoid further extension of
cracks is higher.
In order to avoid such difficulties, a reliable so-
lution was tried in collaboration with microbiology

Corresponding author’s email : poorni.engg@gmail.com

S58
department by means of using bacteria in concrete.
Recent researches conducted on bioconcrete have
given that among all kinds of bacteria, all bacillus
gram-positive strains are more susceptible to an al-
kaline environment which exists in concrete. In this
new era, using microbes in concrete assures precipi-
tation of a certain amount of calcium carbonate
thereby filling the pores and healing the cracks of
micro scale that enhance the durability. This also
checks the further deterioration in hardened con-
crete. The calcium carbonate precipitation by Bacil-
lus licheniformis MTCC 3606 can be achieved by cur-
ing in appropriate medium having a rich calcium
source and urea supplements. Concrete has autog-
enous self-healing capacity without adding any ad-
ditional material but that is limited to only certain
extent that it heals cracks of width 0.2 mm effi-
ciently. But due to adverse effects of environmental
pollutants cracks of width more than 0.2 mm are
expected. So, a new sustainable self-healing mecha-
nism is needed which is possible by incorporating
calcite precipitating bacteria (Jonkers et al., 2011).
The healing capacities of the different bacteria
were checked and it was inferred that Bacillus
Licheniformis AK01 produces more calcite and sus-
tains better in the alkaline conditions which is com-
mon inside concrete. There are a few sources of cal-
cium such as calcium nitrate (Ca(NO3)2), calcium
lactate (C6H10CaO6), calcium acetate (C4H6CaO4) and
calcium chloride (CaCl 2) among which, calcium
chloride was used in the curing media as a supply of
the calcium source since it has better solubility (Ali
Vahabi et al., 2014).
Calcite precipitation is possible in nature by sev-
eral processes, but urease enzyme reaction is used
presently. It was found that in most of the microbes
extracted from soil the urease activity is high. Urea
is a detoxification product which exists naturally in
environment. Bacterial concentration of 105 cells per
ml was used in concrete and significant compressive
strength was achieved (Seshagiri Rao et al., 2013).
The usage of pure culture in mortar and concrete
results in more resistance to capillary water absorp-
tion and little change in the colour aspect compared
to usage of mixed cultures (Willem De Muynck et
al., 2007). The surface of the inferior quality aggre-
gate (recycled aggregate) can be modified by bio
deposition to reduce the water absorption (Grabiec
et al., 2012).
The main focus of this study is to check the com-
patibility of bacteria with different types of
Eco. Env. & Cons. 23 (September Suppl.) : 2017
superplasticizers that are currently in usage in the
field and to compare the strength and durability
properties of the specimens casted with and without
bacteria in M25 and M40 grades.
Conceptual understanding of calcite precipitation
Limited precipitation of calcite in concrete is pos-
sible due to the reaction between Ca(OH)2 and CO2.
This inadequate calcite can be improved by active
contribution of ureolytic bacteria in concrete by its
urease activity which initiates the hydrolysis of urea.
In this process, urea gets converted into carbamate
and ammonia. Carbamate again hydrolyses into car-
bonic acid and ammonia. Afterwards these products
form bicarbonate, ammonium and hydroxide ions
causing increase in local pH value. This leads to for-
mation of more carbonate ions. These carbonate ions
react with sufficient amount of calcium ions from
curing media leading to formation of calcite on cell
surface (Varenyam Achal et al., 2011).
Materials and Methods
Cement
The binding material used was Portland Pozzolana
cement (PPC) containing 25% fly ash. The physical
tests performed on the cement have given results of
fineness as 2.95%, specific gravity as 2.9, and consis-
tency as 36%. It meets requirements of IS 1489-1
(1991).
Fine Aggregate
The locally available clean and dry sand was used.
Sand passed through IS 4.75 sieve was used to cast
all the specimens. The physical tests performed on
the material are given in Table 1.
Table 1. Physical properties of Fine aggregate
Specific Zone Water
gravity absorption
2.8 2 3%
The obtained results conform to IS 383-1970.
Coarse Aggregate
Locally available coarse aggregate was used. Aggre-
gate passed through IS 20 mm sieve and retained on
IS 12 mm sieve was used for casting the specimens.
The physical tests performed on the aggregates are
SODA ET AL S59

given in the Table 2. ent broth and the bacteria was allowed to grow in
this media separately. The amount of bacteria
Table 2. Physical properties of Coarse aggregate grown along with each type of SP was checked by
Specific gravity Water absorption streaking operation in Petri plates. There was no sig-
nificant change in bacterial growth with and with-
2.74 1.8% out SP, therefore this particular bacterial strain is
compatible with three SPs.
The obtained results conform to IS 383-1970.
Water
Potable water was used for casting the specimens.
The tests performed on the water gave the pH value
as 7.8 and the obtained results conform to IS 456-
2000.
Bacteria Fig. 1. Compatibility with SP’s
The particular details of bacteria, given by MTCC
are shown in Table 3. Preparation of Bacterial Solution
The revived bacterial culture was transferred into
Table 3. Particulars of bacteria sterilized liquid growth media. Then, it was incu-
Genus Name Bacillus bated in an incubator shaker for 24 hours at a speed
of 200 rpm. The obtained solution was turbid whit-
Species Name Licheniformis
Type B
ish yellow in colour indicating the presence of bac-
MTCC No. 3606 terial cells (Ravindranatha et al., 2014). The solution
Isolated or Derived from Soil (new Delhi) was checked for number of cells per ml by using Bio
Temperature 370 c Spectrometer which had given the optical density
Growth condition Aerobic value of the solution tested. By substituting the Op-
Incubation 24 hours tical density value in the following equation the
Sub culturing period 20 days amount of cell per ml was calculated as 1011 cells per
mL (Manikandan et al., 2015).
The Bacillus licheniformis MTCC 3606 was re-
ceived in the form of freeze dried condition from Y = 8.59 x 107 X1.3627 .. (1)
MTCC (Microbial Type Culture Collection) Where, X = reading at OD 600 nm; and
Chandigarh (Krishnapriya et al., 2015). The revival Y = concentration of bacterial cells per mL.
of bacteria was done according to procedure men- Serial dilution of high concentration bacterial
tioned by MTCC. The liquid growth media used for solution
growth of bacteria contained beef extract (1.0 g),
yeast extract (2.0 g), peptone (5.0 g), NaCl (5.0 g), Initially, an equal volume of 8 mL nutrient broth
distilled water (1 L). was transferred into 6 test tubes and labeled from A

Compatibility of Bacteria with Superplasticizers

In present scenario most of the works related to con-
crete are using superplasticizers (SPs) for better
workability, so Bacillus licheniformis MTCC 3606 was
checked for compatibility with three SPs namely
Polycarboxylate Ether of density 1.09 kg/L,
Lignosulphonate of density 1.19 kg/L, and Sulpho-
nated Naphthalene Formaldehyde of density 1.2
kg/L (Dhanya Sathyan et al., 2016). A certain per-
centage of SP from each family was added to nutri- Fig. 2. Bacterial solution of high concentration
S60 Eco. Env. & Cons. 23 (September Suppl.) : 2017

to F. Then 1 mL of the high concentration bacterial
solution was transferred into the test tube A and
mixed gently. Again 1 mL of the solution from the
test tube A was transferred into test tube B and
mixed gently. The same procedure is followed till
the last test tube F. The procedure was done in such
a way that the last test tube F will be having the least
concentrated bacterial solution. The test tubes were
then kept inside the incubator shaker and left for 24
hours to get the diluted solution of required concen-
tration. The cell concentration in each test tube was
checked by substituting the OD value of each in
equation (1).
mal concrete mix. The bacterial water (5 mL bacte-
rial solution of concentration 105 cells per mL per li-
ter of water) was used for the preparation of bacte-
rial concrete. Samples of size 100 x 100 x 100 mm
were prepared for both. The curing process was
done in normal water for normal concrete speci-
mens whereas the curing was done in separate cur-
ing media containing 50 g of CaCl 2 and 20 gms of
urea per litre of water for bacterial specimens (James
et al., 2015). The compression testing has been car-
ried out on 7th, 14th, and 28th days. Testing was done
according to IS 516 (1959).
Flexural Strength Test
The specimens of size 500 x 100 x 100 mm were
casted. The curing for the normal and bacterial
specimens was done in the same way as the com-
pression strength samples. Specimens were tested
for flexure on 7th, 14th and 28thday according to IS 516
(1959).
Durability Tests

Water Absorption Test

Specimens of size 100 x 100 x 100 mm were pre-
pared. Curing was done in their respective media
for 28 days. After curing, both normal and bacterial
specimens were taken out from curing media and
kept in the oven at 1050C +/- 50C for 24 hours and
oven dried weight was noted as W1. After that speci-
Fig. 3. Serial Dilution mens were again immersed completely in water at
room temperature for 48 hours. After 48 hours im-
Mix Proportion
mersion in water, the specimens were taken out and
The mix design was done for the two particular allowed to dry surface moisture then the weight was
grades M25 and M40 by following the guide lines noted as W2 .The corresponding percentage incre-
given in the IS 10262-2009. ment of weight gives the percentage of water ab-
sorption value. The procedure was conformed
Laboratory Tests through ASTM C642-06.
Acid Attack Test
Tests based on Strength
Four specimens of cube size 100 mm with each nor-
Compressive Strength Test
mal and bacterial concrete were casted and cured in
Potable water was used for the preparation of nor- their corresponding curing media for 28 days. One
specimen from each type of concrete was tested for
Table 4. Mix proportion for M25 and M40 compressive strength. The rest of the specimens
were checked for compressive strength after immer-
Grade Water Cement Fine Coarse
sion in 5% concentrated sulphuric acid solution for
(Kg/m3) (Kg/m3) Aggregate Aggregate
(Kg/m3 ) (Kg/m3) 7th, 14th and 28th days. The percentage loss in strength
was calculated by using following formula (Hong-
M25 221.04 564 431.08 1184.435 Sam Kim et al., 2007).
M40 223.91 564 582.64 1091.91
SODA ET AL
Si − Sf
Percentage loss in strength = × 100
Si
Where, S i is initial compressive strength (before
immersion) and S f is final compressive strength (af-
ter immersion).
Fig. 4. Acid attack
Microstructure Study
Scanning Electron Microscope (SEM)
The visual examination of the calcium precipitation
was done by using scanning electronic microscope.
A small piece of size having volume less than 1cm3
was taken from the 28th day bacterial specimen after
checking for compressive strength. The specimen
collected was freed from dust and oven dried for 30
minutes then, plasma coating was done using a
sputter coating machine. Coated samples were
placed in SEM and observed for calcium precipita-
tion at different magnification levels in the pores of
the sample.
X-Ray Diffraction (XRD)
The calcium precipitation by bacteria was confirmed
by getting the chemical composition of the sample
from the XRD. This test was done on samples pre-
pared as crystalline powder which was kept inside
the sample holder and placed at an angle of 45 de-
grees. The intensity of beam used was 30 mA and 45
kV. The starting position (02è) for scanning was 100
and end position (02è) was 900.
Results and Discussions
Compression Strength
The results from the tests showed a substantial in-
crease in strength and durability of normal speci-
mens (NS) and bacterial specimens (BS) in both the
grades due to the calcite precipitation of bacteria in
the pores of the concrete. The results were repre-
sented graphically.
S61
Compressive strength results revealed that bac-
terial specimens yielded 12.5%, 13.6%, and 20%
more than normal specimens strength on 7th, 14th,
and 28th days respectively in M25 grade and 10%,
16.6%, and 20% more in M40 grade.
Flexural Strength
Flexural strength of bacterial specimen results
showed 66%, 78.9%, and 79.7% percent more
strength on 7th, 14th, and 28th days respectively
than normal concrete specimens in M25 grade and
93.7%, 87.5%, and 36.6% percent more in M40 grade.
Water Absorption Test
Water absorption test for bacterial specimens
yielded 32.5% reduction in water absorption per-
centage in M25 grade and 21.2% in M40 grade in
comparison with normal concrete specimens.
4.4 Resistance to Acid Attack
Acid attack test results for bacterial specimens gave
% loss in strength as 45.8%, 47.9%, and 52.08% for
7th, 14th, and 28th days respectively for M25 grade.
Whereas normal specimens lost 45%, 50%, and
57.5%. In M40 grade bacterial specimens lost
48.34%, 55%, and 58.34% and normal specimens lost
46%, 56%, and 60% percent of strength on 7th, 14th,
and 28th days respectively.
Scanning Electron Microscope
The calcite precipitation of bacteria inside the bacte-
rial specimen for both M25 & M40 grades can be
observed clearly as shown in Figure 5 & 6.
X Ray Diffraction
The visual examination of calcite precipitation in
bacterial specimen for both M25 & M40 grade was
Fig 5. Results for Compressive Strength
S62
observed from SEM images were confirmed by XRD
as shown in Fig. 7 and 8.
Conclusions
From the work done on the compatibility of Bacillus
Fig. 6. Results for Flexural Strength
Fig. 7. Results for Water Absorption
Fig. 8. Results for Acid Attack
Eco. Env. & Cons. 23 (September Suppl.) : 2017
licheniformis MTCC 3606 with superplasticizers and
comparison of strength and durability properties of
bioconcrete with normal concrete the following con-
clusions can be drawn
1. The bacteria used was found to be compatible
Fig. 9. Image of SEM for BS M25
Fig. 10. Image of SEM for BS M40
Fig. 11. XRD pattern for Calcite in BS M25
SODA ET AL
with super plasticizers namely Auramix 400,
Conplast X421IC, Conplast SP430 which be-
longs to PCE, LS, and SNF families respec-
tively.
2. A significant increase in compressive strength
and flexural strength was achieved by using
Bacillus licheniformis MTCC 3606 in concrete of
grades M25 and M40 compared to normal
concrete.
3. The water absorption capacity and acid attack
on bioconcrete was considerably less com-
pared to normal concrete.
4. The SEM results have shown the sufficient
amount calcite precipitation by bacteria inside
the pores.
5. By XRD results, the calcite precipitation was
confirmed by getting the chemical composi-
tion.
Acknowledgement
We like to thank Dr. Kumutha and Ms. Kavitha
Mary from TNAU (Tamil Nadu Agricultural Uni-
versity) for their support in cultivating the bacterial
solution. We also like to thank Mr. John Stanley for
his support in serial diluting the bacterial solution
from Amrita University.
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