Bacterial Concrete: A Biomimetics based Sustainable Self-healing

Biomaterial
M V Seshagiri Rao 1a, V Srinivasa Reddy 2b, K Mounika 3c
a

Professor, Department of Civil Engineering, JNTUH College of Enginnering,Hyderbad,500085,India

b

Associate Professor, Department of Civil Engineering, GRIET,Hyderbad,500090,India

PG Student ,Department of Civil Engineering, JNTUH College of Enginnering,Hyderbad,500085,India

Abstract

Biomimetics is a field of science that studies biological processes for effectively using them in the development of
innovative engineering materials and systems. Unlike man-made materials, natural materials can interact with the
neighboring environment, defend themselves from it, and repair autonomously when damage occurs. Enthused by
such properties of natural materials, research on the development of innovative and new bio-based sustainable self
healing construction material called Bacterial concrete is in progress and the research findings are presented in
this paper. The idea is that this novel smart composite construction material can self-repair damage resulting in
substantially decreased maintenance and repair costs and increased service life. Lately, interest has been focused
on self-healing applications in cement-based material. One of the major investigations is to provide self-healing
ability by inducing biogenic calcium carbonate precipitation, which is also known as biomineralization. Research
on biomineralization in cement-based systems has shown promising results and studies suggest that microbialinduced calcium carbonate precipitation leading to self-healing of cracks / or pores in the concrete. This bacterial
activity thus reduces the porosity and permeability of concrete. This crack healing potential in concrete based on
phenomena of microbiologically induced mineral precipitation (MICP) improve durability and possibly regain
strength. The bacteria-induced precipitates obtained are characterized using X-ray diffraction (XRD), Fourier
transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Thermal analysis (TGA) is
performed for quantitative characterization of the deposits.
Key words: bacterial concrete, MICP, self-healing concrete, smart concrete, biomineralization
1. INTRODUCTION
In concrete the cracks up to 0.2 mm wide are healed
autogenously. Such micro cracks are acceptable as
these do not directly influence the safety and strength
of the concrete. Research has shown that autogeneous
healing happens due to hydration of non-reacted
cement particles present in the concrete matrix when
comes in contact with ingress water resulting in
closure of micro cracks. However, because of the
variability of autonomous crack healing of concrete
micro cracks can still occur. The inbuilt bacteria-based
self-healing process was found to heal cracks
completely up to 0.5 mm width. Bioengineered Selfrepairing concrete biologically produces calcium
carbonate crystals to seal cracks that appear on the
surface of the concrete structures. Specific spore
forming alkaliphilic bacteria genus Bacillus, supplied
with a calcium-based nutrient are incorporated in to
the concrete suspended in mixing water. When cracks
appear in a concrete structure and water starts to seep
in through, the spores of the bacteria starts microbial
activities on contact with the water and oxygen. In the
process of precipitating calcite crystals through
nitrogen cycle the soluble nutrients are converted to

insoluble CaCO3. The CaCO3 solidifies on the cracked

surface, thereby sealing it up. The consumption of
oxygen during the metabolic biochemical reactions to
form CaCO3 helps in arresting corrosion of steel
because the oxygen is responsible to initiate the
process of corrosion thereby increasing the durability
of steel reinforced concrete structures. In the concrete
technology laboratory, a bacterial concrete mix is
prepared using alkali-resistant soil bacteria Bacillus
subtilis JC3, along with nutrients from which the
bacteria could potentially produce calcite based biominerals. Twenty eight days cured bacterial specimens
are examined visually by Scanning Electron
Microscope to establish evidence for the precipitation
of calcite crystals in concrete. It was found that strains
of the bacteria genus Bacillus were found to thrive in
this high-alkaline environment. Such gram positive
bacteria have extremely thick outer cell membrane
that enables them to remain viable until a suitable
environment is available to grow. They would become
lively when the cracks form on concrete surface
allowing water to ingress into the structure. This
phenomenon will reduce the pH of the concrete
environment where the bacteria incorporated become

activated. In the present study, a peptone based

nutrients supplied along with bacteria in suspension
helps in producing calcite crystals. It is found that this
bio-mineralization process will not interfere with the
setting time of the concrete. The most expensive
ingredient in developing bacterial concrete is
nutrients. So any inexpensive alternative for
laboratory growth media would potentially bring
down the cost of the bacteria based self-healing
sustainable concrete. Only factor need to be checked
is the effect of nutrients media on the setting time of
cement. Potential applications of biological mineral
precipitation are wide such as in sand consolidation
and stabilization, remediation of cracks in concrete,
preservation and restoration of historic heritage
structures, areas where it is not possible to shut down
the plant or hazardous for human beings to reach for
repair work such as nuclear power plants, repair of
waste water sewage pipes etc.
2. MICROBIOLOGICALLY INDUCED CACO3
PRECIPITATION
Bio-mineralization is defined as a biologically induced
precipitation in which an organism creates a local
micro-environment with conditions that allow optimal
extracellular chemical precipitation of mineral phases.
Almost all bacteria are capable of calcium carbonate
precipitation. A novel technique for the remediation of
damaged structural formations has been developed by
employing a selective microbial plugging process, in
which metabolic activities promote precipitation of
calcium carbonate in the form of calcite.
Biomineralization of calcium carbonate is one of the
strategies to remediate cracks in building materials. In
nature, microorganisms can induce calcite mineral
precipitation through nitrogen cycle either by
ammonification of amino acids/ nitrate reduction/
hydrolysis of urea. The binding strength of the
precipitated crystals is highly dependent on the rate of
carbonate formation and under suitable conditions it is
possible to control the reaction to generate hard
binding calcite cement (or bio-cement). Three main
groups of microorganism that can induce the
carbonate precipitation in nature: i. photosynthetic
microorganism such as cyanobacteria and micro-algae
that can remove CO2; ii. sulphate reducing bacteria
that are responsible for dissimilatory reduction of
sulphates; and iii. some species of microorganism
participate in nitrogen cycle by any one of the
methods such as oxidative deamination of amino
acids, nitrate reduction or hydrolysis of urea. The
evidence of microorganism involvement in calcium
carbonate precipitation, has lead the development of
bioprocess technology in the field of construction

material. The involvement of microorganism in

CaCO3 precipitation can be described in three type of
mechanism: i. spontaneous mechanism, usually by
photosynthetic microorganism; ii. Through nitrogen
cycle; iii. Through sulfur cycle.
3. WORKING PRINCIPLE OF BACTERIAL
CONCRETE
Bacterial calcium carbonate precipitation results from
both passive and active nucleation. Passive carbonate
nucleation occurs from metabolically driven changes
in the bulk fluid environment surrounding the
bacterial cells. This increases the mineral saturation
and induces nucleation. In the ammo acid degradation
driven system, this occurs from an increase in pH due
to ammonification. Active carbonate nucleation occurs
when the bacterial cell surface is utilized as the
nucleation site. The cell clusters exhibit a net
electronegative charge which favors the adsorption of
Ca2+ ions. The Ca2+ ions attract CO32- and HCO3- ions,
which will eventually form calcium carbonate
precipitates. Although it is known that there are many
different types of bacteria capable of calcium
carbonate precipitation, it has been hypothesized that
there are specific attributes of certain bacteria that
promote and affect CaCO3 precipitation more than
others. It has already been noted that cell walls have
an inherent electronegative charge that affect the
binding of certain ions, but the extracellular polymeric
substance associated with bio-films may also be
involved. Bio-film cells are contained in the
extracellular polymeric substance matrix and may
exhibit an overall negative charge. This negative
charge is important in trapping metal ions. Strain
Bacillus subtilis JC3, selected for the present study,
was distinguished as aerobic alkaliphilic sporeforming soil bacteria. This strain isolated from soil has
characteristics of incessant precipitation of dense
insoluble calcite crystals and has high negative zetapotential. This spore-forming gram positive strain
bacteria can survive in high pH environment of
concrete sustaining various stresses. Different cell
concentrations were derived from the bacterial growth
culture by serial dilution method. The medium used to
grow Bacillus subtilis JC3 was based on peptone,
NaCl , yeast extract. The pure culture was isolated
from the soil sample of JNTU. Microbiologically
induced calcium carbonate precipitation occurs via
more complicated processes than chemically induced
precipitation. In nutrients medium, it is possible that
individual microorganisms produce ammonia as a
result of amino acids degradation to create an alkaline
micro-environment around the cell. The high pH of
these localized areas, without an initial increase in pH
in the entire medium, commences the growth of
CaCO3 crystals around the cell. Specific proteins
present in biological extracellular polymeric

substances cause the formation of different calcium

carbonate polymorphs. Although all the Bacillus
strains were capable of depositing calcium carbonate,
differences occurred in the amount of precipitated
calcium carbonate on agar plate colonies. Oxidative
deamination of amino acids by Bacillus subtilis JC3 is
temperature dependent and that the highest calcite
precipitation rates occurred near the point of critical
saturation. B. Subtilis JC3 member of the genus
Bacillus is Gram-positive, rod-shaped, endospore
forming bacteria commonly found in soil; precipitate
calcium carbonate (CaCO3) in its micro-environment
by the ammonification of amino acids into ammonium
(NH4+) and carbonate (CO32-) ions. Microbiologically
induced (also called bacteriogenic) calcite
carbonate precipitation by Ammonification (Ammo
acid degradation) comprises of series of complex
biochemical reactions. Amino acids released during
proteolysis (the process of enzymatic breakdown of
proteins by the microorganisms with the help of
proteolysis enzymes) undergo deamination in which
nitrogen containing amino (-NH2) group is removed.
Thus, process of deamination which leads to the
production
of
ammonia
is
termed
as
"ammonification". The process of ammonification is
mediated by Bacillus subtilis JC3. Ammonification
usually occurs under aerobic conditions (known as
oxidative deamination) with the liberation of ammonia
(NH3) or ammonium ions (NH4) when dissolved in
water .The biochemical reactions of ammonification
in peptone based medium is represented as follows
Ca2+ + B.subtilis Cell B.subtilis Cell- Ca2+
CH3CH(NH2)COOH (Peptone) + O2 --------->
C2H2 + H2CO3 + NH3
H2CO3 ----------> H+ + HCO3NH3 + H2O --------> NH4+ + OH- (pH increase)
HCO3- + H+ + OH- ----------> CO32- + H2O
B.subtilis Cell- Ca2+ + CO32- B.subtilis Cell- CaCO3
4. EFFECT OF BIOGENIC TREATMENT ON
CONCRETE PROPERTIES
Experimental investigation reports confirm that
incorporation of bacteria into concrete modifies the
properties of concrete as presented below:

4.1 Optimum bacterial cell concentration for

CaCO3 Precipitation
This investigation was carried out primarily to
understand the effect of bacterial cell concentration on
the quantity of calcium carbonate precipitation. The
appropriate bacterial cell concentration for maximum
calcium carbonate precipitation can be established by
determining the 28 day compressive strengths of
various cement-mortar specimens induced with
different bacterial cell concentrations. The sample
whose 28 day compressive strength was highest
determines the optimum cell concentration for high
amount of crystalline calcite precipitation. Different
cell concentrations were derived from the bacterial
growth culture by serial dilution method. Standard
Cement-mortar cubes incorporated with soil bacteria
Bacillus subtilis JC3 of different cell concentrations
were cast, cured for 28 days and tested for the
compressive strength. The maximum percentage
increase is found to be 17.88 % for 10 5 bacterial cells
induced specimens. This improvement in compressive
strength was mainly due to metabolic deposition of
CaCO3 in the voids /or pores within cementsand
matrix modifying the pore structure of bacteria
induced cement mortar specimens. During bacterial
growth, the calcium precipitation process occurs
continuously, clogging the internal pores with calcium
precipitate. The gradual reduction of compressive
strength of cement mortar cube specimens induced
with bacterial cell concentrations more than 105 cells
per ml of mixing water is attributed to the disruption
of cement-mortar matrix integrity by the presence of
organic matter (biomass) above the permissible limits
as specified by IS 456. Therefore, Bacillus subtilis
JC3 cell concentration of 105 cells/ml of mixing water
generates the greatest reduction in porosity by
precipitating calcite crystals optimally. Reduction in
pores due to such material precipitation (calcium
carbonate) will eventually increase the cement mortar
strength.

Figure 1: Compressive Strengths at various Bacteria Cell Concentrations

Figure 2: Percentage Increase of Compressive Strengths at various Bacteria Cell Concentrations

considered as shown in Table 1 because the voids
and pores are sealed up by mineral precipitation due
to bacteria.

4.2 Compressive Strength

The Compressive Strength of bacteria incorporated
concrete showed significant increase by nearly 25%
in all grades of concrete proposed for all ages

Table-1: Strength Characteristics of Controlled and Bacterial Concrete mixes at various ages
STRENGTH STUDIES
Controlled Concrete

Bacterial Concrete

Grade
M20

M40

Age

M60

M80

M20

M40

M60

M80

Average Compressive Strength (MPa) S.D

28 days

28.1821

52.0196

72.6171

93.888

32.7450

61.0687

94.2196

119.246

60 days

32.4456

56.4756

79.2658

98.3595

37.9709

64.9266

102.980

125.627

90 days

33.2742

57.9655

83.5932

107.5749

40.442

66.8329

108.638

138.1461

4.3 Characterization of CaCO3 Precipitation using

Scanning Electron Microscope (SEM) Analysis
The test is to confirm the presence of bacteria
precipitated calcite by generating high resolution
images of bacteria induced cement mortar samples
and shows spatial variations in chemical
compositions. Scanning Electron Microscope (SEM)
analysis is made on the samples of 28 day old
bacterial cement mortar specimens and control
mortar specimens (without bacteria). Broken pieces
of 28 day old cement mortar cubes from the
compression test were examined under a SEM
Hitachi- S520 using accelerating voltages ranging
from 1to 30 KV in the research centre at CSIRIndian Institute of Chemical Technology. Fig 3 (a)
and (b) shows SEM scanning images of control
cement mortar sample and bacteria induced cement
mortar sample. Improvement in pore structure of
cement-sand mortar samples treated with Bacillus
subtilis JC3 of 105 cell concentration per ml can be
observed in a magnified view (2500x) of SEM
micrograph as shown in Fig 3 (b). The difference
between these two scanning electron micrographs
shows that the in case of sample from bacteria
induced cement mortar, formation of dense CaCO3

Figure 3 (a) Cell Concentration Nil

(Control Specimen)

precipitation spreading over the pores present inside,

with rod-shaped impressions housed by Bacillus
subtilis JC3 can be observed. The morphology of the
newly formed crystals of rhombohedra shape
suggests that the mineral may be CaCO3 and its
formation could be the result of the metabolic
conversion of the nutrients by Bacillus subtilis JC3.
SEM examination shows that in a mortar made with a
105/ml cell concentration, the pores are almost
completely filled with narrow strands of filler
(calcite) and modification of pore size distribution is
noticed. In case of control sample no filler material
was observed.
4.4 Bacteria Viability Test
A piece of bacterial cement mortar of 365 days age
was inoculated in nutrients broth and kept in orbital
shaker for 24 hrs. After 24h incubation, a loop full of
culture is taken from the broth and streaked on agar
plate. Once colonies are formed their morphological
characteristics and microscopic observations match
with Bacillus subtilis JC3. This confirms the presence
of Bacillus subtilis JC3 even after 365 days in cement
mortar. Photo contrast pictures in Figure 4 shows that
bacteria are still viable in cement mortar.

(b) Cell Concentration 105/ml

(Optimum)

Figure 4: Phase contrast microscopic pictures identify microorganisms and white calcium carbonate crystals
formation

5. CONCLUSION
Microbial mineral precipitation resulting from
metabolic activities of some specific microorganisms
in concrete to improve the overall behaviour of
concrete has become an important area of research.
The following are the summary of research outcomes
done at JNTU Hyderabad by Dr M V Seshagiri Rao et
al. on Bioengineered concrete:
1.
An alkaliphilic aerobic microorganism bacillus
subtilis JC3 is induced into cement mortar samples at
various cell concentrations in suspension along with
the mixing water. The greatest improvement in
compressive strength occurs at cell concentrations of
105 cells/ml for all ages. The study showed that a 25%
increase in 28 day compressive strength of cement
mortar was achieved. The strength improvement is
due to growth of filler material within the pores of the
cementsand matrix as shown by the scanning
electron microscopy.
2. Scanning Electron Microscopy (SEM) also
confirmed the role of microbiologically induced
precipitation within the mortar matrix. The growth of
dense calcite fillers are visualized by scanning
electron microscopy (SEM) analysis.
3. The modification in pore size distribution and
total pore volume of cementsand mortar due to such
growth is also noted. The extra cellular growth
produced by the microorganism is expected to
contribute more to the strength of concrete. Even the
dead cells may simply remain in the matrix as organic
fibers.
4. The spores of Bacillus subtilis JC3 are found to
survive even after 365 days. Thus, it is evident that the
minerals
produced
by
the
process
of
biomineralization, which is a normal biological
process in certain types of micro-organism can be
used both as a binder and pore filler in the process of
improving the strength of cement mortar.
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BIOGRAPHIES
M V Seshagiri Rao is Professor
in Civil Engineering at JNTUH
College of Engineering. He has
enormous experience in teaching
and research for nearly 35 years.
He has guided 20 PhDs and
published nearly 175 papers in
various
international/national
journals and conferences. He won best teacher award
in 2009 from State Government of Andhra Pradesh.
He has published one book on Engineering
Mechanics. He is recipient of many more awards and
also associated with various professional bodies.
Srinivasa Reddy V is currently
working as Associate Professor in
Department of Civil Engineering
at GRIET Hyderabad. He has
obtained BTech degree from
Nagarjuna University in 1996,
MTech from JNTU Hyderabad in
2002 and is a doctoral student of
Prof. M V Seshagiri Rao. He has nearly 50
publications in various international/national journals
and conferences.
K Mounika is a post graduate
student in Civil Engineering at
JNTUH College of Engineering.
Her specialization is Structural
Engineering.