Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
ISSN 2250-2149
1 23
Your article is protected by copyright and all
rights are held exclusively by The Institution
of Engineers (India). This e-offprint is for
personal use only and shall not be self-
archived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com.
1 23
Author's personal copy
J. Inst. Eng. India Ser. A
DOI 10.1007/s40030-017-0227-x
ORIGINAL CONTRIBUTION
Abstract Concrete is vulnerable to deterioration, corro- Keywords Bacillus subtilis JC3 Bio-mineralization
sion, and cracks, and the consequent damage and loss of Self healing Bacterial concrete RCPT
strength requires immensely expensive remediation and
repair. So need for special concrete that they would
respond to crack formation with an autonomous self-heal- Introduction
ing action lead to research and development of microbial
concrete. The microbial concrete works on the principle of Crack repair is of crucial importance to concrete since
calcite mineral precipitation by a specific group of alkali- cracks are the main path for the penetration of corrosive
resistant spore-forming bacteria related to the genus substances which greatly decrease the service life of con-
Bacillus called Bacillus subtilis JC3, this phenomenon is crete structures. Nowadays, a promising method to repair
called biomineralization or Microbiologically Induced concrete cracks is being investigated. The idea is that repair
Calcite Crystal Precipitation. Bacillus subtilis JC3, a work should be done by the concrete itself when cracks
common soil bacterium, has inherent ability to precipitate appear, through a self-healing process by a certain kinds of
calcite crystals continuously which enhances the strength natural healing agents released within the concrete on
and durability performance of concrete enormously. This occurrence of cracks [1, 2]. A biological approach of self
microbial concrete can be called as a Self healing Bac- healing in concrete is one such crack remediation methods
terial Concrete because it can remediate its cracks by in which bacteria Bacillus subtilis JC3 can produce or
itself without any human intervention and would make the induce bio-minerals during their growth and metabolism to
concrete more durable and sustainable. This paper discuss fill the cracks and pores in the concrete. Concrete is an
the incorporation of microorganism Bacillus subtilis JC3 alkaline material whose pH is as high as 13. This alkalinity
(developed at JNTU, India) into concrete and presents the imparts concrete the protection against corrosion. Bacillus
results of experimental investigations carried out to study subtilis JC3 is an alkaliphile which can survive in high
the improved durability and sustainability characteristics of alkaline concrete and just needs nutrients for microbial
microbial concrete. activities to form inorganic solids. Bacteria-based self-
healing concrete is produced by incorporating spores of
bacteria of a special kind (Bacillus subtilis JC3), in the
& M. V. Seshagiri Rao concrete matrix at the stage of preparation of the concrete
rao_vs_meduri@yahoo.com by mixing the spore suspension in concrete mixing water.
1
Department of Civil Engineering, Jawaharlal Nehru
When crack is formed water enters the crack subsequently
Technological University Hyderabad College of Engineering, the homogeneously distributed bacterial spores in hardened
Hyderabad, India concrete matrix gets activated and germinate to become
2
Department of Civil Engineering, Gokaraju Rangaraju metabolically active vegetative cells that are able to con-
Institute of Engineering and Technology, Hyderabad, India vert the organic nutrient compounds into insoluble inor-
3
Centre for Environment, Jawaharlal Nehru Technological ganic calcium carbonate based minerals [3]. Copious
University Hyderabad , Hyderabad, India
123
Author's personal copy
J. Inst. Eng. India Ser. A
production of bacteria-mediated minerals results in sealing obtained precipitation without the application of calcino-
off the crack; hence the permeability and porosity of con- genic bacteria. In addition to the bio-deposition treatment,
crete are reduced significantly [4]. The precipitated calcite the use of bacterially induced carbonates as a binder, i.e.
has a coarse crystalline structure and is highly insoluble in bio-cementation, has been addressed. Until now, work on
water. It resists the penetration of harmful agents (chlo- bio-deposition was mainly concentrated in Europe, while
rides, sulphates, carbon dioxide) into the concrete thereby much of the work on remediation of cracks in concrete has
decreasing the deleterious effects they cause [5, 6]. been done in the USA. In China, researchers are investi-
gating the use of microbially induced carbonate precipita-
Bacterial StrainBacillus Subtilis JC3 tion for the restoration of ancient masonry buildings [7]
and the protection of concrete surfaces [8]. Furthermore,
Strain Bacillus subtilis JC3, selected for the present study, the method of producing CaCO3 by bacterial biomineral-
was distinguished as aerobic alkaliphilic spore-forming soil ization has been patented in China by Qian et al. [9]. The
bacteria. The medium used to grow Bacillus subtilis JC3 latter was possible, as the patent by Adolphe et al. [10] was
was based on peptone, NaCl, yeast extract. The pure cul- limited to European countries. In Brazil, Shirakawa et al.
ture was isolated from the soil sample of JNTU, India. [11] are working on bio-deposition on fiber cement roof
tiles. In India, researchers have applied for a patent in
Chemistry of MICCP by Bacillus Subtilis JC3 which the use of Shewanella for the improvement of the
strength of concrete was described [12]. Research and
In nature, microorganisms can induce calcite mineral pre- development of bacterial concrete was happening for the
cipitation through nitrogen cycle either by ammonification past 10 plus years in various universities outside India. In
of amino acids/nitrate reduction/hydrolysis of urea. Bacil- India very limited research was done in the area of biore-
lus subtilis JC3 is able to precipitate calcium carbonate mediation of concrete particularly. The pioneering research
(CaCO3) in its micro-environment by the ammonification was conducted on self-healing phenomena in bio-concrete
of amino acids into ammonium (NH4?) and carbonate by Bang et al. [13] at South Dakota School of Mines and
(CO32-) ions. The precipitated bio-CaCO3 has a great Technology, USA. Willem De Muynck, Nele De Belie,
potential ability to heal concrete cracks because it is nat- Willy Verstraete, Kim Van Tittelboom et al. at Ghent
ural, environmentally friendly and compatible with the University, Belgium has done extensive research on
concrete matrix. Bio-mineralization by Ammonification microbiologically induced calcite precipitation in con-
(Ammo acid degradation) comprises of series of complex struction materials. Henk M. Jonkers et al. at Delft
biochemical reactions. Amino acids released during pro- University of Technology, Netherlands has studied exten-
teolysis (the process of enzymatic breakdown of proteins sively on the self-healing capability of bacteria induced
by the microorganisms with the help of proteolysis cementitious materials. In India, P. Ghosh, S. Mandal, B.
enzymes) undergo deamination in which nitrogen con- D. Chattopadhyay et al. of Jadavpur University, Kolkata,
taining amino (NH2) group is removed. Thus, process of India; V Achal, Abhijeet Mukerjee, Rafat Siddique et al. of
deamination which leads to the production of ammonia is Thapar University, Patiala, India and M V Seshagiri Rao,
termed as ammonification. The process of ammonifica- V S Reddy et al. of Jawaharlal Nehru Technological
tion is mediated by Bacillus subtilis JC3. Ammonification University (JNTU), Hyderabad, India have done extensive
usually occurs under aerobic conditions (known as oxida- research on the development of high performance self-re-
tive deamination) with the liberation of ammonia (NH3) or mediating bacterial concrete. Researchers around the world
ammonium ions (NH4) when dissolved in water. The pro- attempted to study about the application of bio-mineral-
cess of ammonification is represented as follows, ization in civil engineering specifically about the use of
CH3 CHNH2 COOH Peptone 1=2O2 ! C2 H2 H2 CO3 NH3 ammonia particular bacteria in cementitious materials to self-heal
H2 CO3 ! H HCO3
and seal cracks without human intervention. Available
NH3 H2 O ! NH
4 OH
literature has not reported any such suitable self-healing
system which has features such as long-term compatibility,
eco-friendliness, good bonding with surrounding cement
Literature Review matrix, less human intervention, inexpensive and organic
in nature. Though it is reported that the use of specific
Different research groups have searched for alternative alkaliphilic mineral forming bacteria enhances the prop-
approaches to obtain a protective layer of calcium car- erties of cement mortar but there exists little understanding
bonate on the surface of building materials (bio-deposi- of the effect of bacteria on the mechanical and durability
tion). Some authors suggested the use of alternative properties of concrete. The significance of this research
microorganisms or metabolic pathways, while others was to quantitatively understand and assess the role of
123
Author's personal copy
J. Inst. Eng. India Ser. A
Experimental Investigations Yeast extract: 3 gm/l, Beef extract: 1.5 gm/l. Yeast and
beef extracts are nitrogen source for bacteria growth.
Mix Proportions Chemical analysis on 105 cells/ml bacteria suspended
nutrient based solution is done to test for its suitability as
The grades of concrete used in the present investigation are mixing water in concrete and results found are within the
ordinary grade (M20), standard grade (M40) and high Permissible limits as per IS 456-2000.
strength grade concretes (M60 and M80). The mix pro-
portions of M20 and M40 grade concrete are designed Methodology
using IS: 10262-2009 and mix proportions for M60 and
M80 grade concrete are designed using Entroy and Two types of concrete mix of various grades i.e. one
Shacklocks Empirical Graphs. The mix proportions are without bacteria (control sample) and one with bacteria
given in Table 1. (bacterial sample) were prepared for experimental inves-
tigations. The mixes have similar proportions of sand,
Microorganism Used coarse aggregates, cement and water as per mix proportions
shown in Table 1. However, the main difference was in the
Bacillus Subtilis JC3 water component: (1) tap water is used for concrete spec-
imens without bacteria (for M20, M40, M60 and M80
Aerobic alkaliphilic spore-forming microorganism Bacillus grades), (2) while Bacillus subtilis JC3 suspended water is
subtilis strain with accession number JC3 is a laboratory used for specimens with bacteria (for M20B, M40B, M60B
cultured soil bacterium, isolated, deposited, cultured and and M80B grades). For preparation of bacteria-nutrient
grown in distilled water at Bacteria Discovery Laboratory suspension, optimum cell concentration of bacteria
(JNTUH Hyderabad) as shown in Fig. 1. This microor- (Bacillus Subtilis JC3) is grown in nutrient based distilled
ganism is used in preparation of bacterial concrete. Dif- water in sterile conditions at biotechnology laboratory. The
ferent cell concentrations of bacteria were validated using bacterial culture in suspension is added to concrete during
serial dilution method. A member of the genus Bacillus, B. concrete mixing process. Concrete shall be mixed in a
subtilis is rod-shaped, and has the capability to form a mechanical mixer.
sturdy, protective endospore, allowing the organism to
endure extreme environmental conditions. Bacillus subtilis Strength Studies
cells naturally found in soil and vegetation are non-
pathogenic and grows in the mesophilic temperature range Experimental Investigations are carried out to evaluate the
of 2535 C. The Bacillus subtilis JC3 strain isolated from effectiveness of microbiologically induced mineral pre-
soil has characteristics of incessant precipitation of dense cipitation on the mechanical properties of bacteria incor-
insoluble calcite crystals and has high negative zeta-po- porated concrete specimens. Brief summaries of these
tential. Peptone: 5 gm/l., CaCl: 10 gm/l., NaCl: 5 gm/l., investigations are presented below.
123
Author's personal copy
J. Inst. Eng. India Ser. A
Optimum Cell Concentration for Promotion of Maximum Table 2 Properties of Bacterial suspension
Mineral Precipitation Parameter Results Limits as per IS 456-2000
permissible limits (max)
To study the effect of cell concentration on the properties
of concrete, bacteria incorporated cement mortar cubes of pH 8.3 6.58.5
size 70.6 9 70.6 9 70.6 mm were cast using different Chlorides, mg/l 145 2000 (PCC)
cell concentrations of Bacillus subtilis JC3 (1 9 104 cells/ 500 (RCC)
ml, 1 9 105 cells/ml, 1 9 106 cells/ml, 1 9 107 cells/ml) Alkalinity, ml 13 \ 25
and tested as per IS 12269:1987. For each cell concen- Sulphates, mg/l 245 400
tration, six cubes were casted. This investigation was Fluorides, mg/l 0.022 1.5
carried out primarily to understand the effect of bacterial Organic solids, mg/l 31 200
cell concentration on the quantity of calcium carbonate Inorganic solids, mg/l 1380.4 3000
precipitation. More bacteria with enough nutrients will Suspended matter, mg/l 1359 2000
precipitate more calcite in the laboratory conditions but in
the cement-sand environment, bacteria mineral precipi-
tating ability depends on its compatibility with cement- Compressive, Split Tensile and Flexural Strength
sand matrix. Bacteria incorporated into cement-sand
medium should not affect the physio-chemical properties To study the effect of biomineralization, due to Bacillus
of cement-sand. Hence cell concentration of bacteria subtilis JC3 on the compressive, split tensile and flexural
plays a key role in optimizing the performance of strength of concrete treated with 1 9 105 cells/ml bacteria
cementitious materials. The appropriate bacterial cell spore suspension. For each grade, six cubes of size
concentration for maximum calcium carbonate precipita- 100 9 100 9 100 mm are cast and tested for compressive
tion can be established by determining the 28 day com- strength as per IS 516:1959, six cylinders of size
pressive strengths of various cement-mortar specimens 150 9 300 mm are cast and tested for split tensile strength
induced with different bacterial cell concentrations. The as per IS 5816:1999 and six prisms of size 100 9 500 mm
sample whose 28 day compressive strength was highest are cast and tested for flexural strength as per IS 516:1959.
determines the optimum cell concentration for high
amount of crystalline calcite precipitation. The cubes StressStrain Behaviour
having 1 9 105 cells/ml are found to have maximum
strength. Henceforth, Optimum bacterial cell concentra- In this study for each grade, six cylinder specimens of size
tion of 1 9 105 cells per ml of mixing water is used for 150 9 300 mm are cast and are tested under strain control
further research. under uni-axial compression as per IS: 516-1999 to get the
This improvement in compressive strength was mainly stressstrain characteristics. From which peak stress, strain
due to metabolic deposition of CaCO3 in the voids or at peak stress and modulus of elasticity of bacterial con-
pores within cementsand matrix modifying the pore crete specimens are determined.
structure of bacteria induced cement mortar specimens.
During bacterial growth, the calcium precipitation process Ultrasonic Pulse Velocity Measurements
occurs continuously, clogging the internal pores with
calcium precipitate. It is observed that for cement mortar In this study for each grade, bacteria treated concrete
specimens incorporated with Bacillus subtilis JC3 of cell specimens strength and quality is assessed using non-de-
concentration more than 105 cells/ml, the compressive structive testing by Ultrasonic Pulse Velocity on six cube
strength is reduced. The gradual reduction of compressive specimens of size 150 9 150 9 150 mm as per IS: 13311
strength of cement mortar cube specimens induced with (Part 1): 1992.
bacterial cell concentrations more than 105 cells/ml of
mixing water is attributed to the disruption of cement- Durability Studies
mortar matrix integrity by the presence of organic matter
(biomass) above the permissible limits as specified by IS: The understand the durability performance of bacterial
3025-1986 and IS: 456-2000 as shown in Table 2. Max- concrete, the study of key parameters such as water
imum strength is observed for 105 cells/ml of mixing absorption capacity, porosity, sorptivity, water permeabil-
water so this is taken as optimum cell concentration to be ity and chloride-ion penetration resistance studies are
used for further study to investigate the effect of bacteria important. Brief summaries of these investigations are
on properties of concrete. presented.
123
Author's personal copy
J. Inst. Eng. India Ser. A
In this study for each grade, the maximum quantity of For each grade, the tests for permeability were carried out
water absorbed (WAC) by bacteria treated concrete spec- on three 100 mm 9 100 mm cylinders using Concrete
imens (six cube size 100 9 100 9 100) at room tempera- permeability apparatus as per IS: 3085-1965. The total
ture and pressure under conditions of saturation expressed quantity of water percolated through the cylinder speci-
as a percentage of the dry mass of the sample is evaluated mens was collected in a glass bottle for a period of 100 h
as per ASTM C642. using which coefficient of permeability was calculated.
Sorptivity measures the rate of penetration of water into Test Results and Discussions
the pores in concrete by capillary suction. As per
ASTM C1585, for each grade, the test for sorptivity The test results of Strength, Durability characteristics of
was conducted on six numbers of 100 9 100 9 100 mm bacterial concrete are listed in Tables 3, 4 and 5. The
cubes by immersing them to a depth of 510 mm in the results on strength and durability characteristics are sum-
water and then measuring the gain in mass at regular marized as follows:
intervals of 30 min for 72 h until there is no gain in
1. For cell concentration of 105 cells/ml of mixing water
weight. The cumulative volume of water that has pen-
the compressive strength of bacteria incorporated
etrated per unit surface area of exposure Q/A is
cement mortar specimens is maximum (increased by
plotted against the square root of time of exposure Ht.
about 17%) at age 28 days as shown in Table 1 and
The resulting graph could be approximated by a straight
Fig. 2. Chemical analysis on 1 9 104 cells/ml,
line passing through the origin. The slope of this
1 9 105 cells/ml, 1 9 106 cells/ml, 1 9 107 cells/
straight line gives sorptivity coefficient k which is
ml bacteria suspended nutrient based solutions are
considered as a measure of rate of movement of water
done to test to determine the presence of impurities.
through the capillary pores.
Suitability as mixing water in concrete depends on
Table 3 Effect of Bacillus subtilis JC3 JC3 cell concentration on compressive strength (MPa)
Cell concentration/ml of mixing water Effect of optimum cell concentration on compressive strength, MPa
7 days % Increase 14 days % Increase 28 days % Increase
123
Author's personal copy
J. Inst. Eng. India Ser. A
Table 4 Strength characteristics of controlled and Bacterial concrete mixes at various ages
Strength studies
Grade ? Controlled concrete Bacterial concrete
M20 M40 M60 M80 M20 M40 M60 M80
the presence of impurities within permissible limits concrete in hardened state. From the values of
as per IS: 3025-1986 and IS: 456-2000 as shown in stresses and strains, average stressstrain curves for
Table 3. An increase in bacterial cell concentrations different grades of bacterial concretes are plotted.
above 1 9 105 cells/ml of mixing water reduces the The observation made from stressstrain curves is all
strength of cement mortar cube specimens due to grades of bacterial concrete have shown improved
disruption of cement mortar matrix integrity with the stress values for the same strain levels compared to
presence of organic matter in the form of biomass, that of conventional concrete mixes. Ultimate stress,
above the permissible limits specified as per IS: strain at ultimate stress and modulus of elasticity
3025-1986 and IS: 456-2000 in mixing water. (E) are determined from the stressstrain plots as
2. It was observed that with the addition of bacteria, the shown in Fig. 4 and are listed in Table 4. The
Compressive, Split Tensile and Flexural Strength of increase in E value is nearly 2030% for low to high
concrete showed significant increase by nearly 25, 20 grade bacterial concrete.
and 25% respectively in all grades of concrete 4. Ultrasonic Pulse Velocity test conducted on bacterial
proposed for all ages considered as shown in Table 4. concrete specimens revealed that ultrasonic pulse
Higher the grade more increase was noted because velocity values increased with bacteria induction into
the voids and pores are sealed up by mineral concrete due to enhanced pore structure and
precipitation due to bacteria. Reduction in pores microstructure of hardened bacterial concrete of all
due to such material growth has obviously increased grades. The ultrasonic pulse velocity values are listed
the strength characteristics of concrete. Figure 3 in Table 4. The pulse velocity values obtained for
shows variation of compressive strength of controlled bacteria induced concrete were greater than 4.5 km/s
and bacterial concretes of various grades. which classifies as excellent concretes in terms of
3. The relationship between stress and strain is impor- strength and durability point of view. From the
tant in understanding the basic elastic behaviour of results it is observed that the reduction in pores in
123
Author's personal copy
J. Inst. Eng. India Ser. A
Table 5 Durability characteristics of controlled and Bacterial concrete mixes at various ages
Durability studies
Controlled concrete Bacterial concrete
Grade ? M20 M40 M60 M80 M20 M40 M60 M80
123
Author's personal copy
J. Inst. Eng. India Ser. A
the value is in the range of 0.124 to 0.055 mm/min0.5 values are checked against the specifications sug-
for ordinary to high grades at 28 days. gested by MORT&H 4th Revision Clause 1716.5.
8. Very negligible percolation of water has been found Results show that the presence of bacteria resulted in
for higher grades of bacterial concrete specimens lower coefficient of permeability of range
whereas for the ordinary and standard grades there is 0.230.27 9 10-9 m/sec in comparison to controlled
some percolation. Cylinders were removed from the concrete which has coefficient of permeability of
permeability mould and were split to measure the 0.952.31 9 10-9 m/sec for various grades of con-
water penetration depth. This confirmed that use of cretes. Water permeability reduces in bacterial con-
bacteria had resulted in almost impermeable con- crete by nearly 7090% from low to high grades
crete. Table 5 lists the water penetration depths of suggesting that reduction in the permeability is due to
controlled and bacteria incorporated concrete speci- reduction in the porosity of the concrete resulting
mens of different grades at various ages and the from pore filling by calcite crystals.
123
Author's personal copy
J. Inst. Eng. India Ser. A
123
Author's personal copy
J. Inst. Eng. India Ser. A
123