Self-Healing Concrete by Bacteria Approach Research 2022

Research on self-Healing Concrete by bacteria approach
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Self –healing 1

Table of Contents
Abstract.......................................................................................................................................................3
Executive summary.....................................................................................................................................4
Introduction.................................................................................................................................................6
Literature review.........................................................................................................................................9
Grouping of bacteria................................................................................................................................9
Detailed review......................................................................................................................................10
Self-healing concrete and the influencing features of the healing ability...............................................16
Bacterial type used for self-healing...................................................................................................16
Concentration of bacteria...................................................................................................................17
Concentration and types of nutrient...................................................................................................18
Ways to generate a self-healing concrete...............................................................................................18
Concrete self-healing features...............................................................................................................18
Programming through a practical...........................................................................................................19
Reasons for using the bacteria in the concrete.......................................................................................19
Reasons why bacteria should not be used in the concrete......................................................................20
Common process involved in remediation of cracks through bacterial concrete...................................22
Bacterial concrete applications..............................................................................................................22
Methodology.............................................................................................................................................22
Choice and cultivation of spore forming bacteria which are alkali resistant..........................................22
Preparing and strength features of bacterial concrete.............................................................................23
Existence/ viability of concrete motionless spores.................................................................................24
Calcite precipitation ability of bacterial concrete...................................................................................26
Analysis.....................................................................................................................................................27
Choice and cultivation of alkali resistant spore creating bacteria..........................................................27
The strength features of bacterial concrete............................................................................................27
Viability of cement stone motionless spores..........................................................................................28
Calcite precipitation ability of bacterial concrete...................................................................................29
Discussion.................................................................................................................................................29
Conclusion.................................................................................................................................................34
Reference...................................................................................................................................................36
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Abstract
Self-healing capability of the formed small cracks is normally shown by the concrete structures. This is
due to the availability of many non-hydrated particles of cement within the material matrix that actually
takes sometime before the reaction with the permitted water. A new method of self-healing concrete
where the bacteria separate the generation of minerals that closes the immediate cracks formed is
developed in this research. The process is capable of minimizing the permeability of the concrete which
then keeps safe the laid reinforcement of steel from corrosion. Initially, the research conducted indicated
that mixing of particular organic materials compounds that can take part in chemical reaction with
bacteria of spore-forming alkaliphilic acting as agent of self-healing generates up to 100 micrometer of a
calcite particles which can strongly close smaller to even bigger sized cracks. A significant rise of self-
healing ability can portray a new concrete with a higher durability and sustainable range of potential
application through a further growth of this bio-concrete.
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Executive summary
Self-healing concrete is capable of filling cracks autogenously with no intervening from
any outside source. Copious techniques of generating a self-healing concrete are present in the
earlier studies but employing the bacterial approach allows the concrete to be friendly to the
environment. Under this study, we review the elements that are vital in improving a bacterial
self-healing concrete Vijay et al. (2017). These elements involve the types of bacteria and the
technique required in selection of a bacteria as a source of healing agent. There exist several
nutrients and bacteria concentrations being applied inside self-healing concrete which led to a
large range of performance.
Applying concrete as a construction material is expanding. Different structures are constructed
using concrete material because of its adequacy and great performance. Concrete furthermore,
generates a powerful material particularly relating to compressive potency. The wide use of this
in numerous structure components needs periodic checks and maintenance. Concrete have
brittleness which would probably be subjected to cracking, shrinkage, creep or overstressed.
Therefore, maintenance need to be conducted on such faults for prevention of more damages to
the entire system. Consequently, cracks in concretes will accept harmful matter which may set
off carbonation in concrete or corrosion to the steel reinforcement to penetrate. The permeability
of matter would reduce the structures’ appearance. Maintenance and check-ups which require to
be conducted could be convoluted would cracking happen within a region with little access such
as bridge spanning over Wide River or active traffic. Price of mending such system could be
more costly. The project is as well dangerous to the maintenance personnel and those around.
Such a mainetenance problem could be solved using concrete having the capability of occupying
space left because of the formed cracks. Price could be lessened particularly with respect to
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periodical inspection duty. Concrete that has an ability of filling the formed cracked
autogenously is referred to as self-healing concrete; which has been made earlier by making
concrete using extravagant amount of cement in the whole composition Sharma et al. (2017).
The extravagant amount of cement could be non-hydrated as it will execute reaction with water
chemically that enters the strengthened concrete via cracks so as to yield brand cementitious
bonding.
In addition, chemical matter has is also employed as an agent. Among these chemical
products we have a solution of sodium silicate which forms C-S-H gel through a reaction of
calcium hydroxide within the concrete. Nevertheless, the healing agent chemically synthesizes
but never friendly to the environment. Present researches offer another alternative to chemically
synthesized healing agent possessing bacteria as a healing agent. Bacillus bacteria could
precipitate CaCO3 to close the cracks from within the concrete. Self-healing which is bacterial
related was capable of resealing crack of 0.3 mm within 20 days. Application of bacteria as self-
healing agent has embarked since Jonkers et al (2010) employed the bacteria during concrete
mixing other than employing it as a repair material.
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Introduction
Numerous recent studies have discovered the happening of autogenous healing within concrete
and the amount for cracking healing in usual kinds of concrete, though, are seen to be restricted
to small cracks, those having widths ranging from 0.1 to 0.2 mm. Self-healing process
mechanism usually differ, since it largely depends on the constitution of the concrete mixture.
For instance, crack healing has been observed in mortar of centuries old brick structures in the
Amsterdam canals, this process was contributed to re -precipitation and dissolution of calcium
carbonate within the majorly lime based mortar matrix. Water penetrating through the crack
would not only dissolve calcium carbonate but it would also react with the carbon dioxide from
the atmosphere with non-fully hydrated lime components including calcium hydroxide and
calcium oxide in line with the following reactions
H2 O+ ¿ CaO→ Ca(OH )2
Ca(OH )2 +CO2 → H2 O+CaCO3
In the above equations, there are freshly obtained minerals, which are as well produced from re-
crystallized and dissolved calcite minerals, precipitated on the crack surfaces which ended up in
sealing the cracks and accompanying decline in permeability of the mortar. The potential of
healing of this system was much related to the capacity of not reacted lime constituents within
the set mortar Reddy et al. (2012). Recent concrete structures normally based on Portland cement
having variable percentages of cement replacement like silica fume, fly ash or blast furnace slag,
they also contain a particular ability for autonomous crack healing. The set procedures in this
new concrete is though, comparable to the process of lime-based mortars, as more partially
hydrated or non-hydrated cement particles go through secondary or delayed hydration with
Self –healing 6

ingress water. Especially concrete obtained from compositions characterized by a less water to
cement weight ration like high strength concrete or concrete reinforced with polymer fibre may
depict quick crack sealing ability because of the highest amount of non-reactive cement
constituent’s variable within the concrete matrix.
A high crack healing ability of concrete structures is useful as it creates the material to
become stronger and more durable. Consistent healing of especially surface cracks brings about a
decrease in permeability of the material and majorly a lessened risk of degradation of premature
matrix as well as corrosion of the embedded steel reinforcement as a result of aggressive
chemicals and ingress water. The reason as to why traditional concrete is never based on a less
water to cement weight ration and accompanying high self-healing ability is the high charges
involved. Furthermore, new policy is to lessen the amount of cement required in a concrete
mixture, since its manufacture is environmentally unfit because of more energy consumption and
concurrent carbon dioxide emissions. Nevertheless, concrete having less cement content, in the
extent that it is based on a high water to cement ratio, probably it will not possess an important
autogenous crack sealing potential as most cement particles have already underwent complete
hydration during early life Van & Belie. (2013). Therefore, an alternative mechanism of self-
healing has to be probably used so as to improve the durability of such a less costly and
environmentally sustainable concrete. Such a mechanism can be offered by a mineral yielding
bacterium.
Generally, natural soils are the homes of most non-pathogenic bacteria and numerous of those
have been conveyed as potent bio mineral yielders. Additionally, alkali lakes together with some
specific natural stones and minerals store classes of specialized bacteria known as endolithic and
alkaliphilic bacteria. Which involve calcite yielding bacteria (Soundharya & Nirmalkumar
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(2014). The two specialized groups seem significant in the involvement of crack sealing in a
concrete, since the surroundings they come from a given analogy with the matrix of the concrete
that is having a high alkaline Ph and being stony. The main objective of the current study is to
figure out whether concrete motionless calcite yielding bacteria could actively represent another
crack sealing mechanism in a concrete.
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Literature review
Grouping of bacteria
Bacteria are grouped into three categories namely: oxygen requirement, gram strain and shape.
Shape
Shape includes the following;
 Bacilli
 Cocci
 Spirilla
Oxygen requirement
It includes the following;
 Aerobic
 Anaerobic
Gram strain
It comprise of the following;
 Gram positive
 Gram negative
Bacteria involved in concrete
 Bacillus balodurais
 Bacillus cohnii
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 Bacillus pseodofirmus
 Bacillus subtilis
 Bacillnesphaericus
 Bacillus pasteurii
Detailed review
The durability of the bacterial concrete can be increased by collecting a total of six
samples from various sources like that of mangrove area according to Ramakrishnan et al.(2005).
After collecting these samples, the screening of the calcite precipitating organism that precipitate
the carbonate of calcium is conducted through the process of ureolysis. The product coming out
after screening is known as urease and is generating isolates which is then confirmed if they are
having the ability for developing the changing pH. After the incubation period of seven days, a
total number of ten operational taxonomical units are collected from six various samples. Eight
isolates from the ten isolates indicate the activity of urease through showing of media’s color
change of the surrounding colony. Three isolates from the eight isolate indicate the strength
when they are exposed at all temperatures. The in-vitro assay assesses the capability of the
isolates to form the precipitation of the calcium carbonate. Through curing of water, their value
is found to be high after three and seven days respectively.
The next study according to Achal et al. (2011) is based on the effects of bacteria on the
composites of the cement. Here species of two different types of bacteria called
sporosarcinapastuerii and sphaericus are brought from a collection culture form of microbial and
bank of gene, Chandigarh through a condition of a dried freeze. The bacteria is artificially
developed in a media of a solid and then moved to nutrient and given around 48 hours to stay.
Through a combination of grown bacterial culture having different concentration with cement
Self –healing 10

plus mortar, the 5 cm cube is casted. After casting, the cubes is then cured using the tap water at
room temperature and taken for testing after the 7th day and 28th day. In the case of a paste, the
gain of the strength is found to be 39.8% and 33.07% for sphaericus and Sporosarcinapastuerii
respectively. In the other side of the mortar, the gain of the strength is found to be 50% and 28%
for the sphaericus and Sporosarcinapastuerii respectively. Through the study the presence of the
calcite inside the specimen of the cements composite is an indication that they are generated by
means of microbes. The microbes are used to ensure that the durability of the cement composites
and their strength are improved.
A study based on practical hardening of the concrete through the help of deposition from
bacterial mineral by Harkes et al. (2010) is considered. At first combination of the nutrient broth
with some chemicals are put together and enough amount of water added then heated to boil
through the process of autoclaving. The condition of the water boiled is that its color should be
reddish where the needed bacterial cell is moved and through the use of the aluminum foil the
liquid media is covered. After it has been covered, it is then continuously shaken until the light
yellow color is observed indicating the availability of the bacillus subtilis in the liquid mixture.
Through the use of electrically driven mixer by putting together cement, coarse aggregate, fine
aggregate and bacterial water at the required amount, the specimens of concrete are made. The
enhancement in the compressive quality is arranged in the order of 12.32% to 30.05% when
observed at different ages for the concrete grade of m25 through the bacterial addition. For the
split tensile strength, the enhancement percentage value is arranged in the order of 13.80% to
18.45% when observed at different stages. And lastly for the flexural tensile strength, the
enhancement percentage values are arranged in order of 13.19% to 15.56% when observed at
difference ages.
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On the development of bacteria based self-healing concrete according to Jonkers et al.
(2010) different species of bacillus pseudofirmus, bacillus halodurans, and bacillus cohnii are
brought from a microorganism’s collection of German and cultures of cell. It is followed by
cleaning of bacterial cultures which are form the medium residues through configuring, washing
and again suspending the pellet of cell in the water tap. The samples of the cement are made
through the suspension and then testing is carried out. The observation made through the process
is that there is a loss of 10% in the compressive strength due to additional of healing agents like
bacteria. 10% loss in strength a times is allowed if this is recovered through ability of self-
healing. An integral material is formed by the use of both mineral precursor and bacteria mixed
compounds with the paste.
Through the study of the effect of calcite precipitating bacteria by Wikto & Jonkers (2011)
the mechanism of concrete on self-healing is considered. A repair of a crack is improved by a
biological process where a B.sphaericus culture is produced. Silica gel is used to ensure that
there is protection of bacteria against the pH in concrete which is discovered to be having effect
like CaCO3 (calcium carbonate) particles formed inside the matrix. Closing of crack leads to
permeability of water. Formation of the particles improves the durability of the materials. The
quality of the biological process is determined through the use of visual measurements and the
velocity of ultrasonic pulse. By using the discussed method, it is noted that the technique is
natural, free from pollution and highly desirable.
Looking at the analysis made by Alyousif et al. (2015) the study is based on the features
of self-healing concrete. The bacteria of bacillus subtilisaureolytic is used which is aerobic. The
formation of precipitates of CaCO3 in the environment of high alkaline is occurred through the
use of ammonium and carbonate of bacillus subtilis. While viewing through the use of scanning
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electronic microscope the formation of precipitated calcium carbonate is observed. The bio-
based crack closing method in concrete is achieved by the process. Water permeability and
closing of the crack are also achieved through the biological process of the composites of
cement. There is reduction of maintenance costs and repair cost as a benefit of using bio-based
cement composites thereby increasing the durability of the structures.
The study was done by Jonkers et al. (2010) concerning the developing system on the self-
healing where there are bacteria of different species, concrete’s compressive strength changing
above the concentration of bacterial cell, concrete’s self-healing physical features, ability of the
bacteria to behave like the self-healing agent among others are taken into account. A group of
certain alkali resistant spore which forms the bacteria specifically of genus bacillus are picked
and mixed with mortar or concrete paste meant for the growing of self-healing ability in
structures. It is concluded that giving of the nutrients performed an important function for the
bacterial process in the cement mortar, enough nutrients can be provided by the waste water
which act as source of such organic nutrients for the growing of the bacteria. It is observed that
having or not having bacterial concentration for the cement mortar which is cured with water,
including bacteria is very important in that the bacteria increases the strength of the concrete.
And the last conclusion made is that the strength gain of the concrete which are not uniform after
some period of time shows the high bacterial process being relying upon the curing period.
The next study as analyzed by Chahal et al. (2012) focuses on the bacterial effect on the
strengthening and permeating features concrete including the fumes of silica. The cement is
replaced with 5%, 10% and 15% of silica fume but not completely. The partial silica fume is
then mixed with permanent concentration of the bacterial culture plus water of 105 cfu/ml in
volume. After 28 days the concrete is found to be having an increase in compressive strength
Self –healing 13

from almost 10% to 12% when there is an inclusion of bacteria in the concrete of silica fume. It
is also found that when the bacteria is included, the increase of water capillary, absorption of
water and porous state are reduced in the order of 54% to 78%, 42% to 48% and 52% to 56%
respectively for the concrete made of the bacteria as compared to the concrete made without a
bacteria after 28 days. There is also a reduced passage of the chloride for the bacterial concrete in
that the reduced charge is by almost 10% passing through the bacterial concrete as compared to
the concrete samples without the bacteria after 56 days. As viewed through X-ray diffraction
and scanning electron microscope of precipitation of calcite on combining with bacteria, the
study is assumed to be the cause of increasing the features of the concrete. When the bacterial
SF concrete is studied economically it is discovered that there is benefit/cast ratio of bacterial SF
concrete received which lowers the rise of SF number. Bacterial silica fume concrete having the
10% of silica fume indicates the largest advantage in increasing of its features and the
corresponding largest benefit/cast ratio as compared to control concrete.
Practical analysis was done by Siddique et al. (2011). In the analysis, it is indicated that
the spores of the bacterial are first closed against the hydrogels and then taken into the specimens
to monitor the healing capability. The formed particles of calcium carbonate (CaCO3) through
hydrogel-closed spores are indicated via thermo-gravimetric analysis. The distinct self-healing
superiority is indicated by the specimen of mortar having the spores closed with hydrogel. It is
discovered through the analysis that the water passage is reduced by the average of 68% and the
least cured crack width is almost 0.5 mm.
Next is the study done by Siddique & Chaha (2011) dealing with different types of bacteria
used in concrete. The good outcome is obtained through the method of encapsulation than using
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the method of direct application. It is also noted that using the bacteria increases the quality and
the performance of the concrete. Below are the data supporting the analysis;
No Type of bacteria used Obtained outcomes
I. Bacillus Sp. CT-5 40% of compressive strength
greater than the control
concrete.
II. Bacillus megaterium High speed of growing
strength is 24% obtained in
the largest concrete grade of
50 mpa
III. Bacillus subtilis An increase of 12% for the
compressive strength when
with the specimen of the
controlled concrete of low
aggregates of weight.
IV. Bacillus aerius Compressive strength raised
by 11.8% for the concrete
containing bacteria when
compared with the control
concrete.
V. Sporosarcina pasteurii 35% of the compressive
strength greater than the
control concrete
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VI. Akkr5 Raise in compressive strength
by 10% when compared with
control concrete
VII. Species of shewanella Raise in the compressive
strength by 25% when
compared with control
concrete
Self-healing concrete and the influencing features of the healing ability
Bacterial type used for self-healing
According to which type of bacteria is applied, the widely used bacteria as a self-healing
agent are the bacteria from genus bacillus Luo et al. (2015). It increases the quality of the high
alkalinity survival for the medium concrete. Bacillus bacteria are capable to precipitate CaCO3
when the activation by water is done. The laboratory generated of isolated strain of bacteria are
used a times. Cost of production of a self-healing rises depending on the bacterial strain given
from the laboratory. The bacteria of non-axenic that are developed through sub-streaming of
vegetable plant as a source of nutrient are used. As compared with the bacteria from the
laboratory, non-axenic bacteria can work as self-healing agent hence lowering cost. They are
also able to close the crack of 0.45mm width after 28 days. It is almost the same with genus
bacillus which close the crack of 0.3mm after 20 days.
Concentration of bacteria
Working of the sealing relies on the number of bacteria together with nutrient mixed to the
concrete. This number of the bacteria spore added to the concrete raise the speed of crack closing
Self –healing 16

but there is a requirement of nutrients to generate calcium carbonate after the spore’s activation
through water.
The number of nutrients restricts the number of calcium carbonate as shown in the below
equation.
The study by Wang et al. (2014) explains that when some amount of spores are added to the
1m3of the concrete mixture and is 2% within the hydrogels where every hydrogel has spores per
millimeters of 109, the mixture leads to the increase of 80% to 90% healing of crack width
0.3mm and after 28 days the healing percentage improves from 30% to 50% of 0.3mm to 0.7mm
crack width. Hydrogel which works as healing agent has urea and nutrients that helps in
deposition of calcium carbonate. Bacillus magaterium can be used as the healing bacteria and
amount of 105 spores per liter adopted in the composition of the concrete mixture. Almost 186 ×
105 cell/m3 is used for the concrete from the mixture. In summary it is discovered that only 1% to
2% of the cement content should be employed as the amount of self-healing agent in the mixture
of the concrete to lower the bad impact on the mechanical potency of the concrete. The number
of spores added to the mixture of concrete of 108 spores per liter also can generate similar healing
impact as greater spore’s concentration while mixing the solution. This can be as results of the
bacterial ability to generate immediately after being enabled from the spores.
Concentration and types of nutrient
A 0.5% of calcium lactate of the cement weight is used in mixture of concrete that shows the
ability of calcium lactate to live as precipitation agent when compared to urea. Larger volume of
nitrogen and an improved ability of corrosion on the reinforcement of steel can be produced
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through the hydrolysis process of urea. This leads to the confirmation of mineralization of 20μm
to 80μm of particle sized on the surface of the cracked concrete. Through the application of bio-
reagents having calcium nitrate and urea, the mineralization viability procedures in the self-
healing of bacterial concrete is also shown.
Ways to generate a self-healing concrete
Substituting the fresh water part in the mixer of the concrete with the solution of bacteria is a
way producing self-healing of concrete Park et al. (2010). Rather than spore, the bacteria cell is
used which ease the process of closing. This method structure of concrete are increased because
of the continuing deposition through the bacteria. It’s shown through the improved compressive
strength after taking 28 days as compared to 7 days has an advantage in that when the surviving
bacteria are closed the small. The next method is through using the specimen of mortar mixed
with bacteria spore. Larger numbers of spores after 28 days of age are crushed in concrete. This
is because of the process of hydration in concrete which lowers the volume of pores and result to
crushing of closing pores. It lowers the ability of mineral-forming of bacterial cements. It is
therefore important protective device for protecting the spores in the concrete. Other methods
include changing the source of bacteria and nutrient from solution to the powdered form, and
through using the source of calcium and nutrients along with saturated light weight aggregate.
Concrete self-healing features
 Bacterial self-healing compressive power of a concrete
 Remediation of cracks
Programming through a practical
The tensile and compressive strengths of the bacterial concrete are done.
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By the use of Automatic compression testing machine Waimer & Knippers ( 2015) the compressive
power of concrete,mortar and cement paste having bacteria is carried out. -
Velocity of the ultrasonic pulse
Using the circuits of electronic circuits, the duration for the electronic pulse to generate into
concrete is determined.
(SEM) Scanning electron microscopy
With the help of scanning electron microscopy (SEM), the deposition of calcite through small
cracks of the bacterial concrete is measured.
Diffraction of the x-ray
The study of the physical features of cement composite, chemical composition and the crystal
structure can be analyzed
Reasons for using the bacteria in the concrete
 Aids in decreasing maintenance and repair cost Vijay et al. (2017) of the concrete reinforced
structures through self-healing bacterial concrete.
 The attack of the freeze melt has got a good resistance. When the microbial calcite is
used, the opposition to the melting of freeze is lowered because of the activities of the
bacterial chemical. This also lowers the water penetration hence lowering the freezing
process.
 The method reduces the water penetration of the concrete.
 There is controlled emission of carbon dioxide through the concrete production process
 Bacteria have no pollution and are environmental friendly.
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 Production of the concrete is lowered
 There is increased service life of the structure than the expected life when the formation
of crack is cured in the early phase by itself.
 In areas where people may find difficult to access for the maintenance of the concrete, the
self-healing bacteria are used. Through this the dangers of human life in dangerous places
are lowered and the life span of the structures is increased.
 Oxygen being an agent that has the capability of inducing the corrosion, the bacteria
present is feeding on the oxygen and the tendency for the reinforcement of corrosion can
be lowered.
 The concrete can be applied to the already existing structures.
 The beauty look of the building is not interfered with.
 Cracks are corrected as fast as possible. Sand, nutrients and bacteria are filled to the
specimens of concrete. This improves the values of stiffness and compressive strength
when compared to the ones lacking cells.
Reasons why bacteria should not be used in the concrete
 Bacterial concrete is expensive as compared to the convectional concrete.
 Due to different climate condition, some bacteria experience bad growth in some places.
 When the capacity of the self-healing agents such as calcium lactate and bacteria are
combined, they turn to be larger than 20% thereby reducing the strength of the mortar.
 The self-healing agent is held by clay pellets which are about 20% by volume of mortar.
 Lack of IS codes. Because the study to of the bacterial concrete is new, there is no
material proving codes for the research. This makes it uneasy to get the bacterial doses
that can be used in the mortar to obtain the maximum durability.
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 There is a higher cost the investigating activities. Each bacterium has its own properties
to generate the enough calcite precipitation. Finding this investigating amount of the
bacteria is performed through “scanning by electron microscopy” that is expensive since
the method needs a knowledgably person to do the test.
Fig: Diagram of the crack healing by the concrete immobilized bacteria (Chen et al. 2016)
Common process involved in remediation of cracks through bacterial concrete
The cations containing Ca2+ taken from the surrounding are drawn by the microorganism having
negative charge of cell surface to place on the cell surface Van der Zwaag (2010). The role of the
bacterial cell can be summarized the given equation below as the early stage of site phase.
Cell +Ca2+ → Cell – Ca2+
CO32+ + Cell – Ca2+ → Cell – CaCO3
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Bacteria is therefore assumes as the site of nucleation that ease the calcite precipitation which
finally stops the cracks and pores within a concrete.
Bacterial concrete applications
Bacterial concrete application Reddy (2013) has increasingly become very much popular.
1. It is used where there are monuments made of limestone and require some repair.
2. It is used for curing of the cracks within a concrete.
3. Used in constructing of the following;
a) Roads which are durable and are of low cost.
b) Buildings which are of increased durability and strength.
c) Housing which are durable and are of reduced cost.
d) Bank of the rivers.
Methodology
Choice and cultivation of spore forming bacteria which are alkali resistant
Four sets of alkaliphilic spore creating bacteria were bought from German collection of
microorganisms and cell cultures namely; Bacillus pseudofirmus DSM 8715, Sporosarcina
pasteurii DSM 33, Bacillus halodurans DSM 497 and Bacillus cohnii DSM 6307 Sharma et al.
(2017). They were cultivated depending on the supplier’s recommendations. Endospore- creating
ability was established in a mineral medium, where this medium had milli Q ultra-pure water per
litre: 0.02g of KH2PO4, 0.2g of NH4Cl, 0.225g of CaCl2 , 0.2g of MgCl2.6H2O, 0.2g of KCl,
0.1g of yeast extract, 1millilitre trace constituents solution of SL12B, 8.4g of sodium bicarbonate
and 6.45g of citric acid trisodium salt. This medium’s PH was at 9.2. Incubation was conducted
on aerobic batch cultures in 2-1 Erlenmeyer flasks at 150rpm on a shaker table Muhammad et al.
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(2016). Then using microscopy, growth was monitored and the number of cells as well as the
sporulation cells percentage was also quantified using microscopy by a counting chamber known
as a Burger Turk.
Preparing and strength features of bacterial concrete
Preparation of concrete bars having and lacking control added bacteria was done for
tensile and determination of compressive strength. Major aim of such tests was to get whether
the concrete strength was not negatively influenced by the bacteria. Initially, during bacterial
concrete preparation, a packed culture of S.pasteurri was found after growth in DSMZ-2
medium. The total number of cells was quantified using a Burger Turk counting chamber by the
microscopy technique. Afterwards, washing was done on the cells twice by centrifugation and
resuspending the cell pellet in a tap water. Ultimately, these washed cells were then resuspended
in 20millilitres portion of tap water. This suspension of the cell was employed as part of the
required water for the preparation of concrete bar Schlangen et al. (2010).
Determination of compressive and tensile strength for concrete bars were prepared as
below; two sets, with and without control concrete added bacteria, of nine bars each were
constructed using Portland cement, aggregate composition having gravel and sand, and water
ration of 0.5. Every bar had the following dimensions 16×4×4 cm. curing was first conducted on
these bars for about 24 hours at a room temperature in plastic foil sealed moulds, there after they
were uncased and later cured in separate plastic containers filled with tap water at room
temperature. Subclass of three bars was each tested for compressive strength and flexion tensile
Talaiekhozan et al. (2014).
Water, cement and aggregate composition required for constructing nine concrete bars is
tabulated below
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Aggregate/ compound size in mm Weight in g
Water
Cement
Size of aggregate fraction:
0.125-0.25
0.25-0.5
0.5- 1
1-2
2-4
4-8
585
1170
396
730
848
848
1133
1685
Existence/ viability of concrete motionless spores
The ability of alkaliphilic bacterial spores to germinate halted in cement stone was
obtained. Such species include B. pseudofirmus, B. halodurans and B. cohnii. Firstly, the growth
of cultures of these species was done in a mineral medium. They were then washed twice by a
method of centrifugation and resuspending the cell pellets in tap water after formation of some
spores which were then quantified through microscopy Van & Belie (2013). The determined sore
suspensions were divided into two sections; one section was stored at a temperature of 4˚C and
then treated as non-concrete halted control for determining spore viability under storage.
The second section was applied for preparation of cement stone sample, where the spore
suspension was employed as a portion of the makeup water, bacterial and no bacterial control
spore added cement stone specimen were made ready. The quantity of spores coalesced to
cement stone which was figured out by microscopic counting was found to be109 cm−3. For the
Self –healing 24

preparation of cement stone disks having the following dimensions, 4cm diameter and a height
of 1cm, a combination of the ordinary cement and water to cement ratio of 0.5 was employed
where this was casted in plastic vials closed with a plastic lid. They were then cured for about 24
hours at room temperature; additionally the disks were cured at room temperature in tap water.
After ten days curing the disks were taken out from the plastic vials molds, they were then
chipped into sections with a chisel and later crushed into powder form by the use of a strong
pharmaceutical stone mortar. 1.84g of powdered cement stone containing 109 spores were
subsequentially slurred then diluted ten-fold by adding nine volumes of sterile mineral medium.
Cotemporally, the endospore having cell suspensions which were placed in a fridge under a
temperature of 4 degrees Talaiekhozan et al. (2014) were added to a spore density of 109 ml−1
and as well ten-fold diluted by adding 9 millilitres of sterile mineral medium. Furthermore,
original spore suspensions and cement slurries were homogenized by 3 rounds of rapid mixing at
a speed of 2500rpm and twenty seconds treatment in an ultrasonic bath. According to Most
Probable Number dilution technique, the number of viable spores in the slurries of cement stone
and spore suspension were estimated.
In this procedure, a mineral medium was added to 8 by 12 wells of sterile microlitre
plates till full, 180 microliters in each well. Four succeeding wells of the first row were spiked
with 20 microlitre control cell suspension portions or slurry. All these were subsequentially and
serially diluted in 10 fold dilution staged up to a dilution level of about 1011 , setting aside the
12th last row as non-spiked control to investigate for contamination of the medium.as a result,
every cement stone slurry and an equivalent control cell suspension were serially diluted in four
parallel sets. Growth happened in the lower dilution level but not in the higher during the
incubation period, because of dilution -extinction of viable cells available in the samples. Growth
Self –healing 25

could be simply determined by the use of eyes on account of raised turbidity of positive wells
during the succeeding two weeks period of incubation Sharma et al. (2017). Calculation of viable
number of cells in cement stone slurries with their equivalent spore suspensions was carried out
from the number of positive wells by the use of a computer program of Owens and Clerk.
Calcite precipitation ability of bacterial concrete
Incubation of chips of ten days cured cement stone samples was done in a rich medium of
yeast extract and peptone after pasteurization being conducted for thirty minutes at a temperature
of 70 degrees Celsius. Pasteurization of bacterial and control cement stone pieces was done
earlier before incubation so as to deactivate bacteria that actively got in contact with cement
stone samples during a period of curing or non-sterile treatment of the cement stone samples and
pieces after curing Wiktor & Jonkers (2010). Since the bacterial endospores fail to die by
pasteurization procedure, this treated therefore made sure that potential disparity between
bacterial and control concrete samples following incubation were resolved by the addition of
bacteria and not by accidentally introducing contaminants. The rich medium incorporated the
following; 3grams of yeast extract, 5grams of peptone and 8.4grams of sodium bicarbonate
which had a PH of 8.6. Aerobically, individual pieces were incubated in 100 millilitres of
medium aliquots on a shaker table at a speed of 100rpm and at a temperature of 25 degrees
centigrade for a maximum of 12 days. These pieces were rinsed using tap water after incubation
and later stored while wet in closed plastic vials till ESEM analysis, which was carried out
within period of two days following incubation without any more treatment. These chips were
then placed on a 1cm squared metal support and put in place with an adhesive tape and an
observation done using a Philips XL30 series Environmental scanning microscope.
Self –healing 26

Analysis
Choice and cultivation of alkali resistant spore creating bacteria
Out of the four, three of them generated abundant spores in the mineral medium apart
from S. pasteurii, which never grew in this medium. Production of spore was significantly low in
rich yeast extract and peptone based medium. The percentage of cells having endospores was
measured with microscopic counting and they totalled to 50, 75 and 25% for B. halodurans, B.
cohnii and B. pseudofirmus respectively.
The strength features of bacterial concrete
The twenty-millilitre washed cell suspension applied in the manufacture of bacterial
concrete bars, a suspension of S. pasteurii culture, was found to include 3.48×1012cells, which
led to a final density of 1.14× 109 cells cm−3 concrete. As the mean volume of an S. pasteurii cell
is equivalent to 2.5 micrometre cubed, the whole cell volume total to 0.3% of the bacterial
concrete volume. Compressive and tensile strength tests after7, 3 and twenty-eight days curing
displayed no notable difference between bacterial and control concrete.
Organic additives to the paste mixtures can lead to undesired strength loss. Addition of
various organic compounds also influenced splitting of tensile strength of the paste.
Viability of cement stone motionless spores
After 10 days curing, the number of viable spores in cement stone samples and original spore
suspensions, all having a spore density of 109 cm-3 were calculated. Outcomes displayed that
roughly 1% of the spores in the spore suspensions about 107ml-1could be obtained as viable. The
numbers of viable spores in the corresponding cement stone samples appeared considerably
lower that is ranging from 109 to 106 cm-3. Estimated viable spores in cement stone slurries in
Self –healing 27

spore suspensions resulted to 2%, 7% and 1.9% for B. cohnii, B. pseudofirmus and B.
halodurans respectively. Number of viable bacteria in control cement stone samples with no
addition bacterial spores and using tap water for preparation of concrete sample were lower than
detection limit, which is 500 cells cm-3.
Self –healing 28

Calcite precipitation ability of bacterial concrete
Through ESEM analysis, it is discovered that bacterial cement stone in comparison with
control cement stone samples; it precipitated significant quantities of calcite like crystals upon its
surface when placed for incubation in a medium containing yeast extract and peptone. The over
side of control and lactate-based specimens, bunches of small calcite minerals alongside huge
Calcium silicate hydrate -like filaments were created. In contrast, tiny minerals and Calcium
silicate hydrate – like filaments, large in size robust calcite precipitates were produced by paste
specimens having calcium lactate together with bacteria Chen et al. (2016).
Discussion
Applying self-healing concrete, in other words, a concrete that is capable of repairing,
sealing and plugging newly made cracks autogenously Vijay et al. (2017), will not only lead to
more permanent structures but it will also conserve a considerable quantity of money as repair
and labour-intensive check can be reduced. Under this study, we conducted an investigation
concerning the ability of bacteria mediated calcium carbonate generation as a feasible healable
mechanism. Comparing it to other studies where bacteria were applied externally for monument
and concrete crack fixation. In this study we employed bacterial spores, that is resting or dormant
cells within the matrix of the concrete. The outcome of our study is encouraging. The estimated
value of the viable spores obtained from youthful cement stone, following ten days curing, was
ranging between 1.9% and 7% of the total number of viable spores available in the initial spore
suspension employed in the cement stone samples preparations.
Self –healing 29

The table below shows the estimated number of cultivable/viable bacterial spore suspensions
against the cement stone in which spores of the corresponding bacterial species were rendered
motionless.
Suspension of spore and tap water - control Number cm-3
B. halodurans
B. pseudofirmus
B. cohnii
Tap water-control
5.63 E6 (1.74-18.17)
7.98 E6 (2.63-24.24)
5.73 E7 (1.76-18.58)
< 500
Samples of cement stone
B. halodurans
B. pseudofirmus
B. cohnii
Control
1.07 E5 (0.36-3.20)
5.62 E5 (1.74-18.14)
1.15 E6 (3.8-34.8)
<500
Taking into account mechanical forces, grinding, required to suspend and liberate the
cement stone immobilized bacterial spores, these values are substantial. Furthermore, even if the
retrieved percentages reflect accurately viable spores, absolute values are still elevated, ranging
between 1.7 and 7.5 ×107 spores cm-3 cement stone, finding out that one cultivable cell is
theoretically adequate to begin precipitation of calcite and microbial growth, giving that
favourable conditions exist. The mineral precipitation ability of bacterial concrete was
demonstrated by incubation tests with ten days cured samples of cement stone. We believe that
the real precipitation mechanism of bacterial mineral is as follows. The moment it’s in contact
Self –healing 30

with large amounts of growth substrates (peptone and yeast extract) and water, bacterial
endospores sprouts and begin to emit carbon dioxide because of metabolic turnover of growth
substrates. Carbon dioxide which can locally reach extreme concentrations because of quick
metabolic transformation of organic compounds Talaiekhozan et al. (2014), will react chemically
with calcium hydroxide produced from C3S and C2S hydration reactions. The calcium hydroxide
which leaks out of the pore system of the concrete reacts with carbon dioxide and undergoes
precipitation in form of calcite or any other mineral which is calcium carbonate based. This
hypothesis is supported by the calcite like crystals obtained on the bacteria’s surface, however
not on the surface of the samples of control cement stone.
Diagram of a Control cement stone (Wang et al.2014)
Self –healing 31

Self –healing 32

Diagram of the self-sealing Bacteria (Soens et al. 2014)
Tests carried out in this study depict that alkaliphilic endospore creating bacteria included
in the matrix of the concrete can briskly precipitate minerals of calcium carbonate. Water
required for the activation of endospores, can get in the structure of concrete through freshly
created cracks. Additionally, for mineral precipitation, live cells require a substrate which is
organic which can metabolically be transformed to inorganic carbon, what can subsequentially
precipitate with unconstrained calcium to calcium carbonate Jonkers et al. (2010). Unconstrained
calcium is normally available in the concrete matrix, though not organic carbon. In the current
Self –healing 33

experiments organic carbon was employed externally as a section of the incubation medium,
while preferably it should be a section of the concrete matrix.
In that instance external water is required to stimulate the concrete immobilized bacteria
which can transform organic carbon available in the matrix of the concrete to calcium carbonate
and through that it seals the freshly created cracks. At this moment, we examine which certain
type of organic compounds is proper to integrate in the concrete matrix. This is surely not
ignorable as such compounds should be an appropriate source of food from bacteria as well as be
consistent with concrete. Specific groups of organic compounds are less or not appropriate at all,
for example compounds like carbohydrate derivatives which are familiar to restrain the concrete
setting even at concentrations which are low. In addition, we currently examine the long-term
viability alongside potential contingencies to raise the viability of immobilized concrete
endospores to make certain enduring bacterially improved self-healing Wiktor et al. (2011).
Additional ongoing examinations address possible reduction in permeability of concrete and the
modification of mechanical features of sealed cracked concrete due to precipitation of bacterial
calcite.
Conclusion
It can be concluded that it is necessary to protect the bacteria in a concrete in order to
maintain the self-healing process throughout the concrete life span. The bacterial concrete has
the ability to restore physical cracks produced independently from the external cause as
compared to the convectional concrete. Generally application of bacteria in the concrete
improves the compressive and tensile strength and reducing water passage through a concrete as
compared to the convectional concrete. Absorbing water by a concrete and corrosion of
Self –healing 34

reinforcement are also minimized. Bacterial concrete is also called smart bio material due to its
inheritance ability to precipitate calcite continuously. Bacterial concrete is better than
convectional concrete due to the fact that it is environmental friendly and has the self-healing
ability. Bacterial concrete affects the durability features of the building through the healing of
cracks within the building Vekariya et al. 2013). Different kinds of bacteria that are used for
healing cracks, for producing the bacterial isolates like bacillus pasteuri, bacillus subtilis species
in the crack healing of the concrete. It is also discovered that some bacteria are not good for the
health of human being. Such bacteria are bacillus amyloliquefaciesna, shewanella species,
pseudomonas aeruginosa, bacillus megaterium among others while some like bacillus sphaericus,
bacillus pasteurii among other do not affect the health of human being. Lastly it is found that
mixing of bacteria concrete is a complicated process and requires only the skilled labors.
Self –healing 35

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