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CHAPTER 2

REVIEW OF LITERATURE

2.1 INTRODUCTION

The main aim of this literature review is to provide an in-depth


knowledge on different key factors to understand the overall process of
biomineralization. It 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. MICP is
highly desirable, because the calcite precipitation induced as a result of
microbial activities is pollution free and natural. MICP generally results from
a series of complex biochemical reactions involving urease. The highly
potential results of this process encouraged different research groups from the
world to promote MICP. The first part of chapter 2 describes the development
of conventional materials followed by urease producing bacteria and their
characterization. Then assessment of parameters to study on strength and
durability characteristics such as sorptivity, rapid chloride penetration and
acid attack of materials is presented. At the end, MICP had been investigated
for the enhancement of strength and durability of materials and structures
with help of different microorganisms along with various calcium sources.
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2.2 HISTORICAL DEVELOPMENT OF CONVENTIONAL


BUILDING MATERIALS

An artificial hydraulic lime was prepared by calcining an intimate


mixture of limestone and clay. The strength and elasticity of the concrete are
affected due to the presence of total volume of pores that had been reported
by Verbeck (1978) and Powers et al (1959). The macro and micro pores were
more related to dry shrinkage and creep had been observed (Mehta 1993). The
movements of gases, liquids and ions through the concrete had greatly
influenced the integrity of the concrete structure and were reported by Nilsson
et al (1996).

2.3 BACTERIAL SPECIES IN CEMENT MORTAR

The production of urease by the presence of ammonium in


organisms such as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus
megaterium (B. megaterium) and Klebsiella aerogenes had been studied by
Kaltwasser et al (1972), Friedrich & Magasanik (1977). The occurrence of
urease in many bacteria, several species of yeast and a number of higher
plants had been reported by Dixon et al (1980), Kerr et al (1983). Initially,
Bacillus pasteurii (B. pasteurii) was found in the sewage and urinal
deposition was reported (Sneath 1986). Proteus vulgaris, which can produce
urease even in the presence of high concentrations of ammonium, was
reported by Morsdorf & Kaltwasser (1989). Mobley & Hausinger (1989),
Mobley et al (1995) explained the existence of four modes of regulation for
the synthesis of urease in microbial systems. The urease activity involves a
wide range of microorganisms and plants, some of which produce the enzyme
in large quantities had been observed by Mobley et al (1995 a,b), Ciurli et al
(1996). Manoli & Dalas (2000) found that there are six calcium carbonate
(CaCO3) mineral polymorphs including calcite, aragonite, vaterite, CaCO3
monohydrate, CaCO3 hexahydrate and amorphous CaCO3 and among them,
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calcite, aragonite and vaterite are non-hydrated crystals. It had been found that
calcite and aragonite have stable thermodynamic structures, while vaterite is
thermodynamically unstable so that it is easily transformable to calcite or
aragonite in aqueous solution.

The biocementation reaction results in the generation of high


concentrations of ammonium. Those bacterial sources where urease is not
down regulated by the presence of ammonium are useful; also these
organisms include Sporosarcina pasteurii (S. pasteurii) and Proteus vulgaris.
For biocementation purposes, an ideal microbial source of urease should have
the properties such as, high urease production capacity, an ability to produce
urease in the presence of ammonium, stability, reliable production and not
require any further down-stream processing to use in biocementation. The
presence of ammonium inside the cell induces the production of glutamine,
which prevents further hydrolysis of urea by Klebsiella aerogenes species had
been explained by Mulrooney et al (2001). Calcium carbonate precipitation
on the cement mortar specimen had been evaluated by Bang et al (2001).
Nemati & Voordouw (2003) reported that the enzymatic formation of calcium
carbonate (CaCO3) in a batch system was dependent on the concentrations of
enzyme (urease) and reactants (urea and calcium chloride), as well as
temperature.

Another possible explanation such as crystal morphology which


was obtained with different bacterial culture could be due to the level of the
actual urease activity that had been suggested by Hammes et al (2003). The
morphological effect on concrete by the calcium carbonate precipitation had
been analyzed by Braissant et al (2003). Existing of high percentage of salt in
the building materials may damage the structure of the building had been
analyzed by Benavente et al (2003). The pure mineral calcite had high surface
energy and naturally hydrophilic was proved by Ioannou et al (2004). The
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morphology of precipitated CaCO3 crystals changed from regular


rhombohedral to irregular rhombohedral, then to spherical and finally to
peanut-like shape, observed by Liang et al (2004). The influence of calcium
source is limited to the morphology of the crystals, studied by Bottcher et al
(2004). Considering both biocementation and environmental constraints, two
organisms have potential as sources of urease for biocementation namely
(S. pasteurii) and Proteus vulgaris has been studied (Whiffin 2004).

The production of CaCO3 in the media containing urea and calcium


chloride can be induced using bacteria with urease activity had been studied
by Nemati et al (2005). The considerable research on carbonate precipitation
by bacteria had been performed using ureolytic bacteria and reported by Dick
et al (2006). The amino acids play an essential role in the morphology and
mineralogy of bacterially induced carbonate precipitation, observed by Ercole
et al (2007). The high proportion of salt within the porous lattice of building
materials can lead to service damage to the local force of crystallization
process, reported by Cardell et al (2008).

There are morphological differences between treatments with pure


culture Bacillus sphaericus (B. Sphaericus) and mixed ureoltic cultures could
partially be explained by the method of application by Muynck et al (2008).
The structural morphological evolution of the carbonate coating produced on
model plaster sample had been showed by Severine Anne et al (2010). They
also demonstrated that the bio deposition treatment resulted in an increased
resistance of mortar specimens towards carbonation, chloride penetration,
freezing and thawing. The change of calcium salt used in bio deposition result
in modification of morphology of precipitated CaCO3 had been observed by
Wiktior et al (2011).
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2.4 INFLUENCE OF BACTERIAL SPECIES IN pH

The bacteria produce exopolysaccharide film, which contributes to


pore plugging by suspending clay particles, cells and other suspended solids
had been reported by Shaw et al (1985). Douglas & Beveridge (1998),
Castanier et al (1999) reported that the primary role of bacteria in the
precipitation process as subsequently ascribed to their ability to create an
alkaline environment through various physiological activities had been
studied and also they showed that removal of the produced hydrogen sulfide
and resultant pH increase was a prerequisite for carbonate precipitation to
occur.

The high pH hinders the growth of bacteria and the optimum pH


(around 9) for the growth of B. pasteurii was around 9 had been observed by
Ramachandran et al (2001). Hammes & Verstraete (2002) explained that the
key roles of pH and calcium metabolism in the precipitation of CaCO 3 in
bacterial systems such as governed by three parameters like, the calcium
concentration, the carbonate concentration and the pH of the environment.

The B. pasteurii incorporated culture produced maximum amount


of CaCO3 of 52.54% in weight after 120 hours had been observed by Chahal
et al (2011). By their closer observation they found that a small amount of
spherical deposits (vaterite and fluorite) in the culture. But B. sphaericus
bacteria induced CaCO3 precipitation at pH = 8 yields the highest results had
been reported by Arunchalam et al (2010). The highly alkaline pH of concrete
hinders the growth of the bacteria had been reported by Tittelboom et al
(2010). The use of diatomaceous earth to protect the bacteria B. sphaericus
from the high-pH of the concrete matrix had been suggested Wang et al
(2011). The urease catalyzed ureolysis was also temperature dependent and
the same authors reported the optimum temperature range to the growth of
bacteria such as 20 o C to 37o C. Sphaericus pasteurii (S. pasteurii) was found
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to be more efficient in improved urease activity and also showed to be able to


survive at very high pH values had been developed by Achal et al (2009,
2011b).

2.5 BACTERIA EMBEDDED MICP CEMENT MORTAR/


CONCRETE

The successful usage of biomineralization process to induce calcite


precipitation on the cement mortar was reported by many research groups.
Biomineralization technique for the remediation of damaged structural
formations had been developed by employing a selective microbial plugging
process to promote precipitation of CaCO3 in the form of calcite. Numerous
diverse microbial species participate in the precipitation of mineral carbonates
in various natural environments, including soils, geological formations,
freshwater biofilms, oceans, and saline lakes. MICP had been investigated by
many research groups for its potential to improve the strength and durability
of cement mortar/concrete constructions.

The simplest of MICP mechanism is the hydrolysis of urea by the


enzyme urease, which results in the production of carbonate ions in the
presence of ammonium. Use of microorganisms within cement
mortar/concrete leads to the process called biomineralization. It is a potential
field of research in concrete technology. The CaCO 3 precipitation was a
general phenomenon in the bacterial world under appropriate conditions had
been reported by Boquet et al (1973). The microorganism may produce
accumulate inorganic compounds such as phosphorites, carbonates, silicates,
iron and manganese oxides in cytoplasm had been reported by Beveridge et al
(1983), Ghiorse (1984), Knoll (1985), Ruiz et al (1988).

Lowenstan & Weiner (1988) noticed that the biologically


controlled or induced CaCO3 precipitation may be largely dependent on the
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environmental conditions. A large number of bacteria were detectable inside


carbonate crystals, although it was not specified whether they were viable or
not had been noticed (Morita 1980). Geesey & Jang (1990), Buczynski &
Chafetz (1991) results showed that extracelluar polymeric substances (EPS)
play an important role in the formation of carbonate crystals not only by
providing nucleation sites but also by attaching small crystals to each other to
increase the size of the bioliths. Biofilms probably contain the normal
environment for most microbial cells in many natural and artificial habitats
such as concrete surfaces, complex associations of cells (Christensen 1989).
The bacteriogenic mineral plugging and specifically in situ bacterially
induced precipitation of CaCO3 had been shown to reduce the permeability of
porous media was studied by Ferris et al (1996). Different mechanisms, which
could induce precipitation in the natural habitats of bacteria had been
proposed (Erlich 1996).

They also studied about B. pasteurii biomineralization of CaCO3 in


unconsolidated sand columns. The biologically induced mineralization was
also an important geological process that helps in the formation of
microfossil, hot spring deposition and transfer chemical elements had been
reported by Konhauser et al (1996), Jones et al (1997). The carbonate
formation by Pseudomonas fluorescens precipitation was seen to occur
through the activity of a peptide matrix, with aspartic acid, glutamic acid,
alanine, and glycine as the major contributing amino acids had been reported
by Appanna et al (1997). Determining physical and chemical factors, such as
temperature, pH and geography that correlate with differences between
diverse microbial communities will reveal how easily microbes tolerate
different kinds of environmental change and will increase our understanding
of microbial ecology and evolution. The CaCO3 in microbial mats could be
seen as a byproduct of microbial metabolism had been noticed (Des Marais
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1997). The bacteria can produce calcium carbonate in different environment


from the biological process had been noticed by Rivadeneyra et al (1997).

Mc Connaughey & Whelan (1997) reported that the carbonate


precipitation can also function as an energy source through proton production
outside the cell membrane. Due to the sufficiency of the required cations and
anions, the metal salt on the cell surface begins mineral formation by acting as
a nucleation site. The anion (carbonate) in this reaction was a product of the
bacterial metabolism (Erlich 1998). Douglas & Beveridge (1998) reported
that the primary role of bacteria in the precipitation process had been
attributed to their ability to create an alkaline environment through various
physiological activities. The possible biochemical reactions in urea-calcium
chloride medium to precipitate CaCO3 at the cell surface had been discussed
by Stocks-Fischer et al (1999).

The role MICP within the cement mortar matrix through scanning
electron microscope (SEM) had been confirmed by Ramakrishnan et al
(1999). Knorre & Krumbein (2000) observed that the different types of
bacteria, as well as abiotic factors seem to contribute in a variety of ways to
CaCO3 precipitation in a wide range of different environments. Some bacteria
and fungi could induce precipitation of CaCO3 extracellularly through a
number of processes that include photosynthesis, ammonification,
de-nitrification, sulphate reduction and anaerobic sulphide oxidation had been
identified by Castanier et al (2000) and Riding (2000). The six genes involved
in crystal formation in Bacillus subtilis (B. subtilis) PB19 had been identified
by Perito et al (2000). Gonzalez-Munoz et al (2000) investigated that the
precipitation of minerals in different groups of bacteria and the influence of
various environmental factors.

Applications of MICP in various fields like, the treatment of


groundwater contaminated with heavy metals, radio nucleotides and the
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removal of calcium from wastewater were reported by Warren et al (2001).


The biomineralization could occur through active precipitation of carbonate
by microorganisms or passively through bacterially induced changes in the
chemistry of the system had been reported by Fujita et al (2000). The
microbiologically induced calcite could be environmentally safe, compared to
synthetic polymers currently used for concrete repairs had been reported by
Ramachandran et al (2001). Bang & Ramakrishnan (2001) reported that
microbiological process utilizes a biological byproduct, CaCO3, which had
shown a wide range of application potential to make sand as a sealant.

The biofilm produced from B. subtilis was genetically engineered


to secrete polyglutamate or polyaspartate, an additional small increase in
corrosion inhibition on aluminium occurred had been reported by Ornek et al
(2001). Carbonate precipitation by bacteria was a complex process which was
described by Castanier et al (2000) as involving metabolic pathways
associated with photosynthesis, nitrogen and sulfur cycles, and ion exchange
(Ca+2/H+) had been reported by Hammes & Verstraete (2002). The calcite
precipitation by E. coli HB101 (pBU11) was significant; although its
precipitation level was not as high as that by B. pasteurii had been reported by
Bachmeier et al (2002).

Microbially induced carbonate precipitation had received attention


as an environment-friendly method of protecting decayed ornamental
carbonate stone and remediation of cracks in building materials. Some of
MICP bacteria such as, Micrococcus sp., B. subtilis, B. pasteurii, Deleya
halophila, Halomonas eurihalina, and Myxococcus Xanthus had been
investigated by Rivadeneyra et al (1998), Tiano et al (1999), Castanier et al
(2000), Cacchio et al (2003), Rodrigues-Navarro et al (2003). Neemati &
Voordouw (2003) noticed the bio mineralization process by the precipitation
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of CaCO3 crystal particle via microbes. MICP technique had been


successfully used in waste water treatment by Hammes et al (2003).

The biomineralization technique could be used for the remediation


of damaged structural formations had been noticed by Cacchio et al (2003).
Biomineralization 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 had been
noticed (Hamilton 2003). The extracelluar polymeric substances (EPS) play
an important role in the formation of carbonate crystals not only by providing
nucleation sites but also by attaching small crystals to each other to increase
the size of the bioliths had been noticed by Van Lith et al (2003). The use of
MICP had been proposed for the removal of metal ions from waste water by
Simon et al (2004). The microorganisms and microbially mediated
mineralization process are active in almost every environment on the earth
had been noticed by Lopez-Garcia et al (2005). In cell growth and metabolite
production, the selection of the nutrients and determination of its
concentrations in the cultivation media is a very important step.

However, the CaCO3 production by bacteria is generally regarded

specific molecular mechanism had been reported by Barabesi et al (2007).


The S. pasteurii could be used as ecologically sound biological construction
materials had been reported by Chou et al (2011). The most bacterial species
were able to precipitate carbonates given the right conditions, namely, an
alkaline environment rich in Ca2+ ions had been reported by Zamarreno et al
(2009). The B. sphaericus precipitated CaCO3 in the interface between the
medium and concrete specimen leading to the formation bio-film had been
reported by Parks et al (2009). By means of SEM observations, they proved
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that the presence of chloride ions resulted in rhombohedral crystals, while the
presence of acetate ions resulted in spherical crystals.

The bounded metal ions like calcium may react with anions like
carbonate to form an insoluble salt as CaCO3 had been reported by De
Muynck et al (2010) and also they reported that the costs of the MICP
treatments are attributable both to the price of the product and the number of
applications required. Thus, finding out of the cheaper sources of raw
materials are particularly important for the products which are required in
large quantities.

2.6 ROLE OF MICP IN CRACK REMEDIATION

The remediation of damaged structural formation developed by


employing a selective microbial plugging process in which microbial
metabolic activates, promote precipitation of CaCO3 in the form of calcite had
been reported by Kanizas et al (1992). Ferris & Stehmeier (1992) reported
that the usage of bio-deposition technology for consolidation of sand column
and remediation of cracks in concrete. The bacterially or microbiologically
induced CaCO3 technique in fixing crack width environmentally friendly
biological process had been reported by Gollapudi et al (1995). The crack
remediation by MICP of various structural formations such as granite and
concrete had been successively reported by Gollapudi et al (1995), Stocks-
Fischer et al (1999). There were large numbers of products available
commercially in repairing cracks in concrete such as structural epoxy, resins,
epoxy mortar and other synthetic mixtures (Kirley 1999). Currently, these
types of synthetic filler agents are extensively used in concrete crack repair.

The unravel the mechanism, a filling method for crack in concrete


using sand mixed B. pasteurii had been proposed by Ramachandran et al
(2001). A novel approach of microbiologically enhanced crack remediation
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had been noticed by Bang and Ramakrishnan (2001). The physicochemically


versatile polyurethane was an effective enhancement tool in MICP in concrete
cracks had been observed by Bang et al (2001). The effect of MICP on cracks
of various depths on the compressive strength values in cement mortar cubes
and found an increase of the strength in the presence of B. pasteurii had been
studied by Ramachandran et al (2001). B. pasteurii had been used
successfully for remediation of crack in concrete by Santhosh et al (2001).
The immobilization technique for remediation of cracks in concrete where
microbial cells were encapsulated in polymers had been developed and
adopted by Bang et al (2001), it enclosed CaCO 3 precipitation in the cap to
enhance the strength for selective concentration. The precipitation of CaCO3
with the urease enzyme immobilized on polyurethane had been investigated
by Bachmeier et al (2002). In order to protect the cells from the high pH, the
effect of different filler materials on the effectiveness of the crack remediation
had been investigated by Day et al (2003).

Through B. pasteurii species the MICP in crack remediation had


been analyzed by Ramakrishnan et al (2005). Calcium carbonate precipitation
with help of Bacillus lentus and B. sphaericus were used in crack remediation
by Dick et al (2006). The autogenous crack healing of concrete refers to the
ability of concrete to heal the cracks by itself and accepted to be mainly
caused by precipitation of CaCO3 in the crack during concrete carbonation
had been reported by Li & Yang (2008). Zhong & Yao (2008) reported that
the CaCO3 precipitation by bacteria was an important strategy to radiate
cracks in building materials. The technology based Bacillus cereus induced
CaCO3 formation commercialized for repairing cracked surface of ornamental
stones (Mastromai 2008).

B. sphaericus bacteria induced calcite precipitation protect the


surface and heal the cracks of construction materials also reported that the
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presence of CaCO3 crystals decrease the water permeability of cracked


concrete had been observed by Tittelboom et al (2010). The Alkali-resistant
spore-forming bacteria related to the genus Bacillus act as potential healing
agents and autonomous repair of micro (<1mm diameter) cracks of cement
stone at spore concentration of 1 to 6108 cell/ml with w/c 0.5 and the
application of Bacillus pseudofirmus and Bacillus cohnii as self-healing
agents for the development of sustainable concrete had been introduced by
Jonkers et al (2010). Naturally, several bacteria have an ability to precipitate
CaCO3. These bacteria may be found in soil, sand, natural minerals.

The bacterially B. sphaericus precipitated CaCO3 was used to heal


cracks up to 0.15 to 0.17 mm in concrete had been reported by Wang et al
(2011). Wiktor & Jonkers (2011) reported experimental results on the crack-
healing in concrete with lightweight aggregates containing Bacillus
alkalinitrilicus. The healing of cracks in concrete can effectively increase
service life of the structures had been concluded by Achal et al (2013).
Pacheco-Torgal & Labrincha et al (2013) suggested that biomineralization
was low toxic creak repair technique.

2.7 INFLUENCE OF CALCIUM SOURCE ON MICP

Microbially mediated mineral precipitation could result in crack-


plugging, associated decrease in material permeability and increase in
compressive and split tensile strength in the cement mortar/concrete. A
suitable mineral precursor compounds (different calcium sources) as calcium
needs additionally to be incorporated in the material matrix to provide a truly
autonomous healing mechanism. Different calcium sources have been used by
researchers, throughout the world, to help the effective process of microbial
mineral formation. The most soil bacteria are able to precipitate CaCO3
crystals when tested in a medium containing calcium acetate had been
successively showed by Boquet et al (1973). A variety of crystal
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morphologies and big crystals produced by bacteria in medium containing


calcium acetate had been reported by Urzi et al (1999). The enhancement of
durability of building materials with the combination of ureolytic bacteria and
calcium chloride had been reported by Stocks-Fischer et al (1999), Bang et al
(2001). The deposition of CaCO3 improved the durability of building
materials which were influenced by the ureolytic bacteria and calcium salt had
been reported by De Muynck et al 2007. Pacheco-Torgal & Labrincha (2013)
concluded that the existence of current knowledge on the use of bacteria for
concrete with enhanced durability through biomineralization in concrete
surface treatment.

2.8 ROLE OF MICP IN THE STRENGTH OF CEMENT


MORTAR/ CONCRETE

The aerobic microorganism such as B. pasteurii and Pseudomonas


aeruginosa showed a significant improvement (18%) in compressive strength
of cement mortar had been reported by Ramakrishnan et al (1998),
Ramachandran et al (2001). The compressive strength of portland cement
mortar cube was raised 27% in the microbial cement mortar specimen
compared to control at cell concentration of 5.2107 cell/ml and a significant
increase in compressive strength of 7 and 28 days at cell concentration of
5109 cell/ml immobilized cell crack by using B. pasteruii had been reported
by Ramachandran et al (2001). The measurable increase in compressive
strength of S. pasteurii incorporated cement mortar in cubes had been noticed
by Bang et al (2001).

The positive effect on compressive strength of concrete by CaCO3


formations had been reported by Xiao et al (2002), Aguilera et al (2003),
Jerga (2004). The improved mechanical properties of concrete by
incorporating live bacteria into it by the way of MICP had been noticed by
Ghosh et al (2005, 2006). The increase in compressive strength was mainly
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due to consolidation of pores inside the cement mortar cube with MICP had
been reported by Lian et al (2006). The maximum compressive strength at
concentration of 105 cells/ml of water used B. pasteruii treated cement mortar
and concrete had been observed by Ghosh et al (2006). His group observed in
s (28 days) compressive strength raised to 22.62% as
compared with ordinary concrete in the Shewanella Species treated cement
mortar/concrete. From the test result they noticed that the Shewanella species
incorporated cement mortar showed 25% increased compressive strength
compared to ordinary cement mortar.

The maximum strength was observed by Ramakrishnan et al (2005)


at the concentration of 106 to 108cells/ml of water used B. pasteruii
microorganism incorporated concrete. The improvement in compressive
strength (25%) of thermophilic anaerobic microorganism incorporated cement
mortar was reported by Ghosh et al (2005). They also found that there is no
improvement in strength of mortar with the addition of Escherichia coli
bacteria. The effects of Shewanella species on the strength and chemical
composition of hardened cement mortar had been reported by Ghosh et al
(2006).

Jonkers & Schlangen et al (2007) observed that the decrease in


strength (less than 10%) was also noticed addition of a high number of
bacterial spores (108/cm3) using spore forming bacteria. The maximum
compressive strength at the concentration of 105 cells/ml of water used in
cement mortar had been observed by Ghosh et al (2008). Jonkers &
Schlangen et al (2008) noticed that the 28 days, the splitting tensile strength
was significantly increased through spore forming alkaliphilic bacteria at
concentration of 6108cells/cm-3 which in turn 0.1 to 0.2 mm concrete cracks
were arrested with bacterial activity.
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The strength of bacteria modified cement mortar increased due to


the deposition of the new materials gehlenite by the bacterial activity, which
in turn increases the uniformity of SiO2 concentration of mortar had been
reported by Ghosh et al (2009). Due to Arthrobacter crystallopoietes
microorganism, the increased compressive strength of 22% was noticed by
Park et al (2010) in the microbial cement mortars. The characterization of
biosealant properties of B. sphaericus and the bacterial treatment of drilled
cube increased compressive strength of 34% when compared to the drilled
non remediate cube (control) had been studied by Arunachalam et al (2010).
Due to the presence of calcium carbonate precipitation the desirable variation
in mechanical properties of cement mortar had been noticed by Park et al
(2010).

The B. subtilis incorporated concrete showed enhanced


compressive strength of 14.92% compared to ordinary concrete with concrete
grade M40 had been reported by Reddy et al (2010). The Bacillus sp. CT- 5
microorganism showed the higher compressive strength as 36% compared to
ordinary mortar cube had been noticed by Achal et al (2011a). The microbial
mineral precipitation improved the strength of bricks had been noticed by
Kaur dhamia et al (2012). For S. pasteurii incorporated concrete, 28 and 91
days compressive strength showed 38.2 Mpa and 44 Mpa respectively. The
reduction in chloride permeability and water absorption at 105 cells/ml had
been reported by Chahal et al (2012). The microbial biocementation improve
the physico-mechanical properties of cement mortar had been proved by Abo-
EI-Enein et al (2013). Also they noticed 33% improvement of compressive
strength in S. pasteurii incorporated cement mortar.

2.9 EFFECT OF MICP ON DURABILITY PROPERTIES

The influence of water movement in unsaturated porous materials


contained concrete structure had been explained (Rose 1965). The common
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quality in concrete construction varies due to the corrosion inhibiting


properties of the alkaline cement paste pore solution had been adopted
Newman (1986). The decrease in permeability by MICP was believed to be
the result of the in situ growth and metabolism of microorganisms, rather than
abiotic factors had been reported by Raiders et al (1989). They also reported
from their water permeability test that the combination of sol-gel with active
autoclave bacteria may lead to crack filling and decreased water permeability,
also they suggested from Thermo Gravimetric Analysis (TGA) that only
active bacteria will be able to precipitate CaCO3 crystals in the gel matrixes.
The permeability tests to assess the durability of the concrete had been
noticed (Basheer 1991).

Sorptivity is considered as an index for estimating potential


durability of cement mortar/concrete had been reported by Ho & Chirgwin
(1996), Bentz et al (1999). Water penetration reduction about 60% from the
samples treated with bacterial species was observed by Tiano et al (1999).
The bacterial carbonatogenesis for the protection and regeneration of
limestone in buildings, monuments and statues had been studied by Le
Metayer-Levrel et al (1999). Sorptivity increases with increasing water to
cement ratio. Other factors influencing capillary water transport include the
interfacial transition zone Bentz et al (1994), surface finish Senbetta &
Scholer (1984), McCarter (1993), initial moisture content or saturation Hall
(1989), Nokken & Hooton (2002), compaction Leeman et al (2006), curing
time Bai et al (2002) and preconditioning method Dias (2004) had been
noticed.

Khan & Lynsdale (2002) reported the enhanced durability of


concrete by different surface treatments. Bacterially (Myxococcus xanthus)
induced carbonate mineralization proposed as a novel strategy for the
conservation of deteriorated ornamental stone had been reported by
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Rodriguez-Navarro et al (2003). The water cement ratio increases which in


turn indicate the sample to be more permeable had been studied by Abou-Zeid
et al (2003). The deposition of a layer of CaCO3 on the surface of the mortar
specimens resulted in a decrease of water absorption and gas permeability had
been reported by De Muynck et al (2005). The potential drop in migration
testing had been studied by Zhang et al (2005). The performance specification
for durable concrete had been studied by Bickley et al (2006a). Improved
durability of cement mortar/concrete due to MICP had been explained by
Bickley et al (2006b). The CaCO3 deposition on degraded limestone by
Bacillus species had been studied by Dick et al (2006). Nokken & Hooton
(2006) worked towards the permeation of ions to concrete surface.

A decrease in the permeation properties of mortar and concrete


were noticed by De Muynck et al (2007) due to the microbial carbonate
precipitation. It was found that the presence of the bio-deposition treatment
improved the resistance towards chloride penetration with cement mortar
treated by B. Sphaericus. They achieved by 19% decrease of the chloride
migration coefficient by the addition of bacterial biomass compared to
untreated cubes. The surface deposition of CaCO3 crystals decreased the
water absorption by 65-90% depending on the porosity of the specimens,
carbonation rate and chloride migration had been studied by De Muynck et al
(2008) and also they studied that the different durability parameter confirming
biodeposition treatment showed a similar protection towards degradation
process when compared to some of the conventional surface treatment under
investigation. De Belie & Muynck (2008) reported that large reduction of
water permeability could be obtained from B. Sphaericus treated cement
concrete. MICP could greatly improve surface permeability resistance and
resist the attack of the harmful ions on the surface of the cement based
materials protective layer had been studied by Qian et al (2009).
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B. sphaericus and silica gel to repair cracks in concrete had been


observed by Tittelboom et al (2010). Through Bacillus microorganism treated
cement mortar, water absorption was six times less than control had been
noticed by Achal et al (2011a). The Diatomaceous earth specimens with
immobilized bacteria had the lowest water absorption of 30%, which
indicated that the precipitation inside the cracks increased the water
penetration resistance of the cracked specimens had been observed by Wang
et al (2011). The various products such as water repellent, pore blacker and
coating had been applied by Ha et al (2010), Lee et al (2011) to demise the
uptake of water or to produce the chemical deterioration of concrete.

The B. pasteruii incorporated concrete increases the resistance


towards penetration of harmful agents such as chlorides, sulphates and carbon
dioxide into the concrete had been concluded by Patel et al (2011). The
bacteria can help to reduce the water permeability of concrete. Okwadha & Li
(2011) used bacterium S. pasteurii strain ATCC 118519 to create a biosealant
on polychlorinated biphenyls (PCB)-contaminated concrete surface reporting
a reduction on water permeability by 1-5 orders of magnitude. The bacterially
mediated carbonate precipitation on concrete surface reduces capillary water
uptake, leading to the carbonation rate constant to be decreased by 25-40%
had been observed by Li & Qu (2012).

The influence of S. pasteurii bacteria on fly ash concrete had been


studied by Achal et al (2011), Chahal et al (2012). An optimum performance
was achieved for a 105 cells/ml of bacteria concentration. These authors
reported a four time reduction in water absorption and an eight times
reduction in chloride permeability due to calcite deposition and also reported
that the effect of the addition of S. pasteurii with different concentration on
the compressive strength, water absorption and rapid chloride permeability of
fly ash concrete. The surface modification recycled aggregate concrete using
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biodeposition involving the method employing S. pasteurii bacteria had been


presented by Grabiec et al (2012). The biogenic treatments which enhance the
durability of concrete structures had been reported by Achal et al (2013). The
porosity decreased in the presence of B. subtilis significantly by 2.47% and
1.64% on average at 7 days and 28 days of curing respectively had been
reported by Ruoting Pei et al (2013).

One or two dead bacteria inside the carbonate crystals after


dissolution of the crystals with HCl had been noticed (Greenfield 1963).
Later, in 1974, studying aragonite crystals, showed that more than 20 living
bacteria could be found in carbonate crystals up to 90 hours, but after 7 days
they found that they were dead (Krumbein 1974). The rate of acid attack had
been mainly related with the concentration of the solution (Pavlik 1994). It is
known that, cement pastes are more vulnerable to acid attacks than cement
mortars, which incorporates fine aggregate. This result was in conformity
with the result of Pavlik & Uncik (1997). Manoli & Dalas (2001) found that
glutamic acid in supersaturated solution could stabilize CaCO3 polymorph and
precipitate spherical vaterite. Zivica & Bajza (2001) studied that the solubility
of calcium sulfate, calcium chloride and calcium nitrate were 020, 4608 and
5600% (by weight) respectively.

As expected, loss of weight increased with concentration of acids


used which were in conformity with the results. The loss in weight of the
samples that were exposed to sulfuric acid was the smallest because calcium
sulfate (gypsum) is less soluble in water than calcium chloride (CaCl 2) or
calcium nitrate. These results could attribute to the formation of soluble
calcium salts due to the reaction of acids with cements (Zivica 2004).

The glutamic acid could induce the formation of rhombohedral


calcite with a rough surface, studied by Hayashi et al (2008). B. pasteurii act
as a corrosion protection for the surface of cementitious materials had been
30

observed by Qian et al (2009). They also reported that the capillary water
absorption of coefficient of cement paste with water cement ratio of 0.45%
was increased 50-70% by the incorporation of B. pasteurii. Some calcium
salts such as acetate and nitrate could be possibly applied in practice and was
safer for reinforced concrete structures as reported by Tittleboom
et al (2010). The use of B. subtilis bacteria for a cell concentration of 105
cell/ml of mixing water increases the concrete resistance to sulphuric acid
attack, reported by Reddy et al (2010). The aerobic bacteria were hindered
during the development of dome shaped CaCO3 structure on the light weight
concrete specimen, noticed by Kim et al (2013). The same group found that
the density of the CaCO3 crystal of the medium with B. Sphaericus was
higher than that of the cases with other treatment.

2.10 SUMMARY OF LITERATURE REVIEW

The promising results on the use of microorganisms for the


improvement of the strength and durability of materials have drawn the
attention of research groups all over the world. But the work on such
microbial technique was mainly confined in few countries with minimum
attention. A lot of work should be carried out before the technology could be
implemented. The various parameters such as type of mix, concentration of
bacteria, survival of microorganisms in cement mortar, percentage of
enhancement in strength and durability of cement mortar/concrete should be
consolidated. Only limited published work had been described towards
enhancement in the strength and durability of materials using microbes by
their MICP process. Most of the studies based on MICP were carried out to
evaluate the basic parameters like compressive strength, water absorption,
crack remediation and acid attack of mortars/concretes. Different research
groups are working for alternative approaches to obtain a protective layer of
CaCO3 on the surface of the materials.
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2.11 OBJECTIVE OF THESIS WORK

The goal of the present work is to provide an in-depth application


of the different methodologies based on literature to fill the gap of current
research towards the enhancement of the strength and durability of
construction materials. The main objectives of the present doctoral research
work are,

i) to analyze the selected bacteria species under constant pH,


media composition and temperature.
ii) to prepare standard cement mortar specimen for bacterial
species incorporation.
iii) to isolate new urease producing organisms from the cooling
water tower and will be tested for its CaCO3 precipitation.
iv) to investigate suitable calcium source to mix and curing the
microbial cement mortar.
v) to analyze and confirm the precipitation of calcite in the
microbial cement mortar through SEM and X-ray diffraction
studies.
vi) to study the compressive strength of the microbial cement
mortar embedded with different bacterial species.
vii) to analyze the strength characteristics of the microbial cement
mortar.
viii) to study the durability aspects of the microbial cement mortar.
ix) to correlate the mechanical properties and durability
characteristics of the microbial cement mortar.
x) to validate the strength and durability characteristics of the
microbial cement mortars with preceding work.

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