STUDIES ON THE PROPERTIES OF SELF-HEALING

CONCRETE BASED ON MICROBIAL-INDUCED CALCITE

PRECIPITATION BY BACILLUS SUBTILIS JC3

Dr V Srinivasa ReddyProfessor of Civil Engineering

GRIETHyderabad

Research related to Bacterial ConcreteContact me at

vempada@gmail.com

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CONTENTS

Page No.

1.0 Introduction 3

1.1 Research Objectives 8

1.2 Summary of Literature Review 11

1.3 Materials used and Mix proportions 13

1.4 Experimental Investigations 14

1.4.1 Characterization of CaCO3 Precipitation 14

1.4.2 Studies on Mechanical Properties 18

1.4.3 Studies on Durability Properties 20

1.4.4 Temperature Studies 28

1.4.5 Assessment of Crack Repair Efficiency 28

1.5 Important Conclusions 29

1.6 Scope for further studies 33

1.7 List of selected Publications 34

1.8 Selected References 36

Appendix 1: Thesis Organization 38

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1.0 IntroductionAlthough concrete technology has significantly developed in the past decades, fissures, cracks, and steel corrosion remain the potential weakness of any concrete structure. Crack formation is a commonly observed phenomenon in concrete structures. Although micro crack formation hardly affects structural properties, increased permeability due to micro crack networking may substantially reduce the durability of concrete structures due to risk of ingress of aggressive substances particularly in moist environments. Crack repairs can be particularly time consuming and expensive because it is often very difficult to get access to the cracks area to make repairs, especially if they are underground or at a great height. Currently available concrete crack repair systems aims to repair of cracks in aged concrete structures are largely based on environmental unfriendly material systems. This common phenomenon of cracks, if not treated appropriately and instantly, will tend to expand further deep and ultimately increases repair and maintenance costs. One way to circumvent costly manual maintenance and repair is to incorporate an autonomous self-healing mechanism in concrete. Compared with traditional repair methods which follow the procedure of detection, monitoring and repair, the self-healing methods are cheaper over the structure’s life-cycle since the later maintenance would be greatly saved. Concrete has this inherent ability to heal microcracks limiting to 0.2mm width by chemically producing calcium carbonate as a part of hydration. This phenomenon is known as ‘autogenous healing’ or ‘self-healing’ of concrete. Besides autogenous healing, cracks (with width more than 0.2mm) may also be autonomously repaired by incorporating a specific healing agent within the matrix. Various healing agents have been proposed for enhancing the self-healing capacity of concrete. While most healing agents are chemically based inorganic materials, more recently the possible application of bacteria as self-healing agent has also been considered. A

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novel technique for the remediation of damaged structural formations has been developed by employing a selective microbial plugging process, in which metabolic activities promote precipitation of calcium carbonate in the form of calcite. Recently, microbial mineral precipitation resulting from metabolic activities of some specific microorganisms in concrete to improve the overall behavior of concrete has become an important area of research. So the present research is aimed to study the biomimetic process of Bacillus subtilis JC3 developed and cultures in JNTUH Laboratory in the improvement of the mechanical and durability properties of concrete and its related characterization studies.1.0.1 Self-Healing Materials - ConceptsSelf-healing can be defined as the capability of a material to heal (recover/repair) damages automatically and autonomously, that is, without any external intervention. Incorporation of self-healing properties in man-made materials very often cannot perform the self-healing action without an external trigger. Thus, self-healing can be of the following two types:

• Autonomic (without any intervention);• Non-autonomic (needs human intervention/external triggering)

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Currently, self-healing is merely considered as the recovery of mechanical strength through crack healing. However, there are other cases where not only the cracks but also small pores can be filled and healed to have better performance in terms of strength and durability. A composite material ‘manufactured’ to exhibit self-healing healing capabilities due to the release of encapsulated resins or glues, as a result of cracking from the onset of damage, is categorized as having autonomic healing properties. If the healing properties are generic (natural) to that material, then the material could potentially be classed as a smart material, and the corresponding healing process is termed autogenic healing. Cementitious materials have this intrinsic ability to self-repair, because re-hydration of a concrete specimen in water can kick-start, when the water reacts with pockets of un-hydrated cement in the matrix. At times, repair has to be carried out in the areas where it is hazardous for human beings such as nuclear power plants, waste water sewage pipes etc and also in treating surfaces of structures with remarkable heritage significance, self-healing materials could be an ideal alternative.

1.0.2 Problem StatementThe notion of self-healing concrete that can repair itself without human intervention seems to be the matter of science fiction until research conducted on bacteria incorporated concrete realizes the potential of this new field of bio-inspired self-repair innovation. The cracks and fissures in concrete structures not only affect its aesthetics but also have an impact on its strength and durability performance. Research has indicated that a concrete which is low in permeation properties lasts longer without exhibiting any signs of distress and deterioration. Methods currently used for crack remediation often use synthetic polymers which are expensive, incompatible, doubtful long-term performance, needs skilled human assistance and aesthetically

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unpleasant (especially in repairing historic monuments). Because of these drawbacks of conventional surface treatments, attention has been drawn to self-healing bacterial concrete, an alternative technique for the improvement of the durability of concrete. The seepage of aggressive gases and liquids into concrete depends on its permeation characteristics. As the permeation of concrete decreases, its durability performance increases in terms of resistance to physico-chemical degradation. Therefore, permeation of concrete is one of the most significant parameters which governs the concrete durability in aggressive environments. Reinforcement corrosion is one of other key durability problems, mainly when the rebar in the concrete is exposed to the chlorides contributed either from the concrete ingredients or from the surrounding chloride-bearing environment. To thwart this, either the cracks that are formed should be repaired traditionally using epoxy injection/latex treatment or by providing extra reinforcement in the structure to ensure that the crack-width stays within a permissible limit. Use of inorganic synthetic agents such as epoxies/latex for remediation of cracks in these structures introduces a different material system of uncertain long-term performance and furthermore with current steel prices on steep rise, providing extra steel is not also economically feasible. So in search for best crack remediation method without human intervention, many researchers proposed many self healing systems in concrete, each having their own drawbacks. Considering this existing research work an attempt is made to study about biological approach of self-crack healing mechanism through which properties of concrete are enhanced; which resulted in the conception of “Bacterial Concrete”. Bacterial concrete is a new type of smart, sustainable, eco-friendly biomaterial that enhances concrete performance by the microbial deposition of calcium carbonate within its pores through molecular reactions there by refining the pore structure of concrete. 1.0.3 Research Significance

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In the recent past, investigations attempted to study about the application of biomineralization in civil engineering. As a part of those studies, researchers around the world started working on the use of specific bacteria in cementitious materials to self-heal and seal cracks without human intervention. Available literature has not reported any such suitable self-healing system which has features such as long-term compatibility, eco-friendliness, good bonding with surrounding cement matrix, less human intervention, inexpensive and organic in nature. Though it is reported that the use of specific alkaliphilic mineral forming bacteria enhances the properties of cement mortar but there exists little understanding of the effect of bacteria on the mechanical and durability properties of concrete. Hence to address the gaps available in the research, investigations are planned to study the effect of calcite mineral producing bacteria Bacillus subtilis JC3 on the microstructure of the concrete and its impact on its mechanical and durability properties. In the present research work, studies related to characterization of mineral precipitation, permeation properties, resistance to aggressive environment, resistance to corrosion, behaviour at elevated temperature etc of bacteria incorporated concrete has been reported.1.0.3 Microbiologically induced CaCO3 precipitation in Concrete

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Self healing mechanism using biological approach is an emerging topic of research upon which lot of work has been initiated in recent times. In concrete crack-remediation technique by microbiologically induced calcite precipitation (MICP), a highly impermeable calcium carbonate formed in the existing concrete due to microbial activities of the bacteria Bacillus subtilis JC3 (cultured at JNTU) seals the cracks and plugs the pores present in the concrete eventually increasing the strength and durability of concrete. Under prevailing Indian conditions, this process of MICP using environment friendly bacteria to precipitate calcite (CaCO3) during its metabolic chemical reactions is investigated to establish ‘Bacterial Concrete’ as best innovative sustainable self healing biomaterial. This is a special type of concrete in which nutrients associated microorganisms (Bacillus subtilis JC3) are introduced into the concrete during the mixing stage and the influence of its bio-calcification on the properties of concrete are studied.

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1.0.4 Chemical Process in Metabolic activity of Bacillus Subtilis JC3

In nature, microorganisms can induce calcite mineral precipitation (1) through nitrogen cycle either by ammonification of amino acids, (2) nitrate reduction and (3) hydrolysis of urea. In the present adopted peptone based nutrient environment, Bacillus subtilis JC3 is able to precipitate calcium carbonate (CaCO3) in its micro-environment by the process of ammonification, in which amino acids are degraded into ammonium (NH4+) and carbonate (CO32-) ions. The precipitated bio-CaCO3 has a great potential ability to heal concrete cracks and plug the pores present because of its compatibility with the concrete matrix. Bio-mineralization by Ammonification (Ammo acid degradation) comprises of series of complex biochemical reactions. Amino acids released during proteolysis (the process of enzymatic breakdown of proteins by the microorganisms with the help of proteolysis enzymes) undergo de-amination in which nitrogen containing amino (-NH2) group is removed. This process of de-amination which leads to the production of ammonia is termed as "ammonification". Ammonification usually occurs under aerobic conditions (known as oxidative deamination) with the liberation of ammonia (NH3) or ammonium ions (NH4) when dissolved in water. Calcium chloride was used for precipitation of calcium carbonate, while culture medium consists of Peptone: 5 g/lit., NaCl: 5 g/lit., Yeast extract: 3 g/lit. and beef extract to cultivate microorganisms. B.subtilis cell can attract Ca ions (Ca2+), which react with carbonate ions CO32- originating from peptone during oxidative de-amination of amino acids. Simultaneously, ammonia ions NH4+ increase pH value in surrounding medium which improves calcite precipitation efficiency. Once super-saturation is achieved, precipitation of calcium carbonate crystals occurs by heterogeneous nucleation on the bacterial cell wall. The chemical reactions involved in metabolic activity are:

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Ca2+ + B.subtilis Cell → B.subtilis Cell- Ca2+

CH3CH(NH2)COOH (Peptone) + ½O2 ---------> C2H2  + H2CO3 +   NH3

H2CO3 ----------> H+ + HCO3-

NH3 + H2O --------> NH4+ + OH-

B.subtilis Cell- Ca2+ + CO32- → B.subtilis Cell- CaCO3

1.1 Research objectives

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The main research objectives of this research work are to understand the self healing mechanism of bacteria incorporated concrete and the effect of MICP by Bacillus subtilis JC3 on its strength and durability characteristics. To address the gaps in the research, objectives are framed into five categories and can be achieved by conducting the following experimental investigations:

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A. Characterization of CaCO3 Precipitation1. To establish bacteria cell concentration for optimized calcium

carbonate precipitation by measuring the effect of bacteriogenic mineralization on the compressive strength of cement mortar specimens.

2. To perform Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD) and Thermogravimetric (TG) analyses on bacteria incorporated mortar samples to characterize the CaCO3 precipitation by Bacillus subtilis JC3

3. To test for bacteria viability on aged bacteria incorporated mortar samples to check for bacteria survival.

B. Strength related investigations1. To examine the effect of bacteriogenic calcinosis on the compressive

strength, split tensile strength, flexural strength and impact strength of different grades of bacteria incorporated concrete.

2. To assess the concrete quality and integrity using Non Destructive Techniques for different grades of bacteria incorporated concrete.

3. To understand the stress-stain behavior of bacteria incorporated concretes of different grades experimentally and validate it against the analytical stress-strain curves developed by proposed modified stress-strain mathematical model.

4. To evaluate modulus of toughness and modulus of elasticity of bacteria incorporated concretes of different grades from stress-strain curves.

5. To observe the flexural behavior of bacteria incorporated reinforced concrete beams of different grades and to establish load –deflection curves and various parameters such as first crack load, ultimate load carrying capacity, ultimate deflection.

C. Durability related investigations

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1. To investigate the chloride ion penetration ability in bacteria incorporated concrete of different grades.

2. To study the resistance of acid attack and sulphate attack on bacteria incorporated concrete of different grades exposed to different concentrations of HCL, H2SO4, HNO3, H3PO4, Na2SO4, MgSO4

by determining the residual compressive strength and weight loss and also to evaluate their Acid Durability Factors (ADFs) and Acid Attack Factors (AAFs).

3. To learn the corrosion behavior of steel in bacteria incorporated concrete of different grades by accelerated corrosion induced cracking test based on the modified method of constant voltage technique. The “Charge Deterioration Factors” (ChDF) are evaluated to highlight the durability of bacteria incorporated concrete to corrosion.

4. To determine the water penetrability for various grades of bacteria incorporated concrete specimens using the water impermeability tests conducted as per DIN 1048(Part-5) 1991 and MORT&H Clause1761.5 specifications.

5. To find out the water absorption capacity and water absorption rate (sorptivity test) of bacteria treated specimens to estimate the volume of permeable voids and their interconnected pore space distribution.

6. To characterize the pore structure of bacteria incorporated concrete done using Brenauer-Emmett-Teller’s (BET) Nitrogen (N2) nitrogen adsorption method. Porosity of concrete in terms of specific surface area, pore size distribution, and pore volume, was determined using the BET Isotherms.

7. To investigate the resistance to freezing/ thawing and alternative drying/wetting cycles in terms of compressive strength loss and weight loss of bacteria incorporated concrete.

D. Temperature related investigations

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1. To assess residual compressive strength, weight loss of conventional and bacteria incorporated concrete mixes of different grades, exposed to 200ºC, 400ºC and 600ºC temperature for 2 hrs, 4hrs and 6 hrs duration .

2. To evaluate quality of bacteria incorporated concrete exposed to 200ºC, 400ºC and 600ºC temperature for 2 hrs, 4hrs and 6 hrs duration using Non-destructive testing methods.

3. To estimate residual compressive strength and weight loss of control concrete and bacteria incorporated concrete mixes of different grades subjected to 7, 14, 21, 28 thermal cycles of exposed temperatures 50 ºC and 100 ºC.

E. Assessment Of Crack Healing Efficiency1. To assess the crack remediation and strength regain mechanism of

cracked mortar specimens.2. To chemically analyze elemental composition of the microbiologically

induced calcium carbonate precipitate.3. To provide understanding on surface permeability and cost

economics of bacterial concrete.1.2 Summary of literature review An extensive literature review outlined below reveals that the research on bacterial concrete was first pioneered by Venkataswamy Ramakrishnan et al. (2001), Professor Emeritus, South Dakota School of Mines & Technology, USA in remediating cracks and fissures in cement mortar by microbiologically induced calcite (CaCO3) precipitation. Research and development of bacterial concrete was happening for the past 10 plus years in various universities outside India. In India very limited research was done in the area of bioremediation particularly its application in concrete. Willem De Muynck, Nele De Belie , Willy Verstraete, Kim Van Tittelboom et al. at Ghent University, Belgium has done extensive research on microbiologically induced calcite precipitation in construction materials.

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Henk M. Jonkers et al. at Delft University of Technology, Netherlands has studied extensively on the self healing capability of bacteria induced cementitious materials. In India, P. Ghosh, S. Mandal, B.D. Chattopadhyay et al. of Jadavpur University, Kolkata ; V Achal, Abhijeet Mukerjee, Rafat Siddique et al. of Thapar University, Patiala, and M V Seshagiri Rao , Ch Sasikala et al. of Jawaharlal Nehru Technological University Hyderabad are currently doing extensive research on the development of high performance self remediating bacterial concrete. Muynck et al., (2010) developed Biological mortar using microorganism Bacillus cereus. Type of metabolism adopted was oxidative deamination of amino acids and Growth media used was peptone, extract yeast, KNO3, NaCl) + CaCl2.2H2O, Actical, Natamycine. Santhosh et al. (2001) applied this MICP phenomenon in remediating cracks in concrete using Bacillus subtilis. Type of metabolism adopted was Hydrolysis of urea and Growth media used was Nutrient broth, urea, CaCl2.2H2O, NH4Cl, NaHCO3. Belie et al. (2010) also used MICP mechanism in remediating cracks in concrete using Bacillus sphaericus. Type of metabolism adopted was Hydrolysis of urea and Growth media used was yeast extract, urea, CaCl2.2H2O. M V Seshgiri Rao et al., (2010) at JNTU Hyderabad India developed Bacterial concrete using Bacillus subtilis. Type of metabolism adopted was oxidative deamination of amino acids and Growth media used was Peptone, NaCl, Yeast extract and Beef extract. Several bacteria have the ability to precipitate calcium carbonate. These bacteria can be found in soil, sand, natural minerals etc. Jonkers et al.( 2002) used Bacillus cohnii bacteria to precipitate CaCO3. Bacillus pasteurii have been used by V Ramakrishnan, S Bang, Santhosh et al. (2001), while Dick et al. used Bacillus lentus and Bacillus sphaericus. The enhanced performance potential of bacteria B.subtilis is reported by Ch Sasikala et al. (2010) There are a number of species of CaCO3 minerals associated with bacteria, for example calcite by bacillus pasturii, vaterite formation by Acinobacter sp., aragonitic sherulites by

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Deleyahlophila (Rivadeneyra et al., 1996), calcite by Bacillus subtilis (M V Seshagiri Rao et al., 2010) and magnesium calcite spherulites and dumbbells by the slime-producing bacteria, Myxococcus xanthus (González-Muñoz et al., 2000; Holt et al.,1993).

1.3 Materials and Mix proportions1. Ordinary Portland cement of 53 grade, confirming to IS: 12269-1987

was used in this investigation. 2. Locally available clean, well-graded, natural river sand having

fineness modulus of 2.89 conforming to Zone II of IS 383-1970 was used as fine aggregate. Crushed granite angular aggregate of size 20 mm and 10mm nominal size from local source with specific gravity of 2.75 was used as coarse aggregate.

3. Micro silica Grade 92D conforming to IS: 15388 -2003.4. High-performance super plasticizer (SP) based on PCE (polycarboxylic

ether) for concrete conforming IS: 9103-1999.5. Water used for mixing and curing is fresh potable water, confirming

to IS: 3025 – 1986 and IS: 456 – 2000. 6. Aerobic alkaliphilic microorganisms Bacillus subtilis strain with

accession number JC3, a laboratory cultured soil bacterium, which was isolated, deposited, cultured and grown in distilled water at JNTUH Bacteria Discovery Laboratory, was used in preparation of bacterial concrete.

7. The nutrients used for growth of culture are - Peptone: 5 g/lit., NaCl: 5 g/lit., Yeast extract: 3 g/lit.

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8. The grades of concrete used in the present investigation are ordinary grade (M20), standard grade (M40) and high strength grade concretes (M60 and M80). The M20 and M40 mix proportions are designed using BIS method where as high strength grade concretes (M60 and M80) are designed using Entroy and Shacklock's  graphical method and the mix proportions are as follows:

Ordinary grade concrete (M20) 1: 2.27: 3.45: 0.54Standard grade concrete (M40) 1: 1.73: 2.60: 0.42

High Strength Grade(M60) 1:1.25:2.41:0.26(Micro Silica - 6% bwc* ; Superplastisizer -1% bwc*)

High Strength Grade(M80) 1:1.06:1.96:0.23(Micro Silica - 10% bwc* ; Superplastisizer -1.2 % bwc*)

*bwc-by weight of cement

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1.4 Experimental Investigations

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1.4.1 Characterization of CaCO3 Precipitation

1.4.1.1 Bacterial cell concentration for maximum CaCO3 precipitationTo determine the bacterial cell concentration of Bacillus subtilis JC3 for optimum CaCO3 precipitation the standard cement mortar cubes 70.7mm x 70.7mm x 70.7mm are cast with various bacterial cell concentrations (103 cells/ml, 104 cells/ml, 105 cells/ml, 106 cells/ml, 107

cells/ml of mixing water) and tested for compressive strength as per IS: 516-1999. The compressive strength is found to be highest at cell concentration of 105 cells /ml of mixing water. It was observed that increase of compressive strength of cement mortar induced with cell concentration of 105 cells per ml is 17.88% at 28 days. So this is taken as optimum cell concentration to be used for further study to investigate the effect of bacteria on properties of concrete. It was noted that pores of bacteria induced cement sand matrix are filled up by densely grown calcium carbonate. This dense growth of calcite precipitate refines the pore structure of cement mortar specimens resulting in the strength improvement. For cell concentration above 105 cells per ml of mixing water, the strength decreased due to the presence of total solids and suspended matter in the form of biomass, exceeding the permissible limits specified as per IS 456, interfering the cement matrix integrity.

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Nil 103 104 105 106 107

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Fig 1: Compressive Strengths at various Bacteria Cell Concentrations

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1.4.1.2 Scanning Electron Micrograph (SEM) Investigations

Scanning Electron Microscope (SEM) analysis is made on the samples of 28 day old bacterial cement mortar specimens and control mortar specimens (without bacteria). In bacteria induced cement mortar, formation of dense CaCO3 precipitation spreading over the pores present inside, with rod-shaped impressions housed by Bacillus subtilis JC3 can be observed in Fig 2. It is most likely that the microbial activity of Bacillus subtilis JC3 has precipitated dense CaCO3 which may have caused the increase in 28 day compressive strength due to modification of pore size distribution.

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Cell Concentration –Nil (Control Specimen) Cell Concentration – 105/ml (Optimum)

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Fig 2: Magnified SEM Micrographs -Hydrated Structure of Cement-sand Mortar without Bacteria and with Bacteria incorporated

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1.4.1.3 X-Ray Diffraction Analysis (XRD)

Fig 3 depicts the profiles of XRD spectra of control and bacteria incorporated cement mortar samples. From the XRD spectrums, bacteria incorporated cement mortar sample confirms the presence of high amount of calcite mineral. This can be attributed to the copious deposition of calcite (CaCO3) in bacteria induced samples by Bacillus subtilis JC3 during its microbial activity. This deposition of CaCO3 in the pores will maximize the packing density of cement mortar consequently has great impact on the strength produced.

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Fig 3: XRD spectra of control and bacteria incorporated mortar samples

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1.4.1.4 Thermogravimetric (TG) Analysis

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TGA analysis performed on powdered bacteria incorporated cement mortar sample showed an extreme loss of weight at temperature range of 500–700°C confirming the presence of high amount of CaCO3. The descending TGA thermal curve indicates a weight loss occurred. This presence of large amount of CaCO3 can be credited to the precipitation of calcite (CaCO3) in bacteria induced samples by Bacillus subtilis JC3 during its microbial activity.

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0 100 200 300 400 500 600 700 800 900 100075

80

85

90

95

100

105 Sample without bacteriaSample with bacteria

Temperature C

Wei

ght %

Expulsion of Evaporable water

Hydration of the hydrate calcium

silicate

Decomposi-tion of

Ca(OH) 2

CaCO3 Disin-tegrates

Decomposition of hydrate cal-

cium silicate

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0 100 200 300 400 500 600 700 800 900 10000

0.05

0.1

0.15

0.2

0.25

0.3Sample without bacteriaSample with bacteria

Temperature C

Chan

ge in

Wei

ght (

%/

C)

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Fig 4: TGA Results for cement mortar specimens with and without bacteria showing weight loss and Change in weight loss per oC

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1.4.1.4 Bacteria Viability Test

Fig 5 shows Phase contrast microscopic pictures reveal that Bacillus subtilis JC3 spores were produced within vegetative cells (endospores) and white calcium carbonate precipitation (calcite crystals) is formed around its cell. Vegetative Bacillus subtilis JC3 cells were detected viable even after 365 days in mortar samples. This validates that the bacteria Bacillus subtilis JC3 is alkaliphilic and endospore forming soil microorganism.

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Fig 5: Phase contrast microscopic images shows calcite crystals

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1.4.2 Mechanical Properties

1.4.2.1 Strength StudiesTo study the effect of MICP on compressive, split-tensile, flexural and impact strengths of control concrete specimens (M20, M40, M60 and M80 grades) and Bacillus subtilis JC3 incorporated concrete specimens (M20B, M40B, M60B and M80B grades) are cast with optimized cell concentration of bacteria (105 cells/ml). The results of strength studies carried out on specimens of 28, 60, 90,180 and 365 days age and are shown in Fig. 6.

M20

M20B M4

0M4

0B M60

M60B M8

0M8

0B

020406080

100120140160 28 days

60 days90 days180 days365 days

Grade of Concrete

Com

pres

sive

Str

engt

h (M

Pa)

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Fig 6: Compressive Strength development of a normal concrete and bacteria incorporated concrete

1.4.2.2 Stress-Strain Behavior and development of Analytical Stress-Strain ModelStress-strain behavior of control concrete specimens (M20, M40, M60 and M80 grades) and Bacillus subtilis JC3 incorporated concrete specimens (M20B, M40B, M60B and M80B grades) are studied experimentally and validated them against the analytical stress-strain curves developed by proposed new stress-strain mathematical model. After obtaining the stress-strain behavior of controlled and bacterial concrete experimentally, empirical equations are developed to represent uni-axial stress-strain behavior of controlled and bacterial concrete mixes of various grades considered. Appropriate analytic stress-strain mathematical model is developed that can capture the real (observable) stress-strain behavior of bacteria induced concrete. After developing empirical equations for stress-strain curves of controlled and bacterial concrete, theoretical values of stresses are calculated at different values of strains in concrete based on the developed empirical equations. These theoretical stress-strain curves are compared with experimental stress-strain curves and found that, theoretical stress-strain curves have shown good correlation with experimental stress-strain curves for all grades of controlled and bacterial concrete mixes.

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0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.00350

20

40

60

80

100

120

M20Polyno-mial (M20)M20B

Strain

Stre

ss (M

Pa)

Fig 7: Stress- Strain Curves1.4.2.3 Flexural behavior of bacteria incorporated RCC beamsThe flexural behavior of reinforced bacterial concrete beams of M20, M40, M60 and M80 concrete grades was investigated by testing them under symmetrical two-point flexural loading under strain rate control as per IS:9399 – 1979 . Deflections at the central point, the ultimate load (Peak load), and first crack width are measured and development of crack pattern are observed for various grades of bacterial and reference beams. The ultimate flexural strengths of bacterial concrete beams are observed to be higher than normal concrete beams for all grades and at all ages considered for the investigation. The results were presented and discussed in the thesis.1.4.3Durability Properties 1.4.3.1 Rapid Chloride Ion Permeability TestTo investigate chloride ion permeability of bacteria incorporated concrete of M20, M40, M60 and M80 concrete grades, the rapid chloride permeability test method designated in ASTM C 1202(1997) is adopted.

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Char

ge P

asse

d (C

oulo

mbs

)

Chlo

ride

ion

Perm

eabi

lity

as p

er

ASTM

C12

02

Char

ge P

asse

d (C

oulo

mbs

)

Chlo

ride

ion

Perm

eabi

lity

as p

er

ASTM

C12

02

Char

ge P

asse

d (C

oulo

mbs

)

Chlo

ride

ion

Perm

eabi

lity

as p

er

ASTM

C12

02

Char

ge P

asse

d (C

oulo

mbs

)

Chlo

ride

ion

Perm

eabi

lity

as p

er

ASTM

C12

02

Age 28 days 60 days 90 days 180 days

Concrete without Bacteria

M20 2419 Moderate 2213 Moderate 2100 Moderate 2089 Moderate

M40 2008 Moderate 1991 Low 1817 Low 1812 Low

M60 1022 Low 997 Very Low 943 Very Low 941 Very Low

M80 995 Very Low 961 Very Low 937 Very Low 930 Very Low

Concrete with Bacteria

M20 367 Very Low 351 Very Low 327 Very Low 321 Very Low

M40 238 Very Low 222 Very Low 202 Very Low 199 Very Low

M60 173 Very Low 159 Very Low 96 Negligible 93 Negligible

M80 164 Very Low 144 Very Low 89 Negligible 84 Negligible

1.4.3.2 Acid and Sulphate attack resistanceThe effect of aggressive chemical environment on compressive strength loss and weight loss of M20, M40, M60 and M80 bacterial and controlled grade concretes exposed to different concentrations (3%, 5% and 10%) of various acids (HCL, H2SO4, HNO3, H3PO4, Na2SO4, MgSO4) is studied. Corresponding Acid Durability Factors and Acid Attack Factors are evaluated. The results were discussed and presented in the thesis.

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For determining the resistance of concrete specimens to aggressive environment such as acid attack, the durability factors and acid attack factors are proposed by the author, with the philosophy of ASTM C 666 – 1997 as the basis.

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The “Acid Durability Factors” (ADF) can be designed as follows.

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ADF = Sr (N/M)

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where, Sr = relative strength at N days, ( % )

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N = number of days at which the durability factor is needed.

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M = number of days at which the exposure is to be terminated.The extent of deterioration at each corner of the struck face and

the opposite face is measured in terms of the solid diagonals (in mm) for each of the two cubes and the “Acid Attack Factors” (AAF) per face is calculated as follows.

AAF = (Loss in mm on eight corners of each of 2 cubes) / 4Durability studies carried out in the investigation through acid attack test M20, M40, M60 and M80 concrete grades revealed that Bacterial Concrete is more durable in terms of “Acid Durability Factors” and less attacked in terms of “Acid Attack Factors” than the controlled concrete. The results were presented and discussed in the thesis.

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1.4.3.3 Accelerated Corrosion TestThe corrosion behavior of steel in controlled and bacterial concrete of M20, M40, M60 and M80 concrete grades are estimated by accelerated corrosion induced cracking test based on the modified method of constant voltage technique. Specimens for accelerated corrosion cracking studies are beams having the reinforcement of 10 mm diameter HYSD bar at different effective covers ranging from 10 mm to 40 mm at all four corners of each beam. The specimens to be tested are kept in natural sea water in glass tub of dimension 750 mm X 300 mm X 300 mm. All the extended reinforcements are connected to the power supply parallelly and anodic current is impressed at a constant voltage of 20 V. From the current measurements taken at an interval of 12 hours in accelerated corrosion cracking test, the Charge Deterioration Factors (ChDF) are proposed by the author based on the philosophy of ASTM C 666 – 1997. In the present investigation, the author derived the “Charge Deterioration Factors” (ChDF) and presented as follows.

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Charge Deterioration Factor (ChDF) = Cr (N/M)

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where, Cr = Relative Charge, ( % )

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N = Number of hours at which Ch.D.F is required.

M = Number of hours which is taken as referenceThe results were presented and discussed in the thesis.1.4.3.4 Water permeabilityConcrete water permeability test is conducted as per IS 3085 and IS DIN 1048. Since water permeability test as per IS 3085 is not giving correct results for high strength grade concretes so water permeability test as per IS DIN 1048 is conducted. The coefficient of permeability (k, in m/sec) as per IS 3085 is computed at 28 and 90 days as shown in Fig 8.

M20 M20B M40 M40B M60 M60B M80 M80B0

0.5

1

1.5

2

2.528 DAYS90 DAYS

Grade of Concrete

Coeffi

cien

t of

per

mea

bil-

ity

x 10

e-9

m/s

ec

Fig 8: Variation of Coefficient of water permeability with Grade of Concrete

at 28 days and 90 days age

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As per DIN 1048(Part 5):1991 specifications and MORT&H (Ministry of Road transport & Highways) 4th Revision Cl.1716.5, This test gives a measure of the resistance of concrete against the penetration of water exerting pressure. Depth of penetration for various grades of bacteria incorporated concrete specimens is shown in Fig 9.

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M20 Controlle

d

M20 Bacteria

l

M40 Controlle

d

M40 Bacteria

l

M60 Controlle

d

M60 Bacteria

l

M80 Controlle

d

M80 Bacteria

l0

5

10

15

20

25

20

4

14

3

8

1

4

1

Grades of Concrete

Dep

th o

f wat

er p

enet

ratio

n(m

m) a

t 28

days

0

5

10

15

20

25

20

4

14

3

8

1

4

1

Grades of Concrete

Dep

th o

f wat

er p

enet

ratio

n(m

m) a

t 90

days

Fig 9: Depth of penetration for various grades of concrete at 28 and 90 days age

1.4.3.5 Water Absorption Capacity and PorosityTests are conducted as per ASTM C642 -13 “Standard Test Method for Density, Absorption, and Voids in Hardened Concrete” to measure volume of permeable voids and the maximum water absorption capacity.

0

1

2

3

4

5

6M20 M40 M60M80 M20BM40BM60BM80B

time (min)

amou

nt o

f wat

er a

bsor

bed

(%)

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Fig 10: Plot showing amount of water absorption with time for different grades of controlled and bacterial specimens

1.4.3.6 Sorptivity Test

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The objective of this study is to determine the sorpitivity of bacteria incorporated concrete as per ASTM C1585 -13 “Standard Test Method for Measurement of Rate of Absorption of Water by Hydraulic-Cement Concretes”. The gain in mass per unit area over the density of water (I) is plotted against the square root of the elapsed time (√t). The rate of water absorption or sorptivity (k), is the slope of I- √t graph (m / min1/2

or kg/ m2 / √ min).

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0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.000

0.0010.0020.0030.0040.0050.0060.0070.0080.009 M2

0Linear (M20)M40

t min

I x 1

0-3

m

Fig 11: Plot between I- √t to calculate sorptivity coefficient (k)

62

63

M20 M40 M60 M800

0.02

0.04

0.06

0.08

0.1

0.12

0.14

ControlledBacterial

Grade of Concrete

Sorp

tivity

Coe

ffici

ent

(k)

x 10

-3m

/min

0.5

64

Fig 12: Variation of sorptivity of controlled and bacterial specimens for different grades

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1.4.3.7 Pore Structure Analysis using BET Nitrogen (N2) Adsorption methodPorosity of concrete in terms of specific surface area, pore size distribution, and pore volume, was examined using the Brenauer-Emmett-Teller BET nitrogen adsorption method based on DIN 66131 “Determination of Specific Surface Area of Solids by Gas Adsorption using the Method of Brunauer, Emmett and Teller (BET)”.

19.121

3.386

5.084

3.097 3.189 3.095 3.096 3.083

0

5

10

15

20

25

M20 M20B M40 M40B M60 M60B M80 M80B

Grades of Controlled and Bacterial Concretes

Aver

age

Pore

Dia

met

er (n

m)

Fig 13: Pore diameters of controlled and bacteria incorporated concretes of different grades

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0.0161

0.0071

0.0137

0.00570.0052

0.00420.0047

0.0038

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

M20 M20B M40 M40B M60 M60B M80 M80B

Grades of Controlled and Bacterial Concretes

Tota

l por

e vo

lum

e (c

c/g)

Fig 14: Total pore volume of controlled and bacteria incorporated concretes of different grades

1.6

0.7

1.37

0.570.52

0.420.47

0.38

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

M20 M20B M40 M40B M60 M60B M80 M80B

Grades of Controlled and Bacterial Concretes

Poro

sity

( % )

Fig 15: Porosity of controlled and bacteria incorporated concretes of different grades using BET method

1.4.3.8 Freeze and Thaw Durability Studies

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Freezing and thawing was performed according to modified freeze –thaw test based on ASTM C 666. The experimental study of bacteria incorporated concrete specimens subjected to different cycles of freeze-thaw was reported. After 28 days of curing, the specimens were surface dried and then they were kept in freezer at a temperature of -14°C for 24 hours. After 24 hours of freezing, the specimens were taken out and kept at room temperature for 24 hours. This completes one cycle of freezing and thawing. The influences of freeze-thaw cycles on the mechanical properties the compressive strength, tensile strength, weight and Ultrasonic pulse velocity were measured after 0, 7, 14 and 28 cycles of freeze-thaw. The test results were presented and discussed in the thesis.1.4.3.9 Alternate Wetting and Drying CyclesThe specimens of dimensions 150 X 150 X 150mm were subjected alternate wetting and drying for 7, 14 and 28 cycles. Compressive strength, weight loss and Ultrasonic pulse velocity measurements are evaluated when subjected to 7, 14 and 28 cycles of alternate wetting and drying. One cycle of alternate wetting and drying consists of one day drying at room temperature and one day immersing in water. After every wetting and drying cycle, specimens were weighed to find the percentage loss of weight. These specimens were tested for their respective strengths using destructive and non-destructive methods. The test results were presented and discussed in the thesis.1.4.4 Temperature StudiesControlled concrete and bacterial concrete cube specimens of ordinary grade (M20), standard grade (M40) and high strength grade (M60 and M80) of size 100 x 100 x 100 mm were cast and are subjected to different elevated temperatures of 200ºC, 400ºC and 600ºC for exposure periods of 2 hrs, 4hrs and 6 hrs in muffle furnace. Residual compressive strength and weight loss are evaluated as per IS 516-1959. To study the effect of thermal cycles on compressive strength and weight, cubes of

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size 100 x 100 x 100 mm are subjected to 7, 14, 21, 28 thermal cycles at temperature of 50 º C and 100 ºC. One thermal cycle constitute a heating period of 8 hours and subsequent cooling (in air room temperature) period of 16 hours. The test results were presented and discussed in the thesis.1.4.5 Assessment of Crack Healing Efficiency

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1.4.5.1 Crack Remediation and Strength Regain

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Standardized cuts of width 3mm and depth 20 mm were realized in Cement mortar cubes as shown in Fig 15, to simulate cracks. One set of simulated cracked cubes are filled up with Indian standard grade II sand (1mm to 0.5 mm) mixed with water and another set mixed with bacterial culture. The specimens were tested for the compressive strengths after 28 days.

71

72

73

Fig 16: Simulated standard cracks

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Strength gain due to biogenic treatment is mainly due to chemical bonding between CaCO3 precipitated by bacterial cells and sand particles which consolidate the crack space. The gain in strength is found to be 22.6%.

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1.4.5.2 Cost Evaluation

Nutrients broth powder required to prepare one liter of bacterial water is 13 gms. One liter of nutrients mixed bacterial culture costs Rs 60. In this research work, nearly 125 liters of nutrients mixed bacterial culture was used costing nearly 7500 rupees. 1.5 Important Conclusions

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Based on the results and key findings during the experimental investigations, the following conclusions are drawn:

1. The cement mortar cube specimens which contained cell concentration of 1x 105 bacterial cells/ml of mixing water was found to attain higher compressive strength as compared to the control specimen. This improvement of compressive strength is attributed to the deposition of CaCO3 precipitate formed by Bacillus subtilis JC3 which modifies the concrete pore structure by plugging the voids /or the pores within cement–sand matrix, as part of its metabolic activity.2. Characterization of calcite crystal precipitation done using various nano-characterization techniques such as Scanning Electron Microscope (SEM), X-ray diffraction (XRD) and Thermo-gravimetric analysis (TGA) confirms the presence of CaCO3 formed due to complex metabolic mechanism of nitrogen cycle by Bacillus subtilis JC3. 3. It was observed that with the addition of bacteria there is significant increase in compressive strength, split-tensile strength, flexural strength and impact strength of concrete due to pore refinement by bacteriogenic calcite mineral plugging in bacteria induced concrete.4. From the observations made from stress-strain curves, the bacteria incorporated concrete mixes have shown improved stress values for the same strain levels compared to that of controlled concrete mixes.

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Calcite crystal precipitation in bacterial concrete increases the resilience and strain relieving capacity. This resilient character provides the excellent impact resistance and dissipates dynamic loading better than conventional concrete. So toughness or energy absorption capacity of bacteria incorporated concrete grades is high when compared to same grades of conventional concrete. Modulus of Elasticity (E) is comparatively more for all grades of bacteria induced concrete than the controlled concrete. This feature is attributed to the dense pore structure of bacteria incorporated concrete.

5. The ultrasonic pulse velocity values obtained from bacteria induced concrete were greater than 4.5km/sec which denoted that all grades of bacteria induced concrete are classified as excellent concretes in terms of strength and quality where as the controlled concrete is rated as moderate to excellent.6. Due to mineral precipitation in reinforced bacteria incorporated beams, internal micro cracking in the cementitious matrix is prevented through plugging up of cracks by continuous calcite mineral precipitates of Bacillus subtilis JC3. Hence bacteria induced concrete will have dense micro structure with low pore fraction and reduced pore size due to which fatigue strength is increased which in turn increases the first crack load and the ultimate load carrying capacity in case of flexural loading. 7. Chloride ion penetration resistance is more in bacteria incorporated concrete when compared to conventional concrete due to voids being filled with calcite crystals precipitated by bacteria reducing the porosity of the concrete.8. It is found that bacteria incorporated concrete specimens perform better in terms of resistance to acid attack and sulphate attack than conventional concrete specimens due to its improved impermeability. The mineral precipitation in bacteria incorporated concrete reduces the

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interconnectivity of the pore structure by decreasing the pore size which is directly related to durability.9. The “Charge Deterioration Factors” are less for bacteria incorporated concrete than the controlled concrete which indicates that reinforced bacteria incorporated concrete offer better resistance to corrosion than normal reinforced concrete because pores and micro cracks are clogged by calcite minerals.10. Water permeability and Water Absorption Capacity (WAC) of bacteria incorporated concrete specimens reduces by nearly 50 to 80% for low to high grade concretes when compared to controlled concrete. 11. Volume of permeable pores (VPV) of bacteria incorporated concrete specimens are reduced by nearly 50-65%. The possible reason for this is calcite mineral precipitation in the pores reduced the average pore radius of concrete by blocking the large voids (pore discontinuity) in the hydrated cement paste. So bacteria treated concrete samples gave the lower sorptivity (0.091 to 0.041 mm/min0.5) compared to control concrete sorptivity (0.124 to 0.055 mm/min0.5 ). 12. The bacteria incorporated concrete specimens has improved resistance for freeze-thaw cycles and alternate wetting and drying cycles because improved microstructure of concrete induced with bacteria relaxes the expansion / contraction stresses. 13. Bacteria incorporated concrete specimens exhibited much better resistance to elevated temperature than controlled concrete up to 500 ºC. Above 500 ºC, both controlled and bacteria incorporated concretes behave identically due to decomposition of calcite present.14. Bacteriogenic mineral precipitation, contributed to the bonding and regaining of strength of the already cracked specimens. This strength recovery can be attributed to chemical bonding between CaCO3

precipitated by bacterial cells and sand particles which consolidate the crack space.1.6 Summary

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The incorporation of bacteria Bacillus subtilis JC3 found to increase concrete’s compressive strength, split tensile strength and flexural strength in all grades. This improvement in strengths are due to calcite mineral deposition within the pores of cement–sand matrix, making the concrete dense by modifying the pore structure which is characterized using Scanning Electron Micrograph (SEM) and X-ray diffraction analyses. Thermogravemetric analysis confirms the presence of calcite mineral in bacteria incorporated samples. Durability studies showed that, the bacteria incorporated concrete perform better when subjected to aggressive chemical attack, sulphate attack test and sea water attack. Bacteria induced concrete has shown higher resistance against the chloride ion penetration than conventional concrete due to minimum interconnecting voids present. It was established that bacteria incorporated concrete will have the higher resistance to corrosion than conventional concrete. Bacterial concrete was less permeable than conventional concrete due to improved pore structure as a result of precipitation of calcite crystals. Water Absorption Capacity (WAC) of bacteria incorporated concrete specimens is reduced significantly when compared to controlled concrete specimens due to reduction in volume of permeable voids. Pore structure analysis using BET Nitrogen adsorption test designate that there is significant decrease in total pore volume and average pore diameter of bacteria incorporated concrete. The resistance to freezing/thawing and drying/wetting of bacteria incorporated concrete is found to be considerably superior to conventional concrete. Bacteria incorporated concrete specimens exhibited much better resistance to elevated temperatures. To sum up, bacteriogenic calcite mineral precipitation using Bacillus subtilis JC3 mechanism can be used effectively in improving the properties of concrete. This positive impact on both strength and durability properties can be attributed to the activity of Bacillus subtilis JC3 in development of dense and refined microstructure of bacteria incorporated concrete. This

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application of biomineralization in concrete can be considered as a best environmental friendly bio-based durable self -crack remediation technique for Indian conditions.1.7 Scope for further studies

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On-going research in JNTUH laboratories investigates the possibility to use this Calcium carbonate precipitation by bacteria in practical civil engineering applications.

1. To study the micro structure of bacteria induced concrete using various nano-characterization techniques to control the healing mechanism of concrete.

2. To study exhaustively on elasticity, drying shrinkage, creep, thermal and electrical properties of bacteria incorporated concrete.

3. To predict critical conditions for bacterial self healing in concrete4. Application of bio-mineralization can also be tested and evaluated

in concretes with different cementitious materials such as Self compacting concrete and fly-ash based concrete.

5. Study the effect of microbial carbonate precipitation in other cementitious materials.

6. Intensive microbiological studies are to be carried out to characterize the calcium carbonate precipitation

7. To investigate the factors responsible for the quantity and rate of mineral precipitation in concrete.

8. Studies can be extended to self-healing mechanism due to deposition of minerals on the surface of cracked concrete and others factors such as extent of depth and rate of mineral growth in cracks.

1.8 List of selected publications by Scholar

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Journals:

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(a) International

1. “Permeation Properties of Bacterial Concrete”, IOSR Journal of Mechanical and Civil Engineering (IOSR-JMCE) ISSN: 2278-1864, March 2013 Issue pp. 08-16. (DOI No: 10.9790/1684-0560816)2. “Mathematical Model for Predicting Stress-Strain Behaviour of Bacterial Concrete” - International Journal of Engineering Research and Development, e-ISSN: 2278-067X, p-ISSN: 2278-800X, www.ijerd.com Volume 5, Issue 11 (February 2013),pp. 21-29 3. “Strength Enhancement of Cement Mortar Using Microorganisms - An Experimental Study” - International Journal of Earth Sciences and Engineering (IJEE) - CAFET-INNOVA Technical Society, ISSN no: 0974-5904 Volume 04 N0: 06 Spl. October 2011 pp. 933-936 (b) National4. “Quantification and Characterization of CaCO3 Precipitation by Bacillus subtilis JC3 in Bacterial Concrete” – Indian Concrete Institute Journal (Estd:1982) Oct-Dec 2013 pp.25-27 5. “Bioengineered Concrete - A Sustainable Self-Healing Construction Material” - Research Journal of Engineering Sciences - International Science Congress Association -ISSN 2278-9472 Volume 2 Issue 6 June 2013 pp.45-51.Conferences: (a) International1. “Bio-Inspired Solutions for Durable Concrete” – International Conference on Emerging Trends in Civil Engineering (ICETCE 2014) – VNRVJIET – 6th -8th January 2014 Hyderabad pp.158-167.

2. “Studies on Stress-Strain Behaviour of Bacterial Concrete” - The 2nd International Conference on Advancement in Engineering and Management - Royal Institute Of Technology & Science- ICAEM-2013, Feb 2013, pp.27-28

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3. “Studies On Corrosion Resistance Of Bacterial Concrete (B. Subtilis)” -International Conference on Advances In Materials And Techniques For Infrastructure Development AMTID 2011, NIT Calicut, Sep 28-30, 2011 Paper No:S052 pp.55 4. “Strength Enhancement Of Cement Mortar Using Microorganisms - An Experimental Study” - ACE – 2011- International Conference on Advances in Civil Engineering October 21 – 23, 2011 KLU Vijayawada, Andhra Pradesh. ISSN no: 0974-5904, pp.933-936. (b) National

1. “Studies on the flexural behavior of Bacteria incorporated reinforced concrete beams” - National Conference on Recent Advances in Structural Engineering (RASE-2013) 12-14 September 2013, Osmania University College of Engineering Hyderabad, pp.302-308.

2. “Strength and Chloride Ion Permeability Studies Of Bacterial Concrete” - National Conference on Sustainable Construction Materials and Technologies (SCMAT) Mar 15-16, 2013 NIT Warangal, pp.175-184.

3. “Development Of Bacteria-Based Self-Healing Concrete” - National Conference on Recent Advances in Geo-Sciences, Engineering & Technology (NCRAGE) 20th & 21st December 2012 JNTU Kakinada, pp.485-491.

4. Crack Repair in Concrete Structures Using Microorganisms” - Proceeding of National Conference on IPCWM2011, SRIT Coimbatore (20 -21 April 2011), ISBN 81-903838-4-1, pp.1.7-1.17.

Communicated

1. “Performance of microbial concrete developed using Bacillus subtilis JC3” - Journal of The Institution of Engineers (India)- Series A- IEI

85

Springer.( Estd:1920) (communicated on 23 May 2013)(Status: Under Review) ( Manuscript Number IEIA-D-13-00067)

1.9 Selected ReferencesReferred Research Papers:1. De Muynck.W, De Belie.N, Verstraete.W. “Improvement Of Concrete Durability With The Aid Of Bacteria” Proceedings Of The First International Conference On Self Healing Materials April 2007, Noordwijk Aan Zee, The Netherlands.2. Henk M. Jonkers and Erik Schlangen - “Crack Repair By Concrete-Immobilized Bacteria” -Proceedings of the First International Conference on Self Healing Materials 18-20 April 2007, Noordwijk aan Zee, The Netherlands - Delft University of Technology.3. J.Y. Wang, K. Van Tittelboom, N. De Belie And W. Verstraete. “Potential of Applying Bacteria To Heal Cracks In Concrete”. Second International Conference On Sustainable Construction Materials And Technologies 2010, ISBN 978-1-4507-1490-7.4. Kim Van Tittelboom, Nele De Belie, Willem De Muynck, Willy Verstraete – “Use of bacteria to repair cracks in concrete” - Cement and Concrete Research 40 (2010) 157–166.5. M V Seshagiri Rao, Ch Sasikala, S Sunil Pratap Reddy, “Studies on the use of bacteria to improve the performance of cement mortar”, Annual Technical Session on 24th October, 2007 at “The Institute of Engineers (India), Hyderabad.6. P Ghosh, S Mandal, S Pal, G Bandyopadhyaya, B D Chattopadhyay. “Development Of Bioconcrete Material Using An Enrichment Culture Of Novel Thermophilic Anaerobic Bacteria”. Indian Journal of Experiment Biology Vol. 44, April 2006, pp. 336-339.7. R. Siddique, V. Achal, M. Reddy, and A. Mukherjee, "Improvement in the compressive strength of cement mortar by the use of a

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microorganism - Bacillus megaterium," in Excellence in Concrete Construction through Innovation. M. C. Limbachiya and H. Kew, Eds., United Kingdom: Taylor & Francis, 2008, pp. 27-30.8. S. Ghosh, M.Biswas, B.D.Chattopadhyay, S.Mandal-“Microbial activity on the microstructure of bacteria modified mortar” -Cement & Concrete Composites- 31 (2009) pp 93–98.9. Sookie S. Bang and V. Ramakrishnan- “Microbiologically-Enhanced Crack Remediation (MECR)” - South Dakota School of Mines and Technology , 501 E. Saint Joseph Street - Rapid City, SD 57701.10. Varenyam Achal, Abhijit Mukherjee, and M. Sudhakara Reddy-“Microbial Concrete: A Way to Enhance Durability of Building Structures” -ISBN 978-1-4507-1490-711. Victoria S. Whiffin-“Microbial CaCO3 Precipitation for the production of Biocement” –PhD Thesis 2004, School of Biological Sciences & Biotechnology Murdoch University, Western Australia.12. Willem De Muynck, Nele De Belie, Willy Verstraete – “Microbial carbonate precipitation in construction materials: A review” -Ecological Engineering 36 (2010) 118–136.

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Referred Books:

1. “Properties of Concrete”, by Neville. A. M. – Longman, Pearson Education Asia Pvt.Ltd, First Indian reprint 2000.

2. “Concrete – Microstructure, Properties and Materials”, by P. K. Mehta and Paulo J. M. Monteiro, Indian Concrete Institute, Chennai, 1997.

Referred Codes:1. IS: 3085 – 1965 “Method of test for permeability of concrete”

2. DIN 1048-1972 “Test Methods for Concrete – Impermeability to Water”

3. ASTM C1202,“Standard Test Method for Electrical Indication of Concrete’s Ability to Resist Chloride Ion Penetration”

4. ASTM C666 “Standard Test Method for Resistance of Concrete to Rapid Freezing and Thawing”

5. ASTM C642 - 13 “Standard Test Method for Density, Absorption, and Voids in Hardened Concrete”

Appendix 1: Thesis Organization

The main thesis is outlined as following:

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Chapter 1: This chapter gives understanding about self healing approaches, bio-mineralization, bacteria incorporated concrete and its self healing mechanism.

89

Chapter 2: Presents research focus, objective and experimental investigations of the present study.

90

Chapter 3: Presents the review of literature on bacterial concrete

91

Chapter 4: Discusses about materials and their properties along with design mix proportions of ordinary (M20), standard (M40) and high strength (M60 and M80) grade concretes.

92

Chapter 5:Characterizes CaCO3 precipitation using microstructure studies such as Scanning Electron Microscopy (SEM), X-ray diffraction (XRD), Thermo-gravimetric (TG) Analyses.

93

Chapter 6: Presents test methodologies and discusses about studies on mechanical properties of bacterial concrete

94

Chapter 7: Presents test methodologies and discusses about studies on various durability properties of bacterial concrete

95

Chapter 8:Discusses studies on bacterial concrete subjected to elevated temperatures and thermal cycles

96

Chapter 9: Evaluates the crack healing efficiency and strength recovery mechanism

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Chapter 10: Gives summary of conclusions which has emerged from the present investigations.

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Finally, Future research perspectives, list of selected publications by scholar and bibliography follow.