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i
INFLUENCE OF ENTEROCOCCUS FAECALIS AND BACILLUS SP WITH
ADDED CALCIUM LACTATE ON ENGINEERING CONCRETE PROPERTIES
AND ENHANCEMENT OF SELF HEALING IN CONCRETE UNDER
UNSTERILLIZED CONDITION
ANNEZA LUIS HII
A thesis submitted in
fulfillment of the requirement for the award of the
Degree of Master of Civil Engineering
Faculty of Civil and Environmental Engineering
Universiti Tun Hussein Onn Malaysia
2017
iii
For my beloved parents, siblings and friends
iv
ACKNOWLEDGEMENT
for the strength to achieve my goals to obtain a Master's I would like to thank the Lord
Degree. A sincere appreciation that goes out to my supervisor Assoc. Prof. Dr. Mohd
hman. for all the guidancesupervisor Assoc. Prof. Dr. Norzila Ot-Irwan Juki and Co
earch. Last but not least, my loving family whosupport throughout my resand
encouraged me to pursue my studies. Provided me with moral and support when i
need it the most. My friends, whom fight side by side with me. Thank for always being
there
Thank you
v
ABSTRACT
Concrete is one of the most used construction material in the world. The constant
development and research for better concrete has led to bioconcrete, which is concrete
infuse with micro-organisms that benefits the concrete by production of calcium
carbonate. However, the production of calcium carbonate is limited to the calcium
available in the cement. Therefore, the purpose of this study is to add calcium nutrient
source in the form of calcium lactate in bioconcrete and study the engineering concrete
properties and self-healing of micro-cracks. The bacteria used in this study is 3%
Enterococcus faecalis and 5% Bacillus sp. Whereas the calcium lactate that is added
into this study is in concentrations of 0.22 g/L, 1.09 g/L and 2.18 g/L. Concrete with
dimensions of 100 mm × 100 mm × 500 mm for prisms, cylinders of 150 Ø × 300 mm
and cubes of 150 mm × 150 mm ×150 mm are used to test for the engineering concrete
properties at 7th, 14th and 28th day and self-healing of micro-cracks in concrete is in the
range of 0-100 days. This research has contributed significantly to the finding of
overall improvement of concrete properties from the addition of calcium lactate in
bioconcrete. This is confirmed with the improvement of engineering concrete
properties and self-healing of micro-cracks with the addition of 2.18 g/L of calcium
lactate for both bacteria. UPV and micro-structure analysis were conducted to verify
self-healing. Based on overall results, concrete with Bacillus sp with 2.18 g/L is most
ideal.
vi
ABSTRAK
Konkrit adalah salah satu daripada bahan binaan yang paling banyak digunakan.
Pembangunan dan penyelidikan yang berterusan untuk konkrit yang ideal telah
membawa kepada penjumpaan biokonkrit, iaitu konkrit yang mempunyai tambahan
mikro-organisma yang memberi manfaat kepada konkrit dengan pengeluaran kalsium
karbonat. Walau bagaimanapun, pengeluaran kalsium karbonat adalah terhad kepada
kalsium yang terdapat dalam simen. Oleh itu, tujuan kajian ini adalah untuk menambah
sumber nutrient kalsium dalam bentuk kalsium laktat dalam biokonkrit untuk
mengkaji sifat-sifat konkrit kejuruteraan dan pembaikan semulajadi retakan mikro di
dalam biokonkrit. Bakteria yang digunakan dalam kajian ini ialah 3% Enterococcus
faecalis dan 5% Bacillus sp. Selain itu, Kalsium laktat yang ditambah dalam biokonkrit
adalah dalam kepekatan 0.22 g/L, 1.09 g/L dan 2.18 g/L. Konkrit yang keras dengan
dimensi 100 mm × 100 mm × 500 mm untuk prisma, silinder 150 Ø × 300 mm dan
kiub 150 mm × 150 mm × 150 mm telah digunakan untuk menguji sifat-sifat konkrit
kejuruteraan pada 7th, 14th dan 28th hari kematangan dan penyembuhan diri retakan
mikro di dalam konkrit dianalisi dalam tempoh 0-100 hari. Ujikaji ini telah
menyumbang kepada ilmu bahawa penambahan kalsium laktat dalam biokonkrit boleh
menambah baik sifat-sifat kejuruteraan konkrit dan pembaikan semulajadi keretakan
mikro dalam konkrit dengan penambahan 2.18 g/L kalsium laktat untuk kedua-dua
jenis bakteria. Penetuan pembaikan semulajadi keretakan mikro telah dilakukan
dengan UPV dan Analisa mikro-struktur. Berdasarkan keputusan yang didapati,
konkrit dengan Bacillus sp dan 2.18 g/L kalsium laktat adalah paling ideal.
vii
CONTENTS
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
CONTENTS vii
LIST OF TABLES xv
LIST OF FIGURES xvii
LIST OF SYMBOLS AND ABBREVIATIONS xxii
LIST OF APPENDICES xxiv
CHAPTER 1 INTRODUCTION 1
1.1 Introduction 1
1.2 Problem statement 4
1.3 Objectives 6
1.4 Scope of study 6
1.5 Importance of study 7
1.6 Organization of thesis 8
CHAPTER 2 LITERATURE REVIEW 10
viii
2.1 Introduction 10
2.2 Bioconcrete 11
2.3 Bacteria 12
2.3.1 Types of bacteria that are used in concrete 14
2.3.2 Enterococcus faecalis 18
2.3.3 Bacillus sp 18
2.3.4 Function of bacteria in concrete 19
2.4 The importance of calcium carbonate in concrete 21
2.5 Addition of calcium nutrient source 22
2.6 Enzymatic pathway of bacteria 24
2.7 Determination of precipitation of calcium carbonate 26
2.7.1 Micro-structure test 26
2.7.2 Elements in concrete 28
2.7.3 Chemical composition 29
2.7.4 Mineralogy of concrete 30
2.8 Cracks in concrete structures 31
2.8.1 Category of cracks 34
2.8.2 Conventional methods for crack repair 35
2.8.3 Bacteria as self-healing agent for concrete 36
2.8.4 Analyzing crack repairs 38
2.9 Effect of bioconcrete on engineering concrete properties 40
2.9.1 Compressive strength 41
2.9.2 Relationship between compressive strength and 44
splitting tensile strength
2.9.3 Flexural strength with addition of bacteria and 47
Calcium lactate
2.9.4 Relationship between compressive-flexural strength 49
2.9.5 Durability and permeability 50
2.9.5.1 Water penetration 51
2.10 Concluding remark 53
CHAPTER 3 METHODOLOGY
3.1 Introduction 55
3.2 Methodology of study 55
3.3 Material preparation 58
ix
3.3.1 Fine Aggregate 58
3.3.2 Coarse aggregate 59
3.3.3 Cement 60
3.3.4 Bacteria 62
3.3.4.1 Bacteria identification 62
3.3.4.2 Growth curve of bacteria 65
3.3.4.3 Serial dilution and colony count 65
3.4 Calcium lactate 66
3.5 Concrete casting 68
3.5.1 Concrete mix design 68
3.5.2 Batching of concrete 69
3.5.3 Fresh concrete tests 70
3.6 Experimental program 70
3.6.1 Compressive strength test 70
3.6.2 Tensile strength test 72
3.6.3 Flexural strength of prism 74
3.6.4 Water penetration 75
3.7 Self-healing by bacteria with calcium lactate 77
3.7.1 Standardized cracks 77
3.7.2 Realistic cracks 79
3.7.3 Analyzing self-healing 80
3.7.3.1 Ultrasonic pulse velocity (UPV) 80
3.7.3.2 Stereomicroscope 81
3.8 Analyses of concrete 82
3.8.1 Micro-structure analyses 82
3.8.2 Elements of concrete 83
3.8.3 Chemical composition 83
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Introduction 85
4.2 Bacteria identification 85
4.2.1 Gram staining (Morphology observation) 86
4.2.2 DNA quantification 87
4.2.2.1 Polymerase chain reaction (PCR) 88
4.2.2.2 Phylogenetic tree 89
x
4.2.3 Bacteria growth chart 92
4.2.4 Bacteria counting 93
4.3 Concrete properties 94
4.3.1 Workability of fresh concrete 94
4.4 Engineering Concrete properties 94
4.4.1 Compressive strength for concrete specimens 95
4.4.1.1 Compressive strength of control 95
E. faecalis and B. sp
4.4.1.2 Compressive strength results for concrete 96
with E.faecalis and E. faecalis (with 0.22 g/L,
1.09 g/L and 2.18 g/L of calcium lactate)
4.4.1.3 Compressive strength results for concrete 97
with B. sp and with (0.22 g/L, 1.09 g/L and 2.18
g/L of calcium lactate)
4.4.1.4 Comparison between E. faecalis and B. sp 99
4.4.2 Tensile strength test 100
4.4.2.1 Tensile strength of normal and 100
bioconcrete
4.4.2.2 Tensile strength of normal and bioconcrete 101
With calcium lactate
4.4.2.3 Tensile strength of concrete containing 102
B. sp and calcium lactate
4.4.2.4 Comparison between E. Faecalis and B. Sp 104
4.4.3 Flexural strength test 104
4.4.3.1 Flexural strength of control and 105
bioconcrete
4.4.3.2 Flexural strength of control concrete and 106
bioconcrete containing E. Faecalis and
calcium lactate
4.4.3.3 Flexural strength of control and concrete 107
with B. Sp and concentrations of calcium
lactate
4.4.3.4 Comparison between E. faecalis and B. sp 108
with concentrations of calcium lactate
xi
4.4.4 Relationship between compressive-tensile strength 109
4.4.4.1 Relationship between compressive- 110
tensile strength for bioconcrete
4.4.4.2 Relationship between compressive- 111
tensile strength for E. faecalis and
calcium lactate
4.4.4.3 Compressive-tensile strength for B. sp and 113
concentration of calcium lactate
4.4.5 Relationship between compressive-flexural 114
strength.
4.4.5.1 Relationship between compressive-tensile 115
Strength for bioconcrete
4.4.5.2 Relationship between compressive- 116
flexural strength for E. faecalis and
calcium lactate
4.4.5.3 Relationship between compressive-flexural 117
strength for B.sp and calcium lactate
4.4.6 Water penetration 119
4.4.6.1 Water penetration of concrete and 119
bioconcrete
4.4.6.2 Water penetration of E. Faecalis and 120
E. faecalis with concentrations of calcium
lactate
4.4.6.3 Water penetration of B. sp and 122
B. sp with calcium lactate
4.4.6.4 Comparison between E. Faecalis and B. sp 123
4.5 Microstructure and morphology of bioconcrete 124
4.5.1 Chemical composition identification using X-ray 124
Spectroscopy (XRF)
4.5.2 Microstructure analysis 126
4.5.2.1 Microstructure analysis for 126
normal and bioconcrete
4.5.2.2 Microstructure analysis results for 128
concrete with E.faecalis and calcium lactate.
xii
4.5.2.3 Microstructure of concrete specimens with 130
B.sp and calcium lactate.
4.5.2.4 Discussion for micro-structure 132
4.5.3 Element 133
4.5.3.1 Elements in normal and bioconcrete 133
4.5.3.2 Elements in concrete with E. faecalis 135
concentrations of calcium lactate
4.5.3.3 Elements in concrete with B.sp and 138
calcium lactate
4.5.3.4 Comparison between E. faecalis and B.sp 141
4.6 Self-healing properties 142
4.6.1 Ultrasonic pulse velocity (UPV) 142
4.6.1.1Standard crack 142
(Concrete cube with metal strip) of normal
and bioconcrete
4.6.1.2 Standard crack 143
(Concrete cube with metal strip) of concrete
with E. faecalis and calcium lactate
4.6.1.3 Standard crack 145
(Concrete cube with metal strip) of B.sp
and calcium lactate
4.6.1.4 Comparison between E. faecalis and B. sp 146
with concentrations of calcium lactate
4.6.2 Standard crack (Prism) 147
4.6.2.1 Standard crack (Prism) of normal 147
and bioconcrete
4.6.2.2 Standard crack (Prism) of concrete for 148
Normal and concrete with E. faecalis and
Calcium lactate
4.6.2.3 Standard crack (Prism) of normal 150
concrete specimens and bioconcrete
with calcium lactate
4.6.2.4 Comparison between bioconcrete 152
containing E. faecalis and B. sp
xiii
with concentrations of calcium lactate
4.6.3 Stereomicroscope 152
4.6.3.1Standard crack (Prism) of control concrete 153
Specimens and bioconcrete specimens
4.6.3.2Standard crack (Prism) of E.faecalis and E. 154
Faecalis with concentrations of calcium lactate
4.6.3.3 Standard crack (Prism) of B. sp 156
and B. sp with concentrations of calcium lactate
4.6.3.4 Comparison of self-healing 157
between E. faecalis and B. sp
4.6.4 Realistic crack (Cicular disk) 158
4.6.4.1Stereomicroscope of realistic crack 159
(Circular disk) of normal and bioconcrete
4.6.4.2 Stereomicroscope of realistic crack 160
(Circular disk) of E. faecalis with
calcium lactate
4.6.4.3 Stereomicroscope of realistic crack 161
(Circular disk) of B. sp with calcium lactate
4.6.4.4 Comparison of self-healing of 163
E. faecalis and B. sp with
different concentrations of calcium lactate
4.7 Concluding remark 164
CHAPTER 5 CONCLUSION AND RECOMMENDATION
5.1 Introduction 165
5.2 Conclusion 165
5.2.1 Isolation and identification of Enterococcus 165
faecalis and Bacillus sp
5.2.2 Concrete properties 166
5.2.2.1Compressive strength 166
5.2.2.2Tensile strength 166
5.2.2.3Flexuralstrength 167
5.2.2.4Water penetration 167
5.2.3 Micro-structure analysis 167
5.2.3.1 Chemical composition identification using 167
xiv
X-ray spectroscopy (XRF)
5.2.3.2 Micro-structure analysis 168
5.2.3.3 Elements 168
5.2.4 Evaluation of self-healing 169
5.2.4.1UPV (Prism) and standardized crack 169
5.2.4.2 Stereomicroscope 169
5.2.5 Overall conclusion 170
5.3 Research contribution 170
5.4 Recommendation 170
REFERENCES 172
APPENDICES 184
xv
LIST OF TABLES
2.1 Various bacteria used in concrete 15
2.2 Chemical composition of cement and blast furnace slag using XRF 29
2.3 Category and description of cracks (Technical note-No 30, 2010) 34
2.4 Compressive strength of values 7 and 28 days tests with bacteria 41
influence mortar
2.5 Equation for compressive-tensile strength relation 45
2.6 Previous research on bacteria 47
2.7 The result of testing beam for high strength concrete and 49
normal concrete with added fiber
2.8 Relationship between compressive-flexural strength 50
2.9 Treatment available for water Permeability 52
3.1 Chemical composition of cement 61
3.2 Amount of calcium lactate used 66
3.3 Mix proportion used in this study 68
3.4 Fabrication batch 69
3.5 Number of specimens used for compressive strength test 71
3.6 Number of specimens used for tensile strength test 72
3.7 Number of specimens used for flexural strength test 74
3.8 Number of specimens used for water penetration test 76
4.1 The concentration and quality of DNA using Nanodrop 88
4.2 PCR parameter 89
4.3 Slump test for concrete workability 94
4.4 Compressive strength of normal and bioconcrete 95
4.5 Compressive strength of concrete with E. Faecalis and calcium lactate 97
4.6 Compressive strength of concrete with B. sp and calcium lactate 98
xvi
4.7 Tensile strength results for normal and bioconcrete 101
4.8 Tensile strength of concrete with E. faecalis with calcium lactate 102
4.9 Concrete with B. sp and various concentration of calcium lactate 103
4.10 Flexural strength of concrete for normal and bioconcrete 105
4.11 Flexural strength of concrete with E. Faecalis and calcium lactate 107
4.12 Results of bioconcrete with calcium lactate 108
4.13 The relationship between compressive-tensile between bioconcrete 110
4.14 Compressive-tensile relationship of E. Faecalis and calcium lactate 112
4.15 Compressive-tensile of concrete with B.sp and calcium lactate 113
4.16 Relationship between compressive-flexural strength for bioconcrete 115
4.17 Relationship between compressive-flexural strength for E. faecalis 116
4.18 Relationship between compressive-flexural strength for B. Sp 118
4.19 Water penetration of bioconcrete 120
4.20 Water penetration of concrete with E. Faecalis and calcium lactate 121
4.21 Results of water penetration of concrete with B.sp and calcium lactate 122
4.22 Chemical composition of bioconcrete 125
4.23 Ultrasonic pulse velocity of control and bioconcrete 143
4.24 UPV data for control and E. faecalis with different concentrations of 144
calcium lactate
4.25 Data obtained for B. sp and concentrations of calcium lactate 145
4.26 Ultrasonic pulse velocity of concrete prism 148
4.27 Ultrasonic pulse velocity of concrete prism with E. faecalis 149
4.28 Percentage of healing of concrete prism containing B.sp and calcium 151
lactate
4.29 Percentage of healing for control and bioconcrete 153
4.30 Percentage of healing for bioconcrete and calcium lactate 155
4.31 Percentage of healing for bioconcrete specimens containing 156
B. sp and calcium lactate
4.32 Percentage of healing Percentage of healing for Bioconcrete specimens 159
4.33 Percentage of healing for Bioconcrete specimens containing different 160
concentrations of calcium lactate
4.34 Healing of concrete containing B. sp and concentrations of calcium lactate162
xvii
LIST OF FIGURES
1.1 Factors influencing concrete durability 2
2.1 Image of bacteria with calcium carbonate formation of the cell wall 13
2.2 Microstructure analysis of biosealant removed from inside cracks of 27
Concrete: (a), (c) and (e) are concrete with added bacteria, whereas (b),
(d) and (f) are concrete with bacteria and calcium lactate.
2.3 Chemical constituents of precipitation of calcium carbonate. The high 28
Amount of Ca present is evident and the precipitation is inferred
to as calcite (CaCO3) crystals
2.4 Concrete containing fungi with image 28
2.5 Diffractograms of consolidated sand with bacteria 30
2.6 Concrete structure subjected to corrosion 33
2.7 Cracking which initiates the steel corrosion process 33
2.8 Before and after experiment for self-healing of concrete using bacteria 38
2.9 Method to measure UPV (EN12504-4:2004) (a) Direct method 39
(b) and (c) Indirect method
2.10 Image from Stereomicroscope after 100 days 40
2.11 Different bacteria concentration with different amount of silica fume of 42
(a) 28 days (b) 91 days
2.12 The compressive strength of cement mortar of control and addition of 43
Bacterial cells
2.13 Linear relationship between compressive and tensile strength 46
2.14 Flexural strength of mortar beams with addition of bioremediase 48
2.15 The influence of surface treatment on the rate of water absorption versus 51
Time for mortar cubes with water-cement ration of o.6
3.1 Research flowchart 56
xviii
3.2 Bacteria liquid culture and calcium lactate flowchart 57
3.3 Sand used during casting 58
3.4 Sieve analysis result for fine aggregate 59
3.5 Coarse aggregate within the sieve limit 60
3.6 Sieve analysis result for coarse aggregate 60
3.7 Mix Portland cement 61
3.8 Enterococcus faecalis and Bacillus sp used in this study 62
3.9 Bacteria identification procedure 63
3.10 Materials for preparation bacteria with nutrient source
(Enterococcus faecalis / Bacillus sp) 65
3.11 Serial dilution of bacteria 66
3.12 Calcium lactate pre-measured for casting 67
3.13 Calcium lactate dissolved before fabrication 67
3.14 Universal testing machine for compressive strength test 70
3.15 Cubes use for compressive strength test 71
3.16 Cylinder sample placed in UTM machine for splitting tensile test 73
3.17 Fabrication of concrete cylinder sample 73
3.18 Flexural test for concrete conducted using UTM 75
3.19 Mold of prism used to fabricate prism used in flexural strength test 75
3.20 Testing machine for water penetration test 76
3.21 Metal strip pushed into fresh concrete to create cracks 77
3.22 View of prism with steel reinforcement (2R8) 78
3.23 Sample of prism with standard crack 79
3.24 Circular disk sample for self-healing observation and analyses 79
3.25 Ultrasonic pulse velocity set-up 80
3.26 Ultrasonic pulse velocity specimen 81
3.27 Specimens with crack used for monitoring 82
3.28 EDX machine for analysis 83
3.29 Hand-hydraulic press used to create pellets 84
3.30 Pellets formed for XRF analyses 84
4.1 Image of Enterococcus faecalis under microscope 87
4.2 Image of Bacillus sp under microscope 87
4.3 Agarose gel of PCR product 89
4.4 Phylogenetic position of Bacillus sp and closely related taxa (Sulphate 90
xix
Reduction bacteria)
4.5 Phylogenetic position of Enterococcus faecalis and closely related taxa 91
(Ureolytic bacteria)
4.6 The absorbance value of bacteria 92
4.7 Relationship of Absorbance (nm) VS number of cells for E.faecalis 93
4.8 Relationship of Absorbance (nm) VS number of cells for B.sp 93
4.9 Compressive strength of normal and bioconcrete with age of curing 96
4.10 Compressive strength of concrete with E. Faecalis and calcium lactate 97
with time
4.11 Compressive strength of concrete with B.sp and B.sp with calcium lactate 98
4.12 Tensile strength test (left) before and (right) after testing 100
4.13 Tensile strength for control and bioconcrete samples 101
4.14 Tensile strength for control and concrete with E. Faecalis and 102
calcium lactate
4.15 Tensile strength for control and concrete containing B.sp and 103
calcium lactate
4.16 Prism before (left) and after (right) flexural strength test 105
4.17 Comparison of flexural strength of control and concrete containing 106
E.Faecalis and B.sp
4.18 Flexural strength of concrete for control and bioconcrete with 107
calcium lactate
4.19 Flexural strength of concrete with B.sp and calcium lactate 108
4.20 Comparison compressive-tensile between each empirical strength relation111
in bioconcrete
4.21 Comparison of compressive-tensile strength relation in bioconcrete with 112
calcium lactate
4.22 Comparison between compressive-tensile strength relation in bioconcrete 114
containing B.sp and calcium lactate
4.23 Comparison between compressive-flexural relation in bioconcrete 115
4.24 Compressive-flexural strength relation of bioconcrete with calcium lactate117
4.25 Comparison between compressive-flexural strength relation of 118
bioconcrete with calcium lactate
4.26 (a) Shows the water penetration test set-up and (b) shows the 119
water penetration depth after testing
xx
4.27 Relationship of water penetration between bioconcrete 120
4.28 Water penetration of concrete with E. Faecalis and calcium lactate 121
4.29 Relationship between concrete with B.sp and calcium lactate 123
4.30 Comparison between two different bacteria 124
4.31 Microstructure of normal concrete 127
4.32 Concrete sample with E. faecalis 127
4.33 Microstructure of concrete sample with B.sp 128
4.34 Microstructure of E. faecalis and 0.22 g/L of calcium lactate 129
4.35 Concrete sample with E. faecalis and 1.09g/L calcium lactate 129
4.36 Microstructure of E. Faecalis and 2.18g/L calcium lactate 130
4.37 Microstructure of B.sp and 0.22 g/L of calcium lactate 131
4.38 Sample with B. sp and 1.09g/L calcium lactate 131
4.39 Concrete with B. sp and 2.18g/L calcium lactate 132
4.40 The image and graph of elements for control sample by EDX 134
4.41 The image and graph of elements with 3% Enterococcus faecalis by EDX134
4.42 The image and graph of elements for sample with 5% Bacillus sp by EDX135
4.43 Comparison of calcium mass in control and sample with bacteria 135
4.44 EDX image and element composition for 3% Enterococcus faecalis 136
with 0.22g/L of calcium lactate
4.45 Element composition for 3% E. faecalis with 1.09 g/L of calcium lactate 137
4.46 Element composition of 3% E. Faecalis with 2.18g/L calcium lactate 137
4.47 Comparison of calcium mass in control and E. faecalis sample with 138
Calcium lactate
4.48 EDX image and element composition for 5% Bacillus sp with 139
0.22g/L of calcium lactate
4.49 EDX image and element composition for 5% Bacillus sp with 139
1.09 g/L of calcium lactate
4.50 5% B.sp with 2.18g/L Calcium lactate 140
4.51 Amount of calcium in concrete sample of normal and bioconcrete 140
4.52 Comparison of calcium mass of bioconcrete with different 141
Concentrations of calcium lactate
4.53 Comparison of healing percentage of control and bioconcrete samples 143
4.54 Percentage of healing of control, bioconcrete and concrete with 144
calcium lactate
xxi
4.55 Comparison of healing capabilities of control, concrete with B. sp 146
and calcium lactate
4.56 Percentage of healing comparison between, normal E. faecalis 147
and B. sp
4.57 Percentage of healing for control and bioconcrete 148
4.58 Comparison between different concentrations of calcium lactate with 150
bioconcrete containing E. faecalis
4.59 Comparison of concrete containing B. sp with concentrations 151
of calcium lactate
4.60 Percentage of healing for bioconcrete 154
4.61 Percentage of healing for bioconcrete specimens with E. faecalis and 155
calcium lactate
4.62 Comparison of healing between concrete specimens with B. sp and 157
calcium lactate
4.63 Comparison percentage of healing between E. Faecalis and B. sp 158
4.64 Comparison for healing percentages among bioconcrete specimens 159
4.65 Healing capabilities comparison for specimens containing E. Faecalis 161
and calcium lactate
4.66 Comparison between bioconcrete specimens containing different 162
concentrations of calcium lactate
4.67 Comparison of healing between E. Faecalis and B. sp 163
xxii
LIST OF SYMBOLS AND ABBREVIATIONS
E. faecalis - Enterococcus faecalis
B.sp - Bacillus sp
kg - kilogram
m3 - m× m× m or volume
𝑘𝑔/𝑚3- Density
SCC - Self compacting concrete
CaCO3 - Calcium Carbonate
% - Percentage
k - Growth Rate
k1 - Carbonation rate constants
g - Gram
CO2 - Carbon Dioxide
pH - Power of Hydrogen
cells/ml - Cells per 1 ml
FA - Fly Ash
SF - Silica Fume
𝑑𝑞
𝑑𝑡 - The rate of fluid, m3 /s
L - Thickness of sample in m
A - Cross sectional area of the sample in m3
𝛥ℎ - Drop in hydraulic head through the
sample, m
K - Coefficient of permeability in m/s
m/s - Meter per second
xxiii
kPa - Kilo pascal
BS - British Standard
BS EN - British Standard European Norm
g - Gram
mm - Milimeter
DOE - Department of Environment
L - Litre
NaOH - Sodium Hydroxide
ml - Millilitre
NTU - Nephelometric Turbidity Unit
CFU - Colony Forming Unit
CFU/ml - Colony Forming Unit per Mililitre
UB - Ureolytic Bacteria
cm - Centimeter
RPM Revolution Per Minute
NCBI National Centre for Biotechnology
Information
xxiv
LIST OF APPENDICES
APPENDIX TITLE PAGE
A DOE MIX DESIGN 184
B DNA SEQUENCE 186
C RAW DATA FOR UPV 188
D RAW DATA FOR STEREOMICROSCOPE 194
1
CHAPTER 1
INTRODUCTION
1.1 Introduction
Concrete that is made from coarse granular aggregate, sand, cement and water is the
most widely used construction material in the world. Concrete is the preferred
construction material for a wide range of buildings, bridges and other civil
engineering structures. The material has been successful and popular due to the ease
of casting into various shaped and sizes, it is easy to procure in the market and it is
cheaper compared to other building materials. The main strength of concrete lies in
its compressive strength which is higher than conventional building materials such as
bricks and stone masonry (Unnikrishna and Devdas, 2003). The development of
concrete technology had triggered various grade and type of concrete namely, foam
concrete, light-weight concrete, high strength concrete and others. An option of
concrete in the market is concrete infuse with microorganisms known as bioconcrete.
The use of concrete is gaining even more momentum as many more plans and
development are taking place due to the Country’s economic growth and
development. As the demand for concrete increases, the need to create a better,
longer lasting and more durable concrete is much in need. This is due to concrete
shortcomings that are known as concrete degradation which occurs in the form of
cracking, scaling, occurrence of efflorescence and many more. According to Jackson
2
and Dhir (1996), concrete is required to sustain the load and be more durable. The
durability is defined as resistance to deterioration which occurs by means of internal
or external factors. Figure 1.1 shows several factors that influence the durability of
concrete.
Figure 1.1: Factors Influencing Concrete Durability
(Jackson and Dhir, 1996)
Based on Figure 1.1, deterioration in concrete is caused by several factors such as
physical deterioration, chemical deterioration and reinforcement corrosion. These
deteriorations leads to cracking in structures, leaching of chemicals, carbonation and
chlorination. If left untreated, these deteriorations causes the lifespan of a building to
reduce and affect the structural integrity of a building.
In the current market, a lot of methods have been adopted in the construction
industry to minimize the concrete degradation. The concrete degradation is
minimized by adding various chemical admixtures, concrete hardeners, damp-
proofing admixtures and etc (Tittelboom et al., 2012b). These methods used are not
environmentally friendly and pose several drawbacks on the environment such as air
pollution, soil and water contamination (Guadalupe et al., 2014).
There are many studies conducted which focuses on the reduction of concrete
deterioration by adding environmentally friendly materials such as waste from
factory production in order to come out with alternative solution to concrete problem
(Kartini et al., 2010; Panda and Bal, 2013; Bashar et al., 2013; Norlia et al., 2013;
3
Yadhu and Aiswarya, 2015).The common waste and industrial by-products that are
normally being used are rice husk, recycle waste material, fly ash and silica fume.
Kartini et al., (2010) had stated that rice husk ash is able to improve the
durability of concrete in G30 concrete by lowering the permeability, thus lowering
the absorption characteristics and increasing the resistance of concrete to chloride ion
penetration. Apart from that, Panda and Bal (2013) had stated that recycle aggregate
used as partial replacement yielded more desirable compressive strength as compared
to full replacement of course aggregate in self compacting concrete. Conventional
way to increase strength without adding waste in concrete, normally relies on
chemical solution or adding more cement. Development and research are conducted
in order to provide a more environmentally friendly alternative to concrete durability.
There are studies that focus on prolonging the life of concrete structure by inducing
self healing capability. This prevents crack from becoming unmanageable which
leads to expensive repairs and reduction of structural integrity of the building.
Worldwide, researchers have studied on the use of bacteria in concrete with
various improvement in concrete properties and self-healing (Muynck et al., 2008;
Tittelboom et al., 2010; Jonkers., 2011; Abo-El-Enein., 2013; Varenyam et al., 2013;
Parmar et al., 2013). Certain group of bacteria have the capability in prolonging the
life of concrete by using specific enzyme to precipitate calcium carbonate
(Muynck et al., 2008).
Some researchers added calcium based nutrient in bioconcrete to facilitate
with the process of producing calcium carbonate. Introducing additional calcium
nutrient source in the formed of calcium lactate, calcium acetate and calcium
chloride were used and it was discovered that significant improvement was found on
mechanical properties and durability of concrete. Self-healing of concrete were also
found to have increase significantly with addition of calcium based nutrient (Abo-El-
Enein et al., 2013; Xu and Yao., 2014; Xu et al., 2015). Suitability of bacteria in
concrete depends on factors such as the ability of the bacteria to live in little to no
oxygen environment and environment which is high in alkaline. Some researchers
have device methods to seal the bacteria and calcium nutrient source in capsules of
light weight aggregate (LWA) to protect the bacteria from the roughness of concrete
fabrication and protect the bacteria from high alkaline environment of bacteria. The
bacteria and calcium source within the capsules or LWA are released once a crack is
4
formed through external pressure or loading. (Jonkers and Erik., 2008; Tittelboom et
al., 2012b; Wang et al., 2012; Rajesh et al., 2015).
1.2 Problem statement
Concrete is an important element in construction and it is the most used material
worldwide. Concrete is used to construct buildings from foundations to building
structure, road curbs and drainage. Construction of different parts of buildings
requires different standard or strength of concrete. In order to achieve the concrete
standard require of different buildings or sub-structure, different strength of concrete
are used. Methods to increase strength of concrete are to reduce the water ratio of
concrete mixture, increase cement content or adding chemical admixture. Reduction
of water reduces workability thus making it hard to cast while addition of cement
content is costly. Chemicals used in chemical admixture to improve concrete
properties are not as costly compared to adding extra cement but the use of chemicals
itself is harmful towards the environment. Additional natural resources are depleted
in order to manufacture the chemicals used to improve engineering concrete
properties (Mehta, 2002).
Concrete has many shortcomings. These shortcomings come in the form of
high water penetration, cracking, steel reinforcement deterioration and etc.
Constructed building and infrastructures after the second half of the last century has
seen a declined in lifespan due to rapid deterioration. The enhancement of building
durability has been the aim of many researchers as longer lifespan reduces the
amount of raw material used, carbon dioxide emission and energy consumption
related to construction (Schlangen and Sangadji, 2013). New materials are soughtout
to construct new building and the production of cement releases carbon dioxide to
the atmosphere. Regular maintenance could reduce the chance of faster building
deterioration. Cracks are often the trigger to problems, micro-cracks formed which
are not treated could potentially become serious through moisture or water entering
the cracks. The moisture or water that enter the cracks causes the steel reinforcement
to rust and therefore reduce the structure’s integrity (Tittelboom et al., 2010).
The use of chemicals are often the solution to industrial problems. Chemicals
used in order to fix and repair concrete have considerable negative effects on the
5
environment (Tittelboom et al., 2012b). Production of these chemicals requires a vast
amount of resources, thus depletion of the world resources due to usage and high
demands. Apart from that, the use of chemicals on concrete such as epoxy and
synthetic fillers are temporary and often requires re-application when cracks re-
emerges. Alternative methods that are more environmentally friendly need to be
explored for improving degradation of concrete (Muynck et al., 2007).
Ramachandran et al., (2001) had argue that cracks in concrete is a very
common phenomenon. However, cracks in structure have to be treated in order to
prevent the crack from expanding and causing major problems. There are many
available treatments in the market to cope with cracking in structure, among them are
epoxy, resins, epoxy mortar and other synthetic mixtures. Unfortunately, the
synthetic filler which is commonly used acts as a temporary solution and
reapplication is needed depending on the formation of other cracks.
Jonkers., (2011a) had proven that high binder content of concrete mixture
resulted to delay crack formation. Concrete has a certain autonomous healing of
micro crack which is related to the composition of concrete mixtures. Mixtures that
contain high binder content shows high crack healing properties which are due to the
secondary or delayed hydration. Unfortunately, this ability to heal crack is only
limited to cracks which are smaller than 0.2mm and also due to the global sustainable
reasons the use of high binder content cement is not encourage. It is costly to use
more cement in concrete and using more encourages higher production of cement.
This induces carbon dioxide emissions which eventually leads to global warming
Researchers such as Abo-El-Enein et al., (2012) and Xu and Yao (2014) have
researched the possibility of adding calcium source into bioconcrete. In the study
conducted by Xu and Yao (2014), it was found that the type of calcium source added
have a profound effect on the degree of crystallinity of precipitated calcite by
bacteria. Organic and inorganic calcium sources were used, inorganic calcium source
such as calcium chloride were found to have lowest amount of precipitated CaCO3
while using organic calcium source such as calcium lactate were found to achieve a
higher amount of CaCO3.
This research aims to find an environmental alternative for durable concrete
by adding calcium lactate in bioconcrete. The calcium lactate are added in several
different concentrations.The engineering concrete properties and enhancement of
self-healing are studied.
6
1.3 Objective
The objectives of this study are:
(i) To determine the optimum concentration of calcium lactate which are added
into concrete.
(ii) To investigate the effect of calcium lactate together with Enterococcus
faecalis and Bacillus sp towards the engineering concrete properties in
unsterillized condition.
(iii) To study the effect of the addition of calcium lactate with Enterococcus
faecalis and Bacillus sp on the self healing of concrete in unsterillized
condition.
1.4 Scope of study
This study mainly focused on laboratory works, where all laboratory work are
conducted in University Tun Hussein Onn Malaysia (UTHM). The growth of
Enterococcus faecalis and Bacillus sp is conducted in the environmental laboratory,
whereas the casting and testing of concrete is conducted in structural laboratory. The
percentage of Enterococcus faecalis used is 3% whereas the 5% Bacillus sp is used
in this study. The different percentages used is based on previous study (Irwan et al.,
2015). In the previous study, optimum percentage was tested in order to determine
the best percentage that yielded the best results in improving the properties of
concrete. The optimum amount of percentage that is added into the concrete which
achieved the best result is used in this study.
The Enterococcus faecalis and Bacillus sp are both regrown based on the
optimum number of days by the bacteria growth curve. Apart from that, calcium
lactate is added as an addtional calcium source to increase precipitation of calcium
carbonate. The calcium lactate added acts as a catalyst for the bacteria to precipitate
calcium carbonate. Calcium lactate is chosen as the calcium nutrient source due to
the popularity as an organic nutrient source used by Xu et al., (2015), Xu and Yao
(2014) and Jonkers and Erik (2008) in bioconcrete research. Calcium lactate can
7
easily be found in milk and cheese, thus making the use of calcium lactate and
bacteria in concrete innocuous. Several concentrations of calcium lactate (0.22 g/L,
1.09 g/L and 2.18 g/L) are added to determine the optimum percentage which
optimizes the engineering concrete properties in term of compressive strength, tensile
strength, flexural strength and water penetration. Microstructure, chemical
composition and elemental analysis are conducted on concrete samples. Self healing
of concrete with bacteria and calcium lactate is analyzed using Ultrasonic pulse
velocity (UPV) and Stereomicroscope.
1.5 Importance of study
As the world is getting more and more crowded due to the surge of human
population, constant and ever rapid construction should take place to make way for
this growing population. Hence, houses, condominiums, apartments, schools,
University, shopping complexes, shop-lots and so much more are constantly being
built to sustain this growth.
With the ever-growing construction industry, the use and production of
concrete is absolute. Apart from producing concrete for the growing industry,
concrete is also produced for the re-pair and re-built of a demolished building that
has reached the end of its lifespan. The lifespan of a building is due to the concrete
durability which in current standard is only 50 years. Thus, after 50 years the
buildings are either demolished or abandoned due to safety reasons.
Mehta (2002) had stated that during the second half of the twentieth century,
most of the structures that was built were prone to premature deterioration of
concrete, especially for structures that are exposed to industrial and urban
environments. The degradation of concrete was mostly associated with the corrosion
of steel reinforcement due to alkali-aggregate reaction or sulfate attack. The
reinforced concrete structures begin to deteriorate earlier than the design lifespan of
the structure due to the cracks, microcracks which resulted in penetration of water
and therefore cause durability problems.
Present day concrete mixes contain high reactive Portland cement in order to
induce early strength for the purpose of high speed construction. Early strength
concrete undergoes high drying shrinkage and high thermal contraction. This process
8
causes cracks to occur and thus durability problems in concrete. The search for an
environmentally-friendly alternative to cope with the concrete problem is important
more than ever. Natural resources are being depleted faster than it has the chance to
recover due to the chemicals being produced and natural resources being use to
construct new building. The alternative is to build structure that can stand the test of
time, by building structure that are less prone to deterioration or could sustain itself.
Bioconcrete is the solution to this problem, as bacteria with calcium lactate in the
concrete has the ability to produce calcium carbonate which in turns improves
engineering concrete properties. The reduction of water penetration lessen the chance
of steel reinforcement being corroded thus improving the durability of concrete. Self
healing of concrete micro-cracks is also possible by production of calcium carbonate.
Thus, the importance of this study is to determine the effect of Enterococcus faecalis
and Bacillus sp with calcium lactate on the engineering concrete properties and
analyze the potential of these particular bacterium on self healing of concrete micro-
cracks.
1.6 Organization of thesis
This thesis aims to investigate the effect of different concentrations of calcim lactate
and bacteria towards the engineering concrete properties and self-healing of concrete
under unsterrilized condition. Overall, this thesis consists of five chapters with each
chapter focusing on a different subject matter as follow.
Chapter 1 is the introduction for the whole thesis which gives an overview of
introduction, problem statement, objectives, scope of study and importance of study.
It covers a brief overview on the current problems faced by the industry. In addition,
this chapter briefly explains the current method in solving issue with concrete and
suggest a more environmentally-friendly method to solve these issue.
Chapter 2 presents the review of literature related to this study, which includes the
introduction of the type of concrete, the idea of bioconcrete, type of bacteria used by
researchers, the effect of adding calcium source, the enzymatic pathway of these
9
bacteria and the tests conducted by researchers to validate the effect of bacteria
towards concrete.
Chapter 3 focuses on the material used in conducting this research, process of
identifying the bacteria, preparation of concrete samples and procedure for tests
conducted. Futhermore, this chapter also includes the details for preparing samples
for self-healing, analysis for self-healing and analysis of concrete samples.
Chapter 4 presents the findings of this research. This includes the identification of
bacteria, effect of bacteria with different concentration of calcium lactate on
engineering concrete properties and self-healing of concrete. In addition, findings of
Microstructure, chemical composition and elemental analysis is presented and
discussed in this chapter.
The last chapter in this thesis-Chapter 5 gives a summary of the whole result as an
integral part of the study. The recommendations for future works or futher works are
also provided in this chapter.
10
CHAPTER 2
LITERATURE REVIEW
2.1 Introduction
Developments of concrete have been rapid during recent years; this is due to the
demand of the industry. Various types of concrete were developed to cater with the
different structure and environment. Among the types of concrete developed are foam
concrete, self-compacting concrete, geo-polymer concrete and bioconcrete.
Foam concrete is a lightweight concrete that is created using Portland cement
paste or cement filler matrix (mortar) with a uniformly distributed pore structure
produced by adding forming agent. It was created to produce a concrete that is light
but with substantial strength (Ramamurthy et al., 2009).
Self-compacting concrete (SCC) was design and created to ease the concrete
casting in small or hard to reach places. Self-compacting concrete is quite suitable to
be used in structural elements which have dense reinforcement. This characteristic also
enables easier workability, little workmanship and reduces the duration of the concrete
construction stage. Apart from self-compacting concrete (SCC), a non-combustible
and non-flammable concrete that was created is Geo-polymer concrete. Geo-polymer
concrete utilizes the waste material from industries such as fly ash, silica fume,
biomass material and many more to replace cement in concrete. This resulted in a
much greener product for the industrial with the same or better mechanical properties.
As brilliant as the concrete innovations is, it is not without flaw. The main flaw
of concrete is the proneness to cracks. Therefore, development of new concrete to
withstand or self-repair crack is necessary. (Siddique and Chahal, 2011). Presently to
11
reach optimal levels of sustainability, several investigations are being made to reduce
the environmental impact of concrete such as replacing Portland clinker with
alternative cements and increasing concrete durability (Gonsalves, 2011). The use of
bacteria in concrete remediation is a new approach to an old idea that a microbial
mineral deposit constantly occurs in natural environments. Specifically,
microbiologically-induced calcite is environmentally innocuous, compared to
synthetic polymers currently used for concrete repairs (Ramachandran et al., 2001).
2.2 Bioconcrete
The term bioconcrete is a combination of biology (micro-organism) and concrete, Bio-
concrete. Thus, this study focus on an environmental approach in improving
engineering concrete properties and enhancement of self-healing by inducing micro-
organisms mainly Enterococcus faecalis and Bacillus sp.
Ramachandran et al., (2001) stated the bacterial concrete refers to a new type
of concrete in which selective cementation of porous media by microbiologically-
induced CaCO3 has been introduced for remediation of damaged structural formation
or micro cracks.
The field of using bacterial to improve concrete appears to be more beneficial
as bacterial concrete appears to produce more substantially crack plugging minerals
than control specimens (without bacteria). Microbial carbonate precipitation
(biodeposition) decreases the permeation properties of concrete. Hence, a deposited
layer of calcium carbonate on the surface of concrete resulted in the decrease of water
absorption and porosity. The highest decrement of permeation after 140 hours is 24%
compared to control (Siddique and Chahal., 2011).The presence of bacteria which was
the precipitated calcium carbonate by bacteria has resulted in significant decrease of
water uptake of up to 85% compared to concrete without any addition of bacteria
(Muynck et al., 2008).
Bacterial protein bioremediase is bacteria in powdered form. This form of
bacteria was used in the fabrication of concrete mortar and beams. Overall, positive
results in terms of compressive strength, flexural strength of beams and self-healing of
cracks had been found in this study (Chattopadhyay et al., 2011). The addition of
bioremediase in Portland pozzolanic cement has found to have an increase of
12
compressive strength of up to 39.4% as compared to control cube and an increase of
33% of flexural strength of beam as compared to beams without bacteria. It is also
found to have a resistance to environmental pollutants such as water absorption and
sulfate ions. This bioremediase is non-harmful to human beings and is eco-friendly as
well. Apart from that, the use of bioremediase in concrete has shown significant self-
healing properties on concrete cracks. (Chattopadhyay et al., 2011).
Xu and Yao (2014) and Abo-El-Enein et al., (2012), both have similar finding
in which adding calcium source into bioconcrete indeed increase the amount of
precipitated calcium carbonate through the bacteria ezymatic pathway. This increment
of calcium carbonate resulted in improvement of concrete properties. However,
different type of calcium showed different results. It is concluded that organic calcium
source such as calcium lactate is more suited to produce high amount of calcium
carbonate than inorganic calcium source such as calcium chloride and calcium acetate.
Based on the works of previous researchers, bioconcrete has been extensively
studied by researchers conducting study on bacteria in concrete. The addition of
bacteria in concrete has positive results in terms of improving engineering concrete
properties and self healing. A direct method of using bacteria culture during the
concrete mixing process is unpopular because most bacteria are acidic in nature and
do not mix well in alkaline environment such as concrete. Apart from that, most
bacteria are aerobic bacteria thus require oxygen to survive. Concrete has little to no
oxygen, thus different method of adding bacteria into concrete are device such as
encapsulation, bacteria powder and lightweight aggregate in order to sustain the life
of the bacteria. Based on previous study, addition of calcium source increases
precipitation of calcium carbonate significantly, depending on the type of calcium
source (inorganic or organic). In this study, a direct method of adding Enterococcus
faecalis and Bacillus sp with different concentrations of calcium lactate in concrete is
study. The effect towards engineering concrete properties and enhancement in self-
healing is conducted.
2.3 Bacteria
Siddique and Chahal (2011) has define bacteria as unicellular (single cell) micro-
organisms. Bacteria are normally found everywhere and anywhere on earth, growing
13
in soil, acidic hot springs, water and even deep in the Earth’s crust. Bacteria can also
be found in organic matter such as in live bodies of plants and animals. Averagely,
there are 40 million bacterial cells in a gram of soil and a million bacterial cells in a
millimeter of fresh water. The approximate number of bacteria on Earth is five
nonillion (5× 1030) which forms much of the world’s biomass.
According to Chahal et al., (2010) bacteria are able to promote the precipitation
of calcium carbonate in the form of calcite. Calcium carbonate precipitation occurs as
a by-product of a common microbial metabolic process which increases the alkalinity
and produce microbial calcite precipitation. Figure 2.1, shows the image of bacteria
with calcium carbonate formation.
Figure 2.1: Image of bacteria with calcium carbonate formation of the cell wall
(Siddique and Chahal., 2013)
Siddique and Chahal (2013) have stated that there are four phases of bacteria growth:
Phase 1: Lag phase
This is when the bacteria have only begun to adapt to a high nutrient environment and
is preparing for rapid growth. During this phase, proteins necessary for the growth of
bacteria are produced, thus has high biosynthesis rates.
14
Phase 2: Logarithmic Phase (Log Phase)
This phase of the bacterial growth is marked by rapid exponential phase, the growth
rate of this phase is also known as growth rate (k) and the time taken from bacteria to
multiply is known as the generation time (g). During this phase of growth, nutrients
are being depleted by the bacteria due to the rapid growth, after which limiting growth
occurs due to the depleted nutrient.
Phase 3: Stationary Phase
This phase is known as stationary phase due to the stop of growth of bacteria which is
caused by the depletion of nutrient during log phase.
Phase 4: Death Phase
This phase is known as the death phase in which the bacteria starts dying off due to
depleted nutrient.
In this research, the bacteria are used when it reaches log phase, which is when
the bacteria reached optimum growth (highest number of bacteria count). This is
determined through optical density and growth curve of the bacteria. Apart from that,
the bacteria used in this study are Gram positive. Bacteria are either Gram positive or
Gram negative. This is determined by conducting Gram staining tests. Bacteria without
cell walls could not retain stains therefore are Gram negative. The presence of cell
walls has a positive influence towards the compressive strength of concrete. Based on
Pei et al., (2013) study, which compares compressive strength between samples which
contains bacteria with cell walls and without. It was found that bacteria with cell walls
significantly improve the compressive strength of concrete
2.3.1 Types of bacteria used in concrete
There are various species of bacteria that are used in bioconcrete research such as in
Table 2.1. Where, different species of bacteria in bioconcrete are studied through
different applications such as surface treatment and concrete properties.
15
Table 2.1: Various bacteria used in concrete
Researcher Title Bacteria species Application Research result
Ramachandr
an et al.,
(2001)
Remediation
of concrete
using micro-
organisms
(1)Bacillus
Pasteurii
(2)Pseudomonas
Aeruginosa
Microbial mortar
cracks healer and to
increase the
compressive
strength of mortar
This study shows that
live bacteria has higher
increment of
compressive strength
compared to dead
bacteria.
Ghosh and
Mandal
(2006)
Development
of bioconcrete
material using
an enrichment
culture of
novel
thermophilic
anaerobic
bacteria
Escherichia coli To Increase
Strength in
Concrete Mixture
The bacteria used
contributed to the
increment of strength.
Tittelboom
et al.,
(2010)
Use of
bacteria to
repair cracks
in concrete
Bacillus
Sphaericus
Crack repair in
concrete
Bacteria protected in
silica gel healed cracks
in concrete of up to
10mm and 20mm deep.
Arunachala
m et al.,
(2010)
Studies on the
characterizatio
n of
Biosealant
properties of
Bacillus
Sphaericus
Bacillus
Sphaericus
Microbial Concrete
as Surface
Treatment
A maximum calcium
carbonate
concentration by 6µM
concentration of
bacteria was found in
this study
Jonkers
(2011)
Bacteria-based
self-healing
concrete
Bacillus Cohnii Self-healing
concrete
The bio-chemically
mediated process
resulted in efficient
sealing of sub-
milimeter sized cracks
(under 0.15mm width)
There are many types of bacteria that are used by researchers in concrete to test on
concrete properties and self-healing of concrete cracks. These bacteria used by
16
researchers are of various sources, some of soil, hot spring and many more.
Nevertheless, all these bacteria added into concrete could yield positive results not
only in terms of concrete properties, but in the increase of concrete durability.
Ramanchandran et al., (2001) studies the use of Bacillus Pasteurii and
Pseudomonas Aeruginosa which are endospore-forming soil micro-organism. From
this study, it can be concluded that the type of bacteria and amount added into the
mortar mixture influences the compressive strength of the mortar. The range
concentration used for Bacillus Pasteurii are 3.8x103, 3.8x105, 3.8x107 and 7.6x103,
7.6x105, 7.6x107. While the concentration of bacteria used for Pseudomonas
Aeruginosa are 3.8x103, 3.8x105, 3.8x107. Both bacteria were used as live and killed
bacteria culture. It was found that, the used of live bacteria increases the mortar
strength by 6.15% compared to dead bacteria. Bacillus Pasteurii added in lower
concentrations of bacteria, achieved higher compressive strength of up to 15.4%
compared to higher concentration. The bacteria used in this study are more dependent
on oxygen. Therefore, it was found that the shallow cracks healed due to the
availability of oxygen whereas the deeper cracks remained crack.
Jonkers (2011) studies of the use of Bacillus Cohnii in the self-healing of
concrete were found to be quite positive as all specimens were 100% healed with the
addition of bacteria. Relatively to the control cubes where it was found that 2 out of 6
control specimens healed. The control cubes could heal because of autonomous
healing where the 2nd hydration of concrete matrix could self-heal the concrete cube
without the addition of bacteria. In this study, the bacteria used was immobilized in
porous expanded clay particles prior to the concrete mix, this is to protect the bacteria
during the casting and hardening of the concrete. Apart from that, in this study it was
found that the bacteria added into the concrete mix in expanded clay particles were
found to last longer than direct casting.
In the case of Tittelboom et al., (2010), the use of active (live) and autoclaved
(dead) bacteria was used in concrete to compare the effect toward crack sealing. Both
active and autoclaved bacteria were added in concrete with the protection of silica gel,
the silica gel is used to protect the bacteria from the high alkalinity of concrete, as
bacteria naturally are acidic. The autoclaved and active bacteria can seal cracks. By
having active bacteria in the concrete, precipitation of calcium carbonate is seen and
therefore enhances the durability of concrete.
17
The bacteria used in Ghosh and Mandal (2006) was obtained from Jadavpur
University. The bacteria, Escherichia coli is environmentally innocuous and therefore
it is easy to handle. In this study, it was found that the enrichment culture of these
particular bacteria was able to grow inside the matrix of the concrete and the
precipitation process of calcium carbonate resulted in compressive strength
improvement. The anaerobic bacteria can sustain and grow inside the concrete matrix
without the supply of oxygen or food.
Arunachalam et al., (2010) study of Bacillus Sphaericus depicts that the
amount of calcium carbonate that this bacterium can precipitate depends on the pH
level of the medium. This bacterium optimum pH that precipitate optimum calcium
carbonate is pH 8. The result from this study showed that the use of this bacteria as a
crack healer is highly positive as the concrete samples with the use of bacteria were
found to have fully healed.
Overall, many different bacteria are used in bioconcrete study. Most of which
comes from the Bacillus family. Most researchers who study bioconcrete either
encapsulate or immobilize the bacteria to protect from the roughness of concrete
mixing and the high alkaline environment of the concrete. In this study, Enterococcus
faecalis and Bacillus sp were used due to their ability to produce calcium carbonate.
According to Mayur and Jayeshkumar (2013), bacteria generally are able to produce
calcium carbonate through either Sulphur cycle or nitrogen cycle. Enterococcus
faecalis bacterium is isolated from fresh urine. Human urine contains plenty of urea
and thus the bacteria isolated generally can produce calcium carbonate through the
nitrogen cycle. Apart from that, Bacillus sp is isolated from acid mine water which
generally contains plenty of Sulphur. Bacteria isolated from this environment would
produce calcium carbonate through the Sulphur cycle.
Both bacterium was enriched to suit the concrete environment by removing
oxygen and increasing the pH of the bacterium. Based on the Biosafety Guideline
(2010) both bacteria used in this study is in risk group 1 (RG1) which stated that
organisms in this group are unlikely to cause human disease or animal disease.
Therefore, both bacteria are not harmful and are safe to use for study.
18
2.3.2 Enterococcus faecalis
Bacteria generally come in many shapes and sizes such as spherical, rod and comma
shaped. These shapes and sizes of bacteria are commonly known as bacteria
morphology. The sizes of bacteria vary by species; bacteria are commonly 0.5- 5.0 µm
in length. Many bacterial species are either spherical (Cocci) or rod-shaped (Bacillus).
Rod-shaped bacteria, known as vibrio can be slightly curved or comma-shaped. The
variety of shapes is determined by the bacterial cell wall and cytoskeleton. It is
important because the shape of the bacteria can influence the acquirement of nutrients
by bacteria (Siddique and Chahal, 2011). The scientific names of bacteria normally
point out to the shape of the bacteria species. Therefore, Enterococcus faecalis have a
coccus shape. Enterococcus faecalis is Gram positive bacteria occurring in single, pair
or in short chains (Holzapfel and Wood, 2014). The morphology of the bacteria is
confirmed through Gram staining.
A similar species as Enterococcus faecalis is Ureolytic bacteria. This
bacterium is a commonly used in many bioconcrete studies (Dick et al., 2006; Muynck
et al., 2008; Tittelboom et al., 2010; Siddique and Chahal., 2011; Aiko et al., 2011;
Wang et al., 2012). Chahal et al., (2010) had stated that ureolytic bacteria can influence
the precipitation of calcium carbonate by the production of urease enzyme. The
production of enzymes catalyzes the hydrolysis of urea to CO2 and ammonia, which
resulted in an increase of pH and carbonate concentration in the bacterial environment.
The increase of the pH of bacteria is vital as the pH of concrete is high. Therefore, the
increase of pH of ureolytic bacteria enables the bacteria to sustain life in the concrete
matrix.
2.3.3 Bacillus sp
The genus Bacillus includes a wide variety of saprobic bacteria which are widely
distributed in the Earth’s habitats. Saprobic bacteria are bacteria that derived nutrients
from non-living or decaying organic matter. The bacillus species are Gram positive,
rod-shaped bacteria with an average size of 0.5-5 µm and are known for its versatility
in degrading complex macromolecules, Bacillus is also a common source of antibiotic.
19
This species is commonly found in soil, spores are continuously dispersed by means
of dust in water (Talaro and Chess, 2012).
According to Emilio et al., (2008), sulphate reduction bacteria which is a
similar bacterium as Bacillus sp can live in an anaerobic state, which means that this
bacterium is capable of living in environments that has no oxygen, perfect for concrete
which contains little to no oxygen within its concrete matrix. Apart from that, the
average pH range for this bacterium is pH 5.5 to 9, which is not sufficient for concrete
as concrete has high alkalinity. Therefore, for this bacterium to survive in concrete, the
level of pH was slowly raised through adding chemicals. The sulphate reduction
bacteria oxidize the sulphate existing in the water and transform it into hydrogen
sulfides in gaseous state.
Bacteria from the Bacillus species family is commonly used in bioconcrete
studies (Ramakrishnan et al., 2005; Kantha et al., 2010; Wiktor and Jonkers., 2011;
Pei et al., 2013). Ramakrishnan et al., (2005), stated that the ability of this common
soil bacterium to continuously precipitate calcium carbonate is an advantage to
concrete as the impermeable calcite layer produced improves the concrete.
2.3.4 Function of bacteria in concrete
Muynck et al., (2008) and Bang et al., (2001) have both stated in their studies that the
function of the bacteria in the concrete plays an important role by producing urease
which causes hydrolysis of urea to ammonia and carbon dioxide. The ammonia causes
the increment of pH and the hydrolysis process induces calcite precipitation. Naturally,
precipitation of calcium carbonate occurs in concrete as well, but is a lengthy process.
Thus, micro-biologically induce calcium carbonate precipitation is preferred for a
quick and effective precipitation. Salwa, (2011) argues that microbial precipitation is
faster compared to chemical precipitation.
Chemical precipitation of calcium carbonate could be controlled by the
increased or decreased of calcium ions, carbonate concentration, pH value and the
presence of nucleation sites. In terms of micro biological precipitation of calcium
carbonate the nucleation site is irrelevant as the bacteria acts as a nucleation site.
The production of the urea level of bacteria varies according to the condition
in which the bacteria are grown. When Bacillus Subtilis cells is grown in nitrogen poor
20
medium, the production of urea increases 20 to 25 times the original. This causes rapid
precipitation of calcium carbonate (Salwa, 2011). Apart from nitrogen poor
environment, Varenyam et al., (2013) studies the effect of using Bacillus pasteurii and
Pseudomonas aeruginosa in aerobic condition. It was shown to have improved
compressive strength by 18% as compared to control. In Ghosh et al., (2006) studies
on the use of an anaerobic thermophilic microorganism instead of using aerobic
microorganism, it was found to have a profound impact on the compressive strength
of up to 25% increment as compared to the control. The bacteria work by depositing
on the microorganism cell surfaces and within the pores of the cement-sand matrix
thus plugging the gaps in the matrix.
Nemati and Voordouw (2003) states that microbial cell secretes an insoluble
organic compound such as exopolysaccharides which contribute to the cementation
and plugging in natural settings. In the natural setting, the bacterium produces this
organic compound to heal cracks in highly permeable rock formations. This
biomineralization occurs through either active precipitation of carbonate
microorganism or passively by the bacteria induced changes in the chemistry of the
system. The control biological formation of calcium carbonate in the oil reserve
industry is achieved by the decomposition of urea by the catalytic action of urea
enzyme, which is achieved by adding bacteria to produce urea. The effect of
temperature toward the production of calcium carbonate was studied and was found
that temperature has no effect with the production of calcium carbonate. Whereas, it is
the concentration of urease that has an effect towards the production of calcium
carbonate. With high concentration of urea, the plugging was quite rapid and the
permeable as compared to the low concentration of urea.
In Xu and Yao (2014) study, the bacteria added into the concrete for self-
healing were coupled with organic calcium salts such as calcium lactate and calcium
glutamate as precursors to calcium carbonate. In this study, 2 methods were used to
determine the most effective self-healing which are either by external applied
treatment or a 2 component self-healing with G-Ca or L-Ca. From the result of this
study, the external applied self-healing is the most effective. L-Ca produced
significantly good results by 20% compared to control.
Although promising results are reported regarding the use of bacteria in self-
healing of concrete, Schalangen and Sangadji (2013) stated in their study that bacteria
and compounds needed for mineral precipitation are applied externally after crack
21
formation occurred. This is due to the limited lifetime of the enzymatic activity of the
applied bacteria species. In terms of realistic approach, having applied bacteria
externally to self-heal concrete naturally is not the most effective on approach. As
some cracks, may form in hard to reach places or hidden from plain-sight.
Studies have reported that bacteria functions to precipitate calcium carbonate
through enzymatic pathway. This pathway enables the bacteria to utilize the calcium
content within the concrete. Various calcium sources and bacteria are studied to find
the ultimate mix to improve engineering concrete properties and self-healing of
concrete. However, the method in which the bacterium is added into the concrete and
introduction of different concentrations of calcium source is different from researcher
to researcher in the bioconcrete community. The calcium lactate acts as a catalyst and
provides bacteria the additional calcium source to precipitate calcium carbonate.
Therefore, the importance of this study is to determine the optimum concentration
needed to have improvement in terms of engineering concrete properties and self-
healing.
2.4 The importance of calcium carbonate in concrete
Calcium carbonate formation by bacteria is a natural process, this formation aids in the
improvement of concrete. The addition of calcium lactate in bioconcrete is to facilitate
in precipitation of calcium carbonate by bacteria. Calcium carbonate is a common
mineral on earth, calcium carbonate precipitation occurs naturally to form natural
rocks such as limestones, fossiliferous and exists in many different environments
(Salwa, 2011). Limestones are used as part of the ingredient in the production of
cement along with other mixtures. The composition of concrete consists of various
materials of natural origin, such as sand, aggregate and limestone.
According to Stuckrath et al., (2014), calcium carbonate is the focus on
autogenous or self-healing of concrete due to the fact that it can be intentionally
engineered to improve the self-healing capacity of the concrete matrix. There are
several autogenous healings that concrete possesses to close small cracks or normally
cracks less than 0.05 mm wide. These are either by swelling of cement paste, hydration
of remaining unhydrated cement, precipitation of calcium carbonate crystals and crack
filling by impurities in the water or by debris from crack surface. Therefore, many
22
studies that aims to improve self-healing of concrete focuses on precipitation of
calcium carbonate.
Faiz and Steve (2014) study the effect of adding nano-CaCO3 on the
compressive strength. It was found that small percentage of nano-CaCO3 could
improve the compressive strength of the concrete and led to a denser microstructure
which changed the formation of hydration products. This contributed to early strength
and higher durability.
Calcium carbonate is formed during the process of carbonation where calcium
hydroxide chemically reacts with carbon dioxide in the air. Calcium hydroxide is
found within the concrete matrix and is brought up when moisture migrates to the
surface. Carbonation does not only occur at the surface, but also deep within the
concrete matrix. Apart from that, fresh cement paste has normally high pH value of
12.5 to 13, which is beneficial to the structure as it protects the reinforced steel from
rusting (Bjorn, 2006).
Naturally, the production of calcium carbonate in concrete lowers the pH thus
eventually causing corrosion of steel reinforcement. This in turn lowers the structural
integrity of the building. Steel reinforcement corrosion is usually due to water seeping
into cracks that forms due to loading. However, adding bacteria in concrete lessen the
chance for structural deterioration due to the bacteria pore plugging ability which
reduces the water seepage and produces calcium carbonate without reducing the pH of
the concrete. As the bacteria used in this study is enriched in alkaline condition to suit
the concrete environment.
2.5 Additional calcium nutrient source
Naturally, concrete precipitates calcium carbonate through carbonation process which
occurs over time (Bjorn, 2006). To speed up the precipitated calcium carbonate,
bacteria and calcium nutrient source is introduced in concrete. Precipitation of calcium
carbonate is one of the four elements in concrete which can be manipulated to achieve
self-healing of concrete (Stuckrath et al., 2014). Calcium nutrient source acts as an
additional food source for the bacteria to precipitate calcium carbonate at a higher rate.
This higher rate of precipitation increases the strength of concrete and aids in self-
healing of micro-cracks.
23
Several different calcium sources were used together with bacteria by other
researchers such as Xu et al., (2015), Abo-El-Enein et al., (2013) and Xu and Yao
(2014). Abo-El-Enein et al., (2013) studied the use of several different calcium sources
such as calcium chloride, calcium acetate and calcium nitrate. An improvement in
physio-mechanical properties and mortar crack remediation is found with samples
containing calcium than without any calcium source addition. Whereas Xu and Yao
(2014) studied the difference between using organic and inorganic calcium sources. It
was found that the type of calcium source has a profound impact on the crystal, form,
size and morphology of CaCO3.Calcium sources that were used were calcium
glutamate and calcium lactate which are organic calcium source and calcium chloride
which is an inorganic calcium source. It was found that organic calcium source has a
better result in calcium carbonate precipitation compared to inorganic calcium source.
Apart from that, limited amount of durability in terms of surface treatment is achieved
with the aid of bacteria alone. The addition of calcium source resulted in higher amount
of precipitated calcium carbonate, which led to a higher decrease in capillary water
absorption and carbonation.
In Jonkers (2011), expanded clay particle was used as a partial replacement in
concrete. The bacteria and calcium lactate were embedded within the expanded clay
particles. The expanded clay particles served as a partial replacement of aggregate in
normal concrete. The replacement tested in this study was a 50% replacement of
aggregate with expanded clay particle. The huge replacement caused a decrease of
compressive strength after 28th day of curing compared to control (expanded clay
particles with bacteria only) but the self-healing capacity with calcium lactate is better.
Calcium lactate is an organic calcium source. It is used in many milk, cheese
and food products. It is innocuous to human which is important as people are
surrounded in concrete environment almost 24 hours a day. Therefore, it is important
to create a durable concrete which do not have any negative effect towards the
environment and human health. Apart from that, Mayur and Jayeshkumar (2013) have
stated that the presence of calcium lactate added to the additional calcium ions which
are needed to precipitate calcium carbonate. The amount of calcium lactate in this
study was adopted from Xu et al., (2015) which used 0.025 mol/L calcium lactate.
This concentration can produce significant amount of calcium carbonate within a short
period of time. Calcium lactate used in this study are in 0.001 mol/L, 0.05 mol/L and
0.01 mol/L. The effect towards the engineering concrete properties and enhancement
24
in self-healing of concrete is studied. Calcium lactate is in the formed of liquid. It is
added as a supplementary in the water used for concrete mixing. The bacteria liquid
culture and calcium lactate is added directly to the concrete mix.
2.6 Enzymatic pathway of bacteria
Bacteria can precipitate calcium carbonate. However, different types of bacteria and
abiotic factors such as salinity and composition of medium contributes to different
ways to precipitate calcium carbonate. According to Mayur and Jayeshkumar (2013),
there are two pathways for precipitation of calcium carbonate by bacteria. The first
pathway usually involves sulphur cycle and particular sulphate reduction. This
pathway is usually carried out by sulphate reducing bacteria under anoxic condition.
Whereas, the second pathway involved nitrogen cycle which is usually carried out by
ureolytic bacteria. Bosak (2005) had stated that sulphate reduction bacteria could
precipitate calcium carbonate by an increase in alkalinity such as the equation below:
SO42- + 2CH2O HS- + 2HCO3
- + H+ (2.1)
Ca2+ + HCO3- CaCO3 + H+ (2.2)
The precipitation of calcium carbonate by sulphate reduction bacteria increases the
pH. Sulphate reduction bacteria reacts with calcium lactate which aids in increment of
calcium carbonate precipitation (Braissant et al., 2007). The enzymatic pathway of the
bacteria is the chemical reaction which occurs when bacteria is added into concrete. It
describes what the bacterium does in the concrete and the process in which calcium
carbonate is produce as a by-product of the bacteria. According to Tugba and Debora
(2014), Microbially-induced precipitation of calcium carbonate is a chemical reaction
commonly facilitated by microorganisms and is associated with sulfate reduction, urea
hydrolysis and iron reduction. The urea hydrolysis is the most studied and the
stoichiometries reactions are given as:
H2NCONH2 (Urea) + H2O 2NH3 + CO2 (2.3)
2NH3 + CO2 + H2O 2NH4+ + CO3
− (2.4)
172
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