CHARACTERIZATION OF UREOLYTIC

BACTERIA ISOLATED FROM LIMESTONE

CAVES OF SARAWAK AND EVALUATION

OF THEIR EFFICIENCY IN

BIOCEMENTATION

By

ARMSTRONG IGHODALO OMOREGIE

A thesis presented in fulfilment of the requirements for the

degree of Master of Science (Research)

Faculty of Engineering, Computing and Science

SWINBURNE UNIVERSITY OF TECHNOLOGY

2016

i

ABSTRACT

The aim of this study was to isolate, identify and characterise bacteria that are capable

of producing urease enzyme, from limestone cave samples of Sarawak. Little is known

about the diversity of bacteria inhabiting Sarawak’s limestone caves with the ability of

hydrolyzing urea substrate through urease for microbially induced calcite precipitation

(MICP) applications. Several studies have reported that the majority of ureolytic

bacterial species involved in calcite precipitation are pathogenic. However, only a few

non-pathogenic urease-producing bacteria have high urease activities, essential in MICP

treatment for improvement of soil’s shear strength and stiffness.

Enrichment culture technique was used in this study to target highly active urease-

producing bacteria from limestone cave samples of Sarawak collected from Fairy and

Wind Caves Nature Reserves. These isolates were subsequently subjected to an

increased urea concentration for survival ability in conditions containing high urea

substrates. Urea agar base media was used to screen for positive urease producers

among the bacterial isolates. All the ureolytic bacteria were identified with the use of

phenotypic and molecular characterizations. For determination of their respective urease

activities, conductivity method was used and the highly active ureolytic bacteria

isolated comparable with control strain used in this study were selected and used for the

next subsequent experiments in this study. Effects of cultural conditions on urease

activity and evaluation of biocementation potential of these locally selected ureolytic

isolates were also performed.

Out of the ninety bacteria subcultured from enriched cultures containing the cave

samples, thirty-one bacterial isolates were selected based on their respective abilities of

producing urease enzyme by completely turning the colour of urea agar base medium

from yellow to pink in comparison to other isolated urease producing bacteria and the

control strain (Sporosarcina pasteurii, DSM33) used in this study. The microscopic

analysis using Gram staining technique showed that majority of the bacterial isolates

were Gram-positive bacteria while only three of the isolates were Gram-negative

bacteria. In addition, majority of the bacterial cells were rod-shaped except for one

bacterial isolate which was a coccus. Endospore staining test results indicate also

indicated that all except one isolate were spore forming bacteria.

ii

The BLAST results from molecular characterization of the ureolytic isolates suggested

that they were closely related to bacteria from the Sporosarcina pasteurii group,

Pseudogracilibacillus auburnensis group, Staphylococcus aureus group, Bacillus lentus

group, Sporosarcina luteola group and Bacillus fortis group when compared to the 16S

rRNA sequencing data in NCBI nucleotide BLAST database.

Specific urease activity determination from the calculation of conductivity and urease

activity showed that out of all the bacterial cultures, bacterial isolates designated as

NB33, LPB21, NB28, NB30 and the control strain had 19.975, 23.968, 19.275, 20.091

and 17.751 mM urea hydrolysed.min-1.OD-1 respectively, suggesting they had the

highest specific urease activities when compared to the rest isolates. The effect of

cultural conditions on urease activities involving the aforementioned local isolates and

control strain showed that incubated these conditions: at 25 to 30oC; pH 6.5 to 8.0;

incubation period at 24 hr; and urea concentration of 6 to 8%, maximum specific urease

activities for the selected ureolytic bacteria isolates and control strain were obtained.

The biocement treatment test using isolates NB33, LPB21, NB28, NB30 and the control

strain on poorly graded soil clearly showed that MICP is microbially induced and not

chemically induced. The results presented in this study showed that out of all the sand

columns treated, all except the columns containing negative control (only cementation

solution) had calcium carbonate precipitation shown on the top surfaces of their

respective columns. Each column treated with microbial cultures and cementation

solution (containing 1 M or urea and CaCl2) were able to bind the sand particles

together. However, it was observed that there was higher cementation level at positions

close to the injection points which resulting in more calcite contents to be obtained at

this layers of the biocemented sands. Based on the surface strength using penetrometer

test and compressive strength using UCS test, samples treated with isolates LPB21 and

NB28 showed significant strengths when compared to other isolates, consortia, and the

control strain. However, the rest isolates showed similar performance with the control

strain. The application of these newly isolates highly active ureolytic bacteria can be

used to for other MICP treatments in civil and geotechnical industries. The findings in

this study suggest that the isolated ureolytic bacteria (NB28, LPB21, NB33, and NB30)

have the potential to be used as alternative microbial MICP agents for biocement

applications.

iii

ACKNOWLEDGEMENT

Foremost, I would like to express my deepest gratitude to my principal coordinating

supervisor: Assoc. Prof Dr Peter Morin Nissom (Associate Dean, Science) for all the

valuable discussion, brainstorm, helpful advice, critics, challenges and encouragements

throughout this research study. His overwhelming supervision made me develop new

insights and ideas during this research. His quest for “high-quality work”, made me stay

active, focused and enthusiastic. He also provided critical reviews of my experiments

and writing, prompting me to improve problem solving and writing skills. I would also

like to thank my associate supervisor: Dr Irine Runnie Ginjom for her insightful

discussion and comments on my experimental progress. Her invaluable advice, co-

supervision, and encouragement throughout this study helped made this thesis a

success.

I would like to gratefully acknowledge Assoc. Prof Dr Dominic Ek Leong Ong

(Director, Swinburne Sarawak Research Centre for Sustainable Technologies) and Dr

Ngu Lock Hei (Course coordinator, Chemical Engineering Department) for their

financial support (SSRG) used to partially fund my research project. I am thankful for

the continuous moral support and helpful discussion from Assoc. Prof Dr Dominic Ek

Leong, especially with the idea of going to the caves to screen for calcite-precipitating

microorganisms.

I extend my appreciation to Sarawak Biodiversity Centre (SBC) and Sarawak Forestry

Department (SFD) for issuing the permits (SBC-RA-0102-DO and NCCD.907.4.4

[JLD.11]-37) which enabled me to collect samples from Fairy Cave (N 01°22’53.39” E

110°07’02.70”) and Wind Cave (N 01°24’54.20” E 110°08’06.94”) Nature Reserves,

located in Bau, Kuching Division, Sarawak, Malaysia. The collection of the samples

from these extreme environments to conduct biological research stipulated the

potentials of screening, identifying and characterising highly active isolated ureolytic

bacteria. I am thankful to Dr Paul Mathew Neilsen, Associate director of graduate

studies and research education. His thoughtful guidance and warm encouragement,

especially during my confirmation of candidature helped make me achieve my research

goals. I am sincerely grateful for his continual willingness of finding time out of his

busy schedule to meet me and discuss on how I could tackle research challenges and

improve my research study.

iv

I would also like to acknowledge Assist. Prof Salwa Al-Thawadi, Dr Ralf Cord-

Ruwisch, PD Dr David Schleheck and Assist. Prof Leon van Paassen for providing

indispensable guidance on how to measure urease activity, the appropriate way of

determining specific urease activity and selective investigation of cultural conditions on

urease activities. I am very thankful for taking your time to reply my inquiries via

emails and researchgate.net.

I am thankful to the science laboratory officers and technicians: Chua JiaNi, Nurul

Arina Salleh, Cinderella Sio and Marclana Jane Richard, for providing me with

experimental materials and allowing me to make use of some apparatus during the

course of my research study. Without their enormous assistance, my research would not

have been completed on time. An exceptional gratitude goes to Hasina Mohammed

Mkwata for being a helpful research lab mate and an amazing girlfriend. Her assistance

while I carried out my experiment, specifically during the measurement of conductivity,

biomass concentration and effect of cultural conditions on urease activity made my

experiments very convenient. I also extend my appreciation to Ghazaleh

Khoshdelnezamiha for playing a significant role during the in vitro biocement test. Her

efforts and a keen interest in my research made my experiment successful. An extensive

appreciation goes to Dr Noreha Mahidi and Holed Juboi for their vehement assistance

during molecular characterization of the isolated ureolytic bacteria. It was a pleasure

working with her. Big thanks also go to my fellow lab colleagues: Nurnajwani Senian

and Ye Li Phua, for providing assistance during sample collection and when I

conducted my experiments in the laboratory.

I would like to thank my amazing parents: Mr Cletus and Mrs Margaret Omoregie, for

their amazing love, care, patience and their financial supports used to partly fund my

research. Their sacrifices in sponsoring my postgraduate study are forever appreciated. I

also warmly appreciate my siblings: Jennifer, Sharon, and Thelma, for their tender

affection and supports during the years I conducted my experiments and wrote on my

thesis. I am obsequiously grateful to God Almighty for all the blessings and abundances

bestowed on me and for making my MSc research a success.

v

DECLARATION

I hereby declare that this research entitled “Characterization of ureolytic bacteria

isolated from limestone caves of Sarawak and evaluation of their efficiency in

biocementation” is original and contains no material which has been accepted for the

award to the candidate of any other degree or diploma, except where due reference is

made in the text of the examinable outcome; to the best of my knowledge contains no

material previously published or written by another person except where due reference

is made in the text of the examinable outcome; and where work is based on joint

research or publications, discloses the relative contributions of the respective workers or

authors.

(ARMSTRONG IGHODALO OMOREGIE)

DATE: 06 June 2016

In my capacity as the Principal Coordinating Supervisor of the candidate’s thesis,

I hereby certify that the above statements are true to the best of my knowledge.

(ASSOCIATE PROFESSOR DR. PETER MORIN NISSOM)

DATE: 06 June 2016

vi

SCIENTIFIC OUTPUT

PUBLICATIONS

Omoregie, AI, Senian, N, Ye Li, P, Hei, NL, Leong, DOE, Ginjom, IRH & Nissom, PM, 2016, 'Screening for Urease-Producing Bacteria from Limestone Caves of Sarawak', Borneo Journal of Resource Science and Technology, 6 (1): 37-45. Omoregie, AI, Senian, N, Ye Li, P, Hei, NL, Leong, DOE, Ginjom, IRH & Nissom, PM, 2016, ‘Ureolytic Bacteria isolated from Sarawak Limestone Caves show High Urease Enzyme Activity comparable to that of Sporosarcina pasteurii (DSM 33)’, Malaysian Journal of Microbiology. (in press). CONFERENCE PAPERS AND PROCEEDINGS

Omoregie, AI, Senian, N, Li, PY, Hei, NL, Leong, DOE, Ginjom, IRH & Nissom, PM, 2015, 'Isolation and Characterization of Urease Producing Bacteria from Sarawak Caves and Their Role in Calcite Precipitation,' International Congress of the Malaysian Society for Microbiology (ICMSM2015), Malaysian Society for Microbiology, pp. 16-21. Senian, N, Omoregie, AI, Peter Morin Nissom, Ngu, L-H & Ong, DEL, 2014, 'Identification of locally found bacteria for potential use in ground improvement works by microbially induced calcite precipitation (MICP) technique,' The 19th International Conference on Transformative Science and Engineering, Business and Social Innovation, Society for Design and Process Science, pp. 261-266. Omoregie, AI, & Nissom, PM, 2016, ‘Cross disciplinary research: developing biocement applications using local bacteria’, The fourth Borneo Research Education Conference, Universiti Teknologi Mara Sarawak, pp. 1-8. Senian, N, Khoshdelnezamiha, G, Omoregie, AI, Ong, DEL, Ngu, LH, Nissom, PM & Henry-Ginjom, IR, 2016, ‘Development of Bio-Pavers with Microbial Induced Calcite Precipitation Technique Using Sporosarcina Pasteurii,’ 19th Southeast Asian Geotechnical Conference & 2nd Association of Geotechnical Societies in SouthEast Asia Conference, Malaysian Geotechnical Society, pp. 327-331. Phua, YL, Omoregie, AI, Ong, DEL, Ngu, LH, Nissom, PM & Ginjom, IR, 2016, ‘Ground improvement via Microbial-Induced Calcite Precipitation using Push-Pull Injection System’, 19th Southeast Asian Geotechnical Conference & 2nd Association of Geotechnical Societies in SouthEast Asia Conference, Malaysian Geotechnical Society, pp. 495-498.

vii

PRESENTATIONS

Oral presenter, Cross disciplinary research: developing biocement applications using local bacteria, The fourth Borneo Research Education Conference (BREC), 17-18 August 2016, Kota Samarahan, Sarawak, Malaysia Poster presenter, Isolation and Characterization of Urease Producing Bacteria from Sarawak Caves and Their Role in Calcite Precipitation, International Congress of the Malaysian Society for Microbiology, 7-10 December 2015, Batu Ferringhi, Penang, Malaysia. Oral presenter, Isolation of Highly Active Urease Producing Bacteria from Sarawak Limestone Caves, The Regional Taxonomy and Ecology Conference, 1-2 December 2015, Kuching, Sarawak, Malaysia. Poster presenter, Isolation and Characterisation of Urease Producing Bacteria from Sarawak Caves and their Role in Calcite Precipitation, Asian Congress on Biotechnology, 15-19 November 2015, Kuala Lumpur, Selangor, Malaysia. AWARDS

BEST PAPER Awarded for the best paper written at the 4th Borneo Research Education Conference (BREC 2016), organised by Universiti Teknologi Mara Sarawak and Swinburne University of Technology, Sarawak campus. 17-18 August 2016, Kota Samarahan, Sarawak, Malaysia. http://www.sarawak.uitm.edu.my/brec2016 PEOPLE’S CHOICE AWARD Awarded for being one of the best oral presenters at the Three Minute Thesis (3MT) Competition organised by Swinburne University of Technology, Sarawak campus. 17 June 2015, Kuching, Sarawak, Malaysia. http://www.swinburne.edu.my/events/3MT-competition BEST POSTER PRESENTER Awarded best poster presenter for the technical session of environmental biotechnology at the Asian congress on biotechnology organised by Asian federation of biotechnology Malaysia Chapter and Universiti Putra Malaysia. 15-19 December, Kuala Lumpur, Selangor, Malaysia. http://www.acb2015.my/web/list-of-acb2015-winners

viii

TABLE OF CONTENTS Content Page

ABSTRACT i

ACKNOWLEDGEMENT iii

DECLARATION v

SCIENTIFIC OUTPUT vi

TABLE OF CONTENTS viii

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF ABBREVIATIONS xiv

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction 1

1.2 Biomineralisation 3

1.2.1 Biologically induced biomineralisation 4

1.2.2 Biologically controlled biomineralisation 5

1.3 Microbially Induced Calcite Precipitation (MICP) 6

1.3.1. MICP via urea hydrolysis 10

1.3.2. Urease enzyme 12

1.3.3. Mechanism of CaCO3 precipitation 15

1.3.4. Urease Source 17

1.4 Factors Affecting the Efficiency of MICP 18

1.4.1. Concentration of reactants 18

1.4.2. pH 19

1.4.3. Temperature 20

1.4.4. Dissolved inorganic carbon 21

1.4.5. Bacteria size 21

1.4.6. Nutrients 22

1.4.7. Availability of nucleation site 22

1.5 Current Biotechnological Application of MICP 23

1.5.1. Biocementation 24

1.5.2. Creation of biological mortars 24

1.5.3. Bioremediation of cracks in concrete 25

1.5.4. Biodeposition on cementitious materials 27

1.5.5. Biogrout 28

1.5.6. Other essential applications of MICP 30

ix

1.6 Diversities of Microbial Communities in Caves 32

1.7 Screening Sarawak’s Limestone Caves for Ureolytic Bacteria 36

1.8 Aim and Objectives of the Study 39

1.9 Significance of the Study 39

1.10 Thesis Outline 39

CHAPTER 2: ISOLATION, IDENTIFICATION AND CHARACTERISATION OF

UREASE-PRODUCING BACTERIA FROM LIMESTONE CAVES OF SARAWAK

2.1 Introduction 41

2.2 Methods and materials 43

2.2.1. Sampling location and collection 43

2.2.2. Biological material 43

2.2.3. Growth medium and sterilisation 43

2.2.4. Enrichment cultures 44

2.2.5. Isolation of urea degrading bacteria 44

2.2.6. Screening for urease-producing bacteria 45

2.2.7. Preliminary identification 45

2.2.8. Molecular identification 46

2.2.9. Measurement of enzyme activity 48

2.2.10. Evaluation of microbial calcite precipitation 49

2.2.11. Bacterial growth profile and pH profile 50

2.2.12. Statistical analysis 51

2.3 Results 52

2.3.1. Sampling location and sample collection 52

2.3.2. Enrichment culturing and bacterial isolation 54

2.3.3. Selection of urease producing bacteria 55

2.3.4. Phenotypic characterisation 58

2.3.5. Molecular characterization 62

2.3.6. Measurement of conductivity 69

2.3.7. Urease Activity Assay 69

2.3.8. Determination of specific enzyme activity 73

2.3.9. Microbial calcite precipitates 77

2.3.10. Calcite estimation 78

2.3.11. Bacterial growth and pH profiles 80

2.4 Discussion 85

2.5 Conclusion 92

x

CHAPTER 3: EFFECTS OF CULTURAL CONDITIONS ON UREASE ACTIVITY

AND EVALUATION OF BIOCEMENTATION POTENTIALS IN SMALL SCALE

TEST

3.1 Introduction 93

3.2 Methods and Materials 94

3.2.1. The Effect of Cultural Conditions On Urease Activity 94

3.2.2. Small Scale Biocementation Test 95

3.3 Results 100

3.3.1. Temperature (oC) 100

3.3.2. Initial medium pH 102

3.3.3. Incubation period (hr) 104

3.3.4. Effect of urea concentration (%) 106

3.3.5. Biocementation treatment test 108

3.3.6. Soil surface strength 115

3.3.7. Compressive strength 117

3.3.8. Calcite confirmation 119

3.3.9. Calcite content Determination 120

3.4 Discussion 123

3.5 Conclusion 131

CHAPTER 4: GENERAL CONCLUSIONS AND RECOMMENDATIONS

4.1 General Conclusion 132

4.1.1. Aim of the thesis 132

4.1.2. Limestone area as source of ureolytic bacteria 133

4.1.3. Enrichment culture and isolation 134

4.1.4. Screening and identification 134

4.1.5. Measurement of urease activity 135

4.1.6. Biocementation competency of local isolates 136

4.2 Future Directions and Recommendations 136

REFERENCES 138

xi

LIST OF TABLES

Table Page

2.1 Description of samples collected from FCNR and WCNR 52

2.2 Hydrolysis of urea by isolates UAB medium 57

2.3 Morphological characteristics of isolated bacterial colonies 59

2.4 Microscopic characteristics of bacterial isolates 60

2.5 Biochemical characteristics of bacterial isolates 61

2.6 Molecular identification based on 16S rRNA sequencing data using NCBI

nucleotide BLAST database

64

2.7 The nomenclatural taxonomy obtained using Ribosomal Database Project-

II database

66

2.8 Measurement of conductivity variation rate and SEM 71

2.9 Conversion of changes in conductivity to urease activity 72

2.10 t-test results comparing the specific urease activity differences

between individual isolated urease-producing bacteria and control strain

76

2.11 t-test results comparing the calcite precipitate differences between

individual isolated urease-producing bacteria and control strain

79

2.12 Kinetics growth of ureolytic bacteria in batch cultures 81

3.1 Selected ureolytic bacteria for biocement test 95

3.2 Biocement treatment components 96

3.3 Sand characteristics 97

3.4 Sand grain size characteristics 109

3.5 Bacteria concentration and urease activity prior to biocement test 110

3.6 t-test results comparing the strength (psi) differences between the

biocemented sands

116

3.7 Unconfined compressive strength (UCS) of the treated sands 117

3.8 t-test results comparing the unconfined compressive strength (UCS)

differences between the biocemented sands

118

3.9 Summary of calcite content and compressive strength of selected

isolates and consortia

121

xii

LIST OF FIGURES Table Page

1.1 Pathway of biominerals secretion and precipitation in a bacterial cell 11

1.2 Genetic organisation of urease operon in Helicobacter pylori and

Sporosarcina pasteurii

13

1.3 Regulation levels for enzyme activity by microorganisms 14

1.4 A simplified representation of Ureolysis-driven CaCO3 precipitation 16

1.5 An in situ application of bacteria based liquid 25

1.6 Self-healing crack from the addition of bacterial metabolism via urea

hydrolysis

26

1.7 1 mm thick calcite crust formed on the surface of the soil 27

1.8 Set-up for large scale (100m3) soil treatment 29

1.9 Calcified structures of biogenic origin discovered in cave regions 34

1.10 Speleothems samples collected from El Toro and El Zancudo limestone

mines located in Cordillera Central, northeast

of Colombia

35

1.11 Map of Borneo Island showing the geographical divisions and

topographical features of Brunei Darussalam, Indonesia (Kalimantan) and

East Malaysia (Sarawak and Sabah)

38

2.1 Sampling collection site situated in FCNR, Bau, Sarawak 53

2.2 Sampling collection site situated in WCNR, Bau, Sarawak 53

2.3 Microorganisms grown on nutrient agar plates supplemented with 2% urea 54

2.4 Pure colonies of urea degrading bacteria after enrichment culture 55

2.5 Urease production test using UAB medium 56

2.6 Phylogenetic tree based on the bacterial 16S rRNA gene sequence data

sequence from different isolates of the current study along with sequences

available in the GenBank database

68

2.7 Relative conductivity of isolate LPB21 measured for a duration of 5 min 70

2.8 Specific urease activity (mM urea hydrolysed.min-1.OD-1) of urease-

producing bacteria and the control strain

75

2.9 Calcite precipitation media 77

2.10 Comparison of calcite precipitated by selected UPB isolates and the control

strain

78

xiii

2.11 Growth profile of selected ureolytic bacterial isolates and control strain

grown in nutrient broth containing 6% urea for 12 hr

82

2.12 pH profile of selected ureolytic bacterial isolates and control strain grown

in nutrient broth containing 6% urea for 12 hr

83

3.1 The effect of different temperature on urease activity 101

3.2 The effect of different pH on urease activity 103

3.3 The effect of different incubation period on urease activity 105

3.4 The effect of different urea concentration on urease activity 107

3.5 Treatment of sand column using locally isolated bacteria, consortia,

positive and negative controls

111

3.6 Sand columns at the end of treatment using ureolytic bacteria and

cementation solution

112

3.7 Treated sand removed from their respective columns 113

3.8 Treated sand sample held after a curing period and columns were

successfully removed

114

3.9 Surface strength of the biocemented sand samples 115

3.10 Confirming calcite precipitates 119

3.11 Comparison of the relative quantity of calcites in the biocemented sands 120

xiv

LIST OF ABBREVIATIONS MICP Microbially Induced Calcite Precipitation

BIM Biologically Induced Mineralisation

BCM Biologically Controlled Mineralisation

DIC Dissolved Inorganic

IAP Ion Activity Product

UDB Urea Degrading Bacteria

UPB Urease Producing Bacteria

UAB Urea Agar Base

FCNR Fairy Cave Nature Reserve

WCNR Wind Cave Nature Reserve

PCR Polymerase Chain Reaction

TE Trix EDTA

NCBI National Centre for Biotechnology Information

DNA Deoxyribonucleic Acid

BLAST Basic Local Alignment Search Tool

RDP Ribosomal Database Project

MEGA Molecular Evolutionary Genetic Analysis

CPM Calcite Precipitating Media

df Dilution Factor

ATP Adenosine Triphosphate

SUA Specific Urease Activity

UA Urease Activity

HCL Hydrochloric Acid

NaOH Sodium Hydroxide

UCS Unconfirmed Compression Strength

ATSM American Society for Testing and Materials

xv

RH Relative Humidity

SEM Standard Error of Mean

SE Standard Deviation ANOVA

Analysis of Variance

Chapter

1 INTRODUCTION AND LITERATURE REVIEW

1

1.1 Introduction Enzyme technology is a well-established branch of biotechnology undergoing a

development phase (Binod et al., 2013), and their functional significance suggests many

novel application especially for environmentally-friendly industrial purposes (Binod et

al., 2013). Enzymes from microorganisms are an essential source of numerous

industrially relevant enzymes (Ibrahim, 2008). Microbial enzymes are relatively more

stable and properties more diverse than other enzymes derived from plants and animals

(Alves et al., 2014). Enzymes produced from microorganisms can be easily controlled

physiologically, physio-chemically, have quantitative production and mostly extracted

with low production cost extracellularly using downstream processes (Ibrahim, 2008,

Pandey et al., 2010). The industrial usage of the microbial enzymatic process are

classified as (i) Enzymes as final products; (ii) Enzymes as processing aids; (iii)

enzymes in food and beverage production; (iv) Enzymes in genetic engineering and (v)

Enzymes as an industrial biocatalyst (Binod et al., 2013).

Microbially induced calcite precipitation (MICP) is a comparatively innovative soil

improvement technique which requires the production of urease enzyme from bacteria

for soil treatment (Soon, 2013). Modern ground improvement techniques have become

increasingly complex due to sustainability consideration and the expedition of reducing

environmental pollution (Kavazanjian and Hamdan, 2015). Established materials and

methods often require replacement or supplemented by innovative materials which are

environmentally friendly (Kavazanjian and Hamdan, 2015). Existing ground

improvement techniques such a chemical grouting has been proven to have an effective

performance in the increment of soil’s shear strength and stiffness, however,

environmental and human health concerns over their applications have deemed them as

unsustainable materials (DeJong et al., 2010). Portland cement is a major construction

material of choice in building, structure and ground improvement applications in order

to meet the increasing demand of rapid industrialisation and urbanisation (Siddique et

al., 2016). However, the use of Portland cement is associated with certain challenges

such as energy , resource conservation, the cost of production and greenhouse gas

emission (Kavazanjian and Hamdan, 2015). It is estimated that production of Portland

cement clinker solely contributes about 7% global CO2 emission, this makes this

construction material an unsustainable construction material (Jonkers et al., 2010).

2

MICP has been exploited in recent decades as an alternative building material to

Portland cement through either direct substitution or complementary usage

(Kavazanjian and Hamdan, 2015, DeJong et al., 2013). MICP applications require lesser

energy for production, low production cost and no contribution to the greenhouse gas

emission, making it an environmentally friendly construction material (Achal, 2015).

Existing research studies suggests that biocementation technology can be used to

address important geotechnical problems in granular soils which include slope stability,

erosion, stiffness and stress-permeability, tunnelling and liquefaction (van Paassen et

al., 2010, DeJong et al., 2010, DeJong et al., 2011).

Bacteria acts as primary agents of geochemical changes due to their high surface area to

volume ratio, their widespread abundant distribution, evolutionary adaptiveness, diverse

enzymatic and nutritional possibilities (Warren and Haack, 2001). Numerous microbial

species from extremely diverse environments have been linked to the process of

microbial precipitation of calcium carbonate (Hammes, 2003). Calcium carbonate is the

most reactive mineral on earth, composing 4% of the earth’s weight (Whiffin, 2004), it

is constantly involved in processes of dissolution and precipitation (Hammes et al.,

2003b, Hammes and Verstraete, 2002). Carbonaceous minerals are frequently found in

oceans, soils, and geological formations, representing an important segment of the

global carbon pool (Hammes, 2003). The primary role of bacteria in calcium carbonate

precipitation has been subsequently ascribed to their capability to create an alkaline

environment through numerous biological and chemical activities (Fujita et al., 2000,

Castanier et al., 2000, Castanier et al., 1999). Characterisation of microorganisms by

genera and species which were previously unachievable through biochemical methods

alone are now being executed with the use of sequence-classifier algorithms (Ercole et

al., 2007). The ease in microbial identification using traditional and molecular

methodology can aid in understanding and identify wider ranges of the microorganism

of a given community (Rajendhran and Gunasekaran, 2011), with the capability of

producing urease enzyme, and induce microbial calcite sufficient for MICP

applications.

3

1.2 Biomineralisation

Biomineralisation is the reformation of chemicals (Anbu et al., 2016) in a

microenvironment caused by the activity of microorganisms which result in the

precipitation of minerals (Phillips et al., 2013, Barkay and Schaefer, 2001, Stocks-

Fischer et al., 1999). In nature, biomineralisation results in the formation of sixty (or

more) various biological minerals, which exists as extracellular or intracellular

inorganic crystals, although some precipitation of inorganic minerals contains trace

elements of organic compounds (Dhami et al., 2013b, Yoshida et al., 2010, Konishi et

al., 2006). It is anticipated that the number of biominerals formed will continue to

increase (Defarge et al., 2009).

Biominerals are distinguished based on their properties such as size, shape, crystalline

nature and elemental composition (isotopes and trace) (Sarayu et al., 2014). Minerals

which are formed through biologically induced mineralisation, through passive surface-

mediation includes iron (Fe), manganese (Mn), carbonates, phosphonates and silicates.

Calcium carbonate (CaCO3) is a biomineral widely secreted by most microorganisms

(Sarayu et al., 2014, Barabesi et al., 2007). Calcium carbonate mineralisation can be

found in natural formations such as corals, ant hills or caves (Dhami et al., 2013d). Out

of the eight polymorphs of calcium carbonate, seven are crystalline and one is

amorphous (Weiner and Dove, 2003). Calcite, aragonite, and vaterite are pure calcium

carbonate, while two-monohydrocalcite and the stable form of amorphous calcium

carbonate contain one water molecule per calcium carbonate (Weiner and Dove, 2003),

however, the temporary forms of amorphous calcium carbonate do not contain water

(Addadi et al., 2003).

Carbonate minerals precipitated by microorganisms contributes about 50% of the total

biominerals formed, while phosphate minerals contribute 25% of the precipitated

minerals by microbial species (Sarayu et al., 2014). These minerals are usually formed

in high quantities and widespread in nature (Ramesh Kumar and Iyer, 2011, Weiss et

al., 2002). Biominerals have unusual morphologies as they are often defined by the

complexity and variety of secreting microorganisms (Bazylinski and Frankel, 2003).

4

Biomineralisation process is divided into two different fundamental groups which are

based on the degree of their biological control (Sarayu et al., 2014). These groups are

known as biologically induced and biologically controlled mineralisation (Weiner and

Dove, 2003). Lowenstam (1981) introduced these two groups as “biological induced”

and “organic matrix-mediated” mineralisation, however, the latter was renamed by

Mann (1983) to “biologically controlled mineralisation”, recognising that the process of

biomineralisation within these conversions varies with different microorganisms.

1.2.1 Biologically induced biomineralisation

Biologically induced mineralisation (BIM) involves the interaction of the environment

and biological activities resulting in mineral precipitation (Sarayu et al., 2014). In this

type of situation, microbial cell surfaces often act as a causative agent for nucleation

and subsequent growth of the minerals (Weiner and Dove, 2003). These type of

biominerals are often secreted to the metabolism of the microorganisms, and the

systems have little or no control over the minerals which are being deposited (Sarayu et

al., 2014). The precipitation of extracellular by-product of the microbial metabolism can

lead to random crystallisation and non-specific crystal morphologies (Provencio and

Polyak, 2001).

The organelles of these microbes take part in the process of BIM, the cell wall acts as

nucleation sites (Sarayu et al., 2014). Once these biominerals are synthesized, the pH,

CO2, and composition of the microenvironments of the microorganisms are often altered

and any changes in the microorganisms will adversely have an effect on the secreted

biominerals because the whole process of BIM depends primarily on the circumstances

prevailing in the microorganism (Frankel and Bazylinski, 2003, Tebo et al., 1997,

Fortin et al., 1997). BIM process results in engulfment of the whole cell of the

microorganisms by biominerals secretions, which causes an encrustation (Sarayu et al.,

2014). The distinctive feature of BIM is that biominerals, when deposited are usually

formed along the surfaces of the microbial cells where they remain firmly attached to

the cell wall and organic components of the cell wall (lipids, proteins, and

polysaccharide) can influence the process in BIM (Mann, 2001).

5

1.2.2 Biologically controlled biomineralisation

Biologically controlled mineralisation (BCM) due to cellular activities of

microorganism are classified into extracellular, intercellular and intracellular

participations of the microbes (Sarayu et al., 2014). In extracellular participation,

macromolecular matrix (made up of proteins, polysaccharides, and glycoproteins)

situated outside the cell acts as the site of mineralisation, which is related to BIM

(Sarayu et al., 2014). The genes which are responsible play effective roles in

determining the structures and compositions which are integrated with the regulation

and organisation of the composite formation (Weiss et al., 2002). The matrix

composition is unique and contains a high proportion of acidic amino acids (Swift and

Wheeler, 1992).

The structures and compositions are genetically programmed to execute vital regulating

roles which result in composite biominerals formation (Weiner and Dove, 2003). The

intercellular participation is seen in a microorganism that lives as communities (Sarayu

et al., 2014). The minerals which are secreted by these microbes nucleates in the

epithelial cells and fill the intercellular space in a particular orientation which resembles

an exoskeleton (Young and Henriksen, 2003). The intracellular involvement is an

extremely controlled mechanism which precipitates minerals that direct the nucleation

of the biominerals inside the cells, these compositions are then governed by the

environments insides the vesicles or vacuoles usually determined by the specificity of

the species (Rodriguez-Navarro et al., 2012). Some of the species-specific

crystallochemical properties include uniform particle sizes, high level of spatial

organisation, complex morphologies, and well-defined structure and composition

(Mann, 2001).

6

1.3 Microbially Induced Calcite Precipitation (MICP)

Natural lithification of sediment occurs due to physical, chemical and biological

processes (Gadd, 2010) which result in deposition of minerals in the sediments, these

minerals compact the sediments together, reducing pore space together, eliminating

water permeability and causing cementation to occur (Paassen, 2009). However,

production of these minerals which results in a compartment of sediments undergoes a

very slow process (Paassen, 2009). On the other hand, mineralization using biological

process can accelerate cementation, the microorganisms (when supplied with suitable

substrates) are able to catalyse chemical reactions leading to a dissolution or

precipitation of inorganic minerals which aids in changing the properties of soil

(Paassen et al., 2009, Paassen, 2009).

Microbially induced calcite precipitation (MICP) is a process that refers to calcite

precipitation from a supersaturated solution in a microenvironment that occurs due to

the occurrence of microbial and biochemical activities (Hamilton, 2003, Bosak, 2011,

Anbu et al., 2016). MICP utilises the biologically induced pathway of biomineralisation

(Whiffin et al., 2007, Whiffin, 2004). During MICP process, microorganisms are able to

produce metabolic products (CO32-) that react with ions (Ca2+) in the microenvironment

which results in consequent minerals precipitated (Anbu et al., 2016). The ability of

microorganisms to induce biomineralisations, both in natural and laboratory conditions

are influenced by the type of microbes involved (Dhami et al., 2012a), salinity and

compositions of nutrients available in the microenvironments (Rivadeneyra et al., 2004,

Knorre and Krumbein, 2000).

CaCO3 is one of the utmost prevalent minerals on earth, mostly found in rocks, fresh or

marine water and soils (Castanier et al., 1999, Ehrlich, 1998). CaCO3 precipitation

occurs usually when the amount of calcium and carbonate ions in the solution exceeds

the product solubility (Cheng, 2012). Comparing contributions of abiotic change such

as a change in temperature, pressure or evaporation and biotic action which involves

microbial activity, it is suggested that biotic actions have a greater level of contribution

in inducing CaCO3 precipitates in most environments on earth (Castanier et al., 2000).

7

CaCO3 precipitation is a rather straightforward chemical process often governed by four

main key factors (Dhami et al., 2013b): (1) the calcium concentration, (2) the

concentration of dissolved inorganic carbon (DIC), (3) the pH and (4) the availability of

nucleation sites (Hammes and Verstraete, 2002). CaCO3 precipitation requires

sufficient calcium and carbonate ions so that the ion activity product (IAP) exceeds the

solubility constant (Kso) as shown in Equations (1.1) to (1.3) (Dhami et al., 2014, Dhami

et al., 2013b). From the comparison of the IAP with the Kso , the saturation state (Ω) of

the system can be defined; if Ω > 1 (Dhami et al., 2014), then an oversaturation and

precipitation will occur in the system as mentioned below by Morse (1983):

(1.1) Ca2+ + CO3

2- ↔ CaCO3

(1.2) Ω = a (Ca2+) a (CO32-) / Kso

(1.3) with Kso calcite, 25oC = 4.8 x 10-9

As previously mentioned, the concentration of DIC and the pH of the microenvironment

influences the concentration of carbonate ions (Dhami et al., 2014, Dhami et al., 2013b).

However, DIC concentration relies on environmental parameters such as temperature

and partial pressure of carbon dioxide for the systems which are exposed to the

atmosphere (Cheng, 2012, Dhami et al., 2013b). The equilibrium reactions and constant

which governs the DIC concentration in aqueous media (25oC and 1 atm) are given in

Equations (1.4) to (1.8) as suggested by Stumm and Morgan (1981):

(1.4) CO2 (g) ↔ CO2 (aqueous) (pKH = 1.468)

(1.5) CO2 (aqueous) + H2O ↔ H2CO3 (pK= 2.84)

(1.6) H2CO3 ↔ H+ + HCO3- (pK1 = 6.352)

(1.7) HCO3− ↔ CO32− + H+ (pK2 = 10.329)

(1.8) With H2CO3 = CO2(aqueous) + H2CO3

8

CaCO3 precipitation is very slow under normal conditions which require a long

geological time, however, MICP can produce a large amount of carbonate in shorter

duration (Dhami et al., 2013b). Exploratory research involving MICP has gained an

increased interest in the last 20 years, with the primary focus of research in

biotechnology, applied microbiology, geotechnical and civil engineering, due to the

numerous applications of MICP (Dhami et al., 2014). Various bacterial species are

capable of inducing calcite precipitates in alkaline environments rich in Ca2+ ions

(Dhami et al., 2013b) and other mechanisms in natural habitats (Rivadeneyra et al.,

2004, Ehrlich, 1996).

There are mainly four groups of microorganisms which are involved in the MICP

process (Dhami et al., 2013b), namely: (i) photosynthetic microorganisms such as

cyanobacteria and algae, (ii) sulphate reducing bacteria responsible for dissimilatory

reduction of sulphates, (iii) microorganism utilizing organic acids, and (iv)

microorganisms involved in nitrogen cycle either by ammonification of amino

acids/nitrate reduction or hydrolysis of urea (Jargeat et al., 2003, Hammes and

Verstraete, 2002, Stocks-Fischer et al., 1999).

In the aquatic environment, MICP is primarily caused by photosynthetic

microorganisms (McConnaughey and Whelan, 1997). Algae and cyanobacterial

metabolic processes utilize dissolved CO2 (Dhami et al., 2013b) and calcium ions to

induce CaCO3 precipitations as shown in Equation (1.9) to (1.12) (Hammes and

Verstraete, 2002). CaCO3 precipitation (dolomites and aragonite) via this route often

happens in the seawater, geological formations, landfill leachates and during biological

treatment of acid mine drainage (Machel, 2001, Warthmann et al., 2000, Wright, 1999).

(1.9) CO2 + H2O −→ (CH2O) + O2

(1.10) 2HCO3- ↔ CO2 + CO3

2− + H2O

(1.11) CO3 2− + H2O ↔ HCO3

- +OH−

(1.12) Ca2+ + HCO3- + OH− → CaCO3 + 2H2O

9

Heterotrophic microorganisms are also capable of inducing CaCO3 precipitation by the

production of carbonate or bicarbonate and modification of the microenvironment

which favours the precipitations (Castanier et al., 1999). The abiotic dissolution of

gypsum provides an environment that is rich in sulphate and calcium ions, the presence

of organic matter and absence of oxygens allows sulphate reducing bacteria to reduce

sulphate to hydrogen sulphite (Whiffin, 2004) as shown in Equation (1.13) and (1.14)

(Wright, 1999, Castanier et al., 1999, Ehrlich, 1998).

(1.13) CaSO4·2H2O → Ca2+ + SO4 2− + 2H2O

(1.14) 2(CH2O) + SO4 2− → HS−+HCO3- +CO2+H2O

The third pathway involved in CaCO3 precipitation involves bacteria which use organic

acids as their only carbon and energy sources wherein some common soil bacteria

species are included (Dhami et al., 2014). The consumption of these acids results in pH

increase which leads to CaCO3 precipitation in the presence of calcium ions as shown

in Equation (1.15) to (1.17) (Braissant et al., 2002, Knorre and Krumbein, 2000).

(1.15) CH3COO− + 2O2 → CO2 + H2O +OH−

(1.16) 2CO2 + OH− → CO2+ HCO3-

(1.17) 2HCO3-+ Ca2+ → CaCO3 + CO2 + H2O

Various heterogeneous bacterial groups are linked to this pathway for MICP process

(Dhami et al., 2014). Braissant et al. (2002) suggested that this pathway might be

extremely common in natural environment due to the abundance of low molecular

weight acids in soils, especially by fungi and plants. The fourth pathway of MICP

process involves microorganisms in nitrogen cycle via hydrolysis of urea. This pathway

is the easiest and most used method of MICP involving several applications (Dhami et

al., 2013b).This is attributed to the ability of the urea hydrolysis pathway to induce a

high amount of CaCO3 precipitates (Sarayu et al., 2014, Qabany et al., 2012, Siddique

and Chahal, 2011).

10

1.3.1. MICP via urea hydrolysis

CaCO3 precipitation by bacteria through urea hydrolysis is the most straightforward and

easily controlled mechanism of MICP with the ability to induce high amount of CaCO3

in a short duration of time (Dhami et al., 2014).

(1.18) CO(NH2)2 + H2O NH2COOH + NH3

(1.19) NH2COOH + H2O → NH3 + H2CO3

(1.20) H2CO3 → 2H++2CO32-

(1.21) NH3 + H2O → NH4++ OH−

(1.22) Ca2+ + 2CO32- →CaCO3 (KSP = 3.8 × 10−9)

KSP is the solubility product shown in Equation (21).

Stocks-Fischer et al. (1999) suggested that during microbial urease activity, 1 mol of

urea is hydrolyzed intracellularly to 1 mol of carbonate, which spontaneously

hydrolyzes to form an additional 1 mol of ammonia and carbonic ions. The ammonia

and carbonic ions equilibrate in water to form bicarbonates, 1 mol of ammonium and

hydroxide ions which allows an increases the pH of the environment as shown in

Equation (1.18) to (1.22) (Stocks-Fischer et al., 1999). Urease enzyme is responsible for

catalysing the hydrolysis of urea to produce ammonia and carbonate ions (Mobley and

Hausinger, 1989).

microbial urease

11

Figure 1.1: Pathway of biominerals secretion and precipitation in the cell of a bacteria. The bacteria serve a nucleation site for CaCO3 precipitation in the microenvironment (Sarayu et al., 2014). An ATP-generating system coupled with urea hydrolysis process in Sporosarcina pasteurii was suggested by Jahns (1996) and Whiffin (2004). The chemical transport processes which are related to microbial urea hydrolysis was (Mobley and Hausinger, 1989). The leading function of bacteria has been linked to their capability to generate an

alkaline microenvironment (Kumari, 2015) through various biological and chemical

activities as shown in Figure 1.1 (Dhami et al., 2014, Dhami et al., 2013b). The

bacteria’s surface plays an essential role in CaCO3 precipitates (Fortin et al., 1997). Due

to the presence of various negatively charged groups, at a neutral pH, positively charged

metal ions are able to bind to bacteria’s surfaces, favouring heterogeneous nucleation

(Douglas and Beveridge, 1998, Bäuerlein, 2003). The precipitation of CaCO3 on the

external surface of the bacterial cells often occurs by successive stratification, which

makes the cells become embedded in growing CaCO3 crystals (Castanier et al., 1999,

Rivadeneyra et al., 1998).

12

1.3.2. Urease enzyme

Urease and its substrate urea represent an important milestone in the early scientific

investigation (Mora and Arioli, 2014). Urease is produced by many diverse bacterial

species which includes normal flora and non-pathogens (Mobley, 2001). The scientific

interest in microbial urease was previously related to the relevance of this enzymatic

activity in infection (Mora and Arioli, 2014). This interest was strongly stimulated since

the discovery of the association of Helicobacter pylori with gastritis and stomach cancer

(Mobley et al., 1995). Urease has also been demonstrated as a potent virulence factor

for some bacterial species which include Proteus mirabilis, Staphylococcus

saprophyticus and Helicobacter pylori (Eaton et al., 1991, Jones et al., 1990, Gatermann

and Marre, 1989).

1.3.2 (a): Molecular characterisation of urease genes

Microbial ureases are multi-subunit metalloenzymes that hydrolyse urea substrates to

form carbonic acid and two molecules of ammonia (Mobley et al., 1995). The

degradation of urea provides ammonium for integration into intracellular metabolites

and enables the survival of the microorganism in acidic environments (Collins and

D'Orazio, 1993, Mobley et al., 1995). The structure of urease was first explained by

Jabri et al. (1995), showing that ureases may be composed of up to three distinctive

types of subunits, indicating that all the proteins are closely related. The structural genes

that encode both the urease subunits, ureA, ureB, and ureC, and the accessory proteins

required for assembly of the urease nickel metallocenter are typically clustered at a

single locus (Mobley et al., 1995). Different patterns of urease expression have been

observed in various bacteria (Wray et al., 1997).

There are eight genes which are necessary for the synthesis of urease enzyme,

designated as ureA; -B; -I; -E; -F; -G; -H and -I (Hu and Mobley, 1993, Hu et al., 1992,

Cussac et al., 1992, Ernst et al., 2007). Urease genes are evolutionarily related to each

other, sharing a common an ancestor (Ernst et al., 2007). Urease of Helicobacter pylori

is composed of two subunits, UreA (27 kDa) and UreB (62 kDa) and the subunits form

a multimeric enzyme complex with spherical assembly (Labigne et al., 1991, Clayton et

al., 1990, Ernst et al., 2007).

13

Figure 1.2: Genetic organisation of urease operon in Helicobacter pylori and Sporosarcina pasteurii. The ureAB genes of the ancestral urease operon are fused and labelled ureA, the ancestral ureC is labelled ureB in Helicobacter pylori (Ernst et al., 2007). In Helicobacter pylori, ureA and ureB are fused together to create ureA gene, while

ureC gene is labelled as ureB as shown in Figure 1.2. on the other hand, in

Sporosarcina pasteurii, the ancestral genes ureA and ureB are not joined together

(Figure 1.2).The ureEFGH genes codes for urease accessory proteins, which aid in

mediating proper formation of the complex quaternary structure and also transport

nickel ions into the urease enzyme active centre (Ernst et al., 2007). The ureI gene

codes for pH which regulates the urea channel situated in the cytoplasmic membrane

(Akada et al., 2000). ureI and ureA also interact during urea hydrolysis at the cell wall

of bacteria, allowing fast diffusion of ammonia and CO2 to occur (Voland et al., 2003).

1.3.2 (b): Activity of urease enzyme

Urease activity (UA) is the urea hydrolysis activity produced by the enzyme urease per

minute (Alhour, 2013). The process of urease production is illustrated in Figure 1.3

(Whiffin, 2004). Enzyme activity regulation is vital for energy efficiency in cell

function, however not all enzymes are mandatory all the time and their synthesis can

either be turned “off” (repressed) or “on” (induced ) depending the presence or absence

of metabolites (Whiffin, 2004). This type of genetic control is often regulated by the

cell at the transcriptional level where messenger RNA is produced from the DNA

template (Ratledge, 2001, Lewin, 1994). Enzymes such as urease can be controlled at

the transcription (inducible/repressible) level are usually repressed under normal

conditions, which helps to converse energy from unnecessary protein synthesis

(Whiffin, 2004). The presence of an inducer, normally its substrate, can strongly induce

an energy up to 1000-fold its level under non-induced conditions (Lowe, 2001).

14

Figure 1.3: Regulation levels for enzyme activity by microorganisms. The enzyme can be regulated at the transcriptional level or modification level (Whiffin, 2004). The genetic control is regulated by the microorganism’s cell where the messenger RNA (mRNA) codes for the enzyme which is produced from the DNA template (Ratledge, 2001, Lewin, 1994).

Whiffin (2004) determined microbial urease activity by measuring the relative change

in conductivity (mS.cm-1) when exposed to urea under standard conditions of 1.11 M

urea at 25oC. A standard curve was generated by determining the conductivity change

resulting from complete hydrolysis of several concentrations (50mM-250mM) of urea

by purified urease (Sigma Cat. No. U-7127) (Whiffin, 2004). From the standard curve

of changes in conductivity (mS.cm-1.min-1), Whiffin (2004) determined the equations

required to calculate the urease activity (mM urea hydrolysed.min-1) and the specific

urease activity (mM urea hydrolysed.min-1.OD-1) of ureolytic bacteria as shown in

Equation (1.23) and (1.24):

(1.23) Urea hydrolysed (mM) = Conductivity variation rate x (df) x (11.11)

(1.24) Specific urease activity = urease activity /Biomass

15

From Equation (1.23), urease activity (mM urea hydrolysed.min-1) was calculated by

multiplying the conductivity variation rate (mS.cm-1.min-1) by dilution factor (df) and

11.11 (correlation rate). According to Whiffin et al. (2007) 1 mS.cm-1.min-1 corresponds

to a hydrolysis activity of 11 mM urea.min-1 in the measured range of activities

considering the dilution of the culture during the activity measurement by a factor of 10

(Cheng and Cord-Ruwisch, 2013). From Equation (1.24), specific urease activity (mM

urea hydrolysed.min-1.OD-1) was calculated by dividing urease activity (mM urea

hydrolysed.min-1) by biomass (OD600). According to Whiffin (2004), the biomass

concentration was measured at the end of incubation period (overnight cultivation).

1.3.3. Mechanism of CaCO3 precipitation

CaCO3 Precipitation involves: (i) The development of supersaturation solution, (ii)

Nucleation (the formation of new crystals) begins at the point of critical saturation

and (iii) Spontaneous crystal growth on the stable nuclei (Alhour, 2013). CaCO3

precipitation occurs at the bacterial cell surface if there are sufficient concentration of

Ca2+ and CO32− in solution (Figure 1.4) (Anbu et al., 2016). The biochemical reaction

that takes places in the urea-CaCl2 medium leads to precipitation of CaCO3 as shown in

Equation (1.25) to (1.27), act as binders in between the substrate particles was

suggested by Stocks-Fischer et al. (1999).

(1.25) Ca2+ + Cell → Cell − Ca2+ (1.26) Cl − + HCO3− + NH3 → NH4Cl + CO3

2− (1.27) Cell − Ca2++ CO3

2− → Cell − CaCO3

16

Figure 1.4: A simplified representation of Ureolysis-driven CaCO3 precipitation. (A) Bacteria uptake urea and release ammonium (AMM) and dissolved inorganic carbon (DIC), bacterial cells attract calcium ions. (B) A local super-saturation occurs in the presence of calcium ions, resulting in CaCO3 precipitation on the bacterial cell wall. (C)The whole cell is encapsulated (De Muynck et al., 2010b). There are different phases of the CaCO3 precipitated by the bacteria which are: the three

anhydrous polymorphs (calcite, vaterite, and aragonite); two hydrated crystalline phases

(monohydrocalcite and ikaite); and various amorphous phases with different hydration

ranges (Rieger et al., 2007, Gower, 2008, Gebauer et al., 2010). Monohydrocalcite and

aragonite have been reported to be secreted by the bacteria (Gebauer et al., 2010,

Sanchez-Navas et al., 2009), It is also suggested that the proteins of Bacillus firmus and

Bacillus sphaericus are present in the extracellular polymeric substances which controls

the aragonite or calcite polymorph selection and calcium carbonate precipitation

(Kawaguchi and Decho, 2002). Lian et al. (2006) have also suggested that the cells and

the extracellular polymeric substances of Bacillus megaterium have controlled the

precipitation of calcite and vaterite. Similarly, Myxococcus sp. was also been reported to

have precipitated vaterite and calcite with varying morphologies along with other

minerals such as phosphate and sulphate, however depending on the medium that was

being used for culturing (Sarayu et al., 2014)

17

1.3.4. Urease Source

In a review by Sarayu et al. (2014), a list of bacteria that have been reported to induce

CaCO3 precipitates was tabularized. Some of these bacteria listed as Pseudomonas

putida, Arthrobacter sp., Desulfovibrio desulfuricans, Phormidium crobyanum and

Homoeothrix crustaceans (Sarayu et al., 2014). Out of the forty-one bacteria, only a few

are known to produce urease enzyme. Most urease producing bacteria which have been

reported to induce CaCO3 precipitates and have been used for MICP applications are of

Bacillus genus. Ureolytic bacteria which have been reported in literature for MICP

applications are as Bacillus sphaericus and Sporosarcina pasteurii used for to heal

concrete cracks(De-Belie and De-Muynck, 2008, Ramachandran et al., 2001, De-

Muynck et al., 2008); Bacillus pseudifirmus and Bacillus cohnii used to treat surfaces of

concrete (Jonkers and Schlangen, 2007, Jonkers, 2007); and Bacillus cereus and

Shewanella as cement mortar (Achal et al., 2011, Achal and Pan, 2011, Ramachandran

et al., 2001).

The majority of urease producing bacteria which have been reported were mostly from

soils and sludge samples. Alhour (2013) reported to have isolated thirty-two ureolytic

bacteria (closely related to Bacillus licheniformis, Bacillus lentus, Bacillus cereus,

Psuedomonas antarcticus, Psuedomonas apiaries, Bacillus carboniphilus, Bacillus

subtilis, Psuedomonas borealis, Bacillus sporothermodrans, Bacillus lequilensis,

Psuedomonas cellulositropicus, Bacillus mycoides, Lysinbacillus sphaericus,

Panibacillus barcinonesis, Bacillus isabeliae and Bacillus fordii)from soil, sludge and

freshly cut concrete surface samples collected at three locations in Gaza Strip. Al-

Thawadi and Cord-Ruwisch (2012) reported they isolated three ureolytic bacteria

(closely related to Bacillus aqaarimus and Sporosarcina pasteurii) from activated

sludge samples from a wastewater treatment plant collected at different locations in

Woodman Point, Perth, Western Australia. Dhami et al. (2013d) reported they isolated

five ureolytic bacteria (closely related to Bacillus megaterium, Bacillus cereus, Bacillus

thuringiensis, Bacillus subtilis and Lysinibacillus fusiformis) from calcareous soil

samples collected at Anantapur District, Andhra Pradesh, India. Hammes et al. (2003b)

reported they isolated twelve ureolytic bacteria (closesly related to Sporosarcina

pasteurii, Bacillus psychrophilus, Planococcus okeanokoites, Bacillus globisporus and

Filibacter limicola from garden soil, landfill soils, freshly cut concrete surface and a

calcareous sludge from a biocatalytic calcification reactor collected at Ghent, Belgium.

18

Ghashghaei and Emtiazi (2013) reported they isolated twelve ureolytic bacteria (closely

related to Enterobacter ludwigii) from soil, freshwater, chalk, cement and activated

sludge samples. Achal et al. (2010b) reported they isolated two ureolytic bacteria

(closely related to Bacillus cereus and Bacillus fusiformis) from cement samples

collected from commercial bags. Achal and Pan (2011) reported they isolated three

ureolytic bacteria (closely related to Sporosarcina pasteurii, Bacillus megaterium, and

Bacillus simplex) from alkaline soil samples collected at Bhagalpur, India. Stabnikov et

al. (2013) reported they isolated three ureolytic bacteria (closely related to Sporosarcina

pasteurii and Staphylococcus succinus) from tropical beach sand (Singapore), garden

sand soil (Kiev, Ukraine) and water samples (The Dead Sea in Jordan resort, resort).

1.4 Factors Affecting the Efficiency of MICP

Urease activity and the amount of calcite precipitated during MICP process are based on

various environmental factors, including pH, temperature, bacterial size and cell

concentration (Anbu et al., 2016, Qabany et al., 2012, Soon et al., 2012).

1.4.1. Concentration of reactants

Calcium ions in bacteria's environment play a major role in inducing calcite

precipitation (Sarayu et al., 2014). Microbial cell surfaces are negatively charged which

acts as scavengers for cations such as Ca2+ and bind to the cell surfaces in aquatic

environments (Ramachandran et al., 2001, Stocks-Fischer et al., 1999). Bicarbonate

which is produced by bacterial cell gets released when it combines with the calcium

ions available in the environment to precipitate CaCO3 (Sarayu et al., 2014). Hence,

calcium ions involved in this mechanism is supplied either by the medium or may result

from the support material to which the bacterium is attached to (Rodriguez-Navarro et

al., 2012). It safeguards the fixation of the surplus toxic calcium in the environment,

which enables the bacteria to survive in unfavourable conditions (Rodriguez-Navarro et

al., 2012). A reaction between urea and calcium ions results in calcite formation.

However, a solution containing equimolar of 1 mole of calcium chloride and 1 mole of

urea provides better conversion to calcite (Nemati et al., 2005).

19

A lower concentration of cementation reagents adds to a satisfactory level of

ammonium decomposition which might enhance microbial activity (Soon et al., 2012).

Higher concentration of cementation reagents (urea and calcium ions) extends the

precipitation of calcite induced during MICP process (Nemati et al., 2005, Okwadha

and Li, 2010). It was also confirmed in a study conducted by De Muynck et al. (2010b),

whereby the weight of soil samples increased when a higher concentration of

cementation reagents was added compared to the addition of lower concentration.

However, a considerable amount of salinity has an inhibitory effect on microbial

activity, urease production, and calcite precipitation which is mainly contributed by

calcium salts (Soon et al., 2012, Rivadeneyra et al., 1998). In some cases, urease

production is still readily available for MICP process at high salinity. However, the

ratio of actual calcite precipitated and abstract calcite composition decreases when there

is an increase in reactant concentrations (Nemati and Voordouw, 2003, De Muynck et

al., 2010b). Salinity has less inhibitory effects on moderately halophilic bacteria

compare to those non-halophilic bacteria (Soon et al., 2012). Several moderate

halophilic bacteria were studied for calcite precipitation in salinity environment

(Rivadeneyra et al., 2000, Stocks-Fischer et al., 1999, Rivadeneyra et al., 1998).

Moderate halophilic bacteria are capable of growing at a wide range of salinity. Hence,

they should be used for soil treatment during biocementation application if the soil

environment contains high salinity (Rivadeneyra et al., 2004).

1.4.2. pH

The pH environmental of urease-producing bacteria is one of the important aspects of

MICP process. The chemical compositions of the in vivo fluids and adjacent to the sites

of the minerals formation is directly influential to the understanding of

biomineralisation processes (Soon, 2013). The pH of the environment controls the

survival and the metabolic activity of the microorganisms that indirectly monitors the

secretion of the products (Soon et al., 2012). High pH conditions favour the formation

of CO32– from HCO3– which leads to calcification of the generated bicarbonate (Knoll,

2003). Stocks-Fischer et al. (1999) stated that the optimum pH for urease ranges

between 7.0 to 8.0, which was further supported by the research findings of Evans et al.

(1991) and Arunachalam et al. (2010).

20

Stocks-Fischer et al. (1999) also reported that urease activity rapidly increased from pH

6.0 to 8.0, until it reached its peak (pH 8.0) and gradually decreased when at higher pH.

However, Soon et al. (2012) stated that urease activity is still viable at pH 9.0. A recent

study by Gat et al. (2014) showed that urea hydrolysis leads to an increase in the pH of

growth medium due to the production of ammonium as was indeed found in treatment

using Sporosarcina pasteurii. On the other hand, co-culture which included Bacillus

subtilis showed a decrease which correlated in time with the exponential growth phase

of Bacillus subtilis. They suggested that and may, therefore, be attributed to increased

respiration, leading to enrichment in CO2, thus acidifying the medium. A study by Sidik

et al. (2015), which focused on the process of bacterial calcium carbonate precipitation

in organic soil showed that when soils samples were treated with the bacterial solution,

the pH values fluctuated between 9 to 9.4 during the period the sand samples were

being treatment. It indicated that this range, that the treatment medium used was

appropriate for MICP process as suggested by DeJong et al. (2010).

1.4.3. Temperature

Enzymatic reactions such as urea hydrolysis by urease are dependent on temperature

(Anbu et al., 2016). The optimum temperature which favours urease hydrolysis ranges

between 20 to 37oC (Okwadha and Li, 2010, Mitchell and Santamarina, 2005),

however, enzymatic reactions for optimum production is influenced by environmental

conditions and the concentration of reactants in the system (Anbu et al., 2016). A study

performed by Mitchell and Ferris (2005) reported that urease activity increased between

5 to 10 times when temperature increased between 10 to 20oC. Ferris et al. (2003) and

Dhami et al. (2014) investigated the kinetic rate of urease and temperature on

Sporosarcina pasteurii. Their findings showed that urease was very stable at 35oC, but

the enzymatic activity decreased by 47% when the temperature increased to 55oC.

However, other studies reported by Chen et al. (1996) and Liang et al. (2005) on

temperature effects on urease activity showed that optimum 60oC was the optimum

temperature for the production of urease. This temperature for urease activity is

impractical on site for soil treatment using MICP (Soon et al., 2012).

21

1.4.4. Dissolved inorganic carbon

Inorganic carbon present in the environment plays a major role in MICP process (Soon,

2013). Dissolved inorganic carbon (H2CO3 + HCO3−+ CO3

2−), is a major product of

microbial respiration which affects microbial activities and its alkalinity (Sauvage et al.,

2014, D'Hondt et al., 2002). The DIC released from the extracellular polysaccharide of

the microorganisms complexes the calcium ions, thus reducing calcium carbonate

saturation enhancing the calcite precipitation (Tourney and Ngwenya, 2009). A study by

Gat et al. (2011), on stimulation of ureolytic MICP in natural soils, reported that

interaction between ureolytic and non-ureolytic bacteria was affected during ureolysis.

Their finding showed an increase in DIC concentration when ureolytic and non-

ureolytic bacteria co-cultured. This result was supported by a recent study by Gat et al.

(2014) on calcite precipitates using co-culture of ureolytic and non-ureolytic bacteria,

namely, Sporosarcina pasteurii, DSMZ33 and Bacillus subtilis, DSMZ 6397. Their

experiment showed that DIC concentrations were affected by three processes: (1)

hydrolysis of urea to produce bicarbonate, (2) bacterial respiration and mineralization of

the NB by ureolytic and non-ureolytic bacteria to produce dissolved CO2, and (3)

precipitation of CaCO3, which led to a reduction in DIC concentration (Engel et al.,

2004). The decrease in dissolved calcium concentration observed in this experiment

may be attributed to the precipitation of CaCO3. A study by Tobler et al. (2011)

reported a similar phenomenon for the induction of urea hydrolysis in a mixed culture

of indigenous soil bacteria.

1.4.5. Bacteria size

The type of bacteria appropriate for MICP application should be able to catalyst the

urea hydrolysis and they are usually urease positive bacteria (Soon et al., 2012). The

typical urease positive bacteria used for MICP are aerobic bacteria, are often selected

for MICP process because of their ability to release CO2 which is essential for the rise

in pH due to the production of ammonium when urea is being broken down (Soon,

2013). Bacterial sizes found in soil ranges from 0.5 to 3.0 μm microbes can move along

soil particles either through self-propelled manner or via passive diffusion (Mitchell and

Santamarina, 2005, Soon et al., 2012).

22

The geometric compatibility of urease producing bacteria is critical whenever the

transportation of bacteria within the soil is required for soil treatment, and mall pore

throat size would limit their free passage, depending on the size of microbes and soil

composition (Sarayu et al., 2014). A significant amount of silt and clay in the ground

would have an inhibitory effect on bacteria’s movement (Soon et al., 2014). It is

imperative to select appropriate soil and bacteria for MICP treatment (Soon, 2013).

1.4.6. Nutrients

Nutrients are the energy sources for bacteria, providing sufficient nutrient the ureolytic

bacteria is critical for precipitation of calcite (Soon et al., 2012). Nutrients are often

supplied to the bacteria during culture and soil treatment stages (Soon, 2013). The most

common nutrients usually provided to bacterial include Potassium, Sodium, Nitrogen,

Calcium, Iron and Magnesium (Mitchell and Santamarina, 2005). The unavailability of

organic constituents in soil limits bacterial growth, hence the supply of sufficient

nutrient to soil containing ureolytic bacteria can promote bacterial growth which can

enhance calcite precipitation required in achieving the desired level of ground

improvement (Soon et al., 2012).

1.4.7. Availability of nucleation site

A nucleation site is isolated from the environment by a restricting geometry limiting the

diffusion in and out of the system, which enable the modification of the activity of at

least a cation, proton, and other possible ions and ensure electro-neutrality (Sarayu et

al., 2014). The ion movement is enabled by active pumping with organelles or passive

diffusion to enable the microorganisms to use a great variety of anatomical

arrangements (Perry, 2003). The biofilm and the extracellular polysaccharide which is

formed by the microorganisms are effective in binding ions from the environment and

act as a heterogeneous nucleation site for the mineral deposition (Sarayu et al., 2014).

The creation of a strong electrostatic affinity to attract cations and enables the

accumulation of calcium ions on the surface of the cell wall which allows sufficient

supersaturation state of calcium ions to be achieved. Thus binding it to the carbonate

ions and results in the formation of calcium carbonate on the cell wall (Obst et al., 2009,

Tourney and Ngwenya, 2009). This mechanism favours the bacterial growth by

reducing the toxic calcium in the environment (Sarayu et al., 2014).

23

Higher bacterial cell concentration (106 to 108) supplied to soil samples would certainly

increase the amount of calcite precipitated from MICP process (Okwadha and Li, 2010).

Urea hydrolysis rate is directly proportional to a concentration of bacteria cell, provided

there will be enough reagent available for the biocement treatment of sand (Soon et al.,

2012). High concentration of bacteria produces more urease per unit volume to

commence the urea hydrolysis (Soon, 2013). Li et al. (2011b) and Stocks-Fischer et al.

(1999) suggested that the cells of the bacteria served as a nucleation site for MICP

occurrence.

The availability of nucleation sites serves as one of the key factors for microbial calcite

precipitation (Knorre and Krumbein, 2000). Lian et al. (2006) studied the crystallization

by Bacillus megaterium. They showed using scanning electron microscopic images that

nucleation of calcite takes place at bacteria cell walls. Stocks-Fischer et al. (1999) also

demonstrated that calcite precipitation relates with the bacteria concentration used.

Stocks-Fischer et al. (1999) were able to relate calcite induced via MICP efficiency with

chemically induced calcite at pH 9.0. Their findings concluded that about 98% of the

initial concentrations of Ca2+ were precipitated via MICP. On the other hand, only 35 to

54% of chemically induced calcite was observed. It was then suggested that the

bacterial cells provided a nucleation site for calcite to be induced which increased the

environment for further calcite to be induced, was responsible for the differences in

calcite precipitated via MICP and chemical processes.

1.5 Current Biotechnological Application of MICP MICP is highly desirable because of its natural availability and lower production of

pollutants (Al-Thawadi, 2008). MICP process is an effective and environmentally

friendly technology which can be applied to solve various environmental problems such

as soil instability and concrete crack (Anbu et al., 2016). Some of the biological

applications of MICP have been discussed by Whiffin (2004), Al-Thawadi (2008) and

in review articles by Phillips et al. (2013), Sarayu et al. (2014) and Anbu et al. (2016).

24

1.5.1. Biocementation

Biocement or biosandstone was proposed as a novel method for cementing loose sands

to produce structural materials, consisting of Alkaliphilic urease producing bacteria, a

substrate solution (urea), a calcium source and sand (Achal, 2015). However, a typical

set-up for sand consolidation experiment to develop biocementation was simplified by

Reddy et al. (2012), where sand is either mixed with bacterial culture or later injected

directly into the sand columns. The sand was plugged through a plastic column, and the

cementation fluid which consisted of nutrient media, urea, and calcium ions were then

injected at a specific rate in the column using gravimetric free flow direction. Another

study on calcite deposition in sand columns using Sporosarcina pasteurii by Achal et al.

(2009b) found that 40% of calcite deposited in the sandstone resulted and led to a

reduction of porosity and permeability in the sandstone. A study by Qian et al. (2010)

on a sand column of a size of 32.10 and 18.40 mm showed the right amount of

compressive strength, measured up to 2 MPa when CaCl2 was used as a calcium source

for biosandstone. The MICP substance in the biosandstone was confirmed using X-ray

diffraction (XRD) and energy dispersion spectroscopy (EDS), and calcite, which was

precipitated in the sandstone as the main microbial induced substance in the

biosandstone. The results of MICP process on biosandstone lead researchers to carry out

investigation beyond this building material (Achal, 2015).

1.5.2. Creation of biological mortars The knowledge obtained with MICP treatments resulted in the development of

biological mortar for remediation of small cavities on limestone surfaces (De Muynck et

al., 2010a). The purpose of using initiating biological mortars was to avoid some of the

problems related to chemical and physical incompatibilities of commonly used mortars

with the underlying materials, specifically in the case of brittle materials (Castanier et

al., 1999). The resistance of mortar specimens and surface deposition to degradation

process can be improved via microbial calcite precipitation (Siddique and Chahal, 2011,

Al-Thawadi, 2011, Chunxiang et al., 2009).

25

Figure 1.5: An in situ application of bacteria based liquid. Ureolytic bacterial culture was used to repair a system on cracked parking decks (Jonkers et al., 2016). A study by Le Metayer-Levrel et al. (1999) showed that they successfully studied

bacterial cementation which aimed at the creation of biological mortars and patinas on

limestones. Their method solely depended on spraying the entire surface of limestone

with bacteria followed by nutritional medium containing urea and calcium. Rodriguez-

Navarro et al. (2003) reported a relatively low penetration depth of 500 μm by

immersing the limestone sample in cementation media. They reported the use of

Myxocccus xanthus (a slow growing bacterium) resulted in CaCO3 precipitation at the

wall of the porous materials without plugging them. A recent in situ application on

cracked was carried out by Jonkers et al. (2016) as shown in Figure 1.5. Their finding

showed that concrete repair using MICP is inexpensive, improved the durability of the

material and also lowered the environmental impact of civil engineering activities.

1.5.3. Bioremediation of cracks in concrete In concrete, cracking is common due to relatively low tensile strength (De-Belie and

De-Muynck, 2008). Several mechanisms such as shrinkage, freeze-thaw reactions,

mechanical compressive and tensile forces lead to the formation of cracks (Alhour,

2013).

26

Cracking on concrete surfaces also results in enhanced deterioration of embedded steel

through easy ingress of moisture and ions that react with reinforcements in concrete and

expansive stressed which leadings to spalling (Gavimath et al., 2012, Achal et al.,

2013). Thus, it is practical to use adhesive for sealing of concrete cracks so that the

strength and durability of the concrete will be improved (Wong 2015). A conventional

approach used in repairing cracks involves injecting epoxy resin or cement grout into

the concrete. However, they result in various thermal expansion, environmental and

health hazards (De-Belie and De-Muynck, 2008).

Figure 1.6: Self-healing crack from the addition of bacterial metabolism via urea hydrolysis. The ureolytic bacterial culture was able to produce minerals which helped to repair and cover the cracks (Sierra-Beltran et al., 2014). Several research groups have investigated the possibility of using MICP as an

alternative effective repair method for cracks in concrete via bioremediation (Alhour,

2013). Investigation on the potential of using bacteria to act as self-healing agent in

concrete to fix a crack. Specifically, with the use of alkali-resistant spore-forming

bacteria, Bacillus pseudofirmus (type strain DSM 8715) and Bacillus cohnii (type strain

DSM 6307) (Jonkers, 2007, Jonkers and Schlangen, 2007, Jonkers et al., 2010).

27

Their findings showed that bacterial cement stone specimens appeared to produce a

solid result of crack-plugging. Other studies by Abo-El-Enein et al. (2013), Bang et al.

(2010), and Siddique and Chahal (2011) have shown that the cracks in concrete filled

with a mixture of Sporosarcina pasteurii and sand showed a significant increase in

compressive strength and stiffness when compared to cracks without cells. In Figure

1.6, Sierra-Beltran et al. (2014) reported self-healed cracks using MICP.

1.5.4. Biodeposition on cementitious materials

The emergence of microbial involvement in carbonate precipitation has led to the

exploration of this process in a variety of fields, including environmental, civil and

geotechnical engineering (De Muynck et al., 2010a). Among these applications, MICP

has been used for biogenic-carbonate-based surface treatments, a process known as

biodeposition (Figure 1.7) (Le Metayer-Levrel et al., 1999, Rodriguez-Navarro et al.,

2003, Dick et al., 2006).

Figure 1.7: 1 mm thick calcite crust formed on the surface of the soil. A successful percolation treatment with ureolytic bacterial culture, a high concentration of urea and calcium solution resulted in a nearly impermeable crust on the surface of the sample (Achal et al., 2010c).

28

Biodeposition of bacterial calcite is a viable method of surface treatment for cement-

based materials that can be explored in a sustainable approach (Wong, 2015).

Considering the size of bacterial cells are around 1 µm, both the cells and their media

containing the reactants (urea and calcium ions) can permeate deep into the pores and

interface between aggregates or paste of the concrete structure (Ramachandran et al.,

2001). Hence, this enables microbial cementation to take place within and on the

surface of such materials which then provides reinforcement and protection (Wong,

2015).

A study by De-Muynck et al. (2011) using ureolytic biodeposition treatment was

applied to five types of limestones so as to investigate the effect of pore structure on the

protective performance of bigenis carbonate surface treatment. Their findings showed

that in macroporous stone, biogenic carbonate formation occurred to a larger extent and

at greater depths than in microporous stone. Hence, exhibiting a greater protective

performance on macroporous stone compared to microporous stones. Precipitation on

microporous stones was limited to the outer surface of a microporous rock. From this

study, it was clear that biodeposition was very effective and more feasible for

macroporous stones than for microporous stones (De-Muynck et al., 2011). Another

study by Li and Jin (2012) on remediation technique of cracked concrete by bacterially

mediated carbonate deposition showed that bio-deposition was able to make

improvement in concrete compressive strength and flexural load using Sporosarcina

pasteurii. Their findings concluded that this can be used to enhance the strength and

flexural load of a faulty concrete specimen.

1.5.5. Biogrout

Nemati and Voordouw (2003) described the use of urease to cement porous medium.

Their study showed that reducing the permeability of porous medium by enzymatic

CaCO3 precipitation using Canvalia ensiformis was successful. Nemati and Voordouw

(2003) used between 0.1 and 1.0 M (>33 g.L-1) calcite together with high urease activity

for a successful plugging of the sand core. Unfortunately, the strength build-up was not

monitored. Stocks-Fischer et al. (1999) reported that injection of bacteria and reagents

together at low flow rates can result in full clogging of the system near the injection

point. An investigation on Biogrout ground improvement using MICP was also

performed by Paassen (2009).

29

This study was successful in developing an unprecedented 100 m3 field scale

experiment (Figure 1.8), and 40 m3 of the sand were treated using MICP process within

a duration of 12 days Although in both scale up experiments significant increase of the

average strength was obtained, different variable mechanical properties were observed

in the sand. It could be affected by induced flow field, bacteria distribution, the supply

of reagents and crystallization process (Paassen, 2009). Another study by Suer et al.

(2009) investigated the potential of using biogrouting as an alternative approach to jet

grouting to seal the contact between sheet pilling and bedrock. Their finding showed

that biogrouting process was cheaper than jet grouting and had much lower

environmental impact. Biogrouting also consumed less water and produced less

landfilled waste.

Figure 1.8: Set-up for large scale (100 m3) soil treatment. The sand was injected 10 times for 12 days with Sporosarcina pasteurii cell and cementation solution (Paassen, 2009). The scale-up demonstration of MICP in 100 m3 of sand to determine the ground improvement abilities and extent of precipitation (Phillips et al., 2013).

30

1.5.6. Other essential applications of MICP 1.5.6 (a): Removal of calcium ions (Ca2+)

Calcium-rich wastewater is a problem some industries face due to calcification during

downstream processing (Hammes et al., 2003c). High concentration of calcium ions

ranging from 500-1500 mg.L-1 in the wastewater can cause substantial scaling in

pipelines and reactors as a result of calcium formation as carbonate, phosphate, and

gypsum (Al-Thawadi, 2008, Dhami et al., 2013e). A novel application for the process of

MICP as an alternative mechanism for the potential removal of Ca2+ from industrial

wastewater instead of chemical precipitation approach has been developed (Hammes et

al., 2001). MICP process facilitated the removal of soluble calcium from calcium-rich

industrial wastewater via urea hydrolysis pathway, mediated by autochthonous bacteria.

Calcium removal more than 90% was achieved throughout the experimental period

while the effluent pH remained at a reasonable level (Hammes et al., 2001, Hammes et

al., 2003c). A recent study by Isik et al. (2010) showed that a significant parameter,

hydraulic retention time, required an optimum condition of 5-6 hr to hydrolyse calcium

successfully from industrial water using MICP in a biocatalytic calcification reactor.

1.5.6 (b): Removal of polychlorinated biphenyls (PBs)

Polychlorinated biphenyls (PCBs) is a recalcitrant contaminant which surfaces on

concrete when PCBs containing oils leaks from the equipment. (Phillips et al., 2013).

The last two decades have seen an increase in the use of bioremediation for the removal

of contaminants, which includes PCBs (Dhami et al., 2013e). The conventional method

previously used to remove PCBs such as solvent washing, hydro-blasting and epoxy

coating have not been very effective due to resurfacing of the oil over a period of time.

Microbial process using MICP process has been initiated as an alternative measure to

remove PCBs (Dhami et al., 2013e). Okwadha and Li (2010) reported the potential use

of Sporosarcina pasteurii for the treatment of PCB-coated cement cylinders leading to

surficial encapsulation of PCB-containing oils. A study by Okwadha and Li (2011)

stated that when Sporosarcina pasteurii containing urea and calcium ions were applied

on the surficial PCB-containing oil, there was no observation of leaching and there was

a reduction of permeability by 1-5 orders of magnitude.

31

1.5.6 (c): Industrial by-products

Construction materials such as concrete, brick and pavement blocks are all produced

from natural existing resources. Their production has affected our environment due to

continuous exploration limitation of natural resources. It has led researchers to explore

other means of building materials which are environmentally friendly, affordable and

sustainable (Aubert et al., 2006). There are different types of waste such as slag, fly ash,

wheat straw, saw milk waste, cotton stalk, mining waste tailing and waste gypsum

which are currently being recycled for potential utilisation (Pappu et al., 2007). The

production of fly ashes during combustion of coal for energy is one of the industrial by-

product recognised as an environmental pollutant (Dhami et al., 2013e). Rice husk ash

obtained from burning of rice husk is another major agricultural by-product (Dhami et

al., 2013e). Both these materials can be used as construction materials (bricks and

blocks) without any degradation in the quality of products (Nasly and Yassin, 2009).

Despite the previous report of the problems associated with ash bricks such as low

strength, high water adsorption and low resistance to abrasion. Dhami et al. (2012b)

studied the application of bacterial calcite on fly ash and rice rush ash bricks and

reported they were very efficient in reducing permeability and decreasing water

absorption which lead to enhanced durability of ash bricks.

1.5.6 (d): Low energy building materials

The construction sector is responsible for primary input of energy resulting in the

release of CO2 emissions into the atmosphere (Reddy and Jagadish, 2003). Hence, it is

essential to reduce the emission of these gases released into the air (Dhami et al.,

2013e). Energy requirements for production and processing of different building

materials and various implications on the environment have been previously studies

(Oka et al., 1993, Debnath et al., 1995, Suzuki et al., 1995). Reddy and Jagadish (2003)

reported soil blocks with 6–8% cement content uses the moving energy efficient

building material. These materials have low production cost, are easily recyclable and

environmentally friendly as the soils are mixed with additives such as lime (Dhami et

al., 2013e).

32

These building materials do not make use of burning during its production, and these

stable mud blocks were able to converse much energy (Dhami et al., 2013e). Building

materials using low energy by application of ureolytic Bacillus sp. have successfully

been performed by Dhami et al. (2013c) which shows the potential of using MICP

technology to produce sustainable, cheap and durable buildings.

1.6 Diversities of Microbial Communities in Caves Caves are natural geological formations considered as extreme environments,

unfavourable for the development of life due to the severe abiotic conditions present

(Tomczyk-Żak and Zielenkiewicz, 2015). However, cave environments constitute

ecological niches for highly specialised microorganisms (Schabereiter-Gurtner et al.,

2004). The most common types of caves known are karst caves, formed from limestone

rocks and cave created as a result of lava cavities (Tomczyk-Żak and Zielenkiewicz,

2015). Caves constitute oligotrophic ecosystems, which are less than 2 mg of the total

organic carbon per litre. These environments have a low level of light, low, stable

temperature and high humidity (Tomczyk-Żak and Zielenkiewicz, 2015). Despite these

oligotrophic conditions, the average number of microorganisms dwelling in these

ecosystems are 106 cells/g of rock (Barton and Jurado, 2007).

The majority of biological communities are dependent on energy and carbon fixation of

photosynthesis. However, the inhibition of sunlight prevents colonisation of

phototrophs in cave environments (Wu et al., 2015). Only limited energy and nutrients

can enter these caves through sinkholes, underground hydrology and drip water (Barton

et al., 2007). These environments only allow for the survival and functioning of species

adapted to oligotrophic conditions (Wu et al., 2015). The limited access of

photosynthetic activities in caves inhibits the production of primary organic matter

essential for the survival of photosynthetic microorganisms. Hence, these cave

microorganisms make use of alternative methods by synthesising their organic

molecules through carbon dioxide fixation to produce their source of food or energy

(Tomczyk-Żak and Zielenkiewicz, 2015).

33

This condition allows these cave microorganisms to derive their main source of energy

from not only hydrogen, nitrogen or volatile compounds, but they also derive their

energy from the oxidation of inorganic molecules such as iron, sulphur or magnesium

present in caves (Gadd, 2010, Northup and Lavoie, 2001). Other sources of organic

compounds from which these microorganisms derive their energy comes from plant

roots or remains of human or animal activities, these organic matter allows the

developments of a heterotrophic microorganism (Tomczyk-Żak and Zielenkiewicz,

2015).

Studies performed on calcified structures (Figure 1.9) are of biogenic origins, their

study showed that microorganisms interacted with minerals, hence playing an important

role in the formation of these calcified structures (Melim et al., 2009). These

interactions help in shaping cave structures such as stalactites, stalagmites, as well

formations of bristles in surfaces of cave rocks (Tomczyk-Żak and Zielenkiewicz,

2015). Some of these cave microorganism precipitates CaCO3 on the surfaces of their

cells, which contributes to formations of limestones in the caves (Sanchez-Moral et al.,

2003). The occurrence and structure of microbial communities in limestone caves are

influenced by factors such as pH, availability of nutrients, sunlight, oxygen, metal

compounds, humidity and susceptibility of the substrate to colonisation (Tomczyk-Żak

and Zielenkiewicz, 2015). Bacteria and archaea constitute a majority of the biodiversity

in caves, found in numerous cave habitats such as sediments, stream waters and rock

surfaces (Barton and Jurado, 2007, Engel et al., 2004).

Chemoautotrophic microbes are mostly responsible for CO2 fixation and potentially

participate in inorganic nitrogen (Tetu et al., 2013, Diaz-Herraiz et al., 2013). Moreover,

the interactions between microorganisms and limestone caves may contribute to

speleogenesis, for example, in sulfidic caves, microorganisms can oxidise of hydrogen

sulphide to produce sulfuric acid, which then reacts with carbonate and causes rock

dissolution (Macalady et al., 2007, Engel et al., 2004). Bacteria can alter the surfaces of

rocks through oxidation of some metal elements such as iron (Fe2+) and manganese

(Mn2+) which result in the formation of deposits on cave walls (Carmichael et al., 2013).

34

Figure 1.9: Calcified structures of biogenic origin discovered in cave regions. (A) pool fingers formation and (B) U-loops formation (Garcia et al., 2016). Cave microbial communities are often extremely variable depending on the

microhabitats (Wu et al., 2015). A study by Barton et al. (2007) showed there were

significant differences between the diversity of bacteria and its composition observed on

rock walls within one single cave, suggesting this was possibly related to the host rock

geochemistry. An alteration of physiochemical conditions can influence a change of in

the composition of microbial species. For example, in the water mats of streams in the

Kane Cave, which is rich in sulfur compounds, the water flowing directly from the

spring into the cave, contains a high concentration of sulfur and low amounts of oxygen,

dominated by Epsilonproteobacteria. On the other hand, the water flowing out of the

cave to external environments contains large quantities of oxygen and low

concentrations of sulfur, dominated by Gammaproteobacteria (Tomczyk-Żak and

Zielenkiewicz, 2015, Engel, 2010, Jones and Bennett, 2014).

Studies Rusznyak et al. (2012) on the effect of the microbial population in Herrenberg

Cave in Germany, a typical karst cave, showed that the occasional or limited human

presence in cave environments does not necessarily affect the compositions of microbial

diversity of a population. However, a study by Adetutu et al. (2012) indicated that the

presence of human activity in regions of Naracoorte Caves in Australia had a consistent

influence in bacterial diversity which was attributed to the presence of exogenous

organic matter of human origin. Various studies have demonstrated that bacteria from

35

cave environments are capable of inducing calcite precipitates in vitro (Garcia et al.,

2016). Different species and genera of bacteria have been isolated from speleothems

samples in caves which include Sporosarcina pasteurii, Bacillus subtilis, Myxococcus

xanthus, Bacillus amyloliquefaciens, Bacillus cereus, Pseudomonas flurescens,

Micrococcus sp., Rhodocucus sp. and Arthrobacter sp. (Rusznyak et al., 2012, Achal et

al., 2010b, Rivadeneyra et al., 2006).

Figure 1.10: Speleothems samples collected from El Toro and El Zancudo limestone mines located in Cordillera Central, northeast of Colombia. The diversity of bacteria from speleothems samples in Colombia and their ability to precipitate carbonates were studied using conventional microbiological methods and molecular tools, such as temporal temperature gradient electrophoresis (Garcia et al., 2016). In addition, Rusznyak et al. (2012) and Cacchio et al. (2004) have suggested that the

microorganism mentioned above have a direct relationship with calcite depositions and

speleothems developments in limestone caves. Speleothem carbonates formation were

normally considered as inorganic precipitates, but recent studies have demonstrated

biological influence in their formations (Baskar et al., 2007). These discoveries can

advance our understanding of the diversity of bacteria in cave environments (Roesch et

al., 2007).

36

Most researchers regarding the profiles of microbial communities in speleothem

samples (Figure 1.10) make use of culture-dependent study based on partial analysis of

the 16S rRNA gene using clone library methods and genetic fingerprinting techniques

such as denaturing gradient gel electrophoresis (DGGE). Their studies suggested that

the most dominant phyla in cave environments are Firmicutes, Proteobacteria,

Actinobacteria and Acidobacteria (Ortiz et al., 2014, Dhami et al., 2014, Ortiz et al.,

2013). A metagenomics approach on the study of microorganisms in karstic cave Ortiz

et al. (2014) suggested that functional bacterial genes were associated with low nutrient,

high calcium adaptations, and nitrogen-based metabolism.

1.7 Screening Sarawak’s Limestone Caves for Ureolytic Bacteria Sarawak is one of the two Eastern Malaysian states situated on the island of Borneo,

known as the world's third largest island and one of the twelve mega-biodiversity

regions (Lateef et al., 2014, Tan et al., 2009). Borneo has a landmass of nearly 740,000

square kilometres, located in the equatorial region of the Pacific Ocean (Rautner et al.,

2005). The Island consist of the independent Sultanate of Brunei Darussalam, the

Indonesian territory of Kalimantan, and the Malaysian states of Sarawak and Sabah

(Rautner et al., 2005, Sulaiman and Mayden, 2012) as shown in Figure 1.10.

Borneo is widely known for its rich floral and faunal diversity. However, many areas of

the island require further exploration (Clements et al., 2010, Garbutt and Prudente,

2007, Mohd et al., 2003, Koh et al., 2010, Karim et al., 2004). Diverse habitats such as

mangrove swamps, peat swamps, an estimated 15, 000 plant species (5, 000 trees, 17,

000 orchid species and over 50 carnivorous pitcher plants) host a great diversity of

endophytic microorganisms in Borneo (State Planning Unit, 2013). In 2007, the

countries situated in Borneo Island made a declaration to protect 220,000 square

kilometres of pristine rainforest habitats which are now known as the “Heart of

Borneo,” to prevent disturbances such as deforestation and plantation development from

affecting the Island’s biodiversity (Sulaiman and Mayden, 2012).

37

Sarawak is the largest state in Malaysia, containing 37.5% of the country’s total land

(Mahidi, 2015). Also, Sarawak has 512, 387.47 hectares of protected areas comprising

of 18 National Parks, four wildlife sanctuaries, five nature reserves and the largest

peatland area in Malaysia (Van der Meer et al., 2013, Forest Department Sarawak,

2013). The rich mega-biodiversity in Sarawak has attracted the attention of researchers

within and outside of Malaysia. The existing scientific studies have focused on peat

soils, plants, corals, microbes in aquatic and forest environments (Sa'don et al., 2015,

Kuek et al., 2015, Cole et al., 2015). Sarawak’s limestone forest is one of the nine main

types of forest documented in Sarawak, covering about 520 m2 or 0.4% of the total area

(Julaihi, 2004, Banda et al., 2004).

The limestone forest is situated with vast numbers of limestone caves. The caves or

limestone areas in Sarawak have become the main focus for researchers to investigate

the diversity of bats indigenous to Wind and Niah caves (Mohd et al., 2011, Rahman et

al., 2010b, Rahman et al., 2010a). Studies on evolution of limestone formation,

biological influence on formation of stalagmite, investigation of trace metal ratios and

carbon isotopic composition on limestone caves have been carried out in Niah and Mulu

caves, which are also situated in Sarawak (Moseley et al., 2013, Dodge-Wan and Mi,

2013, Cucchi et al., 2009). Despite Malaysia’s abundance of limestone regions situated

in places such as Langkawi Island, Kedah-Perlis, Kinta Valley, Perak, Selangor, Gua

Musang, and Kelantan as reported by Bakhshipouri et al. (2009).

There are limited reported studies on the exploitation of microbial diversity from these

regions. Moreover, there have been recent studies on isolation of calcite forming

bacteria from limestone cave samples of Perak and research on soil improvement using

Bacillus megaterium, ATCC 14581 type strain (Soon et al., 2014, Soon et al., 2013,

Komala and Khun, 2013). To date, there have been no recorded studies in Sarawak on

the isolation of urease producing bacteria from limestone caves samples of Sarawak.

This research gap and the possibility of certain microbes able to induce calcite

precipitates from limestone cave environments initiated the relevance of screening for

urease producing bacteria from two cave regions in Sarawak.

38

Figure 1.11: Map of Borneo Island. showing the geographical divisions and topographical features of Brunei Darussalam, Indonesia (Kalimantan) and East Malaysia (Sarawak and Sabah). The island of Borneo, known as the world's third largest island and one of the twelve mega-biodiversity regions (Lateef et al., 2014, Tan et al., 2009, Tan, 2006).

39

1.8 Aim and Objectives of the Study The aim of this research was to screen and characterise urease-producing bacteria that

are capable of inducing calcite precipitation. The objectives set out to achieve the

research aim are:

i. To isolate urease producing bacteria from limestone cave samples of Sarawak

using enrichment culture technique.

ii. To identify urease-producing bacteria.

iii. To characterise urease activity of bacterial isolates.

iv. To determine the effects of cultural conditions on urease activity.

v. To study biocementation ability of selected bacteria in vitro.

1.9 Significance of the Study This study explores the prospect of using urease-producing bacteria which were isolated

from domestic location rich in microbial diversity for possible biocementation

applications. The advantage of using local isolates is because they are well adapted to

native environments, and they are also less likely to become pathogenic when they are

under stressed conditions. Additionally, studies on the isolation of non-pathogenic

highly active urease-producing bacteria species are very limited. This formed the

necessary initiation of this study which could pave the way for a new frontier in the use

of non-pathogenic bacterial species isolated from Sarawak, Malaysia.

1.10 Thesis Outline This thesis presented is divided into four chapters: Introduction and Literature Review

(Chapter 1). Isolation, Identification, and Characterisation of Urease-Producing Bacteria

from Limestone Caves of Sarawak (Chapter 2). Effects of Cultural Conditions On

Urease Activity, and Evaluation of Biocementation Potentials in Small Scale Test

(Chapter 3). General Conclusion and Recommendations (Chapter 4). Concluding

remarks are shown at the end of Chapter 2 and 3 to summarise the contents of theses

chapters.

40

Chapter 1 provides a brief introductory background of the study and a broad review of

the essential literature regarding MICP, which has been reported by other researchers.

The aim, scope, and significance of the research which was to be performed, were also

conferred in this chapter. Chapter 2, presents a detailed study on the isolation, screening

and identification of the ureolytic bacteria which were obtained using enrichment

culture technique. In this chapter, specific focus was given to the quantitative

measurement of specific urease activity by the local isolates. The enzyme activity of

these isolates was compared with that of the representative strain used in this study. The

isolates capable of producing comparable urease activities with that of the

representative strain were selected and used for subsequent experiments. Chapter 3,

presents the results on the effects of cultural conditions on the urease activity. A

laboratory-scale study concerning the application of ureolytic bacteria for MICP process

to treat poorly graded soil. The sole purpose of this chapter was to access whether

sufficient potential exists to warrant the possible usage of the locally isolated ureolytic

bacteria, serving as alternative MICP agents. This knowledge can lead to further

investigation along this line of research such as large-scale microbial production in a

reactor and field applications using MICP agents. In Chapter 4, a succinct overview of

the most significant findings of the experimental studies is presented and are shown

within the context of one another. Perspectives on future research possibilities within

this field are conferred in this chapter as future directions to be considered.

Chapter

2 ISOLATION, IDENTIFICATION AND

CHARACTERISATION OF UREASE-PRODUCING

BACTERIA FROM LIMESTONE CAVES OF SARAWAK

41

2.1 Introduction Limestone caves, known as natural geological formations are considered as extreme

environments which form an ecological niche for the survival of various

microorganisms (Schabereiter-Gurtner et al., 2004). These environments often excluded

from the outside world with limited in nutrient, may contain novel, diverse microbial

populations (Sugita et al., 2005). Hence, it is imperative to perform pioneering

investigations ventured at exploring and isolating microbial species that indigenous to

cave regions. It’s been known and reported that formation of stalagmite and stalactites

are often as a result of microbial and mineral interactions (Tomczyk-Żak and

Zielenkiewicz, 2015). Some microorganisms are able to induce calcites on the surface

of their respective cells, which promotes limestone formation (Schabereiter-Gurtner et

al., 2004). This chapter reports the investigation of bacterial microorganisms isolated

from limestone caves of Sarawak with potential industrial relevance. These

microorganisms, ureolytic bacteria, prefer to live in alkaline environments, produce an

enzyme which primarily allows calcite precipitation to occur (Achal and Pan, 2011).

This process, microbial-induced calcite precipitation (MICP) is usually directed by

urease enzyme (urea amidohydrolase; EC 3.5.1.5) which is produced by some

microorganisms that relies on urea as their primary source of nitrogen (Zhang et al.,

2015, Achal, 2015).

Urease enzyme was previously studied from clinical evaluation on patients infected

with pathogenic microorganisms (Cheng and Cord-Ruwisch, 2013, Lee and Calhoun,

1997, Mobley et al., 1995). However, the usage of urease on biocementation application

for improvement of soil strengthening has been the subject of various research from the

Microbial biotechnology, geotechnical engineering and civil engineering (Al-Thawadi,

2008, DeJong et al., 2006, Whiffin, 2004). Studies on the alternative source for known

UPB from non-pathogenic bacterial species necessary for urea hydrolysis in

biocementation application are very limited. This research gap forms the basis for, the

initiation of this study. This is the first study elucidating the isolation and identification

of ureolytic bacteria from limestone caves of Sarawak.

42

The objectives of the study in this chapter are as follows:

i. To screen and identify urease-producing bacteria.

ii. To characterise the urease activities of selected isolates.

iii. To test the ability of selected isolates to induce calcium carbonate precipitation

and bacterial growth and pH profiles.

43

2.2 Methods and materials

2.2.1. Sampling location and collection

A field sampling occurred at Fairy Cave Nature Reserve (FCNR) and Wind Cave

Nature Reserve (WCNR) and samples used in this research study were taken from this

sampling site. These caves, Fairy cave (N 01°22’53.39” E 110°07’02.70”) and Wind

cave (N 01°24’54.20” E 110°08’06.94”) are located in Bau, Kuching Division,

Sarawak, East Malaysia, on the island of Borneo. Samples taken from FCNR were

collected at depth of 5-10 cm from regions surrounded by rocks and vegetation, while

samples taken from WCNR were collected from the surfaces of speleothems inside the

cave chamber. Each sample was collected using sterile tools, placed in sterile

polystyrene containers, sealed and stored in an ice box (at the sampling site) before

being transported to Swinburne University of Technology, Sarawak campus for further

microbiological analysis. The samples were then preserved in the refrigerator (4°C)

prior to enrichment culturing.

2.2.2. Biological material

Sporosarcina pasteurii, (DSM33) type strain was purchased from the Leibniz Institute

DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig,

Germany). This bacterial strain was used as a positive control for subsequent

experiments in this study. It was aseptically grown under aerobic batch conditions

according to the DSMZ instruction and stored on Petri plates containing nutrient agar

(HiMedia, Laboratories Pvt. Ltd) at 4oC in the fridge until when usage was necessary.

2.2.3. Growth medium and sterilisation

Nutrient broth (HiMedia) and nutrient agar (HiMedia) were used as a primary growth

medium in this study. All bacterial growth mediums, chemicals (except urea) and

glassware used in this study were sterilised by autoclaving at 121oC, 103.42 kPa for 20

min using an autoclave machine (Hirayama-HVE-50). Urea was sterile filtered through

a 0.45 µm syringe filter.

44

2.2.4. Enrichment cultures

Enrichment of the cave samples was performed as follows: 1 g or 1 mL of each sample

was inoculated into 50 mL growth media (250 mL shaking flasks). The enriched

cultures were placed in an incubation shaker (CERTOMAT® CT plus – Sartorius) under

aerobic batch conditions at 30oC for 120 hr at 130 rpm. The following growth media

were used to enrich the collected cave samples: Nutrient broth (13.0 g.L-1, HiMedia

Laboratories Pvt. Ltd)); Tryptic soy broth (30.0 g.L-1, Merck Millipore); Lactose

peptone broth (35.0 g.L-1, Becton, Dickinson and Company); Luria broth (20.0 g.L-1,

HiMedia Laboratories Pvt. Ltd) and Brain heart infusion broth (37.0 g.L-1, Oxoid

Thermo Scientific Microbiology). Each of the growth culture mediums was

supplemented with C2H3NaO2 (8.2 g.L-1, HiMedia Laboratories Pvt. Ltd), (NH4)2SO4

(10.0 g.L-1, HiMedia Laboratories Pvt. Ltd) and CH4N20 (20.0 g.L-1, Bendosen

Laboratory Chemicals). The initial pH of all media was adjusted to 8.0 using 0.1 M

NaOH or 0.1 M HCL before sterilisation (Reyes et al., 2009). Sterile Urea substrate (by

0.45 µm filter sterilisation) was added post-autoclaving to prevent chemical

decomposition under autoclave condition.

2.2.5. Isolation of urea degrading bacteria

For bacterial isolation, 1 mL of individual enriched culture samples was serially diluted

(sixfold) and plated on nutrient agar (with 6% urea). 0.1 mL aliquot of serially diluted

enrichment samples were inoculated onto Petri plates containing nutrient agar were then

spread using a sterilised L-shaped spreader until the fluid was evenly distributed. The

agar petri plates were then incubated (MMM Incucell ) under aerobic conditions at 32oC

for 42 hr. Upon the growth of isolates capable of hydrolysing 6% urea in petri plates

containing nutrient agar, subsequent sub-culturing was performed until single bacterial

colonies were obtained. Long term storage using glycerol stock was used in this study for

maintenance and preservation of the isolated bacterial isolates. Glycerol stock method

was used for long-term storage of the bacterial isolates by adopting a modified procedure

from Fortier and Moineau (2009).

45

For the maintenance of the bacterial glycerol stock, 500 µL of overnight grown cultures

were inoculated into 2.0 mL cryogenic vials containing sterilised 500 µL of 50%

glycerol to obtain a final glycerol concentration of 25% (v/v). The stocks were mixed

prudently and kept in the refrigerator at -80°C. For the case of reviving stored cells,

sterile toothpick or inoculation loop was used to scrap off the splinters of solid ice and

then onto the nutrient agar medium.

2.2.6. Screening for urease-producing bacteria

Christensen’s medium (Oxoid Thermo Scientific Microbiology Sdn Bhd) also called

urea agar base (UAB) was used to screen for urease producing bacteria (UPB). The

media components contained the following: Peptone (1.0 g.L-1); Glucose (1.0 g.L-1);

Sodium chloride (5.0 g.L-1); Disodium phosphate (1.2 g.L-1); Potassium dihydrogen

phosphate (0.8 g.L-1); Phenol red (0.012 g.L-1) and Agar (15.0 g.L-1). Urea solution, 4%,

(w/v) was separately prepared by filtration with the use of 0.45 µm syringe, and 10 mL

of the urea solution was aseptically introduced into 990 mL of the UAB medium. The

medium was carefully mixed by gentling swirling the Schott bottle containing the UAB

and 10 mL were then distributed into separate sterile test tubes. The bacterial isolates

were heavily inoculated on the surface of the UAB medium and then incubated at 37oC

for 72-120 hr. The urease production test was studied through visual observation for

colour changes. The bacterial isolate able to turn the UAB medium from pale yellow to

pink during the incubation period were selected while others were discarded.

2.2.7. Preliminary identification

Phenotypic analyses were used for a more definitive identification of bacterial isolates.

Morphological, microscopic and biochemical studies were performed under standard

protocols.

2.2.7 (a) Morphological analysis

A loopful of individual UPB cultures was serially subcultured onto Petri plates

containing nutrient agar and incubated for at 32°C for 24 hr. Colony appearance of the

overnight subcultured isolates were recorded with reference to Bergey’s Manual of

Determinative Bacteriology (Holt et al., 1994, Olufunke et al., 2014).

46

2.2.7 (b) Microscopic analysis

Gram staining and endospore staining methods were used to determine and differentiate

the cell morphology of the bacterial isolates. The standard staining protocol used to

differentiate between Gram positive and Gram negative bacteria was adapted from

Moyes, Reynolds and Breakwell (2005). The standard staining protocol used as a

differential stain to determine between the bacterial isolates capable of producing

endospores was adapted from Reynolds et al. (2009).

2.2.7 (c) Biochemical analysis

Motility, oxidase and catalase tests were performed and used for biochemical

characterization of the bacterial isolates. The procedures for these tests were adapted

from standard protocols by Shields and Cathcart (2011), Shields and Cathcart (2013),

and Bisen (2004).

2.2.8. Molecular identification

A Polymerase Chain Reaction (PCR) was used to amplify the 16S rRNA gene of the

unknown isolated urease producing bacteria. The DNA sequences of the 16S rRNA

genes were compared with the generated sequence to a database of a known sequence

which was then used to determine the molecular identification of unknown ureolytic

bacteria isolates.

2.2.8 (a) DNA extraction

A freeze and thaw method was used to lyse bacterial cell of an unknown

microorganism, to prepare Deoxyribonucleic acid (DNA) samples as templates for

DNA amplification. Colonies from 24 hr sub-cultured bacterial isolates were picked

using sterilised toothpicks. Each sample was then placed into individual wells of a

sterile 96 wells plate containing 100 µL Tris-EDTA (TE) buffer solution and then deep

frozen at -80°C for 24 hr as described by (Muramatsu et al., 2003). The 96 wells plate

was then thawed by immersing the plate in a 60°C water bath for 5 min to release DNA

from the microbial cells (Kuek et al., 2015). The lysate was used as a crude DNA

template for PCR.

47

2.2.8 (b) DNA amplification

The PCR technique comprising enzymatic amplification of nucleic acid sequence via

selected repeated denaturing, oligonucleotide annealing and DNA polymerase extension

cycles (Gibbs, 1990), was used in this study. DNA amplification was performed using

MyTaq Red Mix (Biolin) in accordance with the manufacturer’s instructions. PCR

amplification was performed using MyTaq Red Mix (Bioline) according to the

manufacturer’s instructions. The PCR master mix contained the following: template

(200 ng, 2 L); primers (1 L, 20 µm); MyTaq Red Mix (25 L) and sterile ddH2O (22

L). The forward primer, 8F: 5’-AGAGTTTGATCCTGGCTCAG-3’ (Hughes et al.,

2000) and reverse primer, 1525R: 5’-AAGGAGGTGATCCAGCC-3’ (Lane et al.,

1985) were used to amplify the 16S rRNA gene fragment. DNA amplification was

performed using a MasterCycler Gradient Thermal Cycler (Eppendorf 5331). The

cycles consisted of initial denaturation of the template DNA (95°C for 5 min),

denaturation (95°C for 60 sec), annealing (55°C for 60 sec), extension (72°C for 1 min

30 sec) and a final elongation (72°C for 7 min). The process was set to 30 cycles and

the system was held at 4°C.

2.2.8 (c) Visualisation of PCR products

Amplified DNA (PCR product) was visualised on 1% (w/v) agarose gel, stained with 1

L of Midori Green (Nippon Genetics Europe GmbH). The PCR product (5 L) was

loaded into the well of the 1% (w/v) agarose gel. MassRuler™ DNA Ladders (Thermo

Fisher Scientific) was used as a standard to determine the size of the target DNA. The

DNA was separated according to size by gel electrophoresis at 75 volts for 40 min. The

DNA bands were visualised with a gel doc XR system (Biorad) and the image was

captured.

2.2.8 (d) DNA purification and cycle sequencing

PCR purification and cycle sequencing of the products were carried out by First BASE

Laboratory Sdn. Bhd., Malaysia. DNA samples were purified using PCR Cleanup kit

(SS1012/3) with procedures following manufacturer’s instructions. The eluted solutions

(pure DNA) were then stored at -20°C. Sequencing was performed on an Applied

Biosystem 3130xl Genetic Analyzer, using BigDye® Terminator v3. Forward primer,

27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (Heuer et al., 1997) was used while

1525R: 5’-AAGGAGGTGATCCAGCC-3’ was used as a reverse primer.

48

2.2.8 (e) Sequence analysis

The raw DNA chromatogram sequences were viewed using Chromas lite programme,

edited with BioEdit Programme (Hall, 1999) and stored in FASTA format. The forward

and reverse primer sequences were removed before the sequence were blasted with

existing sequences in national centre for biotechnological information (NCBI) GenBank

database (Zhang et al., 2000) using basic local alignment search tool (BLAST)

nucleotide collection database program (Ashelford et al., 2005) to search for closest best

match sequence (Tan et al., 2011). For investigation of the taxonomic composition of

the microbial strains, ribosomal database project (RDP-II) using the SeqMatch tool was

used to search the taxonomy database classification and nomenclature for all of the

organisms in the public sequence databases.

2.2.8 (f) Phylogenetic analysis

Molecular evolutionary genetic analysis (MEGA) version 6 was used to for

phylogenetic analysis (Tamura et al., 2013). Prior to phylogenetic analysis, indefinite

DNA sequences at both ends were removed and the gaps were adjusted to improve the

alignment (Zhao and Cui, 2013). Basic evolutionary distances from the MEGA

programme was used to analyse the DNA sequence (Saitou and Nei, 1987). Bootstrap

replicates (1000) were taken into account to infer the bootstrap consensus tree for the

representation of evolutionary history. The evolutionary distances were then processed

using the maximum composite likelihood method (Tamura et al., 2004, Hanif et al.,

2014).

2.2.8 (g) Nucleotide sequence accession numbers

The nucleotide sequences which were obtained in the present study have been deposited

in NCBI GenBank database (Kaverin et al., 2007). The provided GenBank accession

numbers for the submitted nucleotide sequences are KX212190 to KX212216.

2.2.9. Measurement of urease activity

The conductivity (mS.cm-1) method was used to determine the urease activity (mM urea

hydrolysed.min-1) in this study. For enzyme assay, 1.0 mL of overnight grown bacterial

cultures (0.6-1.0 OD) were inoculated into sterile individual universal bottles (20.0 mL)

containing 9.0 mL of 1.11 M urea solution (Harkes et al., 2010).

49

The changes in conductivity were monitored for a duration of 5 min at 25◦C ±1 and the

respective conductivity values were measured by immersing the probe of the

conductivity meter (Walk LAB conductivity pro meter, Trans Instruments COMPRO)

into the bacterial-urea solution. The conductivity variation rate (mS.cm-1.min-1) was

obtained from the gradient of the graph. The conductivity variation rate was then

multiplied by a dilution factor (df). The df was taken as the ratio of the stock bacteria

culture to the sampling bacteria culture before inoculating into the urea solution (Zhao

et al., 2014). These values were then used to calculate urease activity, by converting the

conductivity variation rate (mS.cm-1.min-1) to urea hydrolysis rate (mM urea

hydrolysed.min-1), based on the correlation that 1 mS.cm-1.min-1 corresponds to a

hydrolysis activity of 11 mM urea.min-1 in the measured range of activities (Paassen,

2009). The urea hydrolysis rate for the urease activity conversion was determined by

(Whiffin, 2004) as described in equation 1.23. Specific urease activity (mM urea

hydrolysed.min-1.OD-1) which reflects the urease catalytic abilities of the urea

hydrolysis (Zhao et al., 2014) was derived by dividing the urease activity (mM urea

hydrolysed.min-1) by the bacterial biomass (OD600). The specific urease activity was

also determined by (Whiffin, 2004) as described in equation 1.24. Biomass

concentration was determined by measuring the optical density of bacterial suspension

with a spectrophotometer (GENESYSTM 20, Thermo Fisher Scientific) at a wavelength

of 600 nm.

2.2.10. Evaluation of microbial calcite precipitation

2.2.10 (a) Testing calcite precipitation

A modified method of Hammes et al. (2003b) was adopted in this study and used to test

the ability of the local isolates to precipitate calcite. The Calcite precipitating media

(CPM) used in this study contains the following components: nutrient broth (3.0 g.L-1,

Oxoid); urea (20.0 g.L-1, Bendosen); NaHCO3 (2.12 g.L-1, Sigma); NH4Cl (10.0 g.L-1,

Sigma); CaCl2 · 2H2O (28.50 g.L-1, Sigma) and agar (20.0 g.L-1, HiMedia). For Calcite

precipitation screening, overnight grown bacterial broth culture were serially diluted

under the sterile condition and spread onto the CPM. The Petri dishes were then

incubated at 30°C for 6 days with the epidermal side facing upwards.

50

2.2.10 (b) Calcite estimation

A modified method of Wei et al. (2015) and Hammad et al. (2013b) were adapted for

this experiment. For a quantitative measurement of calcite precipitation in broth, the

nutrient broth was supplemented with urea 2% (w/v) and calcium chloride 2% (w/v)

solutions. The medium containing overnight grown bacterial cultures were incubated

under shaking condition (150 rpm) at 30°C for duration of 7 days. At the end of the

cultivation, the bacterial cultures were suspended through centrifugation (10,000 g for

60 sec) using centrifuge machine (Eppendorf, 5424R). The pellets which contained the

calcite precipitated and ureolytic bacteria culture were then resuspended centrifuge

tubes containing 50 mL TE buffer (10 mM Tris, 1 mM EDTA pH 8.5). Lysozyme (EC

3.2.1.17), also known as N-acetylmuramide glycanhydrolase was added to the

suspended samples, at a concentration of 1 mg.mL-1 (Wei et al., 2015). The samples

were then incubated at 37°C for 1 hr in order for the lysozyme to properly break down

the cell wall of the ureolytic bacteria. The samples were then centrifuged once more to

separate the cell debris form the calcite precipitates. The supernatants in the centrifuge

tubes were then discarded and dH2O (pH8.5) was added to the centrifuge tubes to wash

the pellets, which were then the air-dried at 37°C for 24 hr. The pellets obtained were

then weighted to estimate the amount of calcite precipitated (Walter et al., 2000).

2.2.11. Bacterial growth profile and pH profile

Ten millilitres (10 mL) of bacterial cultures were grown in universal bottles and

incubated for 24 hr at 32oC under shaking condition (150 rpm). Batch cultures were

prepared by transferring 2.5 mL of the overnight culture into 125 mL of sterile nutrient

broth medium (250 mL capacity conical flasks). The medium was then supplemented

with 6% sterile urea and the batch culture was grown for a total duration of 10 hr. Three

millilitres (3 mL) of the aliquot was sampled from the batch culture at every hour (1 hr)

and transferred into a 10 mm cuvette. A spectrophotometer (Genesys TM 20- Thermo

Scientific) was used to measure the optical density of the bacterial culture at 600 nm. A

pH meter (SevenEasyTM –Mettler Toledo) was also used to study the pH profile of the

bacterial culture by measuring the changes in pH during the incubation period.

51

2.2.12. Statistical analysis

The data were shown as mean ±SE (standard deviation) for three replicates. The results

were subjected to student’s t-test analysis, with statistical significance taken as p<0.05.

GraphPad (Quick Calc) programme was used to analyse the data.

52

2.3 Results

2.3.1. Sampling location and sample collection

Upon the authorization by Sarawak Forest Department and Sarawak Biodiversity

Centre (permit number NCCD.907.4.4 [JLD.11]-37 and SBC-RA-0102-DO)

respectively, to collect samples from nature reserves in Sarawak and conduct biological

research. A total of twelve samples (Table 2.1) were collected in March 2015 from

FCNR (Figure 2.1) and WCNR (Figure 2.2). These caves are about 5 km south-west of

Bau and 30 km from Kuching (Mohd et al., 2011). The caves are part of the nature

reserves protected by environmental laws that preserve the forest, national parks, and

nature reserve. According to Sarawak forestry department (1992), these caves covers 56

and 6.16 hectares respectively and are largely surrounded by forests. FCNR and WCNR

are also part of Bau limestone areas, covering about 150 km2 in Southwest Sarawak

(Mohd et al., 2011). The samples were collected using aseptic techniques and all

samples were stored in the refrigerator at 4oC until the enrichment and isolation

procedures were fully completed.

Table 2.1: Description of samples collected from FCNR and WCNR

WC= Wind cave; FC= Fairy cave; oC= temperature; (%)RH = relative humidity

Sample ID Sample collected Colour Texture oC (%)

RH WC1 Drapery Grey Coarse 29.2 76

WC2 Stalactite White Silk 29.7 78

WC3 Stalactite White Coarse 29.1 80

WC4 Drapery White Coarse 29.2 84

WC5 Stalactite White Fine 30.1 73

WC6 Mudbank Brown Coarse 30.5 76

WC7 Liquid nil nil 28.8 79

FC1 Soil Black Silk 27.2 84

FC2 Soil Grey Coarse 24.8 86

FC3 Soil Brown Clay 26.5 93

FC4 Soil Brown Fine 28.5 90

FC5 Soil Brown Coarse 30.4 89

53

Figure 2.1: Sampling collection site situated in FCNR, Bau, Sarawak. Samples were collected from regions surrounded by rocks and vegetation.

Figure 2.2: Sampling collection site situated in WCNR, Bau, Sarawak. Samples were collected from regions inside the cave chamber.

54

2.3.2. Enrichment culturing and bacterial isolation

In this study, using the aforementioned methods in the enrichment culture and bacterial

isolation process, a total of ninety morphologically distinct urea degrading bacteria

(UDB) colonies were successfully isolated from samples collected from FC and WC in

Sarawak, Malaysia. The samples collected from FCNR and WCNR were subsequently

cultured in different growth medium to target a variety of bacterial species capable of

hydrolyzing urea. Six percentage (6%) of urea was used in the enrichment culture

medium was used in order to screen for microorganisms capable of surviving at high

urea concentration and potentially able to produce high urease enzyme. During

incubation of the enriched samples, which occurred for a total duration of 12 hr, a

pungent smell was observed at 48 hr of incubation which suggests the release of

ammonia as a result of urea degradation by the production of urease from the

microorganisms in the enrichment culture samples. The isolates were selected from

nutrient agar plates containing a variety of microbial colonies as shown in Figure 2.3.

The selected UDB were consequently sub-cultured onto separate nutrient agar with

higher urea substrate percentage, to target isolates capable of degrading 6% urea. Pure

colonies of the UDB are shown in Figure 2.4. All ninety bacterial isolates were able to

grow on nutrient agar (6% urea). The purified bacterial colonies were then preserved as

glycerol (25%) stock at -80oC using a method adapted by Fortier and Moineau (2009).

Figure 2.3: Microorganisms grown on nutrient agar plates supplemented with 2% urea. The plates were incubated for 24-48 hr at 32°C.

55

Figure 2.4: Pure colonies of urea degrading bacteria after enrichment culture. Cave bacterial isolates (A) NB23, (B) TSB55, (C), (D) TSB 46, (E) TSB40 and (F) LB28 grown on nutrient agar plates supplemented with 6% urea and were incubated for 24 hr at 32°C to acquire pure bacterial colonies. 2.3.3. Selection of urease producing bacteria

The screening for UPB was conducted using UAB medium in test tubes as shown in

Figure 2.5. The colour changes of the test tubes from pale yellow to pink-red indicated

positive urease production. Out of the ninety bacteria isolates subcultured from the cave

samples, thirty-one bacterial isolates were selected based on the ability of the isolates to

completely turn the UAB medium pink in comparison to other isolated urease

producing bacteria and the control strain used in this study. The time taken for the

bacterial isolates and the control strain (Sporosarcina pasteurii, DSM33) to turn the

UAB medium pink was observed and noted.

A

D

C

E F

B

56

In Table 2.2, the control strain, bacterial isolates NB33, LPB21, NB28, LPB4, NB30,

NB40 and LPB4 were able to completely turn their respective UAB medium from

yellow to pink between 24-30 hr of incubation period while other UPB isolates were

able to change their respective UAB medium to pink between 36-120 hr of incubation.

The bacterial isolates which were unable to produce urease enzyme by turning the UAB

medium from pale yellow to pink were discarded.

Figure 2.5: Urease production test using UAB medium. The bacterial isolates were incubated at 37oC for 120 hr to test their ability to produce urease enzyme. Out of the 90 bacteria isolates subcultured from the cave samples, 31 bacterial isolates were able to turn the UAB medium from yellow to pink

57

Table 2.2: Hydrolysis of urea by isolates UAB medium

No Isolate ID Time (hr)

1 Control 24 2 NB33 26 3 LPB21 24 4 NB28 28 5 NB40 30 6 LPB4 30 7 TSB21 76 8 NB30 24 9 TSB4 120 10 TSB14 48 11 TSB46 38 12 BHIB17 68 13 BHIB18 70 14 NB23 70 15 TSB55 62 16 TSB31 36 17 TSB40 46 18 TSB29 40 19 TSB12 38 20 TSB8 48 21 LPB22 78 22 BHIB15 60 23 TSB20 120 24 LB6 86 25 LB48 82 26 LB1 70 27 LPB41 60 28 LB31 48 29 TSB2 72 30 A63 60 31 B53 60 32 A62 64

58

2.3.4. Phenotypic characterisation

The thirty-one isolates were initially identified using phenotypic characterizations such

as morphology, microscopic and biochemical analysis. Macroscopic morphological

analysis of the UPB colonies such as shapes, colours of the colonies, the diffusible

pigmentation of the isolates, to name a few, were observed and recorded. The colony

morphology of the bacterial isolates sub-cultured on nutrient agar media is described in

Table 2.3. It was observed that the UPB were isolated from all the samples collected

from FCNR and WCNR except enrichment sample FC1. However, the UPB isolated

from enrichment sample WC6 and WC1 showed the most number of bacterial isolates.

It was also noticeable that most of the UPB colonies had brownish-white and brown

pigmentations, circular forms and smooth surfaces. The microscopic and biochemical

analysis which were used in this study to further classify the analytic bacterial isolates

as detailed in Table 2.4 and Table 2.5 were performed under standard methods. The

majority of the bacterial isolates were Gram-positive bacteria while only three of the

isolates (A63, B53, and A62) were Gram-negative bacteria. Gram staining analysis also

showed the majority of the bacterial cells were rod-shaped except for NB23 which was

a coccus. Endospore staining test results indicate that all except NB23 were spore

forming bacteria. Oxidase and motility test indicated that all bacterial isolates except

TSB21, NB23, TSB14 and LPB41 tested positive. Catalase test showed that all bacterial

isolates except LPB41 and TSB14 tested positive.

59

Table 2.3: Morphological characteristics of isolated bacterial colonies Cave

Origin Isolate

ID Form

(shape) Size

(mm) Optical

Property Pigmentation

WC6 NB33 circular 4 transparent brownish-

white WC6 LPB21 circular 5 opaque brownish-

white WC1 NB28 circular 4 translucent brownish-

white WC1 NB40 circular 6 transparent brown WC2 LPB4 filamentous 12 transparent whitish-

yellow FC4 TSB21 circular 6 transparent white FC5 NB30 circular 3 transparent brownish-

yellow WC1 TSB4 irregular 10 transparent white WC6 TSB14 irregular 3 transparent brown WC3 TSB46 irregular 9 translucent brown FC2 BHIB17 irregular 5 translucent brownish-

yellow FC2 BHIB18 circular 7 opaque brown WC2 NB23 circular 5 transparent brown WC3 TSB55 circular 1 transparent white WC7 TSB31 circular 8 translucent brownish-

yellow FC5 TSB40 irregular 6 transparent brown WC3 TSB29 irregular 4 opaque brownish-

white FC3 TSB12 circular 6 opaque brownish-

white WC5 TSB8 circular 3 translucent brownish-

white WC5 LPB22 irregular 5 transparent brown WC5 BHIB15 circular 2 transparent white FC4 TSB20 irregular 6 transparent brown WC3 LB6 irregular 4 transparent brownish-

yellow WC6 LB48 irregular 3 opaque white WC6 LB1 circular 6 transparent brown FC3 LPB41 circular 2 translucent white FC5 LB31 circular 4 opaque brown WC1 TSB2 irregular 5 transparent brown FC2 A63 circular 1 translucent creamy WC4 B53 circular 1 opaque creamy WC4 A62 circular 1 opaque creamy

60

Table 2.4: Microscopic characteristics of bacterial isolates

+ve= positive; -ve= negative.

Isolate ID

Gram stain

Cell shape

Endospore stain

NB33 +ve, purple rod +ve LPB21 +ve, purple rod +ve NB28 +ve, purple rod +ve NB40 +ve, purple rod +ve LPB4 +ve, purple rod +ve TSB21 +ve, purple rod +ve NB30 +ve, purple rod +ve TSB4 +ve, purple rod +ve TSB14 +ve, purple rod +ve TSB46 +ve, purple rod +ve BHIB17 +ve, purple rod +ve BHIB18 +ve, purple rod +ve NB23 +ve, purple coccus -ve TSB55 +ve, purple rod +ve TSB31 +ve, purple rod +ve TSB40 +ve, purple rod +ve TSB29 +ve, purple rod +ve TSB12 +ve, purple rod +ve TSB8 +ve, purple rod +ve LPB22 +ve, purple rod +ve BHIB15 +ve, purple rod +ve TSB20 +ve, purple rod +ve LB6 +ve, purple rod +ve LB48 +ve, purple rod +ve LB1 +ve, purple rod +ve LPB41 +ve, purple rod +ve LB31 +ve, purple rod +ve TSB2 +ve, purple rod +ve A63 -ve, pink rod +ve B53 -ve, pink rod +ve A62 -ve, pink rod +ve

61

Table 2.5: Biochemical characteristics of bacterial isolates

+ve= positive; -ve= negative.

Isolate ID Oxidase Catalase Motility

NB33 +ve +ve +ve LPB21 +ve +ve +ve NB28 +ve +ve +ve NB40 +ve +ve +ve LPB4 +ve +ve +ve TSB21 -ve -ve -ve NB30 +ve +ve +ve TSB4 +ve +ve +ve TSB14 -ve -ve -ve TSB46 +ve +ve +ve BHIB17 +ve +ve +ve BHIB18 +ve +ve +ve NB23 -ve +ve -ve TSB55 +ve +ve +ve TSB31 +ve +ve +ve TSB40 +ve +ve +ve TSB29 +ve +ve +ve TSB12 +ve +ve +ve TSB8 +ve +ve +ve LPB22 +ve +ve +ve BHIB15 +ve +ve +ve TSB20 +ve +ve +ve LB6 +ve +ve +ve LB48 +ve +ve +ve LB1 +ve +ve +ve LPB41 -ve -ve -ve LB31 +ve +ve +ve TSB2 +ve +ve +ve A63 +ve +ve +ve B53 +ve +ve +ve A62 +ve +ve +ve

62

2.3.5. Molecular characterization

The DNA templates from the thirty-one UPB were successfully extracted using freeze

and thaw method adapted from Muramatsu et al.(2003). In this study, using the

aforementioned method in PCR amplification process, the DNA bands were visualised

on 1% agarose gel containing Midori Green (Nippon Genetics Europe GmbH) using gel

doc XR system (Biorad). The forward and reverse primer sequences were removed

before the sequence were blasted with existing sequences in NCBI GenBank database

(Zhang et al., 2000) using BLAST nucleotide collection database program to search for

closest best match sequences (Tan et al., 2011, Ashelford et al., 2005).

The nucleotide BLAST analysis results of the 16S rRNA region displayed a rational

level of correlation with the physiological characterization, especially the

morphological descriptions of species within the genus (Achal et al., 2011). All

bacterial isolates from limestone caves of Sarawak showed high degrees of similarity

(91-99%) to their respective closest bacterial species as shown in Table 2.6. The

BLAST results suggested that the UPB were closely related to bacteria from the

Sporosarcina pasteurii group, Pseudogracilibacillus auburnensis group,

Staphylococcus aureus group, Bacillus lentus group, Sporosarcina luteola group and

Bacillus fortis group. The result in Table 2.6 also suggest that the isolated ureolytic

bacteria can be classified into their closest relative groups as Sporosarcina pasteurii

(NB33, LPB21, NB28, NB40, LPB4, NB30, TSB4, TSB46, BHIB17, BHIB18, TSB31,

TSB40, TSB29, TSB12, TSB8, LPB22, BHIB15, TSB20, LB6, LB1 and TSB2),

Pseudogracilibacillus auburnensis (TSB21, TSB14 and LPB41), Staphylococcus

aureus (NB23), Bacillus lentus (TSB55), Sporosarcina luteola (LB48 and LB31) and

Bacillus fortis (A63, B53, and A62).

Results from the taxonomic composition of the UPB using ribosomal database project

(RDP-II), specifically with the aid of the SeqMatch tool confirmed the taxonomy

database classification and nomenclature for the UPB in the public sequence databases

as shown in Table 2.7. The findings suggest the majority of the UPB were classified

into the family and genus of Planococcaceae Sporosarcina while the rest UPB were

classified into the family and genus Bacillaceae Bacillus and Staphylococcaceae

Staphylococcus. A phylogenetic tree shown in Figure 2.7 was constructed using a

neighbour-joining method (Liang et al., 2008).

63

The phylogenetic tree suggests that the majority of the isolated UPB were related to

Sporosarcina pasteurii group. However, there was still satisfactory diversity in the 16

rDNA to differentiate the ureolytic bacterial isolates into distinctive clusters (Devos et

al., 2005) which were noticeably acknowledged in Figure 2.6. The largest clusters

observed from the phylogenetic tree suggest that majority of the ureolytic isolates are

closely related to Sporosarcina pasteurii. However, LPB22 and TSB2 were grouped in

a cluster together while TSB20 was grouped as an independent cluster, but they were

derived from a common ancestor member of Sporosarcina pasteurii. Isolate A63, B53,

and A62 were grouped in one cluster while TSB21, TSB14, and LPB41 were grouped

in another cluster. However, isolates TSB55 and NB23 were as independent clusters

and were noticeably distant from others.

64

Table 2.6: Molecular identification based on 16S rRNA sequencing data using NCBI nucleotide BLAST database

No Isolate ID

GenBank Accession number

Closest match Base pair Query Cover

Similarity

1 NB33 KX212190 Sporosarcina pasteurii strain WJ-4 [KC211296] 1198 100% 97%

2 LPB21 KX212191 Sporosarcina pasteurii strain fwzy14 [KF208477] 1385 95% 97%

3 NB28 KX212192 Sporosarcina pasteurii strain WJ-5[KC211297] 1280 90% 96% 4 NB40 KX212193 Sporosarcina pasteurii strain WJ-5 [KC211297] 1200 99% 97%

5 LPB4 KX212194 Sporosarcina pasteurii strain WJ-4 [KC211296] 1298 98% 97%

6 TSB21 KX212195 Pseudogracilibacillus auburnensis [KR153879] 1050 99% 93%

7 NB30 KX212196 Sporosarcina pasteurii strain fwzy14 [KF208477] 1279 99% 98%

8 TSB4 KX212197 Sporosarcina pasteurii strain WJ-4 [KC211296] 599 99% 99%

9 TSB14 - Pseudogracilibacillus auburnensis [KR153879] 1119 93% 94%

10 TSB46 KX212198 Sporosarcina pasteurii strain WJ-4 [KC211296] 1219 98% 96%

11 BHIB17 KX212199 Sporosarcina pasteurii strain WJ-4 [KC211296] 1200 93% 97%

12 BHIB18 - Sporosarcina pasteurii strain WJ-4 [KC211296] 1147 90% 96%

13 NB23 - Staphylococcus aureus strain CICC [KJ643929] 1275 95% 95%

14 TSB55 KX212200 Bacillus lentus strain NBRC 16444 [NR112631] 920 99% 91%

15 TSB31 KX212201 Sporosarcina pasteurii strain WJ-5 [KC211297] 1219 99% 97%

16 TSB40 KX212202 Sporosarcina pasteurii strain WJ-5 [KC211297] 1159 100% 98% 17 TSB29 KX212203 Sporosarcina pasteurii strain WJ-4 [KC211296] 1250 99% 98%

18 TSB12 KX212204 Sporosarcina pasteurii strain fwzy14 [KF208477] 1200 100% 99%

19 TSB8 - Sporosarcina pasteurii strain fwzy14 [KF208477] 1150 81% 97%

65

20 LPB22 KX212205 Sporosarcina pasteurii strain WJ-5 [KC211297] 1151 99% 96%

21 BHIB15 KX212206 Sporosarcina pasteurii strain fwzy14 [KF208477] 1198 100% 99%

22 TSB20 KX212207 Sporosarcina pasteurii strain WJ-4 [KC211296] 1250 99% 95%

23 LB6 KX212208 Sporosarcina pasteurii strain WJ-4 [KC211296] 1110 100% 99%

24 LB48 KX212209 Sporosarcina luteola strain WJ-1 [KF208477] 1269 92% 98%

25 LB1 KX212210 Sporosarcina pasteurii strain WJ-5 [KC211293] 1374 93% 97%

26 LPB41 KX212211 Pseudogracilibacillus auburnensis [KR153879] 1298 99% 95%

27 LB31 KX212212 Sporosarcina luteola strain WJ-1 [KF208477] 1149 99% 99%

28 TSB2 KX212213 Sporosarcina pasteurii strain WJ-3 [KC211295] 1267 98% 97%

29 A63 KX212214 Bacillus fortis strain R-6514 [NR042905] 1250 98% 96%

30 B53 KX212215 Bacillus fortis strain R-6514 [NR042905] 1325 99% 97%

31 A62 KX212216 Bacillus fortis strain R-6514 [NR042905] 1248 100% 97%

66

Table 2.7: The nomenclatural taxonomy obtained using Ribosomal Database Project-II database

No Isolate ID Domain Phylum Class Order Family Genus

1 NB33 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

2 LPB21 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

3 NB28 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

4 NB40 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

5 LPB4 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

6 TSB21 Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus

7 NB30 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

8 TSB4 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

9 TSB14 Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus

10 TSB46 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

11 BHIB17 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

12 BHIB18 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

13 NB23 Bacteria Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus

14 TSB55 Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus

15 TSB31 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

16 TSB40 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

17 TSB29 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

18 TSB12 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

67

19 TSB8 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

20 LPB22 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

21 BHIB15 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

22 TSB20 Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina Firmicutes

23 LB6 Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina Firmicutes

24 LB48 Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina Firmicutes

25 LB1 Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina Firmicutes

26 LPB41 Firmicutes Bacilli Bacillales Bacillaceae Bacillus Firmicutes

27 LB31 Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina Firmicutes

28 TSB2 Bacteria Firmicutes Bacilli Bacillales Planococcaceae Sporosarcina

29 A63 Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus

30 B53 Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus

31 A62 Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus

68

Figure 2.6: Phylogenetic tree based on the bacterial 16S rRNA gene sequence data sequence from different isolates of the current study along with sequences available in the GenBank database (Kang et al., 2014a). The results show that the bacterial isolates were identified as Sporosarcina pasteurii, Pseudogracilibacillus auburnensis, Staphylococcus aureus, Bacillus lentus, Sporosarcina luteola and Bacillus fortis. The tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 6 (Tamura et al., 2013, Tan et al., 2011). Numerical values indicate bootstrap percentile from 1,000 replicates. Bar, 0.005 substitutions per nucleotides (Kang et al., 2014c).

69

2.3.6. Measurement of conductivity

Urease activity was measured through changes in conductivity (mS.cm-1) in the absence of

calcium ions (Whiffin, 2004). The conductivity (mS.cm-1) of each locally isolated

ureolytic bacteria and the control strain were measured for duration of 5 min and the

gradient was obtained from the curve of conductivity (mS.cm-1) against time (hr). Each of

the conductivity measured for individual UPB was performed in three trials, each having

three replicates to obtain data presented as mean ±SE (standard deviation). Figure 2.7

showed a curve of relative conductivity for bacterial culture from LPB21 where the

conductivity measured at 0 min was 6.98 mS.cm-1. The conductivity variation rate for

isolate LPB21 was 0.142 mS.cm-1.min-1 as shown in Figure 2.7. The conductivity variation

rate for the rest of UPB isolates and the control strain are respectively shown in Table 2.8.

The result from table 2.8 showed bacterial isolates NB33, LPB21, NB28, NB30 and

control strain had 0.194, 0.169, 0.132, 0.140 and 0.127 mS.cm-1.min-1 respectively,

suggesting they had the highest conductivity variation rate when compared to the rest

isolates. However, in comparison to all the isolates including the control strain, NB33

showed the highest conductivity variation rate which is 0.194 mS.cm-1.min-1, while TSB4

showed the lowest conductivity variation rate which is 0.010 mS.cm-1.min-1.

2.3.7. Urease Activity Assay

The urease activity of the locally isolated ureolytic bacteria was calculated and compared

to that of the control strain. Table 2.9 showed the conductivity variation rate (mS.cm-

1.min-1) to urease activity (mM urea hydrolysed.min-1)The conductivity was multiplied by

the dilution factor (df) and the constant (11.11) derived by Whiffin (2004), based on the

correlation that 1 mS.cm-1.min-1 corresponds to a hydrolysis activity of 11 mM urea.min-1

in the measured range of activities (Paassen, 2009). The urease activity shown in Table 2.9

showed bacterial isolates NB33, LPB21, NB28, NB30, and control strain had 21.513,

18.768, 14.636, 15.587 and 14.087 mM urea hydrolysed.min-1 respectively, suggesting

they had the highest urease activities when compared to the rest isolates. However, in

comparison to all the isolates including the control strain, NB33 showed the highest urease

activity which is 21.513 mM urea hydrolysed.min-1, while TSB4 showed the lowest urease

activity which is 1.130 mM urea hydrolysed.min-1.

70

Figure 2.7: Relative conductivity of isolate LPB21 measured for a duration of 5 min. Error bars represent standard error of the mean.

y = 0.1415x + 0.0862

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 1 2 3 4 5 6

Co

nd

uct

ivit

y (

mS

.cm

-1)

Time (min)

71

Table 2.8: Measurement of conductivity variation rate and SEM

No Isolate ID

Conductivity variation rate (mS.cm-1.min-1) SEM

1 Control 0.127 0.027 2 NB33 0.194 0.057 3 LPB21 0.169 0.038 4 NB28 0.132 0.058 5 NB40 0.083 0.019 6 LPB4 0.103 0.023 7 TSB21 0.059 0.022 8 NB30 0.14 0.018 9 TSB4 0.01 0.006 10 TSB14 0.069 0.015 11 TSB46 0.091 0.010 12 BHIB17 0.089 0.021 13 BHIB18 0.085 0.008 14 NB23 0.065 0.038 15 TSB55 0.067 0.039 16 TSB31 0.103 0.024 17 TSB40 0.078 0.021 18 TSB29 0.087 0.014 19 TSB12 0.112 0.008 20 TSB8 0.094 0.043 21 LPB22 0.056 0.004 22 BHIB15 0.071 0.027 23 TSB20 0.016 0.001 24 LB6 0.053 0.016 25 LB48 0.052 0.026 26 LB1 0.06 0.011 27 LPB41 0.075 0.011 28 LB31 0.114 0.017 29 TSB2 0.058 0.015 30 A63 0.113 0.033 31 B53 0.089 0.005

72

Table 2.9: Conversion of changes in conductivity to urease activity

No Isolate ID (mS.cm-1.min-1) (mS.cm-1.min-1) *df

mM urea hydrolysed.min-1

1 Control 0.127 1.268 14.087 2 NB33 0.194 1.936 21.513 3 LPB21 0.169 1.689 18.768 4 NB28 0.132 1.317 14.636 5 NB40 0.083 0.826 9.181 6 LPB4 0.103 1.027 11.414 7 TSB21 0.059 0.588 6.529 8 NB30 0.140 1.403 15.587 9 TSB4 0.010 0.102 1.130 10 TSB14 0.069 0.695 7.718 11 TSB46 0.091 0.914 10.158 12 BHIB17 0.089 0.890 9.892 13 BHIB18 0.085 0.848 9.425 14 NB23 0.065 0.654 7.270 15 TSB55 0.067 0.666 7.399 16 TSB31 0.103 1.027 11.406 17 TSB40 0.078 0.783 8.703 18 TSB29 0.087 0.871 9.677 19 TSB12 0.112 1.121 12.451 20 TSB8 0.094 0.941 10.458 21 LPB22 0.056 0.562 6.240 22 BHIB15 0.071 0.706 7.847 23 TSB20 0.016 0.159 1.763 24 LB6 0.053 0.531 5.903 25 LB48 0.052 0.523 5.811 26 LB1 0.060 0.600 6.662 27 LPB41 0.075 0.747 8.299 28 LB31 0.114 1.138 12.647 29 TSB2 0.058 0.576 6.396 30 A63 0.113 1.130 12.554 31 B53 0.089 0.890 9.888 32 A62 0.084 0.840 9.332

df = dilution factor; mS.cm-1.min-1= conductivity variation rate; mM urea hydrolysed.min-1= urease activity.

73

2.3.8. Determination of specific enzyme activity

The SUA of the locally isolated ureolytic bacteria were individually calculated and

compared to that of the control strain. The SUA shown in Figure 2.8 was determined as

the amount of urease activity per unit biomass (Whiffin, 2004). The biomass (OD600)

was determined by measuring the optical density of overnight ureolytic bacterial

cultures. The SUA shown in Figure 2.8 showed bacterial isolates NB33, LPB21, NB28,

NB30 and control strain had 19.975, 23.968, 19.275, 20.091 and 17.751 mM urea

hydrolysed.min-1.OD-1 respectively, suggesting they had the highest specific urease

activities when compared to the rest isolates. However, in comparison to all the isolates

including the control strain, LPB21 showed the highest SUA which is 23.968 mM urea

hydrolysed.min-1.OD-1, while TSB4 showed the lowest urease activity which is 1.594

mM urea hydrolysed.min-1.OD-1. Each of these isolates (NB33, LPB21, NB28, NB30,

and control strain) had biomass OD of 1.072, 0.785, 0.738, 0.775 and 0.783 when

measured at a wavelength of 600 nm.

An independent-samples t-test was conducted to compare the SUA of the UPB isolated

from limestone caves of Sarawak against the SUA of the control strain used in this

study. GraphPad program was used to determine if there is a significant difference

between the mean values. The results in Table 2.10 showed out of thirty-one UPB, there

were no significant differences between the SUA of twenty-two UPB when compared

with the control strain. However, bacterial isolate A62 (M=12.4111, SD=0.979), A63

(12.311, SD=3.947), TSB14 (M=12.052, SD=1.527), LPB41 (M=11.480, SD=0.919),

LPB22 (M=9.227, SD=0.0242), TSB2 (M=9.171, SD=2.096), TSB20 (M=2.497,

SD=0.341) and TSB4 (M=1.594, SD=0.768) showed there were significant differences

between their respective SUA when compared with the control strain (M=17.751,

SD=2.345). In addition, four bacterial isolates with the highest SUA as shown in Figure

2.8 were also compared with the control strain to test for statistical analysis. The result

in Table 2.10 showed that SUA of LPB21 (M=23.968, SD=5.722), NB30 (M=20.091,

SD=1.849), NB (M=19.975, SD=5.227) and NB (M=19.275, SD=5.512) were not

significantly different from the SUA of the control strain (M=17.751, SD=2.345).

74

The rationale of this study was to isolate and screen for UPB with comparable urease

production or SUA with the control strain, Sporosarcina pasteurii, (DSM33) type strain

purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and

Cell Cultures (Braunschweig, Germany). The results from Figure 2.8 and Table 2.10

supports the suggestion that the urease production of ureolytic bacteria LPB21, NB30,

NB33, and NB33 are comparable with the control strain as their respective SUA are

higher than that of the control strain and the t-test analysis also confirms there were no

significant differences between their independently mean values (SUA), thus

confirming suggestion that SUA of the aforementioned bacterial isolates is comparable

with the control strain used in this study.

The effectiveness of these isolates to show comparatively high SUA justify the decision

to confine the selection of these ureolytic bacteria for the subsequent studies in this

chapter and in chapter four. The decision to choose only four ureolytic bacteria (LPB21,

NB30, NB33, and NB28), out of the thirty-one ureolytic bacteria isolated from

limestone caves of Sarawak is because of the SUA these bacteria showed in comparison

to other bacterial isolates and control strain.

75

Figure 2.8: Specific urease activity (mM urea hydrolysed.min-1.OD-1) of urease-producing bacteria and the control strain. Error bars represent standard error of the mean.

0

5

10

15

20

25

30

Spe

cifi

c u

reas

e a

ctiv

ity

(mM

ure

a h

ydro

lyse

d-1

.min

.OD

-1)

76

Table 2.10: t-test results comparing the specific urease activity differences between individual isolated urease-producing bacteria and control strain. (N=3; df=2)

No Isolate ID M SD SE P-value t P <*

1 control 17.751 2.345 nil nil nil nil 2 LPB21 23.968 5.722 4.407 0.294 1.4107 - 3 NB30 20.091 1.849 4.217 0.339 1.2471 - 4 NB33 19.975 5.227 2.667 0.651 0.5272 - 5 NB28 19.275 5.512 2.904 0.625 0.5714 - 6 TSB12 17.400 2.839 1.774 0.9149 0.1207 - 7 TSB31 16.397 2.963 3.174 0.525 0.7634 - 8 LPB4 14.063 3.564 2.823 0.365 1.1624 - 9 TSB29 13.618 2.623 2.190 0.281 1.4641 - 10 B53 13.562 1.989 1.343 0.196 1.9128 - 11 LB31 13.052 0.605 1.555 0.073 3.4989 - 12 NB40 13.003 2.430 3.880 0.093 3.0542 - 13 TSB8 12.714 5.285 4.566 0.324 1.2983 - 14 TSB55 12.715 7.231 2.265 0.3850 1.103 - 15 BHIB15 12.473 2.172 0.880 0.145 2.3299 - 16 A62 12.411 0.979 1.123 0.026 6.0649 + 17 A63 12.311 3.947 2.931 0.040 4.8438 + 18 TSB21 12.089 5.985 0.558 0.193 1.9323 - 19 TSB14 12.052 1.527 2.931 0.010 10.2095 + 20 TSB40 11.994 2.743 0.934 0.1930 1.9323 - 21 LPB41 11.480 0.919 1.745 0.022 6.7172 + 22 BHIB17 11.324 1.921 2.060 0.066 3.6838 - 23 TSB46 10.799 2.047 1.983 0.078 3.3742 - 24 LB1 9.956 1.214 3.755 0.059 3.9304 - 25 NB23 9.813 5.070 1.214 0.1688 2.1142 - 26 LBP22 9.227 0.242 1.593 0.020 7.0212 + 27 TSB2 9.171 2.096 2.296 0.033 5.3851 + 28 BHIB18 8.822 1.646 3.513 0.060 3.8881 - 29 LB48 8.652 4.073 2.450 0.122 2.59 - 30 LB6 7.590 3.133 1.267 0.054 4.1468 - 31 TSB20 2.497 0.341 1.594 0.007 12.0372 + 32 TSB4 1.594 0.768 4.152 0.010 10.1363 +

N= number of sample size; df= degree of freedom; M=mean; SE= standard error; SD= standard deviation; P-value= calculated probability; t= test statistic; += significant; -= not significant; *= P-value is significant at 0.05 level.

77

2.3.9. Microbial calcite precipitates

The CPM was used to test the ability of the UPB to induce calcium carbonate. Bacterial

isolates LPB21, NB28, NB33, NB30, and control strain were selected for this

experiment and the remaining subsequent experiments in this study because they

showed highest enzyme activities in Figure 2.8 and Table 2.9. The bacterial isolates

were cultivated for 24 hr and the serially diluted before being spread on the CPM. The

CPM was studied through visual observation for the formation of precipitates on the

CPM upon addition of bacterial cultures. When tested on CPM, UPB isolates, and

control strain was able to induce precipitate calcite after being incubated at 30°C for 6

days. Milky-white crystal was observed covering the colonies grown on the CPM and

appeared at the 4th day of incubation seen in Figure 2.9. All the precipitates grown on

the CPM appeared as a distinct circular zone around the growth area of the bacterial

colonies. Based on the morphology of the precipitation there was no difference in

crystal formation on the agar plates, all isolates induced the same morphological sizes,

shape and colour of the precipitates.

Figure 2.9: Calcite precipitation media. The appearance of calcite precipitates on bacterial colonies on the 4th day of incubation at 30°C.

78

2.3.10. Calcite estimation

The calcite precipitation induced by LPB21, NB28, NB33, NB30, and control strain

were studied by using a modified method of Wei et al. (2015). Calcite precipitates were

quantified after nutrient broth supplemented with 2% urea and 2% calcium chloride

solutions inoculated with overnight grown cultures under shaking condition (150rpm) at

30°C for incubation period of 7 days.

Figure 2.10: Comparison of calcite precipitated by selected UPB isolates and the control strain. Error bars represent standard error of the mean.

0

2

4

6

8

10

12

14

16

18

20

control NB30 LPB21 NB33 NB28

we

igh

t o

f ca

lcit

e p

reci

pit

ate

s(m

g.m

L-1

)

79

The moment the bacterial cultures were inoculated into the broth media, white

precipitates appeared instantly at the bottom of the conical flasks and its density

increased with incubation. At the end of 7 days incubation, the precipitates were

collected and weighed. All five isolate (including control strain) produced a similar

amount of calcite precipitates at the end of the incubation period. Figure 2.10 showed

the bacterial NB33, LPB21, NB28, NB30, and control strain induced 12.44, 15.82,

10.51, 17.00 and 10.51 mg.mL-1 of calcite precipitates, respectively. This finding

suggests that UPB strain NB30 showed the highest productivity of calcite which was

17.0 mg.mL-1.

Table 2.11: t-test results comparing the calcite precipitate differences between individual isolated urease-producing bacteria and control strain. (N=3; df=2)

No Isolate ID M SD SE P-

value t P <*

1 control 10.511 0.834 nil nil nil nil

2 LPB21 15.822 0.731 0.633 0.014 8.392 +

3 NB30 17.000 0.581 0.774 0.014 8.384 +

4 NB33 12.444 0.269 0.329 0.028 5.879 +

5 NB28 10.511 2.672 1.084 1.000 0.000 -

N= number of sample size; df= degree of freedom; M=mean; SE= standard error; SD= standard deviation; P-value= calculated probability; t= test statistic; += significant; -= not significant; *= P-value is significant at 0.05 level.

80

An independent-samples t-test was conducted to compare the calcite precipitated by the

UPB isolates against that of the control strain used in this study. The results in Table

2.11 showed that there were significant differences between the calcite precipitated by

LPB21 (M=15.822, SD=0.731), NB30 (M=17.000, SD=0.581) and NB33 (M=12.444,

SD=0.269) against the control strain (M=10.511, SD=0.0834). On the other hand, there

was no significant difference between the calcite precipitated by NB28 (M=10.5111,

SD=2.672) against the control strain.

2.3.11. Bacterial growth and pH profiles

Optical density (OD) at a wavelength of 600 nm, indicative of bacterial growth, is

presented in Figure 2.11 which was studied up to 12 hr in a batch culture containing

nutrient broth and 6% urea. It was observed from the graph that the growth of the UPB

cells increased in response to time and all the ureolytic bacteria tested had similar

growth patterns for the total duration of the incubation period. Table 2.12 summarises

the results of the kinetic growth of the ureolytic bacteria during the batch culturing. The

specific growth rate (k), from the experimental result, showed the highest value was

0.398 h-1 from bacteria strain NB30 while the control strain showed the lowest value for

specific growth rate which is 0.254 h-1. Doubling time (td), which refers to the time the

bacterial cells doubles, with shorter times implies more rapid growth. By referring to the

result of td in Table 2.12, isolate NB30 showed the shortest td of 1.741 g to double its

cells, while the control strain showed the longest td of 2.726 g to double its cells. The

result for maximum growth (OD600) of each bacterial culture after being studied for 12

hr showed that the OD values for the ureolytic bacterial cultures ranged between 0.882

to 1.009.

81

Table 2.12: Kinetics growth of ureolytic bacteria in batch cultures

The growth profile shown in Figure 2.11 showed that all the bacterial cultures continued

to have a progressive cell growth, hence, a prolonged stationary phase or death phase

was not observed. It was also observed that the cultures showed similar growth pattern.

The figure suggests all the bacterial isolates showed their maximum growth at the 12 hr

of their incubation period. In table 2.12, LPB21 showed the highest value in the

maximum growth yield [OD600] of 1.076, while the lowest value in the maximum

growth yield [OD600] was observed to be 0.882 by isolate NB28.

Isolate

ID

Specific growth rate,k [h-1]

Doubling time,td [g]

Maximum growth of bacteria [OD600]

Control 0.254 2.726 0.914

NB33 0.274 2.535 0.976

LPB21 0.261 2.653 1.079

NB28 0.319 2.172 0.882

NB30 0.398 1.741 1.009

82

Figure 2.11: Growth profile of selected ureolytic bacterial isolates and control strain grown in nutrient broth containing 6% urea for 12 hr. Error bars represent standard error of the mean.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 2 4 6 8 10 12 14

OD

600

Time (hour)

Control

NB33

LPB21

NB30

NB28

83

Figure 2.12: pH profile of selected ureolytic bacterial isolates and control strain grown in nutrient broth containing 6% urea for 12 hr. Error bars represent standard error of the mean.

8.6

8.7

8.8

8.9

9.0

9.1

9.2

9.3

9.4

9.5

0 1 2 3 4 5 6 7 8 9 10 11 12

pH

Time (hour)

Control

NB33

LPB21

NB30

NB28

84

The pH of the growth medium containing the bacterial culture was also studied.

Figure 2.12 shows that the pH of the medium significantly increased in correspondence

with the growth of the bacterial curve. An increase in the pH medium corresponds to

urea hydrolysis as a result of urea degradation by the ureolytic bacteria. The pH profile

in Figure 2.12 showed similar profiles among the UPB isolates and control strain.

NB33, LPB21, NB28, NB30, and control strain had final maximum pH values of 9.30,

9.32, 9.31, 9.31 and 9.34 respectively. However, all the isolates experienced a

fluctuation in their respective curves during the incubation.

85

2.4 Discussion The study in this chapter aimed at isolating, screening, identifying UPB from limestone

cave (FCNR and WCNR) samples of Sarawak, and to determine their respective urease

activity in comparison to Sporosarcina pasteurii (DSM33), which served as a

representative strain. In order to screen for ureolytic bacteria from cave regions, was

necessary to select appropriate conditions at which desired microorganism would

survive (Al-Thawadi and Cord-Ruwisch, 2012). Hence, samples were collected from

limestone cave samples of Sarawak, Malaysia. The extreme environmental

characteristics cave regions it possesses, make it able to accommodate the unexpected

diversity of microbial communities. (Tomczyk-Żak and Zielenkiewicz, 2015). These

make Sarawak limestone caves suitable sampling locations as microbial communities in

these environments are enriched and exposed to alkaline and limestone conditions. To

screen for highly active urease producing bacteria, enrichment culture technique was

used to instigate a competitive ecosystem among the microorganism for the availability

of growth nutrients (Gorski, 2012b). Enrichment culture technique is widely used to

isolate bacteria in clinical, biotechnological and environmental studies because it brings

about competition among microbiota for available nutrients and against growth

inhibitors by favouring specific bacterial type isolates or subgroups (Gorski, 2012a,

Maite Muniesa et al., 2005).

The present study showed those microorganisms indigenous to cave regions are

promising sources for urease production with the capability of inducing calcite

minerals. This finding is supported by Banks et al. (2010) who previously isolated fifty-

one bacteria from an unnamed cave region in Kentucky, USA. Their results showed that

majority of these microbial species were capable of inducing calcite precipitates.

Stabnikov et al. (2013) suggested that UPB is common inhabitants of soils with the

consistent provision of urea substrate, a final production of amino metabolism (nitrogen

metabolism) of mammals. Hence, enrichment culture designed to select UPB suitable

for MICP ought to be supplemented with an adequate amount of urea substrate

(Burbank et al., 2012, Chu et al., 2011, Hammes et al., 2003a). It was observed that

during the incubation of the enrichment culture samples, there was a unique pungent

smell, indicating the release of ammonia gas.

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The breakdown of urea by the urease enzyme allows the release of ammonium gas to

the bacteria’s environment. This gas can be poisonous to humans if inhaled and can

cause serious respiratory (Gueye et al., 2001, Woto-Gaye et al., 1999). Hence, it is

recommended to work using a facial mask when handling urease producing bacteria

inside the incubation room. Another precaution which should be taken is to incubate

these bacteria in a small incubator (MMM Incucell 55, MMM Medcenter Einrichtungen

Gmbh) and place the incubator inside a fume hood (BASIX 52, LABCRAFT) to

prevent the discharge and spread of ammonia gas in the laboratory. The isolated UDB

capable of growing on nutrient agar supplemented with 6% urea were screened using

UAB medium to test their respective capability of urease production. The incubation

temperature 32oC was chosen because it is the average temperature of Kuching,

Sarawak. Hence it would aid the growth of a wider variety of mesophilic bacteria in the

enrichment cultures.

Typically, aerobic bacteria are often incubated at 30-35oC for a maximum incubation of

72 hr, which is appropriate for cultivation of bacteria from microbiological growth

media (Moldenhauer, 2014). Kielpinski et al. (2005) reported that incubation

temperature of 32oC provided an improvement detection of a microorganism in a sterile

microbiological growth media. Gordon et al. (2014) suggested that this incubation

temperature is appropriate for the recovery of total aerobic microorganism counts from

sample collection using general microbiological growth medium.

UAB is a differentiation medium that tests the ability of a variety of microorganism to

produce urease, an extracellular enzyme which is secreted outside the cells of

microorganisms (Atlas, 2010). Urease test media often contains 2-4% of urea and

phenol red as a pH indicator, which detects an increase in the pH of the medium due to

ammonia production resulting in colour changes from yellow (pH 6.8) to a bright pink

(8.2) (Brink, 2010). Previously Staurt’s urea broth medium was often used to

distinguish urease producers, however only Proteus species were detected as urease-

positive because this medium only contained essential nutrients that facilitated the

growth of only Proteus species and the medium is a highly buffered medium requiring a

large quantity of ammonia production to raise the pH of the medium above o for colour

change to occur(MacFaddin, 2000, Winn et al., 2006).

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On the other hand, Christensen’s urea agar (UAB) contains peptone and glucose which

supports growths of a wider variety of urease-producing microorganisms and it also has

a reduced content of buffer which allows a quicker detection of urea degradation (Brink,

2010). Several studies have reported using urea agar base media as a preferred

qualitative urease assay for isolation and differentiation of ureolytic microorganisms

(Hammad et al., 2013b, Elmanama and Alhour, 2013, Dhami et al., 2013c). When urea

is hydrolysed by the bacteria, ammonia is released and becomes accumulated in the

medium which increases the pH of the environment making it alkaline (Hammad et al.,

2013a). This is the first study to show the presence of at least one cultivable bacterium

from FCNR and WCNR with the production of urease capabilities in the presence of

high concentration of urea. The isolation of a few urease producing bacteria isolates

from the collected samples suggests that a small percentage of environmental bacteria

are capable of participating in the precipitation of calcite through urea hydrolysis

(Burbank et al., 2012).

There were noticeable morphological differences among the isolated urease- producing

bacteria. The limited diversity of the bacterial community in limestone environment is

not surprising because of its extreme alkaline condition, only organisms capable of

growing in these conditions can survive in. such an environment (Achal et al.,

2010b).The close morphology of bacterial isolates was observed among the isolates and

it might be as a result of the dominance species which might occur during enrichment

culturing period since Bacillus species are usually selected by the isolation and

cultivation methods (Shannon Stocks-Fischer et al., 1999). Physiological properties of

the majority of the bacterial isolates resemble those of Bacillus and Sporosarcina

species previously reported (Tominaga et al., 2009, Stocks-Fischer et al., 1999, Gordon

and Hyde, 1982, Gordon et al., 1973) which were then verified via 16S rRNA gene

analysis. The genus bacillus and Sporosarcina appear to be inhabitants of extreme

environments (Achal and Pan, 2011). Achal and Pan (2011) reported in their study that

one of the reasons ureolytic bacteria are capable of enduring alkaline pH might be as a

result of their alkaline habitat of which the aforementioned bacterial isolates were also

isolated from.

88

A study from Aono et al. (1999) on contribution of the cell wall component

teichuronopeptide to pH Homeostasis and alkaliphilic in the alkaliphilic Bacillus lentus

C-125 reported that some of the cell walls of some alkaliphiles such as teichurono-

peptide may play a role in the pH homeostasis at alkaline pH and support the bacteria

isolates to survive in extreme environments.

Based on sequence data of the 16S of the rRNA region, all bacterial isolates from

Sarawak limestone caves showed a high degree of similarity (91-99%) to their closest

species. The results in the phylogenetic tree suggest that the ureolytic bacteria isolated

from samples collected from Sarawak cave were identified as Sporosarcina pasteurii,

Pseudogracilibacillus auburnensis, Staphylococcus aureus, Bacillus lentus,

Sporosarcina luteola and Bacillus fortis. It is noteworthy that the BLAST analysis

showed that most of the local isolates revealed less than 98% similarity to their closest

species. The Sporosarcina comprised 65% of the cultivable ureolytic bacteria isolated

from the cave samples. This isn’t surprising as various studies have reported the

identifying majority of their locally bacterial isolates as Sporosarcina pasteurii. In

addition, a recent study by Wei et al. (2015) reported isolating ureolytic bacteria from

marine sediments of which majority of the identified bacterial isolates were

Sporosarcina sp. However, a few other studies reported isolating bacillus sp.

(Stabnikov et al., 2013, Burbank et al., 2012, Banks et al., 2010).

Sporosarcina pasteurii, majority of the bacteria isolated from FC and WC samples, has

been reported to be non-pathogenic, however sometimes isolated from human faeces with

no pathological reaction happening from its association (Ranganathan et al., 2006), but

it's been suggested it may comprise the immune system of infected patients because

Sporosarcina pasteurii possess abilities to significantly reduce blood urea nitrogen levels

(Arpita et al., 2013, Alhour, 2013, Yoon et al., 2001). Sporosarcina luteola, from the

same genus as Sporosarcina pasteurii, is also non-pathogenic and commonly isolated

from soil samples (Tominaga et al., 2009).

Pseudogracilibacillus auburnensis, which is among the bacteria isolated from FC and

WC samples, has been reported to be a bacterial abundantly available and often isolated

from lakes, desert soil, saline soil and has been reported to be application in controlling

plant pathogens (Mandic-Mulec et al., 2015, Glaeser et al., 2014, Waino et al., 1999).

89

Staphylococcus aureus, one of the bacteria isolated from FC and WC samples, has been

reported to be a virulent pathogen, presently the most common cause of bacterial

infections in hospitalised patients. It's frequently isolated from urine samples obtained

from long-term care patients this pathogenic bacteria’s infection can involve any organ

system has an increasing resistance to antibacterial agents (Rasigade and Vandenesch,

2014, Bien et al., 2011, Archer, 1998). Due to its pathogenicity, it is recommended not to

use this microorganism for biocement application.

According to Stabnikov et al. (2013) to some ureolytic bacteria can be pathogenic,

especially isolates such as Helicobacter pylori, Proteus vulgaris, Staphylococcus aureus,

and Pseudomonas aeruginosa. Due to their level of their pathogenicity, they are not

suitable for biocementation applications. Bacillus lentus and bacillus fortis, which is

among the bacteria isolated from FC and WC samples, are commonly isolated from the

soil, marine waters, and extreme environments. They are considered nonpathogenic and

study has shown the isolation of these microbes have been isolated from dairy farms and

cave environments (Banks et al., 2010, Scheldeman et al., 2004). All the bacterial isolates

except Staphylococcus aureus (NB23) are capable of forming endospore, which has

special resistant dormant structures formed within a cell making the bacteria able to

survive harsh or hostile environments (Krishnapriya et al., 2015). This makes these

isolates suitable for various biocementation applications.

According to Al-Thawadi (2008), conductivity can be used to determine the enzymatic

rate of reaction because the device is robust, easy to operate and an inexpensive assay

system. Conductivity measurement is a suitable method to measure urease activity,

because urease turns the urea molecule (non-conductive) into two charged ions:

ammonium (NH4+, positively charged) and carbonates (CO3

2-, negatively charged)

(Cuzman et al., 2015b).

The release of ammonia as a result of urea hydrolysis can be toxic and detrimental to

most bacterial cells especially when the concentration is high (Cheng and Cord-Ruwisch,

2013). This production of ammonia is advantageous to specific bacteria, such as ureolytic

bacteria which uses the ammonia production for the generation of ATP (Cheng and Cord-

Ruwisch, 2013).

90

The relative changes in conductivity of the urea-bacterial solutions were measured at an

ambient temperature (25◦C ±1) for duration of 5 minutes. These measurement conditions

were also performed by Li et al. (2013), Cheng and Cord-Ruwisch (2013) and Cheng and

Cord-Ruwisch (2012). The use of conductivity to measure bacterial urease activity has

been extensively studied and reported (Krishnapriya et al., 2015, Cuzman et al., 2015b,

Cuzman et al., 2015a). The conductivity variation rate (mS.cm-1.min-1) for Sporosarcina

pasteurii (DSM 33) reported in other studies was in a range of 0.083-0.23 mS.cm-1.min-1

(Cuzman et al., 2015b, Whiffin et al., 2007). A study by Hammad et al. (2013a) reported

using Sporosarcina pasteurii (NCIMB 8841) which had an average change in

conductivity of 0.05 mS.cm-1.min-1. However, another study conducted by (Chu et al.,

2012) reported having an average change in conductivity of 0.06 mS.cm-1.min-1 for

halotolerant and Alkaliphilic urease-producing bacteria which were isolated from tropical

beach sand and later identified as Bacillus sp. The finding by these researchers supports

the results presented in this study on conductivity variation rate of ureolytic bacteria.

Urease activity for Sporosarcina pasteurii (DSM33) reported by Harkes et al. (2010) was

between the range of 5 to 20 mM urea hydrolysed.min-1. Another report from Whiffin

(2004) on the urease activity of Sporosarcina pasteurii (ATCC11859) was between 2.2 to

13.3 mM urea hydrolysed.min-1. However, other studies reported locally isolated Bacillus

strains have urease activity between 3.3 to 8.8 mM urea hydrolysed.min-1 (Stabnikov et

al., 2013, Al-Thawadi and Cord-Ruwisch, 2012). The ability of ureolytic bacteria to

induce calcite precipitation after being incubated for duration of 120 hours was reported

(Hammad et al., 2013b, Hammes et al., 2003b). It is suggested that this biomineralization

is not entirely associated with any specific group of microorganisms, however, it is

relatively associated with a wide variety of microorganism (Boquet et al., 1973). The

capability of bacterial isolates to be able to induce calcite precipitates has been widely

studied and reported, hence proving that ureolytic bacteria are capable of inducing

calcium carbonate (Wei et al., 2015, Krishnapriya et al., 2015, Gat et al., 2014).

Bacterial growth curve of ureolytic isolates has been previously studied and reported. It

shows similar growth pathway to that of the local isolates isolated from samples

collected from Sarawak (Krishnapriya et al., 2015, Achal and Pan, 2014, Stabnikov et

al., 2013, Cheng and Cord-Ruwisch, 2013, Chahal et al., 2011, Achal et al., 2009a).

91

The growth and pH profiles of the local cultures were studied up to 12 hours in nutrient

broth culture supplemented with 6% urea. Figure 2.11 and 2.12 showed that the local

cultures had similar growth profile which confirms they are all of the similar genera

(Sporosarcina pasteurii). The maximum pH observed from the local cultures was

around pH9. Other reports showed that maximum pH profile of Sporosarcina pasteurii

(NCIM 2477, type strain) and Bacillus sp. were at pH11 when grown in nutrient broth

supplement with urea. However, 2% urea was used in their study which suggests the

reason why the growth profile and pH profile were slightly different (Achal and Pan,

2014, Cheng and Cord-Ruwisch, 2013).

Studies by Cheng and Cord-Ruwisch (2013) have shown culturing ureolytic bacteria

using chemostat can maximum their enzyme production and working in non-sterile

conditions would not have a significant impact on the enzyme production or the

bacterial culture. Another study by Achal, Mukherjee and Reddy (2010b), suggested the

use of alternative media such as lactose mother liquor for biocementation applications

but reported there were no significant differences in bacterial growth, urease production

and compressive strength among all media used, however, can serve as a better media

for bacterial growth, support calcite precipitation and reduce the cost in biocementation.

92

2.5 Conclusion The study in this chapter reports the isolation of urease-producing bacteria from

samples collected from limestone caves of Sarawak. Ninety urea degrading bacteria

were successfully isolated from Fairy and Wind cave samples. These bacterial isolates

were tested for their abilities to produce urease enzyme. The experiments performed in

this study indicate the presence of urease-producing bacteria in the cave sample. Thirty-

one bacterial isolates were selected based on their abilities to produce urease. DNA

sequence identification classified the thirty-one urease-producing bacteria isolates as

belonging to the genus of Sporosarcina, Pseudogracilibacillus, Staphylococcus, and

Bacillus. However, the majority of the isolates were similar to Sporosarcina pasteurii

when compared to the 16S rRNA sequencing data in NCBI nucleotide BLAST

database. Conductivity method was used to measure the urease activity of the isolates

and the control. The urease activity detected suggest the potential use of these bacterial

isolates in biocementation. However, results from the specific urease activity, indicates

bacterial isolates LPB21, NB30, NB28 and NB33 produced the highest enzyme activity,

thus were selected as the preferred isolates for the rest subsequent experiments

performed due to their high specific urease activities when compared to other isolates

and also the control strain. The selected aforementioned isolates were then used in the

next chapter. Further studies on LPB21, NB30, NB28 and NB33 were performed in

chapter three which involved studying various cultural conditions that affect the

production of urease and Evaluating these isolates efficiency in biocementation.

Chapter

3 EFFECTS OF CULTURAL CONDITIONS ON UREASE

ACTIVITY AND EVALUATION OF BIOCEMENTATION

POTENTIALS IN SMALL SCALE TEST

93

3.1 Introduction Biocementation is a new ground improvement technique that can be used to improve the

geotechnical properties of soil in a way similar to ordinary cement (Chu et al., 2009).

The use of chemicals agents such as lime, asphalt, sodium silicate, and Portland cement

for soil enhancement has been proven successful (Peethamparan et al., 2009, Basha et

al., 2005, Anagnostopoulo and Hadjispyrou, 2004), however these artificial injection

formulas often alter the pH level of soil, contaminates the soils and groundwater,

attributing to their toxic and hazardous characteristics (DeJong et al., 2006, Karol,

2003). The advantage MICP, a type of biocementation technique has over conventional

ground improvement methods is that it requires a range of ambient conditions with the

diminutive usage of fuel or carbon footprint during production, unlike conventional

cement (Dhami et al., 2016). Studies have also shown that MICP process is able to

significantly improve soil’s shear strength and reduce permeability by filling the pores

of the soil with minerals precipitated (Zhang et al., 2015, Feng and Montoya, 2015,

Bundur et al., 2015).The implementation of MICP as an established ground

improvement method has been partially limited by the need for cultivation and injection

of specific bacteria (Gomez et al., 2014). Although various forms of MICP forms are

available with the use of different bacterial and precursor, however, this chapter

divulges a biological approach for manufacturing biocement using a selected number of

locally isolated urease producing bacteria from limestone caves of Sarawak.

The objectives of the study in this chapter are as follows:

i. To optimise various cultural conditions for maximum urease activity. ii. To study in vitro biocementation potential using single and consortia of

ureolytic bacterial isolates. iii. To determine the calcite contents precipitated in the treated sand specimens.

94

3.2 Methods and Materials 3.2.1. The Effect of Cultural Conditions On Urease Activity 3.2.1 (a) Incubation temperature (oC)

The influence of different temperatures ranging from 20 to 45oC 2 with an interval of

5oC were carried out by incubating the ureolytic bacteria cultures for 24 hr, under

aerobic batch conditions at 32oC with agitation at 130 rpm. The bacterial cultures were

grown in nutrient broth media (13.0 g.L-1, HiMedia Laboratories Pvt. Ltd),

supplemented with 4% urea. The overnight grown bacteria were inoculated (2% v/v)

into separate sterile conical flasks (containing 125 mL nutrient broth). The initial pH of

the growth medium used was attuned to pH 7.5 with the use of 1 N NaOH and 1 N HCl.

The conductivity and OD600 were measured and used to determine the specific urease

activity at the end of the cultivation period.

3.2.1 (b) Initial medium pH

The effect of distinctive pH on the ureolytic activity from the selected isolates was

determined by examining urease activity at different pH ranging from 6.0 to 8.5 with an

interval of 0.5. The bacterial cultures were grown in nutrient broth media (13.0 g.L-1,

HiMedia Laboratories Pvt. Ltd), supplemented with 4% urea. The initial pH of the

growth medium used was attuned with the use of 1 N NaOH and 1 N HCl. The bacterial

cultures were incubated at the optimised incubation temperature for the duration of 24

hr, with agitation at 130 rpm. The conductivity and OD600 were measured and used to

determine the specific urease activity at the end of the cultivation period.

3.2.1 (c) Incubation period (hr)

The optimal incubation period was determined by incubating the ureolytic bacteria

culture at different incubation periods ranging from 24 to 96 hr with an interval of 24 hr,

with agitation at 130 rpm and optimised temperature. The bacterial cultures were grown

in nutrient broth media (13.0 g.L-1, HiMedia Laboratories Pvt. Ltd), supplemented with

4% urea. The initial pH of the growth medium used was attuned with the use of

1 N NaOH and 1 N HCl to maintain the optimised pH medium. The conductivity and

OD600 were measured and used to determine the specific urease activity at the end of the

cultivation period.

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3.2.1 (d) Urea concentration (%)

The influence of urea substrates with varied concentration for enzyme production was

studied. Different urea concentration ranging from 2 to 10% (w/v) with an interval of

2% was selected. The bacterial cultures were grown in nutrient broth media (13.0 g.L-1,

HiMedia Laboratories Pvt. Ltd) at an incubation temperature, initial pH medium and

incubation period previously studied. The conductivity and OD600 were measured and

used to determine the specific urease activity at the end of the cultivation period.

3.2.1 (e) Statistical analysis

The data were presented as mean ±SE (standard deviation) for three replicates. The

optimisation results for different parameters (Incubation temperature, initial medium

pH, incubation period and urea concentration) were analysed using Microsoft Excel

(version 2016) and StatPlus programmes. The analysis of variance (ANOVA) with

Tukey’s procedure was used to compare the variance between different groups with the

variability within each of the groups. The level of significance was set at 0.05.

3.2.2. Small Scale Biocementation Test 3.2.2 (a) Bacteria culture

The selected ureolytic bacteria used in this experiment are shown in Table 3.1. The

ureolytic bacteria were grown under sterile aerobic batch conditions. After incubation,

the bacteria cultures were stored in their growth medium in the fridge at 4oC prior to

use.

Table 3.1: Selected ureolytic bacteria for biocement test Isolate Closest Match

NB33 Sporosarcina pasteurii strain WJ-4 [KC211296]

LPB21 Sporosarcina pasteurii strain fwzy14 [KF208477]

NB28 Sporosarcina pasteurii strain WJ-5[KC211297]

NB30 Sporosarcina pasteurii strain fwzy14 [KF208477] Reference Control Sporosarcina pasteurii strain DSM33

Bacterial Consortia

Comprised of four isolates; Sporosarcina pasteurii LPB21 (SUTS), Sporosarcina pasteurii NB30 (SUTS), Sporosarcina pasteurii NB28 (SUTS) and Sporosarcina pasteurii NB33 (SUTS)

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3.2.2 (b) Cementation solution

The cementation solutions used to treat the sand columns were modified from (Cheng et

al., 2014, Weaver et al., 2011). The constituents and concentration of the cementation

solution are listed in Table 3.2. All the cementation solution components were

autoclaved except urea and CaCl2, which were added after the solution was autoclaved.

Table 3.2: Biocement treatment components

Constituents Concentration Urea

(CO(NH2)2) 1 M

Calcium chloride (CaCl2) 1 M

Sodium acetate (C2H3NaO2)

0.17 M

Ammonium chloride (NH4Cl) 0.0125 M

Nutrient broth 13 g/L

3.2.2 (c) Preparation of sand columns

The characteristics of the sand used in this experiment are summarised in Table 3.3.

Re-informed paper tubes served as the moulds used in this experiment. The moulds had

an internal diameter of 75 mm and length of 49 mm. Each of the column (mould) was

autoclaved and then packed with 294.73 g of sand. All columns were placed on flat

surfaced polypropylene sheet; five holes were drilled on the surfaces of the

polypropylene sheets to allow the effluents of the cementation solution to pass through.

The polypropylene sheets containing drilled holes were later covered with Whatman

filter papers. A plastic container was placed below the polypropylene sheet to

accumulate the effluents. The top of each column was covered with a layer of scouring

pads (Scotch-BriteTM) as filters to prevent disturbance on the surfaces of the sands

during biocement treatments.

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Table 3.3: Sand characteristics

Sand Source

Uniformity coefficient

(Cu)

Coefficient of gradation

(Cc)

D10

(mm) D30

(mm) D60

(mm)

Kuching, Sarawak 1.6 0.907 0.220 0.265 0.352

3.2.2 (d) Biocementation treatment method

Prior to the beginning of the treatment, each of the sand was pre-mixed with bacteria

culture, calcium chloride (1M) and urea (1M) solution before being compacted into

their respective columns. The sand columns were treated with the bacteria and

cementation solutions by percolation (i.e. unrestrained flushing of fluid from top to

bottom). The columns were treated twice daily with the 80 mL ureolytic bacteria culture

(Table 3.1) and 80 mL cementation solution (Table 3.2). However, the treatment was

split into two series of treatment and added twice daily. The Sporosarcina pasteurii

isolates, consortia and control strain were grown in nutrient broth media under aerobic

condition (Table 3.5). The grown cultures, Isolate LPB21 (4.8 X 107), isolate NB30 (4.0

X 107), isolate NB33 (1.5 X 107), isolate NB28 (4.1 X 107), consortia (5.0 X 107) and

control strain (4.7 X 107) were harvested at their respective late exponential phases

before being mixed with the air dried sand specimens. The cementation solution

contained cementation reagents, nutrient broth (13 g.L-1), C2H3NaO (0.17 M), NH4Cl

(0.0125 M). The cementation solution used in this study were urea (CO(NH2)2) and

calcium chloride (CaCl2) which were prepared at a concentration of 1.0 M. The MICP

treatment was performed by introducing 80 mL of bacterial culture and 80 mL of

cementation solution into the sand specimens at an interval of 12 hr for a duration of 96

hrs. The treatments of the sand columns were performed inside a fume hood. Upon

completion of the treatments, all the sand columns were cured at room temperature for a

duration of 14 days before the treated sand were being removed from their respective

mould. Besides the soils being treated with bacteria culture and cementation solution,

another set of control sand specimen was prepared, i.e. sand specimen treated with

cementation solution only.

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3.2.2 (e) Monitoring methods

During the course of treatment and curing time, the environment where the samples

were placed, was monitored by recording its temperature and relative humidity. The

MICP sand treatment was performed inside a fume hood (LabCraft, BASIX 52). The

biomass concentration and urease activity of the bacteria cultures were also measured

via optical density, bacterial viability, and conductivity methods.

3.2.2 (f) Strength measurement The sand specimens that underwent different

treatment conditions (i.e MICP and sand specimen treated with cementation solution

only) were tested for their respective surface strength and shear strength. The surface

strength measurements of the treated sand were obtained by using a pocket

penetrometer (ELE International, 38-2695) as suggested by Al-Thawadi (2008) and

unconfirmed compression strength (UCS) test in reference to American Society for

Testing and Materials (ASTM) C67-07a for conventional bricks and structural clay tile

test (ASTM, 2007). The penetrometer tests were performed by placing the tip of the

instrument on the surface of the cemented sand. Two different penetrometers with

different reading scales were selected for this test. One of the penetrometers had a

reading scale from 0 to 400 kg/cm2 (0 to 441.229 kPa) while the other had a reading

scale from 0 to 700 psi (0 to 4.826 MPa). The pocket penetrometers were used to

measure the surface strength by pushing the tip of the penetrometer into the soil to a

depth of approximately 0.25 inches and three selected surface regions were tested on

each of the cemented sand. The readings of the loaded weight were recorded when the

samples were completely penetrated (breakage). Test for UCS was performed on an

automatic mortar compression / flexural & concrete flexural machine (NL® Scientific

Instruments Sdn. Bhd., NL 3027 X / 002). All the surfaces of the testing apparatus were

cleaned and the sand specimens were placed on it. The tests were performed until the

sand column reached its failure and maximum stress level.

99

3.2.2 (g) Acid quick test

The acid quick test to confirm the presence of calcite precipitate was performed by

using modified procedures from Cordua (2010). A few amount of precipitates found on

the surface of the sand column after the treatment period were collected, weighed and

kept inside sterile test tubes. Each of the test tubes was filled with 10 mL sterile dH20.

The test tubes containing the precipitates were then added with 2 mL of 10% diluted

HCl. The presence of calcite was visually determined by observing for bubble

formation.

3.2.2 (h) Calcite (CaCO3) content measurement

Calcite content measurements were performed and adapted from methods described by

Weaver et al. (2011) and Bernardi et al. (2014). Samples were obtained from the top,

middle and bottom parts of each cemented sands after strength test. The dry weight of

each sample was taken, then washed with 2M HCl, dried and weighed again after

washed with acid to determine the relative amount of calcite present. The samples were

dried for 3 hr at 90°C in an oven before being weighed. The differences in weight

between the dry sands samples prior and after washing with HCl were divided by the

dry weight after washing to determine the percentage of the calcite precipitation by

weight.

3.2.2 (i) Statistical analysis

For statistical analysis, a standard deviation (SE) for each experimental result was

calculated using Excel Spreadsheets available in the Microsoft Excel (version 2016).

The results obtained from the penetrometer tests were analysed with GraphPad (Quick

Calc) program. The data were subjected to student’s t-test analysis, with statistical

significance taken as p<0.05.

100

3.3 Results

3.3.1. Temperature (oC)

The bacteria were allowed to grow in broth media at temperatures ranging from 20 to

45oC 2 with an interval of 5oC. The optimal incubation temperature supporting urease

activity for the UPB is shown in Figure 3.1. Maximum specific urease activity was

observed at 30oC for isolates NB33 (25.32 mM urea hydrolysed.min-1.OD-1), NB30

(41.98 mM urea hydrolysed.min-1.OD-1) and control strain (23.03 mM urea

hydrolysed.min-1.OD-1), while 25oC was observed to be the maximum specific urease

activity for isolates LPB21 (26.96 mM urea hydrolysed.min-1.OD-1) and NB28 (26.26

mM urea hydrolysed.min-1.OD-1). However, isolates NB30 showed the highest specific

urease activity as 41.98 mM urea hydrolysed.min-1.OD-1 when compared to other

isolates and the control strain. A one-way between groups analysis of variance

(ANOVA) was conducted using Statplus program to compare the effects of different

incubation temperature (ranging from 20 to 45oC) on specific urease activity of

individual ureolytic isolates. The ANOVA for the data on specific urease activity as a

function of variation due to different incubation temperature were statistically

significant for isolate LPB21 (F (5,12) = 12.93, P-value = 1.74E-04); NB33 (F (5,12) =

17.30, P-value = 4.06E-05); and isolate NB30 (F (5,12) = 135.35, P-value = 8.91E-07).

On the other hand, the analysis of variance for control strain (F (5,12) = 5.42, P-value =

0.008) and isolate NB28 (F (5,12) = 9.06, P-value = 9.21E-04 were not statistically

significant. A post hoc analysis using the Tukey’s procedure (α=0.05) further revealed

that the effect of different incubation temperature for isolate LPB21, the mean of 25oC

(M= 29.82; SD= 2.98), was significantly higher than the mean of 35oC (M= 15.97; SD=

2.90), 40oC (M= 8.25; SD= 3.75) and 45oC (M= 19.36; SD= 6.60). The post hoc

analysis revealed that for isolate NB33, the mean of 30oC (M= 25.32; SD= 6.88), was

significantly higher than the mean of 25oC (M= 14.60; SD= 1.33), 40oC (M= 2.72; SD=

0.31) and 45oC (M= 6.57; SD= 0.54). The Tukey’s procedure analysis for isolate NB30

revealed that the mean of 30oC (M= 41.98; SD= 2.88), was significantly higher than the

mean of 20oC (M= 31.69; SD= 2.17), 35oC (M= 22.85; SD= 0.72), 35oC (M= 26.42;

SD= 3.74), 40oC (M= 10.31; SD= 3.87) and 45oC (M= 16.86; SD= 4.56).

101

*

Figure 3.1: The effect of different temperature on urease activity. Cultivation of ureolytic bacteria in NB-medium in 250 mL conical flasks incubated at 20 to 45oC for 24 hr. Vertical error bars indicate standard deviation. The analysis of variance (ANOVA) with Tukey’s procedure was used to compare the variance between different groups with the variability within each of the groups. The level of significance was set at 0.05 (*).

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3.3.2. Initial medium pH

The optimum initial medium pH enhancing the activity of urease was performed by

incubating the bacteria cultures in growth medium with varied pH values ranging from

6.0 to 8.5 with an interval of 0.5 as illustrated in Figure 3.2. Maximum specific urease

activity was observed in the medium of pH 6.5 for isolate NB33 (23.71 mM urea

hydrolysed.min-1.OD-1), whereas isolate LPB21 and control strain showed their

respective maximum specific activity at pH 7.5 with 33.74 and 21.43 mM urea

hydrolysed.min-1.OD-1. Isolates NB30 and NB 28 showed their individual maximum

enzyme activities at pH 8.0 with 30.72 and 34.51 mM urea hydrolysed.min-1.OD-1.

However, isolates NB28 showed the highest specific urease activity as 34.51 mM urea

hydrolysed.min-1.OD-1 when compared to other isolates and the control strain. The

ANOVA analysis showed that there were statistical significances in different initial pH

medium (6.5 to 8.5) for the control strain (F (5,12) = 6.35, P-value = 0.004); isolate

LPB21 (F (5,12) = 39.88, P-value = 4.56E-04); NB33 (F (5,12) = 30.59, P-value = 1.97E-

05); isolate NB30 (F (5,12) = 67.80, P-value = 2.24E-07) and isolate NB28 (F (5,12) =

30.99, P-value = 1.84E-04. The post hoc analysis using the Tukey’s procedure (α=0.05)

further revealed that the effect of different initial pH medium for control strain, the

mean of pH 7.5 (M= 21.43; SD= 0.79), was significantly higher than the mean of pH

6.0 (M= 12.52; SD= 4.23), pH 8.0 (M= 12.20; SD= 0.95) and pH 8.5 (M= 12.7; SD=

1.90), for isolate LPB21, the mean of pH 7.5 (M= 33.74; SD= 3.17), was significantly

higher than the mean of pH 6.0 (M=17.88; SD= 1.93), pH 6.5 (M= 15.06; SD= 1.29),

pH 8.0 (M= 16.95; SD= 2.02) and pH 8.5 (M= 14.91; SD= 1.82). The post hoc analysis

revealed that for isolate NB33, the mean of pH 6.5 (M= 23.71; SD= 0.47), having the

maximum specific urease activity, was significantly higher than the mean of pH 6.0

(M= 9.62; SD= 0.54), pH 7.0 (M= 9.75; SD= 3.91), pH 7.5 (M= 4.22; SD= 0.39), pH 8.0

(M= 10.45; SD= 1.10) and pH 8.5 (M= 10.36; SD= 2.77). The Tukey’s procedure analysis

for isolate NB28 revealed that the mean of pH 8.0 (M= 34.51; SD= 3.98), was

significantly higher than the mean of pH 6.0 (M= 19.68; SD=4.37), pH 6.5 (M= 13.24;

SD= 1.92), pH 7.0 (M= 10.19; SD=2.85), pH 7.5 (M= 11.65; SD= 0.39) and pH 8.5 (M=

15.46; SD= 0.20). The analysis for isolate NB30 revealed that the mean of pH 8.0 (M=

30.92; SD= 1.19, was significantly higher than the mean of pH 6.0 (M= 16.41; SD=1.65),

pH 6.5 (M= 20.37; SD= 1.35), pH 7.0 (M= 14.71; SD=0.40), pH 7.5 (M= 23.65; SD= 1.56)

and pH 8.5 (M= 21.22; SD= 0.51).

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Figure 3.2: The effect of different pH on urease activity. Cultivation of ureolytic bacteria in NB-medium in 250 mL conical flasks incubated for 24 hr. Vertical error bars indicate standard deviation. The analysis of variance (ANOVA) with Tukey’s procedure was used to compare the variance between different groups with the variability within each of the groups. The level of significance was set at 0.05 (*).

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3.3.3. Incubation period (hr)

The optimum incubation period for the UPB was performed in growth medium with

varied incubation duration ranging from 24 to 96 hr with an interval of 24 hr as

exemplified in Figure 3.3. Maximum specific urease activity was observed at 24 hr

incubation period for isolates LPB21 (25.98 mM urea hydrolysed.min-1.OD-1), NB33

(27.93 mM urea hydrolysed.min-1.OD-1), isolates NB28 (25.54 mM urea

hydrolysed.min-1.OD-1), NB30 (29.70 mM urea hydrolysed.min-1.OD-1) and control

strain (22.08 mM urea hydrolysed.min-1.OD-1). However, isolates NB30 showed the

highest specific urease activity as 29.70 mM urea hydrolysed.min-1.OD-1 when

compared to other isolates and the control strain. The ANOVA for the data on different

incubation period suggested that there were statistical significances in the effect of

different incubation period for the control strain (F (3,8) = 106.43, P-value = 8.70E-07);

isolate LPB21 (F (3,8) = 20.84, P-value = 3.88E-04); NB33 (F (3,8) = 106.14, P-value =

8.79E-07); isolate NB30 (F (3,8) = 138.04, P-value= 3.15E-04) and isolate NB28 (F (3,8)

= 7.32, P-value = 1.10E-02. Tukey’s procedure (α=0.05) on the effect of different

incubation period showed that for control strain, the mean of 24 hr (M= 22.08; SD=

2.21), was significantly higher than the mean of 48 hr (M= 8.77; SD= 0.92), 72 hr (M=

0.92; SD= 1.17) and 96 hr (M= 4.64; SD= 0.86). The result for isolate LPB21 showed

that the mean of 24 hr (M= 25.98; SD= 3.34), was significantly higher than the mean of

48 hr (M= 6.89; SD= 1.27), 72 hr (M= 9.36; SD= 6.00) and 96 hr (M= 5.34; SD= 1.90).

In addition, the analysis for NB33 that the mean of 24 hr (M= 27.93; SD= 2.03), was

significantly higher than the mean of 48 hr (M= 27.65; SD= 0.70), 72 hr (M=8.86; SD=

2.20) and 96 hr (M= 3.41; SD= 2.00). the Tukey’s test result for Isolate NB28 indicated

that the mean of 24 hr (M= 25.54; SD= 6.09), was significantly higher than the mean of

48 hr (M= 9.24; SD= 1.76), 72 hr (M= 6.69; SD= 3.29) and 96 hr (M= 18.34; SD=

8.47). the test result for Isolate NB30 suggested that the mean of 24 hr (M= 29.70; SD=

2.49), was significantly higher than the mean of 48 hr (M= 7.79; SD= 1.26), 72 hr (M=

65.82; SD= 0.23) and 96 hr (M= 5.86; SD= 1.99).

105

Figure 3.3: The effect of different incubation period on urease activity. Cultivation of ureolytic bacteria in NB-medium in 250 mL conical flasks incubated for 24 hr. Vertical error bars indicate standard deviation. The analysis of variance (ANOVA) with Tukey’s procedure was used to compare the variance between different groups with the variability within each of the groups. The level of significance was set at 0.05 (*).

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3.3.4. Effect of urea concentration (%)

Experimental results showing the enzyme activity at a varying substrate (urea)

concentration ranging from 2 to 10% with an interval of 2% is presented in Figure 3.4.

Maximum specific urease activity was observed at 6% of urea concentration for isolates

LPB21 (32.36 mM urea hydrolysed.min-1.OD-1) and NB28 (25.98 mM urea

hydrolysed.min-1.OD-1), while 8% urea concentration was observed to show the

maximum specific activity for isolates NB33 (33.95 mM urea hydrolysed.min-1.OD-1),

NB30 (39.21 mM urea hydrolysed.min-1.OD-1) and control strain (24.66 mM urea

hydrolysed.min-1.OD-1). However, isolates NB30 showed the highest specific urease

activity as 39.21 mM urea hydrolysed.min-1.OD-1 when compared to other isolates and

the control strain. The ANOVA for the data on different incubation period suggested

that there were statistical significances in the effect of different urea concentration for

the control strain (F (4,10) = 7.91, P-value = 0.00); isolate LPB21 (F (4,10) = 12.60, P-

value = 6.43E-04); isolate NB30 (F (4,10) = 15.67, P-value= 2.62E-04) and isolate NB28

(F (34,10) = 4.176, P-value = 3.00E-02. On the other hand, there was no statistical

significance. isolate NB33 (F (4,10) = 6.65, P-value = 0.007). Tukey’s procedure

(α=0.05) on the effect of different urea concentration showed that for control strain, the

mean of 8% (M= 24.66; SD= 8.91), was significantly higher than the mean of 2% (M=

5.18; SD= 1.48), 4% (M= 9.26; SD= 2.40) and 10% (M= 11.71; SD= 4.18). The result

for isolate LPB21 showed that the mean of 6% (M= 32.36; SD= 6.62), was significantly

higher than the mean of 2% (M= 6.48; SD= 1.51), 4% (M= 15.96; SD= 4.42), 8% (M=

19.81; SD= 12.69) and 10% (M= 18.88; SD= 5.45). The test result for Isolate NB30

suggested that the mean of 8% (M= 39.21; SD= 9.33), was significantly higher than the

mean of 2% (M= 5.11; SD= 1.18), 4% (M= 18.54; SD= 4.27), 6% (M= 20.09; SD=

1.85) and 10% (M= 21.76; SD= 5.62). The Tukey’s test result for Isolate NB28

indicated that the mean of 6% (M= 25.98; SD= 11.40), was significantly higher than the

mean of 2% (M= 5.92; SD= 2.66).

107

Figure 3.4: The effect of different urea concentration on urease activity. Cultivation of ureolytic bacteria in NB-medium in 250 mL conical flasks incubated for 24 hr. Vertical error bars indicate standard deviation. The analysis of variance (ANOVA) with Tukey’s procedure was used to compare the variance between different groups with the variability within each of the groups. The level of significance was set at 0.05 (*).

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3.3.5. Biocementation treatment test

The sand used for biocement test was classified as poorly graded medium sand in

accordance with British Standards, BS5930. The percentage of the particle size

distribution of the type of sands selected is shown in Table 3.4. The particle sizes

selected ranged from fine sand (0.075 mm) to fine gravel (4.75 mm). The sand samples

were selected by sieving for designated particles sizes ranges and the sand that could

pass through sieve number 10 (2 mm) were used for the homogeneity. The selected

samples were later oven dried in 105oC overnight and then allowed to cool to room

temperature. The samples were later autoclaved to eliminate any presence of the

microorganism. In order to immobilise bacteria in the columns for use in subsequent

biocement treatment, 80 mL the ureolytic bacteria were premixed with 294.73 g of sand

and 40 mL of 1M urea and 1M CaCl2. The sands were then immersed in columns and

allowed to sit in a fume hood for 8 hr before subsequent addition of bacteria and

cementation solution. Measurements for optical density, viable cells and enzyme

activity of the bacterial cultures were monitored during the treatments (Table 3.5). The

temperature and relative humidity of the environment where the sand columns were

placed ranged between 23 to 29oC and 74 to 85 %.

In Figure.5 (A), there was no visual observation of calcite on the top layer of the

columns during the initial period of immersion of the bacterial culture and cementation.

Whereas, during the third day of inoculation, white precipitates were seen on all

triplicate samples of the columns containing bacterial cultures as shown in Figure 3.5

(B). On the other hand, none of the columns containing the negative control displayed

any visible precipitation on their respective top layers. Upon completion of the

treatment, the sand columns were then allowed to cure for a total duration of 14 days at

room temperature as presented in Figure 3.6. During the curing period, it was observed

that there was an excessive amount of white precipitates on the surfaces of columns

belonging to Consortia, NB33, NB30, LPB21, and control strain. However, column

belonging to NB28 showed it had a lesser amount of precipitates on the surfaces of its

columns when compared to other columns. On the other hand, the columns containing

negative control showed no white precipitates, despite the continual addition of

cementation solution during the subsequent treatment which occurred for 96 hrs.

109

The columns holding the biocemented sands were carefully removed at the end of the

curing period as shown in Figure 3.7. All the biocemented sand samples appeared to

remain intact after removal from the columns. It was also observed that the scouring

pads (Scotch-BriteTM) which were used to prevent any disturbance of the column’s top

surfaces was not very productive during injection of the cementation solution. However,

the hardness of the biocemented sands was not affected. After the columns were fully

removed from the biocemented sands, any other parts of the columns which remained

on the biocemented sand were then carefully removed (Figure 3.8). The sands were then

kept in an incubator at 37oC for 24 hr to minimise the effect of any differences in water

content remaining in the biocemented sands before their mechanical properties were

evaluated.

Table 3.4: Sand grain size characteristics

Characteristics Percentage (%)

Fine sand 6.72

Medium sand 87.95

Coarse sand 3.96

Fine gravel 1.37

110

Table 3.5: Bacteria concentration and urease activity prior to biocement test

Isolate OD600 CFU.mL-1 mM urea hydrolysed.min-1.OD-1

LPB21 0.79 4.8 X 107 16.6

NB30 0.52 4.0 X 107 17.26

NB33 0.69 1.5 X 107 20.96

NB28 0.76 4.1 X 107 23.49

control 0.64 5.0 X 107 13.65

consortia 0.56 4.7 X 107 12.51

OD600 = optical density; CFU.mL-1 = colony forming unit per millilitre; mM urea hydrolysed.min-1.OD-1 = urease activity.

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Figure 3.5: Treatment of sand column using locally isolated bacteria, consortia, positive and negative controls. [A] setup of sand columns before treated with ureolytic bacteria and cementation solution (Left). [B] sand columns during treatment with bacteria and cementation solution (right). The environment (fume hood) where the MICP treatment occurred had a temperature of 23-29oC and relative humidity of 74-85% during the course of biocement test.

A B

112

Figure 3.6: Sand columns at the end of treatment using ureolytic bacteria and cementation solution. The MICP treatment occurred in a fume hood for a duration of 96 hr with an interval of 12 hr.

Consortia NB28 NB30 NB33 LPB21 Negative control

Positive control

113

Figure 3.7: Treated sand removed from their respective columns. The biocement specimens were allowed to cure for 14 days before being removed from their respective moulds.

Positive control

Consortia LPB21 NB33 NB28 NB30

114

Figure 3.8: Treated sand sample held after a curing period and columns were successfully removed. (A) side view [left], (B) top view [middle] and (C) bottom view [right]. The biocemented specimens were incubated at 37oC for 24 hr to remove any remaining water content before the mechanical properties of the biocement specimens were evaluated.

A C B

115

3.3.6. Soil surface strength

Surface strengths using penetrometer were measured for all the biocemented sand after

curing of the samples. In Figure 3.9, the strength measured for the biocemented sand

treated with different ureolytic bacteria are 582.33 psi for isolate LPB21, 626.67 psi for

isolate NB33, 573.33 psi for isolate NB30, 700 psi for isolate NB28, 533.33 psi for

bacterial consortia and 563.33 for the positive control strain. However, the negative

control was too soft to measure and could not yield any result.

Figure 3.9: Surface strength of the biocemented sand samples. A pocket penetrometer (ELE International, 38-2695) was used to test the surface strength. Vertical error bars indicate standard deviation.

0

100

200

300

400

500

600

700

800

- control + control LPB21 NB33 NB30 NB28 consortia

Str

en

gth

(psi

)

116

The highest strength measured was 700 psi for biocemented sand treated with isolate

NB28 while the lowest strength measured was 533.33 psi for consortia. Among all the

biocemented sand, the sample treated with isolate NB28 reached the maximum reading

of the penetrometer and none of its samples cracked during this surface strength test,

unlike other samples. It was also observed that the sand treated with bacterial cultures

and cementation solutions were slightly more cemented in areas closest to the point of

injection regions. Visual observation after the strength test also indicated that there were

much more precipitates on the surface of the biocemented sands than other areas.

Table 3.6: t-test results comparing the strength (psi) differences between the biocemented sands. (N=3; df=2)

Isolate ID M SD SE P-value t P <*

control 563.33 42.10 nil nil nil nil

LPB21 582.33 67.35 21.221 0.4651 0.8953 -

NB33 626.67 99.81 57.097 0.3829 1.1092 -

NB30 573.33 6.51 26.312 0.7405 0.3801 -

NB28 700.00 0.00 24.306 0.0302 5.6228 +

consortia 533.33 116.93 76.374 0.7324 0.3928 -

(N) number of sample size; (df) degree of freedom; (M) mean; (SE) standard error; (SD) standard deviation; (P-value) calculated probability; (t) test statistic; (+) significant; (-) not significant ;(*<) P-value is significant at 0.05 level.

117

The independent t-test was conducted to compare the surface strength measurement

obtained from biocemented sands for the local isolates and bacterial consortia against

those of the control strain. As illustrated in Table 3.6, there was a significant difference

between the strength of biocemented sand treated with isolate NB28 (M= 700.00; SD=

0.00) against that of the control strain (M= 563.33; SD= 42.10). However, there were no

noticeable significant different between the test results when compared against that of

the control.

3.3.7. Compressive strength

None of the sands treated with the negative control was tested for strength measurement

using the automatic mortar compression machine as the sands were too soft and not

amenable to unconfined compressive testing. During the UCS testing, it was visually

observed that the failure points for all biocemented sands started at their respective

bottom layers. The results from Table 3.7 indicate that the biocemented sands with the

highest test were treated with isolate NB28 (0.219 N/mm2), sustaining a force of 1.020

kN, while sands treated with the lowest strength was treated with the control strain

(0.143 N/mm2), sustain a force of 0.697 kN.

Table 3.7: Unconfined compressive strength (UCS) of the treated sands

Bacteria ID

UCS test

Condition of cemented sand

Force (kN)

Pressure (N/mm2)

- control - - - + control + 0.647 0.143 LPB21 + 0.697 0.152

NB33 + 0.833 0.176

NB30 + 0.647 0.143

NB28 + 1.020 0.219

consortia + 0.623 0.147

(-) the column was not cemented; it was extremely soft and unable to be measured. (+) the cemented column was broken when the maximum strength was applied.

118

The independent t-test was also conducted to compare the UCS test results obtained

from biocemented sands for the local isolates and bacterial consortia against those of the

control strain. The results in Table 3.8 showed that out of all the strength results from

biocemented sands of the isolates and bacterial consortia, there were noticeable

significant differences between the strength results for biocemented sands treated with

isolates LPB21 (M= 0.152; SD= 0.006), NB33 (M=0.176; SD= 0.025) and NB28 (M=

0.219; SD= 0.013) against the control strain (M= 0.143; SD= 0.006).

Table 3.8: t-test results comparing the unconfined compressive strength (UCS) differences between the biocemented sands (N=3; df=2)

Isolate ID M SD SE P-

value t P <*

control 0.143 0.006 nil nil nil nil

LPB21 0.152 0.006 0.005 0.011 9.526 +

NB33 0.176 0.025 0.012 0.072 3.534 +

NB30 0.143 0.002 0.001 0.840 0.229 -

NB28 0.219 0.013 0.000 0.004 16.174 +

consortia 0.147 0.009 0.005 0.374 1.134 -

(N) number of sample size; (df) degree of freedom; (M) mean; (SE) standard error; (SD) standard deviation; (P-value) calculated probability; (t) test statistic; (+) significant; (-) not significant;(*<) P-value is significant at 0.05 level.

119

3.3.8. Calcite confirmation

Figure 3.10: Confirming calcite precipitates. The calcite contents precipitates found on the surfaces of the biocement moulds were tested using quick acid test. (A) before addition of HCl [left]. (B) after addition of HCl [right]. The white precipitates which were seen on the top layer of sand columns were presumed to be calcite precipitates. In order to confirm

this precipitates that were induced by the ureolytic bacteria during the treatment period, some amount of the excess precipitates was

taken and kept in sterile test tubes as shown in Figure 3.10 (A). After the addition of 10% HCl solution, the continual formation of

bubbles was visually observed. The addition of acid (HC)l onto the calcite resulted in bubbles of carbon dioxide gas to be released as

indicated in Figure 3.10 (B). This bubble formation signals the presence of calcite.

A B

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3.3.9. Calcite content Determination

Figure 3.11: Comparison of the relative quantity of calcites in the biocemented sands. The calcite contents were dried for 3 hr at 90°C in an oven before being weighed.

0

2

4

6

8

10

12C

alc

ite

we

igh

t, %

(w

/w

)top middle bottom

+ control LPB21 ConsortiaNB33NB28NB30 - control

121

Table 3.9: Summary of calcite content and compressive strength of selected isolates and consortia

Isolate ID

Weight of calcite, % (w/w) Strength conversion

(MPa)* Top Middle Bottom

- control 0.00 0.00 0.00 0.00

+ control 5.28 3.95 3.19 3.88

LPB21 9.20 2.01 5.59 4.02

NB33 5.86 4.65 7.19 4.32

NB30 6.12 3.09 6.69 3.95

NB28 10.08 7.14 7.09 4.83

consortia 4.70 1.72 1.73 3.68

(*) converted strength (psi) into MPa by knowing that 1 psi = 0.00689476 MPa. The content of the calcite precipitated in the sand specimens as shown in Figure 3.11

were determined by using acid wash method. The average calcite content of the

biocemented sands was determined from samples collected at the top, middle and

bottoms sections of the treated sand as presented in Table 3.9. The top layers of the

biocemented sands treated with the control strain (5.28%), isolates LPB21 (9.20%),

NB28 (10.08%) and bacterial consortia (4.70%) had the highest average calcite

contents, while the sand samples treated with isolates NB33 (5.86%) and NB30 (6.12%)

showed that the bottom layer had the highest average calcite content. The differences in

calcite content on top–middle layers and top-bottom layers for the biocemented sand

samples treated with control strain (3.88 MPa), isolates LPB21 (4.02 MPa), NB28 (4.83

122

MPa) and bacterial consortia (3.68 MPa) were 1.33% and 2.09%; 7.19% and 3.61%;

2.92% and 2.99%; 2.98% and 2.97%, respectively. On the other hand, the differences in

calcite content on bottom–middle layers and bottom-top layers for the biocemented sand

samples treated with NB33 and NB30 were 1.33% and 2.54%; 0.57% and 3.60%,

respectively.

There was not any homogeneity of calcite contents within any layer of the biocemented

sand samples. However, results in Table 3.9 indicated that they were similar calcite

contents between the middle and bottom layers of biocemented sands treated with the

control strain, isolate NB28 and bacterial consortia. This result suggests there was

reasonable precipitation uniformity from middle to bottom layers of these

aforementioned sand samples. In Figure 3.11, among all the biocemented sands, the

highest average calcite content for the top, middle and bottom were determined to be

10.08% (NB28), 7.19% (NB33) and 4.83% (NB28), respectively.

123

3.4 Discussion A study by Hamzah et al. (2012) indicated that physical parameters such as initial

medium pH and incubation temperature play important roles which promote microbial

biomass production. Among all parameters to be studied, the temperature was

considered the most important factor and it is a critical parameter which needs to be

controlled as it usually varies from one organism to another (Delilie et al., 2004, Kumar

and Takagi, 1999). Hence, this physical parameter was first selected among others to

determine the optimum temperature which can facilitate an appropriate production of

urease for the ureolytic bacteria. The result presented in Figure 3.1 suggests that 25oC

and 30oC are the optimum temperature for the selected ureolytic bacteria and the control

strain which produced the most favourable activity of urease enzyme. These

temperatures correspond to the standard temperature of Kuching, Malaysia. It was

perceptible that these bacterial cultures would yield substantial urease activities at a

temperature between 20 to 35oC.

Earlier studies on optimisation of temperatures on urea hydrolysis and calcium

precipitation showed the preferred temperature for bacterial growth was between 30 to

35oC (Helmi et al., 2016, Seshabala and Mukkanti, 2013). Higher temperature

conditions did not have a favourable outcome of enzyme activity when the UPB were

cultured at 40oC and 45oC. However, it was noteworthy that isolate LPB21 showed a

reasonable enzyme activity when cultured at 45oC. Microbial urease strongly relies on

temperature as the rate of urea hydrolysis changes with changes in temperature due to

kinetic energy inducing the collisions between an enzyme and its substrate, and is

capable of stimulating modifying to the cellular membrane of bacteria. (Akgöl et al.,

2002, Rahman et al., 2005). It is well known that proteins conformation changes or

degrades at a higher temperature as the structure of the enzyme may become altered,

thus making the enzyme’s catalytic become eventually destroyed (Seshabala and

Mukkanti, 2013, Abusham et al., 2009).

The pH of a microorganism’s growth medium plays an important role by inducing

morphological changes in microbes, enzyme secretion and affecting the microbe’s

stability in the growth medium (Sethi and Gupta, 2014). Hence, the effect of the initial

medium pH on urease activity for the ureolytic bacteria was studied.

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The results illustrated in Figure.2 indicate that pH 6.5, 7.5 and 8.0 are the optimum pH

values for the selected ureolytic bacteria and the control strain. pH 7.5 and 8.0 produced

the most noticeable activity of urease enzyme when compared to others. On the other

hand, at an acidic level of pH 6.0 and an alkaline level of 8.5, the activities of urease for

the ureolytic bacteria were found to be low and unfavourable. Urea hydrolysis in a

growth medium is expected to lead to an increase in the pH value due to the production

of ammonium (Gat et al., 2014). Previous studies have shown that urease enzyme is

more active at alkaline pH (Prah et al., 2011, Anne et al., 2010, Stocks-Fischer et al.,

1999).

Urea hydrolysis at different alkaline pH was investigated and confirmed by Helmi et al.

(2016)for the previous research outcomes. The primary step of the chemical reaction is

the hydrolysis of urease enzyme as it leads to an induction of calcium carbonate (Millo

et al., 2012, Okwadha and Li, 2010). A study by Helmi et al. (2016) showed that the

hydrolysis of urease enzyme by Bacillus licheniformis and formation of ammonium

increases the pH value up to 8.0, sufficient enough to induce calcite precipitation. A

study by Gat et al. (2014) suggested that the pH values of Sporosarcina pasteurii

(DSM 33) for urea hydrolysis during incubation is at pH 7.39 after being incubation at

28 hr. A study on variation of solution pH for purified urease enzyme from jack bean

meal and microbial urease from Bacillus megaterium by Jiang et al. (2016) showed that

regardless of oxic or anoxic conditions, pH values increases sharply within the initial 1

hr which is ascribed to immediate hydrolysis of urea. Ferris et al. (2004) found that

bacteria cell growth during ureolysis process in anoxic conditions contributes to extra

acidic substances which can reduce the pH values and increase electric conductivity

values.

The incubation period is an essential parameter for enzyme production (Gautam et al.,

2011). The result of the optimum incubation period is presented in Figure 3.3 which

shows that maximum urease activity for all the bacterial isolates and control strain was

found to be at 24 hr incubation period. On the other hand, at an incubation period of 48

to 96 hr, the enzyme activity decreased and was not favourable. This decline is as a

result of saturation of actives sites of the microbial enzyme by the substrate molecules

which is not longer to be involved in the breakdown of it (Fisher, 2001).

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A report by Gat et al. (2014) showed that Sporosarcina pasteurii (DSM 33) grown for

10 days with agitation (100 rpm) at 30oC using nutrient broth supplemented with 2%

w/v urea (333 mM) showed a steady increase in bacterial and enzyme activity its first

80 hr but remained at lag phase at 17 hr of incubation which could be as a result of low

concentration of urea supplemented. The decline in urease activity shown in Figure 3.3

stands in agreements with the finding of previous studies by (Ferris et al., 2004, Dupraz

et al., 2009) that Sporosarcina pasteurii can still hydrolyze urea in the absence of

sufficient organic carbon source, however the number of viable cells and enzyme

activity is likely to decrease significantly with time under these conditions. Another

study by (Achal et al., 2010a) on biocalcification by Sporosarcina pasteurii (NCIM

2477) using corn steep liquor as a nutrient source has grown at 37 oC for a duration of

160 hr with corn steep liquor, nutrient broth and yeast extract, each supplemented with

2% urea. This resulted showed that urease activity and biomass from corn steep liquor

medium was significantly higher than those observed in nutrient broth and yeast extract.

Thus suggesting corn steep liquor is noteworthy a preferred growth medium for enzyme

production and a low-cost nutrient compared to the aforementioned nutrients.

Bacteria needs a source of nitrogen to support their maximal growth because nitrogen is

a key building block of protein, enzymes and nucleic acids (Hamzah et al., 2013).

Hence, this parameter was also studied to determine the optimum urea concentration

(w/v %) since ureolytic bacteria primary requires urea substrate as their source of

nitrogen. The result illustrated in Figure 3.4 suggests that 6% and 8% are the optimum

urea concentration for the selected ureolytic bacteria and the control strain which

produced the most favourable activity of urease enzyme. A study by Mortensen et al.

(2011) on the subject of environmental factors such as ammonium ions and free oxygen

affecting urease activity. Their results showed that since ureolytic activity depends on

the available substrate (urea) and concentration of urease enzyme, production of

ammonium during urea hydrolysis would not alter urease activity. Ammonia, a nitrogen

source for most bacteria can be detrimental or toxic when present in high concentration

due to cytotoxic effect (Hess et al., 2006). On the other hand, a high concentration of

ammonia can be advantageous to particular ureolytic bacteria such as Sporosarcina

pasteurii as it can assist their ATP generation but with an increasing concentration of

urea, a decrease in biomass and specific urease activity can be met (Cheng and Cord-

Ruwisch, 2013).

126

In addition, a study by Cuzman et al. (2015b) suggested that fertilizer urea served as an

effective and cost urea substrate when compared to expensive pure grade urea (Sigma-

Aldrich) as it showed similar behaviour in the presence of commercial urease but there

were no significant differences was observed as regards to microbial urease activity.

The maximum urease ureolytic rate measured for pure grade urea was 0.0892 mS.cm-

1.min-1 and 0.0866 mS.cm-1.min-1 for fertilizer urea. Hence suggesting the use of

fertiliser urea could reduce cost and reduce the environmental impact of urea production

from the release of ammonia by ureolytic bacteria, possible urea substitute such as

poultry manure could be considered (Cuzman et al., 2015b).

An in situ laboratory biocement experiment was conducted in columns containing sand

samples. Three column samples were prepared for each bacterial culture and

cementation solution treatments. The experiment was made in triplicates as presented in

Figure 3.5 to check for repeatability and to quantify the changes in the sand properties

statistically. The negative control contained only the cementation solution and was used

to treat its respective sand columns. This was done to rule out the possibility that

precipitates found in the sand columns were only as results microbial urea hydrolysis

and not any other process. However, during the premixing of sand, bacteria, urea and

calcium chloride solution, the pungent smell was perceived indicating the breakdown of

urea and release of ammonium gas. This treatment method was applied to ensure

bacteria culture and premix urea and calcium chloride solution attach at particle

contacts within the permeable sand matrix. The white precipitates on top layers of the

biocemented sand shown in Figure 3.6 and Figure 3.7 were also reported by Zhao et al.

(2014) and Chu et al. (2012) which Indicates the presence of nucleation sites for MICP

as a result of addition of more bacterial solution in order to promote more urease

enzyme. The precipitates were confirmed to be calcite as shown in Figure 3.10 by

addition diluted HCl onto the precipitates. To confirm the presence of calcite, a quick

acid test can be used by adding drops of HCl on calcite mineral, the reactions allow

bubbles formation and a vigorous effervescence which last for some minutes or seconds

Cordua (2010).

127

The results of the biocemented sands showed that all the samples were found to be

tightly packed except the negative control. The use of penetrometer to test for surface

strength showed that samples treated with NB28 (Figure 3.9) were found to be the most

compact sample, requiring more force to be applied to break the samples. Calcite

precipitation was detected over the entire length of the treated sand samples, which

indicated that the ureolytic bacteria and reactants were present at all locations of the

samples (Whiffin et al., 2007). However, it was observed that the precipitation of the

calcite contents in the biocemented sand samples was not homogeneous and more

calcite contents were found in the top layers of the samples (Figure 3.11). In other

biocementation studies, formation of calcite contents dominantly around surfaces of

their respective columns and with no homogeneity have been reported (Rowshanbakht

et al., 2016, Dhami et al., 2016, Neupane et al., 2015, Feng and Montoya, 2015, Zhao et

al., 2014, Achal et al., 2009b, Achal et al., 2009a). On the other hand, in other similar

studies, the formation of calcite precipitates on the layer of their respective columns was

not mentioned (Harkes et al., 2010, Whiffin et al., 2007, van Paassen et al., 2010).

The reason there was predominantly more calcite formation at the top layers of the sand

samples is mainly that Sporosarcina pasteurii is a facultative anaerobic bacterium,

which grows at a higher rate in the environment containing oxygen and consequently

leading to higher rates of calcites precipitated around the top surface areas (Whiffin et

al., 2007). Additionally, the influence of microbial cementation on granular behaviour is

dependent on the ability of the bacteria to move freely throughout the pore spaces of the

sand and on sufficient particle-particle contact per unit volumes at which cementation

will occur. Hence, this quicker formation of calcite precipitation at injection points of

the bacteria and cementation solution prevents more precipitates from flowing freely

downward the columns and causing a non-uniformity of calcite precipitates (Dhami et

al., 2016, Achal et al., 2009b, Achal et al., 2009a). According to ATSM (D2166-00)

standards, to test for unconfirmed compressive strength of cohesive soil, specimen sizes

are required to have a minimum diameter of 30 mm with a length of one-tenth of the

specimen diameter or 72 mm with a length of one-sixth of the specimen diameter.

However, the diameter and length of column samples used in this experiment were75

mm and length of 49 mm, hence it did not follow the standard of ATSM.

128

The biocement test was primary designed to test the ability of the locally isolated

ureolytic bacteria in treating loose soils by filling their pores and testing the surface

strength of the samples without being in their respective columns. However, the UCS

test was later performed to get an estimated unconfined compressive strength and

understand what state of force will the samples reach their respective failed points.

The results presented in Table 3.7, Table 3.9 and Figure 3.9 suggested that there was a

correlation between the surface strength using penetrometer test, calcite contents, and

compressive strength. Park et al. (2014) stated that increase in calcite precipitation due

to increasing microbial injections may weaken the existing cementation in the cemented

soil. This was supported by Park et al. (2010) and Ghosh et al. (2009) suggesting that

microbes do not always increase the biocement strength, instead, might decrease the

strength. However, other studies on MICP reported an increase in strength as a result of

calcite precipitation with the addition of more bacteria (van Paassen et al., 2010,

DeJong et al., 2006, Ramachandran et al., 2001). Paassen (2009) reported that bacterial

urease activity dropped after 20 days of injected but improved after another injection

batch of bacterial culture was added to the column. It was suggested that the enzyme

activity could have been as a result of a hydraulic construct such as the bacteria being

trapped inside the pores of the sands during precipitation and interruption of chemical

transport which could prevent the flow of required nutrients for growth and more calcite

precipitation from reaching the bacteria. Hence, it was necessary to maintain sufficient

amount of repeated additional bacterial culture to the columns so as to prevent possible

accumulation of metabolic waste which could result in a decrease of urease activity,

cell death and poor precipitation (Stocks-Fischer et al., 1999).

Synergistic microbial communities are abundant in nature, with metabolic capabilities

and robustness. This has inspired fast-growing interest in engineering synthetic

microbial consortia for biotechnology development (Minty et al., 2013). Hence, a

bacterial consortium was included in the biocement test as a comparison with the

individual isolates and control strain and to determine which will provide the best

strength and calcite content. The results in Table 3.9 indicated that the ureolytic bacteria

performed better individually and the consortia showed the lowest performance in terms

of strength and calcite content.

129

The soils samples containing the negative control were unable to bind together, thus, the

solution media flowed freely and the samples fell apart when the columns holding the

samples were removed. This observation was also reported by (Kavazanjian and

Hamdan, 2015). However (Whiffin et al., 2007) reported having the strength of 167 kPa

for samples untreated with bacteria but it was not reported if the samples used were

autoclaved to prevent false results. To rule out the possibility of having false

precipitations such as chemically induced calcite precipitation on any of the samples,

the sand samples used were autoclaved before treated with the ureolytic bacteria. This

suggestion was adopted from Burbank et al. (2011). Their finding showed that the

autoclaved soil samples not treated with ureolytic bacteria did not precipitate calcite

when observed by X-ray powder diffraction analysis. Li et al. (2011a) suggested the use

of soybean meal as it can serve as an alternative source of nutrients to support bacterial

growth and increase urease activity for enhanced supply of calcite precipitation.

The biocement application of bacterial consortia that is well adapted to the

environmental conditions in Sarawak was studied. The advantage of a mixed bacterial

consortium which comprised of Sporosarcina pasteurii LPB21 (SUTS), Sporosarcina

pasteurii NB30 (SUTS), Sporosarcina pasteurii NB28 (SUTS) and Sporosarcina

pasteurii NB33 (SUTS) had the lowest urease activity (12.51 mM urea hydrolysed.min-

1.OD-1) when compared to the single isolates. The low production of urease was seen to

have affected the potential of inducing a high amount of calcium carbonate precipitates

during the in vitro biocement test. The bacterial consortia might have performed better

than the single isolates as a result of a low number of bacteria cell not culture was low.

One of the factors which may have affected the biomass synergy of the bacterial

consortia could be attributed to insufficient oxygen in the microenvironment (Hamzah

et al., 2013). The oxygen demand of an aerobic bacterial culture is influenced by the

bacterial concentration and growth rate (Elsworth et al., 1957). If the oxygen demand by

the bacteria consortia exceeded the oxygen supply, the biomass production will be

affection. Another factor which could affect the ability of the constructed bacterial

consortia from having a performance during the biocement test is the urea (nitrogen

source) concentration.

130

The optimised urea concentration of the four bacterial isolates which were used for the

design of the bacterial consortia were seen to be between 6-8%. sufficient urea can

promote necessary biomass production and urease activity required for binding of soil

particles. The determination of urea concentration for bacterial consortia might help to

enhance the synergistic effectiveness. Findings from this study suggest that the use of

single urease-producing bacteria were more effective than the designed bacterial

consortia for the in vitro biocementation.

131

3.5 Conclusion A series of laboratory test were carried out to determine the cultural conditions required

in improving urease activities for the ureolytic bacteria. It was observed that when

incubated these conditions: at 25 to 30oC; pH 6.5 to 8.0; incubation period at 24 hr; and

urea concentration of 6 to 8%, maximum specific urease activities for the selected

ureolytic bacteria isolates and control strain were obtained. The in situ laboratory

biocement test proved that cement precipitation was observed in all sand columns

except columns treated with negative control. The results presented in this chapter

demonstrated that biocementation using the selected ureolytic bacteria can significantly

improve the engineering properties of poorly graded soils. However, the efficiency of

the MICP process in improving the soil strength varied among the samples which were

treated with different isolates, the bacteria consortia, and the control strain. The results

also showed there was higher cementation level at positions close to the injection points

and more calcite contents were obtained from the top layers of the biocemented sand.

Based on the surface strength using penetrometer test and compressive strength using

UCS test, samples treated with isolate LPB21 and isolate NB28 showed significant

strengths when compared to other isolates, consortia, and the control strain. However,

the rest isolates showed similar performance with the control strain. This comparative

study has shown that the ureolytic bacteria isolated from limestone samples of Sarawak

are capable of improving a poorly graded soil when compared to the control strain. The

results in this chapter also give a basis for further study with large scale fermentation of

the ureolytic bacterial (LPB21, NB30, NB28, and NB33) for application in civil and

geotechnical engineering.

Chapter

4 GENERAL CONCLUSIONS AND RECOMMENDATIONS

132

4.1 General Conclusion 4.1.1. Aim of the thesis

This thesis presents data from our work exploring the biodiversity of Sarawak, Malaysia

for urease producing bacteria. Numerous researchers have selected Sporosarcina

pasteurii as their ideal ureolytic bacteria for biocement applications because of its high

urease activity and inability to cause harmful diseases (Wei et al., 2015, Cuzman et al.,

2015b, Kang et al., 2014b). In addition, many studies have reported purchasing different

type strains of Sporosarcina pasteurii from microorganism culture collection centres

such as National collection of industrial and marine bacteria (Sidik et al., 2014, Raut et

al., 2014, Hammad et al., 2013b), German collection of microorganisms and cell

cultures (Gat et al., 2014, Harkes et al., 2010, Paassen et al., 2009) and American type

culture collection (Zhang et al., 2015, Bundur et al., 2015, Onal and Frigi, 2014) for

their respective MICP investigative studies and field applications.

Some common Sporosarcina pasteurii type stains which have been reported as ideal

MICP agents are ATCC 6453, ATCC 11859, DSMZ 33, MTCC 1761, ATCC 14581,

NCIMB 8841, NCIMB 8221 and NCIM 2477 (Sidik et al., 2014, Abo-El-Enein et al.,

2013, Lee et al., 2012). These strains were selected as bio-agents of biocement

applications because they are able to survive in high alkaline (above pH 8.5)

environments and also a high concentration of calcium ions (i.e. 0.75 M) (Stabnikov

and Ivanov, 2016). On the other hand, apart from the use of Sporosarcina pasteurii for

MICP process, some species of the genus of Bacillus capable of producing urease and

biocement capabilities are Bacillus cereus, Bacillus sphaericus, Bacillus subtilis,

Bacillus licheniformis, Bacillus Cohnii, and Bacillus lentus (da Silva et al., 2015,

Vahabi et al., 2014, Sierra-Beltran et al., 2014).

The current exploitations of isolating alternative ureolytic bacteria species for effective

biocement applications are still very limited (Soon et al., 2014, Dhami et al., 2013d,

Cheng and Cord-Ruwisch, 2012). This drawback spurred the interest of studying

Sarawak’s environment for the possibility to isolate highly active urease-producing

bacteria. Prior to this study, there were no records of any urease producing bacteria

capable of showing high specific urease activity in Sarawak when compared with

representative microbial urease strain, Sporosarcina pasteurii (DSM33).

133

This thesis is thus, a first report on the strategy to screen and characterise urease

producing bacteria isolated from limestone cave samples of Sarawak. This study also

presents the effects of cultural conditions on urease activity for the local ureolytic

isolates, and an evaluation of biocementation potentials in small scale test. This chapter

summarises the findings of chapters two and three presented in this thesis. This chapter

also offers recommendations for imminent works in this study area.

4.1.2. Limestone area as source of ureolytic bacteria

Caves are extreme environments which are unique, unexploited and poorly studied,

making it an ideal region to screen for microorganism capable of producing novel

bioactive compounds (Gabriel and Northup, 2013). Various environmental factors in

caves such as limited sunlight and nutrients influences microorganisms to adapt to such

location, forcing them to search for other carbon and nitrogen sources (Cheeptham,

2013, de Lurdes N. Enes Dapkevicius, 2013, Northup et al., 2011). Energy is essential

for the production of bacterial metabolites, inorganic compounds such as nitrogen,

sulphur and carbon dioxide (Northup et al., 2011). The ability for microorganisms to

survive in as caves, suggests that they are ideal environments that provide diverse

habitats for microbial survival (Alnahdi, 2014). Limestone is a type of carbonate

mineral composed of pure calcite or pure calcite (CaCO3) or dolomite (CaMg(CO3)2) or

a mixture of both (James et al., 2008). Limestone formation can be discovered regions

such as seawater, caves, and coral reefs, these formations usually contain high

concentrations of calcium and bicarbonate ions (James et al., 2008). The possibility of

certain cave microorganisms capable of inducing calcite precipitates on their cell’s

surfaces contributing to the formation of limestone caves initiated the concept of

exploring and isolating indigenous microbial species from extreme environments with

highly active urease producing bacteria. Hence, Fairy and Wind limestone caves of

Sarawak were selected to explore the possibility to attain novel ureolytic bacteria which

are indigenous to Sarawak’s environments.

134

4.1.3. Enrichment culture and isolation

Enrichment and isolation methods were used in this thesis to target microorganisms

capable of surviving on urea as a main source of nitrogen. Nutrient broth (HiMedia),

Tryptic soy broth (Merck), Lactose peptone broth (BD Difco™), Luria broth (HiMedia)

and Brain heart infusion broth (Oxoid) growth medium were used in this thesis because

they contain various compositions of ingredients suitable for growing a substantial

number of bacteria. Each of the enrichment cultures was supplemented with Sodium

acetate (8.2 g.L-1), Ammonium sulphate (10.0 g.L-1) and Urea (40 g.L-1). This selective

enrichment technique facilitated in successfully isolating ninety urea degrading bacteria.

Urea (CO(NH2)2) is a naturally-occurring form of nitrogen present in both aquatic and

terrestrial environments (Fisher, 2014). Urea is part of dissolved organic nitrogen pool

which microorganisms depend on as a valuable nitrogen substrate for survival (Berman

and Bronk, 2003, Altman and Paerl, 2012, Tyler et al., 2003). Urease enzyme raises the

pH of a bacteria’s environment, by allowing it to depend solely on urea as nitrogen

source (Williams et al., 1996). For the purpose of targeting highly active ureolytic

bacteria, 6% urea substrate was selected and used in enrichment culturing of the

samples collected from FCNR and WCNR. Upon successful isolation of urea degrading

bacteria, 6% urea substrate was subsequently used to confirm the ability of the isolated

microorganisms to degrade high urea concentration when subcultured on nutrient agar.

4.1.4. Screening and identification

Christensen’s medium (urea agar base) was used to screen for urease producing

bacteria. Various studies have suggested the use of Christensen’s medium to screen and

detect urease-producing bacteria (Hammad et al., 2013b, Elmanama and Alhour, 2013,

Dhami et al., 2013d). Out of the ninety isolates, thirty-one isolates were capable of

producing urease. Molecular identification showed that these urease producing bacteria

belonged to Sporosarcina, Pseudogracilibacillus, Staphylococcus and Bacillus groups

with 91 to 99% sequence similarity to existing sequences of their respective closest

bacterial species in the GenBank database. However, the majority of the urease-

producing bacteria were similar to Sporosarcina pasteurii when compared to the 16S

rRNA sequencing data in NCBI nucleotide BLAST database.

135

The majority of the bacterial isolates were Gram-positive bacteria while only three of

the isolates (A63, B53, and A62) were Gram-negative bacteria. Gram-positive bacteria

lack an outer cell membrane but are surrounded by layers of thick peptidoglycan cell

wall, while Gram-negative bacteria has a thin peptidoglycan cell wall with an outer

membrane containing lipopolysaccharide (Andrew, 2013, Silhavy et al., 2010). This

membrane in Gram-negative bacteria is responsible for many antigenic properties of

Gram-negative bacterial species, making the majority of species of Gram-negative

bacteria be pathogenic. Hence, for MICP process, Gram-positive bacteria are often

preferred (Wong, 2015).

4.1.5. Measurement of urease activity

Conductivity method was used to determine urease activity of the urease producing

bacteria. This method was suggested as the most preferred urease enzyme assay because

it is easy to use and inexpensive (Al-Thawadi, 2008, Whiffin, 2004). The changes in

conductivity of bacterial-urea solutions were monitored for a duration of 5 min at 25◦C

and the respective conductivity values were measured using a Walk LAB conductivity

pro meter, Trans Instruments COMPRO. The conductivity variation rate (mS.cm-1.min-1)

is obtained from the gradient of the graph. The urea hydrolysis rate for the urease activity

conversion was determined by (Whiffin, 2004) as described in equation 1.23, while the

Specific urease activity (mM urea hydrolysed.min-1.OD-1) which reflects the urease

catalytic abilities of the urea hydrolysis (Zhao et al., 2014) was derived by dividing the

urease activity (mM urea hydrolysed.min-1) by the bacterial biomass (OD600). The

specific urease activity was also determined by (Whiffin, 2004) as described in equation

1.24. The results determined from the enzyme assay showed that isolates NB33 (19.975

mM urea hydrolysed.min-1.OD-1), LPB21 (23.968 mM urea hydrolysed.min-1.OD-1),

NB28 (19.275 mM urea hydrolysed.min-1.OD-1), and NB30 (20.091 mM urea

hydrolysed.min-1.OD-1) had the highest specific urease activities when compared to other

isolates and the representative strain (17.751 mM urea hydrolysed.min-1.OD-1). Due to

their high enzyme activities, the aforementioned isolates were selected and used for rest

subsequent experiments in this thesis.

136

4.1.6. Biocementation competency of local isolates

The effectiveness of MICP treatment on poorly graded sand specimens used in this

thesis was performed by using locally isolated ureolytic bacteria. The implementation of

MICP treatment using these isolates was compared with sand specimens treated with a

bacterial consortium and a representative strain (Sporosarcina pasteurii). The

biocement test results showed the MICP agents were able to induce calcite precipitates

capable of filling the pore particles of the poorly graded sand samples, hence improving

the strength and stiffness of loose sands. The surface strength and compressive strength

test results showed the local ureolytic bacteria had comparative strengths to that of the

representative strain. However, the highest surface and compressive strength results

obtained were 700 psi and 0.231 N/mm2, which was from samples treated with NB28

bacterial culture. The findings from the calcite content for all the samples treated with

microbes showed the distribution of the calcite contents were not uniform. The top layer

of the specimens contained the highest calcite content. The results in this thesis showed

that biocementation treatment using locally isolated ureolytic bacteria were successfully

able to improve the mechanical properties of poorly graded sands comparable with the

representative strain utilised in this thesis.

4.2 Future Directions and Recommendations The findings in this thesis suggest that the isolated ureolytic bacteria (NB28, LPB21,

NB33, and NB30) have the potential to be used as alternative microbial MICP agents

for biocement applications. Future work involving these four isolates may involve

large-scale bacterial production using computerised bioreactor. The large scale bacterial

production can be utilised for MICP treatment involving field application. The use of

alternative growth medium as a carbon source for large-scale bacteria production can be

studied in order to minimise the cost of purchasing nutrient source. A study on how this

alternative medium enhances the production of bacterial growth, urease activity and

calcite precipitation can be investigated. A comparison between the lab grade urea

substrate and industrial grade urea or alternative nitrogen sources can also be

investigated for future work. This will also be essential for field applications and

reduction of cost for MICP treatments. The effect of cementation reagents MICP

process was not investigated in this study.

137

The urea and calcium chloride concentration used were 1 M (w/v%), this might have

limited the precipitation of calcite during biocement treatment. Hence, the effect of the

concentration of these reagents should be further investigated. Additionally, alternative

calcium ions such as eggshells, pearl shells, snail shells, calcium sulphate or calcium

acetate can also be examined in comparison to calcium chloride and determine which

might result in the most amount of calcite content.

The evidence of microbial involvement in calcite precipitation has brought a revolution

in the discipline of biotechnology, geotechnical and civil engineering. However, some

MICP applications involving some expensive materials have resulted in the successful

commercialization of biocement, but it has been at a high cost (Dhami et al., 2013a).

Hence, it is recommended to explore the use of cheap industrial by-products such as fly

ash which can serve a supplementary cementitious material and capable of significantly

reduce cement and concrete carbon footprint (Thomas, 2007). The investigation of

using the mixture of fly ash, sand, and locally isolated ureolytic bacteria could improve

the existing findings which support the use of MICP process as an alternative

economically friendly construction material.

Screening of urease-producing bacteria was from Fairy and Wind Caves Reserves from

Sarawak. A collection of speleothem, calcareous and Guano (from the bat) samples

from other cave regions such as Gunung Mulu National Park, Deer Cave, Lang Cave

and Clearwater Cave situated in other parts of Sarawak should be performed. Screening

of novel highly active ureolytic isolates of various genuses might be attainable. The

discovery of the ureolytic bacteria isolate mentioned in this thesis could be applied in

other MICP applications to solve problems relating to environmental biotechnology,

civil engineering, and geotechnical engineering. The isolated ureolytic bacteria

described in this thesis may hold additional potential in the field MICP and this thesis

can serve as a useful reference resource for researchers in microbial biotechnology and

construction microbial biotechnology sub-disciplines.

138

REFERENCES

Abo-El-Enein, S. A., Ali, A. H., Talkhan, F. N. and Abdel-Gawwad, H. A. (2013)

'Application of microbial biocementation to improve the physico-mechanical

properties of cement mortar', Housing and Building National Research Center,

9(1), pp. 36-40.

Abusham, R. A., Rahman, R. N., Salleh, A. B. and Basri, M. (2009) 'Optimization of

physical factors affecting the production of thermo-stable organic solvent-

tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain

Rand', Microbial Cell Factories, 8(1), pp. 20-30.

Achal, V. (2015) 'Production of Bacteria for Structural Concrete', in Pacheco Torgal, F.,

Labrincha, J.A., Diamanti, M.V., Yu, C.P. & Lee, H.K. (eds.) Biotechnologies

and Biomimetics for Civil Engineering: Springer International Publishing, pp.

309-323.

Achal, V. and Pan, X. (2014) 'Influence of calcium sources on microbially induced

calcium carbonate precipitation by Bacillus sp. CR2', Applied Biochemistry and

Biotechnology, 173(1), pp. 307-17.

Achal, V., Mukerjee, A. and Sudhakara Reddy, S. M. (2013) 'Biogenic treatment

improves the durability and remediates the cracks of concrete structures.',

Construction and Buidling Materials, 48, pp. 1-5.

Achal, V. and Pan, X. (2011) 'Characterization of urease and carbonic anhydrase

producing bacteria and their role in calcite precipitation', Current

Microbiolology, 62(3), pp. 894-902.

Achal, V., Pan, X. and Zhang, D. (2011) 'Remediation of copper-contaminated soil by

Kocuria flava CR1 based on microbially induced calcite precipitation',

Ecological Engineering, 37, pp. 1601–1605.

Achal, V., Mukherjee, A. and Reddy, M. S. (2010a) 'Biocalcification by Sporosarcina

pasteurii using corn steep liquor as the nutrient source', Industrial

Biotechnology, 6(3), pp. 170-174.

Achal, V., Mukherjee, A. and Reddy, M. S. (2010b) 'Characterization of two urease-

producing and calcifying Bacillus spp. isolated from cement', World Journal of

Microbiology and Biotechnology, 20(11), pp. 1571-1576.

139

Achal, V., Mukherjee, A. and Reddy, M. S. (2010c) 'Effect of calcifying bacteria on

permeation properties of concrete structures', Journal of Industrial Microbiology

& Biotechnology, 38(9), pp. 1229-1234.

Achal, V., Mukherjee, A., Basu, P. C. and Reddy, M. S. (2009a) 'Lactose mother liquor

as an alternative nutrient source for microbial concrete production by

Sporosarcina pasteurii', Journal of Industrial Microbiology & Biotechnology,

36(3), pp. 433-438.

Achal, V., Mukherjee, A., Basu, P. C. and Reddy, M. S. (2009b) 'Strain improvement of

Sporosarcina pasteurii for enhanced urease and calcite production', Journal of

Industrial Microbiology & Biotechnology, 36(7), pp. 981-988.

Addadi, L., Raz, S. and Weiner, S. (2003) 'Taking advantage of disorder: amorphous

calcium carbonate and its roles in biomineralization', Advanced Materials, 15,

pp. 959-970

Adetutu, E. M., Thorpe, K., Shahsavari, E., Bourne, S., Cao, X., Fard, M. N. R., Kirby,

G. and Ball, A. S. (2012) 'Bacterial community survey of sediments at

Naracoorte Caves, Australia', International Journal of Speleology, 41, pp. 137–

147.

Akada, J. K., Shirai, M., Takeuchi, H., Tsuda, M. and Nakazawa, T. (2000)

'Identification of the urease operon in Helicobacter pylori and its control by

mRNA decay in response to pH', Molecular Microbiolology, 36(5), pp. 1071-

1084.

Akgöl, S., Yalçınkaya, Y., Bayramoğlu, G., Denizli, A. and Arıca, M. Y. (2002)

'Reversible immobilization of urease onto Procion Brown MX-5BR-Ni(II)

attached polyamide hollow-fibre membranes', Process Biochemistry, 38(5), pp.

675-683.

Al-Thawadi, S. and Cord-Ruwisch, R. (2012) 'Calcium Carbonate Crystals Formation

by Ureolytic Bacteria Isolated from Australian Soil and Sludge', Journal of

Advanced Science and Engineering Research 2, pp. 12-16.

Al-Thawadi, S. M. (2008) High Strength In-Situ Biocementation of Soil by Calcite

Precipitating Locally Isolated Ureolytic Bacteria. Unpublished doctoral thesis,

Murdoch University, Perth, Australia.

Al-Thawadi, S. M. (2011) 'Ureolytic Bacteria and Calcium Carbonate Formation as a

Mechanism of Strength Enhancement of Sand', Journal of Advanced Science

and Engineering Research, 1, pp. 98-114.

140

Alhour, M. T. (2013) Isolation, Characterization and Application of Calcite Producing

Bacteria from Urea Rich Soils. Unpublished MSc thesis, Islamic University of

Gaza, Gaza, Palestine.

Alnahdi, R. (2014) Potential of Cave Bacteria in Drug Discovery: Investigation of

Sphingopyxis terrae and other Bacteria from a BC Cave. Unpublished MSc

thesis, Thompson rivers university, Kamloops, British Columbia, Canada.

Altman, J. C. and Paerl, H. W. (2012) 'Composition of inorganic and organic nutrient

sources influences phytoplankton community structure in the New River

Estuary, North Carolina', Aquatic Ecology, 46(3), pp. 269-282.

Alves, P. D., Siqueira Fde, F., Facchin, S., Horta, C. C., Victoria, J. M. and

Kalapothakis, E. (2014) 'Survey of microbial enzymes in soil, water, and plant

microenvironments', The Open Microbiology Journal, 8, pp. 25-31.

Anagnostopoulo, C. and Hadjispyrou, S. (2004) 'Laboratory study of an epoxy resin

grouted sand', Ground Improvement, 8, pp. 39-45.

Anbu, P., Kang, C. H., Shin, Y. J. and So, J. S. (2016) 'Formations of calcium carbonate

minerals by bacteria and its multiple applications', Springerplus, 5, pp. 250-262.

Andrew, D. S. (2013) 'Applications in Clinical Microbiology', in Nick, A.S. & Martin,

A.L. (eds.) Real-Time PCR: Advanced Technologies and Applications: Horizon

Scientific Press, pp. 284.

Anne, S., Rozenbaum, O., Andreazza, P. and Rouet, J.-L. (2010) 'Evidence of a

bacterial carbonate coating on plaster samples subjected to the Calcite

Bioconcept biomineralization technique', Construction and Building Materials,

24(6), pp. 1036-1042.

Aono, R., Ito, M. and Machida, T. (1999) 'Contribution of the cell wall component

teichuronopeptide to pH homeostasis and alkaliphily in the alkaliphile Bacillus

lentus C-125', Journal of Bacteriology, 181(21), pp. 6600-6606.

Archer, G. L. (1998) 'Staphylococcus aureus: a well-armed pathogen', Clinical

Infectious Diseases, 26(5), pp. 1179-1181.

Arpita, M., Koushik, D., Suchismita, R., Keshab., C. M. and Dilip, K. N. (2013) 'In vivo

assessment of bacteriotherapy on acetaminophen-induced uremic rats', Journal

of Nephrology, 26(1), pp. 228-236.

141

Arunachalam, K. D., Sathyanarayanan, K. S., Darshan, B. S. and Raja, R. B. (2010)

'Studies on the characterisation of bio sealant properties of Bacillus sphearicus',

International Journal of Engineering Science and Technology, 2(3), pp. 270-

277.

Ashelford, K. E., Chuzhanova, N. A., Fry, J. C., Jones, A. J. and Weightman, A. J.

(2005) 'At least 1 in 20 16S rRNA sequence records currently held in public

repositories is estimated to contain substantial anomalies', Applied and

Environmental Microbiology, 71(12), pp. 7724-7736.

Author (2007): Standard test methods for sampling and testing brick and structural clay

tile. United States of America: American Society for Testing and Materials

(ASTM).

Atlas, R., M. (2010) Handbook of Microbiological Media. 4th edn. Boca Raton, Florida,

United States,: ASM press. Taylor and Francis group, LLC.

Aubert, J. E., Husson, B. and Sarramone, N. (2006) 'Utilization of municipal solid waste

incineration (MSWI) fly ash in blended cement Part 1: Processing and

characterization of MSWI fly ash', Journal of Hazardous Materials, 136(3), pp.

624-631.

Bakhshipouri, Z., Omar, H., Yousof, Z. and Ghiasi, V. (2009) 'An overview of

subsurface karst features associated with geological studies in Malaysia',

Electronic Journal of Geotechnical Engineering, 14, pp. 1-15.

Banda, R. M., Gendang, R. and Ambun, A. V. (2004) 'Geology and geochemistry of

limestone in Sarawak', The Sarawak Museum Journal, 6(80), pp. 41-61.

Bang, S. S., Lippert, J. J., Yerra, U., Mulukutla, S. and Ramakrishnan, V. (2010)

'Microbial calcite, a bio-based smart nanomaterial in concrete remediation',

International Journal of Smart and Nano Materials, 1(1), pp. 28-31.

Banks, E. D., Taylor, N. M., Gulley, J., Lubbers, B. R., Giarrizzo, J. G., Bullen, H. A.,

Hoehler, T. M. and Barton, H. A. (2010) 'Bacterial Calcium Carbonate

Precipitation in Cave Environments: A Function of Calcium Homeostasis',

Geomicrobiology Journal, 27(5), pp. 444-454.

Barabesi, C., Galizzi, A., Mastromei, G., Rossi, M., Tamburini, E. and Perito, B. (2007)

'Bacillus subtilis Gene Cluster Involved in Calcium Carbonate

Biomineralization', Journal of Bacteriology, 189(1), pp. 228-235.

142

Barkay, T. and Schaefer, J. (2001) 'Metal and radionuclide bioremediation: issues,

considerations and potentials', Current Opinion in Microbiology, 4, pp. 318–

323.

Barton, H. A. and Jurado, V. (2007) 'What’s up down there? Microbial diversity in

caves', Microbe 2, pp. 132–138.

Barton, H. A., Taylor, N. M., Kreate, M. P., Springer, A. C., Oehrle, S. A. and Bertog.,

J. L. (2007) 'The impact of host rock geochemistry on bacterial community

structure in oligotrophic cave environments', International Journal of

Speleology, 36, pp. 93–104.

Basha, E. A., Hashim, R., Mahmud, H. B. and Muntohar, A. S. (2005) 'Stabilization of

residual soil with rice husk ash and cement', Construction and Buidling

Materials, 19( 448-453).

Baskar, S., Baskar, R. and kaushik, A. (2007) 'Evidences for microbial involvement in

the genesis of speleothem carbonates, Borra Caves, Visakhapatnam, India',

Current Science, 92(3), pp. 350-355.

Bäuerlein, E. (2003) 'Biomineralization of Unicellular Organisms: An Unusual

Membrane Biochemistry for the Production of Inorganic Nano- and

Microstructures', Angewandte Chemie International Edition, 42(6), pp. 614-641.

Bazylinski, D. A. and Frankel, R. B. (2003) 'Biologically controlled mineralization in

prokaryotes', Reviews in Mineralogy and Geochemistry, 54, pp. 217–247.

Berman, T. and Bronk, D. A. (2003) 'Dissolved organic nitrogen: a dynamic participant

in aquatic ecosystems', Aquatic microbial ecology, 31(3), pp. 279-305.

Bernardi, D., DeJong, J. T., Montoya, B. M. and Martinez, B. C. (2014) 'Bio-bricks:

Biologically cemented sandstone bricks', Construction and Building Materials,

55, pp. 462-469.

Bien, J., Sokolova, O. and Bozko, P. (2011) 'Characterization of Virulence Factors of

Staphylococcus aureus: Novel Function of Known Virulence Factors That Are

Implicated in Activation of Airway Epithelial Proinflammatory Response',

Journal of Pathogens, 2011, pp. 601905-601918.

Binod, P., Palkhiwala, P., Gaikaiwari, R., Nampoothiri, K. M., Duggal, A., Dey, K. and

Pandey, A. (2013) 'Industrial Enzymes - Present status and future perspectives

for India', Journal of Scientific & Industrial Research, 72, pp. 271-286.

Bisen, P. S. (2004) 'Microbial Staining ', Microbes in Practice. 1 ed. New Delhi: IK

International pp. 139-155.

143

Boquet, E., Boronat, A. and Ramos-Cormenzana, A. (1973) 'Production of Calcite

(Calcium Carbonate) Crystals by Soil Bacteria is a General Phenomenon',

Nature, 246(5434), pp. 527-529.

Bosak, T. (2011) Calcite precipitation, microbially induced. Encyclopedia of earth

sciences series Netherlands: Springer, p. 223–227.

Braissant, O., Verrecchia, E. P. and Aragno, M. (2002) 'Is the contribution of bacteria to

terrestrial carbon budget greatly underestimated?', Naturwissenschaften, 89(8),

pp. 366-370.

Brink, B. 'Urease Test Protocol', ASM Conference for Undergraduate Educators San

Deigo, California, USA: American Society of microbiology, 1-4.

Bundur, B., Zeynep, K., Mary, J. F. and Raissa, D. (2015) 'Biomineralized cement-

based materials: Impact of inoculating vegetative bacterial cells on hydration

and strength', Cement and Concrete Research, 67, pp. 237-245.

Burbank, M. B., Weaver, T. J., Green, T. L., Williams, B. C. and Crawford, R. L.

(2011) 'Precipitation of calcite by indigenous microorganisms to strengthen

liquefiable soils', Geomicrobiology Journal, 28(4), pp. 301-312.

Burbank, M. B., Weaver, T. J., Williams, B. C. and Crawford, R. L. (2012) 'Urease

Activity of Ureolytic Bacteria Isolated from Six Soils in which Calcite was

Precipitated by Indigenous Bacteria', Geomicrobiology Journal, 29(4), pp. 389-

395.

Cacchio, P., Contento, R., Ercole, C., Cappuccio, G., Martinez, M. P. and Lepidi, A.

(2004) 'Involvement of Microorganisms in the Formation of Carbonate

Speleothems in the Cervo Cave (L'Aquila-Italy)', Geomicrobiology Journal,

21(8), pp. 497-509.

Carmichael, M. J., Carmichael, S. K., Santelli, C. M., Strom, A. and Bräuer, S. L.

(2013) 'Mn(II)-oxidizing bacteria are abundant and environmentally relevant

members of ferromanganese deposits in caves of the Upper Tennessee river

basin', Geomicrobiology Journal 30, pp. 779–800.

Castanier, S., Le Métayer-Levrel, G., Orial, G., Loubière, J.-F. and Perthuisot, J.-P.

(2000) 'Bacterial Carbonatogenesis and Applications to Preservation and

Restoration of Historic Property', in Ciferri, O., Tiano, P. & Mastromei, G.

(eds.) Of Microbes and Art: The Role of Microbial Communities in the

Degradation and Protection of Cultural Heritage. Boston, MA: Springer US,

pp. 203-218.

144

Castanier, S., Le Métayer-Levrel, G. and Perthuisot, J.-P. (1999) 'Ca-carbonates

precipitation and limestone genesis: the microbiogeologist point of view',

Sedimentary Geology, 126(1–4), pp. 9-23.

Chahal, N., Rajor, A. and Siddique, R. (2011) 'Calcium carbonate precipitation by

different bacterial strains ', African Journal of Biotechnology 10(42), pp. 8359-

8372.

Cheeptham, N. (2013) 'Advances and Challenges in Studying Cave Microbial

Diversity', in Cheeptham, N. (ed.) Cave Microbiomes: A Novel Resource for

Drug Discovery: Springer-Verlag New York, pp. 1-34.

Chen, Y. Y., Clancy, K. A. and Burne, R. A. (1996) 'Streptococcus salivarius urease:

genetic and biochemical characterization and expression in a dental plaque

streptococcus', Infection and Immunity, 64, pp. 585-592.

Cheng, L. (2012) Innovative Ground Enhancement by Improved Microbially Induced

CaCO3 Precipitation Technology. Unpublished doctoral thesis, Murdoch

University, Western Australia.

Cheng, L. and Cord-Ruwisch, R. (2012) 'In situ soil cementation with ureolytic bacteria

by surface percolation', Ecological Engineering, 42, pp. 64-72.

Cheng, L. and Cord-Ruwisch, R. (2013) 'Selective enrichment and production of highly

urease active bacteria by non-sterile (open) chemostat culture', Journal of

Industrial Microbiology and Biotechnology, 40(10), pp. 1095-1104.

Cheng, L., Shahin, M. A., Cord-Ruwisch, R., Addis, M., Hartanto, T. and Elms, C.

(2014) 'Soil Stabilisation by Microbial-Induced Calcite Precipitation (MICP):

Investigation into Some Physical and Environmental Aspects'. 7th International

Congress on Environmental Geotechnics Melbourne, Australia. Barton,

Australia: Engineers Australia, 1105-1112.

Chu, J., Ivanov, V., He, J., Naeimi, M., Li, B. and Stabnikov, V. (2011) 'Development

of Microbial Geotechnology in Singapore', Geo-Frontiers 2011, pp. 4070-4078.

Chu, J., Ivanov, V., Lee, M. F., Oh, S. M. and He, J. 'Soil and Waste Treatment Using

Biocement'. International Symposium on Ground Improvement Technologies

and Case Histories, Singapore: Research Publishing Services, 160-166.

Chu, J., Stabnikov, V. and Ivanov, V. (2012) 'Microbially Induced Calcium Carbonate

Precipitation on Surface or in the Bulk of Soil', Geomicrobiology Journal, 29(6),

pp. 544-549.

145

Chunxiang, Q., Jianyun, W., Ruixing, W. and Liang, C. (2009) 'Corrosion protection of

cement-based building materials by surface deposition of CaCO3 by Bacillus

pasteurii', Materials Science and Engineering, 29(4), pp. 1273-1280.

Clayton, C. L., Pallen, M. J., Kleanthous, H., Wren, B. W. and S., T. (1990) 'Nucleotide

sequence of two genes from Helicobacter pylori encoding for urease subunits',

Nucleic Acids Research, 18, pp. 362.

Clements, R., Rayan, D. M., Abdul-Wahab, A. Z., Venkataraman, A., Alfred, R., Payne,

J., Ambu, L. and Sharma, D. S. K. (2010) 'Trio under threat: can we secure the

future of rhinos, elephants and tigers in Malaysia? ', Biodiversity and

Conservation, 19, pp. 1115–1136.

Cole, L. E., Bhagwat, S. A. and Willis, K. J. (2015) 'Long-term disturbance dynamics

and resilience of tropical peat swamp forests', Journal of Ecolology, 103(1), pp.

16-30.

Collins, C. M. and D'Orazio, S. E. (1993) 'Bacterial ureases: structure, regulation of

expression and role in pathogenesis', Molecular Microbiology, 9(5), pp. 907-

913.

Cordua, W. S. (2010) 'Spotlight on Juniors: Doing Acid Tests on Rocks and Minerals',

Rocks & Minerals, 75(4), pp. 276-277.

Cucchi, T., Fujita, M. and Dobney, K. (2009) 'New Insights into Pig Taxonomy,

Domestication and Human Dispersal in Island South East Asia: Molar Shape

Analysis of Sus Remains from Niah Caves, Sarawak.', International Journal of

Osteoarchaeology 19, pp. 508-530.

Cussac, V., Ferrero, R. L. and Labigne, A. (1992) 'Expression of Helicobacter pylori

urease genes in Escherichia coli grown under nitrogen-limiting conditions',

Journal of Bacteriology, 174(8), pp. 2466-2473.

Cuzman, O. A., Rescic, S., Richter, K., Wittig, L. and Tiano, P. (2015a) 'Sporosarcina

pasteurii use in extreme alkaline conditions for recycling solid industrial wastes',

Journal of Biotechnology, 214, pp. 49-56.

Cuzman, O. A., Richter, K., Wittig, L. and Tiano, P. (2015b) 'Alternative nutrient

sources for biotechnological use of Sporosarcina pasteurii', World Journal of

Microbiology and Biotechnology 31(6), pp. 897-906.

Author (2000): Standard Test Method for Unconfined Compressive Strength of

Cohesive Soil. West Conshohocken, Pennsylvania, United States: ASTM

International.

146

D'Hondt, S., Rutherford, S. and Spivack, A. J. (2002) 'Metabolic activity of subsurface

life in deep-sea sediments', Science, 295, pp. 2067–2070.

da Silva, F. B., De Belie, N., Boon, N. and Verstraete, W. (2015) 'Production of non-

axenic ureolytic spores for self-healing concrete applications', Construction and

Building Materials, 93, pp. 1034-1041.

de Lurdes N. Enes Dapkevicius, M. (2013) 'Cave Biofilms and Their Potential for

Novel Antibiotic Discovery', in Cheeptham, N. (ed.) Cave Microbiomes: A

Novel Resource for Drug Discovery. New York, NY: Springer New York, pp.

35-45.

De Muynck, W., De Belie, N. and Verstraete, W. (2010a) 'Microbial carbonate

precipitation in construction materials: A review', Ecological Engineering,

36(2), pp. 118-136.

De Muynck, W., Verbeken, K., De Belie, N. and Verstraete, W. (2010b) 'Influence of

urea and calcium dosage on the effectiveness of bacterially induced carbonate

precipitation on limestone', Ecological Engineering, 36(2), pp. 99-111.

De-Belie, N. and De-Muynck, W. 'Crack repair in concrete using biodeposition'.

Proceedings of the 2nd International Conference on Concrete Repair,

Rehabilitation, and Retrofitting (ICCRRR), Cape Town, South Africa. Leiden,

The Netherlands: CRC Press/Balkema, 777-781.

De-Muynck, W., Debrouwer, D., DeBelie, N. and Verstraete, W. (2008) 'Bacterial

carbonate precipitation improves the durability of cementitious materials',

Cement and Concrete Research, 38, pp. 1005–1014.

De-Muynck, W., Leuridan, S., Van-Loo, D., Verbeken, K., Cnudde, V., De-Belie, N.

and Verstraete, W. (2011) 'Influence of Pore Structure on the Effectiveness of a

Biogenic Carbonate Surface Treatment for Limestone Conservation', Applied

and Environmental Microbiology, 77(19), pp. 6808-6820.

Debnath, A., Singh, S. V. and Singh, Y. P. (1995) 'Comparative assessment of energy

requirements for different types of residential buildings in India', Energy Build,

23, pp. 141–146

Defarge, C., Gautret, P., Reitner, J. and Trichet, J. (2009) 'Defining organominerals:

Comment on ‘Defining biominerals and organominerals: Direct and indirect

indicators of life’ by Perry et al. (2007, Sedimentary Geology, 201, 157-179)',

Sedimentary Geology, 213, pp. 152-155.

147

DeJong, J. T., Fritzges, M. B. and Nüsslein, K. (2006) 'Microbially Induced

Cementation to Control Sand Response to Undrained Shear', Journal of

Geotechical and Geoenvironmental Engineering, 132(11), pp. 1381-1392.

DeJong, J. T., Mortensen, B. M., Martinez, B. C. and Nelson, D. C. (2010) 'Bio-

mediated soil improvement', Ecological Engineering, 36(2), pp. 197-210.

DeJong, J. T., Soga, K., Banwart, S. A., Whalley, W. R., Ginn, T. R., Nelson, D. C.,

Mortensen, B. M., Martinez, B. C. and Barkouki, T. (2011) 'Soil engineering in

vivo: harnessing natural biogeochemical systems for sustainable, multi-

functional engineering solutions', Journal of The Royal Society Interface, 8(54),

pp. 1-15.

DeJong, J. T., Soga, K., Kavazanjian, B. S., van Paassen, L., Al Qabany, A., Aydilek,

A., Bang, S. S., Burbank, M., Caslake, L., Chen, C. Y., Cheng, X., Chu, J.,

Ciurli, S., Fauriel, S., Filet, A. E., Hamdan, N., Hata, T., Inagaki, Y., Jefferis, S.,

Kuo, M., Laloui, L., Larrahondo, J., Manning, D. A. C., Martinez, B., Montoya,

B. M., Nelson, D. C., Palomino, A., Renforth, P., Santamarina, J. C., Seagren, E.

A., Tanyu, B., Tsesarsky, M. and Weaver, T. (2013) 'Biogeochemical Processes

and Geotechnical Applications: Progress, Opportunities and Challenges',

Geotechnique, 63(4), pp. 287-301.

Delilie, D., Coulan, F. and Pelletier, E. (2004) 'Effects of temperature warning during a

bioremediation study of natural and nutrient-amended hydrocarbon-

contaminated sub-Antartic soils', Cold Regions Science and Technology 40, pp.

61-70.

Devos, L., Clymans, K., Boon, N. and Verstraete, W. (2005) 'Evaluation of nested PCR

assays for the detection of Legionella pneumophila in a wide range of aquatic

samples', Journal of Applied Microbiology, 99(4), pp. 916-925.

Dhami, N. K., Mukherjee, A. and Reddy, M. S. (2013a) 'Viability of calcifying bacterial

formulations in fly ash for applications in building materials', Journal of

Industrial Microbiology & Biotechnology, 40(12), pp. 1403-1413.

Dhami, N. K., Reddy, M. S. and Mukerjee, A. (2013b) 'Bimineralization of calcium

carbonates and their engineered applications: a review', front Microbiology, 4,

pp. 1-13.

Dhami, N. K., Reddy, M. S. and Mukherjee, A. (2012a) 'Biofilm and Microbial

Applications in Biomineralized Concrete', in Seto, J. (ed.) Advanced Topics in

Biomineralization. India: Intech, pp. 1-29.

148

Dhami, N. K., Reddy, M. S. and Mukherjee, A. (2012b) 'Improvement in strength

properties of ash bricks by bacterial calcite', Ecological Engineering, 39, pp. 31-

35.

Dhami, N. K., Reddy, M. S. and Mukherjee, A. (2013c) 'Bacillus megaterium mediated

mineralization of calcium carbonate as biogenic surface treatment of green

building materials', World Journal of Microbiology and Biotechnology, 29(12),

pp. 2397-2406.

Dhami, N. K., Reddy, M. S. and Mukherjee, A. (2013d) 'Biomineralization of calcium

carbonate polymorphs by the bacterial strains isolated from calcareous sites',

Journal of Microbiology and Biotechnology, 23(5), pp. 707-714.

Dhami, N. K., Reddy, M. S. and Mukherjee, A. (2013e) 'Biomineralization of calcium

carbonates and their engineered applications: a review', Frontiers in

Microbiology, 4, pp. 314.

Dhami, N. K., Reddy, M. S. and Mukherjee, A. (2014) 'Application of calcifying

bacteria for remediation of stones and cultural heritages', Frontiers in

microbiology, 5, pp. 304.

Dhami, N. K., Reddy, M. S. and Mukherjee, A. (2016) 'Significant indicators for

biomineralisation in sand of varying grain sizes', Construction and Building

Materials, 104, pp. 198-207.

Diaz-Herraiz, M., Jurado, V., Cuezva, S., Laiz, L., Pallecchi, P., Tiano, P., Sanchez-

Moral, S. and Saiz-Jimenez, C. (2013) 'The actinobacterial colonization of

Etruscan paintings', Scientific Reports, 3, pp. 1440.

Dick, J., Windt, W., Graef, B., Saveyn, H., Meeren, P., Belie, N. and Verstraete, W.

(2006) 'Bio-deposition of a calcium carbonate layer on degraded limestone by

Bacillus species', Biodegradation, 17(4), pp. 357-367.

Dodge-Wan, D. and Mi, A. D. H. (2013) 'Biologically influenced stalagmites in Niah

and Mulu caves (Sarawak, Malaysia)', Acta Carsologica, 42(1), pp. 155-163.

Douglas, S. and Beveridge, T. J. (1998) 'Mineral formation by bacteria in natural

microbial communities', FEMS Microbiology Ecology, 26(2), pp. 79-88.

Dupraz, S., Parmentier, M., Ménez, B. and Guyot, F. (2009) 'Experimental and

numerical modeling of bacterially induced pH increase and calcite precipitation

in saline aquifers', Chemical Geology, 265, pp. 44–53.

149

Eaton, K. A., Brooks, C. L., Morgan, D. R. and Krakowka, S. (1991) 'Essential role of

urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic

piglets', Infection and Immunity, 59(7), pp. 2470-2475.

Ehrlich, H. L. (1996) 'How microbes influence mineral growth and dissolution',

Chemical Geology, 132, pp. 5-9.

Ehrlich, H. L. (1998) 'Geomicrobiology: its significance for geology', Earth Science

Reviews, 45, pp. 45-60.

Elmanama, A. A. and Alhour, M. T. (2013) 'Isolation, Characterization and Application

of Calcite Producing Bacteria from Urea Rich Soils', Journal of Advanced

Science and Engineering Research, 3(4), pp. 377-399.

Elsworth, R., Williams, V. and Harris‐Smith, R. (1957) 'The effect of oxygen supply on

the rate of growth of Aerobacter cloacae', Journal of Applied Chemistry, 7(5),

pp. 269-274.

Engel, A. S. (2010) 'Microbial diversity of cave ecosystems.', in Barton, L.L., Mandl,

M. & Loy, A. (eds.) Geomicrobiology: molecular and environmental

perspective. The Netherlands: Springer ScienceC Business Media B.V.

Engel, A. S., Stern, L. A. and Bennett, P. C. (2004) 'Microbial contributions to cave

formation: new insights into sulfuric acid speleogenesis', Geology 32, pp. 369–

372.

Ercole, C., Cacchio, P., Botta, A. L., Centi, V. and Lepidi, A. (2007) 'Bacterially

Induced Mineralization of Calcium Carbonate: The Role of Exopolysaccharides

and Capsular Polysaccharides', Microscopy and Microanalysis, 13(01), pp. 42-

50.

Ernst, F. D., van Vliet, A. H. M., Kist, M., Kusters, J. G. and Bereswill, S. (2007) 'The

Role of Nickel in Environmental Adaptation of the Gastric Pathogen

Helicobacter pylori', in Sigel, A., Sigel, H. & K. O. Sigel, R.K.O. (eds.) Nickel

and Its Surprising Impact in Nature: John Wiley & Sons, Ltd, pp. 545-579.

Evans, D. J., Evans, D. G., Kirkpatrick, S. S. and Graham, D. Y. (1991)

'Characterization of the Helicobacter pylori urease and purification of its

subunits', Microbial Pathogenesis, 10(1), pp. 15-26.

150

Feng, K. and Montoya, B. (2015) 'Drained Shear Strength of MICP Sand at Varying

Cementation Levels', in Iskander, M., Suleiman, T.M., Anderson, J.B. & Laefer,

D.F. (eds.) Proceedings of the International Foundations Congress and

Equipment Expo 2015 (IFCEE 2015): American Society of Civil Engineers, pp.

2242-2251.

Ferris, F. G., Phoenix, V., Fujita, Y. and Smith, R. W. (2003) 'Kinetics of calcite

precipitation induced by ureolytic bacteria at 10 to 20°C in artificial

groundwate', Geochim Cosmochim Acta, 67, pp. 1701–1710.

Ferris, F. G., Phoenix, V., Fujita, Y. and Smith, R. W. (2004) 'Kinetics of calcite

precipitation induced by ureolytic bacteria at 10 to 20°C in artificial

groundwater', Geochimica et Cosmochimica Acta, 68(8), pp. 1701-1710.

Fisher, K. A. (2014) Urea Hydrolysis in Soil Profile Toposequences: Mechanisms

Relevant to Nitrogen Transport and Water Quality. Unpublished doctoral thesis,

University of Maryland, Washington, U.S.A.

Fisher, M. (2001) 'Lehninger Principles of Biochemistry, 3rd edition; By David L.

Nelson and Michael M. Cox', The Chemical Educator, 6(1), pp. 69-70.

Forest Department Sarawak (2013) Annual Report. Kuching, Sarawak Malaysia.

Fortier, L.-C. and Moineau, S. (2009) 'Phage production and maintenance of stocks,

including expected stock lifetimes', Bacteriophages: Springer, pp. 203-219.

Fortin, D., Ferris, F. G. and Beveridge, T. J. (1997) 'Surface-mediated mineral

development by bacteria', Reviews in Mineralogy and Geochemistry, 35, pp.

161-180.

Frankel, R. B. and Bazylinski, D. A. (2003) 'Biologically induced mineralization by

bacteria', Reviews in Mineralogy and Geochemistry, 54, pp. 95–114.

Fujita , Y., Ferris, F. G., Lawson, R. D., Colwell , F. S. and Smith, R. W. (2000)

'Subscribed Content Calcium Carbonate Precipitation by Ureolytic Subsurface

Bacteria', Geomicrobiology Journal, 17(4), pp. 305-318.

Gabriel, C. R. and Northup, D. E. (2013) 'Microbial Ecology: Caves as an Extreme

Habitat', in Cheeptham, N. (ed.) Cave Microbiomes: A Novel Resource for Drug

Discovery: Springer-Verlag New York, pp. 85-102.

Gadd, G. M. (2010) 'Metals, minerals and microbes: geomicrobiology and

bioremediation. Microbiology', Microbiology 156, pp. 609–643.

Garbutt, N. and Prudente, J. C. (2007) Wild Borneo: The Wildlife and Scenery of Sabah,

Sarawak, Brunei, and Kalimantan. Cambridge, Massachusetts: MIT Press.

151

Garcia, G. M., Marquez, G. M. and Moreno, H. C. (2016) 'Characterization of bacterial

diversity associated with calcareous deposits and drip-waters, and isolation of

calcifying bacteria from two Colombian mines', Microbiological Research, 182,

pp. 21-30.

Gat, D., Tsesarsky, M. and Shamir, D. (2011) 'Ureolytic Calcium Carbonate

Precipitation in the Presence of Non-Ureolytic Competing Bacteria', in Han, J. &

Alzamora, D.E. (eds.) Geo-Frontiers 2011: Advances in Geotechnical

Engineering. Dallas, Texas, United States: American Society of Civil Engineers,

pp. 3966-3974.

Gat, D., Tsesarsky, M., Shamir, D. and Ronen, Z. (2014) 'Accelerated microbial-

induced CaCO3 precipitation in a defined coculture of ureolytic and non-

ureolytic bacteria', Biogeosciences, 11(10), pp. 2561-2569.

Gatermann, S. and Marre, R. (1989) 'Cloning and expression of Staphylococcus

saprophyticus urease gene sequences in Staphylococcus carnosus and

contribution of the enzyme to virulence', Infection and Immunity, 57(10), pp.

2998-3002.

Gautam, S. P., Bundela, P. S., Pandey, A. K., Khan, J., Awasthi, M. K. and Sarsaiya, S.

(2011) 'Optimization for the production of cellulase enzyme from municipal

solid waste residue by two novel cellulolytic fungi', Biotechnology Research

International, 2011, pp. 810425-810433.

Gavimath, C., Mali, B., Hooli, V., Mallpur, J., Patil, A., Gaddi, D., Ternikar, C. and

Ravishankera, B. (2012) 'Potential application of bacteria to improve the

strength of cement concrete', International Journal of Advanced Biotechnology

and Research 3(1), pp. 541-544.

Gebauer, D., Gunawidjaja, P. N., Ko, J. Y., Bacsik, Z., Aziz, B., Liu, L., Hu, Y.,

Bergstrom, L., Tai, C. W., Sham, T. K., Eden, M. and Hedin, N. (2010) 'Proto-

calcite and proto-vaterite in amorphous calcium carbonates', Angewandte

Chemie International Edition in English, 49(47), pp. 8889-8891.

Ghashghaei, S. and Emtiazi, G. (2013) 'Production of calcite nanocrystal by a urease-

positive strain of enterobacter ludwigii and study of its structure by SEM',

Current Microbiology, 67(4), pp. 406-13.

Ghosh, S., Biswas, M., Chattopadhyay, B. D. and Mandal, S. (2009) 'Microbial activity

on the microstructure of bacteria modified mortar', Cement and Concrete

Composites, 31(2), pp. 93-98.

152

Gibbs, R. A. (1990) 'DNA amplification by the polymerase chain reaction', Analytical

Chemistry, 62(13), pp. 1202-1214.

Glaeser, S. P., McInroy, J. A., Busse, H. J. and Kampfer, P. (2014)

'Pseudogracilibacillus auburnensis gen. nov., sp. nov., isolated from the

rhizosphere of Zea mays', International Journal of Systematic and Evolutionary

Microbiology, 64(Pt 7), pp. 2442-8.

Gomez, M., Anderson, C., DeJong, J., Nelson, D. and Lau, X. (2014) 'Stimulating In

Situ Soil Bacteria for Bio-Cementation of Sands', in Abu-Farsakh, M., Yu, X. &

Hoyos, L.R. (eds.) Geo-Congress 2014 Technical Papers: American Society of

Civil Engineers, pp. 1674-1682.

Gordon, E. R. and Hyde, L. J. (1982) 'The Bacillusfirmus-Bacillus lentus Complex and

pH 7.0 Variants of Some Alkalophilic Strains', Journal of General

Microbiology, 128, pp. 1109-1116.

Gordon, O., Berchtold, M., Staerk, A. and Roesti, D. (2014) 'Comparison of different

incubation conditions for microbiological environmental monitoring', PDA J

Pharm Sci Technol, 68(5), pp. 394-406.

Gordon, R. E., Harynes, W. C. and Pang, C. H. (1973) The Genus Bacillus.

Washington, United State of America: US Dep Agric. Handbook, p. 427.

Gorski, L. (2012a) 'Selective Enrichment Media Bias the Types of <italic>Salmonella

enterica</italic> Strains Isolated from Mixed Strain Cultures and Complex

Enrichment Broths', PLoS ONE, 7(4), pp. 1-8.

Gorski, L. (2012b) 'Selective enrichment media bias the types of Salmonella enterica

strains isolated from mixed strain cultures and complex enrichment broths',

PLoS One, 7(4), pp. 0034722-34730.

Gower, L. B. (2008) 'Biomimetic Model Systems for Investigating the Amorphous

Precursor Pathway and Its Role in Biomineralization', Chemical Reviews,

108(11), pp. 4551-4627.

Gueye, L., Samb, A., Ciss, M., Ndoye, O., Mbengue-Gaye, A. and Cisse, F. (2001)

'Ammonia-gas poisoning: respiratory troubles evaluated by functional

exploration', Dakar Medical Journal, 46(1), pp. 8-11.

Hall, T. A. (1999) 'BioEdit: a user-friendly biological sequence alignment editor and

analysis program for Windows 95/98/NT', Nucleic Acids Symposium Series, 41,

pp. 95-98.

153

Hamilton, W. A. (2003) 'Microbially influenced corrosion as a model system for the

study of metal microbe interactions: a unifying electron transfer hypothesis',

Biofoulin 19, pp. 65–76.

Hammad, I. A., Talkhan, F. N. and Zoheir, A. E. (2013) 'Urease activity and induction

of calcium carbonate precipitation by Sporosarcina pasteurii NCIMB 8841',

Journal of Applied Sciences Research, 9(3), pp. 1525-1533.

Hammes, F. (2003) Ureolytic Microbial Calcium Carbonate Precipitation. Unpublished

doctoral thesis, Gent University Gent Belgium.

Hammes, F., Boon, N., de Villiers, J., Verstraete, W. and Siciliano, S. D. (2003a)

'Strain-Specific Ureolytic Microbial Calcium Carbonate Precipitation', Applied

and Environmental Microbiology, 69(8), pp. 4901-4909.

Hammes, F., Boon, N., de Villiers, J., Verstraete, W. and Siciliano, S. D. (2003b)

'Strain-specific ureolytic microbial calcium carbonate precipitation', Applied and

Environmental Microbiology, 69(8), pp. 4901-4919.

Hammes, F., de Knijf, S. and Verstraete, W. (2001) 'First steps in microbiological

calcium removal from calcium-rich industrial wastewater', Meded Rijksuniv

Gent Fak Landbouwkd Toegep Biol Wet, 66(3a), pp. 165-168.

Hammes, F., Seka, A., de Knijf, S. and Verstraete, W. (2003c) 'A novel approach to

calcium removal from calcium-rich industrial wastewater', Water Research

37(3), pp. 699-704.

Hammes, F. and Verstraete, W. (2002) 'Key roles of pH and calcium metabolism in

microbial carbonaten precipitation', Reviews in Environmental Science &

Biotechnology 1, pp. 3-7.

Hamzah, A., Phan, C. W., Abu-Bakar, N. F. and Wong, K. K. (2013) 'Biodegradation of

Crude Oil by Constructed Bacterial Consortia and the Constituent Single

Bacteria Isolated From Malaysia', Bioremediation Journal 17(1), pp. 1-10.

Hamzah, A., Zarin, M. A., Hamid, A. A., Omar, O. and Senafi, S. (2012) 'Optical

Physical and Nutrient Parameters for Growth of Trichoderma virens UKMP-1M

for Heavy Crude Oil Degradation', Sains Malaysiana, 41(1), pp. 71-79.

Hanif, M., Khalid, A. N. and Exeter, R. L. (2014) 'Clavariadelphus pakistanicus sp.

nov., a new club fungus (Basidiomycota: Gomphales) from Himalayan moist

temperate forests of Pakistan', Botany, 92(7), pp. 471-476.

154

Harkes, M. P., van Paassen, L. A., Booster, J. L., Whiffin, V. S. and van Loosdrecht, M.

C. M. (2010) 'Fixation and distribution of bacterial activity in sand to induce

carbonate precipitation for ground reinforcement', Ecological Engineering,

36(2), pp. 112-117.

Helmi, F. M., Elmitwalli, H. R., Elnagdy, S. M. and El-Hagrassy, A. F. (2016) 'Calcium

carbonate precipitation induced by ureolytic bacteria Bacillus licheniformis',

Ecological Engineering, 90, pp. 367-371.

Hess, D. C., Lu, W., Rabinowitz, J. D. and Botstein, D. (2006) 'Ammonium toxicity and

potassium limitation in yeast', PLOS Biology, 4(11), pp. 351.

Heuer, H., Krsek, M., Baker, P., Smalla, K. and Wellington, E. M. (1997) 'Analysis of

actinomycete communities by specific amplification of genes encoding 16S

rRNA and gel-electrophoretic separation in denaturing gradients', Applied and

Environmental Microbiology, 63(8), pp. 3233-3241.

Holt, J. G., Noel, R. K., Peter, H. A. S., James, T. S. and Stanley, T. W. (1994)

Determinative Bacteriology. 9 edn. Baltimore, USA.

Hu, L. T., Foxall, P. A., Russell, R. and Mobley, H. L. (1992) 'Purification of

recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB',

Infection and Immunity, 60(7), pp. 2657-2666.

Hu, L. T. and Mobley, H. L. (1993) 'Expression of catalytically active recombinant

Helicobacter pylori urease at wild-type levels in Escherichia coli', Infection and

Immunity, 61(6), pp. 2563-2569.

Hughes, M. S., James, G., Ball, N., Scally, M., Malik, R., Wigney, D. I., Martin, P.,

Chen, S. and Love, D. N. (2000) 'Identification by 16S rRNAgene analyses of a

potential novel mycobacterial species as an etiological agent of canine leoproid

Granuloma syndrome', Journal of Clinical Microbiology, 38(3), pp. 953-959.

Ibrahim, C. O. (2008) 'Development of applications of industrial enzymes from

Malaysian indigenous microbial sources', Bioresource Technology, 99(11), pp.

4572-4582.

Isik, M., Altas, L., Kurmac, Y., Ozcan, S. and Oruc, O. (2010) 'Effect of hydraulic

retention time on continuous biocatalytic calcification reactor', Journal of

Hazardous Materials, 182(1-3), pp. 503-506.

Jabri, E., Carr, M. B., Hausinger, R. P. and Karplus, P. A. (1995) 'The crystal structure

of urease from Klebsiella aerogenes', Science, 268, pp. 998-1004.

155

Jahns, T. (1996) 'Ammonium/urea-dependent generation of a proton electrochemical

potential and synthesis of ATP in Bacillus pasteurii', Journal of Bacteriology,

178(2), pp. 403-409.

James, D. B., Timothy, S. H. and Greta, J. O. (2008) Limestone: A Crucial and

Versatile Industrial Mineral Commodity, Reston, VA.

Jargeat, P., Rekangalt, D., Verner, M.-C., Gay, G., Debaud, J.-C., Marmeisse, R. and

Fraissinet-Tachet, L. (2003) 'Characterisation and expression analysis of a

nitrate transporter and nitrite reductase genes, two members of a gene cluster for

nitrate assimilation from the symbiotic basidiomycete Hebeloma

cylindrosporum', Current Genetics, 43(3), pp. 199-205.

Jiang, N.-J., Yoshioka, H., Yamamoto, K. and Soga, K. (2016) 'Ureolytic activities of a

urease-producing bacterium and purified urease enzyme in the anoxic condition:

Implication for subseafloor sand production control by microbially induced

carbonate precipitation (MICP)', Ecological Engineering, 90, pp. 96-104.

Jones, A. A. and Bennett, P. C. (2014) 'Mineral microniches control the diversity of

subsurface microbial populations', Geomicrobiololgy Journal, 31, pp. 246–261.

Jones, B. D., Lockatell, C. V., Johnson, D. E., Warren, J. W. and Mobley, H. L. (1990)

'Construction of a urease-negative mutant of Proteus mirabilis: analysis of

virulence in a mouse model of ascending urinary tract infection', Infection and

Immunity, 58(4), pp. 1120-3.

Jonkers, H. M. (2007) 'Self healing concrete: a biological approach', in van der Zwaag,

S. (ed.) Self Healing Materials: An alternative Approach to 20 Centuries of

Materials Science. Netherlands: Springer, pp. 195–204.

Jonkers, H. M., Mors, R. M., Sierra-Beltran, M. G. and Wiktor, V. (2016) 'Biotech

solutions for concrete repair with enhanced durability', Biopolymers and

Biotech Admixtures for Eco-Efficient Construction Materials: Woodhead

Publishing, pp. 253-271.

Jonkers, H. M. and Schlangen, E. 'Crack repair by concrete-immobilized bacteria',

Proceedings of the First International Conference on Self Healing Materials,

Noordwijk aan Zee, The Netherlands: Springer 7.

Jonkers, H. M., Thijssen, A., Muyzer, G., Copuroglu, O. and Schlangen, E. (2010)

'Application of bacteria as self-healing agent for the development of sustainable

concrete', Ecological Engineering, 36(2), pp. 230-235.

156

Julaihi, L. C. J. (2004) Altitudinal analyses of limestone vegetation of Gunung Api,

Gunung Mulu National Park, Miri, Sarawak. Unpublished MSc thesis,

Universiti Malaysia Sarawak, Sarawak, Malaysia.

Kang, C.-H., Kim, Y., Oh, S. J., Mok, J.-S., Cho, M.-H. and So, J.-S. (2014a)

'Antibiotic resistance of Vibrio harveyi isolated from seawater in Korea', Marine

Pollution Bulletin, 86(1–2), pp. 261-265.

Kang, C. H., Choi, J. H., Noh, J., Kwak, D. Y., Han, S. H. and So, J. S. (2014b)

'Microbially induced calcite precipitation-based sequestration of strontium by

Sporosarcina pasteurii WJ-2', Applied Biochemistry and Biotechnology, 174(7),

pp. 2482-91.

Kang, C. H., Han, S. H., Shin, Y. J., Oh, S. J. and So, J. S. (2014c) 'Bioremediation of

Cd by microbially induced calcite precipitation', Applied Biochemistry and

Biotechnology, 172, pp. 1929–1937.

Karim, C., Tuen, A. A. and Abdullah, M. T. (2004) 'Mammals', Sarawak Museum

Journal, 6(80), pp. 221–234.

Karol, R. H. (2003) Chemical Grouting and Soil Stabilization. 3rd edn. New York,

United States of America: Marcel Dekker.

Kavazanjian, E. and Hamdan, N. (2015) 'Enzyme Induced Carbonate Precipitation

(EICP) Columns for Ground Improvement', in Iskander, M., Suleiman, T.M.,

Anderson, J.B. & Laefer, D.F. (eds.) Proceedings of the International

Foundations Congress and Equipment Expo 2015 (IFCEE 2015): American

Society of Civil Engineers, pp. 2252-2261.

Kaverin, N. V., Rudneva, I. A., Govorkova, E. A., Timofeeva, T. A., Shilov, A. A.,

Kochergin-Nikitsky, K. S., Krylov, P. S. and Webster, R. G. (2007) 'Epitope

Mapping of the Hemagglutinin Molecule of a Highly Pathogenic H5N1

Influenza Virus by Using Monoclonal Antibodies', Journal of Virology, 81(23),

pp. 12911-12917.

Kawaguchi, T. and Decho, A. W. (2002) 'A laboratory investigation of cyanobacterial

extracellular polymeric secretions (EPS) in influencing CaCO3 polymorphism',

Journal of Crystal Growth, 240(1-2), pp. 230-235.

Kielpinski, G., Prinzi, S., Duguid, J. and du Moulin, G. (2005) 'Roadmap to approval:

use of an automated sterility test method as a lot release test for Carticel,

autologous cultured chondrocytes', Cytotherapy, 7(6), pp. 531-41.

157

Knoll, A. (2003) 'Biomineralization and evolutionary history', Reviews in Mineralogy

and Geochemistry, 54, pp. 329–356.

Knorre, H. V. and Krumbein, W. E. (2000) 'Bacterial Calcification', in Riding, R.E. &

Awramik, S.M. (eds.) Microbial Sediments. Berlin, Heidelberg: Springer Berlin

Heidelberg, pp. 25-31.

Koh, L. P., Ghazoul, J., Butler, R. A., Laurance, W. F., Sodhi, N. S., Mateo-Vega, J.

and Bradshaw, C. J. A. (2010) 'Wash and spin cycle threats to tropical

biodiversity', Biotropica, 42, pp. 67–71.

Komala, T. and Khun, T. C. (2013) 'Calcite-Forming Bacteria Located in Limestone

Area of Malaysia', Journal of Asian Scientific Research, 3(5), pp. 471-484.

Konishi, Y., Tsukiyama, T., Ohno, K., Saitoh, N., Nomura, T. and Nagamine, S. (2006)

'Intracellular recovery of gold by microbial reduction of AuCl4− ions using the

anaerobic bacterium Shewanella algae', Hydrometallurgy, 81, pp. 24–29.

Krishnapriya, S., Venkatesh Babu, D. L. and G, P. A. (2015) 'Isolation and

identification of bacteria to improve the strength of concrete', Microbiology

Spectrum, 174, pp. 48-55.

Kuek, F. W. I., Lim, L.-F., Ngu, L.-H., Mujahid, A., Lim, P.-T., Leaw, C.-P. and

Müller, M. (2015) 'The potential roles of bacterial communities in coral defence:

A case study at Talang-talang reef', Ocean Science Journal, 50(2), pp. 1-14.

Kumar, C. G. and Takagi, H. (1999) 'Microbial alkaline proteases from a bio-industrial

view point', Biotechnology Advances, 17, pp. 561-594.

Kumari, B. (2015) 'Microbial Concrete: A Multi-Purpose Building Material-An

Overview', International Journal of Advances in Engineering & Technology,

7(6), pp. 1608-1619.

Labigne, A., Cussac, V. and Courcoux, P. (1991) 'Shuttle cloning and nucleotide

sequences of Helicobacter pylori genes responsible for urease activity', Journal

of Bacteriology, 173, pp. 1920–1931.

Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. and Pace, N. R. (1985)

'Rapid determination of 16S ribosomal RNA sequences for phylogenetic

analyse', Proceedings of the National Academy of Sciences, 82, pp. 6955-6959.

158

Lateef, A. A., Sepiah, M. and Bolhassan, M. H. (2004) 'Microfungi On Green Leaves

And Leaf Litters From Santubong National Park, Sarawak'. Proceedings of the

International Conference on Beneficial Microbes ICOBM 2014: Microbes for

the Benefits of Mankind, Penang, Malaysia. Malaysia: Universiti Sains

Malaysia, 130-134.

Le Metayer-Levrel, G., Castanier, S., Orial, G., Loubiere, J. and Perthuisot, J. (1999)

'Applications of bacterial carbonatogenesis to the protection and regeneration of

limestones in buildings and historic patrimony', Sedimentary Geology, 126(1-4),

pp. 25-34.

Lee, M. L., Soon, N. W., Tan, C. K. and Hii, S. L. (2012) 'Bio-mediated Soil

Improvement under Various Concentrations of Cementation Reagent', Applied

Mechanics and Materials, 204-208, pp. 326-329.

Lee, S. G. and Calhoun, D. H. (1997) 'Urease from a potentially pathogenic coccoid

isolate: purification, characterization, and comparison to other microbial

ureases', Infection and Immunity, 65(10), pp. 3991-3996.

Lewin, B. (1994) 'Genes V.', American Journal of Physical Anthropology: Wiley

Subscription Services, Inc., A Wiley Company.

Li, M., Cheng, X. and Guo, H. (2013) 'Heavy metal removal by biomineralization of

urease producing bacteria isolated from soil', International Biodeterioration &

Biodegradation, 76, pp. 81-85.

Li, M., Guo, H. and Cheng, X. (2011a) 'Application of Response Surface Methodology

for Carbonate Precipitation Production Induced by a Mutant Strain of

Sporosarcina pasteurii', Geo-Frontiers American Society of Civil Engineers, pp.

4079-4088.

Li, P. H. and Jin, B. 'Healing of Cracked Concrete by Sporosarcina pasteurii Mediated

Carbonate Deposition'. Proceedings of the 2nd International Conference on

Mechanical Engineering, Industry and Manufacturing Engineering

(MEIME2012), Hefei, China: Applied Mechanics, Materials, Industry and

Manufacturing Engineering, 103-106.

Li, W., Liu, L. P., Zhou, P. P., Cao, L., Yu, L. J. and Jiang, S. Y. (2011b) 'Calcite

precipitation induced by bacteria and bacterially produced carbonic anhydrase',

Current Science, 100, pp. 502-508.

159

Lian, B., Hu, Q., Chen, J., Ji, J. and Teng, H. H. (2006) 'Carbonate biomineralization

induced by soil bacterium Bacillus megaterium', Geochimica et Cosmochimica

Acta, 70(22), pp. 5522-5535.

Liang, X., Liang, X., Qiao, D., Huang, M., Yi, X., Bai, L., Xu, H., Wei, L., Zeng, J. and

Cao, Y. (2008) 'Identification of a gene encoding the light-harvesting

chlorophyll a/b proteins of photosystem I in green alga Dunaliella salina', DNA

Sequence, 19(2), pp. 137-145.

Liang, Z. P., Feng, Y. Q., Meng, S. X. and Liang, Z. Y. (2005) 'Preparation and

Properties of Urease Immobilized onto Glutaraldehyde Cross-linked Chitosan

Beads', Chinese Chemistry Letters, 16, pp. 135-138.

Lowe, D. A. (2001) Production of Enzymes. Basic Biotechnology Cambridge

Cambridge University Press, p. 391-408.

Lowenstam, H. A. (1981) 'Minerals formed by organisms', Science, 211(4487), pp.

1126-1131.

Macalady, J. L., Jones, D. S. and Lyon, E. H. (2007) 'Extremely acidic, pendulous cave

wall biofilms from the Frasassi cave system, Italy', Environmental

Microbiology, 9(6), pp. 1402-1414.

MacFaddin, J. F. (2000) Biochemical tests for the identification of medical bacteria. 3rd

edn. Philadelphia, Pennsylvania, USA: Lippincott Williams & Wilkins.

Machel, H. G. (2001) 'Bacterial and thermochemical sulfate reduction in diagenetic

settings — old and new insights', Sedimentary Geology, 140(1–2), pp. 143-175.

Mahidi, N. (2015) Characterization of new Muscodor padawan and Muscodor

sarawak, isolated from Sarawak, Malaysia: evaluation of their potential as a

biological control agent for Ganoderma boninense, a pathogenic fungus of

Elaeis guineensis Unpublished doctoral thesis, Swinburne University of

Technology, Sarawak, Malaysia.

Maite Muniesa, Anicet R. Blanch, Francisco Lucena and Jofre, J. (2005)

'Bacteriophages May Bias Outcome of Bacterial Enrichment Cultures',

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 71(8), pp. 4269–4275.

Mandic-Mulec, I., Stefanic, P. and van Elsas, J. D. (2015) 'Ecology of Bacillaceae',

Microbiology Spectrum, 3(2), pp. 0017.

Mann, S. (1983) 'Mineralization in biological systems', Structure and Bonding, (54), pp.

125-174

160

Mann, S. (2001) Biomineralization Principles and Concepts in Bioinorganic Materials

Chemistry. New York: Oxford University Press, p. 198.

McConnaughey, T. A. and Whelan, J. F. (1997) 'Calcification generates protons for

nutrient and bicarbonate uptake', Earth-Science Reviews, 42(1-2), pp. 95-117.

Melim, L. A., Liescheidt, R., Northup, D. E., Spilde, M. N., Boston, P. J. and Queen, J.

M. (2009) 'A biosignature suite from cave pool precipitates, Cotton- wood cave,

New Mexico', Astrobiology, 9, pp. 907–917.

Millo, C., Dupraz, S., Ader, M., Guyot, F., Thaler, C., Foy, E. and Ménez, B. (2012)

'Carbon isotope fractionation during calcium carbonate precipitation induced by

ureolytic bacteria', Geochimica et Cosmochimica Acta, 98, pp. 107-124.

Minty, J. J., Singer, M. E., Scholz, S. A., Bae, C. H., Ahn, J. H., Foster, C. E., Liao, J.

C. and Lin, X. N. (2013) 'Design and characterization of synthetic fungal-

bacterial consortia for direct production of isobutanol from cellulosic biomass',

Proceedings of the National Academy of Sciences, 110(36), pp. 14592-7.

Mitchell, A. C. and Ferris, F. G. (2005) 'The coprecipitation of Sr into calcite

precipitates induced by bacterial ureolysis in artificial groundwater: temperature

and kinetics dependence', Geochim Gosmochim Acta, 69, pp. 4199–4210.

Mitchell, J. K. and Santamarina, J. C. (2005) 'Biological Considerations in Geotechnical

Engineering', Journal of Geotechnical and Geoenvironmental Engineering,

131(10), pp. 1222-1233.

Mobley, H. L. and Hausinger, R. P. (1989) 'Microbial ureases: significance, regulation,

and molecular characterization', FEMS Microbiology Reviews, 53(1), pp. 85-

108.

Mobley, H. L., Island, M. D. and Hausinger, R. P. (1995) 'Molecular biology of

microbial ureases', FEMS Microbiology Reviews, 59(3), pp. 451-480.

Mobley, H. L. T. (2001) 'Urease', in Mobley, H.L.T., Mendz, G.L. & Hazell, S.L. (eds.)

Helicobacter pylori: Physiology and Genetics. Washington (DC), United States

of America: ASM Press.

Mohd, A. J., Maryanto, I., Kartono, A. P. and Abdullah, M. T. (2003) 'Diversity,

Relative Abundance and Conservation of Chiropterans in Kayan Mentarang

National Park, East Kalimantan, Indonesia', Sarawak Museum Journal, 79, pp.

251–265.

161

Mohd, R. A. R., Roberta, C. T. T., Mohd, I. A., Noor, H. H. and Mohd, T. A. (2011)

'Bats of the Wind Cave Nature Reserve, Sarawak, Malaysian Borneo ', Tropical

Natural History 11(2), pp. 159-175.

Moldenhauer, J. (2014) 'Justification of incubation conditions used for environmental

monitoring', American Pharmaceutical Review 17(2), pp. 1-5.

Mora, D. and Arioli, S. (2014) 'Microbial Urease in Health and Disease', PLoS

Pathogens, 10(12), pp. e1004472.

Morse, J. W. (1983) 'The Kinetics Calcium carbonate dissolution and precipitation',

Review in Mineralogy and Geochemistry, 11, pp. 227–264.

Mortensen, B. M., Haber, M. J., DeJong, J. T., Caslake, L. F. and Nelson, D. C. (2011)

'Effects of environmental factors on microbial induced calcium carbonate

precipitation', Journal of Applied Microbiolology, 111(2), pp. 338-349.

Moseley, G. E., Richards, D. A., Smith, C. J. M., Smart, P. L., Hoffmann, D. L. and

Farrant, A. R. (2013) 'U–Th dating of speleothems to investigate the evolution

of limestone caves in the Gunung Mulu National Park, Sarawak, Malaysia.',

Cave and Karst Science, 40(1), pp. 13-16.

Moyes, R. B., Reynolds, J. and Breakwell, D. P. (2005) 'Differential Staining of

Bacteria: Gram Stain', Current Protocols in Microbiology: John Wiley & Sons,

Inc.

Muramatsu, H., Shahab, N., Tsurumi, Y. and Hino, M. (2003) 'A comparative study of

Malaysian and Japanese actinomycetes using simple identification method based

on partial 16S rRNA sequence', Actinomycetology, 17, pp. 33-43.

Nasly, M. A. and Yassin, A. A. M. 'Sustainable housing using an innovative

interlocking block building system', Kuala Lumpur, Malaysia, 130-138.

Nemati, M., Greene, E. A. and Voordouw, G. (2005) 'Permeability profile modification

using bacterially formed calcium carbonate: comparison with enzymic option',

Process Biochemistry, 40(2), pp. 925-933.

Nemati, M. and Voordouw, G. (2003) 'Modification of Porous Media Permeability,

Using Calcium Carbonate Produced Enzymatically In situ', Enzyme and

Microbial Technology 33, pp. 635-642.

Neupane, D., Yasuhara, H. and Kinoshita, N. 'Evaluation of enzyme mediated calcite

grouting as a possible soil improvement technique', Computer Methods and

Recent Advances in Geomechanics, Kyoto, Japan: Taylor & Francis Group,

London, 1169-1172.

162

Northup, D. E. and Lavoie, K. H. (2001) 'Geomicrobiol of caves: a review',

Geomicrobiology Journal, 18, pp. 199–222.

Northup, D. E., Melim, L. A., Spilde, M. N., Hathaway, J. J. M., Garcia, M. G., Moya,

M., Stone, F. D., Boston, P. J., Dapkevicius, M. and Riquelme, C. (2011) 'Lava

Cave Microbial Communities Within Mats and Secondary Mineral Deposits:

Implications for Life Detection on Other Planets', Astrobiology, 11(7), pp. 601-

618.

Obst, M., Dynes, J. J., Lawrence, J. R., Swerhone, G. D. W., Benzerara, K.,

Karunakaran, C., Kaznatcheev, K., Tyliszczak, T. and Hitchcock, A. T. (2009)

'Precipitation of amorphous CaCO3 (aragonite-like) by cyanobacteria: A STXM

study of the influence of EPS on the nucleation process', Geochimica et

Cosmochimica Acta, 73, pp. 4180–4198.

Oka, T., Suzuki, M. and Konnya, T. (1993) 'The estimation of energy consumption and

amount of pollutants due to the construction of buildings', Energy Build, 19, pp.

303–311.

Okwadha, G. and Li, J. (2011) 'Biocontainment of polychlorinated biphenyls (PCBs) on

flat concrete surfaces by microbial carbonate precipitation', Journal of

Environmental Management 92, pp. 2860–2864.

Okwadha, G. D. and Li, J. (2010) 'Optimum conditions for microbial carbonate

precipitation', Chemosphere, 81, pp. 1143-1148.

Olufunke, O. A., Abiodun, A. O. and Dunah, F. C. (2014) 'Extended Spectrum Beta-

Lactamase- Producing Uropathogenic Escherichia coli in Pregnant Women

Diagnosed With Urinary Tract Infections in South Western Nigeria', Journal of

Molecular Biology Research, 4(34-41).

Onal, O. T. and Frigi, R. D. (2014) 'Optimized carbonate micro-particle production by

Sporosarcina pasteurii using response surface methodology', Ecological

Engineering, 62, pp. 168-174.

Ortiz, M., Legatzki, A., Neilson, J. W., Fryslie, B., Nelson, W. M., Wing, R. A.,

Soderlund, C. A., Pryor, B. M. and Maier, R. M. (2014) 'Making a living while

starving in the dark: metagenomic insights into the energy dynamics of a

carbonate cave', International Society for Microbial Ecology, 8(2), pp. 478-91.

163

Ortiz, M., Neilson, J. W., Nelson, W. M., Legatzki, A., Byrne, A., Yu, Y., Wing, R. A.,

Soderlund, C. A., Pryor, B. M., Pierson, L. S., 3rd and Maier, R. M. (2013)

'Profiling bacterial diversity and taxonomic composition on speleothem surfaces

in Kartchner Caverns, AZ', Microbial Ecology, 65(2), pp. 371-83.

Paassen, L. A. v. (2009) Biogrout Ground Improvement by Microbially Induced

Carbonate Precipitation. Unpublished doctoral thesis, Delft University of

Technology, Delft, Netherlands.

Paassen, L. A. v., Harkes, M. P., Zwieten, G. A. v., Zon, W. H. v. d., Star, W. R. L. v. d.

and Loosdrecht, M. C. M. v. 'Scale up of BioGrout: a biological ground

reinforcement method Agrandissement de BioGrout: méthode biologique pour la

consolidation des sols'. Proceedings of the 17th International Conference on

Soil Mechanics and Geotechnical Engineering, 2328-2333.

Pandey, A., Binod, P., Ushasree, M. V. and Vidya, J. (2010) 'Advanced strategies for

improving industrial enzymes', Chemical Industry Digest, 23, pp. 74-84.

Pappu, A., Saxena, M. and Asolekar, S. R. (2007) 'Solid wastes generation in India and

their recycling potential in building materials', Building and Environment, 42(6),

pp. 2311-2320.

Park, S., Choi, S., Kim, W. and Lee, J. (2014) 'Effect of Microbially Induced Calcite

Precipitation on the Strength of Cemented Sand', in Zhang, G. & Liu, Z. (eds.)

New Frontiers in Geotechnical Engineering: American Society of Civil

Engineers, pp. 47-56.

Park, S. J., Lee, N. Y., Kim, W. J. and Ghim, S. Y. (2010) 'Application of Bacteria

Isolated from Dok-do for Improving Compressive Strength and Crack

Remediation of Cement-sand Mortar ', Korean Journal of Microbiology and

Biotechnoogy, 38(2), pp. 216-221.

Peethamparan, S., Olek, J. and Diamond, S. (2009) 'Mechanism of stabilization of Na-

montmorillonite clay with cement kiln dust', Cement and Concrete Research,

39, pp. 580-589.

Perry, C. C. (2003) 'Silicification: the processes by which organisms capture and

mineralize silica', Review in Mineralogy and Geochemistry, 54, pp. 291–327.

Phillips, A. J., Gerlach, R., Lauchnor, E., Mitchell, A. C., Cunningham, A. B. and

Spangler, L. (2013) 'Engineered applications of ureolytic biomineralization: a

review', Biofouling, 29(6), pp. 715-33.

164

Prah, J., Maček, J. and Dražič, G. (2011) 'Precipitation of calcium carbonate from a

calcium acetate and ammonium carbamate batch system', Journal of Crystal

Growth, 324(1), pp. 229-234.

Provencio, P. and Polyak, V. (2001) 'Iron oxide-rich filaments: possible fossil bacteria

in Lechuguilla Cave, New Mexico', Geomicrobiology Journal 18, pp. 297-309.

Qabany, A. A., Soga, K. and Santamarina, C. (2012) 'Factors affecting efficiency of

microbially induced calcite precipitation', Journal of Geotechnical and

Geoenvironmental Engineering, 138, pp. 992–1001.

Qian, C. X., Pan, Q. F. and Wang, R. X. (2010) 'Cementation of sand grains based on

carbonate precipitation induced by microorganism.', Science China

Technological Sciences, 53, pp. 2198–21206.

Rahman, M. R. A., Achadi, A. S., Tingga, R. C. T. and Hasan, N. H. (2010a) 'A new

distributional record of the rare bat Coelops robinsoni from Sarawak,Malaysian

Borneo', Journal of tropical biology and conservation, 7, pp. 87-92.

Rahman, M. R. A., Tingga, R. C. T., Noor Haliza, H., Sigit, W., Anang, S. A., Eileen,

L., Besar, K., Huzal, I. H. and Abdullah, M. T. (2010b) 'Diversity of bats in two

protected limestone areas in Sarawak, Malaysia', Sarawak Museum Journal, 88,

pp. 209-246.

Rahman, R. N., Geok, L. P., Basri, M. and Salleh, A. B. (2005) 'Physical factors

affecting the production of organic solvent-tolerant protease by Pseudomonas

aeruginosa strain K', Bioresource Technology 96(4), pp. 429-436.

Rajendhran, J. and Gunasekaran, P. (2011) 'Microbial phylogeny and diversity: Small

subunit ribosomal RNA sequence analysis and beyond', Microbiological

Research, `66(2), pp. 99–110.

Ramachandran, S. K., Ramakrishnan, V. and Bang, S. S. (2001) 'Remediation of

Concrete Using Microorganisms', ACI Materials Journal, 98(1), pp. 3-9.

Ramesh Kumar, V., Bhuvaneshwari, B., Maheswaran, S., Palani, G. S., Ravisankar, K.,

and Iyer, N. R. (2011) 'An Overview of Techniques based on Biomimetics for

Sustainable Development of Concrete', Current Science Journal, 101(6), pp.

741-747.

Ranganathan, N., Patel, B. G., Ranganathan, P., Marczely, J., Dheer, R., Pechenyak, B.,

Dunn, S. R., Verstraete, W., Decroos, K., Mehta, R. and Friedman, E. A. (2006)

'In vitro and in vivo assessment of intraintestinal bacteriotherapy in chronic

kidney disease', Asaio Journal, 52(1), pp. 70-90.

165

Rasigade, J. P. and Vandenesch, F. (2014) 'Staphylococcus aureus: a pathogen with still

unresolved issues', Infection, Genetics and Evolution, 21, pp. 510-4.

Ratledge, C. (2001) Biochemistry and physiology of growth and metabolism. Basic

Biotechnology Cambridge Cambridge University Press, p. 17-44.

Raut, S. H., Sarode, D. D. and Lele, S. S. (2014) 'Biocalcification using B. pasteurii for

strengthening brick masonry civil engineering structures', World Journal of

Microbiology and Biotechnology, 30(1), pp. 191-200.

Rautner, M., Hardiono, M. and Alfred, R. J. (2005) Borneo: Treasure Island at Risk,

Germany: The World Wide Fund for Nature.

Reddy, B. V. V. and Jagadish, K. S. (2003) 'Embodied energy of common and

alternative building materials and technologies', Energy and Buildings 35, pp.

129-137.

Reddy, M. S., Achal, V. and Mukherjee, A. 'Microbial concrete, a wonder metabolic

product that remediates the defects in building structures', Microorganisms in

Environmental Management: Microbes and Environment., USA: Springer

Publishers, 547–568.

Reyes, R. G., Lopez, L. L. M. A., Kumakura, K., Kalaw, S. P., Kikukawa, T. and

Eguchi, F. (2009) 'Coprinus comatus, a newly domesticated wild nutriceutical

mushroom in the Philippines', Journal of Agricultural Technology 5(2), pp. 299-

316.

Reynolds, J., Moyes, R. and Breakwell, D. P. (2009) 'Differential staining of bacteria:

endospore stain', Current Protocols in Microbiology, Appendix 3, pp. Appendix

3J.

Rieger, J., Frechen, T., Cox, G., Heckmann, W., Schmidt, C. and Thieme, J. (2007)

'Precursor structures in the crystallization/precipitation processes of CaCO3 and

control of particle formation by polyelectrolytes', Faraday Discussions, 136(0),

pp. 265-277.

Rivadeneyra, M. A., Delgado, G., Ramos-Cormenzana, A. and Delgado, R. (1998)

'Biomineralization of carbonates by Halomonas eurihalina in solid and liquid

media with different salinities: crystal formation sequence', Resource

Microbiology, 149, pp. 277-287.

Rivadeneyra, M. A., Delgado, G., Soriano, M., Ramos-Cormenzana, A. and Delgado,

R. (2000) 'Precipitation of carbonates by Nesterenkonia halobia in liquid

media," Chemosphere', Chemosphere, 41, pp. 617-624.

166

Rivadeneyra, M. A., Martín-Algarra, A., Sánchez-Navas, A. and Martíin-Ramos, D.

(2006) 'Carbonate and Phosphate Precipitation by Chromohalobacter

marismortui', Geomicrobiology Journal, 23(2), pp. 89-101.

Rivadeneyra, M. A., Parraga, J., Delgado, R., Ramos-Cormenzana, A. and Delgado, G.

(2004) 'Biomineralization of carbonates by Halobacillus trueperi in solid and

liquid media with different salinities', FEMS Microbiology Ecology, 48(1), pp.

39-46.

Rodriguez-Navarro, C., Jroundi, F., Schiro, M., Ruiz-Agudo, E. and Gonzalez-Munoz,

M. T. (2012) 'Influence of substrate mineralogy on bacterial mineralization of

calcium carbonate: implications for stone conservation', Applied and

Environmental Microbiology, 78, pp. 4017–4029.

Rodriguez-Navarro, C., Rodriguez-Gallego, M., Ben Chekroun, K. and Gonzalez-

Muñoz, M. T. (2003) 'Conservation of Ornamental Stone by Myxococcus

xanthus-Induced Carbonate Biomineralization', Applied and Environmental

Microbiology, 69(4), pp. 2182-2193.

Roesch, L. F., Fulthorpe, R. R., Riva, A., Casella, G., Hadwin, A. K., Kent, A. D.,

Daroub, S. H., Camargo, F. A., Farmerie, W. G. and Triplett, E. W. (2007)

'Pyrosequencing enumerates and contrasts soil microbial diversity', International

Society for Microbial Ecology, 1(4), pp. 283-290.

Rowshanbakht, K., Khamehchiyan, M., Sajedi, R. H. and Nikudel, M. R. (2016) 'Effect

of injected bacterial suspension volume and relative density on carbonate

precipitation resulting from microbial treatment', Ecological Engineering, 89,

pp. 49-55.

Rusznyak, A., Akob, D. M., Nietzsche, S., Eusterhues, K., Totsche, K. U., Neu, T. R.,

Frosch, T., Popp, J., Keiner, R., Geletneky, J., Katzschmann, L., Schulze, E. D.

and Kusel, K. (2012) 'Calcite biomineralization by bacterial isolates from the

recently discovered pristine karstic herrenberg cave', Applied and Environmental

Microbiology, 78(4), pp. 1157-1167.

Sa'don, N. M., Karim, A. R. A., Jaol, W. and Lili, W. H. W. (2015) 'Sarawak Peat

Characteristics and Heat Treatment', UNIMAS e-Journal of Civil Engineering,

5(3), pp. 6-12.

Saitou, N. and Nei, M. (1987) 'The neighbor-joining method: a new method for

reconstructing phylogenetic trees', FEMS Microbiology Reviews, 4(4), pp. 406-

425.

167

Sanchez-Moral, S., Canaveras, J. C., Laiz, L., Saiz-Jimenez, C., Bedoya, J. and Luque,

L. (2003) 'Biomediated Precipitation of Calcium Carbonate Metastable Phases in

Hypogean Environments: A Short Review', Geomicrobiology Journal, 20(5), pp.

491-500.

Sanchez-Navas, A., Martin-Algarra, A., Rivadeneyra, M. A., Melchor, S. and Marti -

Ramos, J. D. (2009) 'Crystal-Growth Behavior in Ca−Mg Carbonate Bacterial

Spherulites', Crystal Growth & Design, 9, pp. 2690–2699.

Sarawak Forest Department, National Park Wildlife Office Forest Department, S.

(1992) Proposal to constitute Wind cave and Fairy Cave as Nature Reserve.

Sarayu, K., Iyer, N. R. and Murthy, A. R. (2014) 'Exploration on the biotechnological

aspect of the ureolytic bacteria for the production of the cementitious materials--

a review', Applied Biochemistry and Biotechnology, 172(5), pp. 2308-2323.

Sauvage, J., Spivack, A. J., Murray, R. W. and D'Hondt, S. (2014) 'Determination of in

situ dissolved inorganic carbon concentration and alkalinity for marine

sedimentary porewater', Chemical Geology, 387, pp. 66-73.

Schabereiter-Gurtner, C., Saiz-Jimenez, C., Pinar, G., Lubitz, W. and Rolleke, S. (2004)

'Phylogenetic diversity of bacteria associated with Paleolithic paintings and

surrounding rock walls in two Spanish caves (Llonin and La Garma)', FEMS

Microbiology Ecology, 47(2), pp. 2335-2347.

Scheldeman, P., Rodriguez-Diaz, M., Goris, J., Pil, A., De Clerck, E., Herman, L., De

Vos, P., Logan, N. A. and Heyndrickx, M. (2004) 'Bacillus farraginis sp. nov.,

Bacillus fortis sp. nov. and Bacillus fordii sp. nov., isolated at dairy farms',

International Journal of Systematic and Evolutionary Microbiology, 54(Pt 4),

pp. 1355-1364.

Seshabala, P. and Mukkanti, K. (2013) 'Isolation of urease rich bacteria and

determination of its optimal conditions', Indian Journal of Applied Research,

3(7), pp. 336-338.

Sethi, S. and Gupta, S. (2014) 'Optimization Of Cultural Parameters For Cellulase

Enzyme Production From Fungi', BIolife, 2(3), pp. 989-996.

Shannon Stocks-Fischer, Johnna K. Galinat and Bang, S. S. (1999) 'Microbiological

precipitation of CaCO3', Soil Biology and Biochemistry, 31, pp. 1563-1571.

Shields, P. and Cathcart, L. 'Motility Test Medium Protocol', The 18th Annual Americal

Society for Microbiology Conference for Undergraduate Educaters, New

Orleans, Louisiana, United States of America: ASM MicrobeLibrary, 1-7.

168

Shields, P. and Cathcart, L. (2013) 'Oxidase Test Protocol', American Society for

Microbiology pp. 1-10.

Siddique, R. and Chahal, N. K. (2011) 'Effect of ureolytic bacteria on concrete

properties', Construction and Building Materials, 25(10), pp. 3791-3801.

Siddique, R., Nanda, V., Kunal, Kadri, E.-H., Iqbal Khan, M., Singh, M. and Rajor, A.

(2016) 'Influence of bacteria on compressive strength and permeation properties

of concrete made with cement baghouse filter dust', Construction and Building

Materials, 106, pp. 461-469.

Sidik, W., Canakci, H. and Kilic, I. H. (2015) 'An Investigation of Bacterial Calcium

Carbonate Precipitation In Organic Soil For Geotechnical Applications',

International Journal of Science & Technology, 39(1), pp. 201-205.

Sidik, W. S., Canakci, H., Kilic, I. H. and Celik, F. (2014) 'Applicability of

biocementation for organic soil and its effect on permeability', Geomechanics

and Engineering, 7(6), pp. 649-663.

Sierra-Beltran, M. G., Jonkers, H. M. and Schlangen, E. (2014) 'Characterization of

sustainable bio-based mortar for concrete repair', Construction and Building

Materials, 67, Part C, pp. 344-352.

Silhavy, T. J., Kahne, D. and Walker, S. (2010) 'The Bacterial Cell Envelope', Cold

Spring Harbor Perspectives in Biology, 2(5), pp. 000400-000414.

Soon, N. W. (2013) Improvements In Engineering Properties of Tropical Residual Soil

By Microbially-Induced Calcite Precipitation. Unpublished master of

engineering science thesis, Universiti Tunku Abdul Rahman, Perak, Malaysia.

Soon, N. W., Lee, L. M., Khun, T. C. and Ling, H. S. (2013) 'Improvements in

engineering properties of soils through microbial-induced calcite precipitation',

The Korean Soceity of Civil Engineering Journal of Civil Engineering, 17(4),

pp. 718-728.

Soon, N. W., Lee, L. M., Khun, T. C. and Ling, H. S. (2014) 'Factors Affecting

Improvement in Engineering Properties of Residual Soil through Microbial-

Induced Calcite Precipitation', Journal of Geotechnical and Geoenvironmental

Engineering, 140(5), pp. 04014006.

Soon, N. W., Lee, M. L. and Hii, S. L. (2012) 'An Overview of the Factors Affecting

Microbial-Induced Calcite Precipitation and its Potential Application in Soil

Improvement', International Journal of Civil, Environmental, Structural,

Construction and Architectural Engineering, 6(2), pp. 188-194.

169

Stabnikov, V., Chu, J., Ivanov, V. and Li, Y. (2013) 'Halotolerant, alkaliphilic urease-

producing bacteria from different climate zones and their application for

biocementation of sand', World Journal of Microbiology and Biotechnology,

29(8), pp. 1453-1460.

Stabnikov, V. and Ivanov, V. (2016) 'Biotechnological production of biopolymers and

admixturers for eco-efficient construction materials', in Fernando Pacheco-

Torgal, V.I., Niranjan Karak, Henk Jonkers (ed.) Biopolymers and Biotech

Admixtures for Eco-Efficient Construction Materials: Woodhead Publishing, pp.

464

State Planning Unit, Department, C.M.s. (2013) Sarawak fact and figures Kuching,

Sarawak, Malaysia: State planning unit.

Stocks-Fischer, S., Galinat, J. K. and Bang, S. S. (1999) 'Microbiological precipitation

of CaCO3', Soil Biology and Biochemistry, 31, pp. 1563-1571.

Stumm, W. and Morgan, J. J. (1981) Aquatic Chemistry. 2nd edn. New York, United

States of America: John Wiley and Sons, p. 780.

Suer, P., Hallberg, N., Carlsson, C., Bendz, D. and Holm, G. (2009) 'Biogrouting

compared to jet grouting: environmental (LCA) and economical assessment',

Journal of Environmental Science and Health, 44(4), pp. 346-353.

Sugita, T., Kikuchi, K., Makimura, K., Urata, K., Someya, T., Kamei, K. and Uehara,

Y. (2005) 'Trichosporon Species Isolated from Guano Samples Obtained from

Bat-Inhabited Caves in Japan', Applied and Environmental Microbiology,

71(11), pp. 7626–7629.

Sulaiman, Z. H. and Mayden, R. L. (2012) 'Cypriniformes of Borneo (Actinopterygii,

Otophysi): An Extraordinary Fauna for Integrated Studies on Diversity,

Systematics, Evolution, Ecology, and Conservation', Zootaxa Journal, 3586, pp.

359–376.

Suzuki, M., Oka, T. and Okada, K. (1995) 'The estimation of energy consumption and

CO2 emission due to housing construction in Japan.', Energy Build, 22, pp. 165–

169

Swift, D. M. and Wheeler, A. P. (1992) 'Evidence of An Organic Matrix from Diatom

Biosilica', Journal of Phycology, 28(2), pp. 202-209.

Tamura, K., Nei, M. and Kumar, S. (2004) 'Prospects for inferring very large

phylogenies by using the neighbor-joining method.', Proceedings of the National

Academy of Sciences, 101, pp. 11030-11035.

170

Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) 'MEGA 6:

Molecular Evolutionary Genetics Analysis version 6.0', Molecular Biology and

Evolution, 30(12), pp. 2725-2729.

Tan, H. H. (2006) The Borneo Suckers: Revision of the Torrent Loaches of Borneo

(Balitoridae, Gastromyzon, Neogastromyzon). Kota Kinabalu: Natural History

Publications (Borneo).

Tan, H. S., Nadiah, I. and Eryani, S. S. (2009) 'Tree Flora of Sabah and Sarawak project

– progress and future activities', Blumea - Biodiversity, Evolution and

Biogeography of Plants, 54(1), pp. 23-24.

Tan, H. Y., Sieo, C. C., Lee, C. M., Abdullah, N., Liang, J. B. and Ho, Y. W. (2011)

'Diversity of bovine rumen methanogens In vitro in the presence of condensed

tannins, as determined by sequence analysis of 16S rRNA gene library', The

Journal of Microbiology, 49(3), pp. 492-498.

Tebo, B. M., Ghiorse, W. C., van Waasbergen, L. G., Siering, P. L. and Caspi, R.

(1997) 'Bacterially mediated mineral formation: Insights into manganese(II)

oxidation from molecular genetic and biochemical studies', Review in

Mineralogy and Geochemistry, 35, pp. 225-266.

Tetu, S. G., Breakwell, K., Elbourne, L. D., Holmes, A. J., Gillings, M. R. and Paulsen,

I. T. (2013) 'Life in the dark: metagenomic evidence that a microbial slime

community is driven by inorganic nitrogen metabolism', International Society

for Microbial Ecology, 7(6), pp. 1227-1236.

Thomas, M. (2007) Optimizing the Use of Fly Ash in Concrete. Skokie, Illinois, United

States of America: American Society for Testing and Materials (ASTM)

Tobler, D. J., Cuthbert, M. O., Greswell, R. B., Riley, M. S., Renshaw, J. C., Handley-

Sidhu, S. and Phoenix, V. R. (2011) 'Comparison of rates of ureolysis between

Sporosarcina pasteurii and an indigenous groundwater community under

conditions required to precipitate large volumes of calcite', Geochimica et

Cosmochimica Acta 75, pp. 3290–3301.

Tomczyk-Żak, K. and Zielenkiewicz, U. (2015) 'Microbial Diversity in Caves',

Geomicrobiology Journal, 33(1), pp. 20-38.

Tominaga, T., An, S. Y., Oyaizu, H. and Yokota, A. (2009) 'Sporosarcina luteola sp.

nov. isolated from soy sauce production equipment in Japan', The Journal of

General and Applied Microbiology 55(3), pp. 217-223.

171

Tourney, J. and Ngwenya, B. T. (2009) 'Bacterial exopolymeric substances (EPS)

mediate CaCO3 moprhology and polymorphism', Chemical Geology, 262((3-4)),

pp. 138-146.

Tyler, A. C., McGlathery, K. J. and Anderson, I. C. (2003) 'Benthic algae control

sediment—water column fluxes of organic and inorganic nitrogen compounds in

a temperate lagoon', Limnology and Oceanography, 48(6), pp. 2125-2137.

Vahabi, A., Ramezanianpour, A. A., Sharafi, H., Shahbani, H. Z., Vali, H. and

Noghabil, K. A. (2014) 'Calcium carbonate precipitation by strain Bacillus

licheniformis AK01, newly isolated from loamy soil: a promising alternative for

sealing cement-based materials', Journal of Basic Microbiology, 53, pp. 1-7.

Van der Meer, P. J., Demies, M. A., Shabudin, M. S., Verwer, C., Beintema, A.,

Hilligers, P. J. M. and Teo, S. P. (2013) Tropical peat swamp forests of

Sarawak. Alterra, Wageningen UR, Netherland.

van Paassen, L. A., Ghose, R., van der Linden, T. J. M., van der Star, W. R. L. and van

Loos- drecht, M. C. M. (2010) 'Quantifying biomediated ground improvement

by ureol- ysis: large-scale biogrout experiment', Journal of Geotechnical and

Geoenvironmental Engineering, pp. 1721–1728.

Voland, P., Weeks, D. L., Marcus, E. A., Prinz, C., Sachs, G. and Scott, D. (2003)

'Interactions among the seven Helicobacter pylori proteins encoded by the

urease gene cluster', American journal of physiology, Gastrointestinal and liver

physiology, 284(1), pp. 96-106.

Waino, M., Tindall, B., Schumann, P. and Ingvorsen, K. (1999) 'Gracilibacillus gen.

nov., with description of Gracilibacillus halotolerans gen.nov., sp. nov.; transfer

of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus

salexigens to the genus Salibacillus gen.nov., as Salibacillus salexigens comb.

nov.', International Journal of Systematic and Evolutionary Microbiology,

49(2), pp. 821-831.

Walter, J., Tannock, G. W., Tilsala-Timisjarvi, A., Rodtong, S., Loach, D. M., Munro,

K. and Alatossava, T. (2000) 'Detection and Identification of Gastrointestinal

Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and

Species-Specific PCR Primers', Applied and Environmental Microbiology,

66(1), pp. 297-303.

Warren, L. A. and Haack, E. A. (2001) 'Biochemical controls on metal behavior in

freshwater environments', Earth Science Reviews, 54, pp. 261-320.

172

Warthmann, R., van Lith, Y., Vasconcelos, C., McKenzie, J. A. and Karpoff, A. M.

(2000) 'Bacterially induced dolomite precipitation in anoxic culture

experiments', Geology, 28(12), pp. 1091-1094.

Weaver, T., Burbank, M., Lewis, A., Lewis, R., Crawford, R. and Williams, B. (2011)

'Bio-Induced Calcite, Iron, and Manganese Precipitation for Geotechnical

Engineering Applications', Geo-Frontiers pp. 3975-3983.

Wei, S., Cui, H., Jiang, Z., Liu, H., He, H. and Fang, N. (2015) 'Biomineralization

processes of calcite induced by bacteria isolated from marine sediments',

Brazilian Journal of Microbiology, 46(2), pp. 455-464.

Weiner, S. and Dove, P. M. (2003) 'An Overview of Biomineralization Processes and

the Problem of the Vital Effect ', Reviews in Mineralogy and Geochemistry 54,

pp. 1-29.

Weiss, I. M., Tuross, N., Addadi, L. and Weiner, S. (2002) 'Mollusc larval shell

formation: amorphous calcium carbonate is a precursor phase for aragonite',

Journal of Experimental Zoology, 293(5), pp. 478-491.

Whiffin, V. S. (2004) Microbial CaCO3 Precipitation for the production of Biocement.

Unpublished doctoral thesis, Murdoch University, Perth, Australia.

Whiffin, V. S., van Paassen, L. A. and Harkes, M. P. (2007) 'Microbial Carbonate

Precipitation as a Soil Improvement Technique', Geomicrobiology Journal,

24(5), pp. 417-423.

Williams, C. L., Preston, T., Hossack, M., Slater, C. and McColl, K. E. (1996)

'Helicobacter pylori utilises urea for amino acid synthesis', FEMS Immunology

and Medical Microbiology, 13(1), pp. 87-94.

Winn, W., Allen, S., Janda, W., Koneman, E., Procop, G., Schreckenberger, P. and G.,

W. (2006) Koneman’s color atlas and textbook of diagnostic microbiology. 6th

edn. Philadelphia, Pennsylvania, USA: Lippincott Williams & Wilkins.

Wong, L. S. (2015) 'Microbial cementation of ureolytic bacteria from the genus

Bacillus: a review of the bacterial application on cement-based materials for

cleaner production', Journal of Cleaner Production, 93, pp. 5-17.

Woto-Gaye, G., Mendez, V., Boye, I. A. and Ndiaye, P. D. (1999) 'Death from

ammonia poisoning: anatomo-pathologic features', Dakar Medical Journal,

44(2), pp. 199-201.

173

Wray, L. V., Ferson, A. E. and Fisher, S. H. (1997) 'Expression of the Bacillus subtilis

ureABC operon is controlled by multiple regulatory factors including CodY,

GlnR, TnrA, and Spo0H', Journal of Bacteriology, 179(17), pp. 5494-5501.

Wright, D. T. (1999) 'The role of sulphate-reducing bacteria and cyanobacteria in

dolomite formation in distal ephemeral lakes of the Coorong region, South

Australia', Sedimentary Geology, 126(1–4), pp. 147-157.

Wu, Y., Tan, L., Liu, W., Wang, B., Wang, J., Cai, Y. and Lin, X. (2015) 'Profiling

bacterial diversity in a limestone cave of the western Loess Plateau of China',

Frontiers in Microbiology, 6, pp. 244-259.

Yoon, J. H., Lee, K. C., Weiss, N., Kho, Y. H., Kang, K. H. and Park, Y. H. (2001)

'Sporosarcina aquimarina sp. nov., a bacterium isolated from seawater in Korea,

and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus

psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the

genus Sporosarcina as Sporosarcina globispora comb. nov., Sporosarcina

psychrophila comb. nov. and Sporosarcina pasteurii comb. nov., and emended

description of th.', International Journal of Systematic and Evolutionary

Microbiology, 51(Pt 3), pp. 1079-1086.

Yoshida, N., Higashimura, E. and Saeki, Y. (2010) 'Catalytic Biomineralization of

Fluorescent Calcite by the Thermophilic Bacterium Geobacillus

thermoglucosidasius', Applied and Environmental Microbiology, 76(21), pp.

7322-7327.

Young, J. R. and Henriksen, K. (2003) 'Biomineralization within vesicles: the calcite of

coccoliths', Reviews in Mineralogy and Geochemistry, 54, pp. 189–215.

Zhang, Y., Guo, H. X. and Cheng, X. H. (2015) 'Role of calcium sources in the strength

and microstructure of microbial mortar', Construction and Building Materials,

77, pp. 160-167.

Zhang, Z., Schwartz, S., Wagner, L. and Miller, W. (2000) 'A greedy algorithm for

aligning DNA sequences', Journal of Computational Biology 7(1-2), pp. 203-

214.

Zhao, C. and Cui, B. (2013) 'Morphological and molecular identification of four new

resupinate species of Perenniporia (Polyporales) from southern China',

Mycologia, 105(4), pp. 945-958.

174

Zhao, Q., Li, L., Li, C., Li, M., Amini, F. and Zhang, H. (2014) 'Factors Affecting

Improvement of Engineering Properties of MICP-Treated Soil Catalyzed by

Bacteria and Urease', Journal of Materials in Civil Engineering, 26(12), pp.

04014094-04014104.