Innovation, Society for Design and Process Science, pp. 261-266. .... 1.9 Calcified structures of biogenic origin discovered in cave regions. 34 ...... throat size would limit their free passage, depending on the size of microbes and soil .... Cracking on concrete surfaces also results in enhanced deterioration of embedded steel.
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
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 ureaseproducing 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.
i
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. ii
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, cosupervision, 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. iii
I would also like to acknowledge Assist. Prof Salwa Al-Thawadi, Dr Ralf CordRuwisch, 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.
iv
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
v
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,12 (6): 463-470.
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. 1621. 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.
vi
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
vii
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
viii
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
ix
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 o
3.3.1.
Temperature ( C)
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
4.2
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
Future Directions and Recommendations
REFERENCES
136 138
x
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
64
nucleotide BLAST database 2.7
The nomenclatural taxonomy obtained using Ribosomal Database Project-
66
II database 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
76
between individual isolated urease-producing bacteria and control strain 2.11
t-test results comparing the calcite precipitate differences between
79
individual isolated urease-producing bacteria and control strain 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
116
biocemented sands 3.7
Unconfined compressive strength (UCS) of the treated sands
117
3.8
t-test results comparing the unconfined compressive strength (UCS)
118
differences between the biocemented sands 3.9
Summary of calcite content and compressive strength of selected
121
isolates and consortia
xi
LIST OF FIGURES Figures
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
13
Sporosarcina pasteurii 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
26
hydrolysis 1.7
1 mm thick calcite crust formed on the surface of the soil m3
27
1.8
Set-up for large scale (100 ) 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
35
mines located in Cordillera Central, northeast of Colombia 1.11
Map of Borneo Island showing the geographical divisions and
38
topographical features of Brunei Darussalam, Indonesia (Kalimantan) and East Malaysia (Sarawak and Sabah) 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
68
sequence from different isolates of the current study along with sequences available in the GenBank database 2.7 2.8
Relative conductivity of isolate LPB21 measured for a duration of 5 min -1
-1
Specific urease activity (mM urea hydrolysed.min .OD ) of urease-
70 75
producing bacteria and the control strain 2.9
Calcite precipitation media
77
2.10
Comparison of calcite precipitated by selected UPB isolates and the control
78
strain
xii
2.11
Growth profile of selected ureolytic bacterial isolates and control strain
82
grown in nutrient broth containing 6% urea for 12 hr 2.12
pH profile of selected ureolytic bacterial isolates and control strain grown
83
in nutrient broth containing 6% urea for 12 hr 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,
111
positive and negative controls 3.6
Sand columns at the end of treatment using ureolytic bacteria and
112
cementation solution 3.7
Treated sand removed from their respective columns
113
3.8
Treated sand sample held after a curing period and columns were
114
successfully removed 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
xiii
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
xiv
RH
Relative Humidity
SEM
Standard Error of Mean
SE
Standard Deviation
ANOVA
Analysis of Variance
xv
Chapter
1 INTRODUCTION AND LITERATURE REVIEW
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).
1
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.
2
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, StocksFischer 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 surfacemediation 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).
3
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).
4
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).
5
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).
6
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+ + CO32- ↔ 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
7
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
8
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).
9
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).
microbial urease
(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).
10
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).
11
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).
12
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). 13
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
14
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 + CO32−
(1.27)
Cell − Ca2++ CO32− → Cell − CaCO3
15
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, Sánchez-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)
16
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, DeMuynck 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. AlThawadi 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. 17
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).
18
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).
19
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).
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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−+ CO32−), 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 nonureolytic 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).
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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). 22
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).
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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).
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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. RodriguezNavarro 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).
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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 (SierraBeltran 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). 26
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).
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Biodeposition of bacterial calcite is a viable method of surface treatment for cementbased 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). 28
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).
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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.
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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 byproduct 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).
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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).
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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).
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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). Study by 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 34
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).
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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 (Figure 1.11) 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).
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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.
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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).
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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.
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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.
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Chapter
2 ISOLATION, IDENTIFICATION AND CHARACTERISATION OF UREASE-PRODUCING BACTERIA FROM LIMESTONE CAVES OF SARAWAK
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.
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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.
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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.
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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).
44
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).
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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.
46
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 µL); primers (1 µL, 20 µm); MyTaq Red Mix (25 µL) and sterile ddH2O (22 µL). 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 µL of Midori Green (Nippon Genetics Europe GmbH). The PCR product (5 µL) 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. 47
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).
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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.
49
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.
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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
