2
1
A2.5cm
TABLE OF CONTENTS
TITLE CHAPTER
DECLARATION
DEDICATION
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF SYMBOLS
LIST OF APPENDICES
INTRODUCTION
1.1 Introduction
1.2 Rationale of Investigation
1.3 Objectives
LITERATURE REVIEWS
2.1 Xenobiotics: An Overview
2.2 Persistent Organic Pollutants (POPs) and Its Health
Effects
2.3 Persistent Organic Pollutants (POPs) Pesticide in
Malaysia
2.4 Bioaugmentation as a Soil Bioremediation
Approach
2.5 Properties of 2,2-Dichloropropionic Acid (2,2-DCP)
2.6 Chemistry of Halogenated Compound
PAGE
5
1
4 xvi xvii
1 v vi x xiii ii iii iv
6
6
11
14
18
20
22 vi

3
2.7 Microbial Dehalogenation
2.8 Dehalogenation Mechanism
2.9 Dehalogenation of Halogenated Alkanoic Acid
2.10 Classification of 2-Haloalkanoic Acid Hydrolytic
Dehalogenases
2.11 Biochemistry of 2-Haloalkanoic Acid Hydrolytic
Dehalogenases
2.12 Genetics of Haloalkanoic Acid Dehalogenase
MATERIALS AND METHODOLOGY
3.1 Culturing Media Composition
3.1.1 Minimal Media
3.1.2 LB (Lysogeny Broth)
3.1.3 Glycerol Stock Culture
3.2 Bacterial Isolation and Purification
3.2.1 Isolation and Purification of Soil Bacteria
3.2.2 Isolation and Purification of Endophytic
Bacteria from Leaves
3.3 Measurement of Microbial Growth
3.4 Halide Ion Assay (HIA)
3.4.1 HIA Reagent
3.4.2 HIA Standard Curve and Sample Testing
3.5. Cell Free Extract Preparation
3.6 Protein Concentration Determination
3.7 Enzyme Activity Assay
3.8 SDS-PAGE
3.8.1 Chemicals and Preparation
3.8.2 SDS-PAGE Apparatus Assembling, Gel
Loading and Sample Loading
3.8.3 Gel Staining, Destain and Drying
3.9 Molecular Analysis
3.9.1 DNA Extraction
3.9.2 Measurement of DNA Concentration
38
45
47
47
48
41
45
45
48
48
25
28
31
33
49
49
53
54
54
57
49
50
51
52
59
60
60
61 vii

4
5 viii
3.9.3 Gel Electrophoresis
3.9.4 Polymerase Chain Reaction (PCR)
Amplification of 16S rRNA Gene
3.9.5 Dehalogenase Gene PCR Amplification
3.9.6 Phylogenetic Analysis
3.10 Biochemical Characterization
3.11 Cell Fixation for Scanning Electron Microscopy
ISOLATION AND CHARACTERIZATION OF
LABRYS SP. STRAIN WY1 ABLE TO UTILIZE 2,2-
DICHLOROPROPIONATE (2,2-DCP) AS SOLE
SOURCE OF CARBON
4.1 Introduction
4.2 Results
4.2.1 Isolation and Characterization of 2,2-DCP
Degrading Bacteria
4.2.2 Growth Profile
4.2.3 Halide Ion Assay
4.2.4 PCR Amplification of 16S rRNA gene
4.2.5 Sequencing and Analysis of 16S rRNA
Gene
4.2.6 Phylogenetic Study
4.2.7 Biochemical Tests
4.2.8 Amplification of Dehalogenase Gene
4.3 Discussion
ISOLATION AND CHARACTERIZATION OF
ENDOPHYTE FROM AXONOPUS COMPRESSUS
(RUMPUT PARIT) CAPABLE OF UTILIZE 2,2-DCP
AS SOLE CARBON SOURCE
5.1 Introduction
5.2 Results
5.2.1 Bacteria Characterization
86
87
87
61
62
64
66
66
67
68
71
73
74
75
68
69
69
79
81
82
83
86

6
5.2.2 Scanning Electron Microscopy (SEM)
5.2.3 Growth Profile
5.2.4 Halide Ion Assay
5.2.5 PCR Amplification of 16S rRNA Gene
5.2.6 Sequencing and Analysis of 16S rRNA
Gene
5.2.7 Phylogenetic Study
5.2.8 Biochemical Test (API®)
5.2.9 Cell Free Extract (CFE) Analysis
5.2.9.1 CFE Enzyme Activity in Different
Buffers
5.2.9.2 CFE Enzyme Activity in Different
pH and Temperature
5.2.9.3 CFE Enzyme Activity towards
Different Substrate
5.2.9.4 SDS-PAGE
5.2.10 Amplification of Dehalogenase Gene
5.2.10.1 Gel Electrophoresis of PCR Product
5.2.10.2 Sequencing and Analysis of
Dehalogenase Gene
5.2.11 Amplification of Complete deh-wy5 (
D,L
dex -alike) Gene
5.2.11.1 Primers Design based on
D,L
-dex
Gene
5.2.11.2 Gel Electrophoresis of PCR product
5.2.11.3 Sequencing and Analysis of deh-wy5 (
D,L
-dex -alike) Gene
5.3 Discussion
CONCLUSION
6.1 General Conclusion
REFERENCES
Appendix
96
98
100
100
101
102
103
104
104
105
108
108
111
112
116
121
121
123
136
89
91
92
93
94 ix

A2.5cm
LIST OF TABLES
TITLE TABLE
2.1
2.2
2.3
2.4
Estimated annual industrial production of chlorinated hydrocarbons and major applications (Fetzner, 1998)
Some endocrine disrupting effects of pops and selected other chemicals
Part of registered pesticide from October 2005 to
December 2011 listed by Ministry of Agriculture and
Agro-Based Industry Malaysia
Effect of number of chlorine on halogenated organic acid to the pKa value
2.5
2.6
2.7
2.8
3.1
3.2
3.3
Effect of type of halogen on halogenated organic acid to the pKa value
Effect of chlorine position on halogenated butanoic acid to the pKa value
Haloalkanoate dehalogenases produced by miroorganisms and their substrate specificity
Further characterization of 2-haloalkanoic acid hydrolytic dehalogenase into discrete Class according to their substrate specificity and product configuration (Slater et al., 1995). Group categorizing system according to
Weightman et al. (1982) and Hardman (1991) were also included.
Minimal media components
Example – Composition of minimal media
Preparation of chloride standard solution diluted with
PAGE
7~9
13
15
23
24
24
35
36~37
46
46
50 x

5.1
5.2
5.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
3.12
3.13
3.14
3.15
4.1
4.2
4.3
4.4
4.5 minimal media
Standard solutions of BSA 52
Testing media contained 1 mM 2,2-DCP (1 unit)
Chemicals required for SDS-PAGE
Seperating gel preparation
Stacking gel preparation
Primers designed by Weisburg et al . (1991). Only fD1 and rP1 were used in current investigation (Weisburg et al .,
1991)
PCR method for amplification of 16S rRNA gene 63
Touchdown PCR method for “Group I” dehalogenase gene 64
53
54
58
58
63
PCR method for “Group II” dehalogenase gene
“Group I” deh primer sequences, showing comparisons of conserved binding sites from various sources (Hill et al .,
64
65
1999)
“Group II” deh primer sequences, showing comparisons of conserved binding sites from various sources (Hill et al .,
1999)
General fixation schedule for animal cell
Bacterial colony morphology of isolate Wy1 found on 10
65
67
70 mM 2,2-DCP minimal media
Gram stain characteristics of bacteria Wy1
Summary of growth properties of bacteria Wy1 in different concentration of 2,2-DCP
Sequences producing significant alignments with Wy1 in descending order (BLASTn)
Comparison of biochemical test result of Wy1 with related species
Bacterial colony morphology of isolate Wy5 found on 10 mM 2,2-DCP minimal media
Gram stain characteristics of isolate Wy5
Summary of growth properties of isolate Wy5 in different concentration of 2,2-DCP
70
72
76
81
87
88
91 xi

xii
5.4
5.5
5.6
5.7
5.8
5.9
5.10
5.11
5.12
5.13
Summary of chloride ion released by isolate Wy5
List of sequences producing significant alignments with
Wy5 from BLASTn sorted by their “maximum identity” in descending order
API® 20NE test result of isolate Wy5
Summary of CFE dehalogenase activity tested in different buffers. Calculation and method as reported by Ng (2007)
92
95
99
100
CFE dehalogenase activity towards different substrate 102
Dehalogenase genes aligned with partial deh-wy5 sequence 105
Designed primers generated by Primer3
PCR method of designed primers
110
110
Amino acid composition of pH and temperature deh-wy5
Comparison of non-stereospecific dehalogenases’ optima
115
120

A2.5cm
LIST OF FIGURES
TITLE FIGURE
2.1
2.2
2.3
Structure of 2,2-dichloropropionic acid (2,2-DCP)
Dehalogenation mechanisms (Fetzner and Lingens, 1994)
Basic mechanism of hydrolytic dehalogenation (Slater et
2.4
2.5
2.6
2.7
3.1
3.2
3.3
4.1
4.2
4.3 al.
, 1996)
2-haloalkanoic acid halidohydrolase dehalogenation mechanism 1 resulting in inversion of product configuration from
L
-isomer to
D
-isomer (Little and
Williams, 1971)
2-haloalkanoic acid halidohydrolase dehalogenation mechanism 2 resulting in retention of product configuration (Weightman et al., 1982)
Sequence alignment of
L
-haloacid dehalogenases by
Janssen et al.
(1994)
Part of alignment of deduced amino acid sequences of the
Group I deh proteins by Hill et al.
(1999)
SDS-PAGE - stock solutions
SDS-PAGE - working solutions
SDS-PAGE - sample buffers
Bacterial colonies on minimal media (Photo taken after 5 days of incubation)
Gram stained bacteria Wy1 under microscope (1000X)
Growth profile of strain Wy1 in triplicate of minimal media contained four different concentration of 2,2-DCP
PAGE
20
30
32
38
40
42
44
55
56
57
69
70
71 xiii

xiv
4.5
4.6
4.7
4.8
4.4
4.9
4.10
4.11
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
5.10
5.11
Correlation between chloride ion (mM) released in minimal medium containing 30 mM 2,2-DCP and growth profile recorded at A
600nm
Gel electrophoresis of PCR product - 16S rRNA gene fragment of isolate Wy1
Partial 16S rRNA gene sequence of isolate Wy1
Alignment between Wy1 sequence (Query) with Labrys neptuniae strain Liujia-146 16s rRNA sequence (Sbjct)
Information sheet of Labrys sp. Wy1 16S rRNA partial gene sequence from NCBI database
(http://www.ncbi.nlm.nih.gov/nuccore/jf907580)
Phylogeny analysis using MEGA5
Neighbour-Joining phylogeny tree of Labrys sp. Wy1
Amplification of dehalogenase gene from Wy1
Gram staining reveals isolate Wy5 was Gram negative
SEM micrograph of isolate Wy5 visualized at 10,000X,
10kV accelerating voltage
SEM micrograph of isolate Wy5 showing bacterial binary fission
Growth profile of isolate Wy5 in triplicate of minimal media contained 10 mM, 20 mM and 40 mM of 2,2-DCP
Chloride ion released ([Cl
-
]) of isolate Wy5 in triplicate of three different concentration of 2,2-DCP
Gel electrophoresis of PCR product - 16S rRNA gene fragment of isolate Wy5
Partial 16S rRNA gene sequence of isolate Wy5
Neighbour-Joining phylogeny tree of isolate Wy5
Information sheet of API® test profile ID: 1047577 from
API-WEB
API® 20NE test strip of isolate Wy5 after 48 hours of incubation at 30 o
C
Enzyme specific activity from pH5~pH10, 35 o
C; Buffer:
0.1 M Tris-acetate, 1 mM EDTA, 10% (w/v) glycerol
73
74
75
77
78
90
91
92
93
99
101
79
80
82
88
89
94
97
98

5.13
5.14
5.15
5.16
5.12
5.17
5.18
5.19
5.20
5.21 xv
Enzyme specific activity from 25 o
C~50 o
C, pH 7.2; Buffer:
0.1 M Tris-acetate, 1 mM EDTA, 10% (w/v) glycerol
SDS-PAGE of Wy5 CFE
PCR product of “Group I” dehalogenase
ClustalW alignments of 495 bp deh-wy5 with known complete dehalogenase genes (partly shown)
Alignment of deh-wy5 with other dehalogenases displayed in codon form.
Data sheet generated by Primer3
PCR products of deh-wy5 ( DL-DEX -alike) genes
Partial DNA fragment amplified using designed primers
Dxf1 and Dxr1
Chromatogram of sequenced DNA fragment using designed primers Dxf1 and Dxr1
Alignments between extended sequences amplified using primers Dxf1 + Dxr1 and
D,L
-dex displayed in codon form using ClustalW (version 1.6)
101
103
104
106
107
109
111
112
113
114

A2.5cm
LIST OF SYMBOLS
(v/v) - Volume percentage per 100mL volume
(w/v) - Mass percentage per 100mL volume
2,4,5-T - 2,4,5-Trichlorophenoxyacetic acid
2,4-D - 2,4-Dichlorophenoxyacetic acid
A
…nm
- Absorption spectroscopy at …nm light source
BLASTn - Basic local alignment search tool – nucleotide bp - Base pairs
CFE - Cell free extract
DDT - Dichlorodiphenyltrichloroethane dH
2
O - Distilled water
HCH - Hexachlorocyclohexane
HIA - Halide ion assay kb - Kilo bases
MW r
- Relative molecular weight
OD - Optical density
PCB - Polychlorinated biphenyls
PCR - Polymerase chain reaction rpm - Revolution per minute
RT - Room temperature
V - Volts xvi

A2.5cm
LIST OF APPENDICES
TITLE APPENDIX
A
B
C
D
E
F
G
Standard Graphs
Growth Profile and Doubling Time Calculation of Labrys sp. Wy1
Growth Profile and Doubling Time Calculation of
Burkholderia cepacia Wy5
HIA of Labrys sp. Wy1 in 20 mM 2,2-DCP
HIA of Burkholderia cepacia Wy5
Enzyme Assays of Burkholderia cepacia Wy5 CFE
Acid Dilution Calculations
PAGE
136
138
140
141
142
143
145 xvii