vii
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENT
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xiii
LIST OF ABBREVIATION
xv
LIST OF APPENDIX
xvii
INTRODUCTION
1
1.0
Background
1
1.1
Significance of the Work / Problem Statement
2
1.2
Objectives
3
1.3
Study scope
3
LITERATURE REVIEW
4
2.1
4
Heavy Metals Contamination: Global Scenario
2.1.1
Heavy Metals Present in Textile
Industry
5
2.1.2
Toxicity of Heavy Metals
5
2.1.3
Heavy Metal Contamination In Soil
6
viii
2.1.4
Contamination of Heavy Metals at
Aquatic Environment
7
2.2
Organic Compound Contamination
2.3
Sources
of
Heavy
Metals
9
and
Organic
Contaminants
2.4
11
Methods Involved In Removal of Heavy Metals
and Organic Compounds
14
2.4.1
Physical Treatments
14
2.4.1.1
Activated Carbon Adsorption
14
2.4.1.2
Membrane Filtration
15
2.4.2
Chemical Treatments
16
2.4.2.1
Chemical Precipitation
16
2.4.2.2
Ion Exchange
17
2.4.2.3
Chemical
Advanced
Oxidation
2.4.3
Biological Treatments
18
2.4.3.1
Phytoremediation
19
2.4.3.2
Biosorption
20
2.5
Biosorption of Heavy Metals Using Bacteria
21
2.6
Heavy Metal Tolerant Bacteria
22
2.7
The Applications of Heavy Metal Tolerant and
Organic Compounds Degrading Bacteria
3
17
23
MATERIALS AND METHODS
24
3.1
Design Of Work
24
3.2
Medium Preparation
26
3.2.1
Nutrient Broth
26
3.2.2
Nutrient Agar
26
3.2.3
Chemically Defined Medium
26
3.2.4
Heavy Metal Stock Solution Preparation
27
3.2.5
Bacterial Culture Preparation
28
3.2.6
Antimicrobial Reagent Preparation
28
3.3
Growth Profile
28
ix
3.4
Heavy Metals Tolerance Test
29
3.5
Carbon Utilization Experiment
29
3.6
16S Full Sequence PCR Identification
29
3.6.1
Genomic DNA Extraction
29
3.6.2
Gel Electrophoresis
31
3.6.3
Polymerase Chain Reaction
31
3.6.4
PCR Product Clean Up
32
3.6.5
The 16S rRNA Gene Sequencing
Analysis
3.7
4
32
Antimicrobial Reagent Test
33
RESULTS AND DISCUSSION
34
4.1
Growth Profiling
34
4.2
Heavy Metal Tolerance Test
36
4.2.1
Effect of Cobalt Sulfate (CoSO4.7H2O)
on the Growth of CA Strain
4.2.2
36
Effect of Zinc Sulfate (ZnSO4.7H2O) on
the Growth of CA Strain
4.2.3
38
Effect of Aluminium Potassium Sulfate
(AlK (SO4).12H2O) on the Growth of
CA Strain
4.2.4
Effect
40
of
Manganese
Chloride
(MnCl2.4H2O) on the Growth of CA
Strain
4.2.5
Effect of Copper Sulfate (CuSO4.5H2O)
on the Growth of CA Strain
4.3
42
45
Effect of Carbon Source on the Growth of
Bacterium CA
4.3.1
47
Proteinaceous Compounds as Carbon
and Nitrogen Source
52
4.4
Antimicrobial Reagent Test
53
4.5
The 16S rRNA Analysis
56
4.5.1
56
Genomic DNA Extraction
x
5
4.5.2
PCR
57
4.5.3
Analyse of Sequences
58
CONCLUSION AND RECOMMENDATIONS
61
5.1
Conclusion
61
5.2
Recommendations
63
REFERENCES
64
Appendices A-C
74-80
xi
LIST OF TABLES
TABLE NO
TITLE
PAGE
2.1
Source of heavy metals
11
2.2
Source of organic pollutants
13
3.2
Composition of chemically defined medium
27
4.1
The growth of bacterium CA in CDM with different
organic compounds used as carbon source and
casamino acids as carbon and nitrogen source.
48
xii
LIST OF FIGURES
FIGURE NO
3.1
TITLE
PAGE
Description of the steps carried out throughout the
work
4.1
25
Growth pattern of bacterium CA in nutrient broth
for ten hours at 37ºC
4.2
Growth
of
bacterium
35
CA
under
different
concentrations of cobalt sulfate, at 37ºC
4.3
36
The specific growth rate of bacterium CA at 0
mg/L, 20 mg/L, 40 mg/L and 60 mg/L of cobalt
sulfate
4.4
Growth
37
of
bacterium
CA
under
different
concentrations of zinc sulfate, at 37ºC
4.5
38
The specific growth rate of bacterium CA at 0
mg/L, 40 mg/L, 60 mg/L, 80 mg/L and 100 mg/L
of zinc sulfate
4.6
Growth
of
39
bacterium
CA
under
different
concentrations of aluminium potassium sulfate
40
xiii
4.7
The specific growth rate of bacterium CA at 0
mg/L, 100 mg/L, 200 mg/L, 300 mg/L and 400
mg/L of aluminium potassium sulfate
4.8
Growth
of
bacterium
CA
under
41
different
concentrations of manganese chloride
4.9
43
The specific growth rate of bacterium CA under 0
mg/L, 80 mg/L, 100 mg/L, 200 mg/L, 300 mg/L
and 400 mg/L of manganese chloride
4.10
Growth
of
bacterium
CA
under
43
different
concentrations of copper sulfate
4.11
465
The specific growth rate of bacterium CA under 0
mg/L, 60 mg/L, 80 mg/L, 100 mg/L, 200 mg/L and
300 mg/L of copper sulfate
4.12
45
The minimal inhibitory concentration (MIC) of
cobalt sulfate, zinc sulfate, aluminium potassium
sulfate, manganese chloride and copper sulfate on
the growth of bacterium CA
4.13
47
Growth of bacterium CA at CDM provided with
different organic compounds as sole carbon
sources
4.14
The specific growth rate values of bacterium CA
on casamino acids, glucose and glycerol
4.15
49
50
Growth of bacterium CA at CDM with casamino
acids as carbon and/or nitrogen sources
52
xiv
4.16
Antimicrobial agents’ effect on bacterium CA at
37ºC, 20 hours incubation
4.17
Clear zone prevention of growth of bacterium CA
formed by tetracycline hydrochloride
4.18
55
Extracted DNA band and 1kb ladder on agarose
gel
4.20
54
Clear zone prevention of growth of bacterium CA
formed by Kanamycin sulfate
4.19
54
57
Bands of 1kb DNA ladder and PCR product on the
agarose gel
58
4.21
Blastn sheet result of bacterium CA
61
4.22
Phylogenetic tree of bacterium CA done as
Neighbour-joining bootstrap method
60
xv
LIST OF ABBREVIATIONS
AH
-
Aliphatic Hydrocarbon
Bacterium CA -
An isolated bacterium from textile waste water
Cd
-
Cadmium
CDM
-
Chemically Defined Media
CO2
-
Carbon dioxide
Cr
-
Chromium
Cu
-
Copper
DDTs
-
Dichloro Diphenyl Trichloroethanes
DNA
-
Deoxyribonucleic acid
EDTA
-
Ethylenediaminetetraacetic acid
EMP
-
Embden–Meyerhof–Parnas
EOC
-
Emerging Organic Contaminant
GAC
-
Granular Activated Carbon
HBC
-
Hexachlorobenzene
Hg
-
Mercury
HMP
-
Hexose monophosphate
k
-
Specific growth rate
MCL
-
Maximum Contaminant Level
MEGA
-
Molecular Evolutionary Genetics Analysis
MIC
-
Minimal inhibitory concentration
BLASTn
-
nucleotide Basic Local Alignment Search Tool
NCBI
-
National Center for Biotechnology Information
OD
-
Optical Density
PAC
-
Powder Activated Carbon
PAH
-
Polycyclic Aromatic Hydrocarbon
Pb
-
Lead
xvi
PCB
-
Polychlorinated Biphenyl
PCR
-
Polymerase Chain Reaction
rRNA
-
Ribosomal Ribonucleic acid
STP
-
Sewage Treatment Plant
TAE
-
Tris-acetate-EDTA
td
-
Doubling time (Generation time)
USEPA
-
United States Environmental Protection Agency
UV
-
Ultraviolet
WHO
-
World Health Organization
Zn
-
Zinc
xvii
LIST OF APPENDICES
APPENDIX
TITLE
PAGE
A
Single colony of bacterium CA
74
B
Data gained from OD 600nm
75
C
Partial sequence of 16S rRNA of bacterium CA
80