vi
TABLE OF CONTENTS
CHAPTER
TITLE
PAGE
SUPERVISOR’S APPROVAL
THESIS TITLE
i
DECLARATION
ii
ACKNOWLEDGEMENT
iii
ABSTRACT
iv
ABSTRAK
v
TABLE OF CONTENTS
vi
LIST OF TABLES
xii
LIST OF FIGURES
xv
LIST OF ABBREVIATIONS
xx
LIST OF APPENDICES
CHAPTER 1
xxiii
INTRODUCTION
1.1
Bioremediation of Textile Wastewater Using Biofilm
1
1.2
Objective of This Research and Thesis Outline
2
CHAPTER 2
2.1
LITERATURE REVIEW
Introduction to Biofilms
5
2.1.1 Overview of Biofilm Characteristics
5
2.1.2 Physical and Chemical Characteristics of Biofilms
6
vii
2.2
2.3
2.4
2.1.3 Extracellular Polymers Substances (EPS) in Biofilms
7
2.1.4 Bacterial Cell Interactions
8
Bacterial Biofilm Development Process in Textile Wastewater
11
2.2.1 Growth Kinetics of a Mixed Biofilm in Textile Wastewater
11
2.2.2 Net Biofilm Accumulation in Wastewater
12
16 S rRNA Analysis for Identification of Bacteria
15
2.3.1 Analysis of rRNA Molecules
15
2.3.2 16S rRNA Analyses for Gram-negative Bacteria
17
Bacterial Phylogenetic Relationship
18
2.4.1
Measurement of Bacterial Phylogenetic
18
Relationship
2.4.2
2.5
2.6
Meaningful Taxonomy in Bacteria Using rRNA
20
Originality of Colour in Textile Industry Wastewater
21
2.5.1 Basis of Colour Formation in Textile Wastewater
21
2.5.2 Classification of Most Commonly Used Dyes in Textile Industry
23
2.5.2.1
Reactive Dye
23
2.5.2.2
Azo Dyes
24
2.5.2.3
Metal Complex Dyes
26
Heavy Metals Removal from Wastewater
27
2.6.1 Mechanisms of Biosorption and Bioaccumulation in
27
Low Metal Contaminated Wastewater
2.6.2 The Role of Heavy Metals and Their Detoxification in
29
Bacterial Cells
2.7
Evidence of Biofilm Formation during Bioaccumulation of Heavy
35
Metals in Textile Wastewater
2.8
Biological Approach in Textile Wastewater Treatment
36
viii
CHAPTER 3
ISOLATION, SCREENING AND
PAGE
IDENTIFICATION OF BACTERIAL
FORMING BIOFILM
3.1
Introduction
38
3.2
Materials and Method
38
3.2.1 Samples of Textile Wastewater and Biofilm Collection
38
3.2.2 Isolation of Bacteria from Samples of Wastewater and Biofilm
39
3.2.3 Morphology and Biochemical Characterisation of Bacteria
40
3.2.4 Screening of Bacteria
40
3.2.4.1 Screening of Bacteria for Decolourisation
40
3.2.4.1 Screening for Heavy Metals Tolerance
41
3.2.5 Identification of Selected Bacteria Using 16S rRNA Analysis
42
3.2.5.1
Genomic DNA Isolation from Selected Bacteria
42
3.2.5.2
Agarose Gel Electrophoresis
42
3.2.5.3
Polymerase Chain Reaction (PCR) Procedure
43
3.2.5.4
Purification of PCR Product
44
3.2.5.5
Sequencing of the 16S rRNA Gene
45
3.2.5.6
Analyses 16S rRNA Gene and Phylogenetic Tree
45
Construction
3.3
Results and Discussion
45
3.3.1 Isolation of Bacteria from Wastewater and Biofilm Formed
45
in Textile Treatment Pond
3.3.2 Characterization of The Pure Isolates in Chemically Defined
49
Medium (CDM)
3.3.3 Screening Study on Colour Removal of Real Wastewater by
50
Pure Isolates
3.3.4 Screening Study on Decolourisation of Reactive Dye
54
3.3.5 Screening Study on Heavy Metals Tolerance by Selected Isolates 56
3.3.6 16S rRNA Identification of Selected Bacteria
64
3.3.6.1
DNA Extraction of Genomic Interest Isolates
64
3.3.6.2
Polymerase Chain Reaction (PCR)
66
3.3.6.3
Purification of PCR Product
67
3.3.6.4
Sequencing of the 16S rRNA Gene
68
ix
3.3.6.5
Bacterial Phylogeny Revealed by Sequence
69
Analyses of Gene Coding 16S rRNA
3.4
Concluding Remarks
CHAPTER 4
OPTIMIZATION AND KINETIC EVALUATION
PAGE
OF FSTW DECOLOURISATION USING
71
EXOGENOUS BIOFILM
4.1
Introduction
72
4.2
Materials and Method
73
4.2.1 Microorganisms
73
4.2.2 Medium Preparation
73
4.2.3 Support Matrix for Immobilization
74
4.2.4 Experimental Approach in Optimization and Kinetic Study
74
4.2.4.1 Experimental Design
74
4.2.4.2 Chemical Analysis on Colour Removal and
76
Kinetic Study on Batch Growth
4.3 Results and Discussion
77
4.3.1 Effect of pH
77
4.3.2 Effect of Inoculum Size
78
4.3.3 Effect of Temperature
79
4.3.4 Effect of Agitation
80
4.3.5 Effect of Dye Concentration
82
4.3.6 Kinetics Study on Colour Removal and Batch Grow Using
84
Exogenous Biofilm
4.3.6.1
Effect of Nitrogen Sources on Colour Removal
84
4.3.6.2
Effect of Nitrogen Concentration on Colour
86
Removal
4.3.6.3
Effect of Carbon Concentration on
89
Colour Removal
4.3.6.3
Effect of C/N Ratio towards
Decolourisation of FSTW
91
x
4.3.6.4
Kinetics Study of Batch Grow during
93
Decolourisation of FSTW Using Exogenous
Biofilm
4.3.6.5
Summary on Optimization and Kinetics Study
96
in Colour Removal
4.3.7
Microscopic Analysis on Biofilm Formation
100
Under Optimized Condition
4.4
Concluding Remarks on Optimal Decolourisation and
102
Kinetics Study of Exogenous Biofilm using FSTW
CHAPTER 5
BIOREMEDIATION OF TEXTILE
PAGE
WASTEWATER USING EXOGENOUS BIOFILM
5.1
Introduction
104
5.2
Materials and Methods
104
5.2.1 Source of Textile Wastewater
104
5.2.2 Water Quality Measurement in The Field
105
5.2.3 Laboratory Tests on Wastewater Parameters
105
5.2.3.1
Biological Oxygen Demand (BOD)
105
5.2.3.2
Chemical Oxygen Demand (COD)
107
5.2.3.3
Total Suspended Solid (TSS)
107
5.2.3.4
Other parameters
107
5.3
Results and Discussion
108
5.3.1 Textile Wastewater Characteristic
108
5.3.2 Bioremediation of Organic Content of Wastewater Using
109
Biofilm
5.3.3 The Efficiency of Exogenous Biofilm to Remove Total
111
Suspended Solids (TSS)
5.3.4 Biological Nutrient Removal : Sulphate, Nitrate, Phosphate
112
5.3.5 The Efficiency of Exogenous Biofilm to Neutralized pH
116
Of The Wastewater
xi
5.3.6 The Efficiency of Exogenous Biofilm to Remove Colour
117
5.3.7 The Efficiency of Biofilm Growth in the Presence of
118
Exogenus Isolates
5.4
Concluding Remarks
CHAPTER 6
CONCLUSION AND SUGGESTIONS
120
PAGE
6.1
Introduction
122
6.2
Conclusion
122
6.3
Suggestion for Future Work
125
REFERENCES
126
APPENDICES
140
xii
LIST OF TABLES
TABLE NO.
TITLE
PAGE
2.1
The relationship between colour absorbed and colour seen
22
2.2
Examples of plasmid-determined resistance to heavy metals
33
2.3
The efficiencies of current method of dyes and COD removal
36
from industrial effluents
3.1
Morphology of the pure colonies isolated from textile wastewater
46
3.2
Morphology of the pure colonies isolated from biofilm on polyurethane
47
(PU) sheets
3.3
Morphology of the pure colonies isolated from biofilm on
48
polyvinylchloride (PVC) ball
3.4
Single cell observation, Gram stained reaction and new code for pure
50
isolates
3.5
Decolourisation of pure reactive dye (SF Red) by isolates of interest
55
4.1
Decolourisation rate at the optimum pH (% h-1)
78
4.2
Effect of inoculum on maximum colour removal and decolourisation rate 78
(% h-1) by pure and mixed isolates
xiii
4.3
Efficiencies of decolourisation by pure and mixed culture at optimized
82
condition, 100 rpm agitation within 18 h incubation
4.4(a) Effect of dye concentration on decolourisation rate (% h-1) by
83
pure and mixed isolates within 18 h incubation
4.4(b) Effect of dye concentration on maximum decolourisation (%) by
83
pure and mixed isolates within 18 h incubation
4.5
Effect of different nitrogen contents on the maximum decolourisation (%) 85
and the decolourisation rate (% h-1) by pure and mixed culture biofilm
4.6
Effect of different NH4Cl concentrations on the maximum
87
decolourisation (%) and the rate of decolourisation (%h-1) by pure
and mixed culture biofilm
4.7
Effect of different glycerol concentrations on the maximum
90
-1
decolourisation (%) and the decolourisation rate (% h ) by pure and
mixed culture biofilm
4.8
Effect of C/N ratio towards decolourisation rate (% h-1) and maximum
92
decolourisation of FSTW by pure and mixed culture
4.9
Value of constant factor to convert µ of suspended cells as µ of biofilm 94
4.10
Kinetics of growth and decolourisation of FSTW by pure and mix culture 95
4.11
Optimized condition for complete decolourisation (~99.5%) of FSTW
97
5.1
Wastewater parameters tested, program used and wavelength readings
108
5.2
Textile wastewater analysis (Effluent)
108
5.3
Comparison of specific growth rate between suspension and biofilm
118
xiv
5.4
Improved textile wastewater quality after treatment using mixed culture 121
of biofilm system
xv
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
Research Outline
4
2.1
Different growth phases of M(a): bacterial cell; and M(b): biofilm
11
2.2
Fundamental processes governing biofilm formation in wastewater
12
2.3
Possible cell cycle of cells growing on xenobiotics around surfaces
14
in a continuos system
2.4
Gene amplification of 16 S rRNA
17
2.5
The forward primer (pA) and reverse primer (pH’) were used by
18
the isolates of interest (Cb01, Cb02 and Cb15).
2.6
Unrooted tree for four species, A, B, C, and D illustrating the
19
relationship that must hold among six evolutionary distances.
2.7
Proposed structure of Remazol Red
26
2.8 (a) The chemical structure of Cr complex-mono azo in Acid Red 183
27
2.8 (b) The chemical structure of Cu complex-monoazo-vinylsulfone in
27
Remazol Brilliant Violet 5R
xvi
2.9
The active site of oxytyrosinase. N = nitrogen donor of
30
histidyl residues
3.1
3.2
Spectrum of filter sterilized textile wastewater (FSTW) and pure dye
used for screening
51
Screening study on colour removal by 18 pure isolates
51
3.3(a) Pure isolates of Cb01 on FSTW agar
53
3.3(b) Pure isolates of Cb02 on FSTW agar
53
3.3(c) Pure isolates of Cb15 on FSTW agar
53
3.4
3.5
Screening of exogenous culture towards pure dye, SF red 3BS after
7 days incubation.
55
Decolourisation by pure isolates towards reactive dye after 7 days
56
Incubation
3.6(a) Tolerance level of isolated bacteria towards Cu2+ in FSTW
57
3.6(b) Tolerance level of isolated bacteria towards Cr3+ in FSTW
57
3.7
Biofilm formation during copper removal after 84 h of incubation at
10 000x magnification.
3.8
58
Biofilm formation during chromium removal after 84 h of incubation at
10 000x magnification.
59
3.9(a) Micrograph of exogenous biofilm (control) without metals added after
8 h incubation; magnification 500x
3.9(b) EDX spectrum shows the elemental composition measured on the
PU cubes grown at 37°C
60
61
xvii
3.10(a) Micrograph of exogenous biofilm with 200 ppm of Cu (II) after
62
8 h incubation at magnification 500x
3.10(b) EDX spectrum shows the elemental composition measured on the
62
PU cubes grown at 37°C, exposed to Cu (II), 200 ppm
3.11(a) Micrograph of exogenous biofilm with 400 ppm of Cr (III) after
62
8 h incubation; magnification 500x
3.11(b) EDX spectrum shows the elemental composition measured on the
62
PU cubes grown at 37°C, exposed to Cr (III), 400 ppm
3.12
Agarose gel electrophoresis of genomic DNA extracted from selected 65
bacteria Cb01, Cb02, Cb10 and Cb15
3.13 Agarose gel electrophoresis analysis of the 16S rRNA gene fragment
3.14
amplified using universal primers
66
Agarose gel electrophoresis of purified fragments of amplified
68
16S rRNA gene from the genomic DNA of selected isolates
(Cb01, Cb02 and Cb15)
3.15
Dendogram showed phylogenetic relationship of bacteria Cb01, Cb02 70
and Cb15 with other known bacteria based on their 16 S rRNA genes
4.1
Flow chart of experiments on optimization and kinetic evaluation of
75
FSTW decolourisation using exogenous biofilm in detailed
4.2
Effect of pH on decolourisation of FSTW
77
4.3
Effect of temperature on decolourisation of FSTW
79
xviii
4.4
Effect of agitation speed towards decolourisation of FSTW by pure
81
and mixed isolates within 18 h incubation according to
decolourisation rate (% h-1) and decolourisation max (%)
4.5(a) Micrograph of Control after 24 h incubation at
98
low magnification (50x)
4.5(b) Micrograph of biofilm after 8 h incubation at
98
low magnification (50x)
4.5(a) Micrograph of biofilm after 24 h incubation
99
at low magnification (50x)
4.6
Micrograph of biofilm after a month incubation in FSTW at high
100
magnification (12 000x)
4.7
Micrograph of exogenous biofilm development versus time in FSTW 101
under optimized condition
5.1
Schematic diagram of flow chart for wastewater treatment plant
106
at textile factory located in Batu Pahat, Johore
5.2
BOD5 tested during growth of exogenous biofilm within 48 h
110
of incubation in real wastewater
5.3
COD tested during growth of exogenous biofilm within 48 h of
110
incubation in real wastewater
5.4
TSS profile during growth of exogenous biofilm within 48 h of
111
incubation in real wastewater.
5.5(a) Profile of nitrate residual during growth of exogenous biofilm
within 48 h of incubation in real wastewater
113
xix
5.5(b) Profile of sulphate residual during growth of exogenous biofilm
114
within 48 h of incubation in real effluent
5.5(c) Profile of phosphate residual during growth of exogenous biofilm
115
within 48 h of incubation in real wastewater
5.6
Profile of pH changes during growth of exogenous biofilm within
116
48 h of incubation in real wastewater
5.7
Profile of colour removal during growth of exogenous biofilm
117
within 48 h of incubation in real wastewater
5.8
Progression of both liquid phase (suspended cells) and surface
parameters (attached biofilm) during biofilm formation
(pure and mixed culture) in shake flasks within 48 h of incubation.
119
xx
LIST OF ABREVIATIONS
ADMI
-
American Dye Manufacturers Institute
Al
-
alumium
APHA
-
American Public Health Association
AOX
-
adsorbable organic halogens
BOD
-
biological oxygen demand
CaCl2
-
calcium chloride
CDM
-
chemically defined medium
C
-
carbon
Cd
-
cadmium
Cl
-
chloride
C/N ratio
-
ratio of carbon concentration towards nitrogen
COD
-
chemical oxygen demand
Co
-
cobalt
Cr
-
chromium
Cu
-
copper
Cr (III)
-
chromium (III)
Cr (VI)
-
chromium (VI)
Cr(NO3)3.9H2O
-
chromium nitrate nano hydrate
Cu(NO3)3. 2 ½ H2O
-
copper nitrate
-
-
chromates
2-
-
dichromates
Cr(OH)3
-
chromium hydroxide
d-1
-
per day
DF
-
dilution factor
dNTP
-
deoxynucleotide triphosphate
dATP
-
deoxyadenosine 5’-triphosphate
dTTP
-
deoxythymidine 5’-triphosphate
dGTP
-
deoxyguanosine 5’ triphosphate
dCTP
-
deoxycytosine 5’ triphosphate
CrO42
CrO7
xxi
DO
-
dissolve oxygen
DOE
-
Department of Environment
DNA
-
deoxyribonucleic acid
Dye (i)
-
initial concentration of dye
Dye (r)
-
residual dye
EDX
-
energy dispersive X-ray
EPS
-
exopolymeric substances
ESEM
-
environmental scanning electron microscope
FeCl3
-
ferric chloride
fstw
-
filter sterilized textile wastewater
FAS (FeNH4.SO4)
-
ferrous ammonium sulphate
g/g
-
gram per gram
i/i
-
isipadu per isipadu
h-1
-
per hour / per jam
H+
-
hydrogen ion
His
-
histidine (amino acid)
H2 S
-
hydrogen sulphide
-
-
sulphite
H2SO4
-
acid sulphuric
HNO3
-
acid nitric
HRT
-
hydraulic retention time
IR
-
infrared
j-1
-
per jam
K
-
potassium
K2HPO4
-
dipotassium hydrogen phosphate
KH2PO4
-
potassium dihydrogen phosphate
K2CrO4
-
potassium dichromate
λ
-
wavelength
µ
-
specific growth rate
MIC
-
minimal inhibitory concentration
MgCl2
-
magnesium chloride
MgSO4.7H2O
-
magnesium sulphate heptahydrate
mg
-
milligram
mL
-
milliliter
HSO3
xxii
mm
-
milimeter
MWr
-
molecular weight relative
N2
-
nitrogen gas
Na
-
natrium
NaCl
-
sodium chloride
NB
-
nutrient broth
nfstw
-
non-filter sterilized textile wastewater / real wastewater
NH4Cl
-
ammonium chloride
NH4NO3
-
ammonium nitrate
(NH4)2SO4
-
ammonium sulphate
Ni
-
nikel
NO3-
-
nitrate
O2
-
oxygen gas
O
-
oxygen
Pb
-
plumbum
PCR
-
polymerase chain reaction
PHB
-
polyhydroxybutyrate
ppm
-
part per million
PO43-
-
phosphate
Pt-Co
-
platinum cobalt
PVC
-
polyvinylchloride
rpm
-
rotation per minute
rRNA
-
ribosomal RNA
S
-
sulphur
2-
-
sulphate
SEM
-
scanning electron microscope
TAE
-
Tris-amino-ethane
TSS
-
total suspended solid
UV
-
ultraviolet
VPSEM
-
variable pressure scanning electron microscope
v/v
-
volume per volume
w/v
-
weight per volume
Zn
-
zink
SO4
xxiii
LIST OF APPENDICES
APPENDIX
A
TITLE
The Basis of Dye Selection Prior to Screening
PAGE
140
Study on Colour Removal of Most commonly
Used Dyes by the Textile Factory
B
Sequences of The 16S rRNA gene Fragments of
146
Bacteria Cb01, Cb02 AND Cb15
C
Result on BLAST Alignment for Selected
150
Isolates (Cb01, Cb02 & Cb15)
D
C/N Ratio Calculation
160
E
Decolourisation Phenomenon
167
F
Calculation of Yield
169
G
Calculation of COD
170
H
Calculation of TSS Removal
171
I
List of Publication
172