vii
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xvi
LIST OF FIGURES
xviii
LIST OF ABBREVIATIONS
xxi
LIST OF APPENDICES
xxiii
INTRODUCTION
1
1.1
Background of Study
1
1.2
Statement of Problem
2
1.3
Objective
2
1.4
Scope of Study
2
1.5
Significance of Study
3
LITERATURE REVIEW
2.1
4
The Scenario of the Food and Agricultural
Sector in Malaysia
4
2.1.1
6
Pineapple Industry
2.1.1.1 Pineapple Processing
Industry
2.1.1.2 Pineapple Industry
8
viii
Wastewater
9
2.1.1.3 Treatment of Pineapple
Industry Wastewater
2.2
2.3
10
Chemical Oxygen Demand
10
2.2.1
Definition of COD
10
2.2.2
Principle of COD Analysis
11
2.2.3
Methods for COD Determination
12
2.2.4
COD and BOD
13
Methods of COD Reduction
14
2.3.1
Chemical Methods
14
2.3.1.1 Fenton’s Reagent
14
2.3.1.2 Chemical Coagulationflocculation
2.3.2
2.3.3
15
2.3.1.3 Ozonation
15
Physical Methods
15
2.3.2.1 Activated Carbon
16
2.3.2.2 Membrane Filtration
16
Biological Methods
18
2.3.3.1 Biological Treatment by
Bacteria
2.3.4
3
Combined Methods
19
20
2.4
Cell Immobilization
21
2.5
Agricultural Wastes as a Support Material
23
24
EXPERIMENTAL
3.1
Materials and Methods
3.2
Isolation, Characterization and Identification
24
of Efficient COD Reducing Bacteria from
Pineapple Industry Wastewater
3.2.1
Pineapple Industry Wastewater
3.2.2
Characterization of Pineapple
Industry Wastewater
3.2.2.1
pH
24
24
25
25
ix
3.2.2.2
Protein Test (Bradford
Method)
3.2.2.3
25
Total Carbohydrate
(Phenol-Sulphuric Acid
3.2.3
Method)
26
3.2.2.4
Total Nitrogen Test
26
3.2.2.5
Total Organic Carbon
27
3.2.2.6
Total Suspended Solids
27
Chemical Oxygen Demand Analysis
28
3.2.3.1
Materials
28
3.2.3.2
Reagents
28
3.2.3.3
Preparation of Standard
Potassium Dichromate
3.2.3.4
Preparation of Acid
Silver Sulphate
3.2.3.5
Bacteria
3.2.4.1
Starter Culture
3.2.7
30
Bacterial Growth on
Plates
Growth Medium
30
3.2.5.1
Nutrient Broth
30
3.2.5.2
Nutrient Agar
30
3.2.5.3
3.2.6
29
29
3.2.4.2
3.2.5
29
Preparation of Sample for
Analysis
3.2.4
29
30
Luria Broth (LB)
Glycerol
Isolation of Pure Bacterial Culture
31
3.2.6.1
31
Single Colony Isolation
Characterization of Isolated Bacteria
3.2.7.1
31
31
Identification of
Morphological Features
3.2.7.2
3.2.8
of Bacteria
32
Gram Staining
32
Identification of the Efficient COD
Reducing Bacteria using Batch
x
System
32
2.2.8.1
Preparation of Inoculum
2.2.8.2
Preparation of Bacterial
32
Culture for COD
Reduction
3.2.9
Growth of Bacteria
3.2.9.1
33
Growth Profile of Mixed
Bacterial Culture
3.2.9.3
33
Growth Profile of Single
Bacterial Culture
3.2.9.2
33
33
Growth Profile of
Bacteria in Pineapple
Industry Wastewater
3.2. 10 Bacterial Adaptation Studies
3.2.10.1
34
34
Screening for Bacterial
Growth Tolerance in
Pineapple Industry
Wastewater
3.2.10.2
34
Bacterial Survival in
Pineapple Industry
Wastewater
35
3.2. 11 Field Emission Scanning Electron
Microscopy (FESEM) Analysis
36
3.2.12
Identification of Microorganisms
36
3.2.13
COD Reduction of Pineapple
Industry Wastewater using Selected
Bacteria in Batch System
3.2.13.1
37
COD Reduction by
Single Bacteria using
Liquid Culture
3.2.13.1
37
COD Reduction by
Single Bacteria using
Washed Cell Pellet
37
xi
3.3
Chemical Oxygen Demand Reduction in
Pineapple Industry Wastewater using
Efficient COD Reducing Bacteria
Immobilized in Column System
3.3.1
Support Materials
3.3.1.1
38
Sugarcane Bagasse
(SCB)
3.3.2
38
Solid Pineapple Waste
(SPW)
3.3.1.3
38
Rubber Wood Husk
(RWH)
3.3.1.2
38
38
Characterization of Support
Materials
3.3.2.1
39
Brunauer-Emmet-Teller
(BET) Method
3.3.2.2
39
Moisture Content
Analysis
39
3.3.3
FTIR Spectroscopic Analysis
39
3.3.4
Packed-bed Column
40
3.3.4.1
Single Packed-bed
Column
3.3.4.2
Parallel Packed-bed
Columns
3.3.5
40
41
Column Study using Single and
Mixed Bacteria Immobilized in
Various Support Materials
42
3.3.5.1
Column Conditioning
43
3.35.2
Immobilization of Single
and Mixed Bacteria onto
3.3.6
Support Materials
43
3.3.5.3
Serial Dilution Technique
43
3.3.5.4
Dislodging Method
43
Preliminary Selection of
xii
Agricultural Waste Support
Materials for COD Reduction of
Pineapple Industry Wastewater
using Single Packed-bed Glass
Column
3.3.7
44
COD Reduction of Pineapple
Industry Wastewater using Selected
Support Material in Parallel Glass
Columns
3.3.7.1
44
Effect of Initial COD
Concentration of
Pineapple Industry
Wastewater
3.3.8
Reusability of Parallel Packed-bed
Glass Columns
3.3.9
45
FESEM Analysis of Biofilm in the
Sugarcane
3.4
44
45
Application of Parallel Glass Columns
System for Chromium and COD Removal in
ChromebacTM Effluent
46
3.4.1
Bacteria
46
3.4.2
Electroplating
Wastewater
46
3.4.3
Support Materials
46
3.4.4
Experimental Setup
46
3.4.5
Preparation of Bacterial
Inoculum
3.4.6
47
Immobilization of
Bacteria onto Support
Materials
3.4.7
3.4.8
47
Chromium and COD
Reduction System
47
Analytical Method
49
xiii
4
RESULTS AND DISCUSSION
4.1
50
Isolation, Characterization and Identification
of Efficient COD Reducing Bacteria from
Pineapple Industry Wastewater
4.1.1
Characteristics of Pineapple Industry
Wastewater
4.1.2
50
50
Characteristics of Microorganisms
Isolated from Pineapple Industry
Wastewater
4.1.2.1
51
Identification of
Microorganisms Isolated
from Pineapple Industry
4.1.2.2
4.1.3
Wastewater
51
Gram Staining
54
Identification of the Efficient COD
Reducing Bacteria using Batch
System
55
4.1.4
Identification of Microorganisms
56
4.1.5
Growth Profile
58
4.1.5.1
Growth Profile of Single
Cultures
4.1.5.2
Growth Profile of Mixed
Bacterial Culture
4.1.5.3
58
60
Growth Profile of
Bacteria in Pineapple
Industry Wastewater
4.1.6
Bacterial Adaptation Studies
4.1.6.1
61
63
Screening for Bacterial
Tolerance to Pineapple
Industry Wastewater
4.1.5.2
63
Bacterial Survival in
Pineapple Industry
Wastewater
64
xiv
4.1.7
4.1.8
Bacterial Surface of Locally Isolated
Bacteria
66
COD Reduction in Batch System
67
4.1.8.1
COD Reduction using
Single Bacterial Culture
4.1.8.2
COD Reduction using
Bacterial Pellet
4.1.9
67
68
Comparison of COD Reduction
Performance using Single Bacterial
Culture and Bacterial Pellet
4.2
69
Chemical Oxygen Demand Reduction in
Pineapple Industry Wastewater Using
Efficient COD Reducing Bacteria
Immobilized in Column System
70
4.2.1
Characteristics of Support Materials
70
4.2.2
FESEM and SEM Analysis on the
Surface of Support Materials
71
4.2.3
FTIR Spectroscopic Analysis
72
4.2.4
Bacterial Immobilization onto
Support Materials Studies
4.2.5
74
Preliminary Selection of
Agricultural Waste Support
Materials for COD Reduction of
Pineapple Industry Wastewater
using Single Packed-bed Glass
Column
4.2.6
78
COD Reduction of Pineapple
Industry Wastewater using Selected
Support Material in Parallel Glass
Columns
4.2.6.1
81
Effect of Initial COD
Concentration of
Pineapple Industry
xv
Wastewater
4.2.7
Reusability of Parallel Packed-bed
Glass Columns
4.2.8
81
84
FESEM Analysis on the
Development of Biofilm on the
Sugarcane Bagasse in Column
4.3
86
Application of Parallel Packed-bed Glass
Columns System for Chromium and COD
Removal in ChromeBacTM Effluent
4.3.1
Characteristics of Electroplating
Wastewater
4.3.2
89
90
Chromium Removal and COD
Reduction in Synthetic Cr(VI)
Containing Wastewater using
Parallel Packed-bed Glass Columns
90
4.3.3
Chromium Removal and COD
Reduction in Electroplating
Wastewater using Parallel Packedbed Glass Columns
5
96
CONCLUSION
99
5.1
Conclusion
99
5.2
Suggestions
101
REFERENCES
102
APPENDICES
116
xvi
LIST OF TABLES
TABLE NO.
2.1
TITLE
PAGE
Mean chemical composition of pineapple
cannery waste.
9
2.2
Membrane filters characteristics.
17
2.3
Commonly used biological treatment process for
domestic and industrial wastewaters.
2.4
Differences between immobilized cells and free
cells.
2.5
22
Characteristics and selection criteria of support
materials for cell immobilization.
3.1
18
23
The amount of nutrient broth medium and
pineapple industry wastewater used for the
bacterial adaptation studies.
35
4.1
Characteristics of pineapple industry wastewater.
51
4.2
Characteristic features of bacteria isolated from
pineapple industrial wastewater.
52
4.3
Gram Staining.
54
4.4
COD reduction by bacteria.
55
4.5
Identification of K. gibsonii by 16S rRNA gene
sequence analysis.
4.6
Identification of K. pneumonia by 16S rRNA
gene sequence analysis.
4.7
57
Identification of C. tropicalis by 18S rRNA gene
sequence analysis.
4.8
56
CFU of K. gibsonii, K. pneumoniae, and C.
58
xvii
tropicalis grown in different concentrations of
pineapple industry wastewater.
65
4.9
COD reduction using single bacterial culture.
67
4.10
COD reduction using single bacterial pellet.
68
4.11
Characteristics of rubber wood husk, sugarcane
bagasse and solid pineapple waste.
4.12
FTIR spectra of agricultural waste support
materials.
4.13
70
73
Effect of initial concentration of pineapple
industry wastewater (50%) on the COD
percentage reduction.
4.14
82
Effect of initial concentration of pineapple
industry wastewater (100%) on the COD
percentage reduction.
83
4.15
Characteristics of electroplating wastewater.
90
4.16
Characteristics of synthetic Cr(VI) containing
wastewater at different sampling ports of the
integrated biological system.
4.17
94
Characteristics of real electroplating wastewater
at different sampling ports of the integrated
biological and chemical system.
97
xviii
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
2.1
Pineapple production system.
7
2.2
Pineapple cannery process.
8
3.1
Collection of wastewater from the waste processing
section of the pineapple cannery industry; i –
sedimentation tank, ii – liquid waste collection tank.
3.2
25
Schematic representation of the experimental setup for the
single packed-bed column system.
40
Schematic representation of the experimental setup for the
parallel packed-bed columns system.
41
3.4
Packed-bed glass column.
42
3.5
Schematic diagram of the integrated biological system
removal of chromium and chemical oxygen demand in
Cr(VI)-containing wastewater.
3.3
4.1
4.2
Morphology of bacteria isolated from pineapple industry
wastewater.
48
53
Growth profile of K. gibsonii, K. pneumoniae, and C.
tropicalis.
59
4.3
Growth profile of mixed bacteria.
60
4.4
Growth profile of K. gibsonii, K. pneumoniae, and C.
tropicalis. in pineapple industry wastewater.
61
4.5
Colonies of K. gibsonii on nutrient agar plates during 24
hours growth phase a) 0 hour b) 4 hours c) 8 hours d) 12
hours e) 16 hours f) 24 hours.
62
xix
4.6
4.7
Bacterial growth in increasing concentrations of pineapple
industry wastewater.
63
FESEM micrographs of bacteria isolated from pineapple
industry wastewater: (i) K. gibsonii (500x) (ii) K.
pneumoniae (500x) (iii) C. tropicalis (500x).
66
4.8
COD reduction using single bacterial culture.
69
4.9
COD reduction using single bacterial pellet.
69
4.10
FESEM and SEM micrographs of a) raw wood husk –
magnification 1000X, b) solid pineapple waste – 1000X
and c) sugarcane bagasse – 150X.
4.11
Cell count of bacterial colony in bacterial culture before
and after immobilized onto support materials.
4.12
79
(b) COD reduction of pineapple industry wastewater using
bacteria immobilized onto solid pineapple waste.
4.13
77
(a) COD reduction of pineapple industry wastewater using
bacteria immobilized onto rubber wood husk.
4.13
76
Cell count of bacterial colony in bacterial culture attached
to support material after 24 hours.
4.13
72
79
(c) COD reduction of pineapple industry wastewater using
bacteria immobilized onto sugarcane bagasse.
80
4.14
COD reduction of pineapple industry wastewater.
85
4.15
FESEM micrographs of biofilm development on cellulose
support material. A – at 0 h (Magnification 150X), B – at
24 h contact with K. gibsonii (Magnification 500X), C –
after 3 days supplementation with NB (Magnification
500X), D – after 5 days exposure to the LPW
(Magnification 1000X) and E – after 30 days of column
start-up (Magnification 2000X).
4.16
87
Cr(VI) reduction profile by the biofilm system at different
batches of synthetic Cr(VI) containing wastewater ranging
from 100-200 mg/L.
4.17
Schematic diagram of the integrated biological system
removal of chromium and chemical oxygen demand in
91
xx
Cr(VI)-containing wastewater.
4.18
92
COD reduction performance in synthetic Cr(VI)
containing wastewater by immobilized K. gibsonii in
parallel glass column.
4.19
4.20
95
Cr(VI) reduction profile by the biofilm system at different
batches of real electroplating wastewater.
96
COD reduction performance in electroplating wastewater.
98
xxi
LIST OF ABBREVIATIONS
AgSO4
-
Silver Sulphate
APHA
-
American Public Health Association
BET
-
Brunauer-Emmet-Teller
BOD
-
Biochemical Oxygen Demand
BSA
-
Bovine Serum Albumin
CFU
-
Colony Forming Unit
C/N
-
Carbon per nitrogen ratio
COD
-
Chemical Oxygen Demand
Cr(VI)
-
Hexavalent Chromium
Cr(III)
-
Trivalent Chromium
ºC
-
Degree celcius
DPC
-
1,5-diphenylcarbazide
DW
-
Deionized water
EPS
-
Extracellular polymers
FAO
-
Food and Agriculture Organization
FESEM
-
Field Emission Scanning Electron Microscope
FTIR
-
Fourier Transform Infrared
g
-
Gram
g/L
-
Gram per liter
GDP
-
Gross Domestic Product
H2SO4
-
Sulphuric Acid
HgSO4
-
Mercuric Sulphate
i.d.
-
Internal Diameter
IC
-
Inorganic Carbon
IBC
-
Indigenous Bacteria Colony
IUPAC
-
International Union of Pure and Applied Chemistry
xxii
K2Cr2O7
-
Potassium Dichromate
kPa
-
kiloPascal
L
-
Liter
LB
-
Luria Broth
mg
-
Milligrams
mg/L
-
Milligram per liter
mL
-
Millilitres
mL/min
-
Millilitre per minute
NA
-
Nutrient Agar
NaOH
-
Sodium Hydroxide
NAP
-
National Agriculture Policy
NB
-
Nutrient Broth
NIRR
-
Near-Infrared Reflectance
NDIR
-
Nondispersive Infrared Detector
nm
-
Nanometer
o.d.
-
Outer Diameter
OD
-
Optical Density
rpm
-
Rotation per minute
RWH
-
Rubber Wood Husk
SEM
-
Scanning Electron Microscope
SS
-
Suspended Solids
SPW
-
Solid Pineapple Waste
SCB
-
Sugarcane Bagasse
TC
-
Total Carbon
TOC
-
Total Organic Carbon
TSS
-
Total Suspended Solid
UASB
-
Upflow Anaerobic Sludge Bed Bioreactor
v/v
-
Volume per volume
xxiii
LIST OF APPENDICES
APPENDIX
A
TITLE
PAGE
List of publication (journal/article),awards and seminar/
paper presentation during MSc study period between July
2009 to December 2011
B
116
Environmental Quality (Industrial Effluent) Regulation
2009
118