x
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
vi
ABSTRAK
vii
TABLE OF CONTENTS
viii
LIST OF TABLES
xiii
LIST OF FIGURES
xvi
LIST OF ABBREVIATIONS
xxvi
LIST OF SYMBOLS
xxix
LIST OF APPENDICES
xxxi
INTRODUCTION
1
1.1
Background of Research
1
1.2
Objectives of the Study
4
1.3
Scope of the Study
5
1.4
Significance of the Study
6
1.5
Thesis Organization
8
LITERATURE REVIEW
9
2.1
Introduction
9
2.2
Palm Oil
10
xi
2.3
POME (Palm Oil Mill Effluent)
19
2.4
POME Management
22
2.4.1
Pond System
23
2.4.2
Anaerobic Digestion of POME
24
2.4.3
Aerobic Treatment of POME
29
2.5
Granulation Technology
32
2.5.1
Sequencing Batch Reactor
34
2.5.2
Definition of Aerobic Granulation
39
2.5.3
Factors Affecting the Formation of
Aerobic Granular sludge
2.5.3.1
Influence of Feast and Famine
Regimes
2.5.3.2
2.5.4
42
Aggregate Selection by
Settling Times
43
2.5.3.3
Hydrodynamic Shear Force
45
2.5.3.4
Substrate Composition
46
2.5.3.5
Organic Loading Rate
48
2.5.3.6
Aerobic Starvation
49
2.5.3.7
Dissolved Oxygen (DO)
50
2.5.3.8
Reactor Configurations
51
Characteristics of Aerobic Granular
Sludge
2.5.5
41
52
2.5.4.1
Morphology
52
2.5.4.2
Settleability
54
2.5.4.3
Density and Strength
55
2.5.4.4
Fractal Dimension
57
2.5.4.5
Microbial Structure
58
2.5.4.6
Microbial Diversity
59
Applications of Aerobic Granulation
Technology
2.5.5.1
62
Biological Treatment of
Wastewater
62
xii
2.5.5.2
Nitrogen and Phosphorus
Removal
3
65
METHODOLOGY
68
3.1
Introduction
68
3.1.1
Research Design and Procedure
69
3.1.2
The Framework and Outline of Study
70
3.2
Experimental Setup
74
3.3
Raw POME and Seed Sludge Sampling
77
3.4
Reactor Maintenance
80
3.5
General Analytical Procedures
81
3.6
Molecular Analyses
83
3.6.1
DNA Extraction
83
3.6.2
Polymerase Chain Reaction (PCR)
84
3.6.3
Gel Electrophoresis
85
3.6.4
Denaturing Gradient Gel Electrophoresis
(DGGE)
3.6.5
87
Basic Local Alignment Search Tool –
nucleotide (BLASTn) and Sequencing
3.6.6
Multivariate Statistical Analysis of
DGGE Gel
3.7
88
89
Fluorescence in situ Hybridisation Technique
(FISH)
91
3.7.1
Sampling and Fixation
91
3.7.2
Microscopes Slide
92
3.7.3
FISH on Slides Method
93
3.7.4
Microscopy and AOB Detection
94
3.8
Fractal Dimension Analysis
95
3.9
Morphology Characterisation of Aerobic
Granular Sludge Cultivated Using POME
3.9.1
Microscopic Examination using
PAX-IT®
3.9.2
98
Field
Emission
99
Scanning
Microscopy Analysis (FESEM)
Electron
99
xiii
3.10
4
5
Summary
FORMATION OF AEROBIC GRANULAR SLUDGE
WITH POME
100
4.1
Introduction
100
4.2
Preliminary Experimental Results
101
4.3
Dynamics of SBR
111
4.4
Development of Aerobic Granular Sludge
114
4.5
Biomass Profile and Settling Properties
126
4.6
Reactor Performance and Removal Efficiencies
129
4.7
Summary
133
EFFECT OF VOLUMETRIC ORGANIC LOADING
ON AEROBIC GRANULATION
134
5.1
Introduction
134
5.2
Formation of Aerobic Granular Sludge using
POME at Different OLR
5.3
6
99
136
Size and Fractal Dimension of Aerobic Granular
Sludge
144
5.4
Organic Shock Loading Condition
150
5.5
Aerobic Granular Sludge for POME Treatment
152
5.6
Summary
159
MICROBIAL CHARACTERIZATION OF AEROBIC
GRANULAR SLUDGE
160
6.1
Introduction
160
6.2
Identification
162
6.2.1
PCR Amplification of 16S rRNA Gene
162
6.2.2
PCR Products Analyzed by Denaturing
Gradient Gel Electrophoresis (DGGE)
6.2.3
166
AOB Community in Aerobic Granular
Sludge Cultivated using POME
172
xiv
6.2.4
The Distribution of AOB in Granule by
FISH
6.3
6.4
7
178
Microbial Population Dynamics of Aerobic
Granular Sludge
180
6.3.1
188
Similarity Index
Summary
190
CONCLUSIONS AND RECOMMENDATIONS
191
7.1
Conclusions
191
7.2
Recommendations
193
REFERENCES
195
xv
LIST OF TABLES
TABLE NO.
TITLE
2.1
Six different types of palm oil biomass (Source:
Agensi Inovasi Malaysia, 2011)
2.2
28
Examples of POME effluent qualities from
anaerobic treatment studies
2.5
20
The performance of various experimental studies
for POME treatment
2.4
18
Characteristics of POME and ASM estimated
model parameters
2.3
PAGE
29
Description of the operational steps for the
sequencing batch reactor (Adapted from Metcalf
and Eddy, 2003)
2.6
36
The governing factors and characteristics of aerobic
granular sludge for various studies on aerobic
granulation for wastewater treatments
2.7
61
Aerobic granular sludge studies in industrial
wastewater
64
xvi
3.1
Characteristics of POME
78
3.2
Organic loading rates for feed preparation
79
3.3
Laboratory experiment conditions for SBR reactors
79
3.4
Measurements for reactor monitoring,
determination of aerobic granular sludge
morphologies and reactor performance
3.5
81
List of all the primers used for the PCR reaction,
classified according to the oligonucleotide probe
database (Alm et al., 1996)
85
3.6
Hybridization buffer (Manz et al., 1992)
94
3.7
Washing buffer (Amann et al., 1990)
94
4.1
Physical characteristics of POME seeding sludge at
day-0
4.2
Colony characterization of pure isolates from
POME seed sludge
4.3
109
Characteristics of aerobic granular sludge /bioflocs
at different organic loading rates
5.2
102
Properties of selected identified bacteria in POME
seed sludge
5.1
102
146
Aerobic granulation process for the treatment of
industrial wastewater
158
xvii
6.1
Species identification of selected DGGE bands
from the seed sludge and aerobic granular sludge
cultivated at different OLR
6.2
168
Species identification of selected DGGE bands
from the seed sludge and aerobic granular sludge
cultivated using POME
6.3
Properties of identified bacteria in aerobic granular
sludge cultivated using POME
6.4
174
176
Dice coefficients (Cs) comparing the similarities of
PCR–DGGE fingerprints from three reactors
operated at different OLR (S: seed sludge, R1, R2
and R3: reactors operated at OLRs 1.5, 2.5 and 3.5
kgCOD m-3d-1, respectively, numbers in bracket
indicates sampling time in days)
6.5
185
Dominancy of bacterial V3 region analyses based
on Figures 6.3 and 6.5, respectively
187
xviii
LIST OF FIGURES
FIGURE NO.
2.1
TITLE
PAGE
Origin of palm oil. (a) Oil palm trees; (b) Oil palm fruit
(Source: http://palmoil.hubpages.com/hub/palmoil;
Retrieved on 3rd January 2012)
2.2
11
Examples of palm oil‟s various usage
(Source: http://www.forbes.com/2009/09/18/palm-oilmarketing-cmo-network-palmoil.html; Retrieved on 3rd
January 2012)
2.3
Process involved in milling of oil palm (Source: Rupani et
al., 2010)
2.4
14
Average composition of FFB (% in dry weight ) (Adapted
from: Dany and Eliyan, 2010)
2.5
13
15
Illustration of crude palm oil milling process at common
palm oil mills in Malaysia
(Source: http://plotspalmoil.com/plantations.htm;
retrieved on 13th March 2012)
2.6
16
The SBR used in the present study for cultivation of
aerobic granular sludge using POME
37
xix
2.7
Typical operating sequence for a sequencing batch reactor
(Adapted from Metcalf and Eddy, 2003)
2.8
Mechanism of aerobic granular sludge formation
(Adapted from Beun et al., 1999)
2.9
38
39
Photographs of bioflocs and aerobic granular sludge
cultivated at different superficial velocity. (a) Bioflocs
cultivated at a superficial air upflow velocity of 0.008 ms1
; and (b) aerobic granular sludge formed at a superficial
air upflow velocity of 0.025 ms-1 (Liu and Tay, 2002)
2.10
45
Micro-photographs of aerobic granular sludge developed
using different types of wastewater as substrates. (a)
Aerobic granules developed using POME (Abdullah et al.,
2011); (b) Aerobic granules developed using brewery
wastewater (Wang et al., 2007) and (c) Aerobic granules
developed using dairy wastewater (Arrojo et al., 2004)
2.11
FESEM image revealed that AGSP encompasses smooth
and even surface morphology (Dahalan, 2011)
2.12
53
FESEM image revealed that AGS featured an uneven
surface and outer morphology (Dahalan, 2011)
2.13
47
54
Schematic presentation of simultaneous nitrificationdenitrification in aerobic granule (Adapted from: Li et al.,
2008)
2.13
Conceptual aerobic granular structure indicating various
functional zones variation. (Source: Gao et al., 2011)
3.1
65
66
Operational study frameworks for aerobic granular sludge
formation using POME
71
xx
3.2
Overall study outline
73
3.3
Schematic diagram of operational reactor setup
75
3.4
The SBRs used for aerobic granular sludge development
using POME
3.5
76
Imaginary illustration of actual POME ponding system
representing raw POME and seed sludge sampling points
77
3.6
Microbial community analysis protocols
84
3.7
Photographs showing PCR products analysis by gel
electrophoresis
3.8
87
Exhibit showing an electrophoresis of PCR amplified
products on DGGE D-Gene System with marker dyes to
indicate when to stop a DGGE run
3.9
89
Sample of a dendrogram showing Raup and Crick
similarity indices using the UPGMA cluster analysis
method
3.10
90
Photograph of a confocal laser scanning microscope (Axio
Vision Version 4.8.1, Germany) used to analyse FISH
slides and detect AOB community in aerobic granular
sludge samples cultivated using POME
3.11
97
Photograph of the CLSM (Axio Vision Version 4.8.1,
Germany) equipped with an Axio Imager Z-2 (Carl Zeiss,
Germany) camera used to obtain image analysis of
aerobic granular sludge samples
96
xxi
3.12
Comparison between; (a) The actual microscopic image of
a granule and (b) The binary image of the same granule
generated using ImageJ Software (Collins, 2007)
4.1
97
Micrograph of Strain 1 showing moist, slightly raised
elevation of irregular colonies with a distinctively
glistening surface in milky white pigmentation
4.2
103
Colony morphology of Strain 2 showing dry, flat
elevation of round and irregular colonies with glistening
surface in light pink. Bacterial filaments were also
observed in this sample
4.3
Micrograph of Strain 3 showing dry, flat and irregular
colonies with smooth surface
4.4
104
104
Micrograph of Strain 4 indicating a flat elevation and
smooth surface in white colour with wrinkled edges
colony morphology
4.5
105
Micrograph of Strain 5 indicating a flat elevation and
smooth surface in white colour with wrinkled edges
colony morphology
4.6
105
Micrograph of Strain 6 showing an irregular shaped
colony morphology with glistening surface in milky white
colour
4.7
106
Micrograph of gram-positive bacteria from random isolate
in POME seeding sludge indicating gram-positive rods
(Light microscopy magnification: 1000x)
107
xxii
4.8
Micrograph of gram-negative bacteria from Strain 6 in
POME seeding sludge indicating gram-negative rods
(Light microscopy magnification: 1000x)
4.9
SBR bubble column used for the development of aerobic
granular sludge using POME
4.10
112
Dynamic process conditions of the SBR used in the
present study
4.11
108
113
Microscopic observation of aerobic granular sludge
morphology. Photos were taken using light microscope
(Magnification: <200 µm). (a) Seed sludge exhibited nonclear boundary at day-0 (b) Small brown granules with an
average diameter of 1.0 mm sampled at day-20 (c)
Aerobic granular sludge with diameters of between 2.0 to
4.0 mm sampled at day-60. (d) Mature aerobic granular
sludge at day-60 exhibited a spherical shape with a clear
outline boundary
4.12
115
(a) SEM analyses of the sludge used as an inoculum
indicated a typical flocculent activated sludge with a
comparatively feathery structure surrounded by fluffy
edges in which the boundaries of individual particles were
generally uneven and unclear, and (b) The surface of a
mature aerobic granular sludge with dense microbial
composition
4.13
116
The morphology of fresh POME seeding sludge using
PAX-IT®
117
xxiii
4.14
SEM examinations of mature aerobic granular sludge. (a)
The morphology of a mature granule at day-60 of
operation; (b) Surface of mature granule
4.15
118
Different layers accommodating the surface of an aerobic
granular
sludge
for
simultaneous
nitrification-
denitrification process
4.16
120
Scanning electron micrograph of aerobic granular sludge
developed using POME indicating clusters of cocci on
granule surface (Magnification: 25,000x)
4.17
121
Aerobic granular sludge developed using POME also
showing domination of rod-shaped bacteria (size ~ 300 –
500 nm) that are tightly linked and embedded via EPS
(Magnification: 5,000x)
4.18
122
The surface of the granule is layered with some glue-like
substances (Magnification: 500x)
4.19
124
Cavities found on aerobic granular sludge developed
using
POME
resembling
a
channel-like
structure
(Magnification: 10,000x)
4.20
125
Cavities are likely filled with EPS substances which acts
as cohesive material maintaining the bounds between
bacterial cells and colonies (Magnification: 5,000k)
4.21
126
The steady increase in biomass concentrations in SBR
used in the present study (○) MLSS concentration; (●)
MLVSS concentration.
127
xxiv
4.22
The trends of the concentration of reactor MLSS in the
reactor and the SVI values of the aerobic granules (○)
MLSS concentration; (▲) SVI
4.23
129
Profile of COD removal performances of the SBR system.
(∆) Percentage removal; (○) Effluent; (●) Influent
4.24
130
Profile of ammonia removal performances of the SBR
system. (∆) Percentage removal; (○) Effluent; (●) Influent
131
Profile of colour removal performances of the SBR
4.25
system. (∆) Percentage removal; (○) Effluent; (●) Influent
5.1
The change of biomass concentration in SBR at different
132
OLRs, respectively. (●) 2.5 kgCOD m-3d-1; (○) 3.5
kgCOD m-3d-1; (▲) 1.5 kgCOD m-3d-1
5.2
137
Evolution of aerobic granular sludge morphology in R2;
(a) seed sludge obtained from POME facultative pond; (b)
after day-10; (c) after day-17 and (d) mature granules after
day-60 of SBR operation. (Scale bar represents 1 mm)
5.3
138
The MLVSS concentrations in SBRs at different OLR
respectively. (●) 2.5 kgCOD m-3d-1;
(○) 3.5 kgCOD m-3d-1; (▲) 1.5 kgCOD m-3d-1
5.4
Aerobic
granular
sludge
developed
using
140
POME
dominating reactor content at the end of experiments
141
xxv
5.5
Microscopic photographs of the seed sludge and mature
granules in the SBRs; (a) activated seed sludge on day-0;
(b) bioparticles in R1; (c) aerobic granular sludge in R2
and (d) aerobic granular sludge in R3 after day-60 of
experiment
5.6
SEM examination of a mature aerobic granular sludge
featuring a porous structural morphology
5.7
142
147
Fractal dimension vs size profile for aerobic granular
sludge indicating an almost linear relationship between
fractal dimension and equivalent diameter of less than 700
µm
5.8
148
SVI vs size of aerobic granular sludge profile indicating
good and positive association of two important parameters
of granules
5.9
149
(a) Micrograph of aerobic granular sludge cultivated on
day-60 at OLR 2.5 kgCOD m-3d-1 as compared to (b)
disintegration of granule at OLR 19 kgCODm-3d-1
5.10
151
Profile of COD removal efficiencies in reactors (●) 2.5
kgCOD m-3d-1; (○) 3.5 kgCOD m-3d-1; (▲) 1.5 kgCOD
m-3d-1
5.11
Profile of COD removal efficiencies in R2 during shock
load conditions at 19 kgCOD m-3d-1
5.12
153
154
Profile of ammonia and color removal efficiencies in
reactors R2 and R3, respectively. Ammonia removals at
(●) 2.5 kgCOD m-3d-1; (○)3.5 kgCOD m-3d-1; Color
removals at (▲) 2.5 kgCOD m-3d-1 (∆) 3.5 kgCOD m-3d-1
156
xxvi
6.1
PCR products of aerobic granular sludge cultivated using
POME
6.2
PCR products of aerobic granular sludge cultivated using
POME
6.3
164
165
DGGE profiles of the bacterial communities in the three
SBRs namely R1 (OLR 1.5 kgCOD m-3d-1), R2 (OLR 2.5
kgCOD m-3d-1) and R3 (OLR 3.5 kgCOD m-3d-1) during
the aerobic granulation process. The band references are
tabulated in Table 6.1. (MG: Mature Granule; a, b, c, d, e
indicates operation at day - 17, 21, 31, 35 and 41,
respectively)
6.4
167
Scanning electron micrograph of cocci-colonies possibly
Trichococcus sp. found on mature aerobic granular sludge
surface (Magnification: 1000x)
6.5
174
DGGE profiles of the AOB communities in the three
SBRs namely R1, R2 and R3 for mature aerobic granular
sludge cultivated at day-60. The band references are
tabulated in Table 6.2. (OLR is in kgCOD m-3d-1). Species
identification of selected bands is also tabulated in Table
6.2
6.6
173
The distribution of AOB in aerobic granular sludge after
in situ hybridization with a Cy3-labelled probe NSO1225.
(a) AOB colonies in aerobic granule developed at OLR
2.5 kgCOD m-3d-1 and (b) AOB colonies in aerobic
granule developed at OLR 3.5 kgCOD m-3d-1
178
xxvii
6.7
Dendrogram showing Raup-Crick similarity between
microbial communities collected from the various state of
aerobic granular sludge formation using POME (MG:
Mature Granule; a, b, c, d, e indicates operation at day17, 21, 31, 35 and 41, respectively)
6.8
180
Dendrogram generated using BioNumerics® software
indicating similar cluster observation based on DGGE
profiles of aerobic granular sludge samples (MG: Mature
Granule; a, b, c, d, e indicates operation at day- 17, 21,
31, 35 and 41, respectively)
6.9
181
Raup and Crick similarity index calculated from the
DGGE profiles. The labelled data points refer to (a) Pure
seed sludge originated from POME; (b) Bioflocs obtained
at OLR 1.5 kgCOD m-3d-1; (c) Mature granules obtained
at OLR 3.5 kgCOD m-3d-1; (d) Mature granules obtained
at OLR 2.5kgCOD m-3d-1
189
xxviii
LIST OF ABBREVIATIONS
AGS
-
Aerobic granular sludge
AGSP
-
Photosynthetic aerobic granular sludge
AIM
-
Agensi Inovasi Malaysia
AN
-
Ammoniacal nitrogen
AOB
-
Ammonia oxidizing bacteria
APHA
-
American Public Health Association
BAS
-
Biofilm airlift suspension
BLASTn
-
Basic Local Alignment Search Tool - nucleotide
BOD
-
Biochemical oxygen demand
CDM
-
Clean development mechanism
CER
-
Certified emission reduction
CLSM
-
Confocal laser scanning microscopy
COD
-
Chemical oxygen demand
CSTR
-
Continuous stirred tank reactor
DGGE
-
Denaturing gradient gel electrophoresis
DNA
-
Deoxyribonucleic acid
DO
-
Dissolved oxygen
DOE
-
Department of Environment
EBPR
-
Enhanced biological phosphorus removal
EDM
-
Euclidean distance map
EDTA
-
Ethylenediaminetetraacetic acid
EPS
-
Extracellular polymeric substances
EQA
-
Environmental Quality Act
FA
-
Formamide
xxix
FFB
-
Fresh fruit bunches
FISH
-
Fluorescence in situ Hybridization
HB
-
Hybridisation buffer
HRT
-
Hydraulic retention time
MLSS
-
Mixed liquor suspended solid
MLVSS
-
Mixed liquor volatile suspended solid
MPOB
-
Malaysian Palm Oil Board
NMDS
-
Non metric multidimensional scaling
OLR
-
Organic loading rate
PAO
-
Polyphosphate-accumulating bacteria
PBS
-
Phosphate buffered saline
PCR
-
Polymerase chain reaction
PFA
-
Paraformaldehyde
POME
-
Palm oil mill effluent
PORE
-
Palm oil refinery effluent
RNA
-
Ribonucleic acid
SBAR
-
Sequencing batch airlift reactor
SBR
-
Sequencing batch reactor
SDS
-
Sodium dodecyl sulphate
SEM
-
Scanning electron microscopy
SRT
-
Sludge retention time
SS
-
Suspended solid
SVI
-
Sludge volume index
TAE
-
Tris-acetate-EDTA
TEMED
-
Tetramethylethylenediamine
TN
-
Total nitrogen
TSS
-
Total suspended solid
UASB
-
Up-flow anaerobic sludge blanket
UASFF
-
Up-flow anaerobic sludge fixed-film
UFF
-
Up-flow fixed film
UPGMA
-
Unweighted pair group method with Arithmetic Mean
UTM
-
Universiti Teknologi Malaysia
xxx
UV
-
Ultraviolet
VER
-
Volumetric exchange rate
WB
-
Washing buffer
16s rRNA
-
16 Sequencing Ribosomal Ribonucleic Acid
xxxi
LIST OF SYMBOLS
b(S)
-
gray-levels distribution
Ca2+
-
calcium
CaCO3
-
calcium carbonate
CO2
-
carbon dioxide (mgl-1 or mmolL-1)
CODfeed
-
substrate strength in terms of COD concentration (mgL-1)
CTO654r
-
reverse AOB specific primer
CTO189f
-
forward AOB specific primer
ddH2O
-
double sterile water
Df
-
fractal dimension
H/D
-
column height to diameter ratio (mm)
K2HPO4
-
dipotassium phosphate
Mg2+
-
magnesium
MgCl2
-
magnesium chloride
Mw
-
molecular weight
N
-
nitrogen (mgL-1)
NaCl
-
sodium chloride (mL)
NaOH
-
sodium hydroxide
NH4Cl
-
ammonium chloride
NH4-N
-
ammonia
P
-
phosphorus (mgL-1)
PO4-P
-
phosphate
P/COD
-
phosphorus to chemical oxygen demand ratio
P(S)
-
perimeter
Q
-
substrate flow rate (Ld-1)
xxxii
SS0
-
total amount of granular sludge
SSt
-
amount of sludge solids in supernatant after t min
T
-
temperature (°C)
Tris-HCL
-
tris-sodium hydrochloride
Vr
-
reverse PCR primer
Vf
-
forward PCR primer
Vr
-
working volume of reactor (L)
Vpome
-
volume of POME (mL)
Vtapwater
-
volume of tap water (mL)
Vs
-
settling velocity of a particle
§
-
integrity coefficient
dp
-
diameter of a particle
µ
-
viscosity of a solution
ρp
-
density of a particle
ρ
-
density of a solution
xxxiii
LIST OF APPENDICES
APPENDIX
A
TITLE
List of proceedings and publications
PAGE
226