vii
TABLE OF CONTENTS
CHAPTER
TITLE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xiii
LIST OF ABBREVIATIONS
1
2
PAGE
xv
LIST OF SYMBOLS
xvii
LIST OF APPENDICES
xviii
INTRODUCTION
1.1
Background of the Study
1
1.2
Problem Statement
2
1.3
Objectives of the Study
3
1.4
Scope of the Study
4
1.5
Significance of the Study
4
LITERATURE REVIEW
2.1
History of Microalgae
6
2.2
Microalgae Divisions
7
2.3
Cultivation Conditions of Microalgae
10
viii
2.4
2.3.1 Phototrophic Cultivation
10
2.3.2 Heterotrophic Cultivation
15
2.3.3 Mixotrophic Cultivation
16
2.3.4 Photoheterotrophic Cultivation
17
Culture Parameters
17
2.4.1 Environmental Condition
18
2.4.1.1
Light Intensity
18
2.4.1.2
pH
18
2.4.1.3
Temperature
19
2.4.1.4
Mixing
20
2.4.1.5
Salinity
20
2.4.2 Nutrients
2.5
21
2.4.2.1
Carbon Sources
21
2.4.2.2
Phosphorus
21
2.4.2.3
Nitrogen
22
2.4.2.4
Other Elements
22
Growth Cycle of Microalgae
23
2.5.1 Lag Phase
24
2.5.2 Exponential Growth Phase
24
2.5.3 Deceleration Growth Phase
26
2.5.4 Stationary Phase
26
2.5.5 Death Phase
27
2.6
Maintenance of Growth
27
2.7
Importance of Microalgae
28
2.7.1 Microalgae as Feedstock for Biodiesel
28
2.7.2 Lipid Production from Microalgae
31
Microalgae Versus Wastewater
32
2.8.1 Microalgae Growth in Municipal Sewage
36
2.8
Wastewater
2.8.2 Microalgae
Growth
in
Agricultural
36
Artificial
37
Wastewater
2.8.3 Microalgae
Wastewater
Growth
in
ix
2.8.4 Microalgae
Growth
in
Industrial
38
wastewater
2.9
Wastewater from Palm Oil, POME
38
2.9.1 Characteristic of POME
39
2.9.2 POME as Nutrients Source to Culture
40
Microalgae
3
RESEARCH METHODOLOGY
3.1
Introduction
46
3.2
Material and Methods
48
3.2.1 Preparation of the Inoculumns
48
3.2.2 Wastewater Collection
51
3.2.3 Antibiotic Preparation
52
Types of Experimental Parameters
53
3.3.1 Optical Density
53
3.3.2 Chlorophyll a Content
54
3.3.3 Chemical Oxygen Demand
55
3.3.4 Total Nitrogen Measurements
55
3.3.5 Mixed Liquor Suspended Solids and
55
3.3
Mixed Liquor Volatile Suspended Solids
3.4
3.3.6 Cell Dry Weight
56
3.3.7 Lipid Extraction
57
Experimental Procedure
58
3.4.1 Preliminary Study (selection of suitable
58
concentration of microalgae)
3.4.2 Selection of Suitable Strain (First Set up)
59
3.4.3 Comparison POME and Glucose as Sole
61
Carbon Source
3.4.4 Optimization Lipid Content
61
3.5
Inhibition Model
62
3.6
Kinetic and Yield Parameter
63
x
4
RESULT AND DISCUSSION
4.1
4.2
Preliminary Study in The Settled POME
64
4.1.1 Effect of Substrate
65
4.1.2 Substrate Consumption Rate
66
4.1.3 Growth Rate of Microalgae
68
4.1.4 Biomass Productivity
69
4.1.5 Inhibition Model
71
Investigation of Microalgae for High Lipid
73
Content Using POME as Carbon Source
4.3
4.2.1 Microalgae Growth
73
4.2.2 Lipid Content Analysis
75
4.2.3 Carbon Source Consumption
78
Cultivation of Chlorella Sorokiniana Using
79
POME and Glucose as Carbon Source
4.4
Chlorella Sorokiniana From Settled Raw Palm
83
Oil Mill Effluent (POME) for Lipid Production
4.4.1 Correlation of Organic Carbon Substrate
83
and Nutrient Utilization Rate
4.4.2 Relationship of Organic Carbon and
86
Nutrient Utilization with Lipid Production
by Microalgae
4.5
Effect of Photo Cycles (light : dark) on Lipid
88
Production by Chlorella Sorokiniana in POME
5
CONCLUSION
5.1
Conclusion
91
5.2
Recommendation
92
REFERENCES
Appendices A - B
94
102 - 108
xi
LIST OF TABLES
TABLE NO.
TITLE
2.1
The main pigments, storage products, and cell coverings
PAGE
8
of the algal divisions (Barsanti and Gualtieri, 2006)
2.2
The lipid content and productivities of different
11
microalgae species under different cultivation condition
(Chen et al., 2011)
2.3
Comparison of some source of biodiesel (Chisti, 2007)
29
2.4
Oil content of microalgae (Chisti, 2007, Khan et
30
al., 2009, Lam and Lee, 2011)
2.5
Biomass productivity, lipid content and lipid productivity
33
of 30 microalgae strains cultivated in 250 mL flask
(Rodolfi et al., 2009)
2.6
The growth of microalgae under a variation of wastewater
35
condition (Pittman et al., 2011)
2.7
Characteristic of POME (Wong et al., 2009)
40
2.8
Summarizes of previous study about types of microalgae
42
using in any kind of wastewater
3.1
Strain of Microalgae
48
3.2
Bolds Basal Medium (BBM) (Barsanti and Gualtieri,
49
2006)
3.3
Soil Extract Medium
50
3.4
Modified Bold 3N Medium
50
xii
3.5
Characteristic of the sampled POME
52
4.1
COD consumption by Chlorella vulgaris under different
67
concentrations of POME at 28 days
4.2
Value of µ and R2 for every substrate concentration by
69
Chlorella vulgaris
4.3
Results of growth and biomass productivity of microalgae
73
in appropriate medium
4.4
Maximum value for specific growth rate, carbon source
consumption,
chlorophyll
productivity,
82
biomass
productivity and ratio MLVSS/MLSS
4.5
Comparison of nitrogen utilization by Chlorella
sorokiniana
85
xiii
LIST OF FIGURES
FIGURE NO.
2.1
TITLE
Typical growth curve for microorganism population (Bitton,
PAGE
23
1994)
2.2
Chemostat for continuous culture of microorganism (Bitton,
28
1994)
3.1
Steps in the experimental operation
47
3.2
Facultative Ponds
51
3.3
Replicates of antibiotic (Chloramphenicol) Preparation
53
3.4
Optical density measurement at wavelength 600 nm
54
3.5
Sample on freeze dryer
57
3.6
Microalgae cells after have done for extraction
58
3.7
Chlorella vulgaris cultivation in different COD concentration
59
3.8
Flask set up used for all microalgae strains
60
3.9
Light intensity measurement using Lux Meter
61
4.1
Initial with final COD removal in different POME
66
concentration for Chlorella vulgaris
4.2
Variation of substrate consumption with the operation time by
67
Chlorella vulgaris
4.3
Growth rate of microalgae under different initial substrate
68
xiv
concentration by Chlorella vulgaris
4.4
Variation of the Napierian logarithm of the quotient between
70
the MLSS concentrations at any time by Chlorella vulgaris
4.5
Variation of the Napierian logarithm of the quotient between
70
the MLVSS concentrations at any time by Chlorella vulgaris
4.6
Comparison of the model values and experimental data, A
72
(MLSS measurements), B (MLVSS measurements ), C (OD
measurements) by Chlorella vulgaris performance
4.7
Chlorophyll content for five strain of microalgae in batch
74
culture
4.8
Changes of green color for types of microalgae in 20 days
75
4.9
Percentage of lipid content for types of microalgae
76
4.10
Lipid content for five strain of microalgae in batch culture
76
4.11
Correlation of lipid content with MLVSS and chlorophyll
77
content during of batch culture
4.12
Chlorophyll production of microalgae based on carbon
79
sources consumption
4.13
Comparison of biomass between POME and C6H12O6 during
80
of experiment
4.14
Relationship of MLSS with chlorophyll content using POME
80
and C6H12O6
4.15
Comparison of ratio MLVSS/MLSS and lipid content for
82
POME and C6H12O6 in batch culture
4.16
Organic carbon substrate rates during of experiment
84
4.17
Utilization rate of nutrients during of experiment
85
4.18
Relationship between organic carbon substrate rate profile and
86
specific growth rate (Note: A = most of experiment
conducted, B = lipid production potentially)
4.19
(A) Relationship lipid productivity and nutrient utilization
rate, (B) relationship lipid productivity and organic carbon
substrate
87
xv
4.20
The effect of light/dark cycles on specific growth rate profiles
88
4.21
Lipid productivity for three light/dark cycles during of
89
cultivation
4.22
Relationship of chlorophyll production rate and lipid
productivity in varying light/dark cycles
90
xvi
LIST OF ABBREVIATION
ATP
-
Adenosine Triphosphate
BBM
-
Bolds Basal Medium
BOD
-
Biochemical Oxygen Demand
B. sudeticus
-
Botryococcus sudeticus
C. sorokiniana
-
Chlorella sorokiniana
C. pyrenoidosa
-
Chlorella pyrenoidosa
C. vulgaris
-
Chlorella vulgaris
C : TN
-
Carbon to total nitrogen ratio
Ca
-
Calcium
CDW
-
Cell Dry Weight
cells/mL
-
Cell per mili liter
CO2
-
Carbon dioxide
COD
-
Chemical Oxygen Demand
DNA
-
Deoxyribonucleic Acid
Fe
-
Ferrite
g/L
-
Gram per liter
H2O
-
Hidrogen Oxide
H2PO4 -
-
Dihydrogen Phosphate Ion
HPLC
-
High-Performance Liquid Chromatography
HPO42-
-
Hydrogen Phosphate Ion
K
-
Kalium
kJ/g
-
Kilo Joule per gram
L: D
-
Light : dark
Mg
-
Magnesium
mg/L/day
-
Mili gram per liter per day
xvii
MLSS
-
Mixed Liquor Suspended Solid
MLVSS
-
Mixed Liquor Volatile Suspended Solid
N
-
Nitrogen
NADP+
-
Nicotinamide Adenine Dinucleotide Phosphate
NH4+
-
Ammonium
-
-
Nitrite
NO3-
-
Nitrate
OD
-
Optical Density
P
-
Phosphorus
POME
-
Palm Oil Mill Effluent
R2
-
Variance accounted
RNA
-
Ribonucleic Acid
So
-
Substrate concentration
Topt
-
Temperature optima
TAGs
-
Triacylglycerols
TSS
-
Total Suspended Solids
VSS
-
Volatile Suspended Solids
Zn
-
Zinc
NO2
xviii
LIST OF SYMBOLS
µ
-
specific growth rate
µm
-
maximum specific growth rate
A
-
weight of filter +dried residue
B
-
weight of filter
C
-
weight of residue + dish before ignition
D
-
weight of residue + dish or filter after ignition
e
-
exponential
Ki
-
dissociation constant
Ks
-
half saturation constant
L
-
width of cuvette
t
-
time
τd
-
doubling time of cell mass
τ'd
-
doubling time based on the replication rate
V1
-
volume of extract
V2
-
volume of sample
X
-
concentration at beginning
X’
-
growth of microalgae concentration
X’0
-
value of growth concentration
X0
-
concentration at beginning
α
-
alpha
β
-
beta
γ
-
gamma
ε
-
epsilon
xix
LIST OF APPENDICES
APPENDIX
TITLE
PAGE
A
CONVERSION : PPF TO LUX
102
B
EXPERIMENTAL DATA
103