viii
TABLE OF CONTENTS
CHAPTER
TITLE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENT
iv
ABSTRACT
vi
ABSTRAK
vii
TABLE OF CONTENTS
viii
LIST OF TABLES
xiv
LIST OF FIGURES
xvi
LIST OF SYMBOLS
xx
LIST OF ABREVIATIONS
1
2
PAGE
xxiv
INTRODUCTION
1
1.1
Research Background
1
1.2
Problem Statement
5
1.3
Objectives and Scopes
7
1.4
Outline of the Thesis
7
LITERATURE REVIEW
9
2.1
Microalgae
9
2.1.1
10
2.2
Nannochloropsis sp.
Microalgae Cultivation Systems
12
2.2.1 Open Pond System (Raceway)
13
ix
2.2.2 Photobioreactors (Closed System)
2.3
14
2.2.2.1 Tubular Photobioreactor
15
2.2.2.2 Flat Plate Photobioreactors
16
2.2.2.3 Column Photobioreactors
17
Parameter that Affect Microalgae Cultivation
19
2.3.1 Effect of Light
19
2.3.2 Effect of pH
20
2.3.3 Effect of Temperature
20
2.3.4 Effect of Agitation and Carbon Dioxide
21
2.3.5 Effect of Nutrients
21
2.4
Microalgae as Potential Source of Biodiesel
23
2.5
Microalgae Harvesting
25
2.5.1 Filtration
25
2.5.2 Centrifugation
26
2.5.3 Gravity Sedimentation
27
2.5.4 Chemical Flocculation
27
2.5.5 Dissolved Air Flotation (DAF)
29
2.6
2.7
2.8
Microalgae Lipid Extraction
29
2.6.1 Solvent Extraction
30
2.6.2 Supercritical Fluid Extraction
30
2.6.3 Ultrasound Extraction
31
2.6.4 Microwave Assisted Extraction
32
Algal Biodiesel Conversion Technologies
33
2.7.1 Acid Catalyzed Tranesterification
35
2.7.2 Base (Alkali) Catalysed Transesterification
36
2.7.3 Enzymatic Transesterification
38
2.7.4 Supercritical Alcohol Transesterification
38
2.7.5 Direct Transesterification
40
Parameter Affecting Direct Transesterification
42
x
2.9
2.10
2.11
3
2.8.1 Reaction Time
42
2.8.2 Reaction Temperature
43
2.8.3 Catalyst Concentration
43
2.8.4 Alcohol Volume
44
2.8.5 Solvent System
45
History of Ionic Liquids
46
2.9.1 Characteristics of Ionic Liquids
47
2.9.1.1 Structural Features
47
2.9.1.2 Anion
48
2.9.2.3 Cation
50
2.9.2 Application of Ionic Liquids
50
Principle and Properties of Microwave
53
2.10.1 Microwave Heating
54
2.10.1.1
Dielectric
56
2.10.1.2
Dielectric Properties
56
Statistical Analysis on Direct Transesterification
(DT) Process
58
METHODOLOGY
62
3.1
Materials
64
3.2
Microalgae Strain
65
3.3
Culture Media
65
3.3.1 F/2 Medium
66
3.3.2 Walne‟s Medium
66
Microalgae Cultivation
66
3.4.1
67
3.4
Influence of Light and Photoperiod Cycles
3.5
Lipid Extraction
68
3.6
Transesterification Process
69
xi
3.7
DT or Simultaneous ExtractionTransesterification Reaction
69
3.8
Statistical Analysis: Screening Significant Factors
70
3.9
Optimization of Direct Transesterification Process
73
3.10
Experimental Analysis
75
3.10.1 Biomass Concentration
75
3.10.1.1 Cell Concentration
75
3.10.1.2 Dry Cell Weight
75
3.10.2 Measurement of Microalgae Growth
75
3.11
Improved Nile Red Staining Method
77
3.12
Disruption Quantification of Microalgae Cell Wall
77
3.12.1 Surface Structures Morphology using Field
Emission Scanning Electron Microscopy
(FESEM)
4
78
3.13
Determination of Dielectric Properties
78
3.14
Fatty Acid Methyl Esters Composition Analysis
79
CULTIVATION OF NANNOCHLOROPSIS SP.
4.1
Cultivation using F/2 Medium and Walne‟s Medium
80
80
4.1.1 Effect of Light Intensity and Photoperiod
Cycle on Growth Rate
80
4.1.2 Effect of Light Intensity and Photoperiod on
the Lipid Content of Microalgae
4.2
4.3
90
Nannochloropsis sp. Growth and Neutral Lipid
Quantification by Nile Red (NR) Fluorescence
93
Lipid Extraction using Microwave
95
4.3.1 Morphology of Microalgae
98
4.3.2 The Quantification of Lipid Contents
100
4.3.3 Biodiesel (Fatty Acid Methyl Esters)
Composition
101
xii
5
EXTRACTION AND TRANSESTERIFICATION OF
MICROALGAE LIPIDS
5.1
103
Simultaneous Extraction-Transesterification (Direct
Transesterification) using Different Solvents
103
5.1.1 Microscopic Images of Microalgae Cell Wall
103
5.1.2 Effect of Reaction Time on Cell Rupture
104
5.1.3 Percentage of Cell Disruption
110
5.1.4 Enhanced Biodiesel Yield via
Simultaneous Extraction-Transesterification
using Microwave Irradiation
5.1.5 Fatty Acid Methyl Ester Composition
5.2
5.3
112
114
Screening Significant Parameters through Factorial
Design
115
5.2.1 Analysis of Variance (ANOVA) Analysis
119
5.2.2 Model Diagnostic Plots
122
5.2.3 Model Plot
123
5.2.3.1 One-Factor Effect Plots
126
5.2.3.2 Interaction Graph
127
Optimization of Process Parameters for DT
127
5.3.1 Development of Regression Model
129
5.3.2 Main and Interaction Effect Plots
135
5.3.3 Response surface Plots
138
5.3.3.1 Effect of Reaction Time and Wet
Algae to Methanol Ratio
138
5.3.3.2 Effect of Methanol to IL2 and Algae
to Methanol Ratio
141
5.3.3.3 Effect of Methanol to IL2 Ratio and
Reaction Time
144
5.3.4 Comparison Biodiesel Yield and Compositions
via Different Methods
5.3.5 Optimization and Confirmation Experiments
146
148
xiii
5.3.6 Recycle of Ionic Liquid as Co-Solvent Mixture
5.4
149
Investigation of Microwave Dielectric Properties
of Solvents
150
5.4.1 Dielectric Constant of different Types
of Solvents
5
150
5.4.2 Dielectric Loss
152
5.4.3 Tangent Loss, Tan δ
153
CONCLUSIONS AND RECOMMENDATIONS
158
5.1
Conclusions
158
5.2
Recommendations
163
REFERENCES
Appendices A-I
165
184-206
xiv
LIST OF TABLES
TABLE NO.
2.1
TITLE
Classification scheme of the different algal group
(Laura and Paolo, 2006)
2.2
PAGE
10
Advantages and limitation of various microalgae culture
(Giuliano et al. (2010)
18
2.3
Biodiesel source comparison (Chisti, 2007)
23
2.4
Oil Contents of some microalgae (Chisti, 2007)
24
2.5
Advantages and limitation of various extraction methods
for algae oil (Jasvinder and Sai, 2010)
33
2.6
Comparison of transesterification method
40
2.7
Application of ionic liquids
2.8
Loss Tangents of various materials
58
3.1
Chemicals used in this study
64
3.2
The low and high levels for the factors affecting the
biodiesel production
71
3.3
Experimental design of the 24 factorial designs
72
3.4
Experimental design using central composite designs
74
4.1
Maximum cell density, specific growth rate and division
times of Nannochloropsis sp. at different light intensities
and photoperiod cycles when grown in F/2 medium
4.2
88
Maximum cell density, specific growth rate and division
times of Nannochloropsis sp. at different light intensities
and photoperiod cycles grown in Walne‟s medium
4.3
89
Biodiesel yield and composition from wet
Nannochloropsis sp. biomass by conventional heating
(waterbath) and microwave irradiation
102
xv
5.1
Composition of fatty acid methyl ester (% of total FAMEs)
from wet microalgae biomass using hexane as co-solvent
at different reaction time
115
5.2
Experimental design and result of the 24 factorial designs
116
5.3
Analysis of variance (ANOVA) for the selected linear model
120
5.4
Experimental design matrix experimental results of the
response using CCD
128
5.5
Analysis of variance (ANOVA) for model regression
130
5.6
Comparison of biodiesel yield and compositions from wet
biomass by microwave irradiation with different methods
147
5.7
Confirmation runs at optimum condition
149
5.8
Dielectric properties values of different ratio of methanol/IL2
155
5.9
Dielectric properties values of different ratio of algae to
methanol
156
xvi
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
2.1
Nannochloropsis sp.
11
2.2
Arial view of raceway pond (Chisti, 2007)
14
2.3
Tubular photobioreactor
16
2.4
Flat plate bioreactor
17
2.5
Transesterification of triglycerides with alcohol
34
2.6
Mechanism of acid catalysed transesterification of
vegetable oils (Demirbas, 2009)
2.7
35
Mechanism of base catalyzed transesterification of
vegetable oils
37
2.8
Structure of ethylammonium nitrate
47
2.9
Common anion and cation in Ils
49
2.10
Schematic of microwave energy
53
2.11
Electromagnetic spectrum
54
2.12
Changing of polarization in alternating electromagnetic
field
56
2.13
Build-up of central composite design for three factors
61
2.14
Central composite design for 3 design variables at 2 level
61
3.1
Flowchart diagram for overall process study
63
3.2
Nannochloropsis sp. was cultivated in a 5 L
photobioreactor
67
3.3
Microwave irradiation set up for direct transesterification
70
4.1
Increase in cell density under the three light intensities of
(a) 50; (b) 100 and (c) 200 mol m-2 s-1 and different
photoperiod lengths (12:12, 18:06 and 24:0 h
light:dark)
82
xx
4.2
Increase in cell density under the three light intensities of
(a) 50; (b) 100 and (c) 200 mol m-2 s-1 and different
photoperiod lengths (12:12, 18:06 and 24:0 hr light:dark
cycles) when grown in Walne‟s medium
4.3
86
Lipid content under the three light intensities of (a) 50;
(b) 100 and (c) 200 mol m-2 s-1 and different photoperiod
lengths (12:12, 18:06 and 24:0 h light:dark cycles) when
grown in F/2 Media
4.4
91
Relationship between growth rate of microalgae and lipid
content under the light intensity of 100 mol m-2 s-1 and
different photoperiod lengths (12:12, 18:06 and 24:0 h
light:dark cycles) when grown in F/2 Media
4.5
Growth and neutral lipid quantification using NR
fluorescence over 8- day cultivation
4.6
92
94
Total lipid content recovered during the 8-day cultivation
time using microwave irradiation and water bath extraction
96
4.7
Microalgae extraction mechanism using microwave irradiation
97
4.8
SEM images of Nannochloropsis sp.(a) under 1000x (b) 5000x
magnification before extraction; (c) after extraction with water
bath-assisted solvent extraction at 2000x, (d) 5000x
magnification, (e) after extraction with microwave
irradiation at 2000x and (f) 5000x
4.9
The quantitative relationship of fluorescence intensities and
lipid contents in Nannochloropsis sp.
5.1
100
FESEM images of Nannochloropsis sp. (a) under 5000x
(b) 25000x magnification before extraction
5.2
99
105
FESEM images of microalgae biomass under 10000x
magnification after simultaneous extraction-transesterification
with different types of solvents at 5 to 15 min of reaction time
5.3
106
FESEM images of microalgae biomass under 25000x
magnification after simultaneous extraction-transesterification
5.4
with different types of solvents at 5 to 15 min of reaction time
107
Percentage of cell wall disruption at different reaction time
111
xxi
5.5
Biodiesel yield at different reaction time of different type
of solvents under simultaneous extraction-transesterification
via microwave irradiation
113
5.6
The half normal probability plot of biodiesel production
117
5.7
The normal plot probability for biodiesel production
118
5.8
(a) Normal probability plot of residuals, (b) Studentized
Residuals and predicted response plot, (c) Actual and
predicted plots for microalgae biodiesel production,
(d) Outlier t plot
5.9
Schematic diagram for one-factor effects plot for biodiesel
production
5.10
123
125
Schematic diagram for interaction factors in biodiesel
production (a) Methanol to IL2 ratio versus biodiesel production
and (b) Algae to methanol ratio versus biodiesel production
126
5.11
Predicted versus actual biodiesel yield
132
5.12
(a) Normal probability and studentized residual plot
(b) Studentized residuals and predicted response plot
(c) Outlier t plot
5.13
134
Schematic diagram for one-factor effects plot for biodiesel
Production (a) Algae:methanol ratio; b) Reaction time
and c) Methanol:IL2 ratio
5.14
136
Schematic diagram for interaction factors in biodiesel
production (a) Algae: methanol ratio – reaction time (AB);
(b) Algae:methanol ratio – methanol:IL2 ratio (AC) and
(c) Reaction time – methanol:IL2 ratio (BC)
5.15
137
The effect of reaction time and algae:methanol ratio on
biodiesel production using wet microalgae biomass and
microwave irradiation (a) Contour plot (b) Response surface plot 139
5.16
The effect of methanol:IL2 ratio and algae:methanol ratio on
biodiesel production using wet microalgae biomass and
microwave irradiation (a) Contour plot (b) Response surface plot 142
5.17
The effect of reaction time and methanol:IL2 ratio on
biodiesel production using wet microalgae biomass and
microwave irradiation (a) Contour plot
xxii
(b) Response surface plot
145
5.18
Reused of ionic liquid as co-solvent mixture
150
5.19
Dielectric constant of different types of solvent
152
5.20
Dielectric loss of different types of solvent
153
5.21
Tangent loss of different types of solvent
154
xx
LIST OF SYMBOLS
o
-
Constant coefficient
2k
-
Factorial (or fractional factorial )
2k
-
Axial or star runs
gL-1
-
Concentration (gram per liter)
H2SO4
-
Sulphuric acid
HCl
-
Hydrochloric acid
mg
-
Mass, milligram
o
-
Temperature (Degree Celsius)
g
-
Mass, gram
v/v %
-
Volume per volume percentage
ε′
-
Dielectric constant
ρ
-
Density
α
-
Axial point
ε″
-
Dielectric loss
μmolm-2s-1
-
Light intensity, micromole per meter square
C
per second
μ
-
Specific growth rate
tan δ
-
Loss tangen
2
-
Linear regression constant
%
-
Percentage
Kv
-
Kilo volt
O2
-
Oxygen
s
-
Time, second
KOH
-
Potassium hydroxide
sp.
-
Subspecies
R
xxiv
μm
-
Distance, micrometer
mw
-
Microwave
CO2
-
Carbon dioxide
min
-
Time, minute
m
-
Distance, meter
-
Volume, cubic meter
h
-
Time, hour
>
-
More than
mm
-
Distance, milimeter
wt%
-
Weight percentage
CH3COCl
-
Acetyl chloride
cm
-
Distance, centimeter
M
-
Concentration, Molar
L
-
Volume, Liter
nm
-
Distance, nanometer
-
Megahertz
W/m
-
Power, Watt per meter square
% Ct
-
Cell disruption percentage
N1
-
Cell number concentration at t1
N2
-
Cell number concentration at t2
k
-
Division rate
W
-
Watt
f
-
Frequency
Nt
-
Average number intact cells at certain time
D
-
Distribution coefficient
P
-
Power dissipation density
Ei
-
Electric field strength
Cp
-
Specific heat
E
-
Total energy
HCO3
-
Bicarbonate
w/wDW
-
Weight per dry weight
AlCl3
-
Aluminum chloride
BMIM-Cl
-
1-butyl-3-methyl imidazolium chloride
[PF6]-
-
Hexafluorophosphate
m
3
MHz
2
xxv
[BF4]-
-
Tetrafluoroborate,
[CF3SO3]-,
-
Trifluoromethane sulfonate or triflate
[(CF3SO2)2N]-
-
Bis-(trifluoromethanesulfonyl) amide
[NTf2]-
-
Bistriflamide
[N(SO2CF3)2]-
-
N-Phenyl-bis(trifluoromethanesulfonimide)
[(CF3SO2)3]-
-
Ttris {(trifluoromethyl) sulfonyl} methanide
[Ms2N]-
-
Bis (methanesulfonyl) amide
[R1R2IM] +
-
Alkylimidazolium
[RPy] +
-
Alkylpyridinium
[NR4] +
-
Tetraalkylammonium
[PR4] +
-
Tetraalkylphosphonuim
NaOH
-
Sodium hydroxide
SrO
-
Strontium oxide
ml
-
Volume, milliliter
vvm
-
Gas volume flow per unit of liquid volume per
minute, volume per volume per minute
μL
-
Volume, microliter
mgml-1
-
Milligram per milliliter
g
-
Mass, gram
HF
-
Hydrogen fluoride
FeCl3.6H2O
-
Iron (III) Chloride hexahydrate
MnCl2 .4H2O
-
Manganous chloride
H3BO3
-
Boric acid
Na2 EDTA
-
Disodium salt dehydrate
NaH2PO4.2H2O
-
Sodium di-hydrogen orthophosphate
NaNO3
-
Sodium nitrate
ZnCl2
-
Zinc chloride
CoCl2.6H2O
-
Cobalt (II) chloride hexahydrate
NaMoO4.2H2O
-
Sodium molybdate dihydrate
(NH4)6Mo7O24.4H2O -
Ammonium molybdate tetrahydrate
CuSO4.5H2O
-
Copper (II) sulfate pentahydrate
NaCl
-
Sodium chloride,
C14:0
-
Myristic acid methyl ester
C16:0
-
Palmitic acid methyl ester
xxvi
C16:1
-
Palmitoleic acid methyl ester
C17:0
-
Heptadecanoic Acid
C18:0
-
Stearic acid methyl ester
C18:1,cis-9
-
Oleic acid methyl ester
C18:1,trans-9
-
Elaidic acid methyl ester
C18:2,cis-9,12
-
Linoleic acid methyl ester
C18:2,trans-9,12
-
Linolelaidic acid methyl ester
C18:3,cis-9,12,15
-
Linolenic scid methyl ester
C20:0
-
Arachidic acid methyl ester
C22:0
-
Behenic acid methyl ester
ZnSO47H2O
-
Zinc Sulfate Heptahydrate
xxiv
LIST OF ABBREVIATIONS
ANOVA
-
Analysis of variance
CCD
-
Central composite design
FESEM
-
Field emission scanning electron microscope
FCD
-
Face centered design
RSM
-
Response surface methodology
PFD
-
Photon flux density
GHGs
-
Greenhouse gas emissions
GF/C
-
Glass microfiber design
DT
-
Direct transesterification
FFA
-
Free fatty acid
SCM
-
Supercritical methanol
PE
-
Polyethylene
PVC
-
Polyvinyl chloride
TAG
-
Triglyceride
FID
-
Flame ionization detector
SEM
-
Scanning electron microscopy
ID
-
Internal diameter
ATP
-
Adenine tri-phosphate
NADPH
-
Nicotinamide adenine dinucleotide phosphate
L:D
-
Light:dark cycles
NR
-
Nile red
PTFE
-
Polytetrafluoroethylene
IL
-
Ionic liquid
TSIL
-
Task specific ionic liquid
SFE
-
Supercritical fluid extraction
FAMEs
-
Fatty acid methyl esters
mw
-
Microwave
PFA
-
Perfluoroalkoxy alkane
LIST OF APPENDICES
APPENDIX
TITLE
PAGE
A
Chromatogram of standard fatty acid methyl esters
181
B
Fatty acid methyl esters concentration calibration curve
183
C
Determination of cell concentration of Nannochloropsis sp.
188
in F/2 medium
D
Determination of cell concentration of Nannochloropsis sp.
191
in Walne‟s medium
E
Determination of total lipid content
194
F
Total of cell disruption
196
G
Determination of dielectric properties at 2450 MHz
199
H
Biodiesel yield using different types of solvent
201
I
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