i identification of biodiesel yield and its properties...
TRANSCRIPT
i
IDENTIFICATION OF BIODIESEL YIELD AND ITS PROPERTIES FROM
MICROALGAE BOTRYOCOCCUS SP ISOLATED AT SEMBRONG DAM
WELLSON BIN RISIT
A thesis submitted in
Fulfillment of the requirement for the award of the
Degree of Master of Civil Engineering
Faculty of Civil and Environmental Engineering
University Tun Hussein Onn Malaysia
SEPTEMBER 2016
iii
DEDICATION
This thesis is dedicated to my beloved family, my supervisors and friends, for
without their support and guidance none of this would have happened
iv
ACKNOWLEDGEMENT
First and foremost, I would like to Thank to my God for His gracious and kindness
throughout the years of completing this thesis, for without His Guidance I could not
have accomplished anything. Not to forget, my sincerest gratitude to my supervisor
Associate Professor Dr Norzila Binti Othman for being such a good motivator,
supportive and very kindhearted person as well as to my co-supervisor Dr Hazel
Monica Matias-peralta who very supportive and cooperative. Their guidance and
patience throughout these years truly have encouraged, strengthened me in times of
adversity, mold me into what I become today and have utterly contributed to this
study. Without their part in this thesis, this study would not have been completed.
My utmost respect also goes to my friends who always lend a helping hand to
keep me going until the completion of this study. Their present spices up my life and
instilling the positivity in me that stoked to everyday productivity that finally have
my thesis well-presented here. Their invested energy and time are highly appreciated.
Finally, I want to thank to my family for being a super strong supportive who
never give up on, especially in their help through the words of encouragement as
well as financial problem.
v
ABSTRACT
Recently, the unceasing dependency upon fossil-based feedstock has triggered fuel
shortages, volatility of fuel price hike and numerous discharging of carbon dioxide
resulted from the burning of fossil fuel linked to global warming. Years ago there
were many researches tried to find alternatives fuel as to resolve the dwindling of
fuel stocks. The discoveries of high lipids content of microalgae have paved a way of
instigating more research over microalgae. The purpose of this study is to investigate
the potential of Botryococcus sp as an alternative sustainable and renewable biodiesel
to diminish off immoderate dependency over fossil fuel. Botryococcus sp was
isolated from Sembrong Dam water that is located in Johor Malaysia by using
standard plating method and classified morphologically. The growth factors such as
light, initial pH and rotation speed were optimized by adopting techniques from
previous studies. Botryococcus sp biodiesel properties were conducted and compared
with ASTM 6715 for fuel properties. The results show that, Sembrong Dam water
quality is polluted as compared with Interim National Water Quality Standard,
Malaysia (INWQS) and high concentration of chlorophyll-a in Dam Sembrong water
is due to presence of algal blooms. It was found that Botryococcus sp has the best
growth condition under illumination of 6000 lux, initial pH 7 and continuous rotation
speed of 30 rpm. Under optimized and well-monitored cultivation, Botryococcus sp
is able to yield a maximum oil content of 64.8%. The transesterification process has
effectively converted the green crude to biodiesel up to 88%. The microalgae
biodiesel quality is suitable to be used as fuel as it complies with ASTM 6715. On
the basis of the results of this research, it can be concluded that Sembrong Dam
water is polluted and undergoing eutrophication. Botryococcus sp isolated from
Sembrong Dam is a great potential for biodiesel feedstock due its rich oil content.
vi
ABSTRAK
Kebergantungan berterusan terhadap bahan api fosil menyebabkan berlakunya
kekurangan bahan api, ketidakstabilan harga bahan api dan pelepasan karbon
dioksida hasil daripada pembakaran bahan api fosil yang menyumbang kepada
pemanasan global. Banyak kajian telah dijalankan bagi mencari alternatif bahan api
yang lestari bagi menyelesaikan kekurangan stok bahan api fosil. Penemuan
kandungan lipid yang tinggi pada mikroalga telah mendorong lebih banyak
penyelidikan terhadap mikroalga. Tujuan kajian ini adalah untuk menyiasat potensi
Botryococcus sp sebagai alternatif biodiesel yang mampan dan lestari untuk
mengurangkan kebergantungan ke atas bahan api fosil. Botryococcus sp telah
diambil di Empangan Sembrong yang terletak di Johor Malaysia menggunakan
kaedah penyaduran agar dan dikelaskan berdasarkan morfologi. Faktor-faktor yang
mempengaruhi pertumbuhan alga seperti cahaya, pH dan putaran kelajuan telah
dioptimumkan dengan mengadaptasikan kaedah daripada kajian terdahulu. Kualiti
Biodiesel Botryococcus sp telah dikaji dan dibandingkan dengan ASTM 6715 untuk
memenuhi kualiti standard bahan api. Berdasarkan keputusan yang diperolehi, air di
Sembrong Dam tercemar berdasarkan Standard Interim National Water Quality,
Malaysia (INWQS) dan kandungan klorofil-a yang tinggi disebabkan oleh
pertumbuhan alga yang banyak. Botryococcus sp mempunyai pertumbuhan yang
optimum di bawah pencahayaan 6000 lux, pH 7 dan kelajuan putaran 30 rpm.
Botryococcus sp mampu menghasilkan kandungan minyak maksimum 64.8%. Proses
transesterifikasi Berjaya menukarkan minyak mentah hijau kepada biodiesel
sehingga 88%. Kualiti Botryococcus sp biodiesel adalah sesuai untuk digunakan
sebagai bahan api kerana ia mematuhi Standard ASTM 6715. Berdasarkan hasil
kajian, air di Sembrong Dam tercemar dan mengalami eutrofikasi. Botryococcus sp
adalah calon terbaik yang berpontensi sebagai bahan mentah biodiesel kerana
memiliki kandungan minyak yang tinggi.
vii
CONTENTS
CHAPTER 1 TITLE PAGE
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
CONTENTS vii
LIST OF TABLE xii
LIST OF FIGURE xiv
LIST OF ABBREVIATION xvi
LIST OF APPENDICES xIx
INTRODUCTION 1
1.1 Background of study 1
1.2 Problem statement 2
1.3 Objective of study 4
1.4 Scope of study 4
1.5 Importance of study 5
1.6 Limitation of study 6
1.7 Organization of the thesis 6
CHAPTER 2 LITERATURE REVIEW 9
2.1 Introduction 9
2.2 Microalgae 9
2.2.1 The Historical development of
microalgae production system.
11
2.3 Biofuel 12
2.4 Biodiesel 13
viii
2.4.1 Biodiesel in malaysia 14
2.4.2 Microalgae biodiesel 14
2.4.3 Advantages of using microalgae
for biodiesel production
16
2.5 The potential of microalgae as bioenergy 18
2.6 Microalgae lipid content and
productivities
20
2.7 Global outlook on fuel and biodiesel 23
2.8 Sembrong Dam 25
2.9 Eutrophication and its impact on
environmental
27
2.10 Lakes and reservoirs and its water quality
standard
29
2.11 Isolation and identification of microalgae 32
2.12 Morphology 33
2.13 Microalgae cultivation 35
2.14 Microalgae culturing system 36
2.14.1 Open pond system 36
2.14.2 Closed photobioreactors 37
2.15 The environmental factors influencing
the growth of microalgae
38
2.15.1 Nutrient 39
2.15.2 Carbon 39
2.15.3 Nitrogen compound 40
2.15.4 Phosphorus compound 40
2.15.5 Trace metal 40
2.15.6 Temperature 41
2.15.7 Light 42
2.15.8 pH 42
2.15.9 Salinity 43
2.15.10 Mixing 43
2.16 Optimization of microalgae growth
conditions
44
ix
2.17 Extraction of microalgae 48
2.18 Extraction of oil with soxhlet method 48
2.19 Microalgae and GC-MS analysis 50
2.20 Microalgae biodiesel production by
transesterification
52
2.21 Biodiesel fuel testing 54
2.21.1 Flash point 55
2.21.2 Kinematic viscosity 55
2.21.3 Specific gravity/density 56
2.21.4 Acid value 56
2.21.5 Water content 56
2.21.6 Calorific value 57
2.22 Overview of the quality standard for
biodiesel.
57
2.23 Concluding remark 59
CHAPTER 3 METHODOLOGY 61
3.1 Introduction 61
3.2 Flow chart 62
3.2.1 Flow chart phase 1 62
3.2.2 Flow chart phase 2 63
3.3 Dam water sampling 64
3.4 Dam water characterization 64
3.5 Determination of chlorophyll-a 66
3.6 Isolation of single cell for microalgae 66
3.6.1 Washing and cleaning samples 67
3.6.2 Dilution techniques 67
3.6.3 Standard plating agar method 68
3.6.3.1 Agar plate preparation 68
3.6.3.2 Spreading method 68
3.6.3.3 Streaking method 69
3.7 Morphology analysis 69
3.7.1 Light microscopy 70
3.7.2 Scanning electron microscopy 70
x
3.8 Method for cell count 71
3.9 Factor affecting microalgae growth 73
3.9.1 Light intensity optimization 73
3.9.2 pH optimization 74
3.9.3 Shaking speed optimization 75
3.10 The selection of synthetic media grow of
N:P
76
3.10.1 N:P synthetic media preparation
(1.5:1)
76
3.11 The factors affecting N:P as media 78
3.12 Cultivation of microalgae 78
3.13 Harvesting of microalgae 80
3.13.1 Centrifugation procedure 80
3.13.2 Microalgae drying process 81
3.14 Microalgae oil extraction 82
3.14.1 Folch extraction method
procedure
82
3.14.2 The optimization of temperature
in soxhlet extraction
83
3.15 Biodiesel profiling analysis 84
3.16 Transesterification 85
3.17 Biodiesel properties 87
CHAPTER 4 RESULT AND ANALYSIS 91
4.1 Introduction 91
4.2 Water characteristic of Sembrong Dam 91
4.3 Chlorophyll-a 96
4.4 Microalgae isolation 98
4.5 Microscope morphological identification 99
4.6 The SEM morphological identification 101
4.7 Factor affecting the growth of
microalgae using BBM
103
4.7.1 Light intensity factor 104
4.7.2 pH optimization 105
xi
4.7.3 Shaking speed optimization 107
4.8 Modified N:P as a media grow for
Botryococcus sp
110
4.9 Factors affecting N:P as a media grow 111
4.9.1 Light intensity factor on
Botryococcus sp growth
111
4.9.2 pH factor on Botryococcus sp
growth
112
4.9.3 Rotational speed factor on
Botryococcus sp growth
113
4.10 The comparison of cell concentration by
using BBM and synthetic media.
114
4.10.1 Light optimization for BBM and
N:P
114
4.10.2 pH optimization using BBM and
N:P media
115
4.10.3 Rotational speed optimization
using BBM and synthetic.
117
4.11 Optimization of temperature in soxhlet
extraction of Botryococcus sp
118
4.12 Transesterification of Botryococcus sp
lipids
119
4.13 Analysis of fatty acid methyl ester in
Botryococcus sp biodiesel
120
4.14 The Fuel properties of Botryococcus sp
biodiesel
123
CHAPTER 5 CONCLUSION & RECOMMENDATION
5.1 Conclusion 127
5.2 Recommendation 130
REFERENCE 131
APPENDICES 154
xii
LIST OF TABLES
2.1 Oil content of microalgae 19
2.2 The comparison of oil yields between microalgae and
other terrestrial crops
20
2.3 Lipid content and productivities of different microalgae
species
20
2.4 Comparison of microalgae with other biodiesel
feedstock
22
2.5 DOE Water Quality Index Classification 30
2.6 Water classes and uses 31
2.7 DOE Water Quality Classification based on Water
Quality Index
31
2.8 Trophic Status 32
2.9 The comparison between microalgae in open and closed
bioreactors
38
2.10 Review of various microalgae cultures conditions
adapted and optimized.
46
2.11 The chemical composition by using GC-MS analysis 51
2.12 Fatty acid profile of Nannochloropsis salina 52
2.13 Fatty acid profile of Botryococcus sp biodiesel 52
2.14 Overview of microalgae biodiesel properties 58
2.15 Comparison of properties of microalgae oils,
conventional diesel oil fuel and ASTM biodiesel
standard
59
3.1 Standard Method and equipment 65
3.2 Parameter requirement of biodiesel according to
ASTM D6751
88
xiii
4.1 The range, reading and mean values of water quality
parameters at different sampling time
92
4.2 The range, reading and mean values of water quality
parameters at different sampling time compared with
WQI class
93
4.3 Index range for water quality at Sembrong Dam 94
4.4 Trophic state vs chlorophyll-a 97
4.5 Trophic status 908
4.6 The highest reading of chlorophyll-a on 9 N:P ratios. 111
4.7 Effect of temperatures on lipid production 119
4.8 The final extraction and transesterification of
Botryococcus sp
120
4.9 Fatty acid compositions in Botryococcus sp 123
4.10 Botryococcus sp biodiesel analysis 126
xiv
LIST OF FIGURES
2.1 Biofuel production sources 13
2.2 Overview options to covert algal into energy 18
2.3 Molecular structure of tricylglycerols 19
2.4 The comparison of Oil productivity among the four type
of plants
23
2.5 Global Oil Product Demand 25
2.6 Satellite view of Sembrong Dam 26
2.7 Chlorophyll- a map of Sembrong Dam Catchment 27
2.8 Trophic states’s color intensity range 29
2.9 Open ponds photobioreactor 37
2.10 Closed photobioreactors 38
2.11 The overall process of transesterification of triglycerides 54
3.1 Dilution of the sample 67
3.2 Agar plates preparation 68
3.3 Microalgae observation under the microscope 74
3.4 The SEM Carl Zeiss EVO LS 10 71
3.5 Microscope and hemocytometer 70
3.6 Area in hemocytometer 72
3.7 The experiment setup for light optimization 74
3.8 The experiment preparation for pH optimization 75
3.9 The experiment preparation for speed optimization 76
3.10 Microalgae culture 79
3.11 (a) Centrifugation process; b) Botryococcus sp wet
biomass
81
3.12 a) The wet microalgae biomass in plates being dried in
oven: b) Dried microalgae homogenized by using mortal
81
xv
pestle to powder
3.13 Soxhlet extraction set-up 83
3.14 a) Extraction of microalgae: b) Drying the solvent inside
the oven: c) The lipids on the plates were weighed.
84
3.15 GC-MS machine 85
3.16 The mixture of methanol and NaOH 86
3.17 Transesterification’s steps to produce Botryococcus sp
biodiesel.
87
4.1 Chlorophyll-a sample reading within 4 consecutive weeks 96
4.2 Isolation: a) Colonies formed; b) Culturing
microalgae colonies
99
4.3 The comparison the Botryococcus sp cells pictures
captured under microscope
100
4.4 Surface microstructure of a colony Botryococcus sp from
Sembrong Dam
102
4.5 Surface microstructure of a colony Botryococcus sp 102
4.6 Microscopic picture of Botryococcus sp colony 103
4.7 Light optimization using BBM as growth medium 105
4.8 Initial pH optimization using BBM as growth medium 107
4.9 The cell was accumulated in the center of the flask. 108
4.10 Botryococcus brunii cells, grown in 500 mL in conical
Fernbach flasks upon orbital shaking.
109
4.11 Mixing speed optimization using BBM as growth medium 110
4.12 Light optimization using synthetic media 112
4.13 Initial pH optimization using synthetic media 113
4.14 Mixing speed optimization using synthetic media 114
4.15 The comparison of cell concentrations in light
optimization for both BBM and synthetic media.
115
4.16 The comparison of cell concentrations in initial pH
optimization for both BBM and synthetic media.
116
4.17 The comparison of cell concentrations in mixing speed
optimization for both BBM and synthetic media.
117
4.18 GC-MS chromatograph of Botryococcus sp biodiesel 122
xvi
LIST OF SYMBOLS AND ABBREVIATION
APHA - American public health association
ASE - Accelerated solvent extraction
ASTM - American society for testing and
materials
BLPD - Barrels of oil per day
BOD - Biochemical oxygen demand
BBM - Bold’s basal medium
cell/ml - Cell concentration
CLSM Confocal laser scanning microscopy
Co - Cobalt
COD - Chemical oxygen demand
CO2 - Carbon dioxide
Cu - Copper
DME - Dimethyl ether
DNA - Deoxyribonucleic acid
DOE - Department of environment
DO - Dissolved oxygen
EN - European standard
EPA - Environmental protection agency
FAME - Fatty acid methyl ester
Fe - Iron
GC-MS - Gas chromatography-Mass
spectrometry
GHG - Greenhouse gas
GWP - Green wall panel
ha - Hectare
xvii
HCO3 - Bicarbonate
HLPC - High pressure liquid
chromatography
H2SO 4 Sulphuric acid
IEA - International energy agency
IES - International energy statistic
INWQS - Interim national water quality
Standard for Malaysia
K - Potassium
Kg - Kilogram
KH2PO4 - Monopotassium phosphate
KOH - Potassium hyroxide
lux - Illuminance
MAE - Microwave extraction
Mt - Metric ton
Mn - Manganese
MUFA - Monounsaturated fatty acids
N - Nitrogen
NES - The national eutrophication survey
NH4 - H - Ammonium
NH3 - H - Ammoniacal nitrogen
NH4Cl - Ammonium chloride
NaOH - Sodium hydroxide
Ni - Nickel
NSF - National sanitation foundation
N:P - Nitrogen and phosphorus ratio
O2 Oxygen
OECD - Organisation for economic
Cooperation and development
P - Phosphorus
PBCSB - Pahang bio valley corporation Sdn
Bhd
pH - Power of hydrogen
xviii
ppm - parts per million
rpm - Revolutions per minute
RT - Retention time
RNA - Ribonucleic acid
SEM - Scanning electron microscopy
SFE - Supercritical fluid extraction
TAG - Triacylglyceride
TEM - Transmission electron microscopy
TLC - Thin layer chromatography
TP - Total phosphorus
TSS - Total suspended solid
UTHM - Universiti Tun Hussien Onn
Malaysia
U.S - United States
WQI - Water quality index
Zn - Zinc
xix
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Cell Concentration of Botryococcus sp
on Optimization Using BBM and N:P
150
B Process of Converting Botryococcus sp
Into Biodiesel
160
C GC-MS Chromatograph of Botryococcus
sp Biodiesel (fames)
163
1
CHAPTER 1
INTRODUCTION
1.1 Background of study
Nowadays the human population is rapidly increasing that has burst into the
phenomena of over-population which has induced further industrialization, leads into
invention of enormous modes of transportation as well as machineries which have
triggered energy shortages. This great challenge will be more worsening in the near
future and will hit the hydrocarbon-driven society imminently. The demand for
commodities will increase explosively as fossil fuel is the primary source of energy
and currently its rate of depletion is dwindling by time (Scott et al., 2010).
Fossil fuel is non-renewable and unsustainable that has becoming the
impediment for energy sustainability. Not only that, the continual use of fossil fuels
increases the greenhouse gas which is directly related to global warming. The
negative implications of fossil fuels towards the humanity and environmental has
made biodiesel as an attractive source of energy in the future (Ashokkumar et al.,
2014).
According to Hou et al., (2011) instead of working on the same viable
sources for biofuel production such as corn, palm oil, rapeseed, soya bean and many
other, many researchers are inclined to utterly turn to microalgae owing to their most
appealing property. Microalgae can produce more oil compared to the other
feedstock productivity which is 3-35 times higher than terrestrial plants in terms of
oil content (Mata et al., 2010) More than that, the microalgae are absolutely aquatic,
non-edible, highly genetically modifiable and fast-growing. The cultivation of
microalgae needs less water intensive and their species can even grow in brackish
2
water or seawater, thereby avoiding demand for fresh water (Mata et al., 2010), thus
more adaptable to marginal lands.
According to Nagaraja et al., (2014) the growth of microalgae will be
tremendously better in wastewater with enough nutrients from nitrogen and
phosphorus compound. The wastewater is the ideal place for the habitation due to the
high nutrient quantity in wastewater and microalgae is feasible to be grown within a
broad range of environments, started from the open ocean through to land-based
tanks and ponds production farming, therefore makes microalgae to have no limited
areas for cultivation thereby avoiding the highly contentious food versus fuel debate
(Chisti, 2007).
In recent years, scientists have been using various mechanical and chemical
methods to extract oil from microalgae and convert it into fuel (Kong et al., 2012).
The oil extracted is referred to as green crude that is not ready to be used as fuel until
it undergoes transesterification process. The process of converting microalgae into
biofuel in the large scale is indeed costly and expensive but today there are many
researchers trying to make the production more economical and sustainable (Alwi,
2014 & Mata et al., 2010).
Biodiesel is an example of biofuel and a promising source of energy, its
production will be beneficial towards the sustainability of energy production
especially in dealing with the depletion of fossil fuels feedstock by developing a long
term replacement of fossil fuels. Therefore, exploring natural microalgae bloom from
local dam to be extracted as biofuel is an attempt as alternative source of energy.
1.2 Problem statement
Biofuels has staggeringly became the public attention, moved by factors such as oil
price hikes, safeguarding energy security, concern upon greenhouse gas emissions
from fossil fuels, and the continuous support from government subsidies. According
to International Energy Agency (IEA), the global biofuels output reached 120 billion
liters in 2013 and now provides 3.5% of world transport fuel demand and in the near
future the biofuel production is expected to increase 25% to 2018 that will provide
4% of global road transport fuel demand. Fossil fuel remained as the primary source
of energy and if the dependency over it is prolonged the fuel stock would someday
be used up.
3
The price of the fossil fuels is unstable and inconsistent. In near Future, the
price of fuels will be sky rocketing and its volatility could jeopardize the energy
security as well as the whole economy of the world. The approach of sustainable fuel
will be able to control and guarantee the stability of oil price over times.
The burning of fossil fuels over the past 100 years or so, has contributed
trillions tons of CO2. Since around 1920, the large scale of commercial extraction of
petroleum, the CO2 embedded within oil, gas and coal once was permanently store
underground and being there since the era of existence of dinosaurs and owing to
human disruption the CO2 were bringing up to the surface and burning it (Shannon,
2013).
More than that, Ong et al., (2011) stressed that the extreme usage of diesel
and petrol in transportation is the major cause in declining Malaysian oil reserve. In
addition, Malaysia, primarily still relying on using natural gas for electricity
generation which account for about 60 % fuel mix in electricity generation in year
2016 (Eleventh Malaysia Plan).
Upon realising high demand of alternative source for biodiesel, extensive
study on bioresources for biofuel is currently needed and the choice of bioresource
should be easily available for ensuring the sustainability of the source. Therefore,
microalgae are one of the best choices for biofuel.
The algal bloom at Sembrong Dam due to non-points pollution coming from
agricultural source has created unpleasant conditions at the dam. Nevertheless,
besides adopting microalgae treatment method, the unexploited algal could be
isolated as a potential species for biodiesel production. Initial study on dam water
characteristic should also be studied to stimulate favorable condition for microalgae
growth. Other experimental were conducted namely optimization of factor
influencing microalgae growth, biodiesel conversion and biodiesel testing would
beneficial to produce good quality biodiesel.
Microalgae has been highlighted as one of most promising alternative source
of energy due to its high oil yields which is achievable up to 70% and under the
good conditions some species able to reach 90% of dry weight (Sheehan et al.,
1998). Some microalgae species able to produce up to 15,000 gallons of oil
per acre per year, by comparison the microalgae oil able to produce 20-40 % times
more productivity than another type of terrestrial crops such as soya bean, palm trees,
sugar and many others (Sheehan et al., 1998).
4
According to Vicente et al., (2004) the biodiesel production is better than the
fossil-driven diesel, that is renewable, biodegradable and low pollutant emission. It
had been proved long time ago that microalgae could produce up to 70 percent of
oxygen in the atmosphere owing to the huge population of microalgae in ocean, as
the oceans cover about 71% of the earth as stated by Jack (2015).
1.3 Objective of study
Objective of this research is to:
i. To isolate and characterize microalgae Botryococcus sp species from
Sembrong Dam
ii. To optimize the factors influencing the growth of Botryococcsu sp for
higher biomass yielding.
iii. To determine and characterize the feasibility of Botryococcus sp fuel
properties to be used as biodiesel feedstock.
1.4 Scope of study
In this study, microalgae strains were collected at Sembrong Dam water catchment
which is located about 10km from Air Hitam in the state of Johor. The dam water
containing visible algal was collected and brought to the lab in UTHM for the
isolation of the microalgae and water quality characterization. The quality of the
water from Sembrong Dam was analyzed by measuring the chemical properties of
wastewater such as pH, DO, COD, BOD, nitrate, phosphate, conductivity, TSS and
Chlorophyll-a based on the Standard Method and Examination of Wastewater (2012)
prior the isolation of microalgae in order to relate the existing of algal population
Sembrong Dam water catchment as well as to stimulate the growth of Botryococccus
sp based on the actual strength of nutrients measured in Sembrong Dam water. The
isolation and characterization of microalgae were carried out by using preliminary
morphological identification by using light microscope (Metzger and Largeau, 2005).
One of the most dominant species had been successfully isolated. The factors
affecting the growth rates of Botryococcus sp such as light, initial pH and orbital
shaker rotation speed had been experimented. Carbon dioxide is not included due to
limitation of closed bioreactor and it sourced naturally from atmosphere. Once
5
nutrient supplement or microalgae media growth has optimally modified, the mass
cultivation of microalgae were cultivated and harvested after three weeks of
cultivation at optimum growth conditions for three batches. After the harvesting
process, the extraction of microalgae lipids was performed using soxhlet extraction
based on the standard (EPA Method 9071B), the method also conducted by Rawat et
al., (2011). The examination of fatty acid methyl ester (FAMEs) of microalgae
biodiesel was determined using GG-MS screening analysis. The conversion of lipids
into biodiesel was conducted through transesterification by using sodium methoxide
as catalyst (Farooq et al., 2013). Finally, Botryococcus sp biodiesel underwent fuel
properties quality examination which includes the kinematic viscosity, flash point
and density, acid number, water content and moisture content based on the ASTM
D6571.
1.5 Importance of study
The significance of this study is to fully utilize over the third generation biofuels
which is oil-rich microalgae. Problems arising coming from the continual use of
fossil fuels is worsening over times such as supply shortages, pollution and price
hikes. Biodiesel which is a renewable source of energy and sustainable is capable to
control the oil price and to reduce the dependence upon fossil fuels.
More than that, cultivation of microalgae will be able to reduce the CO2 from
atmosphere because microalgae will convert it into biomass energy and thus the
release of oxygen will increase. Decrease in carbon dioxide rates in atmosphere
which in turns will have the greatest effect on the environment and an effective way
to control global warming. Apart from that, the nutrients in wastewater can be
reduced by culturing microalgae species in wastewater. Thus, there is great potential
of microalgae to be used to treat the wastewater to remove the chemical and organic
contaminants, heavy metal and pathogen.
For further development, this study could be applied in Malaysia as the
production of biofuel in Malaysia still negligible because of its small scale
production. Thus, the potential fuel shortage supply in Malaysia can be secured with
the implementation of large scale of microalgae cultivation. Therefore, the utilization
over microalgae will be beneficial solution for sustainable source energy and more
environment friendly biodiesel production
6
1.6 Limitation of study
There are a couple of limitations has been set in this study and one of them is the
exclusion of carbon dioxide factor to be optimized due to limitation of closed
bioreactor as it is expensive to purchase. Thus, carbon dioxide was sourced from the
surrounding atmosphere as this way of supplying could be applied for large scale of
cultivation to meet the cost-effective cultivation as well as to reduce the carbon
dioxide level in atmosphere. The limitation of the ranges of light intensity and initial
pH value was based on previous study on genus Botryococcus as well as other
freshwater green microalgae species.
Furthermore, rotation speed optimization was limited to only 30 rpm which is
the lowest speed settable for laboratory orbital shaker. The growth of Botryocccus sp
grew better as the rotation speeds were reduced. Therefore, due to this limitation the
optimum speed rotation for Botryococcus sp is unknown as the most abundant
biomass yielded observed at speed 30 rpm. By the way, the speed optimization
proved that Botryococcus sp contains higher hydrocarbon as they tend to congregate
and had a slow growth when being continuously shaken at higher speed.
1.7 Organization of the thesis
Chapter 1 of this study introduced the topic research which is the potential of
microalgae as a biodiesel feedstock and highlighting the specific problems arising
from fossil fuel as primary source of fuel. In this part, the thesis is constructed by its
objectives that focusing over isolation of microalgae strains, optimization of the
factor influencing the growth of isolated microalgae, the oil productivity as well as
the fuel properties of microalgae biodiesel. More than that, the scope of study and
limitation of study in this part also constructed the whole thesis as they determine
and limit and scope of the research.
Chapter 2 presents a review of literature and relevant research associated with
the problem addressed in this study, the methods and techniques of experiment
involved in this study such as the isolation techniques, morphological techniques,
extraction method, transesterification method as well as the testing methods for water
quality and biodiesel properties assessment. More than that, the review of advantage
of microalgae as biodiesel feedstock had been highlighted. The results from previous
7
study are very helpful especially to adopt the ranges of value for microalgae growth
optimization and to compare the whole result of the study for further discussion. The
descriptive research questions can be found in the entire of this literature review part.
Chapter 3 presents the methodology and procedures used in the study. The
examination of Dam water quality is conducted following the Standard Method and
Examination of Wastewater 22st edition. The method adopted for chlorophyll-a
concentration is based on ISO 10260 while the isolation of Botryococcus sp was
conducted based on the standard plating method adopted from APHA Method 9215
and previous studies (Lee et al., 2014 & Duong et al., 2012). In addition, the soxhlet
extraction was conducted according to EPA Method 9071B. Since there is no
standard method established for transesterification for algal oil, the methods from
previous study had been adapted from transesterification lab procedure using alkali
catalyst and methanol as well as based on previous study (Farooq et al 2013; Duong
et al., 2012 & Phang and Chu, 1999). Lastly, the fuel properties of microalgae
biodiesel were adopted according to ASTM 6715 and EN 14214.
Chapter 4 contains an analysis of the data and presentation of the whole result
in this study. First and foremost the quality of Sembrong Dam is polluted and needs a
treatment based on INQWS classification. The presence of algal population has
caused the dam water to be in hypereutrophication state based on Us Trophic Status
Index (1974) and Carlson Trophic State index (1977). The isolated strain belongs to
genus Botryococcus after examined under light microscopy and scanning electron
microscopy. Furthermore, the optimal growth of Botryococcus sp is achieved when
illuminated with 6000 lux, cultured with initial pH 7 and its growth was not growing
well when continuously rotated at higher rpm. The FAMEs analysis of Botryococcus
sp has revealed the four major fatty acids which are palmitic acid, oleic adid, linoleic
acid and octadecanoic acid. Apart from that, the lipid production of Botryococcus sp
can be as high as 64.8 % of its dry weight biomass. The transesterification of
microalgae oil into biodiesel can be up to 88 % of microalgae oil weight. Finally, the
biodiesel properties comply with ASTM D6715.
Chapter 5 offers a summary and discussion of the researcher's findings,
implications for practice, and recommendations for future research. Botryococcus sp
is the best choice for microalgae biodiesel feedstock due to its high lipids content. On
top of that, the production of microalgae biodiesel should be commenced in Malaysia
8
as to start the inaugural microalgae biodiesel that will be beneficial towards the
sustainability of energy in Malaysia. Botryococcus sp can be further studied
especially the optimization of the other factors influencing its growth rate as well as
to find the effective way to fully extract the oil content and turn them into biodiesel.
9
CHAPTER 2
LITERATURE REVIEW
2.1 Introduction
Nowadays, the demand of fuel has become a serious concern among the countries
around the globe. As the fossil-driven fuel is still the dominant among another
sources, the adverse effect will be increasing over items and the air pollution will be
worsening that will lead to the increase of global warming. The fossil fuel feedstock
is running low over times in accordance with the advancement of technology and has
attracted many researchers to turn into another alternative feedstock that are
sustainability and renewable. Microalgae had been identified as a plant that produces
the most oil yield as studied by Chisti (2007). In present, the production of biofuel
keeps increasing and United States emerges as a top country producing the largest
biofuel from microalgae with 898 thousand barrels per day (Alwi, 2014). In fact, in
Malaysia there is no implementation of any project for microalgae biodiesel
production but recently Pahang Bio Valley Corporation Sdn Bhd (PBCSB) is started
implementing their large project called “Pahang Bio Valley” for primary production
of microalgae crude oil and microalgae meal that still under construction. In this
study, the information, methods and techniques are collected from different sources
of information.
2.2 Microalgae
Microalgae is known as phytoplankton by many biologist and they are living
organisms that look alike a small-plant organisms that have no roots and leaves and
10
they also called as eukaryotic photosynthetic microorganisms that able to grow
rapidly and can live in harsh conditions due to their unicellular (Chisti, 2007). Apart
from that, they are aquatic biomass same with the seaweeds (Wolkers et al., 2011).
The microalgae is considered as a very common as they existed in hundreds and
thousands species and can live both in freshwater and seawater.
Interestingly, microalgae are living in all existing earth ecosystem, not only
present in aquatic but also terrestrial which makes them as the abundant species
living in a vast range of environmental conditions and there are more than 50,000
species were estimated and of around 30,000 so far have been analysed and studied
(Richard, 2004). With time, microalgae are colonizing the entire planet as they can
be found in everywhere including in marine environments like in the lakes, geysers,
ice, snow, high mountains, wastewater, and soil (Scott et al., 2010). More than that,
the so-called term microalgae is referring into different unicellular eukaryotic
organisms which belong to several classes such as Cyanophyceae (cyanobacteria),
Euglenophyceae, Chlorophyceae, Xanthophyceae, Rhodophyceae, and
Bacillariophyceae which having different photosynthetic assimilatory pigments
(Minowa et al.,1999).
Most of the species of microalgae are containing chlorophyll-a which utilize
over the sunlight to make its own food or the sunlight as their source of energy and
they are capable to convert carbon dioxide (CO2) into biomass (Wolker et al., 2011).
During the photosynthesis takes place the microalgae releases oxygen O2 (Mata et
al., 2010). The microalgae generate about 75 % of oxygen to be consumed for
animals and humans based on a global scale and normally the microalgae cannot be
seen by naked eyes but when they are growing in a eutrophic, massive algal blooms
will occur and turning the colour of water into green, brown, blue, or orange liquid
mass (Wolkers et al., 2011). Microalgae possesses high-quantity oils, partly
consisting out of omega-3 and omega-6 fatty acids, that is particularly used as raw
material for food supplements and a good choice for biofuel feedstock (Mata et al.,
2010). Microalgae was suggested as a good candidate for producing biofuel because
of their high lipid content, high photosynthetic efficiency, high biomass production
and fast growth compared to other energy crops (Chisti, 2007).
11
2.2.1 Historical development of microalgae production system.
For the past 55 years ago, the quest of energy-rich oil from various microalgae
species has been incredibly increased (Channamtum et al., 2015). The researchers
were performing variety of research to fully optimize green microalgae for higher
production of biofuel (Sharma, 2011; Ethier et al., 2011; Dayananda et al., 2005).
The first large cultivation of microalgae was initiated in the early 1960s in Japan by
culturing Chlorella (Spolaore et al., 2006). Afterwards, microalgae started to gain
popularity in 1970s as an alternative source of renewable energy during the first oil
crisis (Spolaore et al., 2006).
More than that, The United State National Renewable Energy Laboratory
(NREL) had launched a Research & Development program through the Aquatic
Species Program (ASP) with interest of alternative renewable fuels and also
comprising the production of biodiesel from microalgae with goals to explore the
biochemistry and physiology of lipid production of oleaginous microalgae and
improve the strain through genetic variability (Sheehan et al., 1998). After being
operated for years, the cost production of biodiesel by using microalgae was
technically feasible, but the R&D with its long term vision to attain the high
productivities as possible. Nevertheless, the Department of Energy reduced the
financial plan allocated in funding this program and this hampered and discontinued
the planned experiments (Sheehan et al., 1998).
The volatility of current crude oil with unstable prices and a push of cutting
down pollutant emissions and greenhouse lead to an increase of microalgae biodiesel
production (Barclay, 2005). Therefore, several emerging companies were observed
selling key process units for photo-bioreactors equipped with optimized designs for
large scale cultivation of microalgae (Behrens et al., 2007 & Kanel, 1999). As
studied by Torrey (2008), there are 37 companies with interest of using microalgae
as fuel source.
Microalgae are also considered as the second generation fuel feedstock with
other crops such as jatropha, lignocellulosic materials, agriculture residues and the
systematically grown energy-source crops (Barclay, 2005) which make them
valuable for better energy sustainability. Although, there is no cost effective or
sufficient budget in competing with fossil fuel from any funding or organizations, yet
12
the research still in the running for better microalgae biofuel production (Yokochi et
al., 2004 & Bijl, 2001).
Furthermore, applying metabolic engineering genetic methods towards
microalgae growth with aims to optimize their productivity for abundance
hydrocarbon evidenced the well-progressing of microalgae development (Rosenberg
et al., 2008 & Barclay, 2005).
2.3 Biofuel
Biofuel can be classified into two categories which are primary and secondary
biofuels (Nigam & Singh, 2011). The primary biofuels normally used for an
unprocessed form such as for heating, cooking, and electricity production like fuel
wood, wood chips and many other. Meanwhile, the secondary biofuel basically
generated from the work of processing the biomass such as ethanol, biodiesel,
dimethyl ether (DME) and many others for vehicles and any industrial processes.
More than that, the secondary biofuels also divided into three categories
namely first, second and third generation biofuel (Dragon et al., 2012). Figure 2.1
shows the basis of raw materials and also technology used for the production of
biofuels. Besides that, biofuels are also categorized based on the source, type and the
origins for example some of them may be derived from forest, fishery, agricultural
products and municipal wastes, also they also originated from ago-industry, food
industry and food services (Gupta et al., 2013 & Dragon et al., 2012).
In fact, the biofuels can be in form of solid such as charcoal, fuel wood, and
wood pellets or they also can be in the form of liquid, such as ethanol, biodiesel and
pyrolysis oils or the third one they may exist in the form of gaseous such as biogas
(methane) (Dragon et al., 2012).
13
Figure 2.1: Biofuel production sources (Dragon et al., 2012).
2.4 Biodiesel
Biodiesel is best defined as an alternative fuel which exhibits the same function as
the fossil fuels (Fangrui & Hanna, 1998). Biodiesel can be generated directly from
vegetable oil, animal oil or fats, plants oil, tallow and waste cooking by
transesterification process which changes the organic group R of an ester with the
organic group R of an alcohol (Horseman et al., 2008). Based on the previous
research, the high potential of oil sources for biodiesel is coming from crops such as
rapeseed, palm oil and soybean and recently microalgae has been the most promising
among all of them (Mata et al., 2010 & Chisti, 2007).
In United Kingdom, rapeseed has become a big deal for biodiesel production
(Marcos & carlos, 2007). Besides, United States produced biodiesel as high as 691
million gallons in 2009, even though the annual production decreased to as low as
490 million gallons in 2010, the amount of overall biodiesel production is considered
plentiful (Ethier et al., 2011).
14
Numerous researches proved that biodiesel has great advantages over fossil
fuel (Dragon et al., 2012; Mata et al., 2010 & Chisti, 2007). As a renewable energy
source, biodiesel is an environmental friendly energy that releases less greenhouse
gas, discharges less carbon dioxide and low content sulphur and carbon monoxide
(Fangrui & Hanna, 1998)
Technically speaking, 90% of air toxicity and 95% of cancers can be
decreased by biodiesel due to their less pollutants emission (Huang et al., 2010).
Furthermore, biodiesel has a great potential in the energy market since the price of
fossil fuel will be tremendously skyrocketed imminently and the high energy demand
(Alwi, 2014). The renewable microalgae energy will not have an issue with price
hike as they can be produced sustainably and the price will be well- balanced by the
intense competing of wide markets around the globe.
2.4.1 Biodiesel in Malaysia
Biofuel production in Malaysia is synonymous with palm oil, a major established
agricultural product in Malaysia. The average national blend of biodiesel in
Malaysia’s transport diesel pool has steadily increased since 2011 by blending the
biodiesel from palm oils with the conventional diesel. From 1.3% in 2011, where
biofuel was only available in the central region of Negeri Sembilan and Selangor
states, it increased to 2.0% in 2012 when biofuel was available in the Southern
region of Malacca and Johore states. When the government fully committed to
implement B5 program in 2014, it increased to 5%. After many delays, the
Government of Malaysia’s (GOM) introduced the B7 mandate in 2015, thus
increased the national blend rate to 7%. In 2015, Malaysia exported 65 % of
biodiesel to Spain, 19% to the Netherlands and 11% to Switzerland (Wahab, 2016).
As far as today Malaysia has not developed the biodiesel based on microalgae
feedstock (Huang et al., 2010). The feasibility of microalgae as biodiesel feedstock is
still under research and has yet to be implemented.
2.4.2 Microalgae biodiesel
Microalgae biodiesel or algal biodiesel is an alternative source of fuels that
uses microalgae as its source of energy-rich oils (Prommuak et al., 2012). Also,
15
microalgae biodiesel are an alternative to common known biofuel sources, such as
corn, sugarcane, soya bean and palm oil. They are several companies and
government agencies are funding efforts to cut down capital and operating costs as
well as make microalgae fuel production commercially viable (Scott et al., 2010).
Like fossil fuel, microalgae biodiesel releases carbon dioxide when burnt, but unlike
fossil fuel, microalgae fuel and other biofuels only release carbon dioxide recently
removed from the atmosphere via photosynthesis as the microalgae or plant grew.
Microalgae biodiesel has attractive characteristics that they can be grown
with minimal impact on fresh water resources, can be also produced using saline
and wastewater, have a high flash point, biodegradable and relatively harmless to the
environment if spilled (Dinh et al., 2009 & Aslan & Kapdan, 2008). Microalgae
costs more per unit mass than other crops due to high capital and operating costs but
are claimed to yield between 10 and 100 times more fuel per unit area (Mata et al.,
2010 & Chisti, 2007)
Lately, researchers are utilizing microalgae to produce biofuels, chemicals,
oils, food, medicine and many other (Lang et al., 2001). Although some researches
are inclined to use crops such as corn, soybean, repressed and palm oil for biodiesel
production and yet some green microalgae are still topping them with possibility of
75 % oil of its dry weight (Metzger & largeau, 2005). Nevertheless, as far as today,
biodiesel cannot meet the increased energy demand since their production is too low
due to the higher cost of cultivation. However, its biodiesel productivity is much
better than crops plants (Chisti, 2007).
More than that, the cultivation area is also an important factor for choosing
the best microalgae biodiesel feedstock. Converting microalgae into biodiesel
requires area of land that is as low as 1% - 2.5% of the total cropland compared to
the other crops that need larger land area for production (Chisti, 2007). What is more,
microalgae biodiesel production can be reached as high as 93% from the lipids
weight (Prasad et al., 2015 & Li et al., 2007). Thus, considering the satiation of
increasing energy demand and cultivation land area needs, the renewable microalgae
biodiesel seems to be the most appropriate source of feedstock. Production of
microalgae biodiesel is hard to meet economic challenges because the price for
harvesting and dewatering are high, and the application for oil extraction is
complicated (Pooja & Himabinda, 2014).
16
2.4.3 Advantages of using microalgae for biodiesel production
According to Sheehan et al., (1998), microalgae can be cultivated easily and can
grow using water that is unsuitable for human consumption such as waste water and
lastly easy to get nutrients. Alwi, (2014) also concluded that microalgae practically
able to grow anywhere including in the dessert and marine environments, thus makes
them so flexible and will having no intense competition with another food crops.
Furthermore, microalgae can grow almost anywhere just needing sunlight and some
simple nutrients and it is possible to accelerate its growth by adding some specific
nutrients and with sufficient aeration as stated by Aslan & Kapdan (2008).
Microalgae is effortless for cultivation, it can grow with little of them even no
attention, using any water that unsuitable for human consumption which is
containing nutrients prerequisite for their growth and microalgae will reproduce
themselves through photosynthesis to convert sunlight energy into chemical energy
in order for completing an entire growth cycle (Li et al., 2008; Reinhardt et al., 2008
& Sheehan et al., 1998). Furthermore, microalgae are incredibly flexible whereby
they can grow to almost everywhere, needing sunlight and with simple nutrients such
as phosphorus and nitrogen compound, however their growth can be accelerated by
adding some specific growth driving nutrients and with sufficient aeration (Aslan and
Kapdan, 2006 & Pratoomyot et al., 2005).
There are thousands species of microalgae, thus, it has a high possibility to
find the right species that suitable for growing with local environment which is not
possible for the other crops and producing biodiesel feedstocks such as soybean,
rapeseed, sunflower and palm oil. The implementation of mass cultivation of
microalgae can be initiated once the most suitable microalgae species containing rich
oil are successfully adapted to local environment conditions (Sharma, 2011).
Microalgae has higher growth rates and productivity than any other plants or crops
pertaining for biodiesel production in which they are requiring less land area, it is up
to 49 to 132 time less land are compare to soybean crops (Chisti, 2007). Thus,
limitation of arable soil for crops would not going to give much impact towards the
cultivation of microalgae which requires less land area compared to the others crops.
Microalgae are able to generate feedstock for numbers of renewable fuels
such as methane, hydrogen, methane, and many others. More than that, microalgae
based biodiesel contain no sulfur but still functioning same as petroleum diesel
17
which able to reduce emissions of particulate matter such as carbon dioxide,
hydrocarbon and sulphur oxides (Pires et al., 2012). The emission of nitrogen
dioxide maybe higher depending on engine types (Delluchi, 2003). The biofuel from
microalgae also serve other purposes such as having a big potential as an agent of
removal CO2 from industrial flue gases through microalgae bio-fixation, which able
to in reducing the GHG emissions (Wang et al., 2008).
Furthermore, microalgae can be used for waste water by removing
ammonium, nitrate, phosphorus and microalgae also can be grown in the waste water
as stated by Wang et al., (2008). After the oil from microalgae is extracted, the
resulting microalgae biomass can be processed for production of ethanol, methane,
livestock fees, fertilizers because of its high N:P ratio, and can be burned for energy
cogeneration either for electricity or heat (Wang et al., 2008). More than that, the
other species of microalgae is also viable to be extracted into variety products which
including a large range of fine chemicals and bulk products such as fats, natural dyes,
oil, sugars, antioxidants, high-value bioactive compounds, polyunsaturated fatty
acids (Raja et al., 2008 & Horseman et al., 2008).
Finally, the present of high-value derivatives can potentially make microalgae
to be revolutionized into the field of biotechnology including biofuel,
pharmaceuticals, cosmetic, nutrition, food additives, and aquaculture (Rosenberg et
al., 2008 & Li et al., 2008). There are many ways of producing biofuel with
microalgae. The overview of the options available is shown in Figure 2.2.
18
Figure 2.2: Overview options to covert algal into energy (Iersel & Flammini, 2010).
2.5 The potential of microalgae as bioenergy
As a matter of fact, microalgae can be fully utilized in generating energy in several
ways. Currently, one of the most sought after of utilization of microalgae is to turn
them into microalgal biodiesel. As far as today, there are many other valuable
products have been introduced from microalgae such as the production of hydrogen
gas under specialized growth conditions (Canakci & Van, 1999). More than that, the
biomass from microalgae can be used in generating electricity and heat when they
are burnt (Wang et al., 2008)
Microalgae biomass has three main components which are carbohydrates,
proteins, lipids or natural oils. Biodiesel can be produced from any oil or lipid source
that contains largely components of tricylglycerol molecules (TAGs) such as shown
in Figure 2.3. The present of bulk natural oil made by microalgae in the form of
Hydrocarbon
Biomass
HTU Fuel gas
Lipids
Gasification
Unique Products
Fermentation
Ethanol
Digestion
Methane
Hydrogen
Heat
Burning Biodiesel
Starch
Esterification
Haydrocracking
Purification
Fermentation
SVO
0il
High Value Products
Hydrogen
Traditional Fuels
Methanol
19
TAGs makes the microalgae oil the right kind of oil for producing biodiesel
(Grobbelaar, 2004).
Figure 2.3: Molecular structure of tricylglycerols (Grobbelaar,2004).
According to Chisti (2007) microalgae are commonly doubled in size for
every 24 hours and at the optimum growth phase, some microalgae able to double
every 3.5 hours. Furthermore, the oil content of microalgae can be between 20 % up
to 75 % of its dry weight as shown in Table 2.1, while some species able to reach up
to 80 % (Spolaore et al., 2006 & Metting, 1996).
Table 2.1: Oil content of microalgae (Chisti, 2007 & Zhiyou, 2009)
Microalga Oil content (% dry weight)
Botryococcus braunii 25–75
Chlorella sp. 28–32
Crypthecodinium cohnii 20
Cylindrotheca sp. 16–37
Nitzschia sp. 45–47
Phaeodactylum tricornutum 20–30
Schizochytrium sp. 50–77
Tetraselmis suecia 15–23
In comparison with the other terrestrial crops, it takes much longer time to
grow and lesser oil yield or particularly, contain roughly of about 5 % of oil yielding.
Thus, makes the green microalgae as the most favourable for biodiesel feedstock.
CH2O-OCHCH2CH2 ………. CH2CH3
CHO-OCHCH2CH2 ………. . CH2CH3
CH2O-OCHCH2CH2 ………. CH2CH3
20
The different oil yield percentages by microalgae compared with the other terrestrial
crops in the open pond cultivation is shown in Table 2.2. Based on the previous
study, the oil yield of microalgae is superior to the other terrestrial crops as shown in
Table 2.2.
Table 2.2: The comparison of oil yields between microalgae and other terrestrial
crops (Mata et al., 2010; Zhiyou, 2009; Chisti, 2007)
2.6 Microalgae lipid content and productivities
Generally, numerous number of microalgae species can be induced to generate
substantial quantities of lipids as studied by Sheehan et al., (1998).The productivity
of oil yield was studied to be as high as 75% and under the good conditions some
species able to reach 90% (Li et al., 2008; Chisti, 2007; Spolaore et al., 2006). Table
2.3 shows the lipid content and biomass productivities between different freshwater
and marine microalgae species that are totally different from previous researches
(Rodolfi et al., 2009; Li et al., 2008; Spolaore et al., 2006; Richmond, 2004; Renaud
et al., 1999).
Furthermore, Table 2.3 evidenced that genus from Botryococcus is able to
produce high oil yield as high as 75% by weight from dry biomass. Thus,
Botryococcus braunii is a good choice for biodiesel feedstock. In selecting the most
adequate species for cultivation there is a need to take into account another factor,
such as the capability of microalgae to be developed by using nutrients available and
under particular environmental conditions for biodiesel production.
Crop Oil yield (gallons/acre)
Corn 18
Soybeans 48
Canola 127
Jatropha 202
Coconut 287
Oil palm 636
Microalgae 6283-14641
21
Table 2.3: Lipid content and productivities of microalgae (Richmond, 2004)
Mata et al., (2010) proved that the microalgae oil yield is supremely greater
than the another vegetable oil crops as shown in Table 2.4 that compares the
biodiesel production efficiencies and land use for microalgae cultivation and other
vegetable oil crops, including the yielding of biodiesel. Generally, Table 2.4
portraying the advantages of microalgae as the best choice for biodiesel as they are
capable to produce the highest oil content up to 70 %, has the slightest land use,
highest oil yield (L oil/ha year) as well as the highest biodiesel productivity (Kg
biodiesel/ha year). Thus, the development of microalgae as a biodiesel feedstock is
very promising to neutralize the imbalance and immoderately usage of fossil fuel.
Marine and
freshwater
microalgae species
Lipid content
(% dry weight
biomass)
Lipid
productivity
(mg/L/day)
Volumetric
productivity of
biomass (g/L/day)
Areal productivity
of biomass (g/m2
/day)
Ankistrodesmus sp. 24.0-31.0 - - 11.5–17.4
Botryococcus braunii 25.0–75.0 - 0.02 3.0
Chaetoceros muelleri 33.6 21.8 0.07 -
Chaetoceros
calcitrans
14.6–16.4/39.8 17.6 0.04 -
Chlorella emersonii 25.0–63.0 10.3–50.0 0.036–0.041 0.91–0.97
Chlorella
protothecoides
14.6–57.8 1214 2.00–7.70 -
Phaeodactylum
tricornutum
18.0–57.0 44.8 0.003–1.9 2.4–21
Porphyridium
cruentum
9.0–18.8/60.7 34.8 0.36–1.50 25
Scenedesmus
obliquus
11.0–55.0 - 0.004–0.74 -
Scenedesmus
quadricauda
1.9–18.4 35.1 0.19 -
Scenedesmus sp. 19.6–21.1 40.8–53.9 0.03–0.26 2.43–13.52
Skeletonema sp. 13.3–31.8 27.3 0.09 -
Skeletonema
costatum
13.5–51.3 17.4 0.08 -
Spirulina platensis 4.0–16.6 0.06–4.3 1.5–14.5/24–51
Spirulina maxima 20.6 17.4 0.08 -
Thalassiosira
pseudonana
8.5–23.0 27.0–36.4 0.12–0.32 19
Tetraselmis suecica 12.6–14.7 43.4 0.30 -
22
Table 2.4: Comparison of microalgae with other biodiesel feedstock (Mata et al.,
2010).
According to a research study by Alwi (2014), microalgae oil production is
indeed much greater than other any terrestrial crops. He created a chart bar to
compare the oil yields among the other crops such as soybean, rapeseed, babassu,
palm oil and microalgae as shown in Figure 2.4. The result shows that among them
microalgae produces more than 150 times fuel compared to soybean.
Plant source Seed oil content (%
oil by wt in
biomass)
Oil yield (L
oil/ha year)
Land use (m2
year/kg
biodiesel)
Biodiesel
productivity (kg
biodiesel/ha year)
Corn/Maize 44 172 66 152
Hemp 33 363 31 321
Soybean 18 636 18 562
Jatropha 28 741 15 656
Camelina 42 915 12 809
Canola/Rapeseed 41 974 12 862
Sunflower 40 1070 11 946
Castor 48 1370 9 1156
Palm oil 36 5366 2 4747
Microalgae 30 58,700 0.2 51927
Microalgae 50 97,800 0.1 86515
Microalgae 70 136,900 0.1 121104
23
Figure 2.4: The comparison of Oil productivity among the four type of plants
(Alwi, 2014)
2.7 Global outlook on fuel and biodiesel
Nowadays, global oil consumption is increasing at an unprecedented rate owing to a
rising in consumption of fuels in some developing nations. Started 1990 to 2012, big
countries such as China, India, and Brazil have an increase in oil consumption by
311%, 186% and 100% respectively and at present consume about 19.6% of the 3590
tonnes (Mt) of the oil generated annually (Enerdata, 2013). In 2002, U.S Geological
had published a survey of 128 territories which is containing 95% of discovered of
potential oil and gas reserves in the world (Balzani & Armaroli, 2007). From that
survey, it was concluded that the remaining undiscovered and discovered reserves of
oil from the unconventional and conventional sources are around 2.3 trillion barrels
of oil equivalent. In 2008, the International Energy Agency (IEA) conducted a
separate study and estimated the recoverable resource with value of 3.5 trillion
barrels of conventional oil left (IEA, 2008) and it shows the oils reserve is being
dwindling.
Furthermore, IEA had reported that a daily global consumption of fuels is
about 85.41 mega barrels for 2008 (Owen et al., 2010). There is possibility that the
huge rate of consumption which is equivalent to 311.2 giga barrels a year and by
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
Soybean Rapeseed Babassu Palm oil Microalgae
Oil
yiel
d (
kg/h
ect
re)
24
assumption of zero growth in the global demand, the complete depletion of oil or
liquid fuels and gas reserves will occur in just over 100 years (Owen et al., 2010).
The earth’s population is increasing, and the poorer countries are struggling for
higher energy consumption and lead to an increase by 2% of the yearly growth of
global energy demand (Balzani and Armaroli, 2007). Some developed countries who
is involved in the Organization for Economic Cooperation and Development
(OECD) are consuming more than half of world’s oil annually and it was predicted
of 12% increase of oil consumption during the next 25 years period started from year
2010 to year 2035, the non-OECD countries are estimated to have about 72%
increase (OECD report, 2009).
Brazil, China and India were spawning hundreds of new opportunity for the
microalgae feedstock development, biofuel production and export (Thurmond, 2008).
In U.S, the marketing of biodiesel is growing rapidly, from 25 million gallons per
year in 2004 and increased until 450+ million gallons in year 2007. Thus, the total
biodiesel being sold in U.S. is less than 1% of all the diesel consumption. More than
that, biodiesel in Europe is 2 to 3% of total transportation consumption and was
targeted to increase by 6% in year 2010. In India, China, Brazil and Europe, They are
currently aiming to increase the consumption of biodiesel instead of importing
petroleum from other countries (Thurmond, 2008). World biodiesel production is
expanding progressively, volatility prices and feedstock shortage in US, Europe and
Asia are causing to a growing delta between capacity and production in many
regions.
According to Browne et al., (2009), United States appears to be the largest
consumer of oil in the world, in 2008 US consumed about 19.5 million barrels of oil
per day and the production is far below its consumption which is only 8.154 million
barrels per day as shown in Figure 2.6. As of 2009 there were 84.9 million barrels of
oil consumed. In the year 2010, the was predicted that the oil demands will far
exceeding the availability by almost 1 million barrels of oil per day, this would cause
the oil price to be skyrocketed.
Throughout the years, there were benchmarks and trends have been set up
and continuous evaluation, surveying of the evolution and transformation as well as
the market opportunity for the production of microalgae biodiesel. Based on the
study by Alwi (2004), year by year it has been an intoxicating result for the
development of microalgae biodiesel production and also it keeps on widening by
131
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