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BIODIESEL PRODUCTION FROM COCONUT OIL
Submitted
by
Samani Carel Tupufia
A thesis submitted as partial fulfillment

of the requirement for the Award of
Masters Degree by Research
in the
School of Biotechnology and Biomolecular Science

University of New South Wales
Sydney, Australia
January, 2012
PLEASE TYPE
THE UNIVERSITY OF NEW SOUTH WALES
Thesis/Dissertation Sheet
Surname or Family name: TUPUFIA
First name: SAMANI Other name/s:
Abbreviation for degree as given in the Master by Research
University calendar:
School: Biotechnology and Biomolecular Science Faculty: Science
Title:
BIODIESEL PRODUCTION FROM COCONUT OIL
Abstract 350 words maximum: (PLEASE TYPE)
Biodiesel production processes using coconut oil containing a relatively high content of free fatty acids (4.5% w/v and higher) have been
characterized using alkali, acid and enzymatic- based catalysts. The use of ethanol (molar ratio of 3:1 of ethanol:triglycerides) instead of methanol
was evaluated in all processes. An alkali-based process resulted in rapid conversion of the triglycerides in the coconut oil to esters with an 80%
conversion in 5-10 min. However pre-treatment with 0.7% H2SO4 v/v at 50oC for 3 h was required before alkali addition to avoid saponification of the
free fatty acids. By comparison, a longer reaction time (50 h) was required for the acid process which also resulted in a lower conversion (67% at
50oC). The use of an acid overcomes the limitations of the alkali process of saponification when high FFA content oils are used, and gave a higher
conversion 70% at 75oC after 300 min. By comparison use of an enzymatic (lipase) catalyst (1% w/v) at 50 oC resulted in a conversion of 80% in 50 h.
The enzyme based process under similar conditions was significantly improved by the use of high frequency sonication which reduced the reaction
time to 3 h and achieved a 92% conversion of triglycerides to esters. To further improve the economics of the enzyme process, an initial study
showed that the enzyme catalyst was able to be recycled with only a 20% decrease in conversion. The use of the glycerol by-product from the
enzyme process as a carbon source for yeast growth resulted in improved kinetics to that for yeast growth on pure glycerol indicating that no
inhibitory by-products were produced during the enzyme process.
The overall economics of the process, and its sensitivity to key process variables was further analysed using a computer-based model of biodiesel
production. It showed that an internal rate of return (IRR) of 30% for a commercial size plant of 4 million litre per annual.
Declaration relating to disposition of project thesis/dissertation
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property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral
theses only).
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i
ORIGINALITY STATEMENT
I hereby declare that this submission is my own work and to the best of my knowledge,
it contains no material previously published or written by another person, or substantial
proportions of material which have been accepted for the award of any degree or
diploma at UNSW or any other educational institution, except where due
acknowledgement is made in the thesis. Any contribution made to the research by
others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged
in the thesis.
I also declare that the intellectual content of this thesis is the product of my own work,
except to the extent that assistance of others in the project’s design and conception or
in style, presentation and linguistic expression is acknowledged.
_______________________________
Samani Carel Tupufia
January 2012
ii
ACKNOWLEDGEMENTS
I would like to first thank my Supervisor, Professor Peter Rogers, for being an
excellent mentor and for giving me the opportunity to be part of the bio-refinery
research group. Also I would like, to thank him for the advice and guidance given
during these two years. I am very grateful and thankful for the encouragement and all
that he has done for me.
I would also like to say thank you to my Co-Supervisor Prof Soji Adesina, for his
support and help throughout my time working on this project, and for his advice and
encouragement.
I would also like to thank very much Dr Young-Jae Jeon, Dr Su Ping and Dr Chris
Marquis all of whom have worked tirelessly to help me to make possible the progress
of my project for their expertise and patience through this project. I am grateful to be
their student.
I would also like to extend my great appreciation to Dr Mathew Lee, Dr Joanna
Koenig, Dr Maria-Luisa Gutierrez from the Centre for Marine Bio-Innovation for their
assistance and guidance when using the Gas Chromatograph.
I would also like to say thank you to the former CEO Faamoetauloa Taito Dr Faale
Tumaalii of SROS for advising me to follow this path of study. I am thankful for the
teachings and challenges that he has given me, and also to colleagues at SROS for their
ongoing assistance whenever I asked for help during my studies.
Lastly, I would like to give a special thanks to my family for their support while away
on studies and to my dear wife and daughter for being with me all the time particularly
during the ups and downs. Thank you so much.
iii
PUBLICATIONS
Conference Proceeding:
Samani Tupufia, Young Jae Jeon, Adesoji Adesina, Chris Marquis and Peter Rogers
(2011) “Evaluation of biodiesel production from coconut oil using both alkali and
enzyme processes” Poster presentation in 2011 Bioenergy Conference of “Towards
2020: Growing our Sustainable Future”, Queensland, Australia 23-25 November 2011.
Publication (in preparation)
Samani Tupufia et al. “Enzymatic process for biodiesel production using coconut oil”
iv
TABLE OF CONTENTS
Acknowledgements........................................................................................................iii
Publications....................................................................................................................iv
Table of Contents............................................................................................................v
List of Figures................................................................................................................ix
List of Tables..................................................................................................................xi
Nomenclature..............................................................................................................xii
Abstract........................................................................................................................xiii
Project Objectives.......................................................................................................xiv
1. Literature review: .......................................................................................................1

1.1. Global Biodiesel Production................................................................................1
1.2. Biodiesel Production Processes......................................... .................................3
1.2.1. Transesterification and esterification reactions...........................................3
1.2.2. Catalysts for transesterification...................................................................4
1.2.2.1. Acid catalysed reactions....................................................................4
1.2.2.2. Alkali catalysed reactions..................................................................5
1.2.2.3. Enzyme catalysed reactions...............................................................7
1.3. Characteristics of Coconut Oil for Biodiesel Production…………………….....7
1.4. Process for used of Coconut oil for Biodiesel......................................................8
1.4.1. Direct blending of coconut oil.....................................................................8
1.4.2. Alkali and acid catalytic transesterification of coconut oil..........................9
1.4.3. Use of enzyme catalysts.............................................................................11
1.4.3.1. Advantages of enzyme catalysts......................................................11
1.4.3.2. Disadvantages of the enzyme catalysts............. .............................12
1.5. Use of Ethanol rather than Methanol for Biodiesel Production.........................13
v
1.6. Possible use of Glycerol By-Product from Biodiesel Production......................14
2. Materials and Methods..............................................................................................16
2.1. List of Chemicals used for this Study................................................................16
2.2. General Solutions...............................................................................................17
2.2.1. Sodium hydroxide titrating solution.........................................................17
2.2.2. Phenolphthalein colour indicator...............................................................17
2.3. Biodiesel Production Processes.........................................................................17
2.3.1. Acid transesterification using coconut oil.................................................17
2.3.2. Enzymatic transesterification of coconut oil.............................................17
2.3.2.1. Ultrasonic assisted transesterification.............................................18
2.3.3. Two step Acid and Alkali transesterification of coconut oil.....................18
2.3.4. Esterification of free fatty acids.............................................. ..................20
2.4. Transesterification and Esterification Sample Analysis................. ..................20
2.4.1. Coconut oil free fatty acid determination.................................................20
2.4.2. Gas liquid chromatography........................................................................21
2.4.3. High performance liquid chromatography.................................................22
2.4.4. Cloud point determination.........................................................................23
2.4.5. Water content.............................................................................................23
2.5. Calculations........................................................................................................25
2.5.1. Carbon balancing.......................................................................................25
2.6. Yeast Growth and By-Product Glycerol............................................................25
2.6.1. Saccharomyces cerevisiae growth cultures...............................................25
2.6.2. Culture growth conditions and culture storage..........................................25
2.6.3. Culture growth seed media........................................................................26
2.7. Batch Fermentation............................................................................................27
2.7.1. Inocula preparation....................................................................................28
2.7.2. Sample collection and analysis..................................................................28
2.7.2.1. Biomass by optical density..............................................................28
2.7.2.2. Dry cell weight determination.........................................................28
vi
2.7.2.3. Glycerol and ethanol concentrations...............................................29
2.7.3. Calculation of kinetic parameters..............................................................29
2.7.3.1. Maximum specific growth rate........................................................29
2.7.3.2. Maximum specific glycerol uptake rate..........................................29
2.7.3.3. Yeast biomass yield.........................................................................30
3. Biodiesel Production.................................................................................................31
3.1. Composition of Coconut Oil..............................................................................31
3.2. Data from the Scientific Research Organisation of Samoa (SROS)..................34
3.2.1. Pilot scale studies.......................................................................................34
3.2.2. Laboratory scale studies.............................................................................38
3.2.2.1. Acid catalysts...................................................................................38
3.2.2.2. Enzyme catalyst...............................................................................39
3.2.3. Summary of the results at SROS...............................................................41
3.2.4. Possible problems identified with the alkali process at SROS..................42
3.3. Detailed Kinetic Studies.....................................................................................42
3.3.1. Alkali catalyst............................................................................................42
3.3.2. Use of acid pre-treatment...........................................................................45
3.3.3. Acid catalysed transesterification of coconut oil.......................................48
3.3.4. Acid esterification of free fatty acids.........................................................48
3.3.5. Enzyme catalysed transesterification of coconut oil..................................51
3.3.6. Enzyme esterification of free fatty acids...................................................52
3.3.7. Improvements of the enzyme process........................................................55
3.3.7.1. Effects of enzyme recycling............................................................55
3.3.7.2. Effect of the use of ultrasound for increased agitation....................57
3.4. Cultivation of S.cerevisiae using Glycerol as a Carbon Source........................58
3.4.1. Composition of glycerol derived from enzyme process............................58
3.4.2. Kinetics of S.cerevisiae growth on glycerol..............................................59
3.5. Discussion of Results.........................................................................................61
4. Summary of the Economic Analysis.........................................................................63
vii
4.1. Process Flowsheet Basis for Economic Analysis...............................................63
4.2. Results of Economic Analysis............................................................................64
4.2.1. Pilot scale process......................................................................................64
4.2.2. Economic feasibility of commercial scale process....................................64
4.2.3. Sensitivity analysis....................................................................................67
5. Conclusions and Future Work...................................................................................69
5.1. Conclusions.........................................................................................................69
5.2. Future Work........................................................................................................70
6. References.................................................................................................................71
7. Appendix’s ...............................................................................................................81
7.1. 200 litre pilot plant information
7.2. Lipase enzyme product data sheet
7.3. Biodiesel B100 and B6-20 Specifications
viii
List of Figures
Page Figure Caption

2 1-1 Development of world biodiesel market
3 1-2 Basic chemistry of the transesterification reaction
4 1-3 Basic chemistry of the esterification reaction
18 2-1 Photo of incubator shaker used for biodiesel experiments
19 2-2 Photo of alkali transesterification experiment setup
24 2-3 Photo of Karl Fischer titration method
27 2-4 Photo of 3 litre bioreactor used for yeast growth studies
31 3-1 Chromatogram of coconut triglycerides by Benzard et al.
(1975)
32 3-2 Chromatogram of coconut triglycerides by Kumar et al. (2010)
35 3-3 Photo of 200 litre biodiesel pilot plant at SROS
35 3-4 Photo of the plant settling and polishing units
36 3-5 Photo of SROS vehicle run on B50 blend
37 3-6 Alkali transesterification : total esters conversion (SROS)
37 3-7 Alkali transesterification major FFA conversion (SROS)
38 3-8 Acid transesterification of coconut oil with ethanol at 75 oC
total ester conversion (SROS)
39 3-9 Acid transesterification; major FFA conversions (SROS)
40 3-10 Enzymatic transesterification of coconut oil with ethanol at
50oC total ester conversion (SROS)
40 3-11 Enzymatic transesterification: major FFA conversion (SROS)
41 3-12 Catalyst comparison (SROS)
43 3-13 Alkali catalyst transesterification of coconut oil with ethanol
1:3 molar ratio with 0.5% w/v catalyst
ix
44 3-14 Alkali transesterification of coconut oil with ethanol 1:3 molar
ratio with 1% w/v catalyst
46 3-15 Stage 1 of two-step acid and alkali process
47 3-16 Stage 2 of two-step acid and alkali process
49 3-17 Acid transesterification of coconut oil with ethanol at 1% v/v
catalyst concentration
51 3-18 Acid esterification of free fatty acids
53 3-19 Enzymatic transesterification of coconut oil with ethanol by 1%
w/v enzyme catalyst
55 3-20 Enzymatic esterification of free fatty acids
56 3-21 Enzymatic transesterification using recycled enzyme
57 3-22 Enzymatic transesterification assisted by ultrasonication
60 3-23 Kinetics growth of S.cerevisiae on glycerol
63 4-1 200 litre pilot plant computer model flow sheet
x
List of Tables
Page Table Caption

11 1-1 Advantages of lipase enzyme
12 1-2 Disadvantages of lipase enzyme
16 2-1 Chemicals used for this study
22 2-2 Gas chromatography standard solutions
23 2-3 High performance liquid chromatography standard solutions
26 2-4 Growth conditions of S.cerevisiae
32 3-1 Fatty acid composition of coconut oil
34 3-2 Coconut oil triglycerides
58 3-3 Properties of glycerol derived from enzymatic biodiesel
process
59 3-4 Summary of batch results run with medium containing 10g l-
1
yeast extract
68 4-1 Cost analysis for two commercial scale plants
xi
NOMENCLATURE
EtOH Ethanol
FFA Free fatty acids
GC Gas chromatography
h Hour/s
HPLC High performance liquid chromatography
FID Flame ionized detector
l Litre
m million
M Molar
mg milligram
ml millilitre
min minute/s
mol moles
mM milliMolar
MQ milli-Q
ppm parts per milliom
ppt parts per trillion
rpm rounds per minute
sec second/s
SROS Scientific Research Organisation of Samoa
RID Refractive Index Detector
μl microlitre
v/v volume to volume
v/w volume to weight
w/v weight to volume
w/w weight to weight
xii
ABSTRACT
Biodiesel production processes using coconut oil containing a relatively high content of
free fatty acids (4.5% w/v and higher) have been characterized using alkali, acid and
enzymatic-based catalysts. The use of ethanol (molar ratio of 3:1 of
ethanol:triglycerides) instead of methanol was evaluated in all processes. An alkali-
based process resulted in rapid conversion of the triglycerides in the coconut oil to
esters with an 80% conversion in 5-10 min. However pre-treatment with 0.7% H2SO4
v/v at 50oC for 3 h was required before alkali addition to avoid saponification of the
free fatty acids. By comparison, a longer reaction time (50 h) was required for the acid
process which also resulted in a lower conversion (67% at 50oC). The use of an acid
overcomes the limitations of the alkali process of saponification when high FFA
content oils are used, and gave a higher conversion 70% at 75 oC after 300 min. By
comparison use of an enzymatic (lipase) catalyst (1% w/v) at 50oC resulted in a
conversion of 80% in 50 h. The enzyme based process under similar conditions was
significantly improved by the use of high frequency sonication which reduced the
reaction time to 3 h and achieved a 92% conversion of triglycerides to esters. To
further improve the economics of the enzyme process, an initial study showed that the
enzyme catalyst was able to be recycled with a 20% decrease in conversion. The use
of the glycerol by-product from the enzyme process as a carbon source for yeast
growth resulted in similar kinetics to that for yeast growth on pure glycerol Indicating
also that no inhibitory by-product were produced during the enzyme process.
The overall economics of the process, and its sensitivity to key process variables was
further analysed using a computer-based model of biodiesel production. It showed that
an internal rate of return (IRR) of 30% may be possible for a commercial size plant 4
million litre per annual.
xiii
PROJECT OBJECTIVES
The project has the following objectives:

1) to compare the kinetics and productivities of three processes, viz. alkali, acid and
enzyme based processes, for the conversion of coconut oil into a suitable
component for biodiesel production,
2) to evaluate strategies which might be used to improve the enzyme-based process
by using techniques such as ultrasonic agitation and enzyme recycling,
3) to determine the potential use of the glycerol residue from the enzyme-based
process for the growth of yeast (Saccharomyces cerevisiae) as a possible source
of a high protein animal feed supplement,
4) to use a computer-based model to determine the possible economic feasibility of
such a biodiesel process using coconut oil, and to evaluate the sensitivity of the
process to key process variables (e.g cost of coconut oil, size of plant, value of
by-products etc).
xiv
1: Introduction and Literature Review
In this Chapter, the current status and research focus for biodiesel production using
various feedstocks with particular reference to use of coconut oil will be described. A
review of the potential use of glycerol by-product derived from the biodiesel
production processes for value added products is also included.
1.1 Global Biodiesel Production

Peak oil and environmental concerns related to climate change have led to the search
for new alternative energy sources including solar, wind, wave, geothermal and bio-
energy, as they are renewable and more environmentally friendly than conventional
fossil-based fuels.
The use of biodiesel as a liquid transportation fuel has been well accepted by the public
as a result of its compatibility with currently available diesel fuels and its use in
engines without any modification, as well as its environmentally friendly
characteristics, such as being renewable, CO2 neutral and resulting in the reduced
emissions of polycyclic aromatic hydrocarbons and sulphur compounds (Roble et al.
2008).
1
Master’s Thesis Samani C Tupufia
Figure 1-1: A projected increase in biodiesel production and biodiesel world trade from 2005
to 2020 taken from OECD-FAO Agricultural Outlook 2011-2020 Biofuels
According to the OECD-FAO Outlook Report (2011 to 2020) (Figure 1-1), 3.8 billion
tonnes of biodiesel were globally produced in 2005 and more than 80% of diesel
produced in the year was produced as well as consumed by European countries.
Germany accounted for 53% of the European biodiesel production in that year. For the
feedstocks used for biodiesel production from 2008-2010, 85% came from vegetable
oils (palm oil, sun flower seed, rapeseed, peanuts etc) with 15% produced from non-
food based oils such as Jatropha cursus and other non-agricultural feedstocks ( e.g
microalgae). It is projected that vegetable oil based biodiesel will decrease from 85%
to 75% by 2020 in developed countries due to the growth of non-agricultural
feedstocks. It is projected also in the Report that crude plant oil prices will continue to
rally in 2011 and could reach USD 107/barrel by 2020, and this may reduce their use in
biofuels unless crude oil prices also continue to rise.
2
1.2 Biodiesel Production Processes

Production of biodiesel is based on four possible processeses, viz. direct use of oil and
blending, micro-emulsion, thermal cracking (pyrolysis) and transesterification.
Transesterification is the most common method used currently for biodiesel production
at the pilot and industrial scale, and will be discussed in detail as it is the focus of the
present research.
1.2.1 Transesterification and esterification reactions

Transesterification is the general term used to describe the chemical reaction for the
production of biodiesel from the various triglycerides. Typical triglycerides react with
an alcohol in the presence of an acid, base, acid/base-hybrid or enzyme catalyst
resulting in a mixture of fatty acid esters and glycerol (Figure 1-2). Among the
alcohols, methanol or ethanol are commonly used due to their low cost and relative
abundance (Yusuf et al. 2011).
The stoichiometric transesterification reaction involves 1 mole of triglyceride and 3
moles of alcohol to produce of 1 mole glycerol and 3 moles of fatty acid esters, while
the associated esterification reaction stoichiometrically produces 1 mole of fatty acid
ester and 1 mole of water from 1 mole of fatty acid and 1 mole of alcohol in the
presence of a catalyst (Figure 1-3).
Figure 1-2 Basic chemistry of the transesterification process.
3
Ethanol is often the preferred alcohol instead of methanol because it can be produced
from a bio-refinery using renewable biomass. However methanol is often used
commercially because of its lower cost.
Figure 1-3 Basic chemistry of an esterification process.
1.2.2 Catalysts for transesterification/esterification

There are commonly three main catalysts used for biodiesel production processes from
various oil feedstocks, viz. acid, alkali and enzyme catalysts. These catalysts facilitate
both the transesterification and esterification processes shown above.
1.2.2.1 Acid catalysed reactions

The most commonly used acid catalysts are sulphuric acid, hydrochloric acid, or
sulfonic acid (Demirbas, 2009). Acid catalysts can be used for both free fatty acid
(FFA) and high FFA oil feedstock conversions using either methanol or ethanol
(Huynh et al. 2011). The acid catalysts have an advantage over alkali catalyst
particularly for high FFA oils, as they avoid unwanted side reactions such as
saponification (soap formation) caused by alkali catalysts. Canakci et al. (1999) have
used alcohol to oil molar ratios ranging from 3:1 to 30:1 to investigate the effectiveness
of an acid catalyst for biodiesel production, and showed that an increase in the molar
ratio resulted in an increase in the ester conversion of the triglycerides with the highest
conversion being 98.4% for a 30:1 molar ratio alcohol to oil. Narasimharao et al.
(2007) reported that the reaction rates with an acid catalyst (1% v/v sulphuric acid))
4
were low and required higher temperatures (78oC) and excess alcohol (1:30 soybean oil
to ethanol) to produce high yields (99%). This result was supported by Vyas (2009)
who showed that an acid catalyst reaction was about 4000 times slower than that using
an alkali catalyst. The longer reaction times with the acid catalyst would make the
process more impractical and less economical Koh et al. 2011), although this could be
improved by increasing the temperature used. For instance, Freedman et al. (1986)
studied the butanolysis of soybean oil using sulphuric acid at temperatures ranging
from 77o C to 117o C and their results showed that the elevated temperatures
significantly increased the reaction rate, with complete conversion in 3 h at 117oC
compared to 20 h in 77oC. Lotero et al. (2005) concluded that at higher temperatures,
the phase separation improved and the rate constants increased and miscibility
improved, leading to substantial shortening of the reaction time.
1.2.2.2 Alkali catalyzed reactions.

Biodiesel production using base catalysts is currently accepted as the most economical
process. It is cheaper and involves a much faster reaction when compared to those with
acid or enzyme catalysts. Such processes function at between 50o C to 60o C at
atmospheric pressure, and result in reaction times of between 1 to 2 h, with conversion
yields of more than 98% (Refaat, 2009). A study by Fillie’res et al .(1995) for
ethanolysis to ethyl esters using sodium ethoxide as catalyst, showed that a reaction
mixture of 6:1 molar ratio ethanol to rapeseed oil and 1% w/v alkali catalyst
concentration resulted in a 94% ester yield after 5 min at 80 oC. Ma et al. (1998)
evaluated the effects of the use of an alkali catalyst for the transesterification of beef
tallow. This study revealed that sodium hydroxide was better than sodium methoxide
as a catalyst. However the presence of water in the reaction mixture showed negative
effects on the transesterification reaction.
Silvia et al. (2009) evaluated castor oil transesterification using ethanol produced
from sugar cane in the presence of sodium ethoxide or sodium hydroxide as catalysts.
5
From this study, the use of a castor oil to ethanol molar ratio of 1:16 with 1% w/v
sodium ethoxide at 30oC produced 99% conversion to esters in 30 min. A similar
conversion yield was obtained with 1% w/v sodium hydroxide and 1:19 molar ratio oil
to ethanol.
Despite, these attractive characteristics of alkali catalysts, some limitations have been
reported. The most important of these is that a base-catalysed reaction cannot
effectively convert oils containing high FFA and high water content into quality
biodiesel (Huynh et al. 2011). The high FFA and water contents in the oil react
undesirably with the alkali catalyst resulting in the formation of unwanted soap (Singh,
2009). This also reduces the catalyst efficiency with a resultant increase in viscosity,
and may lead to gel formation making the glycerol separation more difficult.
To overcome these problems with an alkali catalyst, new strategies have been
investigated. For example, Canakei, (2001) reported the production of biodiesel from
oils and fats with high FFAs using a two step process with initial use of an acid
followed by an alkali catalyst. A synthetic mixture containing 20% palmitic acid with
animal fat and 40% palmitic acid with soybean oil was used. The acid catalyst
(sulphuric acid) esterified the FFAs to their ester forms resulting in lowering the FFA
content to 1%. The intermediate reaction mixture was allowed to settle prior the next
step so that the alcohol phase containing water could be easily removed. In this
demonstration, actual substrates of yellow grease containing up to 12% FFA and brown
grease up to 33% FFA were pre-treated using this two step process. The acid catalysts
lowered the FFA to less than 1% and then the transesterification reaction was
completed with an alkali catalyst. Overall, increasing the acid catalyst to 15% v/v
sulphuric acid was very effective in lowering the FFAs in a mixture containing 1:10
molar ratio oil to alcohol in 30 min. It was reported that ethanol was more effective
than methanol in reducing the FFA of the synthetic mixture and sodium methoxide was
a more effective catalyst than potassium hydroxide in completing the transesterification
reaction.
6
1.2.2.3 Enzyme catalyzed reactions

The use of an enzyme catalyst as the alternative to chemical catalysis has been
investigated to overcome the potential problems associated with chemical catalytic
processes. Lipase enzymes have been used as they can catalyze the hydrolysis of ester
bonds in long chain triglycerides thereby producing esters and glycerol. Although the
enzymes have been defined as glycerol ester hydrolases they not only catalyse carboxyl
ester bonds (hydrolysis) but also catalyze the reverse reaction (esterification and
transesterification) in water restricted systems (Freire et al. 2011).
However, the cost of lipase enzymes may be a major barrier to their use in commercial
scale biodiesel production. To overcome this problem, lipase enzymes have been
intensively investigated to further develop them as efficient and cheap catalysts. It has
been reported also that methanol has a negative effect on their enzyme activity (Abigor
et al. 2000). As a result, Du et al. (2004) investigated the use of methyl acetate as an
acyl acceptor instead of methanol for biodiesel production. It had been reported also
that methanol has a negative effect on the enzyme activity above an oil to methanol
molar ratio of 1:1. However, a methyl acetate to oil molar ratio of 12:1, with 30%
enzyme (w/w) of oil at 40oC gaves a 92% conversion and showed no negative effect on
the enzyme activity. Du et al. (2004) also reported that excess methyl acetate over 1:16
molar ratio led to excessive dilution of oil resulting in a decline in methyl ester yield. It
was reported also after 100 batches (with enzyme recovery) with methyl acetate as the
acyl acceptor that the enzyme activity showed almost no loss in activity, and this would
result in a significant reduction in enzyme costs.
1.3 Characteristics of Coconut oil for Biodiesel Production

Although various oil seeds from different geographical regions have been used as raw
materials for biodiesel production, the coconut is the most abundant oil seed crop in the
Pacific Island Countries and may be the best candidate for biodiesel production in the
Region. The first mature coconuts can be produced after 5-6 years following plantation,
7
and about 50 to 80 fruits per year are produced from a fruit bearing palm with each
endosperm yielding up to 40% oil (Chan 2006). Approximately 8-10 coconuts are
required to produce 1 litre of coconut oil. The productive lifespan of such palms is
about 80 years. The use of coconut oil has not been widely reported in the literature as
a raw material for biodiesel production although it is considered to be one of the most
suitable. Benzard et al. (1975) and Diaz et al. (2002) reported that coconut oil contains
more than 90% saturated fatty acids with the remainder being unsaturated. About 62%
of the saturated fatty acids are medium chain length (C8 –C18) and 29% are
characterized as long chain fatty acids (above C18). Coconut oil has excellent
solubility and solvency, such features make “cocobiodiesel”, a perfect biodiesel for
developing countries (Diaz et al. 2002).
1.4 Processes for Use of Coconut Oil for Biodiesel

1.4.1 Direct blending of coconut oil
The direct use of coconut oil as a liquid fuel has not been widely reported in the
literature. However coconut oil is being evaluated by a number of Pacific Island
Countries as a potential liquid bio-fuel component. Various projects have been
established by National Governments to evaluate the use of local resources and thereby
reduce the quantity of imported fuel. Colin (2007) reported that Kiribati has been
experimenting with the use of 50% fuel blends of coconut oil and diesel, and in Papua
New Guinea coconut oil has also been used as a fuel for rural community
electrification projects, as well as a 40% coconut oil blend being used for ongoing
engine studies. The Samoan Electric Power Cooperation has been experimenting with a
250kW generator using a 20% coconut oil to diesel blend.
Kalam et al. (2003) reported data on performance characteristics and exhaust emissions
in an evaluation of the use of coconut oil in diesel engines. Results with a 30% coconut
oil blend with diesel fuel showed a higher net heat release rate with a net reduction in
exhaust emissions such as HC, NOx, smoke and polycyclic aromatic hydrocarbon
8
when compared with 40% and 50% blends. However exhaust emissions were reduced
with the higher coconut oil blends.
The introduction of coconut oil (triglycerides) as a fuel directly to the engine without
bioconversion has been reported also (Hossain, 2009). However this resulted in a
number of engine combustion problems including carbon deposition, lubrication oil
dilution or thickening, piston ring sticking and injector nozzle coking.
1.4.2 Alkali and acid catalytic transesterification of coconut oil

The transesterification of coconut oil has been the subject of various studies using
different processes. The conversion of palm oil and coconut oil using lipase has been
investigated with various alcohols (Abigor,2000). From this study it was concluded
that the use ethanol or butanol with lipase resulted in conversions of 72% and 62%
respectively for palm oil. However with methanol and coconut oil only 40%
conversion occurred due to the toxicity effects of methanol on the lipase enzyme. The
use of solid catalysts has also been investigated for coconut biodiesel production.
Jitputti et al. (2005) reported that the use of ZnO and SO42-/ZrO2 catalysts resulted in
the highest conversions (more than 90%) with 1% (w/v) catalyst.
The use of coconut oil containing a high free fatty acid content ( more than 12% w/v)
required a two step acid and alkali transesterification to produce a high quality
biodiesel (Nakpong et al. 2009). This involved a first stage esterification with sulphuric
acid which lowered the free fatty acid content to 0.6%. The second step involved the
transesterification of the treated oil with an alkali catalyst. This study successfully
produced high quality biodiesel from a coconut oil containing 12.8% FFA content.
Oliveira et al. (2010) reported that the introduction of a water adsorption material
during the esterification reaction improved the efficiency and quality of the biodiesel
produced from waste coconut oil. The addition of zeolite 3A resulted in a significant
increase in conversion yields (up to 99%) with an acid catalyst when using either
ethanol or methanol. Kumar et al. (2009) studied the transesterification of the coconut
oil using ethanol with potassium hydroxide as a catalyst and with the assistance of
9
ultrasonic vibration. The latter resulted in a significant improvement in the yield and
biodiesel conversion rate with up to 98% conversion to esters after 7 min. It has been
suggested that the enhanced results with ultrasound may be due to one or more of the
following effects:
1) the rapid movement of fluids caused by the variations in sonic pressure resulting
in solvent compression and rarefaction,
2) the formation of cavitations with resultant greater contact between reactants and
enzyme particles,
3) generation of micro-streaming in which a large amount of vibrational energy is
confined in small volumes with little resultant heating.
Many investigators also accept that the formation and collapse of micro-bubbles which
results from the ultrasound vibrations are major contributors to the significant
improvements observed (Kumar et al. 2010).
10
1.4.3 Use of enzyme catalysts

1.4.3.1 Advantages of enzyme catalysts
The use of enzyme catalysts has a number of advantages over the use of chemical
catalysts, possibly making them the preferred catalysts for future biodiesel production.
These advantages of typical lipase enzyme catalysts are summarized in Table 1-1.
Table 1-1 Advantage of lipase enzyme catalyst

Feedstock quality By-product Reaction Enzyme re-use Waste
conditions Management
Lipase enzyme can Glycerol Lipase Immobilized No extra
trans/esterify oil produced enzyme are enzymes can be treatment is
feedstock material from an able to work regenerated and required to
without problems. enzyme at reused several times separate and
No side reactions biodiesel temperatures for biodiesel purify the
were found to occur reaction was from 30oC to production. biodiesel fraction
when oil with high high purity 50oC, Regeneration can be from the glycerol
FFA content was (Ranganathan yielding high as simple as washing fraction
used for biodiesel et al.2008). quality with water although following an
production (Vyas et products, other solvents may enzyme reaction (
al.2009). (Vyas et be needed (Du et Vyas et al.2009).
al.2009). al.2004).
These potential advantages of the use of the enzyme catalysts have made them
attractive and focused research is now being carried out to fully explore their potential.
In general the use of enzymes would save energy as the reaction occurs at relatively
low temperatures (30oC to 50oC). The quality of the transesterified oils should be
suitable for biodiesel production as no side reactions such as saponification occur.
Water usage and management would not be an issue with the enzyme catalyst
transesterification, as there would be little or no washing required associated with
biodiesel production and glycerol by-product recovery. Immobilization of the lipase is
likely to improve enzyme stability and enable its recycling for at least several batches
(Tan et al. 2010, Shahid, 2011).
11
Overall, the use of enzymes may significantly improve the economics of this process.
The more the enzyme can be reused the lower its contribution to the overall cost of the
product (Freire et al. 2011). These advantages indicate that an enzyme-based
transesterification process has significant potential for both improved economic and
environmental outcomes and could be an attractive alternative to the current chemical-
based processes (Lam et al. 2010).
1.4.3.2 Disadvantages of use of enzyme catalysts

Use of an enzyme catalyst overcomes many of the obstacles faced by the use of
chemical catalysts for biodiesel production but also has its disadvantages. Table 1-2
summarizes the main disadvantages of enzyme catalysts which may present a
limitation to their future use in larger scale processes.
Table 1-2 Disadvantages of use of enzyme catalysts.

Reaction Time Cost Inhibition
Use of an enzyme Enzyme costs are Enzyme activity can be
catalyst results in a relatively high and inhibited by high alcohol
relatively slow may be restrictive concentrations (particularly
reaction rate unless enzyme can methanol) and temperatures
compared to alkali be recycled (Lilin above 50oC can denature the
or acid process et al.2006). enzyme. Immobilized enzyme
(Vyas et al.2009). activities may be affected by
glycerol adsorption blocking
its active sites (Al-
Zuhair.2007).
The cost of lipase enzymes, for example Novozyme 435, is quoted at between 300 to
400 USD per kilogram (Novozyme Business Development, 2011) not including the
shipments cost. It is very expensive compared to the cost of alkali or acid catalysts.
Secondly, slower reaction rates have been reported compared to those for alkali and
acid-based catalytic processes, with the reactions taking more than 24 h to produce
acceptable yields (>90 % conversion of triglycerides to esters), (Lam et al. 2010).
12
Royon et al. (2007) reported that an enzyme-based biodiesel process using cotton seed
oil and methanol (as reactants) and t-butanol (solvent) could achieve a 95% conversion
after 24 h. Soumanoua et al. (2003), achieved 91% conversion to esters after 48 h using
methanol (reactant) and hexane as solvent with 4.5:1 molar ratio methanol to oil while
Iso et al. (2001), reported a 91% conversion in 20 h using isopropanol and sunflower
seed in a 3:1 molar ratio. Many other enzymatic biodiesel studies have reported similar
findings that relatively slow reaction rates occur for enzyme-based processes.
Finally, enzyme inhibition may be a major disadvantage for use of an enzyme catalyst.
It has been reported that low pH and acidic substrates can negatively affect the enzyme
activity and alcohols such as methanol can inhibit the enzyme activity even at low
concentrations. For example, Marceiras et al. (2011) reported that the low
concentration of methanol present in a methanol and oil molar ratio (1:1) effectively
deactivated the free enzyme, while the higher concentrations of methanol with a molar
ratio of (3:1) adversely affected the immobilized enzyme activity. Inhibition of enzyme
activity can also be caused by the glycerol by-product and by low pH and high ionic
strength. In addition glycerol production during the transesterification reaction can
reduce the enzyme activity by adsorbing to its surface thereby blocking the active sites
of the lipase and reducing the rate of triglycerides conversion.
From the foregoing review, it is clear that enzyme-based process for biofuels have both
a number of potential advantages as well as disadvantages, and the present study using
lipase for the esterification/transesterification of coconut oil will focus on ways in
which the advantages can be maximized.
1.5 Use of Ethanol rather than Methanol for Biodiesel Production

The current biodiesel process at SROS has mainly use methanol in the past to produce
methyl esters. However recent studies have focused more on the use of ethanol,
because of the potential toxicity of methanol vapour and increased the risk of explosion
(Hamad et al.2008; Brusamarelo et al. 2009; Barbosa et al .2010; Tongboriboon et
13
al.2010; Quintella et al. 2011; Stamenkovic et al. 2011). Further advantages of the use
of ethanol over methanol are as follows:
1) probable reduced inhibition effects on lipase
2) ethanol can be produced from renewable resources (biomass) and could be
produced in an associated process at SROS
3) works well with enzyme catalyst making the process a “greener process”
4) reacts with triglycerides at a similar rates to methanol in the presence of a
catalyst
5) facilitates easy separation of the glycerol and biodiesel phases similar to that
which occurs with methanol.
Producing bio-ethanol from renewable fermentation feedstocks available at SROS
would result in a more sustainable process and minimize environmental impacts.
1.6 Possible use of Glycerol By-Product from Biodiesel Production

The recent increase in biodiesel production has resulted in production of greater
amounts of crude glycerol globally. Current global research has focused on
investigating new ways to utilize this glycerol for the production of value-added
products. This would improve the economics of the biodiesel process. Silva (2009)
reviewed the potential production of higher value fermentation chemicals using
glycerol as a carbon source. These included 1.3-propanediol, dilhyroxyacetone, ethanol
and succinate. Glycerol can also be used as a carbon source to obtain other value-added
products such as proteins and enzymes, medical drugs and antibiotics.
For production of cell biomass, Ochoa-Estopier et al. (2011) investigated the kinetics
of growth of a Saccharomyces cerevisiae strain using glycerol as the sole carbon
source, and the results demonstrated successful growth of the yeast strain CBS 8066-
FL20 on glycerol-based medium. They reported that specific growth rates up to 0.2 h -1
could be achieved with glycerol under aerobic conditions. Nitayavardhana et al. (2011)
investigated the potential of producing an edible fungal species (Rhizopus microspores
14
var. oligosporus) using biodiesel derived crude glycerol and reported that a fungal
biomass yield of 0.83 gg-1 was achieved using glycerol with nutrient supplements and
without pH control. Tang et al. (2008) studied Pichia pastoris fermentation for
phytase production using glucose and glycerol, and reported that under aerobic
conditions the respiratory metabolism was maintained at initial glycerol concentrations
as high as 70g l-1. Under these conditions with dissolved oxygen levels as low as 10%
air saturation, a biomass yield of 0.51 g g-1 was achieved with a maximum specific
growth rates of 0.27 h-1 . Under similar conditions, with glucose as carbon source, a
yield of 0.35 g g-1 and maximum specific growth rate of 0.20 h-1 was achieved showing
that glycerol was a better carbon source than glucose for this yeast. A final cell density
of 146 g l-1 dry cell weight and phytase production of 1125 μL (enzyme activity per
cell-free broth) was attained within 53 h, using a fed-batch strategy.
15
2. Materials and Methods
2.1 List of Chemicals used for this study
Table2- 1: Chemicals used for this study were all Analytical Grade
Chemicals Purity Supplier
Alcohols
Methanol 99% v/v Ajax Finechem Pty Ltd

Ethanol 99% v/v Ajax Finechem Pty Ltd
Isopropanol 99% v/v Ajax Finechem Pty Ltd
Heptane 99& v/v Ajax Finechem Pty Ltd
Pure Glycerol 99% v/v Ajax Finechem Pty Ltd
Free Fatty Acids
Lauric Acid 99% v/v Sigma Aldrich

Myristic Acid 99% v/v Sigma Aldrich
Palmitic Acid 99% v/v Sigma Aldrich
Ethyl Esters
Ethyl Capriate 99% v/v Sigma Aldrich

Ethyl Laureate 99% v/v Sigma Aldrich
Ethyl Myristate 99% v/v Sigma Aldrich
Ethyl Palmitate 99% v/v Sigma Aldrich
Ethyl Oleate 99% v/v Sigma Aldrich
Derivitising Agents
Hexamethyldisilazane 99% v/v Fluka Analytical

Catalysts
Novozyme 435 Activity 100KLU/g Novozyme Australia Pty

Sulphuric Acid 98% Ajax Finechem Pty Ltd
Sodium Hydroxide 97% Pellets Ajax Finechem Pty Ltd
Coconut Oil 95.5% Triglycerides Punjas and Sons Ltd
(Lautoka Fiji)
Phenolphthalein 99% Ajax Finechem Pty Ltd
16
2.2 General Solutions

2.2.1 Sodium Hydroxide 0.1N (titrating solution)
All glassware were dried in an incubator overnight before using to prepare and store
solutions. To prepare 0.1 N NaOH, 0.4 g of NaOH pellets were dissolved in 100 ml of
RO water.
2.2.2 Phenolphthalein colour indicator

Phenolphthalein containing 0.5% phenolphthalein powder was dissolved in 50% (v/v)
ethanol solution.
2.3. Biodiesel Production Processes

2.3.1 Acid transesterification using coconut oil
The acid esterification reactions were conducted in a shaking incubator at 350 rpm
using a 500 ml conical bottle. Prior to the experiment, the frozen coconut oil was
defrosted in an incubator shaker (Figure 2-1) temperature controlled at 30oC. The
required amount of ethanol used was measured into a volumetric flask and 1% (v/v)
sulphuric acid was added. For the initiation of acid catalysis transesterification, 300 ml
coconut oil was added to the reaction vessel and placed in an incubator shaker and
agitated at 350 rpm for 30 min at 50oC, before the addition of the catalyst solution. The
experiment started once catalyst solution was introduced to the pre-warmed coconut
oil.
2.3.2 Enzymatic transesterification of coconut oil

For the enzymatic transesterification, the catalyst used was an immobilized lipase
(Novozyme 435), and no prior preparation was required. The preparation of the
coconut oil was similar to that for the acid transesterification.
For the enzymatic transesterification, 300 ml of coconut oil was added into the reaction
vessel and 60 ml of absolute ethanol was added. The mixture was agitated at 350 rpm
17
for at least 30 min to properly homogenize the mixture before adding 1% (w/v)
immobilized lipase to start the reaction. Samples were taken at required intervals until
the reaction was completed. The mixture was left then overnight at room temperature
to achieve effective separation of the biodiesel fraction from the glycerol fraction.
2.3.2.1 Ultrasonic assisted transesterification

An ultrasonic cleaner Model D80H was used for this experiment, with a container
capacity of 2 litres. The operating frequency was 43 kHz, with output power of 80W
and heater 45W. A 1:3 molar ratio coconut oil to ethanol was used. A 250mL beaker
was used as the reactor; the amount of water in the ultrasonic cleaner was leveled with
reactant mixture in the reactor.
Figure 2 -1 Burnswick Scientific EDISON USA: Controlled Environment Incubator Shaker

used for enzymatic and acid transesterifications.
2.3.3 Two step acid and alkali transesterification reaction of coconut oil
The coconut oil used these studies had a relatively high free fatty acid (FFA) content
and based on this level of FFA it was too high for the single addition of an alkali
catalyst. Previous studies have shown that the FFA content of the oil feedstock should
be less than 1% (w/v) for the alkali catalyst to work properly. In the present study, a
two-step process as reported by Nakpong et al.(2009) to produce biodiesel from 12%
FFA coconut oil, was employed. In the first step (acid esterification) 1 M of ethanol
18
11.7 ml was thoroughly mixed with an estimated 1 M coconut oil (141.1 ml) and
heated to 50oC for up to 30 min. Then 0.1% (v/v) sulphuric acid (based on volume of
oil used) was added to the mixture while stirring at 350 rpm. FFA levels were regularly
checked by titration (see later 2.4.1) until the concentrations of the FFA decreased to a
level at which the alkali catalyst would work. The treated oil was then washed with
warm RO water (60oC) to remove the residual acid and ethanol by centrifuging at 5000
rpm for 30 min at 20oC. This was repeated several times to fully separate the residual
aqueous component from the washed oil. In the second step, for alkali
transesterification, the treated coconut oil was incubated at 50oC and stirred at 350 rpm.
The reaction conditions were an estimated 1 M of concentrated oil (100 ml) and 6 M of
ethanol (56 ml). The catalyst solution containing 0.8% (w/v) sodium hydroxide, based
on the volume of oil, was dissolved in ethanol and added to the oil to initiate the
reaction (Figure 2-2). Samples were taken at regular intervals and when the reaction
was completed, the mixture was left overnight to separate the glycerol (lower layer)
and esters (top layer) fractions.
Figure 2-2 Alkali Transesterification setup: using hotplate and magnetic stirrer for circulation
and temperature controlled sensor and heater probes
19
2.3.4 Esterification of free fatty acids

For further evaluation of esterification reactions, lauric (C12), myristic (C14) and
palmitic (C16) were chosen as model fatty acids for coconut oil. 1 M of pre-warmed
free fatty acids (C12 (22.8 ml), C14 (26.54 ml) and C16 (30.1 ml)) at 50oC were
thoroughly mixed with 3 M of ethanol and the reaction mixture placed in a shaking
incubator. Calculated concentrations of catalysts were then added to initiate the
reaction. The amount of catalyst used was calculated based on % w/v of concentrated
(99 %) FFA used. A 175ml conical plastic bottle was used as the reactor. The reactants
were agitated at 350 rpm in a shaking incubator. The enzymatic esterification reaction
was carried out at 50oC, as this was the maximum temperature that can be used for an
enzyme while 75oC, was the optimum temperature for the acid esterification.
2.4 Transesterification and Esterification Sample Analysis

Before carrying out the reactions, the free fatty acid content based on the coconut oil
major fatty acid (lauric acid) was determined for the coconut oil used. During the
catalysed reactions 2 ml of sample were taken at regular intervals to determine the
concentrations of ethyl esters and glycerol produced, and the triglycerides and ethanol
consumed. After the phase separation, the biodiesel layer (top layer) was analyzed for
its moisture content and cloud point.
2.4.1 Coconut oil free fatty acid determination

Prior to transesterification of coconut oil, the total FFA content was estimated by
titration to identify the percentage of FFAs present in coconut oil. The method was
described in the Official Methods and Recommended Practices of The American Oil
Chemists Society (AOACS Ca 5a-40) (Firestone, 2004).
20
Procedure:
“Weigh sample and add specific amounts of neutralized alcohol and 2ml of indicator.
Titrate with sodium hydroxide, shaking vigorously until the appearance of first
permanent pink colour as evidence of the neutralized alcohol. Colour must persist for
30sec”
FFA Range Sample(g) Alcohol(mL) Strenght of alkali
%
0.00 – 0.2 56.4 50ml 0.1N
0.2 – 1.0 28.2 50ml 0.1N
1.0 – 30.0 7.05 75ml 0.25N
30.0 -50.0 7.05 100ml 0.25/1N
50.0 - 100 3.525 100ml 1N
The FFA percentage was calculated using this formula:
FFA w/w (%) = ml of alkali added x N x 20 X 100 Equation (1)
Gram of sample
* N is Normality, *20 is calculation factor for lauric acid
2.4.2 Gas Liquid Chromatography

Sample preparation
Samples taken at required intervals during a reaction were kept at -20oC freezer for
further analysis. The solvent used was 20% hexamethyldisilazane in 100 ml heptane. A
fifty micro litre sample was added to 10 ml of solvent to achieve a 200 fold dilution
and 1 μL was injected to GLC. This was done consistently with all samples analyzed
by GLC.
21
Analytical conditions
Reaction samples were analysed and the concentrations of FFAs, ethyl esters and
triglycerides determine by Gas Liquid Chromatography (GLC) using the method
previously reported by James et al. (2007). 1μL of sample was injected to GLC column
with a 30 min running time per injection. Separation was achieved using a Zebron ZB-
5HT Inferno column with a 50oC initial temperature rising to 230oC by 10oC per
minute, then increasing to 360oC by 20oC per minute and maintained constant until all
triglycerides was eluted. Helium was used as a carrier gas and detection was achieved
with a flame ionized detector (FID) at 380oC. Peak data values were recorded and
uploaded using Shimadzu GC solution Version 2.0 Workstation software. The
concentrations of the ethyl esters and FFAs were determined by integrating the sample
peak areas and comparing them to known standard (esters/FFAs) calibration curves
using different levels of standard concentrations as shown in Table 2-2
Table 2-2 : GLC Standards

Compound Standard Concentration ppm
Ethyl Caprilate 1 50
Ethyl Laureate 2 100
Ethyl Myristate 3 200
Ethyl Palmitate 4 500
Ethyl Oleate 5 1000
6 2000
Compound Standard Concentration mmol/L
Lauric Acid 1 1
Myristic Acid 2 2
Palmitic Acid 3 5
2.4.3 High Performance Liquid Chromatography

Ethanol and glycerol concentrations were determined by High Performance Liquid
Chromatography (HPLC) using the method previously reported by Jeon et al. (2011).
An Aminex HPX-87H column (Bio-Rad, California, United States) was used and the
separations achieved at 50°C using 5 mM H2SO4 at a flow rate of 0.6 ml min-1. The
peak detection was achieved using a Millipore Waters 410 Differential Refractometer
22
(Millipore, Massachusetts, United States). The data was recorded and uploaded using
Delta Chromatography Data System version 5.0 software. Samples were diluted with
RO water to achieve 5 to 10 fold dilutions and 16 μL of the sample was injected to
HPLC column with a 26 min running time per sample.
Glycerol and ethanol concentrations were determined by integrating the sample peak
areas and comparison of the peaks to known standards concentrations. Standard curves
for ethanol and glycerol concentrations were prepared using 3 different concentration
levels of the analytes as shown in Table 2-3.
Table 2-3: HPLC Standards

Compound Standard Concentration g l-1
Glycerol 1 0.1
2 3
3 8
Ethanol 1 1
2 7
3 40
2.4.4 Cloud point determination

At the end of production the crude biodiesel was tested for its cloud point by
determining the temperature at which wax crystals form, causing the oil to become
cloudy. Wax crystals of sufficient quantity can plug filters used in some fuel systems.
A 50 ml crude biodiesel sample was transferred into a beaker and heated at 130oC for
30 minutes to make sure all moisture was removed. Following cooling, 45ml of this
sample was poured into a clear beaker or bottle and placed in an ice water bath in
which the temperature was maintained between 2oC – 4oC. The cloud point was
determined as the temperature (oC) when the portion of the thermometer immersed in
the oil was no longer visible when viewed horizontally through the bottle.
23
2.4.5 Water content

The water content was analysed using the ASTM D4377 Standard Method (for
determination of water in oil using the Potentiometric Karl Fischer Titration System
(Figure 2-3).
The following procedure was adopted.

A dry clean syringe, (5ml or 10ml) was rinsed with sample several times and the
contents discharged to waste. The sample to be tested was then drawn up and the
syringe and contents weighed to the nearest 0.1 mg. The sample was injected in the
titration vessel, the needle cleaned with paper tissue and the syringe reweighed. The
sample was then automatically titrated to its end point to the nearest 0.01 ml Karl
Fischer reagent.
Calculation: Water (mass) % = (C X F/W) X10 Equation (2)
C = ml of Karl Fischer reagent, F= Water equivalent of KF reagent,
W= sample (g), 10= factor for converting to %
Figure 2-3: Metrohm 756/831 KF Coulometer system used for determining water content in
biodiesel
24
2.5 Calculation
2.5.1 Carbon balancing
Carbon balancing of the reactions was carried out to confirm the accuracy of the
various component measurements. Obtaining a good carbon balance indicates internal
data consistency and provides reasonable confidence in the underlying data (Schell et
al. 2002)
Carbon Balance (%) =Carbon Contents of products (g) x100% Equation (3)
Carbons Contents of reactants (g)
2.6 Yeast Growth and By-Product Glycerol

2.6.1 Saccharomyces cerevisiae growth cultures
The strain of Saccharomyces cerevisiae designated as ATCC26603 was used in these
studies.
All media ingredients were dissolved in RO water. For the preparation of agar medium,
15 g l-1 of bacteriological grade agar was added to 10 g l-1 yeast extract, 20 g l-1 peptone
and 20g l-1 pure glycerol before being autoclaved. When the agar medium reached
room temperature it was then poured into 90 mm diameter disposable Petri dishes for
use as plate culture medium and stored at 4oC. The liquid broth contained the same
medium ingredients without agar.
2.6.2 Culture growth conditions and culture storage

Table 2-4 summarizes the growth conditions for S. cerevisiae on both liquid and solid
medium. For solid culture, 100 μL inoculum from 40% v/v glycerol stock culture was
spread on agar plates and left to grow in an incubator over 24 h. When the yeast was
fully grown, the culture was re-streaked on fresh agar plates and left in the incubator
over 24 h to produce single colonies for future use. All Petri dishes were sealed using
parafilm prior to incubation to prevent liquid evaporation. For liquid cultures, a loopful
25
from the S. cerevisiae plate was added to sterile growth medium (2.6.1) in 50 ml falcon
tubes and left to grow over night.
Yeast cultures on agar plates were kept at 4oC for a period of not more than 3 weeks.
For the long term storage the yeast culture were suspended in 40%v/v glycerol medium
stock and kept at -80oC freezer.
Table 2-4: Growth conditions of yeast S. cerevisiae

Strain Culture type Container Agitation Temperature
S.cerevisiae Solid Petri dish No agitation 30oC
Liquid 50ml Falcon 250 rpm 30oC
tube
2.6.3 Culture growth seed media

For media preparation RO water was used and media were prepared in two separate
stages.
First seed
Medium containing 20 g l-1 peptone, 10 g l-1 yeast extract and 20 g l-1 pure glycerol
(YPG) were used for the first seed culture. The glycerol solution was autoclaved
separately and added aseptically after autoclaving.
Second seed
The second seed medium contained of 10 g l-1 yeast extract, 20 g l-1 pure glycerol, 0.2 g
l-1 MgSO4.7H2O, 1.25 g l-1 KH2PO4 and 1 g l-1 urea. The glycerol was autoclaved
separately from the other medium ingredients prior to mixing aseptically to avoid
caramelization
Media for growth studies in fermentor

The media for the yeast growth contained 20 g l-1 glycerol and 10 g l-1 yeast extract, 0.2
g l-1 MgSO4.7H2O, 1.25 g l-1 KH2PO4 and 1 g l-1 urea. For glycerol derived from an
enzyme transesterification reaction, the medium was filtered through a 0.45 μm syringe
26
filter to remove enzyme beads and other solids. Prior to the growth studies, media were
autoclaved at 121oC for 20 min
2.7 Batch Fermentation

Batch fermentation experiments were carried out in a 3 litre Fermentor (New
Burnswitch BIOFLO 110 Fermenter/Bioreactor, Figure 2-4) using 1 litre culture
medium. The temperature was controlled at 30oC and the pH was controlled at pH 5.0
using 2 M sulphuric acid and 3 M sodium hydroxide. The dissolved oxygen
concentration was maintained at 20% air saturation.
Figure 2- 4: 3 Litre Controlled Fermentor used for this study.
27
2.7.1 Inocula preparation
First seed: A loopful of S.cerecisiae was inoculated into 50ml YPG medium in a 250
ml baffled flask and grown over 24 h at 30oC at 250 rpm.
Second seed: 10 ml of inocula from the first seed was added to 100ml sterile
fermentation medium and grown for another 24 h at 30oC at 250 rpm using a 1000 ml
baffle flask. When the culture had reached a relatively high density, as indicated by an
optical density (OD) of 30.0 at 600 nm, then 100 ml of the second seed culture was
aseptically transferred into1 litre culture medium in the fermentor to start the
experiment.
2.7.2 Sample collection and analysis

During the yeast growth, samples were drawn at various times for determination of
glycerol and cell biomass concentrations.
2.7.2.1 Biomass by optical density:

An Ultrospec 2000 spectrometer (UV/Visible Pharmacia Biotech) was used to measure
the OD of S. cerevisiae culture at 600 nm. The samples were diluted with RO water to
ensure that the measurements were within the linear range of the spectrometer. Cell
biomass concentrations were calculated by multiplying the OD to the cell dry weight
factor of 0.32 as this factor had been derived previously from OD vs dry weight
determinations for S.cerevisiae
2.7.2.2 Dry cell weight determination

Pre-labelled 50 ml falcon tubes were dried in an oven at 60oC overnight and then
maintained at room temperature. The tubes were each weighed using an analytical
balance to 2 decimal places accuracy. At various OD levels, 50 ml of the yeast
fermentation culture were collected and centrifuged at 5000 rpm for 30 min to fully
28
separate the biomass from the medium. The residual medium was discarded and the
concentrated cells were washed with RO water and centrifuged at up to 5000 rpm for
30 min. This was repeated five times to fully remove residual medium in the samples.
The concentrated yeast cells were then maintained in an oven at 105 oC to remove any
residual water. Cell biomass concentrations were then determined using the values of
the initial and final weights from each tube.
2.7.2.3 Glycerol and ethanol concentrations:

Fermentation samples were filtered with 0.45μm (Millex-HP) syringe driven filters,
and 200μL was taken to HPLC for glycerol and ethanol analysis. (see section 2.4.3)
2.7.3 Calculation of kinetic parameters

2.7.3.1 Maximum specific growth rate
The maximum specific growth rates (μmax) were calculated from the dry weight
samples taken during exponential growth phase. The formula used was:
μmax = loge x2 - loge x1 Equation (4)
t2 – t 1
where x2 is the final and x1 is the initial concentration of biomass in the exponential
growth phase, t2 and t1 are the corresponding times.
2.7.3.2 Maximum specific glycerol uptake rate

The maximum specific glycerol uptake rate (qs,max) was determined over the same time
period as that for the maximum specific growth rate (i.e. during the exponential growth
phase)
The formula used was:
qs,max = s2 – s1 x 1 Equation (5).
t2 – t 1 x average
29
where s2 is the glycerol concentration at t2 and s1 is the initial glycerol concentration at

t1, and xaverage is the average biomass over this time period.
2.7.3.3 Yeast biomass yield

The biomass yields (Yx/s) were calculated based on the initial and final concentrations
of biomass and glycerol (g l-1). The unit of Yx/s are gg-1. The formula used was:
Yx/s = xf – xi Equation (6).
s i- s f
where si and xi are the initial and xf and sf are the final biomass concentrations (g l-1)
30
3: Biodiesel Production
Several biodiesel production processes have been investigated to understand the
limitations and advantages of each process and any extra steps which need to be
introduced to overcome such limitations.
3.1 Composition of Coconut Oil

The main components of coconut oil are triglycerides. Benzard et al. (1975),
fractionated the triglycerides of coconut oil by GLC into 13 groups based on carbon
numbers of 28 to 52 (Figure 3-1) and it was reported that these groups represent 99.8%
of total triglycerides of coconut oil. A similar profile of coconut triglycerides
composition was reported by Kurmar et al. (2010) as shown in Figure 3-2, showing a
triglycerides chromatography profile.
Figure 3-1: Coconut oil Triglycerides composition from Benzard et al. (1975)
31
Figure 3-2: The above figure was taken directly from the article Kumar et al., (2010).
Triglycerides composition of coconut oil.
Coconut oil can also contain some low molecular weight saturated fatty acids, with the
most common being lauric acid as reported by Mariana et al. (2009). Details of the
fatty acid composition of coconut oil were reported by Rafael et al. (2002) and show
that 91% are saturated and 9% unsaturated (see Table 3-1). Of the 91% saturated fatty
acids, 62% are medium chain fatty acids and 29% long chain fatty acids. Similar results
were obtained from our GC analysis of coconut oil, showing lauric acid as the major
fatty acid component of coconut oil.
Table 3-1: Fatty Acid composition of coconut oil

Fatty acid Number of % Content Length Molecular
Carbon atoms w/w Weight
Caproic C6 1 Medium Chain 116
Saturated Fatty
Acids (MCFA)
Caprylic C8 8 MCFA 144
Capric C10 6 MCFA 172
32
Lauric C12 47 MCFA 200

Myristic C14 17 Long Chain 228
Saturated Fatty
Acids (LCFA)
Palmitic C16 9 LCFA 256
Stearic C18 3 LCFA 284
Oleic C18:1 7 Unsaturated 282
Linoleic C18:2 2 Unsaturated 280
The free fatty acid concentration of coconut oil depends on its extraction method and
storage duration. The concentrations of the free fatty acids within the oil indicate the
quality of the oil. Oils with high free fatty acid concentrations (3% w/w and above) are
considered lower quality and oils with a lower content of free fatty acids (less than 3%
w/w) are considered higher quality.
The composition of triglycerides as reported by Benzard et al. (1975) is used in the
present study to calculate the average molecular weight of coconut oil. As a result the
average molecular weight was calculated as 647.69 g/mole, and this contains 435
g/mole of carbon, this was calculated based upon carbon numbers and their molecular
weights (see Table 3-2). The coconut oil used for our study has an FFA content of
4.5% w/w. The coconut oil yield was estimated 1 litre of oil for every 9 coconuts (Leo
et al. (2011) unpublished).
33
Table 3-2: Coconut oil triglycerides in carbons and percent composition

Carbon % Molecular Estimated
composition weight grams of
carbon
C28 0.9 560 5.04 336.28 3.02652
C30 4.2 579 24.32 360.3 15.1326
C32 15.8 597 94.33 384.32 60.72256
C34 19 617 117.23 408.34 77.5846
C36 20.3 643 130.53 432.36 86.472
C38 17.2 665 114.38 456.38 78.49736
C40 9.6 690 66.24 480.4 46.1184
C42 6.4 725 46.4 504.42 32.28288
C44 3.2 754 24.13 528.44 16.91008
C46 1.5 765 11.475 552.46 8.2869
C48 1 797 7.97 576.48 5.7648
C50 0.5 809 4.05 600.5 3.0025
C52 0.2 800 1.6 624.52 1.24904
Av
molecular 647.69 Grams of 435.32g
weight g/ mole carbon of carbon
3.2 Data from the Scientific Research Organization of Samoa

3.2.1 Pilot scale studies
The initial investigations on the use of coconut oil as a biodiesel feedstock were
conducted in Samoa at the Scientific Research Organization of Samoa (SROS) and
used 3 catalysts, viz. alkali, acid and enzyme catalysts. The studies with the alkali
catalyst were carried out at pilot scale (200 litres). (See Figures 3-3 to 3-5).
The reactants were mixed by high speed circulating pumps. The methanol and catalyst
are mixed in a different compartment before being added slowly to the reaction vessel
while circulating the liquid at high speed (Figure 3-3). The crude biodiesel was left
overnight in the settling tanks to separate the glycerol (lower phase) and crude
biodiesel (upper phase) before transferring the crude biodiesel to the storage tank
through the polishing unit (Figure 3-4). The biodiesel produced is now being used to
run Government vehicles as shown in Figure 3-5.
34
Figure 3-3: The 200 litre pilot plant used in Samoa to produce coconut biodiesel. Circulation
is achieved by pressure pumps operating at high speed. Temperature is controlled at 50oC.
Figure 3-4: The pilot plant equipment showing the settling tank for phase separation. The
glycerol is removed and crude biodiesel is transferred to the storage tank via the polishing
unit. The polishing unit is designed to separate and filter out residues such as residual
glycerol, methanol and any catalyst particles.(e.g for enzymes)
35
Figure 3-5: One of the Government vehicle now run being using coconut biodiesel produced
from the pilot plant shown in Figures 3-3 and 3-4.
The pilot plant experiments were run using a feedstock with an FFA content of 3%
(w/w) with added methanol. The molar ratio used was coconut oil : methanol of 1:3
based on stoichiometry predicted in section (1.2.1). Under these conditions the alkali
catalyst (approximately 1kg) was effective in converting the oil to be suitable for
biodiesel, finishing the reaction in less than 60 min. The reaction resulted in greater
than 91% conversion triglycerides to esters (Figure 3-6). The changes in the major FFA
components to esters are shown in Figure 3-7.
36
3
Total Ester (g mole l )
-1
0
0 20 40 60
Time (minutes)
Figure 3-6: Conversions to total esters in the pilot plant equipment with alkali catalyst. A 1:3
molar ratio coconut oil to methanol with approximately 1kg sodium hydroxide
1.0
Ethyl esters (g mole l-1)
0.8
0.6
0.4
0.2
0.0
0 20 40 60
Time (minutes)
Ethyl laureate Ethyl myristate Ethyl palmitate
Figure 3-7: Changes in the major FFAs of coconut oil in the pilot plant equipment with alkali
catalyst. The reaction temperature was 50oC with high speed pump circulation.
37
3.2.2 Laboratory scale studies

3.2.2.1 Acid Catalyst
The results for the transesterification of coconut oil with ethanol with an estimated
triglycerides: ethanol molar ratio of 1:3 with 2.5% v/v sulphuric acid are shown in
Figures 3-8 and 3-9 for laboratory scale studies using a total reaction volume of 250
ml. The experiment was conducted at 75oC, the optimum temperature for an acid
catalyst. The results showed a more rapid reaction in the first 120 min, with up to 60%
conversion to esters, then slowing to reach maximum conversion of about 70% at 300
min (Figure 3-8). The results for the major FFAs also showed maximum conversions at
about 300 min (Figure 3-9)
3
Total Ester (g mole l-1)
0
0 100 200 300 400
Time (minutes)
Figure 3-8: Total ester conversion of the acid transesterification of coconut oil to ethanol
molar ratio 1:3 by 2.5% v/v sulphuric acid at 75oC,itation speed 350rpm.
38
Ethyl Ester concentration (g mole l-1)
1.0
0.8
0.6
0.4
0.2
0.0
0 100 200 300 400
Time (minutes)
Figure 3-9: Major fatty acids ester conversion during the acid transesterification of coconut
oil, reaction temperature was 75oC and agitation speed at 350rpm
3.2.2.2 Enzyme catalyst

Laboratory scale enzyme studies were carried out at SROS. The results are shown in
Figures 3-10 and 3-11 with the coconut oil being transesterified using an estimated 1:3
molar ratio of coconut oil: ethanol with 5% w/v catalyst (lipase) at 50 oC. The
concentration of catalyst used was relatively high and a conversion of 60% was
achieved after 300 min (Figure 3-10). The conversions of the main three FFAs are
shown in Figure 3-11.
39
3
Total Ester (g mole l )
-1
0
0 100 200 300 400
Time (minutes)
Figure 3-10: Shows total ester conversion of the enzymatic transesterification of coconut oil
: ethanol molar ratio 1:3 by 5% w/v enzyme at 50oC, agitation speed 350rpm.
Ethyl Ester concentration (g mole l-1)
1.0
0.8
0.6
0.4
0.2
0.0
0 100 200 300 400
Time (minutes)
Figure 3-11: Showing the major fatty acids changes during the enzyme transesterification of
coconut oil with ethanol (1:3) enzyme catalyst was 5% w/v based on oil volume, reaction
temperature 50oC with agitation speed of 350rpm.
40
3.2.3 Summary of the results at SROS

Comparative results with the three catalysts used in the initial investigation of coconut
biodiesel at SROS are shown in Figure 3-12. The results showed the transesterification
reaction with sodium hydroxide catalyst was much faster (90% conversion by 50 min)
compared to the acid (60% conversion by 120 min) and enzyme catalyst (60%
conversion by 300 min). It should be noted however that the results with the alkali
catalyst were at pilot scale (200 litres) while the two latter were at laboratory scale (250
ml) and this may have had some influence on the reaction rates due to different modes
of mixing. Given some of the problems with the alkali process (see later 3.2.4), the
challenge is to improve the conversion levels and rates of the other two processes.
Catalyst Comparison
100
90
80
% Conversion
70
60
50
40
30
20
10
0
0 2 5 10 15 30 50 60 120 180 240 300
Minutes
Acid Catalyst Enzyme Catalyst Alkali Catalyst
Figure 3-12: A comparison of the catalyst conversions with acid, alkali and enzyme base
catalysts
41
3.2.4 Possible problems identified with the alkali process at SROS
The following possible problems have been identified with the alkali process.
1) A relatively high FFA content (3% w/v and above) in the coconut oil which
resulted in saponification and caused partial solidification. Longer term
storage of coconut oil could increase this problem of higher FFAs.
2) The pilot plant alkali catalyst would only work well with high quality (low
FFAs) oils which are more expensive.
3) The open storage system used could introduce moisture into the biodiesel
product, increasing the moisture content of the biodiesel.
4) An accumulation of waste materials which occurred due to the production of
glycerol and solidified materials from failed pilot plant runs.
3.3 Detailed Kinetic Studies

3.3.1 Alkali Catalyst
The experimental work in this section was carried out at UNSW and resulted in a more
detailed kinetic analysis at laboratory scale (300 ml) to identify the capabilities and the
problems likely to be faced when dealing with an alkali or enzyme based process.
Ethanol was used in the trans/esterification process instead of methanol as some
possible toxicity problems have been identified with methanol.
The results using a 1:3 molar ratio of coconut oil to ethanol, with the coconut oil
containing the FFA content of 4.5% w/v, showed similar problems to those observed in
the SROS biodiesel experiments when using an alkali catalyst. The results are shown in
Figure 3-13 using 0.5% w/v sodium hydroxide concentration. For this concentration, it
was found that the reactants became solidified in less than 10 min, with relatively high
amounts of ethanol still trapped within the gel. From the measured values, less than 0.3
mole l-1 of glycerol and 2 mole l-1 of ester were produced, with 0.6 mole l-1 of
triglycerides and 1.5 mole l-1 of ethanol consumed. The reaction doesn’t follow the
predicted stoichiometry (see Section 1.2.1 Chapter 1) presumably due to the difficulties
42
of accurate measurements resulting from gel formation. As a result it was decided to

increase the concentration of the alkali (NaOH) catalyst to see if this gelling problem
could be overcome. The results of this second experiment are shown in Figure 3-14.
Triglyceride concentration (g mole l -1)
1.0 3
Ester concentration (g mole l -1)

0.8
2
0.6
0.4
1
0.2
0.0 0
0 2 4 6
Triglycerides Esters
3 1.0
Glycerol concentration (g mole l -1)
-1
Ethanol concentration (g mole l )
0.8
2
0.6
0.4
1
0.2
0 0.0
0 2 4 6
Time(minutes)
Ethanol Glycerol
Figure 3-13: Alkali reaction using 1:3 molar ratio coconut oil to ethanol with 0.5% w/v
catalyst at 50oC , speed 350 rpm.
43
Triglyceride concentration (g mole l -1) 1.5 3

1.0 2
0.5 1
0.0 0
0 5 10 15 20 25
3 1.0

-1
0.8
2
0.6
0.4
1
0.2
0 0.0
0 5 10 15 20 25
Time(minutes)
Ethanol Glycerol
Figure 3-14: Alkali reaction with 1:3 molar ratio coconut oil to ethanol using 1% w/v catalyst
at 50oC, speed 350 rpm
44
However it was found that there was no significant improvement in the results with
appreciable amounts of reactants being trapped within the gel with these being unable
to be utilized due to solidification resulting from the relatively high FFA content. The
increased amount of alkali appears to quicken the solidification, trapping increased
amounts of ethanol. Overall the stoichiometry of the reaction remained unbalanced due
to this solidification.
The laboratory scale results with the alkali process showed that most of the conversion
occurred in the first 10 min, however similar problems occurred to those reported for
the SROS pilot plant runs. Use of the alkali catalyst proved to result in a fast reaction,
however it was clear that major problems occurred when dealing with a high FFA
content in the coconut oil. The use of an increased alkali catalyst concentration did not
solve the problem.
3.3.2 Use of Acid Pre-treatment

To improve biodiesel production using a high FFA feedstock, a two-step process was
evaluated. This process used an acid catalyst in the first stage to overcome the
solidification problem, and an alkali catalyst in the second stage. The results are shown
in Figure 3-15.
In stage 1, a 0.1% v/v sulphuric acid-based catalyst was mixed with oil and ethanol
(1:1 molar ratio). The results in Figure 3-15 show that the FFA content of the oil was
decreased from 4.5% to 1% (w/v). The reaction temperature used was 50oC and it took
4 h to bring the FFA percent to less than 1% w/v. In addition to the esterification of the
FFAs, some of the triglycerides were converted into esters reducing the triglycerides by
about 0.25 moles l-1 and producing about 0.85 mole l-1 of esters.
In stage 2, the alkali catalyst was introduced and this resulted in complete conversion
of the triglycerides to esters at a much faster reaction rate (Figure 3-16). Full
conversion occurred within 5 min and no gelling problems were evident. This reaction
utilizes 0.60 mole l-1 triglycerides and 2.14 mole l-1 of ethanol, producing 2.17 mole l-1
of esters and 0.58 mole l-1 of glycerol and closely conforms to the predicted reaction
45
stoichiometry (within limits of experimental errors). Excess ethanol and 0.8% w/v
alkali catalyst were used in this stage 2 experiment.
5
Free Fatty acid (% w/v)
0
0 100 200 300
Time(minutes)
Triglyceride concentration (g mole l-1)
1.0 1.0 Ester concentration (g mole l-1)
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 100 200 300
Time(minutes)
Figure 3-15: 2 Step process: Stage 1 using dilute sulphuric acid (0.1% v/v) to reduce the
FFA of the coconut oil using 1:1 molar ratio coconut oil to ethanol at 50 oC.
46

0.8
2
0.6
0.4
1
0.2
0.0 0
0 2 4 6
Esters Triglycerides

6 0.8
Ethanol concentration (g mole l -1)
0.6
4
0.4
2
0.2
0 0.0
0 2 4 6
Time (Minutes)
Ethanol Glycerol
Figure 3-16 2 Step process: Stage 2, transesterification of treated coconut oil to ethanol
(1:6 molar ratio) with 0.8% w/v alkali catalyst at 50oC at 350 rpm
47
3.3.3 Acid Catalyst Transesterification of coconut oil

The use of an acidic catalyst for the complete reaction was also studied to avoid the gel
formation associated with the alkali catalyst process. The results using 1% v/v
sulphuric acid to produce biodiesel from a 4.5% (w/v) FFA coconut oil are shown in
Figure 3-17. The experiment was conducted at 50oC. No gel formation was evident
although use of the acid catalyst resulted in a relatively slow reaction rate, giving 67%
triglycerides conversion after 50 h.
Overall, 2.0 mole l-1 of esters were produced from 0.75 mole l-1 triglycerides, while 1.4
mole l-1 of ethanol was utilized and 0.4 mole l-1 glycerol produced indicating some
deviation from the theoretical stoichiometry.
3.3.4 Acid Esterification of Free Fatty Acids (FFAs)

As shown in Figure 3-18 (A,B,C), in separate experiments with pure samples of the
main FFAs in coconut oil addition of the acid catalyst resulted in the conversion of the
most significant FFAs to their ester forms. The reaction was carried out at temperature
50oC and a 1:3 molar ratio FFA to ethanol was used. This showed a more rapid
reaction rate than that observed when catalysing the FFA’s in coconut oil at 50oC
although the latter was carried out at 0.1% v/v sulphuric acid. For all of these FFAs,
conversion to their esters was completed within 3-4 h at in the presences of 1% v/v
sulphuric acid. The results for the esterification studies showed 92% conversion for
lauric acid, 90% conversion for myristic acid and 90% for palmitic acid.
48

0.8
2
0.6
0.4
1
0.2
0.0 0
0 20 40 60
3 1.0

-1
0.8
2
0.6
0.4
1
0.2
0 0.0
0 20 40 60
Time (hours)
Ethanol Glycerol
Figure 3-17: Acid Catalysed transesterification of coconut oil, using 1% v/v sulphuric acid a
1:3 molar ratio coconut oil to ethanol with reaction temperature at 50 oC at 350 rpm.
49
Ethyl laureate concentration (g mole l-1)

Lauric acid concentration (g mole l-1)
2.5 2.5
2.0 2.0
1.5 1.5
(A)
1.0 1.0
0.5 0.5
0.0 0.0
0 2 4 6 8
Time(Hours)
Lauric Acid Ethyl laureate

Ethyl myristate concentration (g mole l-1)
Myristic Acid concentration (g mole l-1)

2.5 2.5
2.0 2.0
1.5 1.5
1.0 (B) 1.0
0.5 0.5
0.0 0.0
0 2 4 6 8
Time(Hours)
Myristic Acid Ethyl myristate
50
Ethyl Palmitate concentration (g mole l-1)

Palmitic Acid concentration (g mole l-1)
2.5 2.5
2.0 2.0
1.5 1.5
1.0 (C) 1.0
0.5 0.5
0.0 0.0
0 2 4 6 8
Time
Palmitic Acid Ethyl palmitate
Figure 3-18 FFA esterification catalysed by sulphuric acid 1% v/v of lauric acid, myristic acid
and palmitic acid at 50oC using a 1:3 molar ratio FFA to ethanol.
3.3.5 Enzyme catalyzed transesterification of coconut oil

Immobilized lipase enzyme (Novzyme 435) was used for the transesterification
process. As shown in Figure 3-19, close to 90% conversion of the triglycerides to
esters occured at reaction temperature of 50oC with 1% w/v enzyme. The formation of
glycerol follows a similar pattern and maximum production occurs at 50 h. The enzyme
reaction was slower compared to alkali process but slightly faster compared to acid
catalyst (1% v/v sulphuric acid) at this temperature. From the stoichiometry, 0.8 mole l -
1
triglycerides and 2.6 mole l-1 ethanol were converted to 2.5 mole l-1 ester and 0.8 mole
l-1 glycerol. This shows a close conformity with the theoretical reaction stoichiometry
of 1 mole l-1 triglycerides and 3 moles l-1 ethanol giving 3 mole l-1 esters and 1 mole l-1
glycerol (see Section 1.2.1)
51
3.3.6 Enzyme Esterification of fatty acids (FFAs)

The enzymatic esterification of major fatty acids of coconut oil (lauric acid, myristic
acid and palmitic acid) was also studied in separate experiments to determine whether
or not saponification might be a problem in the enzyme process. The results in (Figure
3-20 A,B&C) showed typical Michaelis-Menten kinetics for the conversions of lauric
acid and myristic acid and possibly palmitic acid to their esters.
The results also showed that lauric acid reached 94% conversion by 7 h, while myristic
acid reached 74% by 7 h and a 52% conversion was attained for the palmitic acid after
3 h. The results showed that for the 4.5% w/v concentration of FFAs in the coconut oil
that this would be reduced to less than 2% (w/v) FFA concentration after 3 h, and
therefore any saponification or gel formation would be unlikely to occur or cause
significant problems. This is one of the potential advantages of an enzyme based
process when compared to use of an alkali catalyst.
52
Triglyceride concentration (g mole l -1)
1.0 3

0.8
2
0.6
0.4
1
0.2
0.0 0
0 20 40 60
3 1.0

-1
0.8
2
0.6
0.4
1
0.2
0 0.0
0 20 40 60
Time(hour)
Ethanol Glycerol
Figure 3-19: Enzymatic transesterification of coconut oil using 1% w/v enzyme, 1:3 molar
ratio coconut oil to ethanol at 50oC, agitation speed of 350 rpm
53
Ethyl laureate concentration (g mole l-1)

Lauric acid concentration (g mole l-1)
2.5 2.5
2.0 2.0
1.5 1.5
(A)
1.0 1.0
0.5 0.5
0.0 0.0
0 2 4 6 8
Time (Hours)
Lauric Acid Ethyl laureate
Ethyl myristate concentration (g mole l-1)

Myristic Acid concentration (g mole l-1)
2.5 2.5
2.0 2.0
1.5 ( 1.5
1.0 1.0
(B)
0.5 0.5
0.0 0.0
0 2 4 6 8
Time (Hours)
Myristic Acid Ethyl myristate
54
Ethyl Palmitate concentration (g mole l-1)

Palmitic Acid concentration (g mole l-1) 2.5 1.5
2.0
1.0
1.5
(C)
1.0
0.5
0.5
0.0 0.0
0 1 2 3 4
Time(Hours)
Palmitic Acid Ethyl Palmitate
Figure 3-20: Enzymatic esterification of the main free fatty acids found in coconut oil viz.
lauric acid, myristic acid and palmitic acid using 1:3 molar ratio FFA to ethanol at 50oC
3.3.7 Improvements to the Enzyme Process

3.3.7.1 Effects of enzyme recycling
Recycling the immobilized lipase may be one way to achieve better process economics.
In Figure 3-21, the results are shown when the enzyme beads were recovered from the
previous experiments and reused for the same conditions. Compared to the results
shown in Figure 3-19, the reaction rate with the recycled enzyme resulted in a 20-30%
reduction in the ester production after 50 h. It is possible that some further techniques
for enzyme regeneration are necessary (apart from water washing) to fully regenerate
the lipase. Other researchers have reported that lipase could be re-used for biofuel
production up to 200 times without significant loss of activity following regeneration
with butanol (Lilin et al. 2006).
55

0.8
2
0.6
0.4
1
0.2
0.0 0
0 20 40 60
Triglycerides Ester
3 1.0

-1
2
0.5
0.0
0
0 20 40 60
Time(hours)
Glycerol Ethanol
Figure 3-21 Recycled enzyme transesterification of coconut oil using 1:3 molar ratio coconut
oil: ethanol with 1% w/v enzyme at 50oC
56
3.3.7.2 Effect of use of ultrasound for increased agitation

The relatively slow reaction rate with the enzyme catalyst may make this process less
feasible on an industrial scale and this has led to further investigation of more
economical strategies to improve the enzyme process. To increase the rate of the
enzyme reaction the use of ultrasonics was studied as this technique has been used
successfully by other authors (Yu et al. 2009) to increase reaction rates at laboratory
scale. The results are shown in Figure 3-22, and illustrate the effectiveness of the
ultrasonication in increasing the conversion and reducing the reaction time from 80%
conversion after 50 h to a 92% conversion in 3 h. The ultrasound frequency used was
43 kHz at 50oC with occasional stirring. Higher frequencies were not used as it is
possible that these could denature the enzyme and reduce its activity. The challenge
will now be to develop equipment to use this technique in larger scale systems.
100
80
Esters % Conversion
60
40
20
0
0 2 4 6 8 10
Time (Hours)
Figure 3-22: Improvements of enzymatic transesterification conversions with the

assistance of ultrasound, using 43khz and 50oC; 1% w/v lipase used.
57
3.4: Cultivation of Saccharomyces cerevisiae using Glycerol By-Product

as a Carbon Source
The use of by-product glycerol from biodiesel production has produced the opportunity
to improve the economics of the biodiesel industry and has been the focus of recent and
ongoing research. The present study focuses on growing Saccharomyces cerevisiae on
medium containing glycerol by-product from the enzyme transesterification of coconut
oil with the potential for utilizing S.cerevisiae as a protein source in animal feed.
3.4.1: Composition of glycerol from enzyme-based process

In Table 3-3 the compounds identified in the glycerol derived from enzyme-based
biodiesel production are shown. Chromatographic analysis revealed that the solids
were 78% (w/v) glycerol associated with 22% (w/v) esters, glycerides and free fatty
acids. These latter compounds in the glycerol by-product may provide additional
carbon and energy sources for S.cerevisiae and be beneficial for biomass production.
The calculation of 20 g l-1 of glycerol concentration used for the experiments was based
on this 78% (w/v) concentration in the by-product glycerol.
Table 3.3: Properties of By-product

Glycerol
Properties % (w/v)
Glycerol 78
Esters 10.7
Mono Glycerides 0.78
Di-glycerides 7.01
Triglycerides 1.04
FFA 2.43
58
3.4.2: Kinetics of S.cerevisiae growth on pure glycerol and glycerol derived from
an enzyme transesterification process
The experiment was carried out with standard medium containing 10 g l-1 yeast extract
and 20 g l-1 glycerol to determine the kinetic growth of S.cerevisiae using glycerol as a
sole carbon source (see section 2.6.3). The experimental results are shown in Figures 3-
23 (A,B) for growth on medium containing pure glycerol and by-product glycerol
from the enzyme process respectively. A growth medium containing 10 g l-1 yeast
extract without glycerol showed no yeast growth.
Table 3-4 Summary of batch results in 3L fermentor

By-product
glycerol Pure Glycerol
Maximum specific growth
rate μmax (h-1) 0.13 0.15
Biomass Yields Yx/s (g g-1) 0.77 0.76
Max glycerol uptake rate
qsmax (gg-1 h-1 ) 0.07 0.1
Table 3-4 summarizes the values of the kinetic parameters used for biomass yields and
specific growth rates and the glycerol uptake rates for both batches. The enzyme
derived glycerol showed some reduction in the maximum specific growth rate and
glycerol uptake rate when compared with those for pure glycerol which may indicate
some minor inhibitory effects in the former case. The relatively high biomass yields
(Yx/s = 0.77 g g-1) suggests that the yeast by-product could be a valuable protein
source for future use e.g. in animal feed formulations.
59
20 20
(A)
Dry Cell Weight (g l-1)

15 15
Glycerol (g l-1)
10 10
5 5
0 0
0 10 20 30 40 50
Time(h)
Dry Cell Weight Glycerol
20 20
(B) Dry Cell Weight (g l-1)
15 15
Glycerol (g l-1)
10 10
5 5
0 0
0 10 20 30 40 50
Time(h)
Dry Cell Weight Glycerol
Figure 3.23 : Batch growth kinetics of S.cerevisiae from pure glycerol (A) and glycerol from
enzyme process (B). Temperature 30oCand pH controlled at 5.0
60
3.5 Summary and Discussion of Results

A comparative study of the three processes used for biodiesel production gave the
following results:
1) the alkali process was a lot faster than using either the acid or the enzyme
catalyst,
2) feedstocks with high FFA content (more than 3% w/v) affected the catalytic
activity of the alkali process negatively, resulting in saponification which
decreases the esters yield and causing processing problems,
3) the introduction of a two-step acid/alkali process overcomes the problem of
saponification yielding high ester conversions. A similar outcome has been
reported by Nakpong et al. (2009). in which a two-step process was used to
produce biodiesel from a 12% w/v FFA content coconut oil,
4) the use of 50oC as the reaction temperature was suitable for both the enzyme and
the acid catalyst although conversion rates were relatively slow reaching 67% for
acid and close to 90% for enzyme ester conversion after 50 h. Similar reaction
rates were reported by Watanabe et al.(2001) with a stepwise addition of
methanol with enzyme lipase yielding up to 90% ester conversion after 50 h,
5) an enzyme process using jatropha oil reported by Shah Et al.(2004) produced
similar high yields (92% w/v ester conversion) although higher enzyme
concentrations were used (10% w/v) compared to the 1% w/v enzyme used in
this study,
6) very high ester conversions (99% w/v) were reported by Narasimharao et
al.(2007) using sulphuric acid as catalyst at 78oC as the reaction temperature
after 18 h, whereas in the present study, a reaction temperature of 50oC was used
for the acid-based process.
7) further studies with the enzyme catalyst showed it can be recycled more than
once when the enzyme is immobilized (Novozme 435 lipase used in present
study is in a solid particulate immobilized form). In detailed enzyme kinetic
61
studies Lilin et al.(2006) reported that an immobilized enzyme could be recycled

for up to 200 times with a consistent yield of 95% ester conversion. A similar
outcome was reported by Du et al.(2004) in which recycling the enzyme 100
times gave a consistent yield of 92% ester conversion.
8) the use of ultrasound agitation to increase the enzyme reaction rate was very
effective in achieving a 92% ester conversion in 3 h. A study by Yu et al.(2009)
showed a similar increased conversion with a shortening of the reaction time to 4
h and a 96% ester conversion,
9) good growth of baker’s yeast S. cerevisiae on glycerol was achieved with some
reduction in growth kinetics for glycerol by-product medium compared to the
pure glycerol. The results indicated some evidence of inhibitors in the former.
Specific growth rates were also lower compared to those reported for growth on
glucose based medium (Shafaghat et al. 2009)
10) yields were similar for both pure glycerol and by-product glycerol
(0.76-0.77 g g-1 respectively). These are higher than those for growth on glucose
which are typically 0.40 – 0.50 g g-1 (Rosario et al. 1978),
11) previous studies with yeast growth on glycerol have shown that a period
of adaptation is needed before good growth and high final yeast concentrations
and yields are achieved (Merico et al. 2011). In the present experiments some
residual glycerol remains and additional adaptation of the strain of S.cerevisiae
may be necessary for complete glycerol utilization,
12) previous studies using Pichia pastoris for growth on glycerol derived from
biodiesel production have reported biomass yields of 0.83 g g-1 for phytase
production (Tang et al. 2008). The yield for S.cerevisiae of 0.77 g g-1 with
glycerol derived from enzymatic biodiesel in the present study is similar and
illustrates the potential as a protein source for animal feed.
62
4: Summary of the Economic Analysis
4.1 Process Flow-sheet: Basis for Economics Analysis
The biodiesel transesterification process presented in this flow-sheet is the basis for the
pilot plant at SROS (Figure 4-1). The process is based on a theoretical 1:3 molar ratio
coconut oil triglycerides to methanol which results in 3 moles of esters and 1 mole of
glycerol (see Section 1.2.1). The process is based on this molar ratio and involves the
use of 200 litres of coconut oil plus 40 litres of methanol which is designed to produce
200 litres of crude biodiesel and about 40 litres of glycerol. A washing and polishing
unit is incorporated before the final product storage and usage.
Figure 4-1: Computer model for 200 litre biodiesel pilot plant, developed using a Superpro
Designer 7 software
A computer model using Superpro Designer 7TM (Intelligen) software has been
developed to further characterize the process and conditions for its economic
feasibility.
63
4.2 Results of Economic Analysis

4.2.1 Pilot scale process
An initial economic analysis was based on data from the pilot scale process to produce
200 litre transesterified triglycerides per batch, with two batches per week and two staff
operators working 10 h per batch for each pilot scale run. Based on 50 weeks per year
operation, this would result in 20,000 litres annual production from the SROS pilot
plant. The capital cost of this pilot scale plant was USD 84,000.
The following quantitative reaction describes a typical pilot scale batch operation:
200 l coconut oil + 40 l methanol -Æ 1kg NaOH-Æ 200 l esters + 40 l glycerol.
(catalyst)
This reaction assumes the following:
1) an approximate 3:1 molar ratio of methanol: coconut oil as predicted by the
stoichiometry,
2) close to theoretical conversion from triglycerides to esters in 2-4 h based on the
SROS results for an alkali-based process,
3) glycerol separation achieved by phase separation. It was assumed also that un-
reacted coconut oil and methanol do not affect the quality of the biodiesel blends
produced.
4.2.2 Economic feasibility of commercial scale process

The annual imports to Samoa of diesel can be estimated at 40 x 106 litres per year
(Ministry of Finance, Samoa. 2011) and if it is assumed that this is replaced with
coconut oil derived biodiesel (as B10), then the plant capacity required to supply would
be 4 x 106 litres per year. An estimate of the capital cost of this plant can derived from
the following equation with a scale factor of 0.7 (Buchanan & Sinclair, 1964):
64
C2/C1 = (V2/V1)0.7
Where C2 = Cost (USD) of new plant
C1 = Cost (USD) of the pilot plant
V2 = Annual capacity (litre) of new plant
V1 = Annual capacity (litre) of pilot plant
The capital cost (C2) of the new plant can therefore be estimated as USD $3.4
million for a production capacity of 4 x 106 litres per year as follows:
C2 = 84,000 x (4 x 106 / 2 x 104 ) 0.7
= 84,000 x (200)0.7
= USD 3.4 x 106
An internal rate of return (IRR) can then be estimate for a plant producing 10% of
Samoa’s diesel imports, from the equation:
C = a1/ (1 + r) + a2/ (1 + r)2 + ....... + a10/ (1 + r)10
where
C = Plant capital cost
a1 = profit year 1, a2 = profit year 2, a10 = profit year 10
r = internal rate of return (IRR).
To estimate annual profit (or loss) for the commercial plant, the costs of input per batch
can be calculated and compared to the value of products
1) Raw materials
a. 40,000 litres coconut oil @ USD 1.13 per litre
b. 8000 litres methanol @ USD 1.20 per litre
c. 200 kg NaOH @ USD 1.72 per kg
total cost: = USD 55,144
2) Running Costs
a. Labour (4 workers) @ USD 4.00 per hour
Assuming 10 hours per batch = USD 160.00
b. Electricity and services
65
Assume $500 per batch = USD 500

3) Depreciation
Life of plant estimated of 10 years, therefore an annual depreciation rate of 10%.
This depreciation can then be calculated per batch (with a 100 batches annually)
3.4 x 106 x 10/100 x 1/100 = USD 3,400
Total production cost per batch = USD 59,204.
Total value of products per batch
1) 40,000 litres biodiesel @ USD 1.20 per litre = USD 48,000.
2) 8000 litres glycerol by-product @ USD 1.00 per litre = USD 8000
Total product value of per batch of = USD 56,000.
From this analysis the process would make a loss based on the value for biodiesel
product of USD 1.20 per litre which is the current diesel price per litre in Samoa.
(Ministry of Finance, Samoa. 2011)
A sensitivity analysis could be used to determine at what selling price of biodiesel the
process might have a reasonable level of profitability (e.g. an IRR of 30%)
For example,
1) a biodiesel selling price of USD 1.30 per litre would result in close to break-even
for the process
2) a biodiesel price of USD 1.60 per litre would give an annual profit of
approximately USD 12,000 per batch or USD 1.2 million annually with an IRR
of 30%. This would result in a 4 cents per litre (or SAT 0.1 per litre) increase in
the price to consumers of a B10 product.
From this sensitivity analysis, it is clear that the profitability of the process is very
dependent on the selling price of the biodiesel, and this increased cost would need
to be balanced against the potential social and environmental benefits of such an
initiative, and also have strong Samoan Government support.
66
4.2.3 Sensitivity analysis

For a more complete analysis, the sensitivity of the IRR could be determined for the
following situations:
1) variations in the cost of raw materials e.g for coconut oil, in the range of USD
1.00 per litre to USD 1.30 per litre. Currently the cost of coconut oil is about
70% of total input cost.
2) variation in the selling price of the biodiesel product from USD 1.30 per litre
break-even to USD 1.60 per litre, under changing conditions (e.g. increases in oil
prices)
3) variation in the plant capacity if plant is doubled to give an annual production of
8 X 106 per year on the assumption that a B20 fuel rather than a B10 fuel will be
produced
4) if only 10% of the fuel produced in Samoa is B10, then Table 4-1 shows that the
IRR would reduce to .... for the fuel selling price of USD 1.60
5) effect of changing in depreciation rate from current 10 year period (10% annual
rate) As discussed by Buchanan and Sinclair, (1964), a depreciation rate of 8.3%
over 12 years is also used for some processes, and this would improve the
current process economics,
6) as discussed also by Buchanan and Sinclair. (1964) and Reisman (1988), a scale-
up factor of 0.7 for the capital costs has been used as a general factor,
ie C2/C1 = (V2/V1)0.7 This may change in the range 0.7 – 0.9 depending on the
type of process equipment used.
67
Table 4-1: Cost analysis for two commercial scale plants

B10 by 4 x 105 B10 by 4 x 106
Capital Cost $731,097.0 $3.4 x 106

Raw Materials 4000 l/batch $/ litre 40,000 $/ litre
l/batch
Coconut Oil 4000 l 1.13 40,000 l 1.13
Methanol 800 l 1.20 8000 l 1.20
NaOH 20 kg 1.72 200 kg 1.72
Running Costs
Labour (4 x 10 h at 160 160.00
$4.0 an hour)
Electricity 100 500.00
Depreciation 731.1 3400.00
Total production $6505.5 $59,204.00
cost
Product Value 4000 l/ batch $/ litre

Biodiesel 4000 l 1.60 40,000 l 1.60
Glycerol 400 l 1.00 8000 l 1
Total Product Value $6800.00 $72000.00
Profit per batch $294.5 $12796
Profit per Annum $29,450.00 $1279600
IRR Less than 5% 30%
It should be noted that as well as the potential profitability of the process, it will also
have other socio-economic benefits for Samoa. These include the creation of additional
job opportunities both in process operation and increased coconut oil production,
greater energy independence and reduced greenhouse gas emissions due to the use of
renewable raw materials. The use of glycerol by-product as a substrate for yeast growth
(as animal feed) and the potential for using ethanol (produced from local raw materials)
for the transesterification process, instead of imported methanol, could further
improved the overall process economics. However, the impact of diverting substantial
quantities of coconut oil from food to fuel would need consideration with regards to
social impacts as coconut is the major food product in Samoa.
68
5: Conclusions and Future Work
5.1: Conclusions
Studies on the use of an alkali, acid and an enzyme catalyst to evaluate the potential for
a low quality coconut oil (4.5% w/w FFA) as a biodiesel feedstock have resulted in the
following conclusions:
1) use of an alkali catalyst resulted in a much more rapid conversion compared to

use of an acid or enzyme catalyst,
2) use of a feedstock with an FFA content higher than 4.5% (w/w) with an alkali
catalyst resulted in saponification. Increasing the amount of alkali catalyst did
not improve the overall process,
3) a two-step process (acid esterification followed by an alkali transesterification)
overcame the effects of saponification caused by high FFA content for the alkali
process,
4) the acid and the enzyme catalytic transesterification reactions were not affected
by high FFA contents. The enzyme (lipase) catalytic reaction was carried out at
50oC, while the acid catalyst worked more effectively at temperatures up to
75oC.
5) use of the enzyme catalyst at 50oC resulted in an 80% conversion to esters after
50 h. The use of ultrasonic agitation to assist with the enzymatic reaction
decreased the reaction time 16 fold at laboratory scale with a 92% conversion to
esters in 3 h. Following the initial reaction, the enzyme was able to be recovered
and recycled with only a 20% reduction in rate of conversion to esters,
6) use of the glycerol by-product from an enzymatic transesterification as a carbon
source for yeast growth showed some reduction in kinetics compared to that for
growth on pure glycerol, indicating some degree of inhibition from products
produced during the bioconversion,
69
7) an economic feasibility study based on laboratory scale data and assuming a

larger scale process to replace 10% of Samoa’s diesel imports (4 x 10 6 litre per
annum), projected a capital cost of USD $3.4 million for the process. Current
input and product costs were used and an annual depreciation rate of 10% was
used to evaluate overall process profitability.
8) With an input cost of coconut oil of USD 1.13 per litre, at a 4 x 106 litre per
annum a biodiesel selling price of USD 1.30 per litre was necessary to achieve
break-even. At USD 1.60 per litre an IRR of 30% was calculated.
9) At a USD 1.60 per litre for biodiesel, and with a diesel price in Samoa of USD
1.30 per litre, the price of bio-blend would be increased by about 4 cents per
litre. However this would result in environmental and social benefits such as
reduced GHG emissions, increased local employments and the use of renewable
raw materials for biodiesel production.
5.2: Future Work
1) detailed kinetic studies on enzyme recycling should be carried out to further

improve the process economics,
2) following the significant improvement achieved the enzyme catalyst reaction rate
at laboratory scale will ultrasound the larger scale experiments should be carried
out to determine the feasibility and cost of scaling up this technology,
3) further detailed kinetic studies of the use of glycerol derived from the enzyme
based biodiesel process should be carried out for biomass production as well as
for higher value fermentation products.
4) it was planned to carry out engine experiments on biodiesel blends B10 and B20
with transesterified coconut oil produced using the enzyme process. However the
requisite testing facilities are still under construction in the School of Mechanical
Engineering at UNSW, and studies were not able to be undertaken. It is planned
in future to complete this evaluation and compare the results with the ASTM
standards (see Appendix)
70
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7. Appendixes:
81
SPECIFICATION FOR
BIODIESEL BLENDS B6-B20, ASTM 7467-10
#Biodiesel (B100) and the petroleum diesel must meet their respective ASTM specifications before blending. This
specification covers blend between 5 and 20 percent biodiesel by volume blended with petroleum diesel fuel.
Property ASTM Method Limits Units
Flash Point D 93 52 minimum °C
Water & Sediment D 2709 0.05 maximum % vol.

A 2
Kinematic Viscosity, 40°C D 445 1.9 – 4.1 mm /sec.
Ash Content D 482 0.01 maximum % mass

Sulfur
S 15 Grade D 5453 0.0015 max. (15) % mass (ppm)
Copper Strip Corrosion D 130 No. 3 maximum
Cetane D 613 40 minimum

D 2500, D4539,
Cloud Point, max or LTFT/CFPP, max D6371 report °C
Ramsbottom Carbon Residue on 10%
bottoms D 524 0.35 maximum % mass
Acid Number D 664 0.3 maximum mg KOH/g
Distillation, T90 AET D 86 343 maximum °C
Oxidation Stability EN 15751 6 minimum hours
Lubricity, HFRR at 60 C D 6079 520 maximum microns
Biodiesel Content, % (V/V) D 7371 6. – 20. % volume
BOLD = BQ-9000 Critical Specification Testing Once Production Process Under Control
A If Grade No. 1-D or Blends of No. 1-D and Grade No. 2-D diesel fuel are used the minimum viscosity shall be 1.3
* The carbon residue shall be run on the 100% sample.
# A considerable amount of experience exists in the US with a 20% blend of biodiesel with 80% diesel fuel (B20).
Although biodiesel (B100) can be used, blends of over 20% biodiesel with diesel fuel should be evaluated on a
case-by-case basis until further experience is available.
SPECIFICATION FOR
BIODIESEL (B100) – ASTM 6751-11a
#Biodiesel (B100) and the petroleum diesel must meet their respective ASTM specifications before blending.
Property ASTM Method Limits Units
Calcium & Magnesium, combined EN 14538 5 maximum ppm (μg/g)
Flash Point (closed cup) D 93 93 minimum °C
Alcohol Control (one to be met)
1. Methanol Content EN 14110 0.2 maximum mass %
2. Flash Point D93 130 minimum °C
Water & Sediment D 2709 0.05 maximum % vol.

2
Kinematic Viscosity, 40 C D 445 1.9 – 6.0 mm /sec.
Sulfated Ash D 874 0.02 maximum % mass

Sulfur
Copper Strip Corrosion D 130 No. 3 maximum
Cetane D 613 47 minimum
Cloud Point D 2500 report °C
Carbon Residue 100% sample D 4530* 0.05 maximum % mass
Acid Number D 664 0.5 maximum mg KOH/g
Free Glycerin D 6584 0.020 maximum % mass
Total Glycerin D 6584 0.240 maximum % mass
Phosphorus Content D 4951 0.001 maximum % mass
Distillation D 1160 360 maximum °C
Sodium/Potassium, combined EN 14538 5 maximum ppm (μg/g)
Oxidation Stability EN 15751 3 minimum hours

Cold Soak Filtration D7501 360 maximum seconds
For use in temperatures below -12 °C D7501 200 maximum seconds
BOLD = BQ-9000 Critical Specification Testing Once Production Process Under Control
* The carbon residue shall be run on the 100% sample.

# A considerable amount of experience exists in the US with a 20% blend of biodiesel with 80% diesel fuel (B20).
Although biodiesel (B100) can be used, blends of over 20% biodiesel with diesel fuel should be evaluated on a
case-by-case basis until further experience is available.

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BIODIESEL PRODUCTION FROM COCONUT OIL

A thesis submitted as partial fulfillment

School of Biotechnology and Biomolecular Science

Abstract 350 words maximum: (PLEASE TYPE)

Declaration relating to disposition of project thesis/dissertation

………………………………………………………… ……………………………………..…………… ……….……………………...…….…

FOR OFFICE USE ONLY Date of

THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS

Samani Carel Tupufia

enzyme processes” Poster presentation in 2011 Bioenergy Conference of “Towards

Publication (in preparation)

1. Literature review: .......................................................................................................1

Page Figure Caption

Page Table Caption

The project has the following objectives:

1.1 Global Biodiesel Production

1.2 Biodiesel Production Processes

1.2.1 Transesterification and esterification reactions

Figure 1-2 Basic chemistry of the transesterification process.

Figure 1-3 Basic chemistry of an esterification process.

1.2.2 Catalysts for transesterification/esterification

1.2.2.1 Acid catalysed reactions

1.2.2.2 Alkali catalyzed reactions.

1.2.2.3 Enzyme catalyzed reactions

1.3 Characteristics of Coconut oil for Biodiesel Production

1.4 Processes for Use of Coconut Oil for Biodiesel

1.4.2 Alkali and acid catalytic transesterification of coconut oil

1.4.3 Use of enzyme catalysts

Table 1-1 Advantage of lipase enzyme catalyst

1.4.3.2 Disadvantages of use of enzyme catalysts

Table 1-2 Disadvantages of use of enzyme catalysts.

1.5 Use of Ethanol rather than Methanol for Biodiesel Production

1.6 Possible use of Glycerol By-Product from Biodiesel Production

2. Materials and Methods

2.1 List of Chemicals used for this study

Methanol 99% v/v Ajax Finechem Pty Ltd

Lauric Acid 99% v/v Sigma Aldrich

Ethyl Capriate 99% v/v Sigma Aldrich

Hexamethyldisilazane 99% v/v Fluka Analytical

Novozyme 435 Activity 100KLU/g Novozyme Australia Pty

2.2 General Solutions

2.2.2 Phenolphthalein colour indicator

2.3. Biodiesel Production Processes

2.3.2 Enzymatic transesterification of coconut oil

2.3.2.1 Ultrasonic assisted transesterification

Figure 2 -1 Burnswick Scientific EDISON USA: Controlled Environment Incubator Shaker

2.3.4 Esterification of free fatty acids

2.4 Transesterification and Esterification Sample Analysis

2.4.1 Coconut oil free fatty acid determination

The FFA percentage was calculated using this formula:

FFA w/w (%) = ml of alkali added x N x 20 X 100 Equation (1)

* N is Normality, *20 is calculation factor for lauric acid

2.4.2 Gas Liquid Chromatography

Table 2-2 : GLC Standards

2.4.3 High Performance Liquid Chromatography

Table 2-3: HPLC Standards

2.4.4 Cloud point determination

2.4.5 Water content

The following procedure was adopted.

2.6 Yeast Growth and By-Product Glycerol

2.6.2 Culture growth conditions and culture storage

Table 2-4: Growth conditions of yeast S. cerevisiae

2.6.3 Culture growth seed media