Biodiesel Production by Lipase Mediated

Transesterification of Moringa oleifera Seed

Oil
 Introduction
The world is confronted with
financial and environmental
issues that connected with energy
use that need to be resolved.
Growing concern about carbon
dioxide, C0₂ build-up and other
so-called greenhouse gasses
A solution for the energy
consumption as well as the
environmental issues (Yahya A,
2013).
Safe alternative for internal
combustion engines, that
produced from sources such as
triacylglycerols which present in
vegetable oils (Garba et al.,
2018).
 Literature Review

Moringa seed kernels contain a

Transesterification is the reaction of
significant amount of oil, up to 40%
fats or oils with alcohols to form
with a high-quality of fatty acid
biodiesel. It is a reversible reaction
composition, oleic acid more than 70%
where triacylglycerols are converted to
which could be a good substitute in the
fatty acid alkyl esters and glycerol
diet as well as for non-food
(Garba et al, 2018). Lipases are the
applications, like biodiesel and a
hydrolytic enzymes that can be used in
lubricant for fine machinery. (Leone et
various industrial applications such as
al, 2016).
biodiesel production.
 Problem statement

Alternative sources of
energy for running our
Increase in the demand of generators, automobiles
petroleum products and the etc. are being considered
prices of petrol & diesel worldwide. Both the edible
are increasing worldwide. and non-edible vegetable
oils can be used as the raw
materials for the biodiesel.
 Research Objective
To compare the
fuel properties of
To synthesis produced
biodiesel from biodiesel with
Moringa oleifera standard biodiesel
To extract the oil seeds using properties.
from Moringa transesterification
oleifera seeds using process using lipase
Soxhlet extraction. as a catalyst
 Hypothesis

Moringa seed oil can be extracted by Soxhlet

method.

Biodiesel can be synthesized using a

biological catalyst
Methodology Sample collection and drying process

Extraction process
Iodine value
Saponification value
Free fatty acid test Characterisation of extracted oil
pH value test
Thin Layer Chromatography
Transesterification process

Copper corrosion test

Combustion test Characterisation of biodiesel
Cetane number
 Sample collection and drying process

Collected 30 Moringa
drumsticks. Take the
seeds out of the After fully dried, the
Moringa stick and then seeds were grinded, Percentage of dry
kept on an aluminum weighed and stored in weighed of moringa
foil, let it to dry in an falcon tube until the seed was calculated.
oven at 60-degree extraction to be done.
Celsius for three
consecutive days.
 Extraction process

Kept the
Soxhlet
Poured 50ml Switched on solution for
apparatus Ran process
of hexane into heating solvent
prepared, 15g for 6 hours
column and mantle and set evaporation
of dried until the
250ml into temperature at (Wadud, A.
moringa seeds column is
round bottom 68 degree 2015).
transferred to clear
flask Celsius Calculated the
thimble
oil yield
 Characterisation of extracted oil

Iodine value test

Added 25ml of
Wij’s iodine
solution to the Added 20ml of
Added 0.3g of Titrated against
mixture. potassium iodide Added few drops
seed oil and 0.1N of sodium
Covered the and shake of starch until
dissolved with thiosulfate until
beaker with thoroughly. blue colour
10ml of yellow colour
aluminium foil Added 100ml of disappear
chloroform disappear
and placed in distilled water
dark place for 60
minutes
 Saponification Free fatty acid
value test value test

Mixed 0.3g of oil with

Mixed 25ml of 0.5N Titrated with 0.5N of 10ml of ethanol (96%).
alcoholic hydroxide HCL and record the
into 0.3g of oil sample value when pink colour
and heated under turn into colourless
reflux (Orhevba, et al., 2013). Added 3 drops of
phenolphthalein to the
sample

After heating, allow the

flask to cool and added Prepared blank sample Titrated with 0.05N of
3 drops of without oil potassium hydroxide
phenolphthalein solution until light pink
colour observed
 pH value test TLC test

Mixture prepared chloroform:

Mixed 2g of oil sample with methanol: water with ratio 7:3:1
hot distilled water, stir the
mixture well

Transferred the mobile phase into TLC

chamber

Cool the mixture at to 25

degree Celsius and
immersed the pH electrode. Spot the oil on silica paper and
Recorded the pH transferred to TLC chamber, observe
the presence of double bond
 Lipase mediated transesterification

Biodiesel recovered by evaporating the

4ml of oil mixed with 24ml of
methanol and washed off the glycerol
methanol. Added 6% of hexane and 4%
with water (Zilfa,2017). Calculated the
of lipase into conical flask
yield of biodiesel.

Left the flask at reciprocal shaker for

24 hours at 40 degree Celsius and 150 Separated the mixture from free
rpm. enzyme using separating funnel
 Characterisation of biodiesel
Copper
corrosion
test After 3 hours, observe
Dipped the copper strips the copper strips
into sample fuel and whether the biodiesel
placed in clean test tube produced corrosion or
Covered the mouth of not.
test tube with
Repeat the steps using
aluminium foil and
conventional diesel
placed in water bath at
50 degree Celsius
 Combustio
n test
Soaked cotton thread in
the same fuel that was
burned. Placed under
tripod stand. Placed water bath on
tripod stand and
measured the
temperature rise in the
water Repeat the steps with
conventional diesel,
petrol, methanol, ethanol
and hexane as negative
control
 Result and discussion
Sample Drying & Extraction process
 Moringa oleifera seeds were completely
dried in an oven until a constant dry
mass was achieved
 Moringa seeds composed of crude
carbohydrate, lipid and protein in seeds.
 Therefore particularly suitable to produce
biodiesel (Ivase, et al., 2015).
Wet seeds, 74g Dry seeds, 49g  Soxhlet extraction method was employed
to extract the seed oil and it yielded
42.85% of oil.
 Suitable for oil extraction in laboratories,
Oil yield (%) X 100 whereas leaching may be used in large-
scale industrial preparations because of
its very limited energy consumption
(Ivase, et al., 2015).
 Lipase mediated transesterification
 Lipase catalysed biodiesel production was reported first by Mittelbach, 1990. It
involves hydrolysis of the ester bond and esterification with the second substrate
 Animal lipases were chosen over plant or microbial because they are mostly
exposed in breaking down higher amounts of fats and lipids compared to plant
and microbial lipases.
 Even though enzyme is higher in cost to purchase, by immobilizing the enzyme
it can be reused in the reaction (Yücel.S et al., 2012).
 Lipase mediated transesterification yielded 61.9%.
 Characterisation of biodiesel

Fuel property Unit ASTM Standards Moringa oleifera

biodiesel

Copper corrosion - D130 1a (slight tarnish)

Combustion test J/g D240 1456.1

Cetane number - D613 77.97

 Combustion test
performed in fume hood.
 Diesel, methanol,
ethanol, petrol and
hexane was used as
negative control.
 Slight tarnish was visible
on the copper strips.
 Conclusion
 Transesterification process is vital in the production of any oil into methyl esters
(biodiesel).
 Lipase enzyme was used as the catalyst throughout transesterification process
and it yielded 61. 9% of biodiesel.
 Slight tarnish was observed in the synthesised biodiesel. Average heat of
combustion of biodiesel was 34.07 js-1.
 Cetane value of the sample fuel was 77.97 which indicated that moringa seed oil
can be used as an alternative to conventional diesel.
 Based on the results that have been obtained, it can be concluded that oil from
moringa oleifera seed had an excellent property for biodiesel production.
 Future work
 Researches can be carried out using different types of solvents and extraction method to
maximize the oil yield extracted from the feedstock.
 HPLC analysis must be carried out instead of TLC to determine the triglyceride profile of
the oil.
 GC-MS analysis need to be carried out on the biodiesel synthesized in order to determine
the percentage of methyl esters present in this biodiesel sample.
 Immobilization of the enzyme also must be done to enhance the activity of lipase
enzymes.
 More researches must be carried out on production of biodiesel with better quantity and
quality so it can be replacing the conventional fuel for a pollution - free world.
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