“Direct Production of Biodiesel from Rapeseed by Reactive Extraction/In Situ Transesterification”

Rabitah Zakaria1, Adam P Harvey2

1.
2. School of Chemical Engineering and Advanced Materials, Newcastle University, UK NE1 7RU
adam.harvey@ncl.ac.uk

Keywords: biodiesel, rapeseed, canola, reactive extraction, in situ transesterification

ABSTRACT

Biodiesel is a fuel derived from renewable resources such as edible and inedible oil-bearing seed, algae,
and waste cooking oil. The conventional biodiesel process involves oil extraction, refining and
transesterification. Alternatively, transesterification can actually be performed directly from the oil-bearing
materials without prior extraction. This route which is often termed “reactive extraction” or “in situ
transesterification” has the advantages of simplifying the biodiesel production process as well as potentially
reducing production cost. In this study, the reactive extraction of rapeseed with methanol has been
characterised. The effects of process parameters on the yield, conversion and reaction rate differ
substantially from conventional transesterification due to the dependence on both extraction and reaction.
The rate of ester formation is mainly affected by the catalyst concentration, temperature and particle size
while the equilibrium yield largely depends on the solvent to oil molar ratio. A high yield of ester (> 85%)
can only be achieved at high solvent to oil molar ratios (>475:1). The effect of seed drying and ultrasound
treatment was also investigated. Parametric studies and light microscope images of reactively extracted
seed suggested that reactive extraction occurs by transesterification of the oil inside the seed, followed by
diffusion of the products into the bulk solvent.

1. Introduction

The exploration of new energy resources has become increasingly important in recent years due to the
dwindling fossil fuel reserve and the adverse environmental effects of traditional fuel combustion (Naik et
al., 2009). Consequently, the development of sustainable and “greener” energy resources has taken centre
stage. One of the alternatives of renewable energy is fuel derived from plant and animal matter. Theseso-
called “biofuels” can be produced by various processes, such as fermentation, transesterification, pyrolysis,
gasification, liquefaction, and hydrotreatment. Among these processes, transesterification of vegetable oil
or animal fat to produce biodiesel has been successfully commercialised. Biodiesel is a petroleum diesel

1
substitute and can be readily used in most diesel engines without any modification. Furthermore, engine
performance using biodiesel has been shown to be comparable to that of conventional diesel fuel (Canakei
and Van Gerpen, 2003; Teas et al., 2002). Other merits of biodiesel include reduced emission of carbon
monoxides, particulates and hydrocarbon from the engine (Canakei and Van Gerpen, 2003) and enhanced
engine lubrication (Sinha and Agarwal, 2008; Balat 2006). There are abundant types of resources such as
inedible oilseed (i.e. jatropha, algae, and pongamia) or waste biomass (i.e. waste cooking oil, animal fats,
sewage sludges) that can be utilised to produce biodiesel, directly contributing to the efficient use of
biomass and land resources.

The conventional method for production of biodiesel requires the oil to be extracted from the biomass
before it can be transesterified into ester. The transesterification reaction occurs in a liquid state. The oil
extraction process typically involves the use of a solvent (usually hexane) in a counter current or
percolation extractor, or a mechanical method such as screw press extractor. However, solvent extraction
plant is expensive, complex and poses health and safety hazard due to the handling of flammable and
explosive solvent (Kurki et al., 2008). On the other hand, mechanical pressing often yields less oil than
solvent extraction resulting in a high residual oil in the waste biomass. Alternatively, the need to extract the
oil from the biomass prior to transesterification can be eliminated using a “reactive extraction” method to
produce biodiesel (Harrington and D'Arcy-Evans, 1985). In this method, the oil is simultaneously extracted
and transesterified into alkyl ester in -situ in one single process. Hence, separate extraction and oil refining
steps can be eliminated since extraction is simultaneously carried out by the reactant itself. The The use of
solid raw material rather than refined oil could certainly reduce the feedstock cost. Since the method
directly uses oil-bearing materials rather than pre-extracted oil, it eliminates processes such as crushing,
solvent extraction and degumming, and perhaps drying, which are key processes in conventional biodiesel
production. Furthermore, the elimination of hexane from the overall process provides an added health and
environmental benefit as hexane is classified as a hazardous air pollutant and contributes to the production
of smog and global warming (US EPA, 1990).

Previous studies have demonstrated that biodiesel production via reactive extraction (also referred to as in
situ transesterification) is capable of producing high yields of fatty acid esters from a variety of oil-bearing
raw materials (Shuit et al., 2010 ; Liu and Zhao, 2007; Haas et al., 2007). Increase in extraction yield is
also possible depending on the type of oil in the biomass as certain oil contains high amount of polar
components (i.e. phospholipids), which can be extracted by the alcohol during reactive extraction, whereas
they are non-extractable by conventional methods (Dufreche et al., 2007). The application of this process
to substances with low oil content, such as distillers dried grains with soluble (DDGS) and meat and bone
meal (MBM) seems to offer economic advantages due to the higher wastage associated with a conventional
two-step processes (Haas et al., 2007) . Furthermore, some plant materials, such as algae, are difficult to

2
crush: preparing the biodiesel via reactive extraction may overcome this limitation (Johnson and Wen,
2009).

Reactive extraction substantially differs from the conventional biodiesel process in many ways. Notably,
the system is heterogeneous, involving biological material with an array of biological structures and
components. As pointed out by Poirot et al. (2007), extraction efficiency is influenced by the
microstructure of the vegetable seed matrix, the molecular structure and size of the solute, localization and
link with other components. Hence, the efficiency of reactive extraction could be very different for
different biological materials. This is evident in the differing optimum parameters found in several studies
(Liu and Zhao, 2007; Haas et al., 2004; Qian et al., 2008). Although the method has been shown to be
viable by early researchers, considerable effort needs to be put into fully defining the characteristics of the
process. Most studies reported the yield of ester, but to our knowledge none provided time profiles of the
reaction intermediates (mono- and diglycerides) as well as triglyceride. This information is essential in
order to identify the substance extracted and to fully describe the process kinetics. This work aims to
investigate the factors affecting reactive extraction of oil- bearing biomass to produce biodiesel. The
parameters that can influence the reactive extraction process were studied in considerable depth in order to
understand their effects on the equilibrium yield and the kinetics. In depth study of the characteristics of the
process may enable us to understand the process mechanism, which is critical in predicting the effects of
various process parameters and in providing insight into possible further improvements. The oilseed chosen
was rapeseed (or “canola”) as it is the biodiesel feedstock in Europe.

2. Materials and Methods

2.1 Reagents and Material

The rapeseed was kindly provided by a local farm near Newcastle upon Tyne, UK. The hexane used was of
99.9% purity. The methanol and ethanol used throughout this study were of 99.97% and 99.95% purity
respectively. All solvents were purchased from Fisher Scientific. Methyl heptadeacanoate and tricaprin
internal standards were obtained from Sigma-Aldrich.

2.2 Oil Extraction

The oil seed was first extracted using 4 different solvents namely methanol, ethanol, hexane and a 50:50
mixture of ethanol and methanol. 20 g of the seed were pulverised using a coffee grinder for 1 minute. The
ground seed were transferred into a cylindrical cup, covered with glass wool, and placed in the Soxhlet
extraction unit. 230 ml of solvent was placed in the round bottom flask and heated. The Soxhlet unit was
allowed to run for a pre-determined time i.e. 1, 3, 6, 8, and 12 hours until constant oil yield has been
reached. Solvent was removed from the extracted oil using a vacuum rotary evaporator.

3
2.3 Reactive Extraction
A known amount of ground and sieved seed was placed in a 500 mL round-bottom flask equipped with a
condenser and an overhead stirrer and placed in a constant temperature water bath. Alkaline alcohol,
prepared by dissolving a known amount of sodium hydroxide in methanol, was first heated to the desired
temperature using a heated circulating water bath. Once heated, the alcohol was transferred to the round-
bottom flask and the reaction was carried out at the desired temperature and reaction periods. The stirrer
speed was maintained at 200 rpm for all of the experiment. It was important to ensure that there was no
escape of methanol from the flask as losses of methanol to the atmosphere would distort the outcome of the
parametric study.

After operation for the desired time, a specific amount of glacial acetic acid was added to the mixture to
neutralize the catalyst and prevent further reaction. The mixture was then left to cool for a few minutes.
The liquid was then separated from the seed using vacuum filtration. The solid residue was washed
repeatedly with methanol to recover any product that adhered to the seed. The excess methanol was then
removed from the filtrate using a rotary evaporator. After evaporation, two layers of liquid were formed.
The upper layer contained the ester phase, while the bottom layer contained the glycerol phase. The layers
were separated using a separating funnel. The masses of the phases were recorded and the ester content was
analysed using a gas chromatograph.

The yield of ester extracted after the reaction completion was calculated as:

Ester Yield (wt%) = mass of ester phase *0.995 *100 (1)

mass of triglycerides in the seed* 100

where the factor 0.995 is to correct for the molecular weight difference between the triglyceride and ester.
The concentration of mono-, di- and triglycerides were quantified using a Gas Chromatograph/Mass
spectrometer.

For reaction with ultrasound, the reaction mixture was subjected to ultrasound energy provided by an
ultrasonic probe (VC 750) made by Sonics and Materials Inc (Newtown, USA). The probe operates at 750
W and at a frequency of 20 KHz. The probe tip is 1.3 cm in diameter. The ultrasonic reactor has a
maximum capacity of 300 mL. The ultrasonic probe was directly inserted into the mixture and the depth of
the probe was ensured to be at least 5 cm below the solvent level in order to prevent air from being injected
into the system causing it to foam. The power supplied to the system can be varied from 0% to 100% using
the amplitude controller.

4
2.4 Progress of Reaction
To obtain the time profile of ester and the reaction intermediates, 0.5 mL samples of the reaction mixture
were taken at various time intervals using a syringe and a tube from the sampling pots. 3 μL of glacial
acetic acid was added to stop the reaction. A syringe filter was used to separate the liquid from the rest of
the seed particles. A small portion of the liquid, which contains methanol and the reaction products, was
analysed using a gas chromatograph in order to quantify the amount of ester in the bulk methanol phase.
The rest of the liquid was evaporated to remove the methanol until the sample reached constant weight. The
product was then allowed to settle into two layers and the ester phase was withdrawn from the top and the
concentration of mono-, di- and triglycerides were quantified using a gas chromatograph/ mass
spectrometer (GC-MS).

2.5 Effect of Water

To study the effect of seed moisture, reactive extraction was conducted using dried seed. Seeds were dried
in an oven at 104 oC until they achieved constant weight. The yield of dry and wet seed was compared by
performing the reactive extraction experiment using 25 g of seed, 250 ml methanol, 0.1 molal catalyst
concentrations and at 60 oC. The experiments were carried out in duplicate. The effect of water in methanol
was also studied using methanol containing 1, 2 and 4 wt % water. The experiment was performed using 25
g of both dried and wet seed, 100 ml methanol, and 0.1 molal catalyst concentrations and at 60 oC. The
water concentration in the bulk methanol phase during reactive extraction was also analysed in duplicate
using the Karl Fisher Volumetric Analysis Method (Metrohm 701 KT Titrino). The sample was taken
(about 0.2 g) from the bulk methanol phase using a clean, dry syringe.

2.6 Ester Analysis

Gas chromatography analysis for the determination of individual and total esters was based on the British
Standard BS EN 14103:2003. The internal standard used was methyl heptadecanoate (MHDN). The stock
solution of internal standard was prepared by adding 5.00 g of methyl heptadecanoate to 322.6 g of
analytical grade heptane. About 0.625 g of sample and 1.25 g of the stock solution was weighed accurately
to 4 decimal places on an AND HR-200 analytical balance. The GC used was a HP 5890 Series II, with a
Varian CP wax column with dimensions of 0.53 mm i.d, 10 μm coating thickness and 25 m length. The
carrier gas was helium at a flow rate of 2 mL/min and the column, injector and detector temperature was
maintained at 220 oC. The column separated the esters and the methyl heptadecanoate (MHDN) into
individual peaks.

To analyze the ester concentration in the bulk methanol phase obtained at various reaction times during the
experiment, a modified GC method was employed. The stock solution of the internal standards was
prepared by adding 5 g of methyl heptadecanoate in 322.6 g methanol instead of hexane. A known amount

5
of sample (1-2 g range) was weighed accurately into a sample vial with 1.25 g of the stock solution. The
injection volume used was 0.2 μl. The gas chromatograph temperature program used was the same as
described above.

2.7 Gas Chromatography/Mass Spectroscopy (GCMS) Method for Glyceride Analysis

A GCMS was used to quantify the monoglycerides, diglycerides and triglycerides in the ester phase. The
method was based on BS EN 14105 with modification to the calibration procedure in order to account for
the higher glyceride contents, which occurred early in the reaction. Tricaprin (1,2,3-tricaproylglycerol)
were used as a standard to calibrate the glycerides.

For the analysis of monoglyceride, about 20 mg of sample was accurately weighed and placed in a sample
vial. 20 μl of tricaprin stock solution (12.6 mg/mL ) and 20 μL of MSTFA was added to the sample vial.
The solution was left to silylate for 15 minutes after which 2 mL of heptane was added. For identification
of TG and DG about 20 mg of sample was accurately weighed and place in a sample vial. 100 μL of
tricaprin stock solution (0.5 mg/ml) and 20 μL of MSTFA were added to the sample vial. The solution was
left to silylate for 15 minutes after which 0.5 mL of heptane was added. The GCMS was fitted with Perkin
Elmer Col-elit column (PE-5HT) of 15 m length, 0.25 i.d and 0.1 μm film thickness. The detector type in
the mass spectrometer was electron impact positive. The flow rate of helium was 1 mL/min. The oven
temperature protocol was 50 oC hold for 1 minute, heat to 180 oC at 15 oC/min, then at 7 oC/min to 230 oC
and at 10 oC/min to 370 oC. The temperature was then held at 370 oC for 10 minutes for a total run time of
31.5 min. The inlet line to the MS was kept at 270 oC while the MS source temperature was kept at 250 oC.
The standard used to calibrate the MS was 1.5,7 Triazabicyclo (4,4,0) dec-5–ene purchased from Fluka.

2.8 Light microscope Image

For light microscope observation, the rapeseed particles were rapidly fixed in 1% (wt:vol) osmium
tetroxide and then transferred to 2.5% (vol) glutaraldhyde in 0.1M sodium cacodylate buffer (pH 7.2) and
left overnight. Subsequently, all the samples were washed twice, each time for 30 min, in 0.1 M sodium
cacodylate or phosphate buffer and then post-fixed in 1% osmium tetroxide for 2 hours. Samples were
dehydrated in graded ethanol serial dilutions for 30 minutes for each solution and then finally in 100%
ethanol for 30 minutes 3 times. All samples were impregnated with Spurr resin and embedded in moulds
and polymerized at 60 oC. Sections were cut on Reichert Ultracut ultramicrotome (Lecia Microsystems Ltd,
Milton Keynes, UK), mounted on glass slides. These procedures were performed by the Newcastle
University Electron Microscopy Research Services. Prior to microscopic examination, the slide containing
the fixed sample was immersed in Sudan black B for 5 minutes to stain the lipids. The reagents were then
removed by washing with 90 % ethanol and air-dried. Sudan black B and periodic acid-Schiff reagent was

6
purchased from Sigma Aldrich. The stained samples were examined using an Olympus BX41 light
microscope and digital images were collected using an Altra20 Soft Imaging System.

3.0 Results and Discussion

3.1 Extraction with Different Solvents

The mass of extract from Soxhlet extraction of 20 g of seed for 6 hours, using methanol, ethanol, 50/50
mixtures of ethanol/methanol and hexane yield different amount of oil can be seen in Figure 1.

12

10
Mass of Extract (g)

0
m ethanol ethanol 50/50 hexane
m ethanol/ethanol
TG Phase Non-TG Phase

Figure 1: Soxhlet extraction for 20 g of rapeseed for 6 hours using various solvents.

Methanol extracts some amount of material from the seed but a very little is triglyceride (TG). This was
similar to the findings of several previous studies (Ozgul and Turkay, 1993; Zeng et al., 2009; Kildiran et
al., 1996). For example, Ozgul and Turkay (1993) found that methanol dissolves free fatty acid from rice
bran oil leaving triglycerides in the bran. The poor triglyceride solubility in methanol is as expected, since
methanol is a very polar solvent, whereas most triglycerides are non-polar long chain hydrocarbon
molecules. However, various other compounds in the seed can potentially dissolve in methanol, e.g.
phospholipids, sterols, phenols, and vitamins. Ethanol, on the other hand, is a much better solvent for
triglycerides, although it extracts other polar materials as well. The ability of ethanol to extract considerable
amount of oil has been well-documented (Franco et al., 2007; Johnson and Lusas, 1983; Chien et al.,
1990). A mixture of methanol and ethanol extracts some oil, but not as much as when using ethanol alone.
Despite the poor solubility of triglycerides in methanol, its use in simultaneous extraction and
transesterification results in extraction of most of the oil and transesterification into ester (Ozgul and

7
Turkay, 1993; Zeng et al., 2009; Kildiran et al., 1996). The degree of extraction, however, depends on the
process parameters, which is the subject of the following sections.

3.2 Methanol to Oil Molar Ratio

As can be seen in Figures 2 and 3, increasing the methanol to oil molar ratio increases the equilibrium ester
yield. The yield of ester (as defined by equation 1) improves greatly as the methanol amount is increased
from 0 to 300 molar ratio, but after that, increasing the methanol volume increases the yield only slightly
until a maximum of about 82% of the triglycerides content in the seed is reached at about 600 molar ratio.
It should be noted that increasing the methanol amount to more than 900:1 seems to cause a reduction in
the yield of ester. This is, however, a physical effect whereby at high molar ratio, the catalyst causes the
ester to emulsify with the glycerol and prevent full phase separation, due to the water that is unavoidably
produced upon dissolution of the catalyst. When the emulsified product was extracted with hexane, it was
found that it does contain ester at the same yield as at the lower molar ratio. The time to reach final yield
was similar for high and low methanol to oil molar ratio as shown in Figure 3, indicating that the rate of
extraction was not significantly affected by the solvent amount. Similar finding was observed by the study
of Mondala et al. (2009) on in situ transesterification of municipal sludges, who found that methanol to oil
molar ratio had a weak effect on the overall extraction rate.

100

90

80

70
Yield ( wt %)

60

50

40

30

20

10

0
0 200 400 600 800 1000 1200 1400
Molar Ratio
ester hexane extracted ester

Figure 2: Effect of Methanol to Oil Molar Ratio on the Ester Yield for 1 hour reaction. Reaction conditions:
60 oC, 0.1 m catalyst, 300-1000 µm particle size, 25 g seed.

8
 100
90
80
70
Yield (wt%)

60
50
40
30
20
10
0
0 50 100 150 200
Time (min)
270 Molar Ratio 475 Molar Ratio 150 Molar Ratio

Figure 3: Effect of methanol to oil molar ratio on equilibrium yield. Reaction conditions: 60 oC, 0.1 m
catalyst, 300-1000 µm particle size, 25 g seed

The minimum amount of methanol needed for in situ transesterification using batch type reaction is
presumably the amount that is needed to simply cover the seed and to render the solid phase workable for
agitation. For 25 g of seed used in the study, the amount of methanol needed to adequately cover the seed is
40 ml which is a methanol to oil ratio of 76:1 for the rapeseed used. This is much higher than the optimum
required for pre-extracted oil transesterification of 6:1 (as suggested by Freedman et al., 1986). The amount
of catalyst used here is 1.1 wt % of the oil, which is within the range suggested for conventional
transesterification of 0.5 to 1.0 wt% (Mittelbach and Remschmidt, 2004). Hence these amounts should be
able to provide adequate reactants and catalyst for a significant conversion to be achieved. However, this is
not the case for in situ reaction, as using a methanol to oil ratio of 95:1 and below does not result in any
significant biodiesel being produced.

The fact that a large amount of solvent is needed in in situ transesterification is probably due to the amount
required to drive the extraction rather than the reaction. Solvent extraction of oil from seed can be
explained partly by Fick’s Law of diffusion, in which the yield of extraction depends on the concentration
gradient between the bulk liquid and the internal seed, as well as the oil solubility in the solvent. Early in
the reaction, the yield increases rapidly due to the high concentration gradient between the inside of the
seed and the bulk liquid. As more products are extracted, the concentration gradient starts to decrease,
thereby decreasing the extraction rate. Extraction will stop once the concentration inside the seed is in
equilibrium with that in the bulk liquid. Therefore, the higher the amount of solvent used, the more dilute
the bulk liquid will be, so the higher the concentration gradient, which will allow greater amounts of oil to

9
be extracted. This effect has been observed in several oilseed extraction studies with various solvents. In
the extraction of oil with ethanol for example it was found that the equilibrium yield increased with
increasing ethanol (Franco et al., 2007b). In another study, the extraction of oil from jatropha seed in a
batch reactor was demonstrated to increase with increasing hexane (Sayyar et al., 2009).

The high methanol to oil molar ratio requirement has been observed in most studies for reactive extraction
involving different oil-bearing materials (Kasim et al., 2010). For example Haas found that 226:1 molar
ratio is needed to reactively extract soybeans with methanol (Haas et al., 2004). A 673:1 molar ratio is
required to obtain maximum yield for cottonseed (Georgogianni et al., 2008a) while 476:1 is required for
sunflower Georgogianni et al., 2008b). The high energy required for methanol recovery causes the
production cost of biodiesel by this method to be higher than for the conventional method (Haas, 2005).
Hence, further effort is needed to solve this problem. In this study, the application of ultrasound was
explored to enhance the process efficiency and is discussed in section 3.8

Figure 4 shows the concentration of ester in the ester phase versus methanol to oil molar ratio. The purity
of the ester phase indicates the extent of conversion of the extracted material into ester in the bulk
methanol. The data shows that the conversion to ester is similar at low or high molar ratio. Hence even at
low molar ratio (e.g. at 200:1), the amount of methanol is adequate to convert the extracted material into
fatty acid methyl ester. This shows that the low equilibrium ester yield at lower molar ratio is not the result
of incomplete conversion of triglyceride to ester in the bulk methanol phase, but is probably due to the low
concentration gradient between the seed and the bulk liquid.

100
Ester concentration in ester phase (wt % )

95

90

85

80

75

70

65

60
0 200 400 600 800 1000 1200
Molar Ratio

10
 Figure 4: Concentration of ester in the ester phase at different molar ratios. Reaction conditions: 1 hr, 60
o
C, 0.1 m catalyst, 300-1000 µm particle size, 25 g seed

3.3 Catalyst Concentration

In conventional transesterification, the amount of catalyst is usually 0.5 to 1.0 wt% of the oil. Higher
concentrations cause excessive saponification (Vicente et al., 2005), while lower concentrations cause low
conversion of triglycerides to biodiesel (Mittelbach and Trathnigg, 1990). However, this range may not be
applicable to in situ transesterification due to the physical difference in the reaction phase, such as the
presence of solid and the vast amount of solvent used. The effect of catalyst concentration ranging from
0.03 molal (equivalent to 2.1 wt% oil) to 0.1 molal is shown in Figure 5, below. It can be seen that
increasing the catalyst concentration increases both the final ester yield and the rate of ester formation.
Hence, an adequate amount of catalyst is essential in ensuring complete transesterification of the oil which
consequently allows for the extraction of triglycerides to take place. Increasing the amount of catalyst
causes higher rates of ester extraction, which indicates that the rate of reaction controls the reactive
extraction process.

100
90
80
Ester yield (wt%)

70
60
50
40
30
20
10
0
0 50 100 150 200 250 300 350 400
Time (min)

0.03 molal 0.05 molal 0.1 molal

Figure 5: Effect of catalyst concentration on the ester yield. Reaction conditions: 1 hr, 60 oC, 475:1
methanol to oil molar ratio, 300-500 µm particle size, 25 g seed

Figure 6 shows the interactive effect of molar ratio and catalyst concentration. At a catalyst concentration
of 0.05 molal, the maximum equilibrium yield is achieved at a 1200:1 methanol to oil molar ratio whereas

11
for 0.1 molal, a similar maximum equilibrium yield is achieved at a 600:1 methanol to oil molar ratio.
Increasing the catalyst concentration further to 0.15 molal produces a similar amount of extracted materials
but the ester yield is lower than at 0.1 molal. This is probably due to the adverse effects of the
saponification reaction or dissolution of methyl ester in the glycerol phase associated with excess catalyst.
Clearly, increasing the catalyst concentration reduces the methanol requirement to achieve maximum yield.
However, at 0.05 catalyst concentration and 1200:1 molar ratio, the catalyst to oil ratio is the same as at 0.1
molal and 600:1 molar ratio, which explains how at lower catalyst concentration a similar yield can be
achieved by increasing the solvent amount. Therefore, it appears that it is the catalyst to oil ratio rather than
the catalyst concentration which dictates the amount of triglyceride extracted from the seed. Low catalyst to
oil ratio causes low conversion of the triglycerides to ester and hence low extraction yield. On the contrary,
too much catalyst will reduce the yield presumably due to the excessive saponification reaction. The
optimum amount of catalyst appears to be in the region of 7 wt % of the oil, which is higher than that
required for conventional transesterification. Adequate methanol to molar ratio on the other hand is still
important in order to provide the concentration gradient for extraction and even with enough catalyst the
yield will still be low if the methanol to oil molar ratio is low.

90 18
80 16
70 14
Ester yield (wt %)

Total extract (g)

60 12
50 10
40 8
30 6
20 4
10 2
0 0
0 200 400 600 800 1000 1200 1400 1600

Methanol to oil molar ratio

ester yield 0.1 molal ester yield -0.05 molal
ester yield- 0.15 molal total extract 0.1mola
total extract 0.05 molal total extract 0.15 molal

Figure 6: Effect of catalyst concentration at different molar ratio on ester yield and total extract. Reaction
conditions: 1 hr, 60 oC, 300-500 µm particle size, 25 g seed

12
Table 1 shows the concentration of ester and the equilibrium yield at 0.1 and 0.05 molal catalyst
concentration. The data shows even though the ester yield is lower at 0.05 molal, similar concentration of
ester is achieved for both catalyst concentration. This indicates that even at the low catalyst concentration
sufficient catalyst is available in the bulk phase to convert the extracted material into ester. Therefore, it
could be inferred that the lower ester yield obtained with the lower catalyst concentration is not the result of
incomplete reaction in the bulk phase. Conversely, it is possible that there is insufficient conversion inside
the seed which prevents further extraction of the triglycerides.

Table 1: Ester yield and purity at different catalyst concentrations. Reaction conditions: 1 hr, 60 oC, 475:1
methanol to oil molar ratio, 300-500 µm particle size, 25 g seed
Ester Concentration (wt%) Ester Yield (%)
Catalyst Concentration (molal)
0.1 90.3 ± 1.3 88.8 ± 0.1
0.05 89.7 ± 1.4 59.5 ± 0.8

3.4 Particle Size

The effect of particle size on rate of reactive extraction can be seen in Figure 7:

90
80
Ester yield (wt%)

70
60
50
40
30
20
10
0
0 50 100 150 200 250 300 350
Time (min)

300-500 micron 800-1000 micron 1000-1400 micron

Figure 7: Effect of particle size at 571:1 methanol to oil molar ratio on ester yield. Reaction
conditions: 1 hr, 60 oC, 25 g seed, 0.1 molal catalyst concentration.

13
For particle sizes between 300 and 500 µm, the time to reach maximum ester yield was about 1 hour but it
increased to more than 3 hours when a particle size of 1000 to 1400 µm were used. Despite the longer
extraction time, the final yield of the larger particles was essentially the same as that of the smaller
particles. Reducing the particle size shortens the diffusion path of the oil and solvent, thereby increasing the
extraction rate. It is also possible that the more extensive mechanical grinding needed to reduce the particle
size may also cause a higher degree of distortion of the cell wall causing an increase in the diffusion
coefficient and faster extraction. Although there are several mathematical formulae in the literature that
relate extraction time to particle size (see: Coats and Wingard, 1950; Othmer and Agarwal, 1955), the
extent of improvement in extraction rate may vary between particles of the same size since different seed
preparation methods can result in different degrees of cell wall disruption.

The smaller the particle size, the greater the proportion of broken surface cell walls releasing more oil at the
particle surface. The oil at the surface is not, of course, released to the bulk solvent by internal diffusion,
rather it is solubilised or washed off at a much quicker rate. It is postulated that if most of the oil
accumulates at the particle surface, less solvent is needed for extraction, since the diffusion process is
minimized. Comparing the ester yield at different methanol to oil molar ratios for a low particle size of 300
to 500 μm and a particle size of 1000 to 1400 μm, it can be seen from Figure 8 that similar amounts of
solvent are needed to achieve maximum yield. This indicates that for particle sizes of 300-500 μm, the
extraction of the oil still depends largely on diffusion rather than solubilisation. Different methods of seed
treatment such as ultrasound could however break or distort the cell walls to a greater extent (this effect is
investigated in section 3.8).

100
90
80
70
Ester yield (wt %)

60
50
40
30
20
10
0
571 380 285 190
Molar Ratio

300-500 micron 1400-1000 micron

14
 Figure 8: Effect of particle size on final ester yield at different molar ratios. Reaction conditions: 1 hr, 60
o
C, 25 g seed, 0.1 molal catalyst concentration.

3.5 Temperature

In general, temperature affects both the rate of diffusion and reaction. Examination of Figure 9 shows that
increasing the reaction temperature from 30 to 60 oC increases the rate of ester formation particularly early
in the reaction, but the final equilibrium yield is the same. A number of researchers have shown that
transesterification can be adequately carried out at at temperatures as low as 20 oC without a great deal of
compromise on the rate and conversion (Mittelbach and Trathnigg, 1990; Rashid and Anwar, 2008; Vicente
et al., 2005). Oil extraction, on the other hand, proceeds faster at higher temperatures, as the viscosity of
the oil and the solvent is reduced and diffusivity is increased. The solubility of the oil also increases at high
temperature, which consequently increases the final extraction yield, notably at low solvent to oil ratio
(Lajara, 1989). However, in reactive extraction, the increase in rate is only significant during the early part
of the extraction, as a similar time is needed to achieve maximum yield for lower and higher temperatures.
Other works in in situ transesterification have also shown that minor improvement is gained from
increasing the temperature in the range of 30 to 65 oC (Haas et al., 2007; Qian et al., 2008). The extraction
and subsequent transesterification can be efficiently performed at low temperature, which is favourable for
the process economics.

100
90
80
Ester Yield (wt%)

70
60
50
40
30
20
10
0
0 20 40 60 80 100 120 140
Tim e (m in)

30 deg C 40 deg C 60 deg C

Figure 9: Effect of reaction temperature on ester yield. Reaction conditions: 1 hr, 25 g seed, 0.1 molal
catalyst concentration, 475:1 methanol to oil molar ratio, 300-500 µm particle size.

15
3.6 Drying

As discussed previously, the presence of water can be detrimental to the transesterification reaction as it
causes the formation of hydroxide ions, which induce saponification of free fatty acids, triglycerides and
esters. With respect to extraction, water might further reduce the solubility of triglycerides in methanol due
to its polarity. As the rapeseed contains 6.7 % water, the effect of drying the seed to 0 % water was studied
(Figure 10). It can be seen that drying the seed does not increase the ester yield, nor does it reduce the
amount of methanol needed to achieve maximum yield. T-test analysis (SPSS 17 Statistical Software) gave
a p value of 0.715, which is greater than 0.05, indicating that the difference between yield obtained from
dried and wet seed is not significant. This is in contrast to a study by Haas and Scott (2007) who found that
drying soybeans to 0 % water substantially reduced the amount of methanol needed to achieve maximum
ester yield by 60%. However, soybeans have a lower oil content of about 22 wt % and hence for the same
amount of soybeans the amount of methanol used is lower than that for rapeseed. The higher seed moisture
in less methanol will cause a higher concentration of water in the bulk solvent, which might make the
process apparently less tolerant to water than in situ transesterification using rapeseed.

90
80
70
Ester yield (wt %)

60
50
40
30
20
10
0
190 380 571 666
Molar Ratio ( Methanol to Oil)
Wet Seed Dry Seed ( 0 % Moisture)

Figure 10: Effect of drying on the yield of ester at different methanol to oil molar ratios. Reaction
conditions: 1 hr, 25 g seed, 60 oC, 0.1 molal catalyst concentration, 300-500 µm particle size.

The bulk methanol phase used in reactive extraction using 25 g of rapeseed and 250 mL methanol was
analysed for water content using a Karl Fisher assay, and the results are shown in Figure 11, below. The
fresh alkaline methanol has about 0.6 wt% water initially, since the methanol is only 99.9% pure and the
dissolution of sodium hydroxide in methanol releases water. It can be seen that for the wet seed, the water

16
content of methanol increases quickly to about 1.3 wt % after 1 minute and the water level stays practically
constant thereafter. For dry seed, the amount of water in the bulk phase does not increase and stays almost
constant throughout the reaction. As water in the seed is quickly extracted to the bulk solvent, the process
may have an enhanced water-tolerance if transesterification occurs inside the seed rather than in the bulk
methanol.

1.8
1.6
Water concentration (wt%)

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 10 20 30 40 50 60 70
Time (min)

Wet Seed Dry seed

Figure 11: Water content in bulk solvent phase for 250 mL solvent and 25 g of seed.

In order to identify the level of water that affects the reactive extraction, the reaction was carried out with
25 g seed and 100 mL methanol that contains 1 to 4 wt % water. It can be seen from Figure 12 that when
using completely dried seed, the ester yield was not affected by water until more than 2 wt% water is used.
This amount of water is equivalent to 6.3 wt % of the seed weight, which is approximately the amount of
water that is in the wet rapeseed. Apparently 6.7 wt % of water in the seed is not high enough to cause a
detrimental effect on the process. If more than 6.7 wt% of water in the seed is used, as in the case of
methanol with 4% water for dry seed and 1 wt % for wet seed, the yield of ester substantially decreases.
Hence, as suggested by Haas, a higher amount of methanol is needed in order to dilute the water
concentration and to achieve similar yield as the dry seed (Haas and Scott, 2007).

17
 70

60

50
Ester yield (wt%)

40

30

20

10

0
0 1 2 4
Percent water in methanol (wt%)

Wet seed Dry seed

Figure 12: Effect of water in methanol on the yield of ester at 270:1 methanol to oil molar ratio

3.7 Discussion of the Process Mechanism

It has been shown that methanol was not able to extract triglyceride from rapeseed to any considerable
extent. However the presence of the alkaline catalyst allows significant extraction of triglycerides and
subsequent transesterification to ester. The mechanism of this process is still not clear. Several studies
(Haas et al., 2004; Qian et al., 2008; Kildiran et al., 1996) have speculated on the mechanism although they
have not supported their theories with experimental result nor discussed it in details. One possible
mechanism is that the initial presence of esters causes the remaining triglycerides in the oilseed to be more
soluble in the methanol ester mixture. Kildiran et al. (1996) suggested the extraction of triglycerides is
possible due to the stepwise dissolution and subsequent transesterification of the oil. Haas et al. (2004) and
Qian et al. (2008) suggested that the alkaline catalyst could destroy the cell walls and intracellular
compartmentalization resulting in cellular solubilisation and subsequent transesterification of triglycerides.
However, our light microscope images of the seed particle stained with Sudan Black B after reactive
extraction indicate otherwise (Figure 13). Figure 13a shows that the fresh seed consists of cells of about 10
– 30 m in diameter and the cells were surrounded by cell wall membranes 0.5 to 2 m thick. The lipid
which can be seen as a blue colour was located inside the cells. It can be seen that most of the lipids have
been removed from the cells after reactive extraction (Figure 13b). The cells after reactive extraction
consist of a significant amount of fully intact cells confirming that the addition of sodium hydroxide into
the solvent does not destroy the cell wall structure. The mechanism of extraction would still require the

18
diffusion of some fraction of the oil or ester outward through the cell membrane and into the bulk liquid.
Figure 13c shows the image of particle whereby the oil has not been fully extracted as shown by the blue
colour inside the cells. The remaining oil seems to occur only in the middle of the particle rather than
throughout the seed. This indicates that the lipid does not move radially outward into the bulk liquid.
Rather, it suggests that there was a reaction front that consumes the lipid, while the products of the reaction
dissolve in the methanol and are then extracted into the bulk liquid. This implies that the diffusion of
methanol into the seed is relatively slow (

100 μm

a b

Figure 13: Light micropscope images stained with Sudan Black B. a) typical image prior to extraction
and b) and c) are the images after extraction

In order to have a better insight into the process mechanism, the concentration of mono-, di-, and
triglycerides in the ester phase throughout the reaction was observed. As can be seen, there is only a
maximum of about 6 wt% of mono-, di and triglycerides in total in the ester phase, which occurs very early
in the reaction (1 minute reaction time). During the reactive extraction period, the extracted material is
predominantly ester. It appears that triglycerides and the reaction intermediates (mono- and diglycerides)
do not accumulate to a significant degree in the bulk solvent. Since the extracted materials are

19
predominantly ester, it could be inferred that either i) triglycerides are rapidly reacted to ester once
extracted into the bulk solvent or ii) the transesterification reaction occurred inside the seed and the
resulting ester was extracted into the bulk solvent . The latter mechanism is more likely based on the
images in Figure 13c.

Figure 14: Concentration time profile of mono-, di-, and triglycerides in the ester phase

This mechanism fits well with the behaviour of the process as observed from the parametric studies. As
shown in section 3.2, the yield of ester increases with an increase in the amount of methanol used. If the
reaction occurs first in the seed, the ester will diffuse into the bulk solvent due to the concentration gradient
between the ester in the bulk liquid and in the seed. Increasing the amount of solvent will cause the
concentration gradient to increase, thereby increasing the yield of ester. On the other hand, if triglycerides
were extracted and reacted immediately as they reach the bulk liquid, the concentration of triglycerides in
the bulk liquid will always be zero or near it. Therefore, there would always be a maximum triglyceride
concentration gradient inside the seed and the bulk liquid, which does not explain why higher solvent
(methanol) is needed for higher yield. Also, if it is assumed that triglycerides were extracted since it
becomes more soluble in the mixture of ester and methanol, the rate of extraction should be slower in the
beginning of the reaction and should become more rapid when more ester is produced as the reaction
progresses. In contrast, the rate of extraction is very fast early in the reaction and decreases as the reaction
progresses. Furthermore, from Table 1 it was shown that the ester yield is low at low catalyst concentration,
but the ester concentration in the bulk methanol is similar at higher and lower concentrations. Hence,
inadequate conversion of triglycerides in the bulk methanol phase is not the cause of the lower extraction
yield. On the other hand, it is likely that at low catalyst concentration, there is insufficient conversion of
triglycerides to ester inside the seed, which reduces the yield of ester.

20
3.8 Ultrasound-assisted reactive extraction

Reactive extraction with ultrasound at two different powers was compared with reaction with mechanical
agitation. The ultrasound power was determined by calorimetric measurement (Mason, 1999). 31.2 W was
the maximum power that could be sustained by the available ultrasonic probe without overheating. T-test
analysis (SPSS 17 Statistical Software) gave a p value of 0.815 when comparing between overhead stirrer
and ultrasound experiment (31.2 W), indicating that there was no significant difference betweeen the two
rates. This is a rather surprising result since ultrasound was expected to increase the rate of the extraction
based on its ability to enhance both internal diffusion and the reaction rate. One possible reason for the lack
of enhancement is insufficient ultrasound power on the system. Many studies have shown that the impact of
ultrasound is enhanced by increasing the power or the intensity (Ji et al., 2006, Li et al., 2004, Cravotto et
al., 2008). Cravotto et al. (2008) for example showed that extraction using a double probe of 65 W and 60
W reduces the extraction time by half, as compared to using only a single probe. However, higher intensity
could not be pursued in this study due to the limitations of the available equipment.

100
90
80
70
Ester yield (wt%)

60
50
40
30
20
10
0
0 10 20 30 40 50 60 70
Tim e (m in)

without ultrasound 15.5 W 31.2 W

Figure 15: Effect of ultrasound-assisted reactive extraction on the ester concentration in methanol at
60 oC, 475:1 methanol to oil molar ratio, 0.1 molal catalyst concentration and particle size of 300-500
μm)

Light microscope images of the seed particles that have been treated with ultrasound for 10 minutes and 60
minutes are shown in Figure 16, below. It can be seen that after 10 minutes irradiation, most of the cell
walls were still fully intact and some oil was left, mainly in the centre of the seed. However, after 60

21
minutes irradiation, the cell walls were fully broken and almost all the oil had been removed. This shows
that the ultrasonic impact requires some time to come into full effect. Hence, since almost 75% of the oil
from the reactive process was removed during 10 minutes of the reaction, and almost 90% was removed
after 30 minutes, the lack of extraction rate enhancement could be due to the delayed ultrasonic impact on
the seed’s internal structure.

b
a

Figure 16: Light microscope images stained with Sudan Black B. a) shows the image after 10 minutes
exposure to ultrasound and b) shows the image after 60 minutes exposure to ultrasound.

4. Conclusion

The maximum yield of reactive extraction of rapeseeds using methanol is determined primarily by the
methanol to oil molar ratio. Methanol to oil molar ratios greater than 400:1 were required to obtain ester
yields higher than 80 %. This is a much higher amount of methanol than that needed for conventional
transesterification. In order to make the process economically viable, more research is needed to identify
techniques that can reduce the solvent consumption.

The rate of the process, on the other hand, is mainly controlled by the catalyst concentration and the seed
particle size. Decreasing the catalyst concentration and increasing the particle size reduces the overall rate
of extraction. Temperatures within the range 30 to 60 oC only affect the initial rate of extraction, but the
time to reach the maximum yield is similar. The maximum yield of ester was achieved at a catalyst
concentration of 0.1 mol/kg and 670:1 methanol to oil molar ratio. It appears that the process is not
significantly adversely affected by water when the seed moisture content is less than about 6.7 wt %.
Hence, the drying step could be removed if the moisture content does not exceed this level, which should

22
improve the process economics. It is postulated that the reaction of triglyceride occurs inside the seed and
the resulting ester, mono-, and diglyceride diffuse into the bulk solvent.

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