Production of biodiesel from waste shark liver oil for biofuel applications
Ahmed Said. Al Hatrooshi1, Valentine C. Eze1a, Adam P. Harvey1
1School of Engineering, Newcastle University
Newcastle upon Tyne, NE1 7RU, United Kingdom.
aCorresponding authors: email@example.com; firstname.lastname@example.org
Biodiesel is a renewable alternative to “petro-diesel”. There is an established conventional
production technology based on refined vegetable oils. However, this is always more expensive
than petroleum-based diesel, mainly due to the feedstock cost, and the biodiesel market is based
on subsidies. Use of a cheap non-edible feedstock, such as waste shark liver oil (WSLO), would
reduce the biodiesel production cost and make the process more economically viable.
In this study, production of fatty acid methyl ester (FAME) from WSLO using both acid
(H2SO4) and base (NaOH) catalysts were investigated using a Design of Experiments approach
(response surface methodology). Due to the high levels of FFA (free fatty acids) homogeneous
alkali-catalysed transesterification of WSLO was less effective than the acid-catalysed process,
resulting in WSLO to FAME conversion of 12% after 60 min, with maximum FAME
conversion of about 40% after 15 min. Acid-catalysed WSLO transesterification achieved 99%
FAME conversion at 10.3 molar ratio of methanol to WSLO, 6.5 h reaction time, 60 °C
temperature, and 5.9 wt. % of H2SO4 catalyst.
Keywords: Biodiesel, Waste shark liver oil, Transesterification, Design of experiments,
Homogeneous acid and base catalysis.
Biodiesel, derived from vegetable oils or animal fats, is a renewable replacement for petro-
diesel in compression ignition engines . Its quality is dictated by ASTM D6751  in the
USA & Canada, and EN 14214 in the European union . The main component is fatty acid
methyl esters (FAMEs) of long-chain fatty acids . As a clean-burning alternative to diesel
fuel, biodiesel has various environmental benefits, including biodegradability, very low
toxicity, and reduction in emissions of CO2 and lower particulate matter and sulphur . Due
to the environmental benefits of biodiesel combustion, various government policies have
mandated the blending of biodiesel with petro-diesel, resulting in a growth in biodiesel
production and consumption .
Biodiesel is commonly produced via triglyceride (usually vegetable oil) transesterification
and/or free fatty acid (FFAs) esterification reactions in the presence of alcohols . Many types
of alcohol can be used for this application, including propanol, butanol, ethanol and methanol. ,
Methanol is most commonly used due to its low price and availability . The rate of reaction
is determined by a number of factors, namely: catalyst type and concentration, alcohol/oil
molar ratio, reaction time, temperature, moisture content of the oil and the mixing rate . In
conventional biodiesel production process, refined vegetable oils and homogeneous base
catalysis are frequently used.
The alkali-catalysed transesterification process requires low levels of FFA (<2.5%) to avoid
soap formation and catalyst deactivation , as shown in Equation 1. The oil and alcohol
feedstocks are also required to be substantially anhydrous with total water content less than
0.06 wt.%, as water may favour the saponification reaction . Water also promotes ester
(FAME) hydrolysis to form FFAs as shown in Equation 2, leading to catalyst deactivation and
The ester saponification subsequently reduces the FAME conversion and renders the separation
of ester and glycerol difficult [12-14]. Soap formation decreases FAME conversion,
necessitates the use of more catalyst and alcohol, decreases catalyst efficiency, prevents
product separation and consume more energy . Therefore, alkali catalysis is not suitable
for biodiesel production from feedstock containing high FFA and water contents. Acid-
catalysed transesterification can tolerate high levels of FFAs in the feedstock. The most
commonly used acid catalyst for biodiesel production is H2SO4, due to its higher activity, low
price and availability  . Major disadvantages of acid catalysts are lower reaction rates,
longer reaction times, higher reaction temperature conditions, and higher investment costs for
higher grade materials to withstand the corrosive effect of the acids in the equipment
Notwithstanding the advances in biodiesel production technology, the cost of biodiesel remains
substantially higher than that of petro-diesel. Hence, a major challenge to biodiesel
consumption is high production cost . This is mainly due to the high cost of feedstocks,
which accounts for 70–95% of the total biodiesel production cost . This has caused
increasing research interest in the use of cheap, non-edible feedstocks, such as waste oils to
improve the production cost and make the process economically viable.
Previous studies have shown that for refined vegetable oil transesterification at 6:1 methanol
to oil molar ratio at 1h reaction time and 5250kg/h oil throughput, about 2390MJ/h (5.83MJ/kg)
of energy is required for methanol recovery and 7448MJ/h for downstream biodiesel
purification . Therefore, energy requirements for methanol recovery and biodiesel
purification in the conventional biodiesel process correspond to 23.3% and 72.6% of the total
energy costs (10256MJ/h), respectively. The methanol recovery and biodiesel purification
costs account for over 95% of the total energy consumption in the conventional biodiesel
technology, whiles heating costs for the reaction vessel, pumping and glycerol purification
consume less than 5% of the total energy required . Hence, the estimated energy cost is
1.874MJ per kg of biodiesel produced for transesterification of 5250kg/h of oil using the
conventional biodiesel process. At the optimal FAME conversion of 99.0 ±1.1% and reaction
conditions of 10.3 molar ratio of methanol to WSLO, 6.5 h reaction time and 60 °C temperature,
an equivalent biodiesel throughput would require 5820MJ/h for excess methanol recovery and
approximately 7448MJ/h for the biodiesel purification. Estimated energy cost at the optimal
conditions for the WSLO transesterification was 2.527MJ/kg, which was about 35% higher
than the conventional process using refined vegetable oil. In terms of the actual biodiesel
production, the vegetable oil feedstock can contribute up to 70–95% of the total biodiesel
production costs , while the cost of energy and utility account for less 10% . The 35%
increase in the energy and utility would be negligible compared to cost of the refined vegetable
oil feedstock. Therefore, producing biodiesel for WSLO would reduce the production cost and
improve the process economic.
Sharks’ livers comprise 25-30% of their body weight . The WSLO (Waste Shark Liver Oil)
is obtained by exposing the liver to the sun until it melts, so that the oil can be collected. The
major constituents of WSLO are triglycerides (TG), diacylglycerol ethers (DAGE), and
Generally, sharks form 50% of the by-catch in the deep water fisheries of New Zealand and
Australia, yet most of the sharks are discarded and the liver oil is unutilised . Historically,
the discarded WSLO was used for to proof wooden boats , but now these applications are
no longer required as modern boats are fibreglass. The excess WSLO derived from these
discarded shark livers in the fishing industry could instead be processed to obtain valuable
products like biodiesel, squalene, and omega-3 PUFAs –including EPA and DHA. While the
TG components of the WSLO can be converted to biodiesel using existing biodiesel processing
technologies, the squalene, EPA and DHA can be extracted and sold as value-added products
through a biorefinery process.
This study investigates the productions of FAME from WSLO using H2SO4 and NaOH
catalysis. The biodiesel processes developed in this study was optimised using the Design of
Experiment (DoE), by a response surface method. DoE is a statistical tool used to evaluate, and
optimise, the relations between variables and system responses. The advantage of DoE lies in
minimising the number of experimental trials required to cover a particular parameter space.
2. Materials and Methods
The feedstock used for this study was WSLO obtained from the Carcharhinidae family. The
oil was sourced from Sur, Sultanate of Oman, which is one of the largest collection centres for
WSLO in Oman. Sulfuric acid (H2SO4, 98% purity, Sigma-Aldrich, UK) was used as an acid
catalyst and granulated sodium hydroxide (NaOH, 97% purity, Sigma-Aldrich, UK) as an alkali
catalyst. The alcohols used were anhydrous methanol (99.8% purity, Sigma-Aldrich, UK).
Methyl heptadecanoate (99% purity, Sigma-Aldrich, UK) was used as an internal standard for
gas chromatography (GC) analysis of the FAME produced. Acetic acid (99.5 % purity, Sigma-
Aldrich, UK) and sodium carbonate (99.5% purity, Sigma-Aldrich, UK) were used for
quenching the reactions. A standard grain FAME Mix—10 mg/mL in dichloromethane, part
number: CRM47801, from Sigma-Aldrich, UK—was used for identifying the FAME peaks in
2.2 Characterisations of the waste shark liver oil
The WSLO was characterised for its water and FFA contents, lipid classes, as well as the fatty
acid profile. The water content analysis was performed using a Karl Fischer Titration with
HYDRANAL - coulomat AG as a titration reagent in a C30 Karl Fischer Titrator (Mettler
Toledo, UK). The titration reagent was titrated until a steady background end point of
20μL/min was achieved, followed by additions of about 0.2g of the WSLO sample using a 1
mL syringe. The added samples were then titrated until a stable endpoint was achieved, to
determine the water content of the WSLO. FFAs and lipid classes in the WSLO, such as the
triglycerides (TG), diglycerides (DG) and monoglycerides (MG), were determined using
Iatroscan MK-6s. The Iatroscan thin-layer chromatography (TLC)–flame-ionization detection
is a widely used technique for determination of lipid-classes and FFA contents
The fatty acid profile of the WSLO was obtained by converting it into FAME via complete
transesterification with methanol and then analysing using the BS EN 14103:2003 standard
. The WSLO transesterification was carried in a batch reactor at 18:1 molar ratio of alcohol
to oil, 6 wt. % H2SO4, 60°C and mixing was 720 rpm. Samples were collected at 30min
intervals from the reaction mixture and analysed for FAME profile using a gas chromatography
(GC), with the methyl heptadecanoate as an internal standard. This was continued until no
further increase in FAMEs was observed. The FAME peaks were identified by their retention
times using the grain FAME, and the FAME profiles quantification were done using peak areas
of the internal standards based on the method BS EN 14103:2003  as shown in Equation 3,
where (A) is the peak area of a specified FAME, Ai is the peak area of methyl heptadecanoate,
Ci is the concentration (mg/mL) of the methyl heptadecanoate solution, Vi is the volume (mL)
of the methyl heptadecanoate solution, and m is the mass (mg) of the sample. The average
molecular weight of the WSLO, as well as the average molecular weights of the TG, DG and
MG, were calculated using the obtained fatty acid profile. The calculated WSLO molecular
weight was used to determine the exact amount of alcohol required for the transesterification
2.3 Waste shark liver oil transesterification using homogeneous acid and base catalysts
The homogeneous catalysed transesterification of the WSLO with methanol was conducted in
a 250 mL three-neck round bottom batch reactor, equipped with a condenser, a sampling point
connection, and a thermocouple to measure the temperature. The reactor was seated on a metal
block attached to a heater-stirrer (Stuart UC152), which provided the heating and mixing. For
the homogenous alkali-catalysed transesterification, the reactions were carried out at 6:1
methanol to WSLO molar ratio, reaction temperature of 60°C and mixing speed of 720 rpm. A
mixing speed of 720rpm or above was found to be sufficient to ensure that the reaction was in
the mass-transfer independent regime. 1.5wt% NaOH (based on the WSLO) was dissolved in
a required amount of methanol and heated to the reaction temperature in a separate vessel. The
methanol-catalyst solution was then transferred into the batch reactor containing a desired
amount of WSLO already heated to the reaction temperature inside the reactor. Around 1 mL
sample was collected from the reaction mixture at different time intervals ( 1, 2, 4, 8, 15, 20,
40, 50 and 60 min) using a 1000μL micropipette into a 2 mL pre-weighed vial containing 0.05
mL of 0.1M acetic acid to quench the reaction.
The acid-catalysed transesterification of the WSLO with methanol was investigated using a
Design of experiments (DoE) approach, by a response surface method. The reaction variables
studied were reaction time in the range of 1 to 7 h, H2SO4 catalyst loading from 1 to 6wt. %
and methanol to oil molar ratio from 6:1 to 30:1. The experiments were carried out using the
batch reactor set-up described for the homogeneous alkali-catalysed process and similar
experimental procedure, with reaction temperature at 60°C and the mixing speed of 720 rpm.
The calculated amount of H2SO4 catalyst was dissolved in a required amount of methanol and
heated to the reaction temperature in a separated vessel, and this solution transferred into the
batch reactor containing a desired amount of WSLO already heated to the reaction temperature.
Around 1 mL of sample was collected from the reaction mixture into a 2 mL pre-weighed vial
using a 1000μL micropipette, at sample intervals of 15 min, 30 min, 1h, 1.5h, 2h, and then
every 1h until 6h, and quenched using saturated solution of sodium carbonate. All the collected
samples from the experiments were kept in a freezer at -20°C for GC analysis.
2.4 GC analysis of samples
FAME contents in the samples collected from the transesterification reaction were analysed
using a HP6890 series II gas chromatograph (Hewlett Packard, USA), equipped with a fused
silica (15QC3/BPX5-0.25, SGE Analytics, UK) capillary column of 0.25μm film thickness,
15 m length and 0.32 mm diameter. About 50-80 mg of top layer of each sample, after drying
at 110 ˚C for 2 h, was carefully weighed in a 2mL vial using A&D HR-200 weighing balance
(±1mg), followed by additions of 1 mL of a 10mg/mL of methyl heptadecanoate prepared in
heptane. About 0.5µL of the prepared sample, solution was injected into the GC using a 5µL
micro-syringe (SGE Analytics, UK). The GC analysis was programmed starting with an initial
oven temperature of 120 ˚C (held for 5 min.), ramped to 210 ˚C at rate of 30 ˚C /min, and held
for 17 min (total run time of 25 min). The GC injector and flame ionisation detector (FID)
temperatures were set at 250 ˚C and 260 ˚C, respectively, while the carrier gas used was helium
at pressure of 10.0 psi. FAME contents and yields for the samples were calculated using the
procedure of BS EN 14103:2003 , as shown in Equation 4 and Equation 5, where is
the sum total of the all the peak areas of all the FAMEs in the sample from the GC
3. Results and discussion
3.1 Waste shark liver oil characterisation
The Karl Fischer titration of the WSLO indicated a water content of 0.08 ± 0.01%, and the
measured WSLO density was 0.916 g/mL at 30°C. The glycerides (TG, DG and MG), and
FFAs contents of the WSLO was determined by the Iatroscan MK-6 as shown in table 1.
Table 1: Lipid Classes as measured by Iatroscan MK-6, * with ±1% Iatroscan error 
WSLO content wt.% *
The WSLO composition suggests that this feedstock can be used for biodiesel production.
However, due to the high FFA contents, it is envisaged that homogeneous alkali catalysis
would not be effective. Earlier studies have shown that alkali-catalysed transesterifications
require low levels of FFA <2.5% to avoid soap formation and catalyst deactivation .
Therefore, an acid-catalysed transesterification, which has a tolerance for high FFAs in the
feedstock would be recommended for one-step biodiesel processing from the WSLO. The
water content of the WSLO was slightly higher than 0.06 wt. % required for homogeneous
catalysis, indicating that glyceride and FAME saponification may occur [12-14].
Table 2 shows the fatty acid profile of the WSLO obtained by complete conversion to FAME
via acid-catalysed transesterification with methanol and quantification by the GC and analysed
using the BS EN 14103:2003 standard . The average molecular weights obtained from the
fatty acids profile of the WSLO were 290.7 g/mol for the fatty acid, 910 g/mol for the TG,
698.4 g/mol for the DG, 393.7 g/mol for the MG and 304.7 g/mol for the FAME. The fatty acid
profile in table 2 shows that the WSLO contains about 21% PUFAs, constituting of 15% DHA
and 6.1% EPA. The biodiesel European standard EN 14214 limited the content of
polyunsaturated (≥ 4 double bonds) methyl esters to 1% (m/m). The FAME produced from the
WSLO could be directly used as biodiesel in USA and Canada, as the ASTM D6751 standards
does not specify the percentage of PUFAs. However, it is recommended that the PUFAs in
WSLO be separated to obtain EPA and DHA for pharmaceutical application. The remaining
FAME after EPA and DHA separation meets the EN 14214 standards and can be used as a
Table 2 Fatty acids profile of the WSLO
Type of fatty acids
Weight % of fatty
3.2 Waste shark liver oil transesterification and FAME conversions with the catalysts
The effects of mixing on the WSLO transesterification are shown in Figure 1, for reactions at
60°C, 30:1 methanol to oil molar ratio and reaction time of 6 h using 3 wt. % sulfuric acid
H2SO4 catalyst. The FAME conversions against time for different mixing speeds of 400, 720
and 1080 rpm clearly indicate that the reaction was mixing independent at 720rpm and above,
while at speeds above 400 rpm, there was a mass transfer-controlled reaction. The WSLO
transesterification reaction overcomes the mass transfer dependent region at speeds 720 and
1080 rpm, becoming kinetically controlled.
Figure 1: Effect of mixing intensity on WSLO transesterification at 30:1 methanol to oil
molar ratio, 60°C temperature, 3wt% H2SO4 catalyst and 6 h reaction time
Figure 2 shows the results of the preliminary investigations of the WSLO transesterification
using both acid and base catalysts. The maximum FAME conversion obtained for the
homogeneous alkali-catalysed process was 40%, using the typical transesterification conditions
WSLO conversions to FAME (%)
Reaction time (h)
of 6:1 methanol to oil molar ratio, 1.5 wt. % NaOH, 60 °C temperature, 60 min reaction time
and mixing speed of 720 rpm (Figure 2(a)). The FAME conversions increased only slightly
from 15% at 3 min to reach the maximum after 15 min. This was followed by a period of
continuous decrease in the FAME conversions, with only 12% FAME conversion after 60 min
reaction time. The trend in the FAME conversions for the NaOH catalyst was attributed effect
of high FFA content (19.9%) in the WSLO, which results the catalyst deactivation by reacting
with FFA to form soap and water (saponification) . Moreover, the presence of water in the
reaction mixture also leads glycerides and FAME saponification, resulting in the observed
period of decrease in FAME conversions after achieving the maximum conversion.
Figure 2: FAME conversions for transesterification of WSLO at 720 rpm mixing speed using,
(a) 1.5 wt.% NaOH at 6:1 methanol to oil molar ratio, 60 C temperature and 60 min, (b)
1.5wt % H2SO4 at 30:1 methanol to oil molar ratio, 60 C temperature and 6 h reaction time.
As shown in Figure 2(b), the FAME conversions increased with the reaction time, achieving
93% conversion after 6 h. Acid-catalysed transesterification has an advantage over the base-
catalysed process as it can tolerate high FFAs in the feedstock . It is expected that either a
longer reaction time or an increase in catalyst concentration would be required to reach the
maximum equilibrium FAME conversion for the acid-catalysed WSLO transesterification.
WSLO to FAME conversions (%)
Reaction time (min)
WSLO to FAME conversions (%)
Reaction time (h)
Therefore, a parametric study and optimisation of the acid-catalysed WSLO transesterification
was carried out as discussed in the section 3.3.
3.3 Parametric study and optimisation of acid catalysis of WSLO transesterification
Parametric studies of the reaction conditions for the acid-catalysed transesterification of the
WSLO was evaluated at 1 to 7 h reaction time, H2SO4 catalyst loading from 1 to 6wt. % and
methanol to oil molar ratio from 6:1 to 30:1, using a response surface methodology in DoE
with central composite design [27-29]. Table 3 shows the experimental and predicted
conversion of WSLO to FAME at different parameter spaces. An experimental error of ± 1.1%
was obtained for the data in Table 3, based on the repeat on central points. The main effects
were caused by the reaction time and catalyst concentration, while the molar ratio showed only
moderate effect on FAME conversion. An empirical model for FAME conversions determined
from the experimental data using DoE response method is shown in the Equation 6, where X
is the methanol molar ratio, Y is the catalyst loading (wt.%), and Z is the reaction time (h).
FAME conversion (%) = 0.581 - 0.0385 X + 3.694 Y + 28.164 Z + 0.3190 Cat. Y*Y
- 2.0451 Z*Z + 0.01292 X*Y - 0.7750 Y*Z.
The DoE model predicted that the maximum conversion would occur at: 10.3:1 molar ratio of
methanol to oil, 6.5 h reaction time and 5.9 wt. % H2SO4 catalyst. These conditions were
validated experimentally, and the results showed 99 ±1.1% conversions of the WSLO to FAME.
The interactions of reaction parameters: time, methanol molar ratio and catalyst concentration
on FAME conversion are shown in Figure 3. Clearly, FAME conversions increased with the
methanol molar ratio up to maximum of 18:1, as the increase in methanol molar ratio pushed
equilibrium further toward methoxide formation which favour FAME conversion. This FAME
conversion was also strongly dependent on the reaction time and catalyst concentration. Further
increase in the methanol molar ratio without proportionate addition of the catalyst leads to
reduction in the effective catalyst concentration, requiring longer reaction time to attain
maximum equilibrium FAME conversion.
Table 3: experimental and predicted conversion of WSLO to FAME at different conditions
of WSLO to FAME
Predicted conversion of
Figure 3: effect of the reaction parameters: reaction time (h), methanol molar ratio and
catalyst concentration (wt. %) on conversions of WSLO to FAME.
The increase in catalyst concentration from 1wt% - 3.5wt% resulted in a sharp rise in the FAME
conversions. However, further increases in the catalyst concentration, up to 6 wt.%, did not
improve the FAME conversions. Reaction time also increased conversion over the selected
range, as expected. The p-value obtained from DoE for the molar ratio was 0.039, which is less
than 0.05 so it was statistically significant.
The p-values obtained from DoE for the catalyst loading and reaction time were 0.00, and that
indicates strong evidence for rejecting the null hypothesis. Therefore, the catalyst loading and
reaction time are statistically significant.
Homogeneous alkali-catalysed (NaOH) transesterification of WSLO was not effective in
converting WSLO to FAME, due to the high FFA content. An acid-catalysed (H2SO4)
transesterification of the WSLO was found to be substantially more effective. It can be inferred
that biodiesel can be produced from the WSLO based on the composition. However, the
optimal reaction conditions and effects of the parameters could not be deduced based on the
composition of the WSLO. Carefully design experimental investigations are required to
establish the optimal reaction conditions.
The process parameters (methanol molar ratio, reaction time and catalyst concentration) were
screened using a DoE (Design of Experiments) approach. The FAME conversions increased
with the methanol-to-oil molar ratio, reaction time and catalyst concentration. The maximum
experimental FAME conversion was 99.0 ±1.1%, achieved at 10.3 molar ratio, 6.5h reaction
time and 5.9 wt. % of H2SO4 acid catalyst.
Clearly, it is technically viable to produce biodiesel from WSLO. It is also likely to be
economically viable, as WSLO, as a waste product, is inexpensive. This is of particular
significance in biodiesel manufacture, as the cost of biodiesel is predominantly a function of
the feedstock cost, rather than the processing costs.
WSLO is, therefore, a new feedstock for biodiesel, and should be used as such, as long as it
remains a “waste” product. This would be environmentally beneficial as it converts a waste
into a product. It would not, of course, be desirable that the process created a market for
increased shark fishing
Acknowledgement: The authors would like to thank the Diwan of Royal Court, Sultanate of
Oman for their sponsorship and Oman LNG for facilitating this study.
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