Bioresource Technology 154 (2014) 155–161

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Evaluation of solvent system for the enzymatic synthesis of

ethanol-based biodiesel from sludge palm oil (SPO)
Ricca Rahman Nasaruddin, Md. Zahangir Alam ⇑, Mohammed Saedi Jami
Bioenvironmental Engineering Research Centre (BERC), Department of Biotechnology Engineering, Faculty of Engineering, International Islamic University Malaysia, P.O. Box 10, 50728
Kuala Lumpur, Malaysia

h i g h l i g h t s

 Biodiesel was produced enzymatically from a low-cost sludge palm oil and ethanol.
 Locally-produced Candida cylindracea lipase from POME fermentation was applied.
 The mixture of ethanol and tert-butanol increased the yield of biodiesel.
 The lipase favours esterification than transesterification reaction.

a r t i c l e i n f o a b s t r a c t

Article history: A green technology of biodiesel production focuses on the use of enzymes as the catalyst. In enzymatic
Received 11 September 2013 biodiesel synthesis, suitable solvent system is very essential to reduce the inhibition effects of the solvent
Received in revised form 25 November 2013 to the enzymes. This study produced ethanol-based biodiesel from a low-cost sludge palm oil (SPO) using
Accepted 30 November 2013
locally-produced Candida cylindracea lipase from fermentation of palm oil mill effluent (POME) based
Available online 16 December 2013
medium. The optimum levels of ethanol-to-SPO molar ratio and enzyme loading were found to be 4:1
and 10 U/25 g of SPO respectively with 54.4% w/w SPO yield of biodiesel and 21.7% conversion of free
Keywords:
fatty acid (FFA) into biodiesel. Addition of tert-butanol at 2:1 tert-butanol-to-SPO molar ratio into the
Biodiesel
Sludge palm oil
ethanol–solvent system increased the yield of biodiesel to 71.6% w/w SPO and conversion of FFA into bio-
Lipase diesel to 28.8%. The SPO and ethanol have promising potential for the production of renewable biodiesel
Fatty acid ethyl ester using enzymatic-catalyzed esterification and transesterification.
Ethanol Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction lyst. However, the chemical-catalyzed transesterification requires

Biodiesel is a clean-burning fuel that is now getting higher
attention to solve the problems of climate change and reduce the
dependency on fossil fuels which are facing many issues such as
unstable escalation of price, depleting reserve and higher air pollu-
tants (Röttig et al., 2010; Ganesan et al., 2009). Biodiesel becomes
more attractive because it is biodegradable, clean, non-toxic,
produces less air pollutants and it also has lower sulphur
(0–24 ppm) and aromatic content and higher heat content (Gane-
san et al., 2009; Patel et al., 2013; Moradi et al., 2013; Zheng
et al., 2012; Maceiras et al., 2011; Ghaly et al., 2010; De Paola
et al., 2009).
Most biodiesel are produced conventionally using methanol as
the acyl-acceptor, thus giving the name of fatty acid methyl esters
(FAME). The transesterification reaction uses alkaline as the cata-
⇑ Corresponding author. Tel.: +60 3 61964571; fax: +60 3 61964442.
E-mail addresses: zahangir@yahoo.com, zahangir@iium.edu.my (M.Z. Alam).
multistep separation process which increases the energy consump-
tion and produces lots of toxic wastewater which reduces the
green effects of biodiesel (Ghaly et al., 2010). Thus, as the need
for green technology and sustainable development is increasing,
the application of enzyme as the catalyst is more favorable.
Lipase, the catalyst in biodiesel synthesis can catalyze both
transesterification and esterification reactions in one pot (Yan
et al., 2012). Unlike alkaline catalyst which is limited to low
FFA-content oils, lipase can convert triacylglyceride (TAG), diacyl-
glyceride (DAG), monoacylglyceride (MAG) and FFA into biodiesel
regardless of the initial FFA content of the oils. Moreover, it is also
biodegradable, non-toxic, contributes to easy recovery of biodie-
sel and glycerol and requires moderate alcohol and mild reaction
conditions (Zheng et al., 2012; Yan et al., 2012; Chen et al., 2011).
However, most lipases face stability issue in methanol. Methanol
is a polar solvent which can trip water molecules from the
enzyme surface thus affects its native conformation and reduces
its activity (Sivaramakrishnan and Muthukumar, 2012). Methanol

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.11.095
156 R.R. Nasaruddin et al. / Bioresource Technology 154 (2014) 155–161
which is commonly produced from natural gas, a fossil origin is
also highly toxic and hazardous, thus the methanol based biodie-
sel cannot be regarded as a truly renewable and environmental
friendly biofuel.
The use of ethanol is now getting higher attention in order to
reduce the deactivation of lipase and produce the biodiesel which
is comparable with the FAME. Besides, ethanol is more attractive
economically as compared to methanol because it can now be pro-
duced from the renewable and low cost agricultural biomass
(Ghaly et al., 2010). Thus, ethanol-based biodiesel which is also
called as fatty acid ethyl ester (FAEE) appears as a 100%-renewable
alternative (Rosa et al., 2009). Besides, ethanol was found to give
less inhibition effect to the activity of lipase because it is less
hydrophilic than methanol. Nuylert and Hongpattarakere (2012)
also stated that most lipases studied so far including the lipase se-
creted by Candida cylindracea were very sensitive to methanol.
Although there are limited reports on the application of FAEE,
but as reported by Hamelinck et al. (2007), FAEE has quite similar
properties with methyl esters, thus it is expected that it is in line
with the European biodiesel standard (EN14214) and can perform
well in diesel engine.
Besides adding alcohol which primarily acts as the acyl acceptor
in biodiesel production, other organic solvents were also added to
reduce the inhibitory effects of alcohol and glycerol by-product
(Ghaly et al., 2010). Among all organic solvents, hydrophobic sol-
vents are preferable as compared to hydrophilic solvent in order
to maintain the minimum amount of water in lipase surrounding
(Ghaly et al., 2010). Sivaramakrishnan and Muthukumar (2012) re-
ported that solvents such as tert-butanol, n-hexane, benzene, tolu-
ene and diethyl ether enhanced the lipase activity slightly.
Meanwhile, Park et al. (2008) reported the advantages of using fos-
sil fuel as the additional solvent for FAME production because its
removal is neglected and it can be blended with the FAME for prac-
tical use.
Biodiesel production also faces a big challenge in the cost of its
oil feedstock. The most common edible oils are costly due to the
competition with the food producers (Ghaly et al., 2010; Yan
et al., 2012; Li et al., 2012). Therefore, low-cost waste oils have
been investigated for their potential in biodiesel synthesis. Some
examples are beef tallow (Liu et al., 2011), waste cooking oil (De
Paola et al., 2009; Anastopoulos et al., 2009), winery waste of
grapes seed oil (Fernández et al., 2010), rice bran oil (Kasim
et al., 2009), olive pomace oil (Yücel, 2011), waste coconut oil
(Oliveira et al., 2010), waste animal fats (Encinar et al., 2011),
and grease (Yan et al., 2012; Li et al., 2012). Some of the waste oils
have been studied in enzymatic biodiesel synthesis. Examples are
the production of biodiesel from the brown grease with 21.7%
FFA content using immobilized tandem lipase (Yan et al., 2012)
and waste cooking oil with 33% FFA content with immobilized
Thermomyces lanuginosus (Lipozyme TL IM) (Wang et al., 2008).
Malaysia, as the second largest producer of palm oil in the
world has generated about 40 million tons of sludge palm oil
(SPO) per year. This abundant waste oil has a very high FFA content
which is about 40 to 80% by weight (Wafti et al., 2012). Besides
being used in making animal feed supplementary and low grade
soap, SPO can also be used to produce biodiesel. Currently, the only
study of biodiesel production from SPO used chemical catalyst in
two step processes starting with the esterification reaction using
p-toluensulfonic acid (PTSA) and then transesterification reaction
using sodium hydroxide with the yield of biodiesel over 90% (Hay-
yan et al., 2010).
The current novel research is mainly about the enzymatic bio-
diesel production from SPO and ethanol using locally-produced li-
pase as the catalyst. The lipase was a low cost lipase which has
been produced from Candida cylindracea using palm oil mill efflu-
ent (POME) as the basal medium. This study can add in more infor-
mation on the application of low-cost waste oil especially SPO and
ethanol for the production of renewable biodiesel. At the same
time it addresses some crucial issues such as climate change,
energy security and sustainable development which are highly
important. In this study, the optimum values of influential param-
eters for the ethanol–solvent system were evaluated. The influen-
tial parameters were ethanol-to-SPO molar ratio, enzyme loading
and additional organic solvents while the responses used to deter-
mine their optimum value were the yield of biodiesel and conver-
sion of FFA into biodiesel.
2. Methods
2.1. Raw materials and chemicals
SPO was obtained from West Oil Mill of Sime Darby Plantation
at Carey Island, Selangor, Malaysia. It was preserved in cold room
at 4 °C to avoid any decomposition, oxidation and changes of the
FFA content. The locally-produced Candida cylindracea lipase was
a contribution of an established research of Bioenvironmental
Engineering Research Centre, International Islamic University
Malaysia. The lipase production was done according to Salihu
et al. (2011). All the chemicals such as organic solvents and re-
agents of laboratory and analytical grades with various brands
(Merck, Sigma Aldrich, Supelco, Dr. Ehrenstorfer, HmBG, PC Labo-
ratory, System and Bendosen) were purchased from local suppliers
(Merck Sdn. Bhd, TerasMedikSdn. Bhd and IT-Tech Research (M)
Sdn. Bhd.).
2.2. Preparation and characterisation of SPO
Prior to use, SPO was preheated to 60 °C until it becomes homo-
geneous. The characteristics of the SPO such as acid and saponifica-
tion values, FFA and moisture contents were determined according
to Malaysian Palm Oil Board (MPOB) Test Methods (Ainie et al.,
2005). The fatty acids composition of the SPO was analyzed using
GC/FID (Perkin Elmer Clarus 500), split–splitless mode of injector,
isotherm oven at 250 °C using non-polar stationery phase BPX70
capillary column.
2.3. Production of lipase
The lipase production method was based on Salihu et al. (2011).
The yeast, Candida cylindracea, ATCC 14830 was obtained from
American Type culture collection while POME was obtained from
West Oil Mill of Sime Darby Plantation at Carey Island, Selangor,
Malaysia. The strain was grown on potato dextrose agar plates at
28 °C for 4 days and sub-cultured every 2 weeks. It was maintained
and preserved at 4 °C. The PDA-plated culture of Candida cylindracea
was suspended in 10 mL of sterile distilled water and 1 mL of the
suspension was used as inoculum for pre-cultures. Lipase was pro-
duced in POME at 1% total suspended solid (TSS) as the basal med-
ium containing nutrients (0.45% v/v peptone, and 0.65% v/v Tween-
80) and 2.2% (v/v) inoculum. The initial pH was adjusted to pH 6.0
using 1 M NaOH. The flasks were incubated for 6 days at 28 °C un-
der orbital shaking at 150 rpm. The product was then separated by
microfiltration by using water as the buffer. The cell-free filtrate
was used as a source of extracellular lipase.
2.4. Lipase activity and protein assays
Lipase activity was assayed according to spectrophotometric as-
say method reported by Gopinath et al. (2005) with few modifica-
tions. The phosphate buffer in that method was replaced by
phosphate-citrate buffer because the pH of the buffer can be varied
 R.R. Nasaruddin et al. / Bioresource Technology 154 (2014) 155–161
from 2.0 to 9.0. The phosphate-citrate buffer was prepared accord-
ing to Pearse (1980). 4-Nitrophenyl palmitate (pNPP) was used as
substrate. First, 10 mL isopropanol containing 30 mg pNPP was
mixed with 9 mL phosphate–citrate buffer. A total amount of
2.4 mL freshly prepared substrate solution was mixed with three
different volume of enzyme solution (2, 4 and 6 ll) and then incu-
bated at 37 °C. After 30 min of incubation, absorbance at 410 nm
was measured against an enzyme-free control. One enzyme unit
was defined as 1 lmol of 4-nitrophenol enzymatically released
from the substrate in milliliters per minute (mL/min). Lipase con-
centration was determined using common method of protein assay
which was universal Bradford method where bovine serum albu-
min was used as the standard. The lipase with known activity
and concentration was prepared in aliquots of 40 mL and pre-
served in freezer (20 °C) to avoid rapid reduction of its activity.
2.5. Enzymatic biodiesel production from SPO
All experiments were done in a batch mode and free enzyme
system in 250 mL screw-capped flask. A 25 g of SPO was added
to the flask with 4:1 alcohol-to-SPO molar ratio and 1:10 lipase-
to-SPO volume ratio. The mixture was then incubated in incuba-
tion shaker at 40 °C and 200 rpm agitation speed for 5 h. At the
end of the reaction, the mixture was centrifuged to remove the li-
pase before separating the product in separating funnel. The prod-
uct was washed with recently boiled distilled water to remove
glycerol, excess alcohol and remaining lipase. The upper liquid as
biodiesel in the separating funnel was recovered and dried in oven
to remove the remaining water and alcohol. The product was ana-
lyzed using GC/MS to detect the presence of FAEE. Then, the weight
of product and the final FFA content in the product were measured
to calculate the yield of biodiesel and the conversion of FFA into
biodiesel respectively.
2.6. Evaluation of ethanol–solvent system
The investigation and optimization of ethanol–solvent system
was done using one-factor-at-a-time (OFAT) method. Firstly, the
ethanol-to-SPO molar ratio (2:1, 3:1, 4:1, 6:1, 6:1, and 8:1) and en-
zyme loading (5, 10, 30, 50, 70 and 100 U/25 g of SPO) were varied
and tested. The process conditions of these experiments were
40 °C, 200 rpm agitation speed and 5 h reaction time. Then, the ef-
fects of additional organic solvents (tert-butanol, n-hexane, cyclo-
hexane and iso-octane) were determined using the optimum
amount of ethanol-to-SPO molar ratio and enzyme loading with
the aim to improve the yield and conversion of FFA into biodiesel.
The process conditions for this experiment were 40 °C, 250 rpm
agitation speed and 24 h reaction time. These optimum process
conditions were obtained from the optimization of process condi-
tions (Nasaruddin et al., 2013).
2.7. Analytical analysis
The presence of esters in biodiesel was identified onsite using
GC/MS (Agilent Technologies 7890A gas chromatography equipped
with 5975C mass spectrometer) in the analytical laboratory of Bio-
technology Engineering Department of International Islamic Uni-
versity Malaysia. The GC/MS was operated with split–splitless
mode of injection; the capillary column was DB-wax with a length
of 30 m, film thickness of 0.25 lm and internal diameter of
0.25 mm. Helium was used as carrier gas with a flow rate 30 mL/
min, measured at 50 °C; the run time was 35 min. The temperature
of the injector and detector were 250 and 280 °C. 15 mg sample of
biodiesel was measured in GC vial. The sample was diluted with n-
hexane (AR Grade) to 1 mL final volume prior injection into GC/MS.
157
The chromatogram showed the peak of FAEE to confirm the biodie-
sel production had occurred.
The yield of biodiesel is used to determine the optimum value of
influential parameters. It is defined as the weight percentage of fi-
nal product measured using analytical balance (Mettler Toledo
B204-S) relative to the weight of SPO at the beginning of the exper-
iment as shown by the expression:
Yield ð% w=w SPOÞ ¼ ðweight of biodiesel=weight of SPOÞ  100
ð1Þ
Meanwhile, the conversion of FFA into biodiesel was calculated
using following expression where the FFA content was determined
using titration method described in MPOB Test Methods (Ainie
et al., 2005).
Conversion ð%Þ ¼ 100  ðFinal FFA  Initial FFAÞ=Initial FFA ð2Þ
The analysis of triacylglyceride (TAG), diacylglyceride (DAG),
monoacylglyceride (MAG) and free fatty acids (FFA) contents were
done to the product obtained from the optimum ethanol–solvent
system. The biodiesel was analyzed using GC/FID with the
split–splitless mode of injection at Oils and Fats Laboratory of Sime
Darby Research Sdn. Bhd. Carey Island. The capillary column was
HP-1/SIMDIST with a length of 15 m, film thickness of 0.15 lm
and internal diameter of 0.53 mm. Hydrogen was used as carrier
gas with a flow rate 450 mL/min. The run time was 15 min. The
temperature of the injector and detector were 350 and 360 °C. A
50 mg sample of biodiesel was diluted with 5 mL n-hexane (AR
Grade). 2 mL of the diluted biodiesel was prepared in vial prior
to injection into GC/FID. The injection volume is 0.5 ll. The TAG,
DAG, MAG and FFA content of SPO were also analyzed using the
same method as stated above to compare with the results of bio-
diesel analysis.
3. Results and discussion
3.1. Characteristics of SPO
The chemical properties of the SPO were analyzed to determine
the suitable method for biodiesel production. The FFA content of
SPO analyzed by titration method in this study was found to be
higher (51.64 ± 0.59%) than the FFA content of SPO that was used
by Hayyan et al. (2010) which is 23%. It is too high and unsuitable
for the common alkaline-catalyzed transesterification. The mois-
ture content of SPO is 1.00 ± 0.04% which is low and favourable
for biodiesel production. Water content is very crucial in biodiesel
production. The effects of water contents are further described in
enzyme loading evaluation. The saponification value of
191.92 mg KOH/g oil gave the average molecular weight of
877 g/mol. This molecular weight was used for the calculation of
the organic solvent-to-SPO molar ratio.
The main compositions of SPO analyzed by GC/FID are shown in
Table 1 (column 2). The results show that the SPO contains 31.35%
of TAG, 9.26% of DAG, 1.37% of MAG and 58% of FFA. The percent-
age of FFA in SPO analyzed by GC/FID is slightly higher than the FFA
content analyzed by titration method. However, the difference be-
tween the analytical machine of GC/FID and manual titration
method is small and not much significant. Thus, the initial FFA con-
tent of SPO measured by titration method was used to calculate the
conversion of FFA into biodiesel. The results also indicate that
about 42% of SPO (TAG, DAG and MAG) can be converted into bio-
diesel via transesterification reaction and another 58% (FFA) is via
esterification reaction. Thus, lipase is highly suitable to catalyze
the production of biodiesel from SPO because it is able to catalyze
both transesterification and esterification reactions.
158 R.R. Nasaruddin et al. / Bioresource Technology 154 (2014) 155–161

Table 1 pH of the reaction mixture led to soap production and reduction

Compositions of sludge palm oil (SPO) and biodiesel in comparison with the standard in biodiesel yield while using detergent (Tween-80) to cater the
EN 14214.
soap problem also reduced the biodiesel production. Therefore,
a
Compounds Percentage Percentage Conversion EN 14214 the pH adjustment for the reaction was negligible and the lipase
amount in amount in (%) activity was taken at the average pH of the reaction mixture which
SPO (%) biodiesel (%)
is pH 5.0. At this pH, the lipase has an activity of 4.76 U/mL.
TAG 31.35 27.91 10.97 0.2% (max)
DAG 9.26 14.09 – 0.2% (max)
MAG 1.37 1.91 – 0.8% (max)
3.3. Effects of ethanol-to-SPO molar ratio
FFA 58.00 35.13 39.43 0.1% (max)
FAEE 0.00 20.94 – 96.5% (min) A simple key-and-lock diagram of lipase-catalyzed reactions in
a biodiesel production from SPO was shown and described in Fig. 1.
Biodiesel in above analyses was produced using optimum solvent system (4:1
ethanol-to-SPO molar ratio, 10 U/25 g of SPO and 2:1 tert-butanol-to-SPO molar There are three main reactions that occurred during the enzymatic
ratio) at 40 °C, 250 rpm agitation speed and 24 h reaction time. The Conversion biodiesel production. The desired transesterification and esterifica-
(%) = (WSPO  WBiodiesel)/WBiodiesel  100) where WSPO and WBiodiesel is the percentage tion reactions are favourable when the concentration of alcohol is
amount of the compound in SPO and biodiesel respectively.
higher than water. However, when water content is higher due to
the increasing water by-product of esterification reaction, hydroly-
sis and reversible esterification reaction, biodiesel production be-
The fatty acid compositions analyzed by GC/FID are shown in came low and FFA content increased in the reaction mixture.
Table 2. Based on the results, it can be seen that the highest The evaluation of the ethanol–solvent system is described in the
amount of fatty acids in SPO is palmitic acid with 42.12 ± 1.02%, following section beginning with the effects of ethanol-to-SPO mo-
followed by oleic acid with 40.31 ± 1.03% and linoleic acid with lar ratio. The effects of ethanol-to-SPO molar ratio are illustrated in
10.49 ± 0.81%. Hayyan et al. (2010) and Chow and Ho (2002) also Fig. 2. Based on the results, it was found that the maximum yield
reported that palmitic acid is the most abundant fatty acid in (43.8% w/w SPO) was achieved at 4:1 ethanol-to-SPO molar ratio.
SPO with 42.84% and 51% respectively. SPO has semisolid appear- The optimum level of 4:1 alcohol-to-oil molar ratio was also re-
ance at room temperature because the palmitic acid exists as solid ported by Li et al. (2012). Based on stoichiometry in Figs. 1 and 3
while oleic and linoleic acids as liquid at room temperature. As the mols of alcohol are required to convert 1 mol of TAG into 3 mols
fatty acids composition determines the type of esters in the biodie- of FAEE and 1 mol of glycerol by-product. Since the transesterifica-
sel (Ruan et al., 2012), it was found using GC/MS that the main tion reaction is a reversible or an equilibrium-limited reaction
FAEE in the biodiesel are palmitic acid ethyl ester (PAEE) and oleic (Röttig et al., 2010; Patel et al., 2013), excess ethanol is used to fa-
acid ethyl ester (OAEE). The retention time of the PAEE and OAEE vour the forward reaction which is the formation of esters. How-
are 10.27 and 12.73 min respectively. ever, excessive ethanol from the stoichiometry requirement can
cause the conformational change of the lipase protein structure
thus inhibits its activity and reduce the yield of biodiesel (Patel
3.2. Characteristics of locally-produced lipase et al., 2013). This effect can be seen when the ethanol-to-SPO mo-
lar ratio was increased to 6:1.
The lipase used in this study was recovered from microfiltration Meanwhile, the maximum conversion (16.7%) was achieved at
after the fermentation of palm oil mill effluent (POME) based med- 3:1 ethanol-to-SPO molar ratio. The conversion analysis indicates
ium. Thus, it has a low purity and existed in water solution. The only the conversion of FFA into biodiesel via esterification.
concentration of the lipase was also low (0.15 mg/mL). Since, this Although only 1 mol of ethanol is needed to convert 1 mol of FFA
is an initial study to evaluate the potential of the lipase, so free li- into biodiesel, but since the esterification is also a reversible or
pase system was used in the enzymatic biodiesel production. Be- an equilibrium-limited reaction, thus more ethanol is needed to
sides, the low purity of the lipase is not suitable for shift the reaction forward. Higher ethanol content is also needed
immobilization. The study also excluded the reused and recyclabil- to cater for the increasing amount of water by-product which
ity evaluation of the lipase because of the low purity and concen- can promote the reversible reaction of esterification. Also, the high
tration of the lipase in the water solution. amount of water by-product led to the hydrolysis of TAG, DAG and
The activity of the lipase was examined at pH 4, 5, 6, 7, 8 and 9. MAG rather than transesterification reaction. Therefore, 4:1 was
It was found that the lipase had an optimum activity at pH 7 and taken as the optimum ethanol-to-SPO molar ratio and the small
pH 8. However, the FFA content of SPO is too high, and it made reduction of the conversion at this optimum level can be neglected.
the mixture of the reactants more acidic. An investigation of using It is important to note that the results of yield of biodiesel is far
alkalis (calcium carbonate, sodium hydrogen carbonate, sodium higher than the results of conversion because the yield includes the
hydroxide, and di-sodium hydrogen phosphate) to increase the biodiesel produced by transesterification of TAG and the esterifica-
tion of FFA into biodiesel. However, the conversion is only for the
esterification of FFA into biodiesel. Besides, these two responses
were measured by different methods and have different units.
Table 2
Fatty acids compositions of SPO.
3.4. Effects of enzyme loading
Fatty acids Structure Composition (%)
Capric acid C10:0 0.04 ± 0.05 One of the challenges in this study was the use of free lipase
Lauric acid C12:0 0.62 ± 0.82
which was obtained from cell-free filtrate. It contained water that
Myristic acid C14:0 1.25 ± 0.24
Palmitic acid C16:0 42.12 ± 1.02 was used as buffer during the microfiltration process. The amount
Palmitoleic acid C16:1 0.15 ± 0.02 of water was limited by enzyme loading. The higher enzyme load-
Stearic acid C18:0 4.26 ± 0.07 ing means the higher amount of water. Higher amount of enzyme
Oleic acid C18:1 40.31 ± 1.03 is favourable for the biodiesel synthesis but higher amount of
Linoleic acid C18:2 10.49 ± 0.81
Alpha-linoleic acid C18:3 0.26 ± 0.16
water is unfavourable for the biodiesel synthesis. The effects of en-
Arachidic acid C20:0 0.43 ± 0.44 zyme loading in this study are illustrated in Fig. 3. Based on the re-
sults, it was found that the highest yield (54.4% w/w SPO) was
 R.R. Nasaruddin et al. / Bioresource Technology 154 (2014) 155–161 159

Fig. 1. Key-and-lock diagram of lipase-catalyzed reactions in biodiesel production from SPO. The initial mixture of reactants (SPO and Ethanol) with the lipase contains TAG,
DAG, MAG, FFA, and water from the lipase solution. (a) A complete transesterification reaction convert 1 mol of TAG with 3 mols of alcohol (acyl acceptor) into 2 mols of alkyl
esters (biodiesel) and 1 mol glycerol by-product. However, incomplete transesterification yields in DAG or MAG. (b) A complete hydrolysis reaction converts 1 mol of TAG and
3 mols of water into 3 mols of FFA and 1 mol glycerol by-product. In hydrolysis reaction, water competes with alcohol and disturbs the transesterification reaction. (c) The
esterification reaction converts 1 mol of FFA and 1 mol of alcohol into 1 mol of alkyl ester and 1 mol water by-product. All three reactions are reversible reactions.

Fig. 2. Effects of ethanol-to-SPO molar ratio. (The process conditions: 40 °C,

200 rpm agitation speed and 5 h reaction time.)

achieved at 10 U/25 g of SPO. More than 10 U/25 g of SPO caused
the reduction of biodiesel yield due to the high amount of water
that favours the reversible reaction.
Meanwhile, the highest conversion (21.7%) was achieved at
100 U/25 g of SPO which has the highest amount of lipase and
highest water content. This result shows disagreement with the
yield of biodiesel and the main reason is from the method of ana-
lyzing the FFA content. The final FFA content is measured after
5 days which was after the removal of solidified-non-converted
SPO which could be some of FFAs, TAGs, DAGs, and MAGs. The high
removal of FFAs occurred at 100 U/25 g SPO because the reaction
Fig. 3. Effects of enzyme loading. (The process conditions: 40 °C, 200 rpm agitation
speed and 5 h reaction time).
mixture contained the highest amount of water which promotes
more hydrolysis and reversible esterification reactions, thus yield
in highest amount of FFA at the end of reaction. Most of the FFAs
at the end of the reaction were probably palmitic acid that solidi-
fied and removed after 5 days thus gave lower final FFA content
which led to highest conversion but lowest yield of biodiesel. This
result can be improved by analyzing the final FFA content immedi-
ately after the reaction. The optimum level of enzyme loading was
160 R.R. Nasaruddin et al. / Bioresource Technology 154 (2014) 155–161
determined according to the highest yield of biodiesel which was
at 10 U/25 g of SPO. At this level, the water content was small
(6.5% v/v of the total reaction mixture). Although water can inhibit
the lipase activity and lipase can react in non-aqueous environ-
ment, but lipase still needs a minimum amount of water molecule
to maintain its native conformation and its activity (De Paola et al.,
2009).
3.5. Effects of additional organic solvents
The most common hydrophobic organic solvents used in biodie-
sel production are isooctane, n-heptane, n-hexane, petroleum
ether, cylohexane, 2-butanol and tert-butanol (Ghaly et al.,
2010). Hydrophobic organic solvent can stabilize the enzyme and
improve the solubility of oil and the homogeneity of the reaction
mixture. In this study, tert-butanol, n-hexane, cyclohexane and iso-
octane were examined. The organic solvent-to-SPO molar ratio was
varied at 1:1, 2:1 and 3:1. The effects of the additional solvent are
illustrated in Fig. 4. According to the results, it was found that the
addition of tert-butanol at 2:1 tert-butanol-to-SPO molar ratio into
the ethanol–solvent system gave the highest conversion of FFA into
biodiesel (28.8%) and highest yield of biodiesel (71.6% w/w SPO).
Based on these findings, the conversion of FFA into biodiesel and
the total yield of biodiesel were improved using the additional
tert-butanol at 2:1 tert-butanol-to-SPO molar ratio. However, fur-
ther increasing in the amount of tert-butanol caused the reduction
in the conversion of FFA into biodiesel and the total yield of biodie-
sel. Excess organic solvent could reduce the water activity around
the enzyme, thus promoting the loss of enzyme activity (Chaiyaso
et al., 2012).
(a)
(b)
1:1 organic solvent/SPO molar ratio, 2:1 organic solvent/SPO molar ratio and
3:1 organic solvent/SPO molar ratio.
Fig. 4. Effects of additional organic solvent: (a) Conversion (b) Yield of FAEE. (The
process conditions: 40 °C, 250 rpm agitation speed and 24 h reaction time).
The significance of the use of tert-butanol as co-solvent was
also reported by Chen et al. (2011) and Lu et al. (2008). Tert-
butanol has a moderate hydrophobic and hydrophilic properties,
thus the addition of tert-butanol to the mixture enhances the misci-
bility of alcohol with oil and therefore protects lipase from deactiva-
tion (Lu et al., 2008). Tert-butanol also enhances the mass transfer by
reducing the viscosity of oils (Chen et al., 2011). Besides, tert-butanol
is not a substrate for the lipase. Lipase does not act on tertiary alco-
hols. The short chain alcohol and glycerol by products are also solu-
ble in tert-butanol, thus reducing their inhibitory effects on lipase
activity. However, it is more practical if the use of additional organic
solvent can be avoided in making biodiesel because after completion
of the reaction, the solvent must be removed by distillation, extrac-
tion and so on, which need additional energy and effort.
3.6. Characterisation of biodiesel
The biodiesel produced using ethanol–tert-butanol solvent sys-
tem (4:1 ethanol-to-SPO molar ratio, 10 U/25 g of SPO and 2:1 tert-
butanol-to-SPO molar ratio) was then characterized based on TAG,
DAG, MAG, FFA and fatty acid ethyl ester (FAEE) contents using GC/
FID. The comparisons of their contents with the original SPO are
tabulated in Table 1. Based on the results in Table 1, it was found
that the SPO has 58% of FFA which is higher than the amount of
TAG. When comparing the percentage amount of FFA and TAG in
SPO and biodiesel, it was found that the conversion of FFA into
FAEE is higher (39.43%) than the conversion of TAG into FAEE
(10.97%). These findings suggest that locally-produced Candida
cylindracea lipase is favouring esterification reaction as compared
to transesterification reaction. The conversion of FFA into biodiesel
measured using GC/FID is also higher than the conversion of FFA
into biodiesel measured by titration method (28.8%). Since titration
is a manual method and the FFA content is expressed as palmitic
acid while GC/FID is based on the weight of the free fatty acids,
the difference values for the conversion from these two methods
are not a major concern.
The results in Table 1 also show that the percentage amount of
DAG and MAG is higher in biodiesel as compared to the SPO. The
reason might be due to the incomplete transesterification and
hydrolysis reactions of TAG that release MAG and DAG thus
increasing their contents in biodiesel. Meanwhile, the ester content
of the biodiesel analyzed by GC/FID is lower (20.94%) as compared
to the final yield of biodiesel measured by analytical balance
(71.6% w/w SPO). The different method of analysis is the main rea-
son for the big difference of these two results. Also, the higher
weight of biodiesel measured by analytical balance was also due
to the biodiesel itself which was still in crude form and had some
non-converted unsaturated fatty acids especially oleic acid. Oleic
acid was still in liquid form on the fifth days after the end of reac-
tion. Since the objective of the study is to determine the optimum
level of each parameter in ethanol–solvent system, the yield of the
biodiesel measured by analytical balance can still be accepted and
referred. However, using GC/FID can give better analysis because
each composition was measured at their highest purity. Therefore,
this study can have further improvement by using GC/FID to ana-
lyze the compositions of the biodiesel.
The comparisons of the TAG, DAG, MAG, FFA and FAEE contents
in the biodiesel produced in this study with the International Stan-
dards (EN14214) are also shown in Table 1. Unfortunately, the re-
sults show that the TAG, DAG, MAG, FFA and FAEE contents were
not within the limit suggesting that the transesterification and
esterification reaction catalyzed by locally produced lipase are
not completed and need further improvement in order to increase
the amount of FAEE and reduce as many as possible TAG, DAG,
MAG and FFA content.
 R.R. Nasaruddin et al. / Bioresource Technology 154 (2014) 155–161
4. Conclusions
The present study shows that ethanol-based biodiesel (FAEE)
was produced from low cost waste oil, SPO using the locally-pro-
duced lipase as the catalyst. The percentage amount of FAEE pro-
duced from the ethanol–tert-butanol solvent system (20.94%)
shows that the utilization of SPO with ethanol using the locally
produced lipase from POME has a promising potential if the activ-
ity of the lipase can be improved in further study. Apart from
reducing the cost of biodiesel production, this study also promotes
the green technology because the use of renewable materials such
as SPO, ethanol and locally-produced lipase will reduce the envi-
ronmental pollution.
Acknowledgements
The authors would like to thank the personnel of Sime Darby
Research Sdn. Bhd. for supplying the SPO. Highest appreciation
was also given to the Department of Biotechnology Engineering,
International Islamic University Malaysia (IIUM) for providing the
facilities to undertake this research. This research was funded by
the Ministry of Higher Education using Exploratory Research Grant
Scheme (ERGS-11-002-0002).
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