Renewable Energy 146 (2020) 901e906

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Renewable Energy
jo urn a l h omepage: w ww.els ev ier. co m/locate/renen e

Combination of fatty acids extraction and enzymatic esterification for

biodiesel production using sludge palm oil as a low-cost substrate
Papasanee Muanruksa a, c, Pakawadee Kaewkannetra b, c, *

a
Graduate School of Khon Kaen University, Khon Kaen, 40002, Thailand
b
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand
c
Centre for Alternative Energy Research and Development (AERD), Faculty of Engineering, Khon Kaen University, Khon Kaen, 40002, Thailand

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

Article history: The alternative approach to the production of biodiesel from sludge palm oil (SPO) via two steps of
Received 2 November 2018 extraction and enzymatic esterification was performed. Free fatty acids (FFAs) from SPO were obtained
Received in revised form through saponification reaction and subsequent extraction. The FFAs were further converted to biodiesel
17 May 2019
(fatty acids methyl esters) via esterification reaction catalysed by immobilised Rhizopus oryzae lipase on
Accepted 3 July 2019
alginate-polyvinyl alcohol (PVA) beads. The esterification reaction was investigated by variations of
Available online 4 July 2019
temperature (30e50 C), molar ratio of methanol to FFAs (1:1 to 5:1), agitation rate (100e300 rpm) and
for 4 h. The results were revealed that optimal condition for biodiesel production reached at reaction
Keywords:
Sludge palm oil
temperature of 40 C, methanol to FFAs molar ratio of 3:1 and agitation rate of 200 rpm. Under the
Fatty acids extraction optimum condition, the highest biodiesel yield of 91.30% was achieved. Successfully, biodiesel production
Esterification from SPO gave high conversion rate. In addition, the biocatalyst also showed in high catalytic efficiency
Immobilised alginate-PVA lipase bead and stability up to 15 cycles. The process cost could be reduced cost effectively for biodiesel production
Biodiesel when a low cost substrate of SPO as well as the capable biocatalyst were used.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction processing plant is called as palm oil mill effluent (POME). It

Biodiesel, a renewable energy, is defined as fatty acids alkyl ester
or fatty acid methyl esters (FAMEs). It can be used for diesel engine
with many advantages such as biodegradable, non-toxicity, less
regulated exhausted emission (exception of nitrogen oxide (NOx)),
higher flash point than petro diesel, excellent lubricity and envi-
ronmental friendly. Typically, biodiesel can produce from various
vegetable oils via transesterification or esterification reactions.
According to high cost of vegetable oil causes the production cost of
biodiesel is higher than petroleum diesel. Therefore, biodiesel
production from alternative cheap raw materials of waste oil or
low-grade oil such as waste cooking oil, waste beef tallow, waste
animal fats and grease could be solved this problem [1]. However,
the major drawback of these feedstocks for biodiesel production is
unstable production rate per year.
Typically, waste water discharged from palm oil refinery
* Corresponding author. Department of Biotechnology, Faculty of Technology,
Khon Kaen University, Khon Kaen, 40002, Thailand.
E-mail addresses: pakawadee.kku@gmail.com, paknar@kku.ac.th
(P. Kaewkannetra).
annually produced 3.86 million ton [2]. However, it was found that
sludge palm oil (SPO) is the main residue oil on the upper layer of
POME. It is separated from the initial stage of POME discharge [3].
In addition, the price of SPO is very low (1 USD/Ton) due to its low
grade. Currently, SPO is used as animal feed a supplementary in
animal feed and low-grade soap. Previously, many research works
have focused to use SPO on biogas productions [4e7]. Besides these
applications, SPO can also be used to produce biodiesel. Conversion
of SPO to renewable energy as biodiesel leads to meet effective
waste utilization and reduction of biodiesel production cost. The
challenge aspect of SPO for biodiesel production is high concen-
tration of free fatty acids (FFAs). According to biodiesel production
via transesterification catalysed by potassium hydroxide (KOH),
high FFAs content results in low biodiesel yield due to soap for-
mation [8,9]. Therefore, SPO might be not converted to biodiesel
using alkaline catalyst. Similarly, conversion of FFAs to biodiesel by
using acid catalyst also has some hinder aspect such as low reaction
rate, corrosive and low biodiesel yield [10].
Hydroesterification, hydrolysis coupling with esterification re-
actions catalysed by lipase, was reported as suitable process for
biodiesel production from low quality oil because of high yield,

https://doi.org/10.1016/j.renene.2019.07.027
0960-1481/© 2019 Elsevier Ltd. All rights reserved.
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high purity of glycerol and environment friendly process [11].

However, long reaction time of enzymatic hydrolysis reaction
(12e24 h) was hinder aspect for the process [12,13]. Furthermore,
oil hydrolysis via physical method also required high energy con-
sumption; high pressure of 40 bars, 250 C and 2 h [14]. To over-
come the problem, hydrolysis reaction can be substituted by
chemical fatty acids extraction to obtain FFAs in 1 h [15]. Moreover,
in the second step, enzymatic esterification presents more advan-
tages than chemical catalysts such as no soap formation, low re-
action temperature, no corrosion and high purity of biodiesel. As a
result, lipase is proper for using as a biocatalyst to produce biodiesel
via esterification reaction.
There are some hinder aspects of lipase for various industrial
applications such as high cost, difficult to recovery in downstream
process, low stability and etc. However, immobilised lipase on matrix
support could be figured these problems. A substance of alginate is
also widely used as a matrix for enzyme entrapment in biotechnology
industry especially food and beverage. Since, it has many desirable
properties such as mild conditions for entrapment, high porosity,
relative inert aqueous environment within the matrix, easy to control
porosity with simple coating procedures, retaining most catalytic
activity of enzyme and biodegradation under normal physiological
conditions. However, alginate matrix can be faster degradation and
reduction of alginate molecule when exposed to high temperature,
low pH and organic solvents [16,17]. Previous works [18] reported
that mixing alginate with polyvinyl alcohol (PVA) for invertase
immobilisation could increase efficiency and stability of enzyme.
Therefore in this study, the production of biodiesel from SPO as a
low cost substrate was investigated. The FFAs from SPO were ob-
tained via saponification followed by solvent extraction. The second
reaction, esterification of FFAs from SPO catalysed by immobilised
alginate-PVA lipase bead as biocatalyst was performed. The optimal
condition for esterification reaction was considered by varying in
several effects of reaction temperature, molar ratio between
Fig. 1. Flow diagram of fatty acids (FFAs) obtained from sludge palm oil (SPO).
methanol and fatty acids, and agitation speed. In addition, reus-
ability of immobilised alginate-PVA lipase bead was also evaluated.
hydroalcoholic and hexane phases were separated by separating
2. Materials and methods funnel and the hexanic phase was washed twice with water. Finally,
hexane was vapored and FFAs solution was washed 3 times before
2.1. Raw materials using in further experiments.

Samples of sludge palm oil (SPO) were collected from Esarn

2.3. Lipase immobilisation
palm oil (ESP) company, Sakon Nakhon province, North-eastern of
Thailand. Then, SPO was heated at 80 C and filtered through cotton
The mixture solution of lipase (5% w/v) in phosphate buffer (pH
sheet prior to use. In addition, SPO sample and their fatty acids
7.0) 10 mL was mixed with 90 mL of matrix solution between
composition were characterised. Most of chemicals used in this
alginate and alginate and PVA (ratio of 1:1). Then, the mixture
work such as calcium chloride, sodium hydroxide, monobasic so-
solution was dropped into 0.1 M CaCl2 by sterile syringe and kept at
dium phosphate, phenolphthalein, bovine serum albumin, copper
4 C for 24 h to form immobilised beads. Lastly, the alginate-PVA
sulfate, sodium potassium tartrate, ethanol, methanol, hydrochloric
beads were washed with distilled water three times and stored at
acid and polyvinyl alcohol are analytical grade. Only alginate is food
4 C for further experiments.
grade. Lipase powder of Rhizopus oryzae lipase and methyl hepta-
decaoate were purchased from Sigma Co., Ltd, company, Bangkok,
Thailand. Helium gas is industrial grade. 2.4. Esterification reaction

2.2. Fatty acids extraction
FFAs from SPO were obtained by direct saponification according
to modified method from previous study [19]. There were three
main steps for fatty acids extraction as shown in Fig. 1. In brief,
50 mL of SPO was mixed with 5 M KOH in ethanol (ratio of 2:1). The
saponification was conducted by shaking this mixture reaction at
250 rpm, 60 C for 30 min. The reaction mixture was then cooled
down at room temperature followed by adding 30% water by vol-
ume and pH was adjusted to 1 using HCl to form FFAs. The FFAs
were extracted with hexane at 250 rpm, 30 C for 30 min. Then,
Esterification of FFAs from SPO was performed in a 250 ml
Erlenmeyer flask containing of 50 mL FFAs, methanol and biocata-
lyst (2% w/v). The reaction was optimised by temperature
(30e50 C) methanol to fatty acids molar ratio (1:1 to 5:1), and
agitation speed (100e300 rpm). The analytical procedure was
described in Section 2.5.4.
2.5. Analytical techniques
2.5.1. Determination of lipase activity
Lipase activity was determined by olive oil emulsion method
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[19]. Briefly, immobilised lipase was mixed with mixture solution of Table 2
Fatty acids compositions of sludge palm oil (SPO).
olive oil and PVA (4% w/v) in ratio of 1:3 and incubated at 37 C for
30 min. The reaction was stopped by adding mixture solution of Fatty acids Structures Wt. (%)
acetone and ethanol (ratio of 1:1). Finally, sample was taken to Capric acid C10:0 0.29 ± 0.02
analyse FFAs content (2.4.3). One unit of lipase activity was defined Lauric acid C12:0 0.21 ± 0.03
as the release of 1 mmol oleic acid per minute under the assay Myristic acid C14:0 0.83 ± 0.07
conditions. Specific activity was defined as the number of enzyme Palmitic acid C16:0 18.00 ± 0.04
Palmitoleic acid C16:1 32.05 ± 0.05
units per milligram protein or milligram support.
Stearic acid C18:0 12.82 ± 0.08
Oleic acid C18:1 33.85 ± 0.04
Linoleic acid C18:2 0.59 ± 0.05
2.5.2. Determination of immobilised yield (%)
Linolenic acid C18:3 0.08 ± 0.01
Immobilised yield was defined as reduction of protein concen- Arachidic acid C20:0 0.71 ± 0.05
tration in solution following the previous work [19]. Gadoleic acid C20:1 0.21 ± 0.02
Behenic acid C22:0 0.16 ± 0.07
Erucic acid C22:1 0.11 ± 0.07
2.5.3. Determination of FFAs content (%) Total 100.00
The percentage of FFAs in the oil sample was determined ac-
cording to an official method of the American Oil Chemistry Society
(Ca 5a-40) [20]. 3.2. Lipase activity and immobilisation yield (%)

2.5.4. Determination of esterification degree (%)
Esterification degree (%) was determined as the reduction of
FFAs content in oil sample. The degree of the reaction can be
calculated using Eq. 3 [21].
2.5.5. Analysis of biodiesel composition
The FAMEs product was evaluated by the European Standard
method (EN 14103:2003). It is suitable for FAMES which contains of
C14eC24. Firstly, 250 mg sample was filled into 10 mL vial followed
by adding 5 mL of internal standard (methyl haptadecanoate so-
lution 10 mg/ml). Subsequently, the sample was analysed by Gas
Chromatography e Mass Spectroscopy (GC-MS) (GC-2010, Shi-
madzu, Japan) equipped with 30, length and diameter 0.25 mm of
capillary column lined with a 0.25 mm (Rtx-5 ms, Rextex). Samples
were injected in split/column flow ratio 24:1. Helium used as the
carrier gas (flow rate 1 ml/min). The injection temperature was
250 C and column oven 250 C (programmed to start at 120
C, held at this temperature for 5 min and heated at a rate of 3
C/min to 250 C).
2.5.6. Statistical analysis
All experiments were carried out at least in triplication for
reproducibility and the data obtained was reported as mean ± SD.
The data was analysed using single factor ANOVA with Microsoft
excel 2010. The results were considered statistically significant for P
value less than 0.05.
3. Results and discussion
3.1. Characteristics of sludge palm oil (SPO)
SPO samples and their fatty acids composition before and after
reaction were also characterised and illustrated in Tables 1 and 2.
Table 1
Characteristics of sludge palm oil (SPO).
Parameters Test Methods
FFAs content (%) AOCS Ca 5a-40
Moisture content (%) AOAC, 1999
Acid value (mg KOH/g) AOAC, 1999
Density (g/cm 3) [22]
Kinematic viscosity at room temp. (mm 2/s) [22]
Saponification value (mg KOH/g oil) AOAC, 1999
Average molecular of fatty acids (g/mol) GC-MS
At the beginning, specific lipase activity was tested and reached
3.47 U/mg proteins. After lipase on alginate-PVA was immobilised
as a bead, specific activity of 89.33 U/g support was obtained. It was
implied that 89.33 unit of lipase was adsorbed in matrix support
1 g. Furthermore, high immobilisation efficiency (93.52%) was also
achieved. The results showed that immobilisation lipase within
alginate-PVA matrix showed in higher lipase efficiency than free
lipase. Moreover, it was due to active centre of lipase became stable
open form and explored to substrate in the presence of hydro-
phobic support [23].
3.3. Effect of temperature on esterification reaction
According to the results as shown in Fig. 2, esterification degree
of fatty acids from SPO increased rapidly between 30 C to 40 C. On
the other hand, lower esterification degree was observed at tem-
perature reaction of 45 C and higher FFAs content was also ob-
tained because higher temperature caused enzyme deactivation.
The maximum esterification degree of 90.97% and minimum FFAs
of 1.85% were obtained at 40 C. Therefore, the reaction tempera-
ture was fixed at 40 C in further experiments. The results obtained
were in agreement with previous study [2] which reported that
optimal temperature for enzymatic biodiesel production from
POME via transesterification catalysed by lipase from Pacific white
shrimp was 40 C.
3.4. Effect of methanol to fatty acids molar ratio on esterification
reaction
Based on the results, esterification degree increased sharply, on
the other hand, FFAs content in mixture reaction decreased when
methanol to fatty acids molar ratio increased from 1:1 to 3:1. The
reaction could be driven more completion with the molar ratio was
higher than the stoimetric ratio (1:1) resulting in higher biodiesel
yield. However, much large more amount of methanol might be not
increase biodiesel yield due to denaturation of lipase as seen in
Fig. 3. The esterification degree showed a downward trend with
using molar ratio higher than 3:1 and FFAs content trended to
Values
slightly increased. The highest esterification degree of FFAs from
49.26 ± 1.30
SPO (91.30%) and the lowest FFAs content in mixture reaction
1.53 ± 0.09
104.34 ± 2.75
(1.79%) were achieved at 3:1 methanol to fatty acids molar ratio. As
0.91 ± 0.06 a result, methanol to fatty acids molar ratio of 3:1 was selected. In
43 ± 0.09 addition, the results obtained were agreed with previous work [24]
201.03 ± 6.61 which reported that optimal alcohol to fatty acid molar ratio for
264.85 ± 0.08
conversion of fatty acids in sludge palm oil into biodiesel via
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Fig. 2. Esterification degree of biodiesel as a function of temperature and FFAs content. Fig. 4. Esterification degree of biodiesel as a function of agitation speed and FFAs
content.

showed that the immobilised lipase bead could catalyse esterifi-

cation reaction with high efficiency (91.30%) up to 15 cycles and
after that it trended to slightly decrease (Fig. 5). It might be due to
lower methanol stability of lipase occurred with repetitive using
and lipase desorption from matrix support [27,28]. In Table 3,
comparing among biodiesel yield obtained via the novel process in
this work was high as same as previous studies [2,26]. Furthermore,
the catalyst used was immobilised lipase while others used free
enzyme. Therefore, only the reusability of catalyst in this work was
done and showed remarkable of catalyst circle up to 15. Moreover,
capability of immobilised lipase obtained was similar to previous
work [18] which reported that immobilised invertase on alginate-
PVA bead could reuse with high catalytic efficiency of 100% for 14
cycles.

3.7. Composition of biodiesel

Fig. 3. Esterification degree of biodiesel as a function of methanol to fatty acids molar
ratio and FFAs content.
The biodiesel composition was analysed by GC-MS. The results
obtained were similar to Table 2. In addition, the proportion of
transesterification was 4:1. monounsaturated fatty acids such as palmitoleic acid (C16:1) and
oleic acid (C18:1) found in SPO biodiesel was reported that it could
reduce viscosity, solidification, oxidative stability and cold flow
3.5. Effect of agitation speed on esterification reaction

It was clearly seen that esterification degree of FFAs from SPO

increased sharply at the beginning of agitation rate form
100 rpme200 rpm (Fig. 4). High agitation speed leads to high effi-
ciency mass transfer and high conversion rate. However, increasing
agitation speed higher than 200 rpm could not increase esterifi-
cation degree as shown in Fig. 3. It was due to high agitation speed
caused insufficient interaction between reactants [25]. The
maximum esterification degree of FFAs (91.29%) and minimum
fatty acids content (1.79%) were achieved at agitation speed of
200 rpm. Thus, agitation speed of 200 rpm was selected as the
optimal rate for the reaction. The results obtained were also similar
to previous study [26].They reported that optimal agitation speed
for biodiesel production from palm oil mill effluent via trans-
esterification catalysed by lipase from oil palm fruit was 200 rpm.

3.6. Reusability test of immobilised lipase for esterification reaction

For reusability of immobilised alginate-PVA lipase bead was

tested under optimal conditions (40 C, 3:1 M ratio of methanol to
FFAs and 200 rpm agitation speed) for 20 cycles. The results Fig. 5. Reusability test of immobilized lipase during esterification reaction.
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Table 3
Comparative biodiesel yield from sludge palm oil (SPO) via different processes.

References Processes Catalysts Biodiesel yield (%) Reusability of catalyst

This study Fatty acids extraction/Esterification Immobilised Rhizopus Oryzae lipase 91.30 15
[2] Transesterification Lipase from pacific white shrimp 91.45 e
[24] Transesterification Locally-produced Canida cylindracealipase 71.60 e
[25] Transesterification Crude lipase from oil palm fruit 92.07 e
[29] Transesterification NaOH 83.19 e
[30] Transesterification Alum and KOH 93.00 e
[31] Esterification/Transesterification Trifluoromethanessulfonic (TFMSA)/KOH 84.00 e
[32] Transesterification Canida cylindracea lipase 62.30 e
[19] Esterification/Transesterification H2SO4/KOH 83.72 e
[33] Esterification/Transesterification P-toluene-4-sulfonic monohydrate acid (PTSA)/KOH 76.62 e
[34] Esterification P-toluene-4-sulfonic monohydrate acid (PTSA) 96.00 e

Table 4
Comparative fatty acids methyl esters (FAMEs) compositions from different sources of SPO.

References Wt. (%)

Palmitic acid (C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2)

This study 18.00 ± 0.05 32.05 ± 0.08 12.82 ± 0.06 33.85 ± 0.05 0.59 ± 0.07
[23,31] 42.12 ± 1.02 0.15 ± 0.02 4.62 ± 0.07 40.31 ± 1.03 10.49 ± 0.81
[28] 34.50 0.14 5.99 41.89 15.84
[29] 23.40 e 1.20 75.50 e
[21] 46.2 ± 0.39 e 4.45 ± 0.17 37.99 ± 0.92 9.85 ± 0.08
[32] 46.20 ± 2.38 e 4.45 ± 0.14 37.99 ± 1.07 9.85 ± 0.05
[33] 42.84 ± 3.91 0.13 ± 0.01 4.21 ± 0.25 39.58 ± 1.57 9.92 ± 1.39

problems in biodiesel quality. Moreover, saturated fatty acids of
palmitic acid (C16:0) and stearic acid (C18:0) found in biodiesel
resulted in high cetane number and less nitrogen oxides (NOx)
exhaust emissions [35,36]. The SPO biodiesel obtained in this work
presented in high contents of both palmitoleic and oleic acids. On
the other hand, biodiesel from other works were mainly consisted
of palmitic acids and oleic acids (See Table 4). It should be noted
that there are many factors which affecting on fatty acid compo-
sitions of natural palm oils such as climate conditions, growing
season, plant maturity, and plant [28]. Furthermore, linoleic
showed quite low compare to other works. It was proven in good
oxidative stability. Therefore, it could be confirmed in high quality
biodiesel.
4. Conclusion
All in all, biodiesel production from SPO via chemical fatty acids
extraction followed by enzymatic esterification could reach high
conversion rate of 91.30% under the optimal condition. The bio-
catalyst could keep in high catalytic efficiency and stability up to 15
cycles. Both short reaction and high reusability were clearly shown
that this process could reduce cost effectively. Interestingly, low
purity of SPO should be considered as alternative choice of low cost
substrates for biodiesel production. Our work is the first group who
applied immobilised lipase on alginate-polyvinyl alcohol (PVA)
bead for biodiesel production from SPO. Further work should be
focused on using immobilised lipase/immobilised cell and
improvement of supporting material. The matrix support should be
assisted to get high reusability and low cost.
Acknowledgements
The authors would like to sincere gratefully acknowledge for all
sponsors; Royal Golden Jubilee (RGJ) Ph.D. Programme [Contract
no. PHD/0018/2557], Bangkok, Thailand for financial support;
Graduate School of Khon Kaen University and Centre for Alternative
Energy Research and Development (AERD), Faculty of Engineering,
Khon Kaen University, Khon Kaen, Thailand are also acknowledged
for travel bursary.
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