Fuel 195 (2017) 82–87

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Fuel
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Full Length Article

The production of biodiesel using residual oil from palm oil mill effluent
and crude lipase from oil palm fruit as an alternative substrate and
catalyst
Saowakon Suwanno a, Thanaphorn Rakkan a, Tewan Yunu a, Nisa Paichid a, Pattarawadee Kimtun a,
Poonsuk Prasertsan b, Kanokphorn Sangkharak a,⇑
a
Department of Chemistry, Faculty of Science, Thaksin University, Phatthalung 93210, Thailand
b
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, 90112, Thailand

h i g h l i g h t s

High biodiesel was obtained from POME oil and crude lipase under optimal conditions.

The properties of POME biodiesel are acceptable, according to ASTM standards.

The production of biodiesel can be done at a low investment cost.

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

Article history: Biodiesel production using residual oil from palm oil mill effluent (POME) and crude lipase from oil palm
Received 10 October 2016 fruit as the substrate and catalyst had a high biodiesel yield (92.07 ± 1.04%) under optimal conditions.
Received in revised form 10 January 2017 POME is considered as an alternative source for oil because it contained high oil and grease content
Accepted 12 January 2017
(5569.82 mg/L). Oil was extracted from POME by the soxhlet method using a mixture of hexane, metha-
nol and acetone. Eighty percent of residual oil (4455 mg/L) was recovered from POME. Biodiesel produc-
tion from crude lipase catalyst is an alternative method that is simple to perform and can be done at a low
Keywords:
investment cost. In addition, biodiesel from residual oil using crude lipase catalyst was characterized
Lipase
Methyl ester
according to ASTM standards. Most properties of biodiesel from crude lipase are acceptable, according
Palm oil mill effluent to Thai biodiesel and ASTM standards. Low free fatty acid (0.07%) content was observed in enzymatic bio-
Transesterification diesel. A high cloud point (10–13 °C) and cetane number (59.0–60.0) were also illustrated since a high
cetane number is an important property used to qualify high quality biodiesel, POME biodiesel may pos-
sibly be used as a sole biofuel or blended with fossil fuels.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Palm oil mills are abundant in Southern Thailand. Currently,
more than 72 palm oil industries are in operation in Thailand. Most
of them are oil palm crushing mills (>60 mills) and the others are
palm refineries mills. In 2010, production of crude palm oil (CPO)
in Thailand reached 1.29 million tons [1]. In the process of palm
oil milling, effluent is mainly generated through the sterilization
and clarification process. Due to an increase in palm oil produc-
tions in the last few years, palm oil waste have increased rapidly.
According to Borja and Bank [2], more than 3.86 million tons of
palm oil mill effluent (POME) is obtained annually. POME contains
⇑ Corresponding author.
E-mail address: skanokphorn@yahoo.com (K. Sangkharak).
a high amount of organic matter, oil and grease, total solids and
suspended solids. Therefore, a suitable treatment should be uti-
lized to keep the surrounding environment safe from damage. In
addition, looking for an alternative method for utilizing and treat-
ing POME at the same time is still the main challenge. POME from
oil palm crushing mills (Thailand) contained COD and oil in the
range of 45000 and 6000 mg/L, respectively. After oil recovery,
more than 70% of COD was significantly decreased and 78% of oil
was easily recovered from POME using n-hexane [3]. Therefore,
the recovery of oil from POME shows a great potential to reduce
high COD from POME and an adequate amount of oil from POME
may be utilized as substrate for valuable products. Due to the
reduction of fossil fuel, many countries encourage and support pro-
jects which produce energy from renewable resources. Therefore,
POME shows a potential to be utilized as a novel material for bio-

http://dx.doi.org/10.1016/j.fuel.2017.01.049
0016-2361/Ó 2017 Elsevier Ltd. All rights reserved.
 S. Suwanno et al. / Fuel 195 (2017) 82–87
diesel production due to its high content of oil and grease which
could be converted into biodiesel via chemical and bio-catalytic
transesterification.
Currently, sodium hydroxide is mostly utilized as a chemical
catalyst for biodiesel production. However, high FFA and acid value
in oil from POME are considered drawbacks of chemical biodiesel
[3,4]. Due to the limit of FFA, content for chemical method is 1%
or 2.5% [5,6]. The chemical catalyst can easily promote soap forma-
tion. Thus, it reduces the yield of biodiesel and complicates the
separation process [6,7]. Hayyan et al. [5] reported the production
of biodiesel from sludge palm oil (SPO). SPO, a byproduct from
palm oil mill using distillation method, also contained high FFA.
Therefore, both acid- and alkali-catalyst were suggested for biodie-
sel production from SPO. Two steps of biodiesel production began
with acid catalyst to reduced FFA content in SPO. Afterwards, bio-
diesel was produced via the transesterification process. High yield
of biodiesel (76.62%) with 96% ester content was obtained from
SPO. But, it consumed more energy and effort for the separation
process. Therefore, the utilization of enzyme instant of chemical
transesterification was considered in this study. Lipase is
biodegradable, nontoxic, helps to ease recovery of product and
glycerol and requires moderate alcohol and mild reaction condi-
tions [8–10]. It can also catalyze both esterification and transester-
ification [9]. Thus, it is suitable for high FFA feedstock like POME.
However, lipase-catalyzed biodiesel production is still less attrac-
tive for commercialization because commercial lipase is very
expensive [11–13]. From our knowledge, crude lipase from oil
palm fruit after 120 h of harvesting contained high lipase activity
(1.38 Unit/mg protein) using Tris-HCl (pH 8) extraction. After-
wards, lipase activity increased significantly to 4.76 Unit/mg pro-
tein after the separation process using PEG and NaH2PO4. Palm
lipase was already evaluated for its feasibility for biodiesel produc-
tion using cooked palm oil [12]. The results indicated that biodiesel
from palm lipase were not significantly different from commercial
biodiesel and it also passed Thailand’s fuel standards. The objective
of this research was to investigate the potential of residual oil from
POME and crude palm lipase as a novel substrate and catalyst for
biodiesel production. The optimal conditions for biodiesel produc-
tion including molar ratio, enzyme ratio, incubation temperature,
mixing speed and reaction time were also determined. Biodiesel
from crude palm lipase was characterized and compared with
the production from NaOH, partial purified lipase and commercial
lipase. In addition, the properties of biodiesel from crude lipase
were also compared with Thai biodiesel and ASTM standards.
2. Materials and methods
2.1. Raw materials and chemicals
POME was collected from the Krabi Oil Palm Farmers Coopera-
tives Federation Limited (Krabi, Thailand) and stored at 4 °C to
avoid any decomposition, oxidation and changes to the FFA con-
tent. All the chemicals such as organic solvents and reagents of lab-
oratory and analytical grades were employed in this study. Lipase
(EC 3.1.1.3) from Candida rugosa was obtained from Sigma-Aldrich.
The Candida sp. lipase was prepared in 0.1 M Tris-HCl buffer fol-
lowed the instruction from Kimtun et al. [12].
2.2. Oil extraction from POME
POME was firstly centrifuged at 5000 rpm for 60 min before use.
Only sediment was collected and utilized. Furthermore, oil was
extracted from the sediment by the soxhlet extraction technique
using an organic solvent containing hexane:methanol:acetone
(6:2:2). The sample was mixed with the solvent under an optimal
83
ratio at 1:6 (w/w). The process was maintained at 100 °C for 1 h,
afterward oil was collected and recovered by evaporation [14].
Afterward, the residual oil was utilized as a substrate for biodiesel
production.
2.3. Lipase extraction from oil palm fruit
2.3.1. Plant collection
Ripen palm fruit (E. guineensis) were collected randomly accord-
ing to ARDA (Thailand) standards and left for 120 h after harvesting
under room temperature (28 ± 2 °C). Afterward, 1 g of lipase was
extracted from 50 g of ripen palm fruit using 0.1 M Tris-HCl buffer
(pH 8.0) as described by Kimtun et al. [12]. The supernatant was
freeze-dried and kept at 4 °C until used. The enzyme after buffer
extraction was called ‘‘crude lipase”.
2.3.2. Partial purification of lipase from oil palm fruit
Crude lipase from oil palm fruit was purified by using the aque-
ous two-phase systems (ATPS) method, 20% of polyethylene glycol
(PEG)-1000 and 15% of NaH2PO4. Lyophilized lipase (1 g) was
firstly mixed with 5 g of distilled water. Thereafter, the sample
was loaded into the ATPS system. A bottom layer, an enzyme con-
taining layer, was collected and characterized [12]. The super-
natant was dialyzed with 12000 Da membrane and left overnight
against distilled water. The protein and protease activity after dial-
ysis were determined. Afterwards, the supernatant was freeze-
dried and kept at 4 °C until used. The enzyme after ATPS was called
‘‘partial purified lipase”.
2.3.3. Determination of protein and lipase activity
The determination of protein was followed by the Lowry
method. The activity of lipase was determined by adding 200 lL
samples in 0.1 mM Tris-HCl buffer (pH 8.0) 2.45 mL containing
0.15 M NaCl and 0.5% Triton X-100. The determination was oper-
ated under 40 °C for 5 min, then 50 mM p-nitrophenylpalmitate
200 lL was added. p-nitrophenol was used as a standard and the
sample was analyzed at 410 nm [12].
2.4. Enzymatic biodiesel production from POME and optimization
study
All experiments were determined in a 250 mL screw-capped
flask under batch condition. Crude lipase from palm oil fruit was
used as the catalyst. The investigation and optimization of enzy-
matic biodiesel from POME was varied using one-factor-at-a-
time (OFAT) method. Ten grams of residual oil from POME were
added to the flask with a methanol-to-oil ratio from 2:1 to 12:1
and enzyme loading at 14 to 91 U/10 g of oil were varied and
tested. The process conditions of these experiments including incu-
bation temperature (35–70 °C), mixing speed (100–400 rpm) and
reaction time (6–72 h) were also varied. At the end of the reaction,
centrifugation was used to remove glycerol and lipase before sep-
arating the product in a separating funnel. The sample was left for
1–2 h to separate in a separating funnel. Biodiesel (upper layer)
was taken, washed twice with 20 mL of deionized water at 50 °C
and dried in an oven (105 °C, 24 h) to get rid of the remaining
water and alcohol. Fatty acid methyl ester (FAME) content was
evaluated by GC/MS [16]. The yield of biodiesel and the conversion
of FFA into biodiesel were calculated by weight and the final FFA
content in the product. Biodiesel yield from crude lipase was also
compared with NaOH, partial purified and commercial lipase.
2.5. Analytical analysis
Table 1 shows the full list of parameters for the characterization
of POME, while the methodologies for each of the analyzed
84 S. Suwanno et al. / Fuel 195 (2017) 82–87
Table 1
Analyzed parameters and methodologies employed for the characterization of POME.
Parameters Unit
pH
Temperature °C
Chemical oxygen demand (COD) mg/L
Biochemical oxygen demand (BOD) mg/L
Total nitrogen (TN) mg/L
Ammonium-nitrogen (NH3-N) mg/L
Oil and grease
Total solid (TS) mg/L
Suspended solids (SS) mg/L
parameters in the POME characteristic study are also indicated.
The density of the samples was determined at 20 °C using an
Anton-Paar DMA 4500 density meter. A Brookfield RV-I instrument
with an S03 spindle was utilized for viscosity analysis. The speed
was set at 100 rpm and conducted at room temperature [15].
The chemical properties of residual oil were determined accord-
ing to the AOAC Official Method: free fatty acid content (%FFA)
(AOAC 969.33), saponification value (sv) (AOAC 920.160), iodine
value (AOAC 930.15), moisture content (air-oven method) and
unsaponifiable matter (AOAC 933.08). The analysis of FFA content
was done to the product obtained from the optimum condition.
The biodiesel was analyzed using a GC/Frame Ionization Detector
(FID) with the split-splitless mode of injection. The capillary col-
umn contained a length of 15 m, a film thickness of 1.5 lm and
an internal diameter of 0.53 mm. Hydrogen was used as a carrier
gas with a flow rate of 450 mL/min. The run time was 15 min.
The temperatures of the injector and detector were 350 and
360 °C. A 50 mg sample of biodiesel was diluted with 5 mL of n-
hexane. 2 mL of the diluted biodiesel was prepared in a vial prior
to injection into GC/FID. The injection volume was 0.5 lL. The
FFA content of POME was also analyzed using the same method
as stated above to compare with the results of the biodiesel analy-
sis [16]. Each sample was determined in triplicate. GC/MS was also
utilized for determined the esters content in biodiesel. It was oper-
ated with a split-splitless mode of injection. DB-wax with a length,
a film thickness and an internal diameter of 30 m, 0.25 lm and
0.25 mm was used, respectively. Helium, a carrier gas, was set with
30 mL/min flow rate. The experiment was measured at 50 °C for
35 min with the injector temperature at 250 °C and the detector
temperature at 280 °C. A 15 mg sample of biodiesel was weighted
and diluted with n-hexane in GC/vial before injection. The presence
of FAEE peak in GC/MS chromatogram was determined [16,17].
The yield of biodiesel is used to determine the optimum value of
influential parameters. It is defined as the weight percentage of the
final product measured using an analytical balance (Mettler
Toledo) relative to the weight of POME at the beginning of the
experiment as shown by the expression:
Yield ð% w=w POMEÞ ¼ ðweight of biodiesel=weight of POMEÞ  100
For the conversion of FFA to biodiesel, initial and final FFA was
firstly determined using the MPOB Test method. Afterwards, the
conversion (%) was determined as described below:
Conversion ð%Þ ¼ 100  ðFinal FFA  Initial FFAÞ=Initial FFA
3. Results and discussion
3.1. POME characterization and oil extraction
The characteristics of POME are indicated in Table 1. Among all
properties, only 4 factors including FFA, saponification value, acid
value and moisture content were affected in biodiesel production
[18]. High FFA in residual oil (27.67%) is unfavorable for the
Methodology Content
pH meter 4.82
Thermometer 57 ± 10
HACH method 8000 48900 ± 27.00
3 day BOD test at 30 °C 19560 ± 1.01
HACH method 10072 460 ± 9.90
HACH Nessler’s method 8038 51 ± 24.00
APHA 5220 B 5569.82 ± 10.59
APHA 2540 B 22666.01 ± 1.41
HACH method 8006 (Photometric) 20200.78 ± 2.345
alkaline-catalyzed transesterification. However, it can be con-
verted to biodiesel via enzymatic transesterification reaction. Thus
lipase is highly suitable to catalyze the production of biodiesel
from residual oil from POME because it is able to catalyze both
esterification and transesterification reactions [18]. Low nitrogen
content in POME (460 mg/L) was correlated with the presence of
51 ppm of ammonium-nitrogen. The BOD of POME was estimated
to be around 19560 mg/L while the COD concentration was
approximately 48900 mg/L. pH of POME was acidic (4.82).
High oil and grease content (5569.82 mg/L) was observed in
POME. It indicated that POME was a great oil source. Content of
oil in POME showed the possibility for oil extraction. After charac-
terization, oil was extracted from POME by the soxhlet method
using a mixture of hexane, methanol and acetone. The highest oil
and grease content that could be recovered from POME was
4455 mg/L which means that 80% of the oil and grease could be
recovered from POME. Therefore, residual oil from POME showed
a high potential to be utilize as substrate for biodiesel production.
Normally, the potential for the production of biodiesel seems to be
limited by the availability and costs of substrates. Therefore, POME,
a high volume and costless waste, seems to be a great substitute
for normal biodiesel substrates such as edible oil. POME is pro-
duced from a mixture of sterilizer condensate, separator sludge
and hydrocyclone wastewater. It is abundant and available. About
3.86 tons of this waste is produced for every ton of oil extracted in
an oil mill [2,20].
Residual oil from POME was further characterized for moisture
content, saponification value and average molecular weight. The
moisture content of residual oil was 2.20 ± 0.07%. Water was
removed before enzymatic biodiesel production because it pro-
motes the reversible reaction of esterification. In addition, a high
amount of water was already present in the lipase solution [19].
Therefore, residual oil was firstly pretreated by heat before use.
Primandari [15] reported the similar composition of fatty acid in
POME oil. The composition of fatty acids is very important in iden-
tifying carbon chains and its properties. In addition, it can deter-
mine the type of esters in biodiesel [21]. Palmitic acid (45.3%)
was the highest FFA in residual oil followed by oleic acid (38.0%)
and linoleic acid (10.1%). The saponification value of
201.96 ± 0.21 mg KOH/g oil gave the average molecular weight of
833 g/mol.
3.2. Biodiesel yields
Biodiesel was produced via an enzyme-catalyst. Crude, partial
purified and commercial lipase was utilized as the catalyst. Each
enzyme was dissolved in buffer and the concentration was fixed
at 36 U/10 g oil. 4.42, 10.19 and 4.64 mg of crude, partial purified
and commercial lipase was dissolved in 5 mL of 0.1 M Tris-HCl buf-
fer (pH 8.0). The total aqueous volume of the reaction mixture was
18.18 mL, comprise 10.59 mL (10 g) oil, 5 mL enzyme solution and
2.59 mL methanol (methanol-to-oil ratio at 6:1). Residual oil from
POME was used as the sole substrate for the transesterification
 S. Suwanno et al. / Fuel 195 (2017) 82–87
Table 2
Biodiesel yield (%) from extracted oil after 24 h transesterification with alkali- and
enzyme-catalyst. Each enzyme was dissolved in 5 mL of 0.1 M Tris-HCl buffer (pH 8.0)
and the concentration was fixed at 36 U/10 g oil. The total aqueous volume of the
reaction mixture was 18.18 mL.
Catalyst Enzyme activity (U/g) Biodiesel yield (% ± S.D.)
NaOH – 80.30 ± 2.51
Commercial lipase 7753 91.75 ± 1.58
Oil palm lipase
Crude lipase 8138 90.37 ± 1.43
Partial purified lipase 3533 91.04 ± 1.98
process. Biodiesel yields from all lipase tested in this study were
not significantly different (Table 2). The highest biodiesel yield
(91.75 ± 2.51%) was obtained from commercial lipase-catalyst fol-
lowed by partial purified lipase (91.04 ± 1.98%) and crude lipase
(90.37 ± 1.43%). The lowest biodiesel yield (89.30 ± 2.51%) was
achieved from NaOH-catalyst. The alkali transesterification shows
a very fast kinetic of reaction but also a collateral saponification
reaction that reduces the biodiesel production efficiency [22].
Therefore, the enzyme catalyst gave higher biodiesel yield than
the alkali-catalyst.
This study indicated that POME is suitable for biodiesel produc-
tion. Using POME as a substrate has many advantages such as the
added value ensuing from their re-utilization and their simple pro-
cess, which reduces waste and creates green energies. From the
economic point of view in term of the cost of biodiesel from resid-
ual oil, those of commercial biodiesel and diesel were also com-
pared. Since almost 90% of commercial biodiesel in Thailand was
produced from crude palm oil, (CPO), CPO biodiesel is chosen as
the subject of cost analysis. In December 2016, CPO biodiesel
obtained by the chemical approach was $0.982/kg while diesel
was only $0.672/kg (NESTE Oil Corporation, December 2016). From
the operation in this study that was analyzed including the cost of
chemical, electricity and labor, the biodiesel cost was reduced to
$0.266/kg. The cost of POME biodiesel was 3.69 and 2.52-times
lower than CPO biodiesel and diesel price, respectively. In addition,
utilization of palm lipase showed the great attractive. Various
sources of lipases have been reported including animal, microbial
and vegetable, with differences in their catalytic properties
[23,24]. Bacterial and fungal lipases have been employed to pro-
duce biodiesel. High yield (90–99%) of FFAE was obtained under
24–90 h of incubation times, making the process more economi-
cally feasible [25,26]. To date, microbial lipases are the most stud-
ied. Nevertheless, utilizing microbial enzymes on a large scale is
still limited. The major drawback of these enzymes is their high
production costs. Some enzymes are still economically produced
from plant and animal sources. This is possible because of suffi-
ciently high amounts of this enzyme in such sources and also as
a means to convert inexpensive, renewable material like agricul-
tural and slaughter waste into value added products. Therefore,
the search for other sources of these enzymes is an important issue
[27]. From our study, partial purified lipase from palm oil fruit was
already used for biodiesel production using cooked oil as substrate
[12]. However, crude lipase has never been demonstrated before.
The data indicate that the crude enzyme treatment is less effective
but competitive to the commercial enzymes. The usage of crude
enzyme in place of the commercial enzymes will be cost effective
and will reduce the cost of the process to the proportion of 60–70%.
Cost of crude lipase from palm fruit was 0.01 USD/g while commer-
cial lipase (C. rugose, Sigma) was 406.30 USD/g (data not shown).
Crude enzymes may have been selected, rather than purified and
commercial enzymes, in many cases in the past for reasons based
on economy and convenience. Crude lipases from oil palm fruit
are an alternative method that are simple to perform, and can be
done at a low investment cost.
85
3.3. Optimum process for biodiesel production
Crude lipase from oil palm fruit was selected and used as the
catalyst throughout this study. The OFAT method was used to eval-
uate the optimization conditions. The methanol-to-oil ratio was
firstly assessed. The biodiesel production was increased when the
ratio was increased. The maximum yield of biodiesel
(90.01 ± 1.00%) was achieved at a 6:1 methanol-to-oil ratio
(Fig. 1a). The enzyme concentration was the variable that had
the highest influence. The enzyme loading was varied from 14–
91 U/10 g oil. Higher enzyme loading is favorable for the biodiesel
synthesis while high biodiesel yield was obtained. The optimum
enzyme loading at 36 U/10 g oil yielded the highest yield of biodie-
sel at 90.62% w/w residual oil (Fig. 1b). Biodiesel yield was slightly
constant with an increase of enzyme higher than 36 U/10 g oil.
Incubation temperature is also important for enzymatic bioiesel
production. The highest yield (91.02 ± 0.90%) was obtained at 35 °C
of incubation. Lower yield was obtained when incubation temper-
ature was higher than 35 °C (Fig. 1c). The optimum mixing speed of
200 rpm was sufficient for the reaction completion. The highest
yield of biodiesel (92.07 ± 1.04%) was achieved under this condi-
tion (Fig. 1d). The yield of biodiesel increased with an increase in
reaction time. The yield of biodiesel during reaction time between
36 and 72 h was not significantly different. Therefore, 36 h of incu-
bation time was selected in order to save energy and to decrease
the cost of the process. The yield of biodiesel at 92.07 ± 1.04%
was obtained after 36 h of reaction time (Fig. 1e). The analysis
shows that by choosing of incubation time at 36 h instead of
72 h, the energy will on average save up to 500 kW h annually. This
corresponds to a 50% reduction in energy consumption, leading to
optimized financial gains, corresponds to a 30% saving (data not
shown). A suitable reaction time is needed to complete the biodie-
sel reaction. Therefore, sufficient contact time must be provided
[28]. Crude lipase required the longest reaction time. The results
suggested that crude enzyme are capable of converting triacylglyc-
erols, diacylglycerols, monoacylglycerols and free fatty acids to
FAME in addition to fat hydrolysis, but could not completely the
process within 1–24 h.
The production of biodiesel from the enzymatic process was
also compared with the commercial enzyme and chemical process.
The optimal conditions for biodiesel production from POME using
crude lipase containing a 6:1 level of methanol-to-oil using
36 U/10 g oil of lipase under incubation temperature, a mixing
speed and reaction time of 35 °C, 200 rpm and 36 h, respectively.
92.07 ± 1.04% of biodiesel yield was achieved under these optimal
conditions. Similar yield (92.11 ± 1.54%) was also obtained from
commercial lipase under the same condition. However, alkali-
catalysts required a higher level of methanol-to-oil (Table 3).
3.4. Biodiesel quality
Biodiesel from residual oil and crude lipase as the substrate and
catalyst was characterized according to ASTM standards. Table 4
shows the fuel properties and the stability of biodiesel from POME.
The results show that most properties of biodiesel from crude
lipase are acceptable, according to Thai biodiesel and ASTM stan-
dards. Low free fatty acid (0.07%) content was observed in POME
methyl ester catalyzed by crude lipase. However, FFA content of
POME oil was <0.5% before transesterification.
A high cloud point (10–13 °C) observed in enzymatic biodiesel
may be used to qualify high quality biodiesel. The higher the
cetane number the more easily the fuel will combust in a compres-
sion setting. A high cetane number in biodiesel may pose problems
if the engine is used during cooler seasons but it can be used suc-
cessfully in tropical and desert regions. However, a cloud point is
not specified in Thailand’s biodiesel standards. The cloud point
86 S. Suwanno et al. / Fuel 195 (2017) 82–87

Yield (%), Conversion (%) (a) (b)

100

Yield (%), Conversion (%)

100
80
80
60 60

40 40

20 20

0 0
2:1 4:1 6:1 8:1 10:1 12:1 14 25 36 47 58 69 80 91
Methanol-to-oil molar ratio Enzyme loading (U/10 g oil)
(c) (d)
Yield (%), Conversion (%)

100

Yield (%), Conversion (%)

100

80 80

60 60

40 40

20 20

0 0
35 45 55 70 100 200 300 400
o
Incubation temperature ( C) Mixing speed (rpm)
(e)
Yield (%), Conversion (%)

100

80

60

40

20

0
6 12 24 36 48 60 72
Reaction time (h)

Fig. 1. The effect of methanol-to-oil ratio (a), enzyme loading (b), incubation temperature (c), mixing speed (d) and reaction time (e) on biodiesel yield ( ) and% conversion of
fatty acid to FAME ( ).

Table 3
Optimal condition for biodiesel production from palm oil mill effluent using enzymatic- and chemical-catalyst.

Conditions Catalyst
Alkali- Crude lipase from oil palm Commercial lipase from C. rugosa
A level of methanol-to-oil 9:1 6:1 6:1
Enzyme activity (U/10 g oil) – 36 36
Alkali catalyst (%w/v) 1 – –
Incubation temperature (°C) 60 35 35
A mixing speed (rpm) 800 200 200
Reaction time (h) 1 36 24
Biodiesel yield (%) 91.31 ± 1.56a 92.07 ± 1.04b 92.11 ± 1.54b

Different letter(s) in the same row indicate significant differences (p < 0.05).

Table 4
Partial fuel properties of biodiesel from POME.

Catalyst Free fatty acid, FFA (mg KOH/g) Viscosity (mm2/s) Cloud point (°C) Methyl ester content (%) Cetane number
NaOH 0.06 ± 0.20 4.0 ± 0.14 4.0 ± 1.0 96.5 ± 1.01 59.0 ± 0.3
Crude lipase 0.07 ± 0.10 5.0 ± 0.42 13.0 ± 1.0 96.5 ± 0.90 60.0 ± 0.1
Commercial lipase 0.06 ± 0.12 4.0 ± 0.25 10.0 ± 1.0 98.0 ± 1.02 59.0 ± 0.3
Thailand’s specification 0.50 (max) 3.5–5.0 Reported >96.5 51 (min)
ASTM specification 0.50 (max) – Reported >96.5 47 (min)

depends on the chain length and saturated fatty acid compound point of 30–33 °C, its pour point of 10–13 °C is quite high, which
[29]. Since POME methyl ester contains a majority of saturated could be trouble in the Northern part of Thailand during times of
methyl ester, palmitic acid methyl ester, C16:0 with high melting cool weather or in the mountains [29]. Higher saturated fatty acids
 S. Suwanno et al. / Fuel 195 (2017) 82–87
(51.8%) in residual oils give a higher cetane number and the oil is
less prone to oxidation [28]. A fuel quality is primary indicated
by the cetane number. The cetane number indicated the ease of
its self-ignition. Most diesels for general vehicles have a cetane
number in the range of 45–55. A high cetane number (59–60)
was determined from POME biodiesel (this study). Nevertheless,
dimethyl ether is a high quality fuel for diesel engines and is pro-
duced by converting syngas into methanol followed by dehydra-
tion of methanol to dimethyl ether. Dimethyl ether has a high
cetane rating (55–60) and can be utilized as a biofuel [30]. There-
fore, POME biodiesel may possibly be used as sole biofuel or
blended it with fossil fuels depending on the policy objectives
and desires.
4. Conclusion
Residual oil from POME was an alternative source for biodiesel.
Oil was extracted from POME using the soxhlet method and 80% oil
(4455 mg/L) was recovered. High FFA content (27.67 ± 0.10%) was
observed in POME. The moisture content, saponification value
and molecular weight of residual oil were 2.20 ± 0.07%,
201.96 ± 0.21 mg KOH/g oil and 833 g/mol, respectively. The opti-
mal conditions for biodiesel production from POME oil using crude
lipase containing a 6:1 level of methanol-to-oil using 36 U/10 g oil
of lipase under incubation temperature, a mixing speed and reac-
tion time of 35 °C, 200 rpm and 36 h, respectively. The highest bio-
diesel yield (92.07 ± 1.04%) was achieved under this optimal
condition. Biodiesel from POME oil using an enzymatic-catalyst
were characterized according to ASTM standards. The results show
that most properties of biodiesel from residual oil from palm oil
mill effluent are acceptable, according to Thai biodiesel and ASTM
standards. The biodiesel from crude lipase catalyst has similar
properties to that of biodiesel catalyzed by commercial lipase.
POME oil and crude lipase showed a good potential for biodiesel
production. In order to assure that scaling of the processes, systems
and procedures can be directly transferred to the full scale unit, a
more detail for understanding of scale-up process is still required.
Acknowledgements
The authors would like to thank Thailand Research Fund (TRF)
Grant for Researcher (project number RTA 5780002), Energy Policy
and Planning Office (Ministry of Energy, Thailand-Project number
AE-PSU-59-03) and the Research and Development Institute at
Thaksin University for financial support. Finally, we would like to
thank Mr. Christopher Joseph Forti (English adviser and English
proof-reader, Thaksin University) for useful comments and sugges-
tions on the language and structure of our manuscript.
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