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 a high amount of organic matter, oil and grease, total solids and
suspended solids. Therefore, a suitable treatment should be uti-
Palm oil mills are abundant in Southern Thailand. Currently, lized to keep the surrounding environment safe from damage. In
more than 72 palm oil industries are in operation in Thailand. Most addition, looking for an alternative method for utilizing and treat-
of them are oil palm crushing mills (>60 mills) and the others are ing POME at the same time is still the main challenge. POME from
palm refineries mills. In 2010, production of crude palm oil (CPO) oil palm crushing mills (Thailand) contained COD and oil in the
in Thailand reached 1.29 million tons [1]. In the process of palm range of 45000 and 6000 mg/L, respectively. After oil recovery,
oil milling, effluent is mainly generated through the sterilization more than 70% of COD was significantly decreased and 78% of oil
and clarification process. Due to an increase in palm oil produc- was easily recovered from POME using n-hexane [3]. Therefore,
tions in the last few years, palm oil waste have increased rapidly. the recovery of oil from POME shows a great potential to reduce
According to Borja and Bank [2], more than 3.86 million tons of high COD from POME and an adequate amount of oil from POME
palm oil mill effluent (POME) is obtained annually. POME contains may be utilized as substrate for valuable products. Due to the
reduction of fossil fuel, many countries encourage and support pro-
⇑ Corresponding author. jects which produce energy from renewable resources. Therefore,
E-mail address: skanokphorn@yahoo.com (K. Sangkharak). 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 83

diesel production due to its high content of oil and grease which ratio at 1:6 (w/w). The process was maintained at 100 °C for 1 h,
could be converted into biodiesel via chemical and bio-catalytic afterward oil was collected and recovered by evaporation [14].
transesterification. Afterward, the residual oil was utilized as a substrate for biodiesel
Currently, sodium hydroxide is mostly utilized as a chemical production.
catalyst for biodiesel production. However, high FFA and acid value
in oil from POME are considered drawbacks of chemical biodiesel 2.3. Lipase extraction from oil palm fruit
[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- 2.3.1. Plant collection
tion. Thus, it reduces the yield of biodiesel and complicates the Ripen palm fruit (E. guineensis) were collected randomly accord-
separation process [6,7]. Hayyan et al. [5] reported the production ing to ARDA (Thailand) standards and left for 120 h after harvesting
of biodiesel from sludge palm oil (SPO). SPO, a byproduct from under room temperature (28 ± 2 °C). Afterward, 1 g of lipase was
palm oil mill using distillation method, also contained high FFA. extracted from 50 g of ripen palm fruit using 0.1 M Tris-HCl buffer
Therefore, both acid- and alkali-catalyst were suggested for biodie- (pH 8.0) as described by Kimtun et al. [12]. The supernatant was
sel production from SPO. Two steps of biodiesel production began freeze-dried and kept at 4 °C until used. The enzyme after buffer
with acid catalyst to reduced FFA content in SPO. Afterwards, bio- extraction was called ‘‘crude lipase”.
diesel was produced via the transesterification process. High yield
of biodiesel (76.62%) with 96% ester content was obtained from 2.3.2. Partial purification of lipase from oil palm fruit
SPO. But, it consumed more energy and effort for the separation Crude lipase from oil palm fruit was purified by using the aque-
process. Therefore, the utilization of enzyme instant of chemical ous two-phase systems (ATPS) method, 20% of polyethylene glycol
transesterification was considered in this study. Lipase is (PEG)-1000 and 15% of NaH2PO4. Lyophilized lipase (1 g) was
biodegradable, nontoxic, helps to ease recovery of product and firstly mixed with 5 g of distilled water. Thereafter, the sample
glycerol and requires moderate alcohol and mild reaction condi- was loaded into the ATPS system. A bottom layer, an enzyme con-
tions [8–10]. It can also catalyze both esterification and transester- taining layer, was collected and characterized [12]. The super-
ification [9]. Thus, it is suitable for high FFA feedstock like POME. natant was dialyzed with 12000 Da membrane and left overnight
However, lipase-catalyzed biodiesel production is still less attrac- against distilled water. The protein and protease activity after dial-
tive for commercialization because commercial lipase is very ysis were determined. Afterwards, the supernatant was freeze-
expensive [11–13]. From our knowledge, crude lipase from oil dried and kept at 4 °C until used. The enzyme after ATPS was called
palm fruit after 120 h of harvesting contained high lipase activity ‘‘partial purified lipase”.
(1.38 Unit/mg protein) using Tris-HCl (pH 8) extraction. After-
wards, lipase activity increased significantly to 4.76 Unit/mg pro- 2.3.3. Determination of protein and lipase activity
tein after the separation process using PEG and NaH2PO4. Palm The determination of protein was followed by the Lowry
lipase was already evaluated for its feasibility for biodiesel produc- method. The activity of lipase was determined by adding 200 lL
tion using cooked palm oil [12]. The results indicated that biodiesel samples in 0.1 mM Tris-HCl buffer (pH 8.0) 2.45 mL containing
from palm lipase were not significantly different from commercial 0.15 M NaCl and 0.5% Triton X-100. The determination was oper-
biodiesel and it also passed Thailand’s fuel standards. The objective ated under 40 °C for 5 min, then 50 mM p-nitrophenylpalmitate
of this research was to investigate the potential of residual oil from 200 lL was added. p-nitrophenol was used as a standard and the
POME and crude palm lipase as a novel substrate and catalyst for sample was analyzed at 410 nm [12].
biodiesel production. The optimal conditions for biodiesel produc-
tion including molar ratio, enzyme ratio, incubation temperature, 2.4. Enzymatic biodiesel production from POME and optimization
mixing speed and reaction time were also determined. Biodiesel study
from crude palm lipase was characterized and compared with
the production from NaOH, partial purified lipase and commercial All experiments were determined in a 250 mL screw-capped
lipase. In addition, the properties of biodiesel from crude lipase flask under batch condition. Crude lipase from palm oil fruit was
were also compared with Thai biodiesel and ASTM standards. 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
2. Materials and methods
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
2.1. Raw materials and chemicals
tested. The process conditions of these experiments including incu-
bation temperature (35–70 °C), mixing speed (100–400 rpm) and
POME was collected from the Krabi Oil Palm Farmers Coopera-
reaction time (6–72 h) were also varied. At the end of the reaction,
tives Federation Limited (Krabi, Thailand) and stored at 4 °C to
centrifugation was used to remove glycerol and lipase before sep-
avoid any decomposition, oxidation and changes to the FFA con-
arating the product in a separating funnel. The sample was left for
tent. All the chemicals such as organic solvents and reagents of lab-
1–2 h to separate in a separating funnel. Biodiesel (upper layer)
oratory and analytical grades were employed in this study. Lipase
was taken, washed twice with 20 mL of deionized water at 50 °C
(EC 3.1.1.3) from Candida rugosa was obtained from Sigma-Aldrich.
and dried in an oven (105 °C, 24 h) to get rid of the remaining
The Candida sp. lipase was prepared in 0.1 M Tris-HCl buffer fol-
water and alcohol. Fatty acid methyl ester (FAME) content was
lowed the instruction from Kimtun et al. [12].
evaluated by GC/MS [16]. The yield of biodiesel and the conversion
of FFA into biodiesel were calculated by weight and the final FFA
2.2. Oil extraction from POME content in the product. Biodiesel yield from crude lipase was also
compared with NaOH, partial purified and commercial lipase.
POME was firstly centrifuged at 5000 rpm for 60 min before use.
Only sediment was collected and utilized. Furthermore, oil was 2.5. Analytical analysis
extracted from the sediment by the soxhlet extraction technique
using an organic solvent containing hexane:methanol:acetone Table 1 shows the full list of parameters for the characterization
(6:2:2). The sample was mixed with the solvent under an optimal 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 Methodology Content

pH pH meter 4.82
Temperature °C Thermometer 57 ± 10
Chemical oxygen demand (COD) mg/L HACH method 8000 48900 ± 27.00
Biochemical oxygen demand (BOD) mg/L 3 day BOD test at 30 °C 19560 ± 1.01
Total nitrogen (TN) mg/L HACH method 10072 460 ± 9.90
Ammonium-nitrogen (NH3-N) mg/L HACH Nessler’s method 8038 51 ± 24.00
Oil and grease APHA 5220 B 5569.82 ± 10.59
Total solid (TS) mg/L APHA 2540 B 22666.01 ± 1.41
Suspended solids (SS) mg/L HACH method 8006 (Photometric) 20200.78 ± 2.345

parameters in the POME characteristic study are also indicated. alkaline-catalyzed transesterification. However, it can be con-
The density of the samples was determined at 20 °C using an verted to biodiesel via enzymatic transesterification reaction. Thus
Anton-Paar DMA 4500 density meter. A Brookfield RV-I instrument lipase is highly suitable to catalyze the production of biodiesel
with an S03 spindle was utilized for viscosity analysis. The speed from residual oil from POME because it is able to catalyze both
was set at 100 rpm and conducted at room temperature [15]. esterification and transesterification reactions [18]. Low nitrogen
The chemical properties of residual oil were determined accord- content in POME (460 mg/L) was correlated with the presence of
ing to the AOAC Official Method: free fatty acid content (%FFA) 51 ppm of ammonium-nitrogen. The BOD of POME was estimated
(AOAC 969.33), saponification value (sv) (AOAC 920.160), iodine to be around 19560 mg/L while the COD concentration was
value (AOAC 930.15), moisture content (air-oven method) and approximately 48900 mg/L. pH of POME was acidic (4.82).
unsaponifiable matter (AOAC 933.08). The analysis of FFA content High oil and grease content (5569.82 mg/L) was observed in
was done to the product obtained from the optimum condition. POME. It indicated that POME was a great oil source. Content of
The biodiesel was analyzed using a GC/Frame Ionization Detector oil in POME showed the possibility for oil extraction. After charac-
(FID) with the split-splitless mode of injection. The capillary col- terization, oil was extracted from POME by the soxhlet method
umn contained a length of 15 m, a film thickness of 1.5 lm and using a mixture of hexane, methanol and acetone. The highest oil
an internal diameter of 0.53 mm. Hydrogen was used as a carrier and grease content that could be recovered from POME was
gas with a flow rate of 450 mL/min. The run time was 15 min. 4455 mg/L which means that 80% of the oil and grease could be
The temperatures of the injector and detector were 350 and recovered from POME. Therefore, residual oil from POME showed
360 °C. A 50 mg sample of biodiesel was diluted with 5 mL of n- a high potential to be utilize as substrate for biodiesel production.
hexane. 2 mL of the diluted biodiesel was prepared in a vial prior Normally, the potential for the production of biodiesel seems to be
to injection into GC/FID. The injection volume was 0.5 lL. The limited by the availability and costs of substrates. Therefore, POME,
FFA content of POME was also analyzed using the same method a high volume and costless waste, seems to be a great substitute
as stated above to compare with the results of the biodiesel analy- for normal biodiesel substrates such as edible oil. POME is pro-
sis [16]. Each sample was determined in triplicate. GC/MS was also duced from a mixture of sterilizer condensate, separator sludge
utilized for determined the esters content in biodiesel. It was oper- and hydrocyclone wastewater. It is abundant and available. About
ated with a split-splitless mode of injection. DB-wax with a length, 3.86 tons of this waste is produced for every ton of oil extracted in
a film thickness and an internal diameter of 30 m, 0.25 lm and an oil mill [2,20].
0.25 mm was used, respectively. Helium, a carrier gas, was set with Residual oil from POME was further characterized for moisture
30 mL/min flow rate. The experiment was measured at 50 °C for content, saponification value and average molecular weight. The
35 min with the injector temperature at 250 °C and the detector moisture content of residual oil was 2.20 ± 0.07%. Water was
temperature at 280 °C. A 15 mg sample of biodiesel was weighted removed before enzymatic biodiesel production because it pro-
and diluted with n-hexane in GC/vial before injection. The presence motes the reversible reaction of esterification. In addition, a high
of FAEE peak in GC/MS chromatogram was determined [16,17]. amount of water was already present in the lipase solution [19].
The yield of biodiesel is used to determine the optimum value of Therefore, residual oil was firstly pretreated by heat before use.
influential parameters. It is defined as the weight percentage of the Primandari [15] reported the similar composition of fatty acid in
final product measured using an analytical balance (Mettler POME oil. The composition of fatty acids is very important in iden-
Toledo) relative to the weight of POME at the beginning of the tifying carbon chains and its properties. In addition, it can deter-
experiment as shown by the expression: 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%)
Yield ð% w=w POMEÞ ¼ ðweight of biodiesel=weight of POMEÞ  100
and linoleic acid (10.1%). The saponification value of
201.96 ± 0.21 mg KOH/g oil gave the average molecular weight of
For the conversion of FFA to biodiesel, initial and final FFA was
833 g/mol.
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 3.2. Biodiesel yields

Biodiesel was produced via an enzyme-catalyst. Crude, partial

3. Results and discussion purified and commercial lipase was utilized as the catalyst. Each
enzyme was dissolved in buffer and the concentration was fixed
3.1. POME characterization and oil extraction 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-
The characteristics of POME are indicated in Table 1. Among all fer (pH 8.0). The total aqueous volume of the reaction mixture was
properties, only 4 factors including FFA, saponification value, acid 18.18 mL, comprise 10.59 mL (10 g) oil, 5 mL enzyme solution and
value and moisture content were affected in biodiesel production 2.59 mL methanol (methanol-to-oil ratio at 6:1). Residual oil from
[18]. High FFA in residual oil (27.67%) is unfavorable for the POME was used as the sole substrate for the transesterification
 S. Suwanno et al. / Fuel 195 (2017) 82–87 85

Table 2 3.3. Optimum process for biodiesel production

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
Crude lipase from oil palm fruit was selected and used as the
reaction mixture was 18.18 mL. catalyst throughout this study. The OFAT method was used to eval-
uate the optimization conditions. The methanol-to-oil ratio was
Catalyst Enzyme activity (U/g) Biodiesel yield (% ± S.D.)
firstly assessed. The biodiesel production was increased when the
NaOH – 80.30 ± 2.51 ratio was increased. The maximum yield of biodiesel
Commercial lipase 7753 91.75 ± 1.58
Oil palm lipase
(90.01 ± 1.00%) was achieved at a 6:1 methanol-to-oil ratio
Crude lipase 8138 90.37 ± 1.43 (Fig. 1a). The enzyme concentration was the variable that had
Partial purified lipase 3533 91.04 ± 1.98 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
process. Biodiesel yields from all lipase tested in this study were enzyme loading at 36 U/10 g oil yielded the highest yield of biodie-
not significantly different (Table 2). The highest biodiesel yield sel at 90.62% w/w residual oil (Fig. 1b). Biodiesel yield was slightly
(91.75 ± 2.51%) was obtained from commercial lipase-catalyst fol- constant with an increase of enzyme higher than 36 U/10 g oil.
lowed by partial purified lipase (91.04 ± 1.98%) and crude lipase Incubation temperature is also important for enzymatic bioiesel
(90.37 ± 1.43%). The lowest biodiesel yield (89.30 ± 2.51%) was production. The highest yield (91.02 ± 0.90%) was obtained at 35 °C
achieved from NaOH-catalyst. The alkali transesterification shows of incubation. Lower yield was obtained when incubation temper-
a very fast kinetic of reaction but also a collateral saponification ature was higher than 35 °C (Fig. 1c). The optimum mixing speed of
reaction that reduces the biodiesel production efficiency [22]. 200 rpm was sufficient for the reaction completion. The highest
Therefore, the enzyme catalyst gave higher biodiesel yield than yield of biodiesel (92.07 ± 1.04%) was achieved under this condi-
the alkali-catalyst. tion (Fig. 1d). The yield of biodiesel increased with an increase in
This study indicated that POME is suitable for biodiesel produc- reaction time. The yield of biodiesel during reaction time between
tion. Using POME as a substrate has many advantages such as the 36 and 72 h was not significantly different. Therefore, 36 h of incu-
added value ensuing from their re-utilization and their simple pro- bation time was selected in order to save energy and to decrease
cess, which reduces waste and creates green energies. From the the cost of the process. The yield of biodiesel at 92.07 ± 1.04%
economic point of view in term of the cost of biodiesel from resid- was obtained after 36 h of reaction time (Fig. 1e). The analysis
ual oil, those of commercial biodiesel and diesel were also com- shows that by choosing of incubation time at 36 h instead of
pared. Since almost 90% of commercial biodiesel in Thailand was 72 h, the energy will on average save up to 500 kW h annually. This
produced from crude palm oil, (CPO), CPO biodiesel is chosen as corresponds to a 50% reduction in energy consumption, leading to
the subject of cost analysis. In December 2016, CPO biodiesel optimized financial gains, corresponds to a 30% saving (data not
obtained by the chemical approach was $0.982/kg while diesel shown). A suitable reaction time is needed to complete the biodie-
was only $0.672/kg (NESTE Oil Corporation, December 2016). From sel reaction. Therefore, sufficient contact time must be provided
the operation in this study that was analyzed including the cost of [28]. Crude lipase required the longest reaction time. The results
chemical, electricity and labor, the biodiesel cost was reduced to suggested that crude enzyme are capable of converting triacylglyc-
$0.266/kg. The cost of POME biodiesel was 3.69 and 2.52-times erols, diacylglycerols, monoacylglycerols and free fatty acids to
lower than CPO biodiesel and diesel price, respectively. In addition, FAME in addition to fat hydrolysis, but could not completely the
utilization of palm lipase showed the great attractive. Various process within 1–24 h.
sources of lipases have been reported including animal, microbial The production of biodiesel from the enzymatic process was
and vegetable, with differences in their catalytic properties also compared with the commercial enzyme and chemical process.
[23,24]. Bacterial and fungal lipases have been employed to pro- The optimal conditions for biodiesel production from POME using
duce biodiesel. High yield (90–99%) of FFAE was obtained under crude lipase containing a 6:1 level of methanol-to-oil using
24–90 h of incubation times, making the process more economi- 36 U/10 g oil of lipase under incubation temperature, a mixing
cally feasible [25,26]. To date, microbial lipases are the most stud- speed and reaction time of 35 °C, 200 rpm and 36 h, respectively.
ied. Nevertheless, utilizing microbial enzymes on a large scale is 92.07 ± 1.04% of biodiesel yield was achieved under these optimal
still limited. The major drawback of these enzymes is their high conditions. Similar yield (92.11 ± 1.54%) was also obtained from
production costs. Some enzymes are still economically produced commercial lipase under the same condition. However, alkali-
from plant and animal sources. This is possible because of suffi- catalysts required a higher level of methanol-to-oil (Table 3).
ciently high amounts of this enzyme in such sources and also as
a means to convert inexpensive, renewable material like agricul- 3.4. Biodiesel quality
tural and slaughter waste into value added products. Therefore,
the search for other sources of these enzymes is an important issue Biodiesel from residual oil and crude lipase as the substrate and
[27]. From our study, partial purified lipase from palm oil fruit was catalyst was characterized according to ASTM standards. Table 4
already used for biodiesel production using cooked oil as substrate shows the fuel properties and the stability of biodiesel from POME.
[12]. However, crude lipase has never been demonstrated before. The results show that most properties of biodiesel from crude
The data indicate that the crude enzyme treatment is less effective lipase are acceptable, according to Thai biodiesel and ASTM stan-
but competitive to the commercial enzymes. The usage of crude dards. Low free fatty acid (0.07%) content was observed in POME
enzyme in place of the commercial enzymes will be cost effective methyl ester catalyzed by crude lipase. However, FFA content of
and will reduce the cost of the process to the proportion of 60–70%. POME oil was <0.5% before transesterification.
Cost of crude lipase from palm fruit was 0.01 USD/g while commer- A high cloud point (10–13 °C) observed in enzymatic biodiesel
cial lipase (C. rugose, Sigma) was 406.30 USD/g (data not shown). may be used to qualify high quality biodiesel. The higher the
Crude enzymes may have been selected, rather than purified and cetane number the more easily the fuel will combust in a compres-
commercial enzymes, in many cases in the past for reasons based sion setting. A high cetane number in biodiesel may pose problems
on economy and convenience. Crude lipases from oil palm fruit if the engine is used during cooler seasons but it can be used suc-
are an alternative method that are simple to perform, and can be cessfully in tropical and desert regions. However, a cloud point is
done at a low investment cost. 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 87

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