Biochemical Engineering Journal 49 (2010) 207–212

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Biochemical Engineering Journal

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Two-step lipase catalysis for production of biodiesel

Md. Mahabubur Rahman Talukder ∗ , Jin Chuan Wu, Ng Mei Fen, Yeo Li Shi Melissa
Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore

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

Article history: Lipase-catalyzed methanolysis of vegetable oils has attracted considerable interests for the production
Received 3 July 2009 of biodiesel (BD). However, the activity of lipase such as Novozym 435 (immobilized Candida antarctica
Received in revised form 27 October 2009 lipase B) is negatively affected by methanol. To minimize this problem, two-step lipase catalysis was
Accepted 23 December 2009
investigated. Crude palm oil (CPO), which is relatively cheaper because of avoiding refining cost, was
used as the source of BD. CPO was first hydrolysed to fatty acids (FA), which was then esterified to BD.
Candida rugosa and Novozym 435 lipases were used as biocatalysts for the hydrolysis of CPO and the
Keywords:
esterification of FA, respectively. The complete conversion of CPO to FA was achieved under an optimal
Biodiesel
Lipase
condition of buffer to CPO ratio 1:1 (v/v), buffer pH 7.0, lipase 0.1 wt.% of CPO, isooctane to CPO ratio 1:1
Two-step catalysis (v/v), temperature 30 ◦ C, shaking speed 250 rpm and time 4 h. The methyl esterification of FA with 1.2-fold
Crude palm oil stoichiometric excess of methanol reached the equilibrium after 2 h at which BD yield was 98%. C. rugosa
Methanolysis and Novozym 435 lipases were repeatedly used for 10 and 50 cycles, respectively without significant loss
of their activities. The developed two-step process is very promising because of its feedstock flexibility:
it can be used for production of BD and FA from crude, refined and waste oils.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction and methanolysis of triglycerides [8,9]. However, acid-catalyzed

Alkali-catalyzed methanolysis is usually adopted in conven-
tional chemical process for the production of fatty acid methyl
esters, which are collectively called BD [1–3]. However, the con-
ventional alkali process produces salt containing glycerol, and also
suffers from complicated downstream processes. The alkali process
also requires feedstock (e.g. vegetable oils), which must comply
with rigorous specifications [4]. For example, it must be essen-
tially anhydrous and the free FA content must not exceed 0.5 wt.%.
The presence of these impurities generates soap, which reduces
BD yield. To conform to such demanding feedstock specifications
necessitates the use of highly refined but costly vegetable oils,
which accounts for 70–80% of the total production cost of BD. There-
fore, the exploration of low cost feedstock generally associated with
high content of free FA and water, is of interest in recent BD research
[5–8].
The aforementioned drawbacks of alkali process have led
researchers to seek catalytic and processing alternatives that could
ease the technical difficulties of utilizing low cost feedstocks such
as crude vegetable oil, used frying oil, trap grease, soapstock
and acid oil. Methodologies based on acid-catalyzed reactions
have the potential to convert the feedstock with free FA to BD
since acid catalyst can simultaneously catalyze esterification of FA
∗ Corresponding author. Tel.: +65 67963826; fax: +65 63166182.
E-mail address: talukder@ices.a-star.edu.sg (Md.M.R. Talukder).
methanolysis is relatively slow and the side reactions such as
methanol etherification [4] cause difficulties in products purifi-
cation. Lipase-catalyzed methanolysis has attracted considerable
attention for the production of BD [10–14] as it is considered to
be an effective mean of circumventing drawbacks involved in the
chemical processes. Unfortunately, lipase such as Novozym 435
is deactivated when more than one-third stoichiometric amount
of methanol present in the reaction [10–14]. This deactivation
was caused by the contact between Novozym 435 and insoluble
methanol, which exists as droplets in the oil. Different techniques
have been employed to overcome the methanol deactivation of
lipases. Organic solvents have been used to improve the solubility
of methanol [12,15,16]. A very low concentration of methanol dur-
ing the reaction was maintained by stepwise addition of methanol
[10–14]. Methanol has been replaced by methyl acetate [17]. Salts
saturated solutions [18] and silica-gel [19] based controlled release
system for methanol have also been applied. However, these tech-
niques have some inherent problems. The methanolysis in organic
solvent such as hexane is relatively slower. Although a noticeable
increase in reaction rate using t-butanol has been reported, a large
molar excess of methanol (methanol to oil ratio 6:1) is required
to obtain a higher BD yield. Maintaining methanol concentration
at a very low level by stepwise addition is not an appropriate
approach for the large-scale production of BD. When methanol is
replaced by methyl acetate, the byproduct triacetylglycerol causes
difficulties in product purification. Furthermore, methyl acetate
is more expensive than methanol. The presence of salt saturated

1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2009.12.015
208 Md.M.R. Talukder et al. / Biochemical Engineering Journal 49 (2010) 207–212
Table 1
Specifications of CPO.
Parameters Composition (wt.%)
Free fatty acids (as palmitic) 5.97
Moisture 0.25
Iodine value 52.4
Gum (as phospholipids) 0.05
Carotene 0.04
Vitamin E 0.06
solution or silica-gel causes difficulties in downstream separa-
tion.
In this study, the production of BD from CPO via Candida rugosa
lipase-catalyzed hydrolysis followed by Novozym 435-catalyzed
esterification was investigated. The effect of different parameters
on the hydrolysis of CPO and the esterification of FA were examined.
The results showed that the developed two-step process is more
efficient than the single-step Novozym 435-catalyzed methanoly-
sis, and produces a higher BD yield. Since methanol solubility in FA
is higher than that in oil, the methanol deactivation of Novozym
435 can be easily minimized in the two-step process.
2. Materials and methods
2.1. Materials
Novozym 435 (Candida antarctica lipase B immobilized on
acrylic resin), t-butanol (GC) and standard methyl esters (98–99%)
were from Sigma. C. rugosa lipase was from Meito Sangyo Co. Ltd.,
Osaka, Japan. CPO was purchased from Wawsan Tebrau SDN BHD,
Johor, Malaysia. The saponification value of CPO was 195 mg KOH/g.
The saponification value was determined by a method reported
previously [20]. The other specifications of the CPO provided by the
suppliers are shown in Table 1. HPLC grade methanol, isopropanol,
isooctane and hexane were from J.T. Baker, USA. All chemicals
unless mentioned otherwise were of analytical grade and used as-
received.
2.2. Hydrolysis of CPO
2 g CPO with or without isooctane was preheated in a glass bottle
(80 ml size) at a reaction temperature of 30–40 ◦ C and 250 rpm for
about 30 min by a shaker incubator. The reaction was initiated by
adding an appropriate amount of lipase solution. This lipase solu-
tion was prepared by dissolving C. rugosa lipase in 0.1 M sodium
phosphate buffer (pH 7.0) and directly used. The lipase concentra-
tion was varied according to 0.01–0.1 wt.% of CPO. The buffer to CPO
ratio was varied in the range of 0.25:1 to 2:1 (v/v). The isooctane to
CPO ratio was varied in the range of 0.05:1 to 5:1 (v/v).
2.3. Separation of FA after hydrolysis step
20 ml isooctane was added to the reaction mixture and mixed
at 30 ◦ C and 250 rpm for 15 min. The mixture was centrifuged at
4000 rpm and 25 ◦ C for 10 min. The upper layer containing FA in
isooctane was separated and used as feedstock for Novozym 435-
catalyzed esterification. To measure FA concentration, 5 ml of upper
layer was mixed with 50 ml ethanol–acetone (50/50, v/v) solu-
tion and titrated against 0.2N NaOH using phenolphthalein (1% in
95% ethanol) as an indicator. To determine the average molecular
weight of FA, isooctane was completely evaporated from FA solu-
tion (0.35–0.36 M) and gravimetric analysis was done. The average
molecular weight of FA was 270.8. FA yield is calculated as the
amount of FA produced (actual value in mole) divided by theoretical
value in mole.
2.4. Esterification of FA or methanolysis of CPO in the presence of
isooctane
10 ml FA solution (0.35–0.36 M) and an appropriate amount of
methanol according to methanol to FA molar ratio of 1:1 to 2:1
were mixed in a glass bottle (80 ml size) at a reaction temperature
of 30–60 ◦ C for about 15 min by a shaker incubator. The reaction
was initiated by adding 0.04 g Novozym 435. Methanolysis of CPO
was performed under the same conditions described above except
that molar concentration of CPO was 0.12 M, 3-fold lower than that
of FA as 1 mol of CPO gives 3 mol of FA or BD.
2.5. Esterification of FA or methanolysis of CPO in the absence of
isooctane
Isooctane was completely removed from stock solution of FA by
a rotary evaporator. 5 g FA was preheated in a glass bottle (80 ml
size) at 60 ◦ C and 250 rpm for about 30 min by a shaker incubator.
1.2-Fold stoichiometric excess of methanol was mixed with pre-
heated FA for about 15 min. The reaction was initiated by adding
0.2 g Novozym 435. Methanolysis of CPO were performed under the
same conditions described above except that FA was replaced by
CPO.
2.6. HPLC analysis of BD
After the specified time of methyl esterification or methanolysis
in isooctane, whole sample was centrifuged at 4000 rpm and 25 ◦ C
for 10 min, and the BD in upper layer was analyzed. For reactions
in the absence of isooctane, whole sample was first mixed with
40 ml isooctane at 60 ◦ C for 15 min, and centrifuged. The method
used for HPLC analysis of BD was previously reported [21,22]. HPLC
(Waters 2695, USA) was equipped with a UV detector (Waters 2487,
USA) and a prevail-C18 5u column (4.6 mm × 250 mm, Altech Inc.,
USA). The UV wavelength and the column temperature were set at
210 nm and 40 ◦ C, respectively. The mobile phase consisted of three
different components: hexane, isopropanol and methanol. Reser-
voir A contained methanol and reservoir B contained a mixture of
isopropanol and hexane (5:4, v/v). The gradient went from 100% A
to 50% A + 50% B linearly over 30 min. The flow rate of the mobile
phase was 1 ml/min and the sample injection volume was 10 ␮l.
BD was quantified according to the external calibration curves.
Methyl oleate, methyl palmitate, methyl stearate, methyl linoleate
and methyl myristate were used as standards for BD because oleic,
palmitic, stearic, linoleic and myristic acids are major fatty acids in
palm oil. BD yield was based on CPO and calculated as the amount
of BD produced (actual mass in gram) divided by theoretical mass
in gram.
3. Results and discussion
3.1. C. rugosa lipase-catalyzed hydrolysis of CPO
Among the lipases from various sources, C. rugosa lipase has
been reported as one of the most active and versatile enzyme
[23–25]. Hence, this lipase was chosen for converting CPO to FA
as an intermediate for BD production. The time course of the CPO
hydrolysis at different lipase concentrations (wt.% of CPO) is shown
in Fig. 1. The complete conversion of CPO to FA was achieved after
4, 10 and 20 h for lipase loading of 0.1, 0.05 and 0.02 wt.% of CPO,
respectively. The reaction at a low lipase loading (e.g. 0.01 wt.%
of CPO) progressed slowly and could not reach the equilibrium
because of a time limit of 20 h. Lipase is a surface active enzyme and
binds with substrate at oil–water interface [26,27]. Since interfacial
area depends on water to oil ratio (v/v) [28,29], the optimal water
(buffer) content was investigated at a lipase loading of 0.1 wt.% of
 Md.M.R. Talukder et al. / Biochemical Engineering Journal 49 (2010) 207–212 209

Fig. 1. Effect of Candida rugosa lipase loading on hydrolysis of crude palm oil (CPO).
Reaction conditions: 2 g CPO, 2 ml lipase solution in buffer (pH 7.0), buffer to CPO Fig. 3. Effect of isooctane on hydrolysis of crude palm oil (CPO) at different tem-
ratio 1:1 (v/v), temperature 40 ◦ C and shaking speed 250 rpm. peratures. Reaction conditions: 2 g CPO, 2 ml lipase solution (1 mg/ml) in buffer (pH
7.0), isooctane to CPO ratio 1:1 (v/v) and shaking speed 250 rpm.

CPO. Fig. 2 shows that the optimal water to CPO ratio was 1:1 (v/v).
3.2. Recycling of C. rugosa lipase
The low water content shifts the reaction equilibrium to the left giv-
ing a lower BD yield of 90 and 80% at a water to CPO ratio of 0.5:1
For investigating the recyclability of C. rugosa lipase, the hydrol-
and 0.25:1 (v/v), respectively. The excess water reduces interfacial
ysis of CPO was performed under the following conditions: 2 g CPO,
area and dilutes lipase solution. Hence, the rate of reaction at water
2 ml isooctane, 4 ml lipase solution in buffer (0.25 mg/ml), temper-
to oil ratio of 2:1 decreased.
ature 30 ◦ C, time 24 h and shaking speed 250 rpm. After each cycle
Lipase-catalyzed hydrolysis of vegetable oil has been improved
of reaction, the sample was mixed with 20 ml isooctane at 30 ◦ C and
by different water immiscible organic solvents and isooctane was
250 rpm for 15 min. The mixture was centrifuged at 4000 rpm and
among the best [30,31]. Fig. 3 shows that the presence of isooc-
25 ◦ C for 10 min. The organic phase was analyzed for FA concen-
tane significantly improved the hydrolysis of CPO particularly at
tration, and the aqueous phase containing lipase and glycerol was
a temperature of 30 ◦ C. FA yield with isooctane at 30 ◦ C reached
subsequently used without further treatment. Fig. 4 shows that C.
almost 100% after 4 h while without isooctane it was 80% after 7 h.
rugosa lipase is quite stable: FA yield over 10 cycles is 92%. It has
The addition of isooctane reduced the viscosity of CPO and conse-
been reported that lipase can be stabilized by maintaining the glyc-
quently increased mass transfer. Since an increase in temperature
erol concentration in the aqueous phase within the range of 10–40%
reduced the viscosity of CPO, the effect of isooctane at 40 ◦ C was
by weight throughout the hydrolysis of oils or fats [33]. Therefore,
not as significant as at 30 ◦ C. Since C. rugosa lipase is most active at
the byproduct glycerol might have a positive effect on the recycling
35–40 ◦ C [32], the hydrolysis of CPO at higher reaction temperature
of C. rugosa lipase.
was not investigated. The optimal isooctane to CPO ratio (v/v) was
investigated at 30 ◦ C, and found to be 0.5:1 to 1:1 (v/v).
3.3. Novozym 435-catalyzed methyl esterification of FA

The time course of the methyl esterification of FA in isooctane

at different methanol concentrations is shown in Fig. 5. The reac-

Fig. 4. Repeated use of C. rugosa lipase-catalyzed hydrolysis of crude palm oil (CPO).
Fig. 2. Effect of buffer to CPO ratio (v/v) on hydrolysis of crude palm oil (CPO). The Reaction conditions: 2 g CPO, 4 ml lipase solution (0.25 mg/ml) in buffer (pH 7.0),
ratio was varied by changing the volume of buffer only. Reaction conditions: 2 g CPO, isooctane to CPO ratio 1:1 (v/v), temperature 30 ◦ C, reaction time 24 h and shaking
buffer pH 7.0, lipase 0.1 wt.% of CPO, temperature 40 ◦ C and shaking speed 250 rpm. speed 250 rpm.
210 Md.M.R. Talukder et al. / Biochemical Engineering Journal 49 (2010) 207–212

Fig. 7. Repeated use of Novozym 435-catalyzed methyl esterification of fatty acids

(FA). Reaction conditions: 10 ml FA solution (0.35–0.36 M) in isooctane, methanol
Fig. 5. Effect of methanol concentration on Novozym 435-catalyzed esterification to FA molar ratio 1.2:1, 0.04 g Novozym 435, temperature 40 ◦ C, reaction time 3 h
of fatty acids (FA). Reaction conditions: 10 ml FA solution (0.35–0.36 M) in isooctane, and shaking speed 250 rpm.
0.04 g Novozym 435, temperature 40 ◦ C and shaking speed 250 rpm.

3.4. Recycling of Novozym 435

tion with a stoichiometric amount of methanol (i.e. methanol to FA
After each cycle (3 h), Novozym 435 was recovered by filtration
molar ratio 1:1) reached the equilibrium after 1.5–2 h at which BD
(Whatman 125), washed with 10 ml t-butanol, freeze dried for 24 h
yield was 85%. With an increase in methanol concentration from 1:1
and subsequently reused. Fig. 7 shows that when Novozym 435 was
to 1.2:1 molar ratio, the reaction equilibrium shifted to the right giv-
washed with t-butanol and freeze dried, it could be repeatedly used
ing a higher BD yield of 98% after 2 h. Further increase in methanol
over 50 cycles without losing its activity. However, BD yield without
concentration (e.g. methanol to FA ratio 2:1) led to the decrease in
washing and freeze drying dropped drastically and was only 55%
BD yield. The high methanol concentration deactivated Novozym
after 10 cycles. The byproduct water adsorbed onto Novozym 435
435, hence the reaction progressed slowly and could not reach the
and facilitated hydrolysis over synthesis resulted in the decrease in
equilibrium because of a time limit of 3 h.
BD yield. BD yield with washing alone dropped from 98 to 88% after
Fig. 6 shows the effect of different temperatures on Novozym
10 cycles. Similarly, BD yield with freeze drying alone dropped to
435-catalyzed methyl esterification. Although the initial reaction
82%. The results suggest that both washing and freeze drying are
rate was highest at 60 ◦ C, the maximum BD yield at 60 ◦ C (94%)
essential for maintaining a high BD yield over an extended period
was slightly lower than that at 40 ◦ C (98%). It has been found in
of time.
our previous study that the optimal temperature for Novozym
The effect of solvent types on regeneration of Novozym 435
435-catalyzed methanolysis of refined palm oil is about 60 ◦ C [18].
was also examined. Fig. 8 shows that among the solvents tested, t-
Therefore, the slight decrease in BD yield at 60 ◦ C might not be due
butanol was the most effective. t-Butanol has also been reported as
to the thermal deactivation of Novozym 435 but rather the loss of
an ideal solvent in regenerating Novozym 435-catalyzed methanol-
methanol by evaporation as the reaction temperature (60 ◦ C) was
ysis of triglycerides [21]. The very polar solvent like DMSO
close to the boiling point (65 ◦ C) of methanol.

Fig. 6. Effect of temperature on methyl esterification of fatty acids (FA). Reaction Fig. 8. Effect of solvent type on regeneration of Novozym 435-catalyzed methyl
conditions: 10 ml FA solution (0.35–0.36 M) in isooctane, methanol to FA molar ratio esterification of fatty acids (FA). Reaction conditions are the same as those men-
1.2:1, 0.04 g Novozym 435 and shaking speed 250 rpm. tioned in Fig. 7.
 Md.M.R. Talukder et al. / Biochemical Engineering Journal 49 (2010) 207–212
Fig. 9. Comparison of Novozym 435-catalyzed methyl esterification of FA and
methanolysis of CPO in presence and absence of isooctane. Reaction conditions in
presence of isooctane: 10 ml FA solution (0.35–0.36 M) or CPO (0.12 M) in isooctane,
1.2-fold stoichiometric excess of methanol, 0.04 g Novozym 435, temperature 40 ◦ C
and shaking speed 250 rpm. Reaction conditions in absence of isooctane: 5 g FA or
CPO, 0.2 g Novozym 435, 1.2-fold stoichiometric excess of methanol, temperature
60 ◦ C and shaking speed 250 rpm.
deactivates Novozym 435 [34], hence BD yield dropped signif-
icantly. Non-polar solvents such as isooctane and hexane were
better than DMSO but less effective than t-butanol. This result
suggests that the solvent with moderate polarity can be used in
regenerating Novozym 435.
3.5. Comparison of Novozym 435-catalyzed methyl esterification
of FA and methanolysis of CPO
Fig. 9 shows that methyl esterification of FA progresses much
faster than methanolysis of CPO. In the presence of isooctane, BD
yield reached 98% after 2 h in methyl esterification of FA while it was
only 52% after 24 h in methanolysis of CPO. Isooctane is, however,
not as good as t-butanol for methanolysis of vegetable oils [16,35].
We have recently reported that methanolysis of CPO in t-butanol
mediated system reached the equilibrium yield (92%) after 10–12 h
[36]. In the same report, it is also demonstrated that methyl esteri-
fication of oleic acid progresses faster than methanolysis of triolein.
In the absence of isooctane, Novozym 435 was almost completely
deactivated in methanolysis of CPO (BD yield 10% after 15 h) (Fig. 9).
On the contrary, BD yield in methyl esterification of FA reached 94%
after 3 h. The stepwise addition of methanol is generally employed
for methanolysis of vegetable oil in solvent free system [10–14].
The maximum BD yield from CPO in solvent free system reached
92% after 30 h when 1.2-fold stoichiometric access of methanol was
added by three successive additions [36]. The present study thus
reveals that two-step lipase catalysis gives a higher BD yield than
the single-step Novozym 435-catalyzed methanolysis in t-butanol
mediated system or solvent free system with three successive addi-
tions of methanol.
4. Conclusions
The production of BD from CPO via C. rugosa lipase-catalyzed
hydrolysis followed by Novozym 435-catalyzed esterification was
investigated. The presence of isooctane significantly improved the
hydrolysis of CPO and the methyl esterification of FA. Methanol
deactivation of Novozym 435, which is common in methanol-
ysis of oils or fats, was minimized in two-step lipase catalysis.
211
Novozym 435-catalyzed methyl esterification of FA progressed
much faster than methanolysis of CPO. The two-step process gave
higher BD yield (98%) than the single-step Novozym 435-catalyzed
methanolysis in t-butanol mediated system (BD yield 92%) and the
solvent-free system with three successive additions of methanol
(BD yield 92%). A key attribute of the developed two-step process
is its ability to utilize feedstock with any percentage of free FA and
water.
Acknowledgement
Financial support from the Agency for Science Technology and
Research (A*STAR) of Singapore is gratefully acknowledged.
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