Process Biochemistry 42 (2007) 951960

www.elsevier.com/locate/procbio

Proposed kinetic mechanism of the production of biodiesel from

palm oil using lipase
Sulaiman Al-Zuhair a,*, Fan Wei Ling b, Lim Song Jun b
a
Chemical and Petroleum Engineering Department, United Arab Emirates University, P.O. Box 17555, Al-Ain, United Arab Emirates
b
School of Chemical Engineering, Faculty of Engineering, The University of Nottingham Malaysia Campus, Semenyeh, Malaysia
Received 30 November 2006; received in revised form 14 February 2007; accepted 5 March 2007

Abstract
Experimental determination of the separate effects of palm oil and methanol concentrations on the rate of their enzymatic transesterification
was used to propose suitable mechanismic steps and to test the generated kinetic model. The reaction took place in n-hexane organic medium and
the lipase used was from Mucor miehei. At a constant methanol concentration of 300 mol m3, it was found that, initially as the palm oil
concentration increased, the initial reaction rate increased. However, the initial rate dropped sharply at substrate concentrations larger than
1250 mol m3. Similar behaviour was observed for methanol concentration effect, where at a constant substrate concentration of 1000 mol m3,
the initial rate of reaction dropped at methanol concentrations larger than 3000 mol m3. Ping Pong Bi Bi mechanism with inhibition by both
reactants was adopted as it best explains the experimental findings. A mathematical model was developed from a proposed kinetic mechanism and
was used to identify the regions where the effect of inhibition by both substrates arised. The proposed model equation is essential for predicting the
rate of methanolysis of palm oil in a batch or a continuous reactor and for determining the optimal conditions for biodiesel production.
# 2007 Elsevier Ltd. All rights reserved.

Keywords: Biodiesel; Lipase; Palm oil; Transterification; Ping-Pong Bi Bi kinetics

1. Introduction production of biodiesel has received less attention because it is

Biodiesel has been widely researched using many different
types of vegetable oils. However, interest of this study was
focused on palm oil, due to its higher yield of around 5000 kg
per hectare, compared to that of other vegetable oils, the highest
of which, for coconut oil, is around 2250 kg per hectare.
Therefore, it would be economically intuitive to consider palm
oil as the feedstock for biodiesel production [1].
Biodiesel can be produced by pyrolysis, which involves
heating in the absence of air or oxygen and the presence of
catalysts such as metallic salts. However, the equipments used
in pyrolysis are expensive for modest throughputs. In addition,
the removal of oxygen during the thermal processing removes
any environmental benefits of using an oxygenated fuel and
produces some low value materials. On the other hand,
biodiesel is typically produced by alkaline or acidic catalysed
transesterification of triglyceride (oil). The acid catalysed
* Corresponding author. Tel.: +971 37133636; fax: +971 37624262.
E-mail address: s.alzuhair@uaeu.ac.ae (S. Al-Zuhair).
1359-5113/$ see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2007.03.002
400 times slower than that of alkali catalyst and more
hazardous. Nevertheless, acid catalysed approach is less
sensitive to free fatty acids in the feedstock oil compared to
the alkali-catalysed one. The transesterification by either
chemical catalyst has several drawbacks, which include being
energy intensive, difficult to recover glycerol and requires
wastewater treatment. In addition, free fatty acids present in the
oil interfere with the reaction, especially for the alkaline
catalyst case, leading to undesirable side products. A less
energy intensive and environmental friendly procedure would
be using enzyme as a catalyst for oil transesterification.
Enzymatic transesterification can overcome the problems
facing conventional chemical methods without compromising
their advantages. Most importantly, glycerol can be easily
recovered without any complex process, free fatty acids
contained in the oils can be completely converted to methyl
esters and subsequent wastewater treatment is not required
[25].
A major part of the studies done on the enzymatic
production of biodiesel from vegetable oils was on the effect
of alcohol. In absence of organic solvents, Samukawa et al. [6]
952 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960
and Shimada et al. [7] have both reported that fatty acids methyl
esters (FAME) production increased when methanol concen-
tration increased up to an oil to methanol ratio of 3:1
equivalents and then decreased when methanol concentration
was further increased. This was also found by Noureddini et al.
[8] although the ratio was much lower (7.5:1, equivalents).
Lipases efficiently catalysed reactions when the substrates
dissolved each other. It was believed that when methanol
amount exceeded its solubility limits, lipase was deactivated by
the insoluble methanol that existed as drops in the oil, as
proteins in general are unstable in short-chain alcohols.
In addition to substrates concentrations, the temperature was
also reported to affect the rate of reaction considerably. It was
found that the reaction rate initially increased as the
temperature increased. That was mainly due to the increase
in rate constants with temperature and partly due to decrease in
mass transfer resistances. However, the reaction rate decreased
sharply at the onset of denaturation of the enzyme [9]. This
trend was observed in all the studies that investigated the effect
of temperature on the production of biodiesel by lipase. The
optimum operating temperature were of lipases from different
sources, including the one used in this work (Mucor miehei)
were determined to be close to 40 8C [2,3,8].
Although the application of lipase in the production of
biodiesel from vegetable oils has been thoroughly addressed in
the literature [2,4,6,1015], these studies were purely experi-
mental. On the other hand, all the kinetic studies found in the
literature considered the esterification of free fatty acids rather
than the transesterification of vegetable oil [1623]. The main
industrial interest, however, is on the production of biodiesel
from the triacylglyceride (oil), not the free fatty acids.
Therefore, it was desired to develop a reliable kinetic model
for this important reaction. An earlier attempt to model
vegetable oil transesterification was done by the author,
assuming that the reaction took place in two consecutive steps
[24,25]. In the first step, triglycerides were hydrolysed to
produce free fatty acids, and in the second step, the free fatty
acids produced in the first step were esterified to produce fatty
acids methyl esters. However, in the current work it was shown
that it was more accurate to assume that transesterification takes
place by direct alcoholysis of the triglycerides.
The main difference between esterification of free fatty acids
and transesterification of triglycerides (oils) is that in the first
OH bonds are broken, whereas in the second ester bonds are
the ones that are broken. In addition, the by-product of
esterification is water whereas it is glycerol in transesterifica-
tion. In this study, experimental determination of the separate
effects of palm oil and methanol concentrations on the rate of
enzymatic transesterification were used to understand the
reaction behaviour and propose suitable mechanismic steps.
On the other hand, most of the experimental data reported in
literature were found using reactors of small volumes in the
range of 2 ml [2] to 50 ml [7,12,13,1518,2629]. The mixing
was achieved by orbital shakers or magnetic stirrers. It was
obvious that these experimental results could not represent the
large scale industrial reactors, and therefore it was desired to
generate more reliable experimental results using a more
practical experimental set-up. A standard 1.5 l agitated
fermentor having a working volume of 1 l to determine the
separate effects of substrate and alcohol concentrations was
used, which constitute a promising industrial expansion. The
work also included proposing a suitable kinetic model and
numerical determination of the constants found in this model,
using the collected experimental data.
2. Materials and methods
2.1. Materials
Palm oil was obtained from Yee Lee Edible Oils Private Limited, Malaysia.
The substrate considered was the ester bond on the triglyceride. Analytical
grade n-hexane and methanol were obtained from Fisher Scientific, UK. Liquid
lipase (EC 3.1.1.3) from M. miehei (claimed activity 100 kLU ml1) was
obtained from Novo Nordisk, Denmark. The enzyme chosen was the one that
maintains its activity under the highest water content, in view to use it for waste
oil in the future [30,31]. The reuse of lipase is essential from the economic point
of view, which can be achieved by using the lipase in immobilised form.
However, soluble enzyme and not immobilised was purposely used in this study
to avoid the mass transfer limitations or clogging problems, which could
complicate the kinetic model. Using soluble lipase has the advantage of having
the interaction with the substrate be at molecular level, with no mass transfer
limitations or clogging problems. Therefore, it was justified to assume negli-
gible external mass transfer resistance in the subsequent analysis. The model
derived in this study could be used to determine a mass transfer resistance of
immobilised enzyme of the same strain, however, this is beyond the scope of
this paper.
2.2. Experimental set-up
The reactor used was a 1.5 l batch stirred fermentor with four baffle plates
provided to reduce vertex effects and enhance the mixing (Miniforce,
INFORCE, Switzerland). Two rushton impellers attached to the same shaft
were used for agitation. A schematic diagram of the experimental set-up is
shown in Fig. 1. A stirring of 150 rpm was kept constant throughout the
progress of all experiments. The temperature of the reaction system was kept at
40 8C using a controlled heating metal jacket. The temperature used was that
presented in the literature to be the optimum [2,3] and the agitation speed was
chosen to provide suitable mixing without affecting the activity of the enzyme.
The working volume at the beginning of each run was 1 l, consisting of
different methanol concentrations in the range of 3005000 mol m3 and
substrate (i.e., the ester bond on the triglyceride palm oil) concentrations in the
Fig. 1. Experimental set-up.
 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960 953

range of 1001250 mol m3 in n-hexane. The reactor was covered tightly
throughout the progress of the experiments to prevent evaporation. After the
desired conditions were reached in the bioreactor, enzyme solution with an
activity of 20 LU was added to initiate the reaction. The enzyme activity was
chosen low enough to ensure a slow reaction rate to obtain more accurate
determination of the initial rate of reaction. If the rate was fast, the substrate
concentration would drop considerably within short times and result in
inaccurate estimation of the initial rate of reaction. In addition, if the enzyme
concentration was high, the effect of substrate concentrations would not be
clear, especially at enzyme concentrations close to the critical saturation
concentration [9].
At regular intervals, 5 ml sample was withdrawn from the reactor by a
special sampling port, using a syringe, for subsequent analysis. Directly after
being withdrawn, the sample was placed in a screw-capped bottle and heated to
100 8C for at least 5 min to denature the enzyme and stop the reaction. The
sample was then analysed for fatty acids methyl esters content. In the range of
substrate and alcohol concentrations considered in this study, the reaction
Fig. 2. Total fatty acids methyl esters concentration vs. time (initial substrate
mixture was completely homogeneous and was well agitated in the reactor.
and methanol concentrations of 250 and 300 mol m3, respectively).

2.3. Sample analysis

was however, reported in works involving the esterification of
Gas chromatograph was used to determine the fatty acid methyl esters
free fatty acids [12,13,26,28].
composition of the withdrawn samples. One microlitre was extracted from the
sample bottle using 10 ml syringe and then injected into the gas chromatograph
(Perkin-Elmer, Clarus 500), equipped with a flame ionisation detector (FID) 3.2. Effect of methanol concentration
with Supelco fused silica capillary column (30 m  0.25 mm  0.1 m film
thickness). The carrier gas used was Helium at 11 psig. Throughout the Additional runs using methanol concentrations in the range
analysis, oven temperature was kept at 185 8C and injector and detector
temperatures were kept at 220 8C. The areas of each peak were determined
of 10005000 mol m3, at constant substrate concentration of
and compared with those obtained with two different dilutions of known 1000 mol m3 were done to confirm the inhibition effect of
amounts of standards, consisted of oleic, linoleic, stearic and palmitic acid alcohol, which has been thoroughly investigated in earlier
methyl esters in n-hexane. The fatty acids methyl esters considered were the studies using soybean oil [8], palm oil [4,6] and others. Fig. 4
ones found in the palm oil. shows the effect of methanol concentration on the initial
reaction rate. The solid line shown in the figure is a connection
3. Results and discussions between the experimental data, shown to highlight the trend.
Methanol inhibition was observed at concentrations higher than
3.1. Effect of palm oil ester bonds concentration 3000 mol m3. This confirmed the findings of Soumanou and
Bornscheuer [2] who predicted inhibition by methanol in
To determine the sole effect of substrate concentration, the organic media at alcohol:oil ratios above 3:1. It was interesting
experiment was run using substrate concentrations in the range to notice that, outside methanol inhibition region, increasing its
of 1001250 mol m3, at a constant methanol concentration of concentration 3.33 times from 300 to 1000 mol m3 resulted in
300 mol m3. The methanol concentration was chosen to keep increasing initial reaction rate 1.27 times from 1.1  104 to
its ratio to substrate below 1:3 to ensure operating outside 1.4  104 mol m3 min1. In contrast, outside substrate
alcohol inhibition region [6,8]. The time course for the
production of FAMEs using an initial substrate and methanol
concentrations of 250 and 300 mol m3, respectively, is shown
in Fig. 2 which was similar to other concentrations. The initial
rate of reaction at each initial substrate concentration was
determined from the slope at time zero of the straight line that
best fit product concentration versus time data. The best fit line
was found by the least square method using Excel. The
relationship between initial substrate concentration and the
initial reaction rate is shown in Fig. 3. The solid line shown in
the figure is a connection between the experimental data, shown
to highlight the trend. Fig. 3 shows that the initial rate of
reaction increased with initial palm oil concentration up to
1000 mol m3 and then abruptly dropped at 1250 mol m3,
which indicated the presence of substrate inhibition. The onset
of this inhibition was obeserved at substrate to alcohol ratio of
around 4:1. Substrate inhibition was not reported in previous
works done on the transesterification of vegetable oils, as more Fig. 3. Initial rate of reaction vs. initial palm oil ester bond concentration at
focus was always on the alcohol inhibition. Substrate inhibition constant methanol concentration of 300 mol m3.
954 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960
Fig. 4. Initial rate of reaction vs. initial methanol concentration at constant
palm oil ester bond concentration of 1000 mol m3.
inhibition region, increasing its concentration three times
from 250 to 750 mol m3 resulted in a higher increase in the
initial reaction rate of 1.67 times from 0.6  104 to
1.0  104 mol m3 min1. It was therefore concluded that
the transesterification reaction was more sensitive to changes in
substrate concentration in comparison to methanol concentra-
tion.
3.3. Fatty acids methyl esters profile
In the previous analysis and the subsequent kinetic study, all
the FAMEs produced were considered as a one product. In
order to do so, it was essential first to prove that the rate of
production of each individual FAME was the same. Therefore,
the rate of production of the main saturated and unsaturated
FAMEs, namely, palmitic acid methyl ester and oleic acid
methyl ester, was compared. Fig. 5 shows the results for
substrate concentrations in the range of 1001250 mol m3, at
Fig. 5. Initial rate of FAME production vs. initial palm oil ester bond con-
centration at constant methanol concentration of 300 mol m3.
a constant methanol concentration of 300 mol m3. The figure
shows that the ratio of concentration of palm oil methyl ester to
oleic acid methyl ester remained almost constant at 1.5:1,
which was almost equal to the ratio of their respective fatty
acids found in the palm oil itself, which is 1.3:1 [32].
4. Kinetic model
The proposed mechanism of alcoholysis of oils was based on
the enzymatic hydrolysis mechanism [33]. Acidic or basic
functional groups found at specific locations in the active sites
of the enzyme catalyse the reaction by donating or accepting
protons during the course of the reaction. Conjugate acids of
amines, for example, are proton donors, whereas amines and
carboxylate ions are proton acceptors. By transferring protons
to and from these groups to the substrate, an enzyme carries out
acid and base catalysed reactions within the active sites. The
active sites of the lipase have been studied by chemical and X-
ray techniques [34]. Two functional groups that are part of the
active sites have been identified as being particularly important
to the catalytic process. One is hydroxyl group that acts as a
nucleophile, and the other is the nitrogen atom of an amine
group, which accepts a proton and then gives it back during the
reaction. The mechanism of esterase-catalysed alcoholysis
consists of following steps as shown in Fig. 6. (a) Nucleophilic
addition to form enzymesubstrate complex, where the
nucleophile is the oxygen in the OH group on the enzyme.
(b) Proton transferred from the conjugate acid of the amine to
the alkyl oxygen atom of the substrate, and a glycerol moiety is
formed. If a triacylglyceride was the initial substrate, then a
diacylglyceride would form, whereas if diacylglyceride was the
substrate, then monoacylglyceride would form and so on. (c)
The oxygen atom from a methanol molecule is added to the
carbon atom of the C O of the acyl enzyme intermediate to
form acylated enzymealcohol complex. Finally, (d) the
enzyme oxygen atom of the complex is eliminated and a
proton is transferred from the conjugate acid of the amine,
resulting in fatty acid methyl ester, i.e., biodiesel. These steps
represent a Ping-Pong Bi Bi mechanism, which agrees with
most of the earlier kinetic studies on lipase-catalysed
esterifications of long-chain fatty acids [1823]. However, in
all of these studies, the first product that comes out of the
reaction was assumed to be the FAME, then followed by
glycerol moiety (in these studies, water was the second product,
as free fatty acid was the substrate used). On the other hand, it
has been shown by the current proposed mechanism that the
first product should be the glycerol moiety, followed by the
FAME as a final product. The mechanismic steps explained
above, were represented by Eqs. (1)(6):
k1
E S $ E:S (1)
k1
E:S $ E:Ac:G (2)
k2
E:Ac:G $ E:Ac G (3)
k2
 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960 955

Fig. 6. The mechanism of enzymatic production of FAME from triacylglycerides.

956 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960
k3
E:Ac A $ E:Ac:A (4)
k3

E:Ac:A $ E:Bd (5)

k4
E:Bd $ E Bd (6)
k4

where, k1 and k1, k2 and k2 and k3 and k3 are the rate
constants for the reversible formation of enzymesubstrate
complex, E.S, acylated enzymeglycerol moiety complex,
E.Ac.G, acylated enzymealcohol complex, E.Ac.A, respec-
tively and k4 and k4 are the rate constants for the product
formation and enzyme regeneration.
To account for the inhibition by alcohol, competitive
inhibition was assumed when an alcohol molecule reacts with
Fig. 7. Comparison between the experimental values of the initial rate of lipase
the enzyme directly to produce a dead-end enzymealcohol methanolysis of palm oil and those predicted by Eq. (11) at constant alcohol
complex (E.A). And to account for the inhibition by the concentration of 300 mol m3 and different substrate concentrations.
substrate, competitive inhibition was also assumed when a
substrate molecule reacts with the acylated enzyme to produce
an equation to be used when the water [W], taken as one of the
another dead-end complex, namely, acylated enzymesubstrate
products, was assumed to inhibit the reaction. This modification
complex (E-Ac.S). The two competitive inhibition reactions
was applicable when free fatty acids were considered as the
mentioned above were presented by the following equations,
substrate. However, when the substrate was the triglyceride,
respectively:
water was replaced with monoglyceride, diglyceride or glycerol.
k5 And unlike water which is usually present in the reaction medium
E A $ E:A (7)
k5 at time zero, these products were not. Therefore, the product
inhibition was neglected, especially when considering the initial
k6
E:Ac S $ E:Ac:S (8) rate of reaction, as done in this study.
k6
Eq. (9) describes the rate of transesterification reaction of
where, k5 and k5 and k6 and k6 are the rate constants for the triglycerides using lipase, which was derived from the
reversible formation of dead-end complexes, enzymealcohol, mechanismic steps of the reaction (Eqs. (1)(8)). The equation
E.S, and acylated enzymesubstrate, respectively. is applicable for oil, alcohol and enzyme concentrations in the
With the above mechanism and assumptions, the reaction region of complete homogeneity and could be used to predict
rate presented in the following equation was derived: the optimal operating conditions. The kinetic model obtained
(Eq. (9)) is non-monotonic which have important implications
V max
y on optimal design. Under specific condition, the non-
1 K IA =A1 S=K S  K IS =S1 A=K A  monotonic kinetics might give rise to complex bifurcation
(9) behaviour when applied in a continuous processes; the simplest
where y is the initial reaction rate, Vmax is the maximum reaction of it is multiplicity of the steady states. This bifurcation occurs
rate, KS and KA are the dissociation constants for the substrate (S) when a small smooth change made to the parameter values
and the alcohol (A), respectively, and KIS and KIA are the
inhibition constants for the substrate and the alcohol, respec-
tively. Eq. (9) describes the initial reaction rate in the absence of
any product inhibition, which is similar to the one proposed by
Krishna and Karanth [12] for the esterification of short chain fatty
acids with alcohol. On the other hand, Janssen et al. [17] derived

Table 1
Comparison between the values of Vmax, KS, KA, KIS and KIA, from different
references
Reference
[12] [17] Current work
3 1
Vmax (mol m min ) 11.72 6.0 0.041
KS (mol m3) 3.03  10 6 44 430
KA (mol m3) 3.06  10 6 61 350
KIS (mol m3) 1050 N/A 4.45  104 Fig. 8. Comparison between the experimental values of the initial rate of lipase
KIA (mol m3) 6550 107 3.3  104 methanolysis of palm oil and those predicted by Eq. (11) at constant substrate
concentration of 1000 mol m3 and different alcohol concentrations.
 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960 957

(e.g., substrate concentrations) of a system causes a sudden
qualitative or topological change in the systems long-term
dynamical behaviour. The development of reaction kinetic
model, such as Eq. (9) would sturdily assist in predicting the
dynamic behaviour of the system and avoid any unexpected
responses.
5. Determination of kinetic parameters
In this study, it was proposed to consider the changes of both
methanol and substrate concentrations to demonstrate the
significance of their effects. The results shown in Figs. 3 and 4
were used to determine the numerical values of the parameters
found in Eq. (9). The kinetic parameters found for esterification
of free fatty acids using lipase from similar strain [12], were
first assumed. The values were then optimised using Excel
solver to find the minimum objective function (Eq. (10)) that
compares the measured rate of reaction with those predicted by
the proposed kinetic equation (Eq. (9)):
X
OF ypred  yexpt 2 (10)

Fig. 9. Effect of kinetic parameters on the initial rate of reaction predicted by Eq. (11) at different substrate concentrations.
958 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960

The estimated kinetic parameters for the transesterification of It was found that the dissociation constants for the substrate,
triglycerides compared to those for the esterification of free KS, and alcohol, KA, found in this study, were in the same
fatty acids (Table 1) [12,17]. The proposed model equation order of magnitude, which agreed with the previous results
(Eq. (9)) with the estimated constants is presented in the [12,17]. However, the inhibition constant for the substrate,
following equation: KIS, was found to be slightly higher than that for the alcohol,
KIA, which disagreed with that found earlier [12]. It is
0:041
y 4
 2:8  105 important to notice that the comparison was done with
1 3:3  10 =A1 S=430 values extracted from experiments done on free fatty acids,
4:45  105 =S1 A=350 namely butyric acid, rather than the triglyceride, since the
(11) kinetic studies on triglycerides transesterification is lacking.

Fig. 10. Effect of kinetic parameters on the initial rate of reaction predicted by Eq. (11) at different alcohol concentrations.
 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960
Nevertheless, these values [12,16] provide reasonable basis
for comparison.
A comparison between the experimental results and the
proposed kinetic model predictions was done to measure how
closely the experimental results were presented. The compar-
ison was done at different substrate and alcohol concentrations,
and the results are shown in Figs. 7 and 8, respectively. The
figures show that the kinetic model curve predicted fairly well
the inhibition by both reactants. It can be seen however, that the
model underestimated the inhibition effect of both substrates.
The deviation between the values of the initial rate of reaction
determined experimentally and those predicted by the model
were presented by the standard deviation value shown in
Eq. (11). It should be noted that the kinetics parameters could
be further improved, and the model curve would be closer to the
results if more experimental data were available. Nevertheless,
the model equation derived still predicted the initial rate of
reaction trend and the substrate and alcohol inhibitions.
A sensitivity analysis was done to measure how the model
responded to the values of the kinetic parameters found in
Eq. (9). Figs. 9 and 10 show these effects on the predicted initial
rate of reaction at different substrate and alcohol concentra-
tions, respectively. The figures show that as the value of Vmax
increased, the rate of reaction and the local maximum
increased, however, the concentration at which the local
maximum occurred was not affected. This behaviour was
similar at different substrate and alcohol concentrations. In
addition, the rate of reaction increased when KS and/or KA were
increased and/or KIS and/or KIA, were decreased. However,
these effects were diminished when the values of the
parameters were increased. The figures also show that at
different substrate concentrations, the effects of increasing KS
and decreasing KIA were similar, whereas the effects of
increasing KA and decreasing KIS were similar. As the value of
KS increased (or KIA decreased), the rate of reaction increased
and the concentration at which the local maximum occurred
also increased. On the other hand, as the value of KA increased
(or KIS decreased), the rate of reaction increased whereas the
concentration at which the local maximum occurred decreased.
Similar behaviours were observed for different alcohol
concentrations. However, in these cases, the effects of
increasing KA and decreasing KIS were similar, and the effects
of increasing KS and decreasing KIA were similar. As the value
of KA increased (or KIS decreased), the rate of reaction
increased and the concentration at which the local maximum
occurred also increased. And as the value of KA increased (or
KIS decreased), the rate of reaction increased whereas the
concentration at which the local maximum occurred decreased.
The kinetics proposed for the transesterification of oils was a
double non-monotonic, with regards to both oil and alcohol. A
3D figure of the reaction rate versus the concentration of both
reactants was drawn to show the non-monotonic surface
(Fig. 11). It can be seen that the effect of inhibition by one
reactant was most significant at low concentration of the other.
This is because at reduced concentration of one reactant, a low
concentration of the other would be enough to dominate the
reaction medium and cause the inhibition effect. It should be
959
Fig. 11. The combined effects of substrate and alcohol concentrations on the
initial rate of reaction.
noted though, that the irreversible inactivation of the enzyme at
very high concentrations of alcohol and low concentrations of
substrate was not considered in this model, and therefore, not
presented in the graph. The non-monotonic surface shown in
Fig. 11 illustrates that the local maximum point with respect to
substrate concentration increased as the alcohol concentration
increased. Similar behaviour was observed for the maximum
point with respect to alcohol concentration, which was found to
increase as substrate concentration increased.
6. Conclusions
The transesterification of palm oil with methanol catalysed
by lipase from M. miehei in n-hexane microaqueous system has
been sussessfully decribed by a Ping-Pong Bi Bi model with
competitive inhibition by both reactants. Experimental results
showed that the reaction was more sensitive to substrate
concentration but more inhibited by the alcohol. The values of
the parameters found in the proposed kinetic model equation
were determined by fitting the experimental results. It was
shown that the effect of inhibition by one reactant was most
significant at low concentration of the other. Further, the local
maximum point with respect to the concentration of one
reactant increased as the concentration of the other increased.
The proposed model equation can be used to determine the
optimal substrate and alcohol concentrations and to design a
batch or a continuous bioreactor.
Appendix A
Notation
[A] alcohol concentration (mol m3)
[Bd] fatty acid ester (i.e., biodiesel) concentration
(mol m3)
[E]0 total enzyme activity (LU m3)
[G] glycerol moiety concentration (mol m3)
KA binding constants for the alcohol (mol m3)
KIA inhibition constant for the alcohol (mol m3)
KIS inhibition constant for the substrate (mol m3)
KS dissociation constant for the substrate (mol m3)
960 S. Al-Zuhair et al. / Process Biochemistry 42 (2007) 951960
[S] substrate concentration (i.e., the ester bond on the
triglyceride) (mol m3)
Vmax maximum reaction rate (mol m3 min1)
Greek letter
y initial reaction rate (m3 mol1 min1)
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