Biochemical Engineering Journal 41 (2008) 87–94

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Biochemical Engineering Journal

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Kinetic studies on the Rhizomucor miehei lipase catalyzed esterification reaction

of oleic acid with 1-butanol in a biphasic system
G.N. Kraai a , J.G.M. Winkelman a , J.G. de Vries b , H.J. Heeres a,∗
a
Department of Chemical Engineering, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
b
Department of Organic and Molecular Inorganic Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

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

Article history: The kinetics of the esterification of oleic acid with 1-butanol catalyzed by free Rhizomucor miehei lipase in
Received 17 January 2008 a biphasic system was studied in a batch reactor. The reaction appeared to proceed via a Ping Pong bi–bi
Received in revised form 14 March 2008 mechanism with 1-butanol inhibition. The kinetic constants of the model were determined from exper-
Accepted 22 March 2008
iments at 30 ◦ C with initial concentrations of 0.1–0.95 mol L−1 −1
org oleic acid and 0.1–1 mol Lorg 1-butanol
in the organic phase and 0.05–0.2 g L−1 enzyme in the aqueous phase. The model was used to simulate
Keywords:
the batch concentration profiles of the product as well as the initial reaction rates. Agreement of the
Biphasic catalysis
model with both the batch concentration profiles (average error of 7.2%) and the initial reaction rate per
Esterification reaction
Rhizomucor miehei lipase
experiment (average error of 16.0%) was good.
Dynamic modeling © 2008 Elsevier B.V. All rights reserved.
Substrate inhibition
Kinetic parameters

1. Introduction in enzyme substrate and/or product inhibition, the solubilization

Lipases, also known as triacylglycerol ester hydrolases, are
enzymes that cleave ester bonds of triacylglycerols with the subse-
quent release of free fatty acids, diacylglycerols, monoacylglycerols
and glycerol. Lipases are also able to catalyze the reverse reactions
(esterification, interesterification and transesterification) provided
that the aqueous medium is replaced by an organic or a biphasic
aqueous/organic medium. Although ester synthesis can be chem-
ically catalyzed by acids or bases, the use of enzyme technology
offers environmental advantages and a reduction in energy con-
sumption. Furthermore, lipases show high selectivity including
stereo-selectivity and give products of high purity and improved
quality [1].
A range of fatty acid esters is now being produced commercially
using immobilized lipase in non-aqueous solvents. For example,
esters produced from long-chain fatty acids (12–20 carbon atoms)
as well as short-chain fatty acids (2–8 carbon atoms) are used
increasingly in the food, detergent, plasticizer, lubricant, cosmetic
and pharmaceutical industries [2].
The use of enzymes in biphasic systems instead of aqueous
media offers several important advantages such as a reduction
∗ Corresponding author. Tel.: +31 50 363 4174; fax: +31 50 363 4479.
E-mail address: h.j.heeres@rug.nl (H.J. Heeres).
of hydrophobic compounds, the possibility of shifting thermody-
namic equilibria towards the desired reaction [3] and the possibility
of re-using the separate phases to increase enzyme turnover num-
bers.
Several lipases of potential industrial interest have been stud-
ied intensively over the last 10–15 years, however, rarely in biphasic
systems. One of these well-studied lipases is the Rhizomucor miehei
lipase. This single-polypeptide chain protein is an ␣/␤ type pro-
tein, which is made up of 269 residues and has a molecular weight
of 29,472. Its active site is composed of the catalytic triad Ser144,
His257 and Asp203 [4]. The enzyme activity is greatly enhanced in
the presence of a lipid–water interface, a phenomenon known as
interfacial activation [5].
We have applied the biphasic R. miehei lipase esterification
of long chain fatty acids with alcohols in a biphasic system as a
model reaction to prove the concept of process intensification in
continuous centrifugal contactor-separators (CCS) [6]. To develop
reactor models for CCS devices, kinetic expressions are imper-
ative. Although R. miehei catalyzed esterifications of fatty acids
with alcohols have been studied by several groups [7–10], kinetic
data in biphasic systems are scarce. Al-Zuhair et al. [11] have
determined the kinetics of the esterification of butyric acid with
methanol catalyzed by a free R. miehei lipase in a micro aque-
ous system containing enzyme in a suspension in hexane and
a biphasic system (n-hexane and water). A kinetic model was

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.03.011
88 G.N. Kraai et al. / Biochemical Engineering Journal 41 (2008) 87–94

reaction mixture consisted of two phases: an organic phase (3.4 g

Nomenclature (12 mmol) oleic acid and 1.3 g (18 mmol) 1-butanol dissolved in
14 mL n-heptane) and an aqueous phase (40 mg enzyme in 18 mL
a specific contact area (m2 m−3 ) phosphate buffer solution (pH 5.6)). The reaction mixture was agi-
A interfacial area (m2 ) tated at room temperature at a stirrer speed of 1000 rpm. Samples
c concentration (mol L−1 ) were withdrawn from the organic phase periodically and were
ds Sauter mean diameter of the drops (m) directly centrifuged. The samples obtained were analyzed by gas
kL mass transfer coefficient (m s−1 ) chromatography (GC).
KI inhibition constant (mol L−1 ) The experimental setup for the kinetic modeling experiments
KM Michaels Menten constant (mol L−1 ) consisted of a glass reactor (6.2 cm i.d., 300 mL), equipped with
m partition coefficient four baffles and a six-bladed turbine impeller (diameter 3.5 cm). To
n number of moles (mol) operate the reactor isothermally at the desired temperature, it was
R volumetric reaction rate (mol L−1 s−1 ) equipped with a heating/cooling jacket connected to a temperature
R reaction rate per unit interfacial area (mol m−2 s−1 ) controlled water bath (accuracy of ±0.01 ◦ C).
R2 coefficient of correlation A typical reaction mixture consisted of an organic phase (10.3 g
t time (s) (36 mmol) oleic acid and 4.0 g (54 mmol) 1-butanol dissolved in
V volume (L) 43.5 mL (n-heptane)) and an aqueous phase consisting of 6 mg
enzyme in 60 mL phosphate buffer solution (pH 5.6). The reac-
Greek letter
tion mixture was agitated at 30 ◦ C for 60 min at a stirrer speed of
ε dispersed volume fraction
1500 rpm. Samples were withdrawn from the organic phase peri-
odically and were directly centrifuged. The samples were analyzed
Subscripts
by GC.
alc alcohol
aq aqueous phase
2.3. Analytical methods
BO n-butyl oleate
BuOH 1-butanol
The concentration of n-butyl oleate in the organic phase was
enz enzyme
analyzed using GC. The samples were prepared by diluting 70 ␮L
FA fatty acid (oleic acid)
of the organic product phase with 1 mL of n-heptane containing
org organic phase
hexadecane (2%, v/v) as internal standard. Analysis was performed
0 initial
with an Interscience Trace G.C. Ultra equipped with a flame ioniza-
tion detector and an HP-5 column (30 m length, 0.25 mm internal
diameter and 0.25 ␮m film thickness). A split of 1:200 was used. The
determined for the micro aqueous phase but not for the biphasic injector and detector temperature were set at 275 ◦ C. The oven was
system. set to 250 ◦ C. The gas flow rate was set to 1 mL min−1 . A Chrom-Card
Oliveira et al. [3] have studied the reaction of oleic acid with data system version 2.3␤ was used for data analysis.
ethanol using a free R. miehei lipase and immobilized R. miehei
lipase on accurel EP700 in a hexane and a phosphate buffer sys- 2.4. Determination of the interfacial area
tem. However, the rate expression for the free enzyme was based
on the total liquid volume, without taking into account the phase 0.6 mol L−1 −1
org oleic acid and 0.9 mol Lorg 1-butanol in n-heptane
ratio’s and the enzyme concentration. This limits the applicability and an aqueous phosphate buffer solution (pH 5.6) containing the
of this model considerably. Baron et al. [12] reported the esterifica- enzyme (0.2 g L−1 aq ) were mixed in a batch reactor and the drop size
tion of oleic acid with n-butanol using a crude lipolytic extract of distribution was measured online using a Lasentech S400A FBRM
Penicillum coryophilum in AOT/n-heptane reversed micelles. system equipped with a PI 14/206 probe. Drop size distributions
In this study, we report an exploratory screening study on the R. were determined by the number of counts for a certain drop size
miehei lipase esterification of oleic acid with various alcohols in a interval. In total 90 drop size intervals were measured, ranging from
biphasic system consisting of an organic phase with a hydrocarbon 1 to 1000 ␮m. The counts per drop size interval were normalized
diluent and a buffered aqueous phase. Furthermore, a kinetic and by multiplying the fraction of drops per interval by the drop size
modeling study on the biphasic esterification of oleic acid with 1- interval.
butanol catalyzed by free R. miehei lipase will be provided.
2.5. Kinetic modeling
2. Experimental
The initial reaction rate was obtained from the experimental
2.1. Chemicals concentration–time profiles by linear regression of the data over
the first 2 min. The experimental concentration profiles were mod-
n-Heptane (p.a.) and 1-butanol (99%) were obtained from Acros, eled using the Matlab® programming platform using the numerical
Belgium. The R. miehei lipase, produced in Asperigillus oryzae, was integration toolbox ode45. The kinetic parameter values were deter-
procured from Sigma, Germany. Potassium dihydrogen phosphate mined by minimization of the sum of squared errors between all
(p.a.) and disodium hydrogen phosphate dodecahydrate (p.a.) were experimental data and the simulated data from the kinetic model.
obtained from Merck, Germany. Hexadecane 99% and oleic acid Error minimization was performed using the Matlab® toolbox
(technical grade) were acquired from Aldrich, Germany. lsqnonlin, which is based on the interior-reflective Newton method.

2.2. Experimental set-up and procedures 2.6. Determination of the disperse phase

The exploratory experiments were conducted in a batch setup The nature of the dispersed phase (aqueous or organic) in
(100 mL) with magnetic stirring. For these experiments a typical the biphasic system was determined by performing conductivity
 G.N. Kraai et al. / Biochemical Engineering Journal 41 (2008) 87–94
measurements. The experimental setup for the conductivity exper-
iments was similar to the setup used for the kinetic experiments
(Section 2.2). The conductivity experiments were performed for a
biphasic system consisting of an organic phase (0.6 mol L−1
org oleic
acid and 0.9 mol L−1
org 1-butanol in n-heptane) and an aqueous phos-
phate buffer solution (pH 5.6). The system was agitated at 30 ◦ C at
a stirrer speed of 1500 rpm. The conductivity set-up consisted of a
direct current (10 V) power source and a 10 k resistance connected
in series with the reactor. The electric potential was measured over
the reactor using two electrodes submerged in the liquid at a fixed
distance.
3. Results and discussion
Initial screening studies on the esterification of oleic acid with
different alcohols and organic solvents were performed using a
biphasic system consisting of an organic phase with substrates
(oleic acid, alcohols) dissolved in an organic solvent and a buffered
aqueous phase with R. miehei enzyme. Several parameters (type of
alcohol, organic solvent, stirrer speed, enzyme concentration and
substrate concentration) were varied to determine their influence
on the reaction rate.
3.1. Effect of different alcoholic substrates on enzyme activity
Various alcohols, such as ethanol, 1-butanol, 1-pentanol and 2-
ethyl-1-hexanol were tested. The oleic acid conversion is a clear
function of the type of alcohol. After 1 h of reaction time, 100% oleic
acid conversion was observed for both 1-butanol and 1-pentanol,
as opposed to only 60% and 10% for ethanol and 2-ethyl-1-hexanol,
respectively. The initial reaction rate of oleic acid versus the carbon
number of the alcohol is provided in Fig. 1.
The graph shows a clear optimum and the highest initial reac-
tion rate was observed for 1-butanol. These trends are in accordance
with the results by Gandhi et al. [13] on the esterification of lauric
acid and oleic acid (with n-propanol, n-butanol, iso-amylalcohol,
n-hexanol, n-octanol, 2-ethyl-1-hexanol, n-decanol and lauryl alco-
hol) using an immobilized lipase in a solvent free system and the
data reported by Yadav et al. [14] on the transesterification of
vinyl acetate (with n-decanol, 2-ethyl-1-hexanol, benzyl alcohol,
cinamyl alcohol, 1-phenylethyl alcohol and 2-phenylethyl alcohol)
using an immobilized lipase in n-heptane. In both studies straight
chain alcohols give higher conversions than aromatic and branched
Fig. 1. Initial reaction rate vs. the carbon number of the alcohol: cFA,0 =
0.6 mol L−1 −1 −1
org , calc,0 = 0.9 mol Lorg , cenz,0 = 0.2 g Laq , Vorg = 20 mL, Vaq = 20 mL, stirrer
speed = 1000 rpm, T = 294 K.
89
alcohols and optima for the activity as a function of the carbon
length of the straight chain alcohols were observed. Due to the high
reactivity, further experiments were carried out using 1-butanol as
the alcoholic substrate.
3.2. Effect of organic solvent on enzyme activity
Enzyme activity and stability are known to be a function of the
properties of the organic solvent. A number of hydrocarbon sol-
vents were tested (n-heptane, hexane, toluene and decane) for the
biphasic esterification of oleic acid with butanol (Table 1).
Clearly, enzyme activity is a function of the organic solvent. The
oleic acid conversion after 15 min was highest in decane and n-
heptane and by far the lowest in toluene. To quantify the effect
of the organic solvent on the enzyme activity, the log P concept
was applied. The log P of a solvent is defined as the logarithm of
the partition coefficient of the solvent in a standard mixture of
1-octanol and water [15]. The initial reaction rate is a clear func-
tion of the log P value of the solvent (Table 1). The highest value
was found for decane, the solvent with the highest log P value. The
activity is considerably lower in toluene, the solvent in this study
with the lowest log P value. This trend is in line with literature data
on solvent effects on enzyme activity. Solvents with a log P > 4 are
known to have a positive effect on enzyme activity. It is assumed
that the essential water coat around the enzyme is not distorted
by these solvents, thereby leaving the enzyme in an active state.
Solvents with a log P < 2 are known to reduce enzyme activity [16].
Although the initial reaction rate in decane is slightly higher than
in n-heptane, further experiments were performed in n-heptane on
the basis of cost considerations.
3.3. Effect of agitating speed on enzyme activity
The conversions in biphasic liquid–liquid systems may be
affected by the rate of mass transfer of reactants from the bulk
phase to the locus of the reaction. To probe possible mass trans-
fer effects on the reaction rates, a number of experiments were
performed using different agitating speeds while keeping all other
variables at standard values. Two different enzyme concentrations
were applied, namely 0.05 and 0.2 g L−1 aq . The effect of the agitating
speed was studied in the range of 250–2000 rpm. The results are
provided in Fig. 2. It is clear that the initial reaction rate is a function
Fig. 2. The initial reaction rate vs. the agitating speed at different enzyme concentra-
tions: cFA,0 = 0.6 mol L−1 −1
org , cBuOH,0 = 0.9 mol Lorg , Vorg = 60 mL, Vaq = 60 mL, T = 304 K;
() 0.05 g L−1 −1
aq enzyme, (䊉) 0.2 g Laq enzyme; lines are for illustrative purposes only.
90 G.N. Kraai et al. / Biochemical Engineering Journal 41 (2008) 87–94

Table 1
Initial reaction rate and oleic acid conversion as a function of the organic solvent

Solvent Log P value Initial reaction rate (×10−4 mol L−1 −1

org s ) Oleic acid conversion after 15 min (%)

Decane 5.6 9.33 98

n-Heptane 4 8.17 95
Hexane 3.5 3.33 79
Toluene 2.5 2 49

rate increased linearly with the lipase concentration in the reaction

mixture (Fig. 3).
The observed linear relation between the initial reaction rate
and the enzyme concentration indicates that the reaction is first
order in enzyme in the concentration range between 0.05 and
0.2 g L−1
aq .

3.5. Effect of the initial substrate concentration on enzyme

activity

To probe the occurrence of substrate inhibition, the effects of the

Fig. 3. The initial reaction rate vs. the concentration of enzyme: cFA,0 = 0.1 mol L−1
org ,
cBuOH,0 = 0.2 mol L−1org , Vorg = 60 mL, Vaq = 60 mL, stirrer speed = 1500 rpm, T = 304 K;
line is linear fit through origin.
of the agitating speed when the speed is less than 1500 rpm. Above
this value, the initial reaction rate remains essentially constant. It
clearly implies that the overall reaction rate is affected by mass
transfer effects when the agitating speed is less than 1500 rpm.
Therefore, all experiments to determine the kinetics of the reaction
were performed at a stirring speed of 1500 rpm.
3.4. Effect of enzyme concentration
The effect of the enzyme concentration was studied for the
esterification of oleic acid with 1-butanol in a biphasic system
consisting of water and n-heptane. It was found that within the
enzyme concentration range (0.01–0.2 g L−1
aq ) the initial reaction
initial concentrations of both 1-butanol and oleic acid on the initial
reaction rate were investigated. At low 1-butanol concentrations,
the reaction rate is proportional with the initial concentration of
1-butanol (Fig. 4). At higher 1-butanol concentrations, the initial
reaction rate levels off and reaches a maximum. A further increase
in the 1-butanol concentration leads to a decrease in the initial reac-
tion rate. This dependency clearly implies that enzyme inhibition
by 1-butanol takes place.
For oleic acid, a different concentration dependency of the initial
reaction rate was observed (Fig. 5). Here, the initial reaction rate
does not show a clear optimum, indicating the absence of substrate
inhibition by oleic acid at the conditions investigated in this study.
4. Kinetic experiments and modeling
A total of 49 experiments were performed in a batch reactor at
30 ◦ C to be used as input for kinetic modeling activities. The initial
oleic acid concentration was varied from 0.1 to 0.95 mol L−1 org . The
initial 1-butanol concentration ranged from 0.1 to 1 mol L−1 org , and
was always in excess compared to the oleic acid concentration. The
enzyme concentration was varied between 0.05 and 0.2 g L−1 aq .
A kinetic model for the esterification of oleic acid with 1-butanol
in a biphasic system (n-heptane–water) and using the R. miehei

Fig. 4. The initial reaction rate vs. the initial concentration of 1-butanol at dif- Fig. 5. The initial reaction rate vs. the initial concentration of oleic acid at dif-
ferent enzyme concentrations: cFA,0 = 0.1 mol L−1 org , Vorg = 60 mL, Vaq = 60 mL, stirrer ferent enzyme concentrations: cBuOH,0 = 1 mol L−1 org , Vorg = 60 mL, Vaq = 60 mL, stirrer
speed = 1500 rpm, T = 304 K; () 0.2 g L−1 −1
aq enzyme, (䊉) 0.05 g Laq enzyme; lines are speed = 1500 rpm, T = 304 K; () 0.2 g L−1 −1
aq enzyme, (䊉) 0.05 g Laq enzyme; lines are
based on the kinetic model, vide infra. based on the kinetic model, vide infra.
 G.N. Kraai et al. / Biochemical Engineering Journal 41 (2008) 87–94 91

lipase was developed on the basis of the following considerations 4. In a batch reactor setup with no density- and volume changes,
and assumptions: the concentration of n-butyl oleate as a function of time may be
represented by the following ordinary differential equation:
1. Conductivity measurements revealed that, within the range of dcBO
experimental conditions applied in this study, the organic phase = RFA,org (4)
dt
is the dispersed phase and the aqueous phase is the continu-
5. Esterifications of fatty acids with alcohols using a R. miehei
ous phase. In line with literature data for R. miehei lipase, it is
lipase are often modeled using the so-called Ping Pong bi–bi
assumed that the enzyme has a preference for the liquid–liquid
mechanism, and in most cases include substrate inhibition
interface and that this is the locus of the reaction.
[3,20–31]. The Ping Pong bi–bi mechanism is schematically
2. To measure intrinsic kinetics, mass transfer of the substrates to
presented in Fig. 6.
the locus of the reaction should be much faster than chemical
The reaction rate for the Ping Pong bi–bi mechanism is given
reaction. Both mass transfer of substrates in the dispersed phase
by [32]
(internal mass transfer) as well as mass transfer of enzyme and
substrates in the continuous phase (external mass transfer) may kenz · cenz,aq
RFA,org = − (5)
affect the overall reaction rate. 1 + (KM,FA /cFA,org ) · (1 + (cBuOH,org /KI,BuOH ))
The experiments performed at different stirrer speeds (Fig. 2) +(KM,BuOH /cBuOH,org ) · (1 + (cFA,org /KI,FA ))
give information on the extent of external mass transfer lim-
itations. Evidently, external mass transfer effects do not affect where RFA,org is the rate of reaction for oleic acid (mol L−1 −1
org s ),
the overall reaction rate provided that the stirrer speed exceeds cFA,org and cBuOH,org are concentrations of oleic acid and 1-butanol
1500 rpm. Therefore, all experiments were performed using a (mol L−1 −1
org ), respectively, cenz the concentration of enzyme (g Laq ),
−1 −1
stirrer speed of 1500 rpm. kenz the enzymatic constant (mol Laq g−1
enz Lorg s ), KM,FA and
The possible effects of internal mass transfer limitations may KM,BuOH are Michaelis Menten constants (mol L−1 org ) for oleic
be estimated by using the approach of characteristic times for acid and 1-butanol, respectively, and KI,FA and KI,BuOH are the
reaction and mass transfer. The characteristic time for reaction inhibition constants for oleic acid and 1-butanol (mol L−1 org ),
may be defined as the time required for 66% conversion of oleic respectively.
acid and is about 120 s for the fastest reaction. The characteristic
time for internal mass transfer may be defined as (kL ·a)−1 . The 6. Considering that enzyme inhibition was only observed for 1-
diffusion coefficients of 1-butanol and oleic acid were calculated butanol and not for oleic acid (Figs. 4 and 5), Eq. (5) can be
using the Wilke–Chang correlation [17]. The diffusion coefficient simplified to
of 1-butanol (≈1 × 10−10 m2 s−1 ) is twice that of oleic acid and
therefore oleic acid is the limiting component. The value for the kenz · cenz,aq
RFA,org = − (6)
kL of oleic acid in a droplet may be obtained using the expres- 1 + (KM,FA /cFA,org ) · (1 + (cBuOH,org /KI,BuOH ))
sions provided by Wesselingh et al. [18] and was calculated to +(KM,BuOH /cBuOH,org )
be 5.4 × 10−4 m s−1 . In combination with an average drop size of
24 um of the dispersed phase (vide infra), the characteristic time A total of 49 batch experiments were performed and provided
for internal mass transfer may be calculated and is in the order a total of 995 data points. The experimental concentration profiles
of 0.01 s. This estimation indicates that internal mass transfer were modeled using Eqs. (3), (4) and (6) using the Matlab® pro-
effects may also be neglected in our experiments. gramming platform. These equations were solved using the numer-
3. It is assumed that the solubility of water and enzyme in the ical integration toolbox ode45 in the Matlab® software package.
organic phase is very low and can be neglected. It is also assumed The kinetic parameter values were determined by minimization of
that the solubility of n-heptane, oleic acid and the product n- the sum of squared errors between all experimental data and the
butyl oleate into the aqueous phase is negligible. These assump- simulated data from the kinetic model. Error minimization was per-
tions are confirmed by solubility experiments [19]. 1-Butanol is formed using the Matlab® toolbox lsqnonlin, which is based on the
soluble both in the organic phase and the aqueous phase and dis- interior-reflective Newton method. The optimized kinetic parame-
tributes between both phases. The partitioning coefficient m at ters and their 95% confidence limits are shown in Table 2.
equilibrium is defined as the ratio of the 1-butanol concentration Fig. 7 shows a number of typical experimental and modeled
in the organic and aqueous phases (Eq. (1)): n-butyl oleate batch concentration profiles. Agreement between
cBuOH,org experimental and modeled data is very good as is illustrated by a
m= (1) parity plot (Fig. 8). An average error of 7.2% was observed.
cBuOH,aq
According to Berg et al. [33] the Michaelis Menten constant for
The partition coefficient of 1-butanol was determined most enzymes are in the range of 10−1 to 10−7 M. The kinetic con-
experimentally and found to be 1.83 at 30 ◦ C [19]. stants found in this study are within this range.
The concentration of 1-butanol in the organic phase may be With the kinetic rate expression available, it is also possible to
calculated using a mass balance for butanol: model the initial oleic acid reaction rates using Eqs. (3), (4) and
(6) and to compare these with the experimentally observed val-
nBuOH = cBuOH,aq · Vaq + cBuOH,org · Vorg (2)
ues. Fig. 9 shows the parity plot of the modeled initial reaction rate
Rearrangement and taking into account the reaction
stoichiometry leads to Table 2
Modeled kinetic constants
nBuOH,0 − (cBO · Vorg )
cBuOH,org = (3) Parameter Value
(Vaq /m) + Vorg
−3 −1
kenz (×10 mol Laq g−1enz Lorg
−1
s ) 7.384 ± 0.001
where nBuOH,0 is the intake of 1-butanol (mol), cBO the concen- KM,FA (×10−2 mol L−1
org ) 6.776 ± 0.026
tration of n-butyl oleate (mol L−1 KM,BuOH (×10−1 mol L−1
org ) 2.536 ± 0.011
org ), Vaq and Vorg are the volumes
KI,BuOH (×10−1 mol L−1
org ) 1.277 ± 0.004
of the aqueous phase and the organic phase, respectively (L).
92 G.N. Kraai et al. / Biochemical Engineering Journal 41 (2008) 87–94

Fig. 6. Graphical representation of the master pattern of the Ping Pong bi–bi mechanism with competitive inhibition by both substrates.

Fig. 9. Parity plot of the initial reaction rate.

Fig. 7. Representative n-butyl oleate batch profiles. Symbols: experimental results.
Lines: calculated from Eqs. (3), (4) and (6) using the parameters of Table 2. Con-
ditions: stirrer speed = 1500 rpm; T = 304 K; Vorg = 60 mL; Vaq = 60 mL; cFA,0 = 0.797,
 ) is more
0.599, 0.443, 0.118, 0.121 mol L−1 −1
org ; cBuOH,0 = 1.002, 0.937, 1.000, 0.999, 0.233 mol Lorg ;
Therefore, a rate expression based on interfacial area (RFA
cenz,0 = 0.049, 0.202, 0.204, 0.050, 0.010 gL−1
aq , respectively for lines 1–5. appropriate for further application in reactor modeling and design.
The following relation holds

versus experimental initial reaction rate for all 49 experiments. An RFA · A = RFA · Vorg (7)
average error of 16.0% was observed. Thus we can conclude that the
 is the reaction rate per unit interfacial area (mol m−2 s−1 )
where RFA
model also predicts the experimental initial reaction rates well.
The kinetic model derived in the previous paragraph is based and A is the total interfacial area (m2 ). Eq. (7) can be rewritten as
on the concentration of the substrates in the organic phase. Litera-
RFA
 6 · ε −1
ture precedents indicate that the enzymatic reaction takes place at 
RFA = 103 · = 103 · RFA · (8)
the interface of the organic phase and the aqueous phase [4,34,35]. a ds

Fig. 10. Drop size distribution of a system containing 0.6 M oleic acid, 0.9 M 1-
Fig. 8. Parity plot of the n-butyl oleate concentrations. butanol in n-heptane and 0.2 g L−1
aq enzyme in an aqueous phosphate buffer.
 G.N. Kraai et al. / Biochemical Engineering Journal 41 (2008) 87–94
where a is the specific contact area (m2 m−3 ) [36], ds the Sauter
mean drop size diameter (m) and ε is the dispersed volume
fraction.
To gain insights in the interfacial area in the reactor, an online
laser method was applied. For a system containing of 0.6 mol L−1 org
oleic acid and 0.9 mol L−1
org 1-butanol in n-heptane and an aqueous
phosphate buffer solution containing the enzyme (0.2 g L−1 aq ) the
drop size distribution curve was determined and the results are
given in Fig. 10.
With this information available, the Sauter mean drop size diam-
eter may be calculated using Eq. (9):
 [9]
N · d2
ds =  i i i (9)
i
(Ni /6) · di3
where the summations are over 90 drop size intervals.
By applying the experimental data to Eq. (9), the ds for the
organic phase was calculated as 24 ␮m. With this information avail-
able, the reaction rate in our experimental setup per m2 interfacial
area may be calculated using Eq. (8) with RFA according to Eq. (6).
This expression is the most appropriate for reactor modeling pur-
poses.
5. Conclusions
The esterification of 1-butanol and oleic acid using free R. miehei
lipase in a biphasic system was studied. Of the four alcohols tested
in this work, 1-butanol was found to give the highest enzyme activ-
ity. Of the four hydrocarbon solvents tested, decane showed the
highest reaction rate, however due to cost considerations n-heptane
was chosen as the most appropriate solvent.
Analyses of the kinetic data indicates that the esterification of
1-butanol with oleic acid catalyzed by free R. miehei lipase can
be described adequately by a Ping Pong bi–bi mechanism with 1-
butanol inhibition. This model was used to simulate the kinetic
experimental data as well as the initial reaction rates with good
results. Reaction rates in terms of m2 interfacial area could be
obtained by on-line measuring the average drop size of the dis-
persed organic phase using a laser technique. The model and the
kinetic constants obtained in this study can be used for novel reac-
tor design and process optimization. The application of the model
for the design and optimization of CCS reactors will be provided in
separate papers by our group.
Acknowledgements
The research described in this paper is financially sponsored by
the IBOS Program (Integration of Biosynthesis & Organic Synthesis)
of the Netherlands Organization for Scientific Research (NWO) and
Advanced Chemical Technologies for Sustainability (ACTS). John
Janssen from Mettler Toledo is gratefully acknowledged for mea-
suring the drop size distributions.
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