HUNGARIAN JOURNAL

OF INDUSTRY AND CHEMISTRY

VESZPRM
Vol. 40(1) pp. 3944 (2012)

ETHYL-ACETATE SYNTHESIS IN GAS PHASE BY IMMOBILISED LIPASE

Z. CSANDI1 , R. KURDI2, K. BLAFI-BAK1
1

Research Institute on Bioengineering, Membrane Technology and Energetics

E-mail: csanadi@almos.uni-pannon.hu
2
University of Pannonia, Institute of Environmental Engineering, 10 Egyetem Str., 8200 Veszprm, HUNGARY
Gas-solid phase biocatalytic reactions offer economic and environmentally sound ways to produce ester compounds,
which can be used as natural flavour components, and other types of value-added products. Therefore, the aim of this
work was first to study the continuous gas-solid phase manufacture of ethyl-acetate (EtAc), which is an important fruit
flavour compound, from ethanol (EtOH) and acetic acid (AcAc) applying immobilised Candida antarctica lipase B
enzyme in a self-constructed bioreactor and then to determine the effects of initial substrate composition, applied
temperature, and the amount of used enzyme on the yield. It can be concluded that there was a well-defined connection
between the yield of the ethyl-acetate product, the temperature and the amount of used enzyme, while the correlation
between the initial substrate composition and the product yield could not be described so easily. The activation energy of
the esterification was found to be much lower in our system than that of the same enzymatic reaction carried out in other
reaction media, such as organic solvent system, ionic liquid, etc.
Keywords: solid/gas phase, enzymatic reaction, lipase, ethyl-acetate

Introduction
Organic flavour compounds can be defined as natural
when they are produced either from natural sources [1]
or in natural processes, such as physical treatment,
fermentation [2], or enzymatic reaction [3]. Although
these natural compounds are healthier and more
attractive to the consumers [4], their extraction from
natural sources, e.g. various plants and fruits, is not only
expensive but also results in extensive waste production.
Therefore, the production of natural aroma compounds,
especially esters by fermentation and enzymatic
reactions in aqueous [5], organic solvent [6], or novel
environmentally friendly solvent media [7] has become
a widely-studied field of research in the last few years.
Solid-gas phase biocatalysis, where biocatalysts are
in solid form while the substrates are in gaseous state or
can be easily vaporized [8], offers the following advantages
over solid-liquid systems and organic liquid medium [911]:
-

higher biocatalyst stability

thermo-denaturation of the (partially) dehydrated
biocatalyst is limited
mass transfer of components is more efficient in the
gas phase
diffusion limitation is reduced due to the low
viscosity in the gas phase
production of by-products is reduced or avoided
very high conversion yields can be achieved
products and unconverted substrates can be easily
recovered with condensation
risk of microbial contamination is lower

Recently, both enzymes (mainly lipases in esterification

reactions [12, 13]) and whole cells (e.g. bakers yeast)
have been studied in solid-gas systems. The experiments
were carried out either by immobilized or soluble
biocatalyst [14, 15].
Barzana [16] studied the gas-phase oxidation of
ethanol vapour with molecular oxygen and dehydrated,
immobilized alcohol dehydrogenase from Pichia
pastoris cells and they found that dry alcohol oxidase
was more thermostable in gas-phase than in aqueous
solution.
In the works of Mikolajek [17] and Spiess [18]
immobilized enzyme preparations were used successfully
in carboligation reactions, where benzaldehyde was
converted to benzoin using thiamine diphosphatedependent enzymes.
Gas phase ethyl acetate production was studied by
Hwang and Park [19] in a batch bioreactor applying
porcine pancreatic lipase in a powder form. In the
experiments, effects of reactant concentration, amount
of enzyme, and reaction temperature on the performance
of the bioreactor were investigated.
Letisse and co-workers [20] used a continuous gas
phase reactor to study the effect of organic molecules on
the kinetic parameters of the alcoholysis of methyl
propionate by 1-propanol catalyzed by immobilized
Candida antarctica lipase B.
The gas phase continuous production of acetaldehyde
from ethanol and the production of hexanal from
hexanol using dried bakers yeast were studied in a
continuous operational system and it was found that
after 20 hours long experiments the hexanal conversion
was as high as 32% without a decrease in enzyme

40
activity [21]. The results suggested that dehydrated
enzymes may have potential advantages in solid-gas
phase bioreactors.
The aim of this work was first to construct a solidgas phase laboratory scale experimental set-up and then
to carry out experiments on the continuous esterification
of ethyl-acetate from acetic acid and ethanol applying
Candida antarctica lipase B enzyme in this bioreactor
and to determine the effects of initial substrate
composition, applied temperature, and amount of used
enzyme on the esterification reaction. The synthesis of
ethyl acetate is considered to be a quite important
process since it can be extended to the gas-solid phase
synthesis of other flavour esters, such as isopropyl
acetate, isobutyl acetate, ethyl propionate, and butyrate.
Furthermore, it was planned to determine the activation
energy of the reaction and to compare the results to the
data in the literature concerning the same esterification
reaction carried out in other types of reaction media.
Material and methods
Materials
The immobilized biocatalyst, Novozym 435 Candida
antarctica lipase B was purchased from Novozysmes
(Bagsvaerd, Denmark).
Acetic acid (99.7%), ethanol (99.7%), and ethylacetate were of the highest purity and provided by
Sigma-Aldrich, Germany.
Nitrogen (N2) gas was provided by Linde, Hungary.
All other chemicals were analytical grade and purchased
from Sigma-Aldrich, Germany.

ethanol solution (-18C), then removed from the N2

stream by cooling, while N2 was recycled.

Figure 1: Scheme of the experimental set-up

1. thermostate, 2. flow meter, 3. substrate saturation
unit, 4. septum for gaseous sampling, 5. bioreactor, 6.,7.
vapour condensation units, 8. condensated sample
reservoir, 9. gas outlet, 10. cryostat
In the first two functional units the constant
temperature (which was lower in the case of unit I than
that of unit II in order to avoid occasional condensation)
was maintained by thermostates. Gas phase samples
were taken both from the substrate separation unit and
the bioreactor through the septums.
Analytical method
The gas samples were analysed by gas chromatography
using a HP4890 type gas chromatograph equipped with
a FID detector and a FFAP fused silica capillary column
(Macherey Nagel, Germany). The temperature program
was the following: 3 min isothermal period at 60C,
then temperature was raised to 250C at 10Cmin-1,
while the injector and detector were maintained at
250C.

Experimental set-up
The experimental set-up, shown in Figure 1, is
composed of three main functional units; I. substrate
saturation module, II. bioreactor, III. product and
remaining substrate recovery module. It is built up of
the following parts; two thermostates (1), flow meter
(2), substrate separation unit (3), septums for gas
sampling (4), bioreactor with the immobilized biocatalyst
(5), two vapour condensation units (6,7), condensated
sample reservoir (8), N2 gas outlet (9), and a cryostat (10).
Substrate was continuously fed to the bioreactor via
the flow meter by passing N2 carrier gas through the
mixed ethanol and acetic acid substrate solution with a
rate of 2 dm3(N2)h-1. The carrier gas first was saturated
by the substrates in the substrate saturation unit then
was passing through the thermostated glass spiral tube
bioreactor packed with approximately 11 g of immobilized
lipase operating under atmospheric pressure and with a
mean residence time of 72 s.
After the enzymatic synthesis of ethyl-acetate took
place in the bioreactor, the remaining substrates and
products were first condensated and trapped into cold

Optimization of carrier gas amount

Optimal flow rate, hence optimal amount of the carrier
N2 gas - which affects the residence time of the gaseous
reactant mixture and productivity of the whole system was determined in the experimental set-up by measuring
the conversion of the acetic acid substrate and the yield
of ethyl-acetate product at various carrier gas flow rates
(2, 3, 4, 5 dm3h-1). In all cases the ratio of acetic acid
and ethanol was 80:20 cm3cm-3 (3.86 gg-1), the amount
of used enzyme was 11 g, and the temperature of the
bioreactor and unit I were 50C and 30C, respectively.

Effect of temperature on the esterification reaction

Selection of the optimal reaction temperature and the
investigation of the effect of temperature were realized
by the determination of the acetic acid substrate
conversion and ethyl-acetate product yield at different
bioreactor temperatures (30C, 40C, 50C, and 60C).

41
The temperature of unit I was always higher than that of
unit II in order to avoid occasional condensation, and
the temperature of unit I and III were continuously
detected. The ratio of acetic acid and ethanol was
80:20 cm3cm-3 (3.86 gg-1), the amount of used enzyme
was 11 g, the carrier gas flow rate was 2 dm3h-1 and the
experiments were 9 hours long, in all cases.
Effect of initial substrate composition on the
esterification reaction
Effect of initial substrate composition was studied and
the optimal substrate composition was selected by
measuring the acetic acid conversion and the ethylacetate yield applying various acetic acid and ethanol
ratios (80:20, 75:25, 65:35, 50:50, 25:75 in cm3cm-3).
The amount of enzyme was 11 g, the carrier gas flow
rate was 2 dm3h-1, the bioreactor and the substrate
saturation temperatures were 50C and 30C,
respectively.

Figure 2: Effect of carrier gas flow rate on ethyl-acetate

yield as a function of time
It can be seen that both higher conversion and yield
can be achieved at lower flow rate, hence longer
reaction time provided for the production of ethylacetate can be obtained. Therefore, 2 dm3h-1 carrier gas
flow rate was selected for further experiments.

Effect of the amount of enzyme on the esterification

reaction
Experiments were carried out applying different
amounts of Novozym 435 lipase enzyme (3.7 g, 6.7 g,
11 g) to determine the effect of enzyme quantity on the
ethyl-acetate production. Temperature of the bioreactor
was kept at 50C, the N2 carrier gas flow rate was
2 dm3h-1, and the acetic acid and ethanol ratio was
80:20 cm3cm-3 (3.86 gg-1).

Figure 3: Effect of carrier gas flow rate on acetic acid

conversion

Calculation of the activation energy

Effect of temperature
The activation energy of the reaction was calculated
from the Arrhenius-equation (Eq. 1), and the logarithmic
Arrhenius-equation (Eq. 2), where reaction rate is
proportional to the activation energy.
v = A exp(-Ea/RT),

(1)

ln v = ln A - Ea/RT.

(2)

Results of the experiments concerning the effect of

temperature on the enzymatic esterification and the
selection of the optimal reaction temperature are shown
in Figure 4 and Figure 5.

where:
A Arrhenius constant
R 8.314 (Jmol-1K-1)
Ea activation energy (Jmol-1)
T temperature (K)
Results and discussion
Optimization of carrier gas amount
Ethyl-acetate yields and acetic acid conversions as a
function of carrier N2 gas flow rate are represented in
Figure 2 and Figure 3, respectively.

Figure 4: Effect of temperature on ethyl-acetate yield as

a function of time
Fig. 3 shows that the amount of produced ethylacetate increases relatively fast at the beginning of the
esterification until it reaches equilibrium, hence, the

42
curves follow the trend of enzymatic reactions. The
system can be considered continuous after it reaches
equilibrium, which happens approximately after
36 hours.

Figure 6: Effect of initial substrate composition on

ethyl-acetate yield as a function of time

Figure 5: Effect of temperature on acetic acid

conversion
It is reasonable that higher coversion and yield
values belong to higher temperatures; the highest values
were measured at 50C, up till 60C, where the enzyme
activity decreased due to the temperature sensitivity of
enzyme proteins. At the selected optimal temperature of
50C quite high conversion was achieved and the yield
of ethyl-acetate was higher than 110 mgdm-3h-1.
Effect of initial substrate composition
The values of ethyl-acetate yield and acetic acid
conversion in the case of different substrate mixture
compositions are shown in Figure 6 and Figure 7. It is
important to mention that due to the difference in the
vapour pressure of the two substrate components
(b.p. acetic acid = 11.8C, b.p. ethanol = 78.4C), higher
amount of liquid acetic acid in the mixture would mean
lower amount of acetic acid vapour and higher amount
of ethanol vapour and vica versa. The exact values for
the substrate composition in liquid and gaseous phases
are shown in Table 1.
Table 1: Comparison of the initial substrate composition
in liquid and gaseous phase
Ratio of substrates in
liquid phase
AcAc:EtOH
(cm3cm-3)
80:20
75:25
65:35
50:50
25:75

Ratio of substrates in
gaseous phase
EtOH:AcAc
(gg-1)
2.0 0.04
3.5 0.05
1.0 0.03
0.9 0.05
0.2 0.06

Figure 7: Effect of initial substrate composition on

acetic acid conversion
In Fig. 6 it can be seen that the enzymatic
esterification has reached equilibrium independent of
the initial substrate composition. In the case of 25:75
acetic acid and ethanol ratio, there was so small amount
of acetic acid vapour in unit I that the reaction started
only after one hour reaction time. The highest conversion
and yield values were obtained by the experiment in
which the acetic acid and ethanol ratio of 75:25 mlml-1
was used, hence this value is considered to be the
optimal initial substrate ratio.
The highest conversion values are obtained for 75:25
liquid acetic acid and ethanol ratio, while the lowest
value belongs to its opposite, the ratio of 25:75. This
can be explained by the fact that the substrate mixture
has inhibition effect on the enzymatic reaction mainly
caused by acetic acid. Therefore, the lower is the acetic
acid vapour proportion in the bioreactor is, the less
significant of inhibition and more efficient the
enzymatic reaction will be.
Effect of the amount of enzyme
The effect of the used enzyme quantity on the
conversion is shown in Figure 8 and on the yield of
ethyl-acetate product is represented in Figure 9.

43
Table 2: Activation energy of esterification in different
reaction media
Activation energy
(kJmol-1)
solid-gas phase
9.2
[bmim][PF6] ionic liquid [22]
21.7
n-hexane [23]
30.6
solvent-free system [23]
52.9
Reaction media

Figure 8: Effect of the amount of enzyme on ethylacetate yield as a function of time

Figure 9: Effect of the amount of enzyme on acetic acid

conversion
Although both the conversion and yield results
showed an increase with the increase in the amount of
enzyme, it is quite surprising to note that there is only a
small difference between the conversion values for 3.7 g
and 6.7 g of enzymes, while the conversion was more
than four times higher when the amount of enzyme was
increased from 6.7 g to 11 g. Probably, further enzyme
addition would further increase the conversion and the
product yield but taking the relatively high price of the
enzyme into account, further enzyme addition was not
studied due economical considerations.
Calculation of the activation energy
The calculated activation energy of the gas-solid phase
ethyl-acetate production reaction was Ea = 9.2 kJmol-1,
which is in good agreement with the value reported by
Perez [12]. Our result and activation energies determined
for the same esterification reaction in other reaction
media are summarized in Table 2. It can be seen that our
result is lower than the ones found in the corresponding
literature. Therefore, it can be stated that the studied
enzyme catalyzed esterification reaction can be carried
out more easily and efficiently in gas phase than it other
reaction media due to the much more effective mass
transfer, low viscosities, and high diffusion coefficients
in the gas phase.

Conclusion
In this work, first the Candida antarctica lipase B enzyme
catalyzed esterification of ethyl-acetate from acetic acid
and ethanol was investigated in a self-designed and selfconstructed continuous gas-solid phase system. Then,
the effect of temperature, enzyme quantity, and initial
substrate composition on the conversion of acetic acid
and on the yield of ethyl-acetate was determined and the
activation energy of the biocatalytic reaction was
calculated.
It was found that both conversion of acetic acid
substrate and yield of ethyl-acetate increased with the
increase of temperature and the amount of enzyme and
that there was no such linear correlation between these
values and the initial substrate ratio. The optimal
reaction conditions were found to be described by the
following parameters:
-

flow rate of N2 carrier gas: 2 dm3h-1,

temperature in the bioreactor: 50C,
amount of used enzyme: 11 g,
initial acetic acid and ethanol substrate ratio:
75:25 mlml-1.

The activation energy was found to be

Ea = 9.2 kJmol-1, which is much lower than that of the
same reaction carried out in other reaction media.
Therefore, it can be stated that it is easier and much
more effective to carry out this type of enzymatic
esterification in the gas-solid phase bioreactor
constructed and studied by our research group.
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