Bioprocess and Biosystems Engineering

https://doi.org/10.1007/s00449-019-02250-2

RESEARCH PAPER

Synthesis of n‑butyl acetate via reactive distillation column using

Candida Antarctica lipase as catalyst
Honghai Wang1,2 · Wenjing Liu1,2 · Liya Gao2 · Yifan Lu1,2 · Erxuan Chen3 · Yuchao Xu3 · Hongli Liu1,2

Received: 8 January 2019 / Accepted: 8 November 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
The reactive distillation process for the synthesis of n-butyl acetate via transesterification of ethyl acetate with n-butyl alco-
hol catalyzed by immobilized lipase was simulated and experimentally tested in this work. Based on the reaction kinetics,
a reactive distillation process model was developed. The effects of theoretical stages number in the reaction section, the
rectifying section and stripping section, reflux ratio, feed molar ratio and relative feed position on the transesterification
distillation process were investigated. The transesterification of ethyl acetate with n-butyl alcohol was carried out in a small-
scale reactive distillation column. The results showed that the optimal operating conditions are as follows: reaction section
stages were 13, rectifying section stages were six, stripping section stages were five, reflux ratio was 1, mole ratio of ethyl
acetate and n-butanol was 3:1, the feeding positions of n-butanol and ethyl acetate were at the top and bottom of the reac-
tion section, respectively. Compared to the batch reaction with only 60% conversion of n-butanol, the reactive distillation
column can improve the conversion of n-butanol (up to 93.6%).At the same time, the experiment verified that the conversion
of n-butanol could still reach 72.5%, after the lipase-loaded packing storage in the reaction system at 70 °C for 120 days.

Keywords  Lipase · Reactive distillation · Transesterification · Simulation

Introduction internal components for catalytic separation, new process

Reactive distillation is a unit operation in which chemi-
cal reaction and product separation are coupled in a single
tower. It can improve the conversion rate and separation
efficiency of the reaction and achieve the purpose of sav-
ing investment and increasing yield [1–3]. As an important
means of process strengthening, reactive distillation should
not only follow the reaction law, but also follow the distilla-
tion principle; the interaction between the two has a different
impact on the process [4, 5]. In recent years, the research
of reactive distillation has focused on the development of
* Honghai Wang
ctstwhh@163.com
1
School of Chemical Engineering, Hebei University
of Technology, Tianjin 300130, China
2
National‑Local Joint Engineering Laboratory for Energy
Conservation of Chemical Process Integration and Resources
Utilization, Hebei University of Technology, Tianjin 300130,
China
3
Tianjin Tianou Zhengan Detection Technology Co., Ltd,
Tianjin 300130, China
design and computer simulation [6–8].
The catalysts used in traditional reaction distillation are
strong acid, strong base, solid acid, acidic or alkaline ionic
liquids. When hydrochloric acid, sulfuric acid, and other
liquid proton acids are used as catalysts for transesterifica-
tion, there are some problems such as low yield, long reac-
tion time, difficulty in recovery and utilization of catalyst,
serious pollution of wastewater and so on [9]. The synthesis
of butyl levulinate catalyzed by organic sulfonic acid sup-
ported on titanate nanotubes was studied by Zhou et al. [10].
The results showed that the conversion of levulinic acid was
86.8%. The synthesis of ethyl acetate by ionic liquid reactive
and extractive distillation was studied by Jiehuimin et al.
[11]. The experiment used ionic liquids 1-sulfobutyl-3-meth-
ylimidazolium hydrogen sulfate ­([HSO3bmin][HSO4]) and
1-butyl-3-methylimidazolium bis[(trifluoromethyl) sulfonyl]
imide ­([BMIM][Tf2N]) as catalyst and entrainer, respec-
tively. The extractive distillation process for the preparation
of ethyl acetate and methanol from methyl acetate with etha-
nol was studied. The conversion rate of methyl acetate was
99.22%. However, the price of ionic liquids on the market is
relatively high. Although these acid and alkali catalysts have

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achieved some results in transesterification, the use of these
catalysts will inevitably lead to environmental pollution.
Immobilized enzyme catalysis has attracted more and
more attention, because of its mild reaction conditions,
strong specificity and high efficiency [12–14]. As early as
2007, Santos et al. [15] studied the immobilization of lipase
on poly N-hydroxymethyl acrylamide for the synthesis of
butyl butyrate. The results showed that the stability of lipase
on poly N-hydroxymethyl acrylamide was good. Jiang Yan-
jun et al. [16] formed silica coating on the surface of P/
CLEAs, and the lipase CLEAs (Coated-CLEAs) showed
good stability. Gao et al. [17] have studied the immobiliza-
tion of lipase on the surface of magnetic nano silica. The
results showed that lipase immobilized on nano-flowers for
catalytic reaction processes showed good recoverability and
stability, but the reaction time was too long. Because reac-
tion distillation technology can realize chemical reaction and
product separation simultaneously, it is more advantageous
to realize the enhancement of the process. Li et al. [18]
studied the synthesis of n-amyl acetate by reactive distilla-
tion with a new type of catalytic packing, Seepage Catalytic
Packing Internal (SCPI), and discussed the effect of different
operating parameters on the performance of the reactive dis-
tillation process. The conversion of amyl alcohol can reach
more than 95% under the optimum operating conditions.
Paiva et al. [19] studied the effectiveness of an integrated
reactive distillation setup in the lipase-mediated production
of butyl butyrate via alcoholysis and conclude that reactive
distillation setup behaves as an integrated system, and that
it can effectively be used to promote lipase-catalyzed alco-
holysis reactions. Heils et al. [20–23] studied the integration
of a biocatalytic reaction into a reactive distillation column
with structured wire gauze packings and the feasibility for an
enzymatic reaction in a continuously operated reactive dis-
tillation column. The enantioselective biocatalytic reaction
was carried out for the first time in a fully integrated batch-
reactive distillation setup, the in situ production method for
biocatalytic coatings on structured packings, which enables
production and renewal of the catalytic structures in place.
These studies offer new possibilities for the application of
biocatalysts in the thermal integrated processes of reactive
distillation. Wierschem et al. [24] have verified the feasibil-
ity of the pilot-scale experiment on the production of butyl
butyrate by enzymatic catalytic reactive distillation. Heils
and Wierschem’s work is groundbreaking, we have extended
on this basis. Kühn et al. [25] studied the in situ separation
of the chiral target compound (s)-2-pentanol in biocatalytic
reactive distillation. The results showed that the accumula-
tion of a chiral target compound in the top of the reactive
distillation column allowed a one-step synthesis of chiral
molecules in reactive distillation, which offered the oppor-
tunity of broadening the substrate scope in an integrated
distillation approach.
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Bioprocess and Biosystems Engineering
To highlight the respective advantages of enzymatic
catalysis and reactive distillation, the transesterification of
ethyl acetate with n-butanol was used as a model reaction
and the immobilized lipase catalytic reactive distillation was
studied in detail. The aim of this paper is to provide data
support and theoretical basis for the industrial practice of
immobilized enzyme catalytic reactive distillation.
Experimental section
Materials
Lipase B from Candida Antarctica (CALB) was
kindly provided by Novozymes (Bagsvaerd, Den-
mark) as CALB L solution. Ethanol(EtOH), ethyl
acetate(EtAC), n-butanol(BuOH), n-butyl acetate(BuAC),
methanol(MeOH), polyethylene glycol(PEG, MW = 400),
acetonitrile, and methyl trimethoxysilane (MTMS) were
purchased from Damao Chemical Reagent Factory (Tian-
jin, China); sodium fluoride was obtained from Fengchuan
Chemical Reagent Technology Co., Ltd (Tianjin, China);
tetramethyl orthosilicate (TMOS) was obtained from
HEOWNS Biochemistry Technology Co., Ltd (Tianjin,
China). All chemicals were pro-analysis grade and used
without further purification. The bulk packings are θ ring
316 L stainless steel packing with a size of 3 × 3 mm.
Methods
Preparation of lipase‑loaded packing
First, the immobilized lipase was prepared by sol–gel
method, and then the sols were combined with the bulk
packing by dip-coating method and spray-coating method,
respectively. By comparing the advantages and disadvan-
tages of the two methods, the preparation method of lipase-
loaded packing with better performance was obtained.
Preparation of  immobilized enzyme First, the mixture
of tetramethyl orthosilicate, methyl trimethoxysilane and
methanol, which has a mass ratio of 1:3:3, was used as
crosslinking agent and stirred in an ice bath. Then, the liquid
mixture of polyethylene glycol, sodium fluoride, deionized
water, and CALB with mass ratio of 1:4:9:15 was used as
solvent. Finally, the solvent was poured into the crosslinking
agent and the reaction system was stirred in ice bath. After
that, the reaction system was brought to room temperature,
and then the sol containing CALB could be obtained.
The enzyme activity was measured by transesterification
of ethyl acetate and n-butanol. The experimental condi-
tions were as follows: the molar ratio of ethyl acetate and
n-butanol was 1:1, the reaction temperature was 60  °C,
Bioprocess and Biosystems Engineering
and the rotational speed was 300 r·min−1. After the reac-
tion, the sample was centrifuged for 1.5 min, then analyzed
by gas chromatography. The enzyme mass required for
catalytic consumption of 1 μmol of ethyl acetate per min-
ute was defined as an activity unit (U). The activity of the
free enzyme was 510 ± 17 U·g−1 by the above method and
the immobilized enzyme was 484 ± 12 U·g−1. The results
showed no significant loss after immobilization [26]. The
activity of the new immobilized enzyme was regarded as
100%, then stored at different temperatures to observe the
loss of activity as shown in the Fig. 1.
It can be seen from Fig. 1 that the relative activity of
immobilized enzyme decreases with the increase of stor-
age time, when the storage temperatures were − 20 °C, 3 °C,
25 °C, and 50 °C and when the storage temperature was
70 °C, the relative activity of the immobilized enzyme did
Fig. 1  Loss of activity of immobilized enzyme at different tempera-
tures
Fig. 2  Ordinary packing and
lipase-loaded packing. a Ordi-
nary packing. b Lipase-loaded
packing
not change obviously with the increase of storage time, indi-
cating that 70 °C was a suitable preservation temperature
for the immobilized enzyme. Therefore, the optimum tem-
perature for preservation or reaction of immobilized enzyme
was 70 °C.
Preparation of  lipase‑loaded packing by  dip‑coating
method  In the dip-coating method, the sol was prepared in
a vessel, and the wire gauze packing was immersed into the
prepared immobilized enzyme sol for about half a minute,
then dried at room temperature for a short time, and then
immersed again in the sol. Above steps were repeated for
10–15 times until all of the sol was loaded onto the pack-
ing and solidified into gel on the surface of the packing.
The packing coated with immobilized CALB was dried at
room temperature for 48 h until the weight became constant
and the packing with biocatalytic activity was obtained and
stored at room temperature. Ordinary packing and lipase-
loaded packing are shown in Fig. 2.
As shown in the Fig. 2, the lipase-loaded packing pre-
pared by dip-coating method was loaded uneven. Some
packing surfaces were heavily impregnated, some packing
surfaces have almost no gel adhesion and there was a phe-
nomenon that the internal pores of the packing were blocked
by gel. The microstructure of lipase-loaded packing prepared
by dip-coating method was characterized by scanning elec-
tron microscope (SEM), as shown in the Fig. 3.
The uneven coating on the packing surface can be
reflected from the diagram. As the dip-coating method was
to immerse the packing in the sol for coating, most of the sol
is stacked in the middle pore of the packing. Finally, gelation
occurs after drying. The volume of the sol shrinks during the
gelation process, causing the immobilized enzyme to easily
fall off the pores of the packing. In the structural characteri-
zation of this kind of packing, the three-dimensional annular
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Fig. 3  Micromorphology of lipase-loaded packing prepared by dip-coating method (b is a partial enlargement of a)
packing should be scanned in a plane. At this time, due to the
weak adhesion of the immobilized enzyme to the packing,
most of the immobilized enzymes fall off from the packing
pore. There were only some strong adhesion positions on
the packing surface. As shown in the Fig. 3b, the 1, 2, and 3
positions were still loosely attached. Therefore, it was nec-
essary to improve the preparation method to minimize the
defects of the dip-coating method.
Preparation of  lipase‑loaded packing by  spray‑coating
method  In view of the shortcomings and defects of the
dip-coating method, a spray-coating setup was developed to
gain control over the production process of the coating such
as the coating thickness, the extension to larger packing
pieces, and enhance the results’ reproducibility. The setup
consists of a commercially available paint spray gun driven
by pressurized air (0.1  MPa). The prepared crosslinking
agent A solution (the mixture of tetramethyl orthosilicate,
methyl trimethoxysilane, and methanol with mass ratio of
1:3:3) and solvent B solution (the mixture of polyethylene
glycol, sodium fluoride, deionized water, and CALB with
mass ratio of 1:4:9:15) were stirred 3 min in ice bath, then
introduced into the spray gun. The sol solution was mixed
within the nozzle of the spray gun and sputtered with the air
stream (0.1 MPa). For the application of the spray coatings,
the bulk packing was tiled into the petri dish, a wire gauze
was placed over the petri dish, the coating was then coated
on the surface of the packing with a distance of approxi-
mately 15 cm from the pistol to the surface of the petri dish.
The spray flow rate of the pistol was 1.82 kg/h. An interme-
diate drying step for 10  min (blower assisted drying) pre-
vented the removal of freshly sprayed sol by the air stream
of the spray gun. After reaching the desired loading with
coating, the bulk packing was dried under atmospheric con-
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Bioprocess and Biosystems Engineering
ditions, until constant mass was achieved. The lipase-loaded
packing prepared by the spray-coating method is shown
below in Fig. 4a.
It can be seen from the above image, the surface of the
lipase-loaded packing prepared by the spray-coating method
(Fig. 4a) is more uniform than the one prepared by the dip-
coating method (Fig. 4b) and the sol was almost uniformly
attached to the surface of the packing. The microstructure
of the lipase-loaded packing was characterized by scanning
electron microscope (SEM), as shown in the Fig. 5.
It can be seen from the Fig. 5a that the surface of the
lipase-loaded packing prepared by the spray-coating method
was relatively uniform and the sol does not accumulate in
a large amount in the pores of the annular packing. It uni-
formly attached to the surface layer of the packing, and when
the annular packing was flattened for characterization, there
was no obvious phenomenon of immobilized enzyme shed-
ding. As shown in the partial enlargement Fig. 5b that the
gel was flat attached to the surface of the wire mesh pack-
ing. It indicated that the lipase-loaded packing prepared by
the spray-coating method had stronger adhesion and better
stability than that prepared by the dip-coating method.
Column setup
The experimental setup for enzymatic reactive distilla-
tion is shown in the Fig. 6. The setup consisted of a glass
column with 30 mm inner diameter and a packing height
of 1500 mm. The column was connected to a tempera-
ture-controlled 1 L reaction vessel at the bottom and a
condenser and reflux valve at the top. The reactive dis-
tillation column was divided into two sections, one was
the reactive section filled with lipase-loaded packing and
the other was the separated section filled with ordinary
Bioprocess and Biosystems Engineering
Fig. 4  Lipase-loaded pack-
ing prepared by spray-coating
method and dip-coating method,
respectively. a Prepared by
spray-coating method. b Pre-
pared by dip-coating method
Fig. 5  Microstructure of lipase-loaded packing prepared by spray-coating method (b is a partial enlargement of a)
packing. The reactive distillation column consists of three
different zones, the rectifying zone at the top, the reactive
zone in the middle and the stripping zone at the bottom.
The thermocouples at the top of the column and the bot-
tom of the column were used to measure the temperature
in real time and the temperature sensors were provided at
different locations of the column to record the temperature
change of the reaction section. All the sensors were con-
nected to a digital main control board. Asbestos rope was
used as insulator to reduce the heat losses. The reboiler
was equipped with an external electrical heater, the top
of the column was cooled by circulating cooling water,
and the reflux ratio was controlled by relays and electro-
magnets. Peristaltic pumps were used to supply the feeds
to the column. The rotameters were used to measure the
feed flow rates.
Experimental procedure
To control the temperature distribution in the column, the
experiment adopts vacuum distillation, the pressure was
about 65 kPa (absolute pressure). To start the experiment,
the reboiler was filled with ethyl acetate up to 500 mL. Then
the heater was switched on. As the reboiler started heating,
the vapors of ethyl acetate from the reboiler started filling the
sections one after another, and finally reached the condenser,
where the distillate started collecting. When the temperatures
along the column height reached constant values, the reflux
was started by adjusting its valve and maintained the full reflux
operation for about 0.5 h. The peristaltic pumps were then
started to feed ethyl acetate and n-butanol at desired flow rates
and the heavy-boiling feed component n-butanol was fed above
the reactive section, the low-boiling feed component ethyl
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Fig. 6  Reactive distillation column setup. 1: heating jacket, 2:
reboiler, 3,12: thermometer, 4,8: feeds tank, 5,9: peristaltic pump,
6,10: rotameters, 7,11: feed position, 13: condenser, 14: reflux ratio
acetate was fed below the reactive section. After the column
reached steady state, samples were collected from the vacuum
sampler at regular intervals. Simultaneously, the temperatures
along the column were recorded. The light component product
ethanol was separated from the top of the column to promote
the transesterification in the positive direction. In addition, due
to the whole column being under vacuum operation and to
prevent the gas leakage caused by the continuous collection
of the column kettle, the heavy component product of n-butyl
acetate was stored in the reactor until the end of the experi-
ment. The samples, distillate and bottom, were analyzed for
their composition. This was done by GC. The material balance
was verified to ensure that the mass flow rate in equaled the
mass flow rate out of the reactive distillation column. The sam-
ples from the distillate and column kettle were collected and
weighed accurately with an accuracy of ± 0.1 g error. In each
run, the reaction distillation column ran for 2–3 h at steady
state to collect sufficient data. At the end of the experiment, the
top of the column was stopped, then the heater was cut off, and
when the temperature of the column kettle dropped to room
temperature, the experiment was completed.
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Bioprocess and Biosystems Engineering
controller, 15: vacuum sampler, 16: column body, 17: U tube differ-
ential pressure gauge, 18: vacuum pump
Analytical methods—Gas chromatography
The samples were analyzed by gas chromatography (GC-
2014, Shimadzu, Japan). For GC analysis, a capillary col-
umn CB-FFAP with a length of 30 m, ID = 0.32 mm, and
film thickness of 1.2 μm was used. Samples were detected
by a flame ionization detector (FID) (T detector = 210 °C,
­Tinjector = 200 °C, T column = 80 °C). Gas flow was 17.7 mL/
min for N ­ 2, the sample amount was 0.2  μL. The reten-
tion time of ethyl acetate, ethanol, n-butyl acetate, and
n-butanol was 2.103 min, 2.281 min, 3.760 min, 4.868 min,
respectively.
Simulation study of enzymatic reactive
distillation
Model selection
The correct selection of the model has a great influence
on the accuracy of the simulation results. For simulation,
Bioprocess and Biosystems Engineering
the Radfrac of Aspen plus was used to simulate the enzy-
matic reaction distillation column. As the ethyl acetate,
n-butanol, n-butyl acetate, and ethanol in the simulation
are strong non-ideal systems, the NRTL model was chosen
for calculation. Because the NRTL is not only suitable for
non-ideal systems, but also easy to converge in calculation.
The reaction kinetics data can be found in reference 26.
Simulation process
The setup shown in Fig. 7 was chosen for the simulation
study presented in this article. The two reactants are fed
separately at two different feed positions, the heavy-boil-
ing feed component was fed above the reactive section
(F1) and the low-boiling feed component was fed below
the reactive section (F2), the product ethanol was collected
from the top of the column (DIS), n-butyl acetate was col-
lected from the column kettle (BOT).
The simulation used the above technological process
in the calculations. Based on the experimental parameters
and a large number of experimental experiences in the ini-
tial exploration phase of the enzymatic reaction distillation
column, a group of operating conditions was preliminarily
given and used as a reference. The initial operating condi-
tions are given in Table 1. Because the data in Table 1 are
selected within a reasonable range according to a large
amount of experience, it is not the optimal condition and
the optimal result, but only as a set of initial values. So,
the next step is to optimize to get the best result with the
closest 100% conversion.
Fig. 7  Flow sheet of the reactive distillation setup
Table 1  Preliminarily given simulated operating conditions
Operating condition Initial value
Theoretical stages of reaction section 10
Theoretical stages of rectifying section 5
Theoretical stages of stripping section 5
Total theoretical stages 20
Reflux ratio 2
Feed molar ratio 2:1
Mass fraction of BuAC 0.931
Conversion of BuOH 89.6%
Results and discussions
Simulation results
Influence of theoretical stages number in reaction section
The transesterification model of ethyl acetate with n-butanol
was carried out in the reaction section of distillation column
and the theoretical stages number in the reaction section
have a certain influence on the transesterification process. If
the theoretical stages number is not enough, the reaction will
be insufficient and will not meet the requirements of reac-
tion purity and conversion; too many theoretical stages will
lead to excessive investment in equipment. Figure 8 shows
the effect of increasing the theoretical stages number from
5 to 15 on the purity of n-butyl acetate and the conversion
of n-butanol.
It can be seen from Fig. 8, when the theoretical stages
number in the reaction section increases from 5 to 11, the
purity of the product n-butyl acetate and the conversion of
the reactant n-butanol increase with the theoretical stages
Fig. 8  Effects of theoretical stages in reaction section on purity of
BuAC and conversion of BuOH
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increase. This was because appropriate increase the theo-
retical stages number in the reaction section, the gas–liquid
phase contact time of the two reactants in the reaction sec-
tion was correspondingly lengthened, which was beneficial
to the reaction to proceed sufficiently, thereby improving the
purity of n-butyl acetate and the conversion of n-butanol.
If continue to increase the theoretical stages number in the
reaction section, we can see that the purity and conversion
no longer increase significantly. This was because after 11,
the change of stage number has little effect on the results,
so further adjustments are needed to optimize the results
later, but at this point, the equipment cost will be increased
correspondingly. Therefore, considering economic factors
and reaction effect, the suitable theoretical stages number
in reaction section was 11.
In general, when other operating conditions are
unchanged, only that changed was the number of theoretical
stages of the reaction section, the results will reach a limit
value as the number of stages increases. If the number of
stages continues to increase, the results will change a little,
but the equipment cost will obviously increase. Therefore,
comprehensive consideration is needed. Since the reactive
distillation process is carried out in the rectification column
equipment, it is a small-scale experiment. Therefore, it is
difficult to achieve complete conversion in the rectification
column under the process conditions, but the conversion can
be improved by optimizing various operating conditions.
Influence of theoretical stages number in rectifying section
In the reactive distillation column, the function of the rec-
tifying section was to refine the light component products
produced by the reaction section, that is, to remove the heavy
constituent, and then to collect the light component prod-
ucts at the top of the column. However, separating the light
component products was conducive to promote the transes-
terification in the positive direction, so the theoretical stages
number in the rectifying section was related to the effect of
reaction and separation of the distillation column. Figure 9
shows the effect of increasing the theoretical stages in rec-
tifying section from 3 to 9 on the purity of n-butyl acetate
and the conversion of n-butanol.
It can be seen from the Fig. 9 that the purity of n-butyl
acetate and the conversion of n-butanol increase with the
increase of theoretical stages number in the rectifying sec-
tion. When the theoretical stages number was less than 6,
the main reason for the lower purity of the product and the
conversion of reactants was that the fewer theoretical stages
number in the rectifying section, the closer the reaction
section was to the top of the column. A large amount of
the rising vapor will bring a part of n-butanol to the top of
the column, and thus being collected with the light compo-
nent, so the purity of the product and the conversion of the
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Bioprocess and Biosystems Engineering
Fig. 9  Effects of theoretical stages in rectifying section on purity of
BuAC and conversion of BuOH
reactant will be lower. When the theoretical stages number
was greater than 6, the reaction section position gradually
moves down in the whole column, which leads to a con-
tinuous increase of the reaction temperature, and the higher
temperature may damage the lipase. Therefore, the optimal
theoretical stages number in rectifying section was 6.
Influence of theoretical stages number in stripping section
In the reactive distillation column, the function of the strip-
ping section is to concentrate the heavy component produced
by the reaction section, so that the heavy component prod-
ucts can be gathered in the column kettle and high purity
heavy component products can be obtained in the column
kettle. In addition, the separation of the products was ben-
eficial to promote the transesterification reaction in the posi-
tive reaction direction. Therefore, the stages number in the
stripping section was related to the reaction and separation
efficiency of the distillation column. Figure 10 shows the
effects of increasing the theoretical stages in the stripping
section from 1 to 9 on the purity of n-butyl acetate and the
conversion of n-butanol.
It can be seen from the Fig. 10 that when the theoretical
stages in the stripping section was 3, the reaction and separa-
tion achieved the best results. But at this time, the relative
position of reaction section and column kettle was close, the
temperature of reaction section may be higher. Therefore,
by increasing the stages in the stripping section the reaction
section temperature is reduced. When the theoretical stages
in the stripping section was increased to 7, the temperature
of the reaction section was maintained at an optimum tem-
perature of about 70 °C of the immobilized enzyme. Thus,
the theoretical stages number of the stripping section was
selected to be 7.
Bioprocess and Biosystems Engineering
Fig. 10  Effects of theoretical stages in stripping section on purity of
BuAC and conversion of BuOH
In summary, the theoretical stages in reaction section was
11, the theoretical stages in rectifying section was 6, and the
theoretical stages in stripping section was 7, the total number
of theoretical stages was 24.
Influence of reflux ratio
The reflux ratio is an important parameter for the distillation
and reactive distillation process. It not only relates to the
stable operation of the column setup, but also to the initial
investment and the later operation cost. It is very important
to the reaction and separation effect and energy consump-
tion. Figure 11 shows the influence of the change in reflux
ratio on the purity of n-butyl acetate and the conversion of
n-butanol.
It can be seen from the Fig. 11 that increasing the reflux
ratio in the beginning was advantageous to the reaction and
separation effect. In a certain range, with the increase of the
Fig. 11  Effects of reflux ratio on purity of BuAC and conversion of
BuOH
reflux ratio, the purity of the product and the conversion of
the reactant also increase. When the reflux ratio was greater
than 1, the purity of n-butyl acetate and the conversion of
n-butanol no longer increase significantly, so the most suit-
able reflux ratio was 1.
Influence of feed molar ratio
The transesterification of ethyl acetate with n-butanol is a
reversible reaction. In general, increasing the concentra-
tion of one reactant increases the conversion of the other
reactant. To study the effect of the feed molar ratio of ethyl
acetate and n-butanol on the reaction and separation effect,
the molar ratio of ethyl acetate and n-butanol was changed
and its effect on the purity of n-butyl acetate and the con-
version of n-butanol was analyzed, as shown in the Fig. 12.
It can be seen from Fig. 12 that when the molar ratio of
ethyl acetate and n-butanol was lower than 3, the purity of
n-butyl acetate and the conversion of n-butanol increase with
the increase of the molar ratio of ethyl acetate and n-butanol.
When the feed molar ratio of ethyl acetate and n-butanol
was greater than 3, the purity of n-butyl acetate and the con-
version of n-butanol were no longer increased significantly.
Therefore, 3 was selected as feed molar ratio of ethyl acetate
and n-butanol.
Influence of relative feed position
The relative feed positions of n-butanol and ethyl acetate
affect the adequacy of the reaction by influencing the con-
centration distribution of the materials in the column. The
influence of the relative feed position of n-butanol and ethyl
acetate on the purity of n-butyl acetate and the conversion
of n-butanol is investigated in Table 2.
It can be seen from Table 2 that the purity of n-butyl
acetate and the conversion of n-butanol gradually decreased
Fig. 12  Effects of feed molar ratio on purity of BuAC and conversion
of BuOH
13
 Bioprocess and Biosystems Engineering

Table 2  The effects of relative feed position Comparison of results

BuOH EtAC Mass fraction of Conversion
BuAC of BuOH Comparison of experimental values and simulated values
in the distillation column
6 17 0.994 99.3
8 15 0.986 98.4
Based on the simulated and optimized operating conditions,
10 13 0.978 96.8
experimental verification was carried out in a small-scale
12 12 0.957 93.4
reactive distillation column. In the range of the experiment,
the basic conditions of the column are as follows: the theo-
retical stages in the reaction section was 13; the theoretical
stages in the rectifying section was 6; the theoretical stages
in the stripping section was 5; the reflux ratio was 1; the
feed molar ratio of ethyl acetate and n-butanol was 3:1; and
the feed position of n-butanol and ethyl acetate was at the
top and bottom of the reaction section, respectively. At this
time, the mass fraction of n-butyl acetate in the column was
0.924 and the conversion of the n-butanol was 93.6%. Above
experimental values were compared with the simulated val-
ues. After calculation, the experimental results were in good
agreement with the simulated results and the maximum error
was within 8% and the average error was within 6%. It veri-
fies the accuracy of the simulation and provides theoretical
guidance for its industrialization.

Fig. 13  Temperature distribution in the column Comparison of results in batch reactor and distillation

column

with the relative feed position of n-butanol and ethyl acetate
moved away from the upper and lower ends of the reaction
section. This is because the gas–liquid phase countercur-
rent contact time of the two reactants in the reaction section
was gradually shortened, as the two feed positions gradu-
ally approach each other, resulting in insufficient reaction.
Therefore, the relative feed position for n-butanol and ethyl
acetate was chosen to be the top and bottom ends of the reac-
tion section, respectively.
Based on the above optimization results, the temperature
distribution inside the column is shown below (Fig. 13).
In previous work, the transesterification of ethyl acetate
with n-butanol catalyzed by immobilized lipase in the batch
reactor was studied [26], and under these conditions, the
conversion of n-butanol was 60%. In this paper, the samples
were analyzed by gas chromatography during the experi-
ment. The conversion of n-butanol was 93.6% according to
the conservation of matter. The comparison results highlight
the advantages of enzymatic reactive distillation.
Stability of the lipase‑loaded packing

The catalyst in the reactive distillation process was applied

Optimization conditions and results
According to the analysis of the above influencing factors, a
group of optimal operation conditions is obtained. The theo-
retical stages of the reaction section was 11, the theoretical
stages of the rectifying section was 6, the theoretical stages
of the stripping section was 7, the reflux ratio was 1, the feed
molar ratio of ethyl acetate and n-butanol was 3:1, the feed
position of n-butanol was 6, and the feed position of ethyl
acetate was 17. The mass fraction of n-butyl acetate in the
column kettle was simulated to be 0.994, and the conversion
of the n-butanol was 99.3%.
to the column as a coating combined with the bulk packing,
so the stability of the lipase-loaded packing was critical to
the process conditions. In this study, the long-term stability
of the lipase-loaded packing was examined by the follow-
ing method: The lipase-loaded packing used in the enzy-
matic reactive distillation experiment was removed from the
column, then it was placed in a batch reaction system at
70 °C and the reaction system was stirred using an electric
stirrer. Thus, the lipase-loaded packing in the reactor was
approximately simulated as the environment of the reaction
section in the column, the device is shown in the Fig. 14.
After a period, it was loaded into the column for testing.

13
Bioprocess and Biosystems Engineering

Conclusion

1. The lipase-loaded packings were prepared by the dip-

coating method and the spray-coating method, respec-
tively, and the advantages and disadvantages of the two
methods were compared by SEM. Finally, the lipase-
loaded packing was prepared by spray-coating method.
2. The immobilized lipase was used as a catalyst and the
transesterification was a model reaction. The effects of
operation conditions on the enzymatic reactive distil-
lation process were investigated by Aspen Plus and
obtained a group of optimal operation conditions. Opti-
mization results obtained the purity of n-butyl acetate
0.994, the conversion of n-butanol was 99.3%.
3. Based on the simulated and optimized operating con-
ditions, experimental verification was carried out in a
small-scale reactive distillation column. By calcula-
tions, the experimental results were in good agreement
with the simulation results and the maximum error was
Fig. 14  Experimental setup
within 8% and the average error was within 6%.
4. The stability experiment of the lipase-loaded packing
showed that after the lipase-loaded packing was stored in
the reaction system at 70 °C for 120 days, the conversion
of n-butanol in the reactive distillation column could
still reach 72.5%.

Acknowledgements  This work was supported by the National Natural

Science Foundation of China (Grant no. 21878066) and the Special
Correspondent Project of Tianjin(Grant no. 18JCTPJC56500). The
authors wish to thank all the partners of our laboratory.

Compliance with ethical standards 

Conflict of interest  The authors declare that they have no conflict of

interest.

Fig. 15  Effects of lipase-loaded packing which soaked for 20  days

each time on the conversion of n-butanol
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The transesterification distillation experiment was carried
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results indicated that the lipase-loaded packing prepared
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time in the reaction system at 70 °C and the conversion of
n-butanol in the model reaction could still reach 72.5% after
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