Eng. Life Sci. 2012, 12, No.

1, 4956 49

Ana Tusek1 Research Article

Anita Salic2
Zelimir Kurtanjek1 Modeling and kinetic parameter estimation
Bruno Zelic2
of alcohol dehydrogenase-catalyzed hexanol
1
Faculty of Food Technology oxidation in a microreactor
and Biotechnology,
University of Zagreb,
Zagreb, Croatia A mathematical model for hexanol oxidation catalyzed by NAD1-dependent
2
alcohol dehydrogenase from bakers yeast in a microreactor was developed and
Faculty of Chemical
compared with the model when the reaction takes place in a macroscopic reactor.
Engineering and
The enzyme kinetics was modeled as a pseudo-homogeneous process with the
Technology, University of
double substrate MichaelisMenten rate expression. In comparison with the
Zagreb, Zagreb, Croatia
kinetic parameters estimated in the cuvette, a 30-fold higher maximum reaction
rate and a relatively small change in the saturation constants are observed for the
kinetic parameters estimated in the continuously operated tubular microreactor
(Vm1 5 197.275 U/mg, Khexanol
m 5 9.420 mmol/L, and KNAD1m1 5 0.187 mmol/L).
Kinetic measurements performed in the microreactor, estimated from the initial
reaction rate experiments at the residence time of 36 s, showed no product
inhibition, which could be explained by hydrodynamic effects and the continuous
removal of inhibiting products. The Fourier amplitude sensitivity test method was
applied for global kinetic parameter analysis, which shows a significant increase in
the sensitivity of KNAD1
m1 in the microreactor. Independent experiments performed
in the microreactor were used to validate and to verify the developed mathe-
matical model.

Keywords: Alcohol dehydrogenase / Hexanol oxidation / Mathematical modeling /

Microreactor

Received: March 1, 2011; revised: May 3, 2011; accepted: August 10, 2011

DOI: 10.1002/elsc.201100020

1 Introduction characterization [3]. Modeling of the biotransformations as a

Microreaction technology is gaining an important place in the
different fields of application, from scientific research to
industrial production. Large surface-to-volume area ratios
provide very effective heat and mass transfer. Better process
control and new production concepts by numbering-up
instead of scale-up are advantages that make the microreaction
technology so interesting [1]. The main advantages of micro-
reactors are their small dimensions, typically 10100 mm,
which could reduce the diffusion limitations of enzyme and
substrates. According to Swarts et al. [2], in a system where the
substrate has to bridge a large distance to the active site, the
effective reaction rate could be limited. In the recent years,
great efforts have been made to develop microreactors for
screening substrates and enzymes and for kinetic enzyme
Correspondence: Professor Bruno Zelic (bzelic@fkit.hr), University of
Zagreb, Faculty of Chemical Engineering and Technology, Marulicev
trg 19, HR-10000 Zagreb, Croatia.
Abbreviations: ADH, alcohol dehydrogenase; FAST, Fourier ampli-
tude sensitivity test; SA, sensitivity analysis
tool for enzyme reaction engineering represents an important
role in the development of enzyme-catalyzed reactions for
large-scale production and for microreactors. Knowledge of
the enzyme kinetics enables to find the optimal operating
conditions and facilitates the identification of the most effec-
tive mode of process operation [4]. Tadepalli et al. [5] claimed
that, for fast chemical reactions, an estimation of the kinetics
in a microreactor would give more precise results. This is
because, for fast reactions (i.e. hydrogenation reactions) in a
macroscopic reactor, mass transfer effects become predomi-
nant over the intrinsic kinetics. As a result, mass transfer
limitations that mask the true kinetic rates are a problem that
can be overcome in a microreactor.
Detailed knowledge of the reaction kinetics is essential for
reaction development and for collecting information about the
catalyst under operating conditions [6]. Considering the fact
that the conditions in microreactors differ from those in the
conventional macroreactors (high mass transfer rate, short
residence time, large volume-to-surface area ratio [7]), it is
necessary to establish the kinetic parameter values of enzyme
reactions also for microreactors.

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com
50 A. Tusek et al. Eng. Life Sci. 2012, 12, No. 1, 4956

Up to now, mathematical modeling of enzymatic reactions
in an immiscible two-phase system in a microreactor, which
would enable process description and optimization, is poorly
described. Most investigations on biocatalysts and enzyme
characterization are directed towards studies on supported
enzymes [813]. Using microreactors, problems like the diffi-
culty of mixing of the solid particles containing the supported
enzyme with the substrate solution can be overcome and the
characterization of new enzymes can be facilitated [3].
Tisma et al. [14] developed a mathematical model for
laccase-catalyzed L-DOPA oxidation in a microreactor. They
used kinetic parameters previously estimated in a cuvette for
modeling the process. The same approach was used for
modeling and experimental studies on lipase-catalyzed isoamyl
acetate synthesis in a microreactor [1].
In this study, the mathematical model for hexanal
production catalyzed by alcohol dehydrogenase (ADH) from
bakers yeast in a microreactor was developed. Hexanal is part
of the group of natural volatile chemicals, the so-called green
notes. They are used to impart a green sign associated with
freshness [15] and their organoleptically fresh note is what
makes them so interesting for consumers in the pharmaceu-
tical, agrochemical and aroma industries [16]. Nowadays,
several methods are applied for green chemical production:
halogenation of alkyl aromatics followed by hydrolysis, vapor
phase oxidation of alkyl aromatics and reduction of acid
halides, hydrogenation of carboxylic acids by molecular
hydrogen, and oxidation by dehydrogenation of aromatic
alcohols [17]. The main problem of these traditional methods
is that, due to low yields, the formation of unwanted by-
products, and large quantities of wastes, they cannot provide
sufficient amounts of green chemicals [18]. The application of
microreactors could be the next generation of production
processes, and biotransformations in microreactors, after
process optimization, could be a good alternative to the clas-
sical chemical synthesis processes [19]. Nevertheless, for the
commercial application of microreactors, it is important to use
a catalyst with high performance regarding the volumetric
productivity relative to the catalyst mass [20].
Evaluation of the parameters of the MichaelisMenten
kinetics for hexanol oxidation was first performed in a
cuvette experiment [21]. Unfortunately, the obtained kinetic
parameters were not suitable for the description of hexanol
oxidation in a microreactor, and no other data regarding this
problem could be found in the literature. Kinetic parameters
were then estimated from experimental results achieved in the
microreactor experiments. The Fourier amplitude sensitivity
test (FAST) method was applied for the analysis of the esti-
mated kinetic parameters. The model prediction results were
proven and verified on a set of independent experiments
performed in the microreactor.
2 Materials and methods
2.1 Materials
2.1.1 Chemicals
NAD1 and hexanal were purchased from Fluka (Switzerland).
Hexanol was from Merck (Germany) and cyclohexanone from
Carlo Erba (Italy), while hexane was purchased from Sigma
(Germany). HCl, glycine and Na2P2O7  10H2O were
purchased from Kemika (Croatia).
2.1.2 Enzyme
ADH from bakers yeast with a commercial activity of 451
U/mg, where 1 U is the amount of enzyme that catalyzes the
conversion of 1 mmol of substrate per minute under standard
conditions (pH 5 7, T 5 251C), was purchased from Sigma.
2.2 Methods
2.2.1 Microreactor experiments
ADH-catalyzed oxidation was carried out at room temperature
(251C) in two borosilicate glass microreactors: (i) 332 mm
length, 150 mm width and 150 mm height with an internal
volume of 6 mL; (ii) 676 mm length, 150 mm width and 150 mm
height with an internal volume of 13 mL; with two inlets
(Y shaped) and one outlet (Micronit Microfluidics B.V.,
The Netherlands) (Fig. 1B). The enzyme and different
concentrations of coenzyme (ci,NAD1 5 04.4 mmol/L,
ci,NADH 5 05.5 mmol/L) dissolved in aqueous buffer

Syringe pump for coenzyme

(NAD+) and enzyme
dissolved in buffer
Fused silica
connections

Syringe pump for hexanol

dissolved in hexane

Figure 1. Schematic diagram of

A Channel width Sample
collection
the reaction system used for
Channel the kinetic measurements:
vials
p
depth
(A) reaction system, (B) cross-
C section of the microchannel,
B Aqueous phase
Organic phase and (C) segmented flow in the
microreactor.

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com
Eng. Life Sci. 2012, 12, No. 1, 4956 Modeling and kinetic parameter estimation 51

(75 mmol/L glycine-pyrophosphate buffer, pH 5 9) were fed The model of a steady-state plug flow reactor with
from one inflow and the substrates (ci,hexanol 5 0105 mmol/L, negligible axial dispersion was used for the description
ci,hexanal 5 024.5 mmol/L) dissolved in hexane from the of ADH-catalyzed hexanol oxidation in the microreactors. It
second inflow, using two syringe pumps (Fig. 1A; PHD 4400 was assumed that there were no radial variations in
syringe pump series, Harvard Apparatus, USA) equipped with velocity, concentration, or reaction rate. This description does
high-pressure stainless-steel syringes (8 mL; Harvard Appara- not take into account the liquidliquid slug flow in the
tus, USA). The solutions were fed at a 1:1 volumetric flow microchannels (Fig. 1C). Mass balances for the hexanol
ratio. Outflows from the microreactors containing substrate, oxidation are based on the assumption of constant reaction
product, enzyme, NAD1 and NADH were gathered in 0.1 mol/ volume. Additionally, the change in the mass transfer coeffi-
L HCl to stop the reaction by enzyme disruption. cient along the reactor was assumed to be negligible. Mass
Fluid flow in the microreactors was observed using a micro- balances for ADH-catalyzed oxidation in the microreactor are
scope (Motic B1-220A, binocular; Wetzlar, Germany) at given by equations for hexanol, hexanal, NAD1, and NADH
magnifications of 40 and 100 (eyepiece magnification 510; (Eqs. 25):
objective magnification 5 4 and 10). dchexanol 1
All experimental measurements shown in this paper were  r 2
dx v
performed in triplicate, and in the 95% confidence range the
results showed no statistical difference. dchexanal 1
r 3
dx v
2.2.2 Analytics dcNAD1 1
Hexanol and hexanal concentrations in the organic phase were  r 4
dx v
determined by a Shimadzu GC-2014 (Kyoto, Japan) gas
chromatograph equipped with a flame ionization detector. A dcNADH 1
r 5
polar ZB-WAX column (Phenomenex, Torrance, USA) and dx v
helium as gas carrier were used. Compound concentrations where x is the microreactor length, v is the linear velocity and
were measured under flowing conditions: splitless injector r is the reaction rate in U/mg with the boundary condition
2801C, linear velocity 25 cm/s, detector 2401C, initial c(x 5 0) 5 ci. A mathematical model for hexanol oxidation
temperature 501C, initial time 1 min, rate 101C/min to 1801C, performed in a macroreactor has been described elsewhere
final temperature 1801C, and final time 2 min [22], and 1-mL [21].
samples were injected. Samples for analysis were prepared by
mixing an equal volume of sample with the internal standard
(1% solution of cyclohexanone in hexane) for 1 min. Hexane 2.4 Fourier amplitude sensitivity test
was used to extract the hexanol and hexanal from the aqueous
solution. To separate the aqueous from the organic phase, To analyze the importance of individual kinetic parameters
the samples were centrifuged (Hettich, Universal 320R, included in the model describing hexanol oxidation in a
Andreas Hettich GmbH, KG, Germany) for 3 min at 41C and macroreactor and a microreactor, the FAST was applied. It is a
9000 rpm. After filtration (Filter Chromafils AO-20/3; 0.2 mm, global sensitivity method based on variance contribution [23].
3 mmo100; Macherey-Nagel, Germany), the upper layer was Application of FAST provides sensitivities to large and
used for analysis [21]. The observed retention times of hexane, simultaneous changes of the complete set of model parameters.
hexanal, cyclohexanone and hexanol were 2.1, 5.9, 9.2 and The method is based on Fourier transformation of
9.7 min. uncertain model parameters into a frequency domain, thus
reducing the multi-dimensional model into a single-dimen-
sional one (Eq. 6):
2.3 Mathematical model
1 1
ki 1  arcsinsinoi  S1ji 6
The mathematical model for hexanol oxidation performed in 2 p
the microreactor includes the kinetic model and the reactor where ki is the analyzed kinetic parameter, S is the sampling
model. The reaction rate of hexanol oxidation was modeled as parameter in the range of [1, 1], oi are selected frequencies,
pseudo-homogeneous process (as if the organic and aqueous and ji are randomly selected phase angles. The output model
phases were homogeneously mixed) with the double substrate functions (reaction rate, concentration of hexanal) were
MichaelisMenten equation (r 5 f(chexanol, cNAD1)) (Eq. 1). decomposed into Fourier series with the coefficients Ao
The reaction rates were measured for a residence time of (Eq. 7) and Bo (Eq. 8):
t 5 36 s and the reactor content was considered to be homo- Zp
1
geneous. Owing to the short residence time of o180 s, product Ao  f S  coso  S  dS 7
2p
inhibition and effects of reverse reaction were assumed to be p
negligible [21].
Zp
Vm1  gADH  chexanol  cNAD1 1
r 1 Bo  f S  sino  S  dS 8
NAD1 1c
Kmhexanol 1chexanol  Km1 NAD1
2p
p

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com
52 A. Tusek et al. Eng. Life Sci. 2012, 12, No. 1, 4956

The total dispersion, DT, is determined from the Fourier A

coefficients (Eq. 9):
X
1
DT 2  A2o 1B2o 9
o1

The individual parameter contribution in the total disper-

sion, Di, is calculated by the corresponding parameter
harmonics (Eq. 10).
X
1
Di 2  A2o 1B2o 10
okoi

First-order sensitivity coefficients, Si, are calculated as

follows:
Di B
Si 11
DT

2.5 Data processing

The kinetic parameters were estimated by non-linear regres-

sion analysis from the reactor experiments. The least-squares
method implemented in the SCIENTIST package software was
used. The numerical values of the parameters were evaluated
by fitting the kinetic model to the experimental data. The
calculated data were compared with the experimental data and
variance was defined by the sum of squares of the differences
between the experimental and calculated data.
For the reactor model simulation and verification, batch
macroreactor and continuous microreactor comparison, and
Figure 2. Dependence of the initial reaction rate on the
FAST parameter analysis Mathematica 7 codes were developed. concentration of (A) hexanol (ci,NAD1 5 4.4 mmol/L) and (B)
NAD1 (ci,hexanol 5 100 mmol/L); gADH 5 0.092 g/L, 75 mmol/L
glycine-pyrophosphate buffer pH 9; T 5 251C.
3 Results and discussion
3.1 Parameter estimation Table 1. Comparison of the kinetic parameters estimated from
experimental results obtained in a cuvette and in the
The kinetic parameters of the mathematical model (Eq. 1) continuously operated microreactor
were estimated from the independent experimental data from Parameter Cuvette [21] (V 5 1 mL) Microreactor (V 5 6 mL)
a microreactor (V 5 6 mL; Fig. 2). For estimation of the kinetic
parameters, the concentration of the first reactant was varied Hexanol oxidation
while the second one was kept constant in saturation. The Vm1 (U/mg) 6.75570.089 197.27679.334
parameters were estimated using double substrate Michae- Khexanol
m (mmol/L) 10.06770.853 9.42071.809
lisMenten kinetics and are presented in Table 1. Experiments KNAD1
m1 (mmol/L) 0.83570.078 0.18770.037
hexanal
for the kinetic parameter estimation were performed at a Ki1 (mmol/L) 0.48270.045
NADH
Ki1 (mmol/L) 0.09170.005
residence time of t 5 36 s in the continuously operated
Hexanal reduction
microreactor. This residence time was chosen because previous
Vm2 (U/mg) 35.42170.561
experiments showed that longer residence times had no effect Khexanal (mmol/L) 3.39970.758
m
on the conversion [19]. The effects of product inhibition KNADH
m2 (mmol/L) 0.15270.060
(hexanal and NADH) and reverse reaction (hexanal reduction) hexanol
Ki1 (mmol/L) 25.92572.568
were tested in the previously defined concentration range. An KNAD1
m2 (mmol/L) 1.78870.103
inhibitory effect of the products was not observed, and there
was no reverse reaction. A possible explanation for this effect
could be based on the fluid flow regime in the microreactor. As enzyme and coenzyme were in the aqueous phase. Owing to
mentioned before, all experiments were performed in the slug the low concentration range, there was no inhibition effect.
flow regime where the reaction takes place only at the interface. Additionally, inhibiting products were continuously removed
Substrate and product were kept in the organic phase while from the phase interface, because the kinetic measurements

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Eng. Life Sci. 2012, 12, No. 1, 4956 Modeling and kinetic parameter estimation 53

were performed in a continuously operated tubular micro- A

reactor. The kinetic parameters estimated in the continuously
operated microreactor were compared with the kinetic para-
meters estimated from the measurements performed in a
cuvette (V 5 1 mL, [21]). It can be seen from the values of the
estimated parameters that the maximal initial rate
(197.27679.334 U/mg) detected in the microreactor is
significantly higher than the one observed in the cuvette (6.755
7 0.089 U/mg). This could be explained by two of the
main characteristics of the microreactors: the fast mass transfer
and the high surface-to-volume area ratio, which make the
reaction performed in the microreactor faster, with higher
yields and productivities. Owing to the absence of radial
diffusion limitations in the microchannels (small diameter
compared with the channel length), they are suitable for
the determination of the kinetic data of enzyme-catalyzed B
reactions.
The mass transfer effects on the overall reaction rate are
negligible, and the kinetics estimated in the microreator is just
the result of the reaction at the enzyme active site. On the
other hand, the kinetic measurements performed in the
cuvette, due its geometry and the properties of the aqueous
and organic phases, were strongly influenced by insufficient
phase mixing. For this type of reactor configuration, the
kinetic measurements in the two-phase system resulted in the
apparent kinetic parameter estimates.
The results also indicate that the enzyme ADH is specific for
hexanol and NAD1 as substrates (low Km values). As was
expected, the MichaelisMenten constants for hexanol are
practically the same when estimated in a cuvette and in a
microreactor, because this parameter is a characteristic of the C
enzyme used. On the other hand, the MichaelisMenten
constant for NAD1 is 4.46-fold lower when measured in the
continuously operated microreactor, meaning that the enzyme
and the coenzyme form their complex faster than in the
cuvette (Table 1).

3.2 Model simulations

Model simulations were performed for the same initial/inlet

concentrations of hexanol, ADH and NAD1 (ci,NAD1
5 0.55 mmol/L, gi,ADH 5 0.092 g/L, ci,hexanol 5 5.5 mmol/L).
The kinetic parameters estimated in the cuvette were used for
the simulation of an ideally mixed 10-mL batch reactor, while
the kinetic parameters estimated in the continuously operated
Figure 3. Simulation of hexanol oxidation performed in:
microreactor were used for the simulation of a 6-mL tubular
(A) 10-mL ideally mixed batch reactor using kinetic parameters
microreactor (Table 1, Fig. 3). When the kinetic parameters obtained in a cuvette (ci,hexanol 5 5.5 mmol/L, ci,NAD1
estimated in the microreactor were used for the simulation of 5 0.55 mmol/L, gi,ADH 5 0.092 g/L); (B) 6-mL tubular micro-
hexanal oxidation in the 6-mL tubular microreactor, maximal reactor using kinetic parameters obtained in a continuously
conversion of 10.08% was achieved after 7.8 s (Fig. 3B). The operated microreactor (ci,hexanol 5 5.5 mmol/L, ci,NAD1
model of the batch-operated macroreactor considers reverse 5 0.55 mmol/L, gi,ADH 5 0.092 g/L); (C) 6-mL tubular micro-
reaction and inhibition effects [21]. For the same initial reactor using kinetic parameters obtained in a continuously
concentrations, the simulation of the ideally mixed 10-mL operated microreactor, for equimolar inlet concentrations of
batch reactor resulted in a maximal conversion of 2.13%, substrates (ci,hexanol 5 5.5 mmol/L, ci,NAD1 5 5.5 mmol/L,
which was achieved after 480 s (Fig. 3A). gi,ADH 5 0.092 g/L); () hexanol oxidation, (- - - - ) hexanal
reduction.
When equimolar concentrations of coenzyme and substrate
(ci,NAD1 5 5.5 mmol/L, gi,ADH 5 0.092 g/L, ci,hexanol 5 5.5
mmol/L) were used for the simulation of hexanol oxidation in

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com
54 A. Tusek et al. Eng. Life Sci. 2012, 12, No. 1, 4956

the 6-mL tubular microreactor, the predicted conversion was A

100% (Fig. 3C). Because of the coenzyme price, usage of an
equimolar amount is not economically viable. To ensure the
economics of the process, coenzyme regeneration should be
considered.
The simulation results obtained for the macroreactor and
the microreactor were compared in terms of volumetric
productivities. For the same initial/inlet concentrations, the
simulation of the ideally mixed 10-mL batch reactor resulted
in a volumetric productivity of 1.69 g L1 d1 while the
simulation of the 6-mL tubular microreactor resulted in a
volumetric productivity of 3.97 g L1 d1. When simulation
was performed for equimolar concentrations of coenzyme
and substrate (ci,NAD1 5 5.5 mmol/L, gi,ADH 5 0.092 g/L,
ci,hexanol 5 5.5 mmol/L) in the 6-mL tubular microreactor, a
volumetric productivity of 99.16 g L1 d1 was achieved. This
clearly demonstrates the potential of microreactors as good B
alternative to macroreactors for the enzyme reactions
performed in systems of two immiscible phases.

3.3 Sensitivity analysis

In order to qualify the sensitivity of the model predictions with

respect to the estimated parameter errors, a sensitivity analysis
(SA) was performed. SA studies the relations between the
input and the output of the model. The objective of an SA is to
identify the critical inputs of a model and to quantify how the
input uncertainty impacts on the model outcome(s). When
input factors such as parameters or initial conditions are
known with little certainty, we can examine the partial deri-
vative of the output function with respect to the input factors
[24]. To identify the most important parameters in the
mathematical models of hexanol oxidation in the macro- and
microreactors, FAST analysis was applied. In the case of the
macroreactor ten parameters, and in the case of microreactor
only three parameters, were simultaneously sampled from
interval of values of one order of magnitude around the esti-
mated value. Totally, 2000 samples were generated using
Eq. (6), with uniformly distributed sampling parameters S in
the range of sA[1, 1].
The effects of the kinetic parameters on the reaction rate, r,
and those of the kinetic parameters on the hexanal concen-
tration, as the model output functions, were analyzed. For the
macroreactor model, Vm1, Khexanol m , and KNAD1
m1 have the
highest effect on the reaction rate of approximately 20%
(Fig. 4). Other kinetic parameters have significantly lower
effects (o5%), which clearly indicates the insignificant influ-
ence of the kinetic parameters estimated in a cuvette on the
model simulation results and renders the applicability of the
kinetic measurements in cuvettes questionable.
In contrast, all parameters used for the description of the
assumed kinetics in the microreactor show significant influ-
ence on the process. The kinetic parameter KNAD1
m1 estimated in
the microreactor has the most significant effect on the reaction
rate. When the effects of the kinetic parameters on the hexanal
concentration were analyzed, Vm1 showed the highest effects
in both reactor configurations, the macroreactor and the
microreactor.
Figure 4. First-order sensitivities of (A) reaction rate and (B)
hexanal concentration at the outflow of the reactor. () Kinetic
parameters estimated in a cuvette; (&) kinetic parameters
estimated in the microreactor (ci,hexanol 5 5.5 mmol/L,
ci,NAD1 5 0.55 mmol/L, gi,ADH 5 0.092 g/L). (1) Vm1, (2) Khexanol
m ,
(3) KNAD1
m1 , (4) Khexanal
i1 , (5) KNADH
i1 , (6) Vm2, (7) Khexanal
m ,
(8) KNADH
m2 , (9) Khexanol
i1 , (10) KNAD1
m2 .
3.4 Model validation
In order to validate the developed mathematical model of
hexanol oxidation in the microreactor, experiments were
performed in two different microreactor types with the same
microchannel width and height dimensions (150 mm  150
mm), but with different lengths (323 and 676 mm). Beside the
microreactor type, the inlet concentrations of the substrates
were also altered (experimental conditions 1: ci,NAD1
5 0.55 mmol/L, gi,ADH 5 0.092 g/L, ci,hexanol 5 5.5 mmol/L,
75 mmol/L glycine-pyrophosphate buffer pH 9; T 5 251C;
experimental conditions 2: ci,NAD1 5 4.4 mmol/L, gi,ADH 5
0.092 g/L, ci,hexanol 5 4.4 mmol/L, 75 mmol/L glycine-pyro-
phosphate buffer pH 9; T 5 251C). As shown in Fig. 5, the
developed model well describes the experimental data in all
experiments (experiments in 6 mL, R2 5 0.963; experiments in
13 mL, R2 5 0.904; experiments in 6 mL with equimolar
amounts of hexanol and NAD1, R2 5 0.998), showing that the

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com
Eng. Life Sci. 2012, 12, No. 1, 4956
B Figure 6. Hexanol oxidation simulation performed for different
Figure 5. Conversion of hexanol. (A) In two different types of
microreactors for different residence times; (K) experimental
results for the 6-mL microreactor, (J) experimental results for
the 13-mL microreactor, () model (ci,hexanol 5 5.5 mmol/L,
ci,NAD1 5 0.55 mmol/L, gi,ADH 5 0.092 g/L, 75 mmol/L glycine-
pyrophosphate buffer pH 9; T 5 251C). (B) For different inlet
NAD1 concentrations in the 6-mL microreactor: (K) ci,NAD1
5 0.55 mmol/L, ci,hexanol 5 5.5 mmol/L, (.) ci,NAD1 5 4.4 mmol/
L, ci,hexanol 5 4.4 mmol/L, () model.
kinetic measurements in the microreactors are necessary to
define the exact kinetic conditions. Model simulations
predicted that a conversion of approximately 100% could be
achieved when equimolar amounts of hexanol and coenzyme
are fed into the microreactor, which was also confirmed
experimentally. Although Vrsalovic Presecki and Vasic-Racki
[21] reported that the hexanol oxidation reaction is signifi-
cantly inhibited by the products and by the reverse reaction,
the results obtained for the microreactor did not show this
effect, indicating that the assumption considering the micro-
reactor hydrodynamic conditions due to the slug flow regime
could be a good explanation. It is important to mention again
that a simple steady-state plug flow reactor model was used for
the process simulations, allowing quite simple experiment
planning and result predictions. Despite the model simplicity,
it ensures very good experimental data description.
The validated model and the obtained results were used to
predict the experimental results and hexanol conversion in
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Modeling and kinetic parameter estimation 55
NAD1 inlet concentrations in the microreactor: ()
ci,NAD1 5 0.55 mmol/L, ( ) ci,NAD1 5 1.1 mmol/L, (- - - -)
ci,NAD1 5 2.2 mmol/L, (       ) ci,NAD1 5 4.4 mmol/L,
( ) ci,NAD1 5 5.5 mmol/L. (ci,hexanol 5 5.5 mmol/L,
gi,ADH 5 0.092 g/L, 75 mmol/L glycine-pyrophosphate buffer pH
9; T 5 251C).
correlation with the coenzyme concentration (ci,NAD1
5 0.55.5 mmol/L) (Fig. 6). According to the model, even at
low coenzyme concentrations (ci,NAD1 5 0.5 mmol/L), higher
conversions in comparison with the batch process [21] could
be achieved.
4 Concluding remarks
A mathematical model for ADH-catalyzed hexanol oxidation
in a microreactor was developed. The parameters of the kinetic
model were estimated from the experimental results obtained
in the microreactor. It was observed that the kinetic parameters
obtained in the microreactor under slug flow conditions differ
significantly from the kinetic parameters obtained in the
cuvette batch experiments, which was especially significant for
the estimated maximal reaction rate. The maximal reaction
rate of hexanol oxidation calculated from the results obtained
in the micoreactor was approximately 33-fold higher than the
maximal reaction rate obtained from the experiment in the
cuvettes. Also, in the experiments performed in the micro-
reactor, no product inhibition and reverse reaction were
noticed. These results show that microreactor measurements,
in comparison with cuvette measurements, are more suitable
for the determination of kinetic data of enzyme-catalyzed
reactions.
Nomenclature
Ao [] Fourier coefficient
Bo [] Fourier coefficient
c [mmol/L] Concentration
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56 A. Tusek et al. Eng. Life Sci. 2012, 12, No. 1, 4956

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