Heterogeneous Catalysts “on the Move”: Flow Chemistry with Fluid Immobilised (Bio)Catalysts

Article abstract of DOI:10.1002/ejoc.202000705

As both, continuous synthesis and (bio-)catalysis gained increasing interest in research as well as for industrial applications, ways for merging these fields enables novel opportunities for modern sustainable process development. In this contribution, an alternative approach for the application of immobilized enzymes in continuous flow processes is presented utilizing heterogeneous biocatalysts as a mobile phase. Based on superabsorber-entrapped enzymes and whole cells as a “fluid heterogeneous phase”, a segmented hydrogel/organic solvent system was developed. Its applicability was investigated with two entirely different model reaction systems, namely the alcohol dehydrogenase (ADH)-catalysed reduction of acetophenone, and the aldoxime dehydratase (Oxd)-catalysed dehydration of octanal oxime. In particular for solvent labile catalytic systems, this approach offers an alternative for the application of immobilized biocatalysts in a continuously running process beyond the “classic” packed bed and wall coated reactors.

Full text of DOI:10.1002/ejoc.202000705

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Heterogeneous catalysts “on the move”: flow chemistry with fluid
immobilised (bio-)catalysts
Authors: Niklas Adebar and Harald Gröger
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000705
Link to VoR: https://doi.org/10.1002/ejoc.202000705
0
1/2020

European Journal of Organic Chemistry
10.1002/ejoc.202000705
RESEARCH ARTICLE
Heterogeneous catalysts “on the move”: flow chemistry with fluid
immobilised (bio-)catalysts
[
a]
[a]
Niklas Adebar and Harald Gröger*
A major limitation of all such types of heterogeneous catalysts in flow
is their applicability only for very stable catalytic systems. When
catalysts loose activity over a certain operating time, constant product
quality cannot be ensured. Especially biocatalysts can suffer under
stability issues, particularly in the presence of organic solvents.
Exceptions for extremely long-term stable biocatalysts are, e.g.,
Abstract: As both, continuous synthesis and (bio-)catalysis gained
increasing interest in research as well as for industrial applications,
ways for merging these fields enables novel opportunities for modern
sustainable process development. In this contribution, an alternative
approach for the application of immobilised enzymes in continuous
flow processes is presented utilizing heterogeneous biocatalysts as a
mobile phase. Based on superabsorber-entrapped enzymes and
15
lipases like the widely applied lipase from Candida antarctica B (CAL-
16,17
B), which can be used in organic solvents.
whole cells as
a “fluid heterogeneous phase”, a segmented
hydrogel/organic solvent system was developed. Its applicability was
investigated with two entirely different model reaction systems,
namely the alcohol dehydrogenase (ADH)-catalysed reduction of
acetophenone, and the aldoxime dehydratase (Oxd)-catalysed
dehydration of octanal oxime. In particular for solvent labile catalytic
systems, this approach offers an alternative for the application of
immobilised biocatalysts in a continuously running process beyond
the “classic” packed bed and wall coated reactors.
Figure 1. Approaches for application of immobilised catalysts in flow mode.
A: Standard approaches such as packed bed, monolithic or wall coated reactors.
B: Our approach based on catalytic hydrogel segmented flow.
Introduction
Over the past years, flow chemistry has gained increasing interest in
An alternative concept in flow chemistry is the use of homogeneous
catalysts in the fluid phase. In case of biocatalysis, segmented flow
processes consisting of alternating aqueous and organic phase
compartments became very popular and a range of such continuous
processes for enzymatic catalysis by means of segmented flow have
1-4
academia as well as industry.
As pointed out from regulatory
agencies such as the FDA or EMA, one desired key feature of such
continuous flow processes in the pharmaceutical industry is to ensure
5,6
a
constant product quality.
In particular the combination of
18-21
extremely selective catalysis and continuous manufacturing can yield
in high-quality products with high reliability. As these kinds of systems
offer advantages over classical batch processes, this research area
been recently described.
Such a concept would also fulfil the
regulatory issues for constant product quality by definition because all
components of the reaction are continuously pumped in and removed
from the reactor. However, in this case a concern in the field of
biocatalysis is often limited stability of the enzymes in particular in the
1-4
flourishes. When it comes to the use of catalysis in flow, in particular
heterogenized catalysts turned out in many cases as preferred form
of the catalysts due to, e.g., increases stability and simple separation
from the product mixture. However, at the same time applications with
such heterogeneous catalysts are surprisingly limited to a few
established concepts. The most prominent ones of them are
22
presence of an organic solvent. By immobilising biocatalysts, the
stability towards organic solvents often can be improved
23
significantly. In this context, the superabsorber-based encapsulation
24,25
of enzymes or whole cells in a packed bed reactor
has recently
7
visualized in Figure 1 and briefly summarized in the following.
been investigated among many other systems based on, for example,
26-31
32,33
carrier-immobilization
or immobilization in alginate beads.
Packed bed reactors are among the most popular and successfully
8,9
applied systems. Major drawbacks of the immobilised, and therefore
often highly modified catalysts, is the limitation to very stable
catalysts. In addition, technical disadvantages like pressure drops,
which might complicate scale-up enormously, are known. Monolithic
reactors are another commonly used methods for application of
heterogeneous catalysts in microreactor chemistry. However, the
technique does not significantly differ from packed bed reactors,
What would be desired is to make use of both advantages, namely on
the one hand the use of a biocatalyst being stabilized through
heterogenization and on the other hand its utilization as a fluid phase
in a segmented flow fashion (as it known for homogeneous catalysts).
Although on the first glance such two catalytic concepts appear not to
be compatible with each other, we envisioned that this gap could be
closed by immobilising the entire aqueous phase including the
catalyst(s) as a hydrogel and by utilizing the resulting hydrogel
catalytic phase subsequently in a segmented flow process (Figure 2).
In detail, the biocatalyst is immobilized in a superabsorber-gel matrix,
which can be considered as compartmentalized aqueous phase. It is
noteworthy that this superabsorber-gel can then be utilized as a
mobile phase, leading to a segmented flow process when being
utilized together with another mobile, organic phase containing the
hydrophobic substrate.
10
except for the usually much smaller diameter of the reactor tube. Yet
11,12
another method is coating
and modification of the reactor wall
surface, or even the use of a catalytic active metal as reactor material.
Recently, copper-catalysed click reactions have been employed in a
13
copper tube, which acts as a catalyst. Coated wall reactors are also
14
commonly used for several biocatalytic systems.
In the last time many catalytic hydrogel systems have been reported,
emphasising the potentially broad applicability of this system.
[
a]
N. Adebar, Prof. Dr. H. Gröger
Faculty of Chemistry, Industrial Organic Chemistry and Biotechnology
Bielefeld University
Universitätsstraße 25, 33615 Bielefeld (Germany)
E-mail: harald.groeger@uni-bielefeld.de
32 -37
Despite probably being as well exciting, we did not investigate the
catalyst recyclability in this study, as we were rather interested in a
process delivering constant product quality. If the catalysts were
reused, it would not necessarily perform equally and would be
changed from the original catalyst formulation.
Supporting information for this article is given via a link at the end of the
document.
1
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000705
RESEARCH ARTICLE
water bath. Fractions of the reaction mixture were collected in glass
vials (containing an HCl solution for instant quenching of the reaction
mixture). Apart from phase separation by decantation and solvent
removal under reduced pressure, no further workup to isolate the
products was necessary. For continuous use, a gravity phase
separator could be used as an option in the future since membrane
separators are likely to clog. Traditionally, the phase separation of
biocatalytic reaction mixtures in batch (based on whole cells as well
as isolated enzymes) is tedious due to intensive emulsification, and
significant improvement addressing this issue has been shown when
applying segmented flow processes. A further advantage of the this
superabsorber-based segmented flow system is the simple reactor
set-up.
Wet whole cells (containing the OxdB enzyme as a “model system” in
initial experiments) were dispensed in buffer and immobilised by
adding superabsorber powder (ground Evonik Favor SXM 9155,
particle size < 100 µm) while strongly mixing the solution with a
spatula. Initial tests with the superabsorber have shown the
importance of adjustment of the superabsorber amount in relation to
the biomass loading, buffer concentration and amount of aqueous
solution as these aspects affect the hydrogels consistency. As the
absorbent polymer consists of partially neutralised, cross-linked
polyacrylic acid, enzymes and whole cells can be embedded in this
polymer matrix. The basic principle of this set-up is shown in Scheme
1
. Compared to traditional methods for immobilization the applied
procedure is very fast and the superabsorber polymer is economically
superior to most polymer-based carriers on the marked. Furthermore,
the reactor itself is very cost-attractive.
hydrogel
catalyst
Figure 2. Illustration of the concept of flow chemistry with fluid immobilised
(
bio-)catalysts.
product
We herein introduce the realization of this flow concept exemplified
for two (bio-)catalytic model processes (shown in Figure 3). Firstly, an
enzyme-catalysed reduction of acetophenone (1) to (R)-1-
phenylethanol (2) in the presence of an alcohol dehydrogenase
starting
material
organic
38-40
hydrogel organic
(
ADH),
and, secondly, an enzyme-catalysed dehydration of
octanal oxime (3) to octanenitrile (4) in the presence of aldoxime
41-44
dehydratase (Oxd) containing whole-cells.
Both types of reactions
gained broad interest in biocatalysis and led, in case of the
biotransformations with alcohol dehydrogenases, already to industrial
applications.
Scheme 1. Schematic setup of the developed hydrogel/organic
segmented flow system consisting of two syringe pump feeds, a PFE tube
reactor and a reservoir for product recovery, respectively quenching.
44,45
A | Ketone reduction
Furthermore, the inner diameter of the tubular reactor, as well as the
Y-mixer have shown to be crucial. Initial experiments on separation
were carried out using a colourised superabsorber/MTBE segmented
flow system (Figure 4).
O
OH
alcohol dehydrogenase (ADH)
1
(R)-2
It is noteworthy, that in these experiments it could be demonstrated
that by means of such a set-up the superabsorber hydrogel can be
utilized as a “standard mobile phase”. Therefore, it can potentially
serve as a technology basis for the subsequent extension of this set-
up towards other desired biotransformations with an “immobilized”
enzyme-containing aqueous phase being entrapped in the
superabsorber and an immiscible organic mobile phase containing the
substrate.
B | Aldoxime dehydration
N
OH
N
aldoxime
dehydratase (Oxd)
3
4
Figure 3. Investigated model systems: A) ADH-catalysed reduction of
acetophenone (1) to (R)-1-penylethanol ((R)-2) and B) OxdB-catalysed
dehydration of octanal oxime (3) to octanenitrile (4).
Results and Discussion
The first step consisted in the proof-of-concept for the general
feasibility of such a concept utilizing a superabsorber-hydrogel matrix
with an entrapped biocatalyst as a fluid hydrogel phase in a
segmented flow process with an immiscible organic phase. The
developed system addressing this concept consists of two syringes
containing either organic or hydrogel reaction solution mounted on a
syringe pump. The two streams were fed to a Y-mixer (ID = 1 mm) in
which segments were produced and led to a tubular poly(fluorenylene
ethynlene) (PFE) reactor (ID = 1.01 mm), which was tempered by a
Figure 4. Superabsorber/MTBE segmented flow system with colourised
superabsorber gel (using Coomassie blue) in a PFE tube (ID = 1/32 “).
2
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European Journal of Organic Chemistry
10.1002/ejoc.202000705
RESEARCH ARTICLE
After these successful initial experiments regarding general behaviour
of both phases, the first reaction system, namely the reduction of
acetophenone (1) to (R)-1-phenylethanol (2) catalysed by an alcohol
indicated with dashed vertical lines (residence time = 2 h, cat. loading =
2 U mL , T = rt, csubstr./overall volume = 50 mM).
-1
38-40
dehydrogenase from Lactobacillus brevis (LB-ADH),
investigated. Over the past years, in general alcohol dehydrogenases
ADHs) have turned out to be an efficient tool for the synthesis of chiral
was
Compared to the batch process under identical conditions (apart from
different hydrogel to organic phase ratios), the conversion utilizing this
segmented flow system with a superabsorber-immobilised ADH and
GDH as a mobile phase turned out to be the same (Figure 6), thus
(
3
7
alcohols via ketone reduction. One popular and quite robust
representative of this enzyme class is this LB-ADH, which was
reported by the Hummel group.
proofing the feasibility of this segmented flow-concept with
a
3
8,39
heterogenized mobile (bio-)catalyst phase. In addition, also the
efficiency (product per catalyst) and volumetric productivity (space-
time-yield; product per reactor volume and time) of batch and flow are
identical. The volumetric productivity of the process (based on the
average conversion per reactor volume and time) has been calculated
As the cofactor NADP(H) is cost intensive, an efficient cofactor
regeneration system utilising glucose dehydrogenase (GDH) was
used. Catalysed by the GDH, glucose is converted to gluconolactone,
which irreversibly opens to gluconic acid, thus shifting the equilibrium
towards the desired product (Scheme 2). As the hydrogel itself acts
as a buffer system in our experiments, however, the pH value is
difficult to analyse and predict.
-1 -1
to be 1.2 g h L .
O
OH
ADH from L. brevis
1
(R)-2
NADPH
NADP+
glucose
gluconolactone
gluconic acid
GDH
Scheme 2. Schematic hydrogel/organic segmented flow process for the
enzymatic reduction of acetophenone (1) to 1-phenylethanol (R)-2.
The amount of superabsorber had to be carefully adjusted to the
reaction system as ions, such as the used potassium phosphate
buffer (PPB), have a huge influence on the viscosity of the hydrogel.
The more ions, the more superabsorber is needed for absorbing the
aqueous solution. In an exemplary reaction, a catalyst loading of
-1
2
U mL and a residence time of 2 h was used (Figure 5). As shown
in Figure 5, the system could be operated stable for at least 6.5 h with
an average conversion of 40%, after an equilibration time of two
residence times, to ensure steady state.
Figure 6. Conversion of acetophenone (1) to 1-phenylethanol (R)-2 using
superabsorber-immobilised ADH with co-immobilised GDH-based cofactor
regeneration system in a superabsorber/MTBE segmented flow system vs.
batch approach using the same conditions (residence time = 2 h, cat.
-1
loading = 2 U mL , T = rt, csubstr./overall volume = 50 mM).
With these results in hand, another catalytic system, namely the
dehydration of oximes catalysed by an aldoxime dehydratase (Oxd)
41-44
under formation of nitriles,
was investigated. Oxd-type enzymes
recently gained much attention as catalysts enabling a cyanide-free
synthesis of nitriles. They can be used efficiently not only for the
synthesis of chiral nitriles, but also for bulk chemicals such as n-alkyl
nitriles or adiponitrile. Starting from the corresponding aldoximes
being accessible via simple condensation of aldehydes with
41-44
hydroxylamine, the nitriles are formed via dehydration.
At the
same time, this Oxd enzyme class is an example for a highly solvent
40
44
labile type of biocatalyst. In detail, batch experiments in biphasic
systems of the OxdB-catalysed dehydration of n-octanal oxime (3)
have shown to be very challenging due to instability of OxdB
containing whole cells in organic media without immobilisation. When
this system was applied as superabsorber immobilised cells in a
44
packed bed reactor, the stability of the system was limited.
Therefore, an alternative solution for a continuous application of the
reaction system must be developed. To face this challenge of solvent
instability whilst running the process continuously, we became
interested to explore if our new system based on mobile phases would
be an alternative to standard approaches such as packed bed
reactors. The reaction was conducted using the same setup as
previously described with only minor adjustments of the amount of
superabsorber. Since the system is not cofactor-dependent, only wet
Figure 5. Conversion of acetophenone (1) to 1-phenylethanol (R)-2 using
superabsorber-immobilised ADH with co-immobilised GDH-based cofactor
regeneration system in a superabsorber/MTBE segmented flow system.
Horizontal lines show conversion for fractions collected between times
3
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000705
RESEARCH ARTICLE
cells containing OxdB were immobilised in presence of buffer. In this
system, the liquid catalyst phase appeared to be more viscous then
observed previously, causing inconsistent segment sizes.
A
residence time of 30 min together with a catalyst loading of 33 mg wet
cells per mL was chosen as well as a substrate concentration of 100
mM for the organic phase (thus, leading to an overall substrate
concentration of 50 mM when considering both phases). We were
pleased to find that under such conditions, a stable process was
obtained over an operating time of 6 h, leading to an average
conversion to the desired nitrile 4 of 53% after initial equilibration
(
Figure 7). Despite a non-uniform segment size in this initial
experiment utilizing the Oxd whole cells, the conversion appeared to
be very stable over the entire investigated time. The average absolute
deviation is only 1%, which indicates a reliable working process over
the course of a 10-fold residence time (after equilibration).
Figure 8. OxdB-catalysed dehydration of octanal oxime (3) to octanenitrile
(
4) in a cyclohexane/superabsorber segmented flow system (residence
-1
time = 30 min, T = 30 °C, cat. loading = 250 mgwcm mL ).
Conclusion
In conclusion, for the first time a flow process has been reported,
which merges the fields of using an immobilised biocatalyst being
stabilized through heterogenization with its utilization as a fluid phase
in a segmented flow fashion (as it is known for homogeneous
catalysts). The entire aqueous phase including the biocatalyst has
been immobilised within a superabsorber-based hydrogel, which then
was used as a mobile catalytic phase in a segmented flow together
with an immiscible organic phase containing the hydrophobic
substrate. The developed system was proven for two fundamentally
different biocatalytic reactions, namely an ADH-catalysed ketone
reduction and an Oxd-catalysed nitrile synthesis. Simple downstream
processing, high operational stability, fast and simple setup, and
economical attractiveness are among the advantages of the
developed system. Especially, for instable heterogeneous catalysts it
offers an alternative to standard approaches such as packed bed or
wall coated reactors. Compared to batch approaches, the system
does not lack behind in conversion. In current work, we are focusing
on the expansion of this flow system towards further
biotransformations as well as on the extension towards
chemocatalytic applications. Furthermore, recyclability via a catalyst
recycling loop would be an interesting option, in particular in
combination with a continuous phase separation, and will be studied
in future work.
Figure 7. OxdB-catalysed dehydration of octanal oxime (3) to octanenitrile
(
4) in a cyclohexane/superabsorber segmented flow system (residence
-1
time = 30 min, T = 30 °C, cat. loading = 33 mgwcm mL ).
After demonstrating the general applicability of the developed system,
we tried to push the limits of the investigated OxdB-catalysed
dehydration of n-octanal oxime (3). Toward this end, we increased the
catalyst loading and improved the segmentation by using a tube with
wider inner diameter (1 mm) and grinding the superabsorber finer
(
< 80 µm). At the same time, the reaction temperature, substrate
concentration and residence time were kept constant. To our delight,
the process turned out to be robust and could be successfully
optimized also in terms of conversions (Figure 8). In detail, the
conversion of this whole cell-catalysed biotransformation leading to
the corresponding nitrile 4 was improved to > 90%. The volumetric
productivity based on the average conversion was calculated to be
-1
-1
-1 -1
7
.6 g h L and could be optimized to 12.9 g h L , which shows that
the productivity can be increased by scale up at least to some extent.
Furthermore, the overall productivity could be further increased by a
numbering up of the reactor channels. Regarding the theoretical
catalyst productivity (calculated from average conversion), 0.1 mg of
product could be obtained from 1 mg of well cell mass.
Acknowledgements
N.A. and H.G. gratefully acknowledge generous support from the
European Union (EU) within the EU-Research Project ONE-FLOW
(
“Catalyst
Cascade
Reactions
in
‘One-Flow’
within
a
Compartmentalized, Green-Solvent ‘Digital Synthesis Machinery’ –
End-to-End Green Process Design for Pharmaceuticals”), Work
programme: EU proposal 737266. We furthermore thank Manuel
Warkentin and Yannick Günzel for technical assistance.
Keywords: flow chemistry • immobilisation • heterogenous
catalysis • biocatalysis
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10.1002/ejoc.202000705
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