C=3

Enzymatic membrane bioreactors

and their applications
D. M. F. Prazeres and J. M. S. Cabral

Laborat6rio de Engenhana Bioqulmica, Instituto Superior TOcnico, Lisbon, Portugal

The basic concepts, advantages, problems, and applications of enzymatic membrane reactors are reviewed. A broad
classification of this type of reactor is proposed based on the type of contact between enzyme and substrates that occurs
in these devices. Applications of membrane reactors for enzymatic reactions published in the scientific literature in the
last decade are presented and discussed. Some comments on the state of the art and future prospects of this area are
also outlined.

Keywords: Membrane reactor; ultrafiltration; enzymatic reactors; process integration; bioreactors

Introduction Membrane reactor concept

Biocatalysis involves the enzyme-promoted transformation The basic concept of membrane reactors is based on the
of a substrate into useful products, in either a homogeneous separation of enzyme and products (or substrates) by a
or a heterogeneous system. As in all other chemical pro- semipermeable membrane that creates a selective barrier.
cesses, the separation of reactants from products and the Permeable solutes can be separated from the reaction mix-
recovery and reuse of the catalyst from the reaction mixture ture by the action of a driving force (chemical potential,
is an important step which significantly adds to the cost of pressure, electric field) that is present across the mem-
the process. Membrane reactors constitute an attempt to brane. Membranes can also be used in a reactor exclusively
integrate catalytic conversion, product separation and/or as a matrix for immobilization of the enzyme, without any
concentration, and catalyst recovery into a single operation. separation intentions. 7-1° In recent years, the functions of
Most enzymatic processes currently in industrial use are the membrane have been extended with the systematic use
carried out in batch reactors. 1,2 However, batch reactors of these reactors in two-phase bioconversions. Here, the
suffer from a number of well-documented limitations, such membrane acts as a support for the interface between two
as batch-to-batch oscillations, high labor costs, frequent distinct liquid phases. The membrane not only separates
startup and shut-down procedures, and the need to recover the phases, but also provides interfacial contact area and,
the enzyme, or enzyme preparation, after each batch.1 together with the enzyme, acts as an interfacial catalyst. 11-14
By immobilizing the enzyme, it is possible to operate A complete retention of the enzyme within the system is
enzymatic processes continuously with all the attendant ad- the first and most important requirement for a successful
vantages, such as better process control, higher productiv- continuous operation of a membrane reactor. Upon this
ity, more uniform products, and the integration of a purifi- retention, the enzyme becomes confined to a defined region
cation step in the process. 1,3 Enzyme immobilization has of the membrane reactor, where reaction with the substrate
been accomplished by chemical and physical attachment to occurs.
solid surfaces. 4,5 The advantages of using a membrane as The enzyme is usually present in two forms: soluble or
this solid surface were first pointed out by Rony. 6 Since that insolubilized at the surface, or inside the pores or mem-
time, a number of membrane reactor configurations have brane matrix. If the enzyme is in the free form, the immo-
been suggested, with the membrane offering additional ser- bilization is achieved by confining the enzyme to one side of
vices, such as product separation, phase separation, etc. the membrane. This can be done by size exclusion, 15-19elec-
trostatic repulsion, 2°,21 or enlargement through chemical or
physical immobilization onto an intermediate support (inert
protein, 22 gels, 23 liposomes 24 ).
Address reprint requests to Dr. Cabral at the Laborat6rio de Engenharia
The immobilization of the enzyme directly onto the
Bioquimica, Instituto Superior T6cnico, 1000 Lisbon, Portugal membrane can be achieved by chemical binding, 25-29
Received 9 June 1993; accepted 31 January 1994 physical adsorption, 10,30-36 or electrostatic attraction. 7,8

738 Enzyme Microb. Technol., 1994, vol. 16, September © 1994Butterworth-Heinemann

 Enzymatic membrane reactors: D. M. F. Prazeres and J. M. S. Cabra/
In multiphase reactors the enzyme is usually found im- solute rejection coefficient, which should be zero for the
mobilized on the side of the membrane that faces the hy- products to facilitate permeation, and should be one for
drophilic phase, or alternatively, inside the membrane. In the enzyme to insure a complete retention of the catalyst
certain cases (e.g,, lipases), the enzyme is immobilized on inside the reaction system.
the hydrophobic side of the membrane. 37-4° The selectivity of a membrane toward a solute is nor-
The products resulting from catalysis should permeate mally associated with a discrimination according to size:
through the membrane pores, either by diffusion (induced molecules smaller than the micropores are not retained.
by concentration gradient) or convection (usually induced Apart from this effect, a steric exclusion process may be
by a pressure gradient). In this way a continuous removal of present for those molecules with sizes inferior but close to
products from the reaction media is attained. This perme- the dimensions of the pores. 43 However, the chemical na-
ation of products in membrane reactors has always been ture of the membrane can interfere with solute perme-
regarded as an essential requirement in this kind of reactor ation due to nonspecific interactions (electrostatic, hy-
and is traditionally part of the membrane reactor concept. drophobic, and hydrophilicz4) which lead to the
However, in the case of low-solubility precipitating prod- formation of a second layer (gel layer) that decreases the
ucts, complete rejection of solid products might turn out to permeation (concentration polarization phenomena).
be an unexpected advantage of these reactors. In such a This is especially true when chemical reaction and mem-
case, the product of interest is recovered behind the mem- brane separation occur simultaneously.
brane and remains in the reactor during the course of op- The majority of commercially available UF membranes
eration. A membrane reactor of this kind for the enzymatic are asymmetric, i.e., the pore size varies continuouslyin one
synthesis of peptides by a-chymotrypsin is currently under direction. The surface of these membranes is formed by an
investigation in our laboratory. 41 Substrate molecules may ultrathin layer that lies upon a sublayer of higher porosity.
be either freely permeable or impermeable. Due to their unique structure, these membranes are less
Ultrafiltration (UF) membranes, due to the range of susceptible to clogging, allow higher permeate flow rates,
pore size distribution displayed (1-100 nm or NMWCO and are easier to clean. 45,46The materials most often used
500-100,000 daltons), are most adequate for the retention in the manufacture of UF membranes are synthetic poly-
of the majority of enzymes (10,000-100,000 daltons), mod- mers and certain ceramic materials (Table 1). 44,47Ceramic
ified or native. These membranes are usually classified ac- membranes, compared with polymeric membranes are
cording to nominal molecular weight cutoff (NMWCO), generally more resistant to high temperatures and chem-
which is defined as the molecular weight of a macrosolute icals, and mechanically stronger under pressure. Partic-
capable of being rejected at 90% under well-defined filtra- ular attention should be paid to the selection of the mem-
tion conditions. 42 brane material for a particular enzymatic process, since it
The selection of a membrane to be used in enzymatic can significantly affect enzyme stability. 46,48 This choice is
membrane reactors should take into account the size of the usually made by trial and error and is based on character-
enzyme, substrate(s), and product(s) as well as the chemical istics such as morphology, porosity, pore size distribution,
nature of the species in solution and of the membrane itself. molecular weight cutoff, chemical resistance, temperature,
An important parameter to be used in this selection is the pH and pressure tolerance, and price. 42,45 The particula,

Table 1 Characteristics of membrane reactor concept: alternatives and representative references

Function of Retention of Location of Product separation Membrane UF membrane

membrane enzyme enzyme mechanism modules a material

Semipermeable Size exclusion 1s-19 Solution Sieving 81,82 Plate and frame 32 Ceramic 25,41,44
barrierS0,81 (free) 54
Immobilization Electrostatic Solution Solution Tubular 44 Cellulose 37,39
matrix 7-10 repulsion20, 21 (enlarged) 24 diffusion 16,18
Phase Electrostatic Membrane matrix Precipitation 4~ Hollow Fibers TM Nylon 26
separator 11-13,32 attraction 7,8
Physical Membrane surface 31 Electrical charge b Spiral w o u n d 1° Polypropylene 1°,32
adsorption 10,30-36
Chemical Dynamic module b Polyamide 15,1°°
binding 25-29
Enlargement 22-24 Ultrafiltration Polysulfone 24,54,8°
cell~5 Polytetrafluor-
ethylene7, TM
Cellulose acetate 73

aSee Tables 5 - 7 for additional references

bNot described in the literature

Enzyme Microb. Technol., 1994, vol. 16, September 739

Review

case of multiphase reactors has been discussed by Vaidya et a)

al. 49 who present some rules of thumb for the choice of S P
material based on the necessity to maintain a stable inter-
face between the two phases.
U F membranes are usually assembled and associated in
a module with a determined geometry offering distinct flow
U'F unit
zones for the feed and permeate streams 47,5° (Table 1). The
modules can be combined and integrated with pumps,
valves, and tanks in numerous ways, thus forming a mem-
brane reactor.
Table 1 lists some of the important characteristics of
membrane reactors described in this section.

Classification of membrane reactors b)

P
One of the criteria classically used for the classification of
membrane reactors is based on the type of configuration/ S
hydrodynamics of the system. 1,3 According to this classifi-
cation, membrane reactors are globally divided into CSTR LTF unit
(continuous stirred-tank reactor) and plug flow types, each
of these categories being further subdivided. The mem-
brane reactors in each of the above-mentioned classes also
share another characteristic1,3: in plug flow reactors, the
c)
substrate molecules have to diffuse across the membrane to
reach the enzyme, but in the CSTR type, the substrate
P
molecules become directly available to the catalyst as soon
as they enter the system.
Some membrane reactors, however, with regard to the
particular flow regime displayed in the ultrafiltration unit,
fall outside this classification (e.g., flat membrane, U F mod- S
ules operated in dead-end mode). For this reason and also
because of the advent of multiphase membrane reactors,
we propose a broader classification of membrane reactors, LTY unit
based on the mechanism by which enzymes and substrates
are brought into contact (Table 2).

Direct contact membrane reactors Figure 1 Direct contact membrane reactors: (a) CSTR recycle, (b)
dead-end and (c) dialysis membrane reactors. O, Enzyme; S, sub-
A first type of membrane reactor promotes what might be strate; P, product
called direct contact between the enzymes and the sub-
strate (i.e., substrates are introduced on the side of the
membrane containing the enzyme). In this type of reactor, A recycle membrane reactor is typically formed by a
soluble enzymes can act directly on the substrate as soon as stirred vessel coupled to an ultrafiltration module in a semi-
it enters the system. The enzyme may be either immobilized closed loop configuration (see Figure la). The enzymatic
or in the free form. This class of reactors can be further solution (if adsorption of the enzyme is not present), to-
divided into recycle, dead-end, and dialysis reactors (Table gether with the substrate, is continuously recycled from and
2). Table 5 lists several applications of direct contact mem- to the vessel through the ultrafiltration unit. If the enzyme is
brane reactors described in the literature. immobilized on the membrane surface (or interior), exter-
nal diffusional limitations can be reduced by high recycling
flow rates. 26 The membrane modules commonly used in
this type of reactor include tubular membranes and hollow
Table 2 Classification of membrane reactors based on the mecha- fibers. In spite of the presence of a membrane module,
nism by which enzymes and substrates establish contact (see Fig- studies on the residence time distribution in these recycle
ures 1-3 for a complete description of each subclass)
reactors indicate that the whole system can be considered
as a single entity behaving like an ideal CSTR. 5a-53 This
Contact by configuration is widely used and referred to in the
Direct contact diffusion Interracial contact literature. 22,26,35,36,41,44,45,51-56
Dead-end membrane reactors, which include the wide-
Recycle Single pass Dual single-pass
Dead-end Single-pass/recycle Single-pass/recycle spread ultrafiltration cells, are also included in this class. In
Dialysis Dual-recycle Dual-recycle this case, separation and reaction occur in the same com-
partment and the reaction media is pressurized against the

740 Enzyme Microb. Technol., 1994, vol. 16, September

 Enzymatic membrane reactors: D. M. F. Prazeres and J, M. S. Cabral
membrane and forced to flow through the micropores (see a)
Figure lb). This type of operation can be carried out in a S
common ultrafiltration module by sealing the appropriate
ends of the unit. 27 Ultrafiltration cells are essentially oper-
ated as CSTRs, and the permeation of solutes is achieved by
conventional filtration through a flat membrane placed per- UT unit
pendicularly to the reactor bottom. Despite a very low ratio
of membrane surface area to reactor volume and low per-
meation fluxes caused by concentration polarization, this
configuration is extensively used in laboratory-scale studies
to test operation concepts and investigate mechanisms of
enzyme action, probably due to the simplicity of
operation. 15,21,24,48,54,57-66 b)
Dialysis reactors are operated as dialyzers: the substrate
is introduced on the side of the membrane containing the
enzyme, and the products formed are transferred to the
other side of the membrane as a result of a concentration
gradient (solution-diffusion). The process streams in each S
side of the membrane are circulated at approximately the
same flow rate, thus minimizing convective flow (see Figure
lc). The disadvantages of this type of configuration are
mainly a consequence of a less efficient mass transport due
to an operating mode based on diffusion. One example of
direct contact, dialysis reactors, has been described in the
literature. 67

Diffusion membrane reactors

A second class of reactors promotes enzyme-substrate con-
tact after a simple passive diffusion step of substrate mole-
cules through the membrane micropores to the adjacent
compartment where the enzyme is located (soluble or im-
mobilized) (see Figure 2). This means that these reactors
(diffusion membrane reactors) can only process low-
molecular-weight substrates. After catalysis, the products
diffuse back to the unreacted substrate stream that circu- S
lates by. Hollow-fiber modules are mostly used in this type
of reactor, with the enzyme usually placed on the shell side UT~it
of the module. These reactors act essentially as dialyzers, as
solutes are transferred through the membrane under a con-
centration driving force, rather than pressure. This class of
diffusion membrane reactor, when compared with the pre-
vious one, presents some disadvantages. These are related
Figure 2 Diffusion membrane reactors: (a) single-pass, (b) single-
to the fact that diffusion is the dominant transport mecha- pass/recycle, and (c) dual-recycle membrane reactors. 0, Enzyme; S,
nism for substrates. For instance, the kinetic behavior of substrate; P, product
enzymes in these reactors is inferior when compared with a
free enzyme because the permeation of the substrates
across the membrane is often a rate-limiting step. In addi-
tion, the control of the environmental conditions in the vicinity conversion degrees by operating the reactors with a periodic
of the entrapped enzyme is also limited by the slow transport pulsating inlet flow. The effectiveness of the operation was
of other chemical species in the media. 68,69 The relatively found to be dependent on the relative increase of the convec-
small number of publications reporting the experimental op- tive transport due to the induced ultrafiltration over the
eration of such a type of membrane reactor 16.18
, ,19,70- 73 prob- diffusion-moderated transport. 77
ably reflects these disadvantages (see Table6 for a description Subdivisions in the class of diffusion membrane reactors
of applications). Theoretical investigations of these reactors can be made according to the trajectory of the process
have also been made by several authors. 17,69,74,75 streams adjacent to the membrane (see Table 2 and Figure
Some authors have investigated methods to induce ultrafil- 2). The enzyme-containing stream is either confined 19,75
tration convective fluxes in this type of diffusion reactor in (see Figure 2a) or recirculated through the system in a
order to overcome the above-mentioned mass transfer closed loop coupled with an external vessel 16.18 , ,71,73 (see
disadvantages. 16,76,77 Experimental 16,77 and theoretical 76 Figure 2b). The substrate-containing stream can flow
studies showed that significant increases could be obtained in through the membrane module in a single pass 18,19,71,73

Enzyme Microb. Technol., 1994, vol. 16, September 741

Review
Table 3 Advantages and disadvantages of membrane reactors

Advantages Disadvantages

Opportunity to develop continuous processes Unfavorable adsorption and poisoning of enzyme

Higher productivity Deactivation of enzyme by shear-related effects
Better control possibilities ProductJsubstrate inhibition at the membrane surface
Integration of unit operations Loss of enzyme activators or cofactors
Shift of chemical equilibrium Concentration polarization
Improved rates in product-inhibited reactions Fouling
Enrichment and concentration of products in process streams Leakage of enzyme
Control of the molecular weight of hydrolyzates
Possibility of conducting multiphase reactions
Refined research tool for enzyme mechanisms

(Figure 2a and b) or, alternatively, be recirculated using an productivity and possibly to the economic viability of the
external vessel 16,75 (Figure 2c). process. This is common to all continuous reactors when
compared to batch systems.
Multiphase membrane reactors Other important advantages stem from the continuous
Finally, multiphase membrane reactors are included in a selective removal of products from the reaction media. For
class of reactors capable of promoting interfacial contact instance, in those cases where chemical equilibrium affects
between enzyme and substrates at the membrane matrix the yield of reactions, a selective separation of products
(Figure3). Substrates and products may be located in either against the substrates (i.e., the substrate is retained behind
phase, and diffusion is again the dominant transport mech- the membrane in a higher degree when compared to the
anism together with interracial transport. In these cases, the product) can contribute to a favorable shift of the equilib-
membrane usually acts as a support for the interface be- rium towards the product side. 79
tween two distinct liquid phases (polar and apolar) that There is also the generalized idea that in membrane
constitute reservoirs for substrates and/or products. The reactors, as opposed to batch reactors, higher conversion
hydraulic pressure caused by circulation of the two phases and reaction rates can be obtained as a result of the contin-
is, in principle, sufficient to maintain a good phase separa- uous removal of inhibitory products from the reaction me-
tion at the membrane. 78 In some cases, a slight positive dia through the membrane pores. 15,79
pressure may have to be applied in order to keep the inter- Membrane reactors may display additional advantages
face in the plane of the membrane and prevent the phases in multiproduct systems. In such cases, if the membrane
from mixing. exhibits some selectivity toward the products, an enrich-
Figure 3 represents three different configurations, in the ment of the product that is less rejected can be obtained in
multiphase membrane reactor class, for a hypothetical case the outlet process stream.t1 On the other hand, if a product
of a two-substrate, two-product reaction. Each of the pro- is rejected by the membrane, it can be concentrated inside
cess streams adjacent to the membrane may flow through the system. This may be disadvantageous if product inhibi-
the module in a single pass 31-33,4° (Figure 3a) or be recircu- tion is present.
lated with external vessels 12,3° (Figure 3c). Configurations One of the early-recognized advantages of membrane
with a single pass of one of the streams and recirculation of reactors was detected in the hydrolysis of macromolecules.
the other are also used 34 (Figure 3b). The membrane not In these cases, a membrane with the correct cutoff usually
only separates the two immiscible phases but also provides permits some control of the molecular weight of the hydrol-
interfacial contact area and, together with the enzyme, acts yzates; this leads to an increase in lower-molecular-weight
as an interfacial catalyst. These multiphase membrane re- components in the permeate stream and to a concentration
actors have been mostly used, and seem specially indicated of the heavier products behind the membrane.l.8°-82
when interfacial activation is needed for the enzyme action As already mentioned above, membrane reactors also
(e.g., lipases and phospholipases). Table 7 lists several ap- offer the possibility of conducting two-phase reactions,
plications that have been described in the literature. without the emulsification problems, namely, the inactiva-
tion of the enzyme related to the intensive agitation neces-
Advantages and disadvantages of membrane reactors sary to make and maintain emulsions and the high power
requirements (hence, energy costs).
Advantages At a laboratory scale, ultrafiltration membrane reactors
Membrane reactors have several intrinsic advantages that have also proved themselves as a useful research tool for the
make them a possible alternative system when compared study of phenomena related to enzyme mechanisms, such
with other, more conventional, enzymatic reactors (e.g., as reaction kinetics, product inhibition effects, and enzyme
batch, fixed, and fluidized beds) (see Table 3). deactivation processes. 48,57,83
One of the great advantages of membrane reactors is The use of a membrane as an immobilization technique
undoubtedly the possibility of a continuous, and thus inten- presents advantages compared to enzyme supports, such as
sive, use of enzymes, which contributes to an increase in porous particles or beads, that may overcome the higher

742 EnzymeMicrob. Technol., 1994, vol. 16, September

 Enzymatic membrane reactors: 13. M. F. Prazeres and J. M. S. Cabral

P2 substrates and products can be readily transported to and

swept away from the enzymes. This may be especially im-
portant in product-inhibited reactions. 84 This enhance-
ment of mass transfer may be responsible for increased
productivity in membrane reactors compared to traditional
column reactors with enzymes immobilized on beads (see,
for instance, Nakajima and co-workers25,29).
Conventional column reactors may have further disad-
vantages not found in membrane reactors, such as plugging
(pore blockage) caused by impurities in substrates and
channeling (nonuniform flow during operation). 25,29
$1
Disadvantages
The disadvantages of membrane reactors are associated
with decreases in performance during operation, usually
b) caused by the loss of catalytic and mass transfer efficiencies.
P2
The operational kinetic stability of an enzyme in a mem-
T

~
brane reactor can be affected by several factors apart from
thermal inactivation of the enzyme. 51 For instance, enzyme
1"1 leakage may occur depending on the form of the enzyme
molecules and distribution of pore size, even when the mo-
lecular weight of the enzyme is higher than the membrane
UTunit cutoff. This loss of enzyme causes a gradual decrease in the
activity (if the enzyme is not present in excess). 8°,85 Small
enzyme activators, such as metal ions or cofactors, 59,63 may
also escape through the membrane, with consequent de-
crease in enzymatic activity. 51 In such cases, supplementa-
tion of the component leaking must be provided.
When the enzyme is used in a free form, unfavorable
adsorption to the membrane, with eventual alterations in
the enzyme conformation, may contribute to a decrease in
c) P2 activity. Even if no structural changes occur, the enzyme
may be poisoned upon contact with the membrane. This
means that the type of membrane material may influence
the stability of the enzyme. 46,48
Enzyme molecules in ultrafiltration systems and mem-
1"1 brane reactors are subjected to shear forces and friction
generated near the walls. This shear is claimed to cause
inactivation due to the sometimes observed correlation of
decrease in activity with increase in recirculating rates. 26,86
'l UT unit However, there is evidence that secondary effects occurring
together with high shear fields (e.g., interfacial inactivation,
adsorption, local heating, air entrainment) are important
and connected to the observed deactivation. 86- 88 These ef-
fects may be significant in recycle reactors that usually
Figure 3 Multiphase membrane reactors: (a) dual single-pass, (b)
single-pass/recycle, and (c) dual-recycle 0, Enzyme; $1, $2, sub- operate with high recirculating flow rates. In the case of
strates; P1, P2, products membrane reactors using stirrers, shearing-related ef-
fects associated with rotation may also contribute to
deactivation. 48
The usual enzyme inhibition caused by products and
substrates may be increased in membrane reactors using
costs usually associated with membranes. 84 Some of the enzymes immobilized on the membrane surface, due to
advantages are related to the separating possibilities of- gradual accumulation of these species in the form of a gel
fered by a membrane reactor, which permit an integration layer adjacent to the membrane.
of the catalytic process with separating steps. Furthermore, Whenever a decrease in operational kinetic stability
in certain membrane reactor configurations, mass transfer caused by one of the factors listed above is present, fresh
limitations that often occur in conventional fixed- or enzyme should be added to maintain constant productivity
fluidized-bed reactors due to diffusion in particles can be in the reactor.
reduced, since it is possible to operate the system with con- The loss of mass transfer efficiency (convection or diffu-
vective mechanisms rather than diffusive ones. In this way, sion) during the separation processes can also limit the use

Enzyme Microb. Technol., 1994, vol. 16, September 743

Review
of a membrane reactor. Two distinct phenomena are usu- Table 4 General applications of membrane reactors
ally responsible for the reduction in membrane filtration
capacity during an operation: concentration polarization Group of applications References
and fouling.
Concentration polarization is induced by the accumula- Hydrolysis of macromolecules 15,26,28,46,51-54,56,57,62,64,66,75,80-82,90
Cofactor regenerating reactions 18-21,45,58,59,63,65,70,91-97
tion of solutes at the boundary layer adjacent to the mem- 10,12,24,30-40,44,55,60,67,78,79,99-104
Hydrolysis and synthesis
brane with the consequent formation of a concentration
catalyzed by lipases
gradient. The gel layers formed at the interface act as a Catalysis in reversed micelles 24,35,36,41,44,67,107109
second membrane and originate a diffusion flux from the Other 9,16,22,23,25,27,29,61,71-73,77,85,98,110-112
membrane towards the bulk of the solvent that decreases
the flux of solutes across the membrane. The solvent pas-
sage through the membrane is also restricted due to the macromolecular substrates is always a major objective to be
intrinsic hydrodynamic resistance of this solute layer, caus- achieved in this type of reactor. This requirement can only
ing a decrease in the solvent filtration flux. 5° In many cases, be met in membrane reactors that promote a direct contact
concentration polarization-related phenomena (adsorp- between the enzyme and the substrate. This is confirmed by
tion, deposition, solute/membrane interactions) increase checking that all the above-mentioned applications are
the rejection capability of the membrane. 89 Concentration found in Table 5.
polarization is a reversible phenomenon that can be re-
duced by manipulating some operational conditions,
namely, by increasing recirculation flow rates or by intro-
Cofactor-regenerating reactions
ducing cyclic backflushing or pulsating flow. Moreover, if an Membrane reactors have also been increasingly used for
efficient cleaning procedure is used between consecutive the synthesis of bulk and fine chemicals with different pu-
operations, a complete restoration of original permeation rities, including optically active compounds.
fluxes can be obtained. A group of reactions that has been the subject of atten-
The second limiting phenomenon that may interfere tion is the one that involves a second substrate as a cofactor
during a filtration operation is fouling. Fouling is associated (or coenzyme), such as NAD +, NADP +, coenzyme A, or
with a modification of the filtration properties of a mem- ATE These coenzyme-dependent enzymatic transforma-
brane as a result of the deposition or adsorption of particles tions, such as covalent bond synthesis, energy transfer,
at the surface or inside the pores. This is an irreversible group transfer, and redox reactions, are attractive from the
process and leads to a progressive reduction in the effi- point of view of industry because of the wide variety of
ciency of a membrane, essentially due to a reduction in the products that can be synthesized. 1 Unfortunately, the cost
permeate flux and an increase in the rejection of of cofactors is usually so high that practical applications can
solutes. 47,5° It is usually minimized by pretreatments of the only be developed if a regenerating system for those com-
membrane and substrate feed. pounds is designed. The application of membrane reactors
A good control of concentration polarization and fouling to the regeneration of cofactors is presently considered a
effects is thus essential to maintain constant mass fluxes, solution for this problem, constituting an interesting re-
and hence productivity, in membrane reactors. search topic that has originated a significant number of
publications in recent years. 18- 21 .4.5. 5. 8. .5.9 6 3 6 5 7 0 9 1 - 97
Membrane reactors offer the possibility of regenerating
Applications of enzyme membrane reactors cofactors by using a coupled enzyme system or, alterna-
The membrane reactor concept can be applied to any en- tively, a coupled substrate approach. 2° In either case, the
zymatic reaction with the aim of developing continuous coenzyme is required only in catalytic amounts.
processes with high productivities. The applications that The first solution requires the use of a cofactor-regen-
were found in a survey of the scientific literature can be erating enzyme and an inexpensive sacrificial substrate.
broadly divided into five groups that share some common This enzyme uses the extra substrate to catalyze the regen-
characteristics (Table 4). A description of each specific ap- eration of the cofactor that was worn out in the main reac-
plication is presented in Tables 5-7 that separates each case tion. The criterion used for the choice of the cofactor-
according to the type of membrane reactor used. regenerating enzyme is based on the ease of removal of
by-products and on equilibrium considerations. Dehydrog-
enases (alcohol, formate, glucose, lactate, etc.) have been
Hydrolysis of macromolecules systematically used as regenerating enzymes.
In the first applications described, a great emphasis was In the second approach, the enzyme that synthesizes the
put on the hydrolysis of macromolecules, such as proteins product of interest also catalyzes the regeneration of the
and carbohydrates (starch and cellulose) (see Cheryan cofactor by using another substrate. 20 - 4 5 For instance, in
and Mehaia I for an earlier review of applications). This the synthesis of pheromone sulcatol catalyzed by alcohol
type of reaction still shows some interest, and in recent dehydrogenase, NADP + was regenerated by the same en-
years several publications have appeared dealing with zyme at the expense of isopropanol as a secondary
the hydrolysis of macromolecules: proteins, 46 ,51 -52 ,56 ,75 substrate. 21
peptides, 81 ,82 cellulose, 15,57 ,62,64 pectin, 26 ,90 maltodextrins, 80 Depending on the type of reactor configuration and
and starch. 28,53,54,66 The separation of the low-molecular- membrane porosity, the cofactor may be enlarged by covalent
weight products and, more important, the retention of the binding to polymers [e.g., polyethylene glycol (PEG)] in order

744 EnzymeMicrob. Technol., 1994, vol. 16, September

 Enzymatic membrane reactors: D. M. F. Prazeres and J. M. S. Cabral
Table 5 Applications in direct contact membrane reactors (in chronological order starting from the oldest)

Membrane/UF unity Enzyme reaction description Reference

Hollow fibers Hydrolysis of soy proteins catalyzed by pronase 51,56

Tubular membrane Investigation of acid phosphataseactivity over p-nitrophenyl phosphate 22
Thin-channel cell Hydrolysis of urea catalyzed by urease 111
Flat membranes Study of the activity of amyloglucosidase, urease, and aspartase immobilized by electro- 7,8
static forces
Flat sheet membrane Production of maltose corn syrups by (~-amylase/glucoamylase 28
Ultrafiltration cell Hydrolysis of sodium-hydroxide-pretreated sallow catalyzed by cellulase 15
Hollow fibers (pilot plant) Synthesis of L-methionine and L-phenylalanine from the N-acetyI-DL-derivatives by acylase 45
Hollow fibers (pilot plant) Synthesis of L-malic acid by the fumarase stereoselective addition of water to fumaric acid 45,95
Hollow fibers Synthesis of L-leucine with NADH (PEG bound) regeneration by leucine and formate de- 45
hydrogenases
Hollow fibers (?) Transformation of LD-lactatevia pyruvate to L-alanine with NADH (PEG bound) 91
regeneration
Hollow fibers (?) Synthesis of L- and D-2hydroxyisocaproic acid by dehydrogenases with NADH (PEG 92
bound) regeneration
Thin channel Synthesis of glucose 6-phosphate by hexokinase with ATP regeneration by acetate kinase 96
Hollow fibers Hydrolysis of lactose catalyzed by l~-galactosidase 85
Hollow fibers Peptide synthesis catalyzed by (x-chymotrypsin in reversed micelles 107
Ultrafiltration cell Synthesis of L-lactate with NAD (PEG bound) regeneration by lactate and malate 63
dehydrogenases
Diffusion cell Study of diffusion close to membrane containing glucose oxidase 112
Capillary tubes Production of glycerol and fatty acids by the hydrolysis of triglycerides by lipase in 55
emulsion
UItrafiltration cell Hydrolysis of olive oil by lipase in an emulsion system 60
Hollow fibers Hydrolysis of bovine plasma proteins catalyzed by alcalase 52
Tubular membrane Sucrose conversion into glucose and fructose catalyzed by invertase 23,25,29
Ultrafiltration cell Hydrolysis of cassava starch catalyzed by glucoamylase 66
Ultrafiltration cell Study of product inhibition in the hydrolysis of cellobiose catalyzed by 13-glucosidase 48
Nylon membrane Pectin hydrolysis catalyzed by pectolytic enzymes 26,90
Ultrafiltration cell (?) Synthesis of optically pure (R)-mandelonitrile by (R)-oxynitrilase 61
Ultrafiltration cell (R)-(-)-mandelic acid from benzoylformate with NADH regeneration by formate 97
dehydrogenase
Flat membrane Production of amino acids, hydroxyacids, alcohols, aldehydes, and lactones with 94
dehydrogenases
Ultrafiltration cell Production of sulcatol with simultaneous NADPH regeneration by alcohol dehydrogenase 21
Ultrafiltration cell Synthesis of mannitol by mannitol dehydrogenase with NADH regeneration by glucose 20
dehydrogenase
Tubular membrane Hydrolysis of maltodextrins by glucoamylase 80
Ultrafiltration cell Synthesis of NADPH with alcohol dehydrogenase 58
Ultrafiltration cell Hydrolysis of cellulose catalyzed by cellulases 57,62,64
Ultrafiltration cell Transesterification of glycerol with olive oil by lipase in liposomes/AOT-isooctane reversed 24
micelles
Diaphragm cell Hydrolysis of olive oil catalyzed by lipase encapsulated in AOThsooctane reversed micelles 67,108
Hollow fibers Saccharification of liquefied corn starch catalyzed by glucoamylase 53
Spiral wound Hydrolysis of lactose in skim milk by 13-galactosidase 9
Spiral wound Hydrolysis of milkfat trigylcerides catalyzed by lipase in an emulsion system 10
Tubular membrane Investigation of penicillinase and lactate dehydrogenase activity 27
Tubular membrane Hydrolysis of olive oil catalyzed by lipase encapsulated in AOT/isooctane reversed micelles 35,36,44
Spiral wound Bioactive peptides by the hydrolysis of macropeptides and proteins catayzed by trypsin 81,82
Hollow fibers Hydrolysis of fish proteins catalyzed by protease 46
Synthesis of N-acetylneuraminic acid by the conjugated action of epimerase and lyase 97,98
UItrafiltration cell Hydrolysis of starch to maltose syrups by simultaneous use of B-amylaseand isoamylase 54
Ultrafiltration cell (?) Synthesis of 12-ketoursodeoxycholic acid by dehydrogenases with NADH and NADPH 65
regeneration
Tubular membrane Dipeptide synthesis catalyzed by c~-chymotrypsinin TI-AB/octanol/heptane reversed 41
micelles
Flat membrane Large-scalesynthesis of dipeptide (kyotorphin) catalyzed by (~-chymotrypsin 110

to be retained by the membrane, 21,45,63,91,92,95,96 immobi- phogluconate, 7° lactate, 63,7° aldehydes, and lactones. 94 A
lized by size exclusion 97 or electrostatic repulsion, 2°,21,59 or large-scale process was even developed at DEGUSSA AG for
used as a permeable solute by immobilized enzyme. 17-19,70 the production of L-alanine from pyruvic acid catalyzed by
These cofactor-regenerating membrane reactors were I.-alanine dehydrogenase, with N A D + being regenerated back
used for the production of several different compounds: to N A D H by using formate dehydrogenase. 45 In this case
NADPH, 58 amino acids, 19.45 . . .91 94 95 hydroxyacids,92.94
, alco- N A D H was maintained in the system by increasing
hols, 20,21,70,94 acids, 45,65,97 glucose-6phosphate, 17,18,96 6-phos- its molecular weight upon binding to PEG. This use of

Enzyme Microb. Technol., 1994, vol. 16, September 745

Review
Table 6 Applications in diffusion membrane reactors (in chronological order starting from the oldest)

Membrane/UF unity Enzyme reaction description References

Hollow fibers Conversions of O-nitrophenyl-[3-D-galactopyranoside by [3-galactosidase 77

Hollow fibers Lactose conversion catalyzed by [3-galactosidase in a membrane reactor with pulsatile flow 16
Hollow fibers Synthesis of 2-naphthol glucuronide by UDP-glucuronyltransferase with cofactor (UDPGA) 73
addition
Hollow fibers Synthesis of glucose 6-phosphate by g lucokinase with ATP regeneration by acetate kinase 17,18
Flat membrane Study of diffusion reaction in the hydrolysis of urea catalyzed by urease 72
Hollow fibers Coenzyme-dependent reactions catalyzed by dehydrogenases (production of propyl alcohol, 70
lactate, and 6-phosphogluconoate)
Hollow fibers Process optimization of a membrane reactor for lactose conversion catalyzed by [3-galactosidase 71
Hollow fibers Synthesis of L-alanine by alanine dehydrogenase with NADH regeneration by lactate 19
dehydrogenase

Table 7 Applications in multiphase membrane reactors (in chronological order starting from the oldest)

Membrane/UF unity Enzyme reaction description Reference

Flat sheet membrane Production of glycerol and fatty acids by the hydrolysis of olive oil catalyzed by lipase 32,102
Hollow fibers Production of glycerol and fatty acids by the hydrolysis of lipids catalyzed by lipase 33,40,78,101
Flat sheet membrane Phosphatidylglycerol synthesis catalyzed by phospholipase D 104
Flat sheet membrane Synthesis of glycerides catalyzed by lipase 34
Flat sheet membrane Production of glycerol and fatty acids by the hydrolysis of tallow catalyzed by lipase 103
Hollow fibers Synthesis of (R)-glycidol by the lipase-catalyzed resolution of racemic glycidyl butyrate 12
Hollow fibers Hydrolysis of the alkyl ester ethyl butyrate catalyzed by porcine liver esterase 12
Hollow fibers Synthesis of n-butyl oleate catalyzed by lipase 30
Hollow fibers Hydrolysis of triacetin catalyzed by lipase 100
Hollow fibers Resolution of a racemic mixture of N-benzoyl tyrosine ethyl ester by (x-chymotrypsin 13
Flat sheet membrane Hydrolysis of butter oil catalyzed by lipase 31
Hollow fibers Esterification of sorbitol and fatty acids catalyzed by lipase 39
Hollow fibers Synthesis of mono-, di-, and triglycerides catalyzed by lipase 37,38,79
Flat sheet membrane Hydrolysis of soybean oil by lipase in a reactor with 2 different membranes 99

membrane reactors as a process strategy for regenerating research effort (scientific and economic) that has been
cofactors has permitted cycle numbers (defined as the num- devoted to these enzymes in recent years, the particular
ber of product molecules per cofactor molecule) as high as structure and unusual mode of action of lipases is also re-
500,000 (reported by Wandrey 93). sponsible for this trend. The fact that lipases are activated
Many of the membrane reactor systems developed for co- by, and act at, interfaces probably makes them the perfect
factor regeneration use two l7-2070.929697
, , , , or even three 6591 , biocatalyst to use in membrane reactors, and particularly in
enzymes acting synergistically. This possibility of easily immo- multiphase membrane reactors, which promote interracial
bilizing different enzyme molecules is a particularly attrac- contact between enzymes and substrate (see Table 7).
tive feature of membrane reactors for the conduction of The majority of the examples deal eitherwith the hydrolysis
enzyme-catalyzed sequential reactions. Some authors have of lipids and fats for the production of fatty acids, mono- and
described further applications (other than cofactor regen- diglycerides, and glycerol 10,31-33,35,36,40,44,55,60,67,7s,99-103 or
erating) of membrane reactors using the conjugated ac- with the synthesis of esters, 30'34'37-39'79including transesterifi-
tion of two enzymes. Kragl and co-workers 98 used an epi- cation reaction~ 24,1°4
merase for the isomerization of N-acetylglucosamine to Usually in these reactors, the lipid (or organic) phase is
N-acetylmannosamine, which was then further converted passed in one side of the membrane, while a buffer solution
to the final product, N-acetylneuraminic acid, by the addi- flows tangentially on the other side. The membrane will
tion of pyruvic acid catalyzed by a lyase. Membrane reac- become wetted by the lipid phase if the membrane material
tors using two enzymes, 13-amylase/isoamylase54 and is hydrophobic, or by the aqueous phase if it is hydrophilic,
et-amylase/glucoamylase, 28were also investigated for the pro- and the reaction then takes place at the interface. The
duction of maltose syrups from starch. lipase may be immobilized on either side of the membrane.
Tanigaki and co-workers 99 developed a slightly different
Hydrokysis and synthesis catalyzed by lipases approach by using two different types of flat membranes in
the reactor: hydrophilic and hydrophobic. The hydrolysis
A significant number of the recent applications of mem- of soybean oil was conducted in an enzyme chamber sep-
brane enzyme reactors (see Tables 5 and 7) use lipases. arated by the two membranes, with water permeating
Although this fact might be a consequence of the large from one side through the hydrophilic membrane and oil

746 EnzymeMicrob. Technol., 1994,vol. 16, September

 Enzymatic membrane reactors: D. M. F. Prazeres and J. M. S. Cabral
permeating from the other through the hydrophobic mem- in dynamic equilibrium with micelles by an ultrafiltration
brane. The products formed, glycerol and fatty acids, dif- membrane is highly improbable. Additionally, it is neces-
fused back through the membranes to the recirculating sary to consider the stability of reversed micelles to shear
aqueous and oil phase. force usually found in membrane devices. An eventual de-
Lipases have also gained enormous popularity among struction of the micellar structure integrity, or even its defor-
chemists (together with other enzymes such as esterases, mation, might decrease their retention by ultrafiltration. 44
penicillin acylase, and adenosine deaminase) as biocatalysts Khmelnitsky and co-workers 1°9 used a different ap-
for the synthesis of optically pure drugs. The ability of these proach by polymerizing the reversed micelles containing
enzymes to discriminate between enantiomers of racemic the enzyme (~x-chymotrypsin). This enzymatic system was
substrates has made enzymatic synthesis an alternative to used in a membrane reactor, permitting the occlusion of the
conventional chemical synthesis in the preparation of chiral enzyme and the separation of products without the pres-
pharmaceuticals, l°s This situation, however, has not been ence of contaminating surfactant.
accompanied by a parallel development and use of enzyme
reactors, probably because in these early stages of product Other
development, small amounts of drug (on a gram scale) are
Apart from the example described above, other applica-
needed. Eventually, larger-scale processes, and hence suit-
tions (not including cofactor regeneration) of membrane
able enzyme reactors, will have to be developed in order to
reactors at a pilot plant scale were also investigated. 45 A
increase production. Membrane reactors, due to their spe-
40-1 loop reactor coupled to 0.5-m 2 hollow-fiber modules
cific characteristics and advantages, will probably play an
was used in a recycle configuration for the production of
important role in this process development of enzyme syn-
L-amino acids (L-methionine and L-phenylalanine) by en-
thesis of chiral products.
zymatic (acylase) resolution of N-acetyl-D,L-derivatives.
At present, two applications of optical resolutions in
In a 1400-h pilot plant experiment, an approximate pro-
membrane reactors have been described in the literature.
ductivity of 10 kg of L-methionine per day was achieved
These were developed at SEPRACOR and use hollow fiber
(69% conversion), and in a combined 2300-h experiment,
modules for the racemic resolution of an ester (glycidyl
366 kg of L-methionine and 179 kg of L-phenylalanine
butyrate) catalyzed by a lipase, 12 and for the resolution of
were prepared consecutively (82-86% conversion). A
racemic amino acids by ~-chymotrypsin.13
production plant has even been conceived and con-
structed for the acylase-catalyzed resolution of acetyl-
Catalysis in reversed micelles D,L-amino acids with a capacity of 15-20 tons of L-amino
acids per month. 45
Membrane reactors have also been used to carry out catal- The fumarase-catalyzed stereoselective production of
ysis by enzymes microencapsulated in reversed micellar sys-
L-malic acid from fumaric acid was also conducted in a
tems. The applications of these reversed micellar systems in
similar 10-1 enzyme membrane reactor. In an 800-h exper-
biocatalysis, in spite of several advantages, are strongly lim- iment, 300 kg of L-malic acid was produced with an approx-
ited by the problem of contamination of the reaction media
imately constant conversion of 70%, maintained by addi-
with the surfactant. This constitutes a drawback for the tion of fresh enzyme. 45
separation and purification of products and the recovery of
An enzymatic peptide synthesis has also been scaled up
enzymes. Therefore, the development of reversed micellar
by using a 0.2-1 enzyme membrane (flat sheet) reactor.
reactors that permit reactions in continuous mode while
About 270 g of kyotorphin, an analgesic peptide, was con-
simultaneously allowing for a partial separation of products
tinuously synthesized by using a-chymotrypsin (followed by
has been pointed out as one of the priorities in reversed
acidic hydrolysis), during a 380-h operation.110
miceUes technology.I°6
Additional applications of membrane reactors in-
The examples found in the literature have reinforced the
clude the transformation of disaccharides (lactose and
general idea that ultrafiltration membrane reactors offer
sucrose), 9,16.23
. . .25. 29 48 71 the hydrolysis of urea, 72 ,111 the
the most appropriate configuration for the confinement of synthesis of (R)-mandelonitrile, 61 and the synthesis of
microencapsulated enzymes in reversed micellar media.
glucoronides. 97 Other enzymes and reactions were used
According to this concept, synthetic semipermeable mem- in membrane reactors mainly as model systems. 22,27,77,112
branes could retain reversed micelles (either containing or
not containing enzyme molecules) and separate products,
Conclusions and future prospects
while maintaining the unique advantages of catalysis in re-
versed micelles. 106 In recent years, membrane reactors have been established
A group of papers describe the attempt to use mem- as an alternative configuration for enzymatic reactors. The
brane reactors to carry out enzymatic reactions in reversed unique advantages offered by these reactors, together with
micelles. 24,35-36,4l ,44.67.107
, , - 109 Despite the expectations, the the wide variety of membrane shapes, modules, and mate-
membranes used were not capable of completely retaining rials commercially available at reduced costs, have made
the surfactant monomers and the hydrated micelles, there- them a serious alternative to more conventional reactors,
fore contaminating the product streams. 35,36,44This fact is such as fixed or fiuidized beds.
not surprising if one considers the specific dynamic charac- These reactors seem particularly suited to carry out com-
teristics of reversed micellar systems. Even if size exclusion plex enzymatic transformations, involving, for instance, sev-
of whole reversed micelles seems reasonable, the retention eral enzymes and cofactor regeneration. They also display
of the rather small surfactant monomers usually found unusual geometries which are particularly appropriate for

Enzyme Microb. Technol., 1994, vol. 16, September 747

Review
nonconventional types of media such as organic-aqueous 11 Matson, S. L. and Quinn, J. A., Membrane reactors in bioprocessing.
two-phase systems and reversed micelles. Further, the sep- Ann. NYAcad. Sci. 1986, 469, 152-165
aration possibilities offered by these reactors open up new 12 Lopez, J. L., Wald, S. A., Matson, S. L. and Quinn, J. A. Multiphase
membrane reactor for separating stereoisomers.Ann. NYAcad. Sci.
perspectives of applications in enzyme engineering. 1990, 613, 155-166
Considering that process integration is becoming more 13 L6pez, J. L., Matson, S.L., Stanley, T. J. and Quinn, J. A., Liquid-
and more a guiding philosophy in the development of new liquid extractive membrane reactors. In: Extractive Bioconversions
bioreactor configurations, membrane reactors therefore (Mattiasson, B. and Holst, O., eds.). Marcel Dekker, New York,
1991, pp. 27-66
constitute ideal systems for developing biotechnology ap- 14 Matson, S. L., and Lopez, J. L. Multiphase membrane reactors for
plications with the coupling of bioconversion with separa- enzymatic resolution: Diffusional effects on stereoselectivity. In:
tion process. Frontiers in Bioprocessing (Sikdar, S. K., Bier, M. and Todd, P., eds.).
In general terms, membrane reactors represent an evo- CRC Press, Florida, 1991, pp. 391-403
lutionary step in the development of bioreactors and con- 15 Ohlson, I., Trfig~rdh, G. and Hahn-Hfigerdal, B. Enzymatic hydro-
lysis of sodium hydroxide pretreated sallow in an ultrafiltradon
stitute a new generation of enzymatic reactors. membrane reactor. BiotechnoL Bioeng.1984, 26, 647-653
At this stage, however, membrane reactor possibilities 16 Park, T. H., Kim, I. H. and Chang, H. N. Recycle hollow fiber enzyme
have not been explored to their full extent. The future will reactor with flow swing. Biotechnol. Bioeng. 1985, 27, 1185-1191
probably bring the development of even more complex en- 17 I shikawa, H., Tanaka, T., Takase, S. and Hikita, H. Theoretical anal-
ysis of G6P production and simultaneous ATP regeneration by con-
zymatic systems and elaborate configurations based on jugated enzymes in an ultrafiltration hollow-fiber reactor. Biotech-
these types of reactors. The following are some suggested nol. Bioeng. 1989, 34, 357-368
topics where research efforts in the area of membrane re- 18 Ishikawa, H., Tanaka, T., Takase, S. and Hikita, H. Experimental
actors may be concentrated in the coming years. investigation of G6P production and simultaneous ATP regenera-
tion by conjugated enzymes in an ultrafiltration hollow-fiber reactor.
Biotechnol. Bioeng.1989, 34, 369-379
• Exploration of electric potential as a driving force for 19 Fujii, T., Miyawaki, O. and Yano, T. Modeling of hollow-fiber capil-
transport of solutes through membranes and eventually lary reactor for the production of L-alanine with coenzyme regener-
as an alternative cofactor-regenerating technique in ation. Biotechnol. Bioeng. 1991, 38, 1166-1172
membrane reactors 20 Kulbe, K. D., Howaldt, M. W., Schmidt, K., Rothig, T. R. and
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coenzyme NAD(P)H in a charged ultra filtration membrane enzyme
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750 EnzymeMicrob. Technol., 1994,vol. 16, September