TOPIC 5

DESIGN OF BIOCATALYTIC
PROCESSES

The most common definition for immobilized enzymes is that

proposed
by Katchalski-Katzir
the 1960s:
1. Heterogenization
of thein
soluble
enzyme by coupling to
an insoluble support by adsorption or covalent binding, by
Enzymes
physically
confined
oror
localized
in a certain
defined
cross-linking
of the
enzyme
entrapment
in a lattice
or inregion
of space
with retention
catalytic
activities, which can be
microcapsules
suchof
astheir
alginate
beads
used repeatedly and continuously.
2. Retention of the enzyme by means of ultrafiltration
According
to this definition, three types of immobilized enzymes can
membranes
be distinguished:
3. Use of whole cells for biotransformations using their
enzyme apparatus

Advantages over suspension

cultures
(1). Immobilisation provides high cell concentration
(2). Immobilisation provides cell reuse and eliminates
the costly processes of cell recovery and cell recycle
(3). Immobilisation eliminates cell washout problems at
high dilution rates
(4). Combination of high cell concentrations and high
flow rates allows high volumetric productivities
(5). Favourable microenvironmental conditions
(6). Improves genetic stability
(7). Protects against shear damage

Advantages of immobilised cell

reactors
Being able to maintain high cell concentrations in

the reactor at high dilution rates provides

immobilised cell bioreactors with advantages over
chemostats.

More biomass means that the fermenter

contains more biocatalysts, thereby high
bioconversion rates can be achieved.
Immobilised cell bioreactors are also more
stable than chemostats.

A higher cell concentration in the immobilised bioreactor

prevents the microbial population from completely washing
out.

Inhibitor enters
inlet feed

Immobilised
bioreactor

Biomass

cells are not as easily washed out

of an immobilized cell reactor
recovery time will be more
quicker and fall in biomass
concentration will be smaller

Chemostat
Time

cause a rapid drop in cell number

take time for the cells
numbers to build up again

Limitations
(1). Often the product of interest has to be excreted
from the cell
(2). Complications with diffusional limitations
(3). Control of microenvironment conditions is
difficult due to heterogeneity in the system
(4). Growth and gas evolution can lead to
mechanical
disruption of the immobilised matrix

Active immobilisation
Is entrapment or binding of cells by physical or

chemical forces
Physical entrapment within porous matrices is the

most widely used method of cell immobilisation

Immobilised beads should be porous enough to

allow transport of substrates and products in and

out of the beads

Active immobilisation
Beads can be prepared by
1)
2)
3)
4)
5)
6)

Gelation of polymers
Precipitation of polymers
Ion exchange gelation
Polycondensation
Polymerisation
Encapsulation

Passive immobilisation
Biological films
The multilayered growth of cells on solid support

surfaces
The support material can be inert or biologically

active
Biofilm formation is common in natural and

industrial fermentation systems, i.e biological

wastewater treatment and mold fermentations

Inactivation factors in designing

immobilized cell reactor
The abrasion effect due to shear forces by stirrer or mixing mechanism,

impeller type and particle-particle collisions in reactors can cause unfolding on

enzymes must be considered.
The bimolecular reactions of immobilized enzyme with substrates or products
at high concentrations used in enzyme processes must be considered which
can be controlled through flow rate. The substrate and product in enzyme
reactor will affect reactor operation.
Mass transfer limitations of reactor designed due to adsorption of molecules
or microorganisms in or on support also need to be considered, where it can
reduce the specific activity of enzymes from the immobilized cell.
The temperature controlled in the reactor also must be considered wherein
increasing and drecreasing in temperature which is not suitable can cause the
unfolding of chemical modification and dissociation of oligomeric enzymes,
respectively.
The pH controlled in reactor must be considered due to increasing and
decreasing of pH can cause unfolding and instability of enzyme from
microorganism which might denatured the protein.

Description of support
material
The Hydrogels
Natural
Carrageenan
Alginate
Agar
Gelatin
Synthetic
Polyvinyl alcohol
Polyurethane
Polyethylene glycol

Types of immobilized cell

reactors

There are many types of immobilized cell

reactors either in use or under development.

In this section we will look at four major
classes of immobilized cell reactors:

Cell recycle systems

Fixed bed reactors / packed bed
Fluidized bed reactors
Flocculated cell systems

Cell recycle
system
Fresh feed

In a fermenter with cell recycle the cells are

separated from the effluent and then
recycled back to the fermenter; thus
minimizing cell removal from the fermenter:

Biomass
separation
system
Fermenter

Biomass recycle

Effluent

Cell recycle systems

Cell recycle is used in activated sludge systems. A

portion of the cells are separated in a settling tank and

returned to the activated sludge fermenter.
Biomass recycling for product or biomass production is

more difficult due to the need for maintaining sterility

during cell separation. Centrifugation which is a
faster process than settling would be used to separate
the cells.
Biomass recycle systems can be easily modelled.

Fixed bed reactors

In fixed bed fermenters, the cells are immobilized by

absorption on or entrapment in solid, non-moving

solid surfaces.

Fixed bed reactors

In these fermenters, the cells are physically trapped

inside the pores of the gels and thus giving better cell
retention and a large effective surface area for cell
entrapment.
In order to increase the surface area for cell
immobilization, some researchers have investigated the
use of hollow fibres and pleated membranes as
immobilization surfaces.
Industrial applications of fixed bed reactors include
waste water treatment
production of enzymes and amino acids
steroid transformations

Fixed bed reactors

The liquid feed is either pumped through or allowed

to trickle over the surface of the solids where the

immobilized cells convert the substrates into
products.
Once steady state has been reached there will be a
continuous cell loss from the solid surfaces.
In other types of fixed-bed fermenters, the cells are
immobilized in solidified gels such as agar or
carrageenan

Fixed bed reactors

One advantage fixed bed reactors is that non-

growing cells can be used.

In such systems, the cells enzymatically act on

substrates in the feed.

The cells can be either inactivated or not fed

nutrients required for growth.

Fluidised bed reactors

In fluidized-bed fermenters

the cells are immobilized on

or in small particles.
The use of small particles

increases the surface area

for cell immobilization and
mass transfer.
Because the particles are

small and light, they can be

easily made to flow with the
liquid (ie. fluidised).

Small
moving
particles

Fluidized bed reactors

The fluidisation of the particles in the reactor

leads to the surface of the particles being

continuously turned over. This also increases the
mass transfer rate.
Fluidised beds are typically categorized as either
being a
2 phase system which are not aerated and
3 phase system which is aerated by
sparging
Fluidized bed bioreactors are used widely in
wastewater treatment.

Fluidized bed reactors

Fluidized bed bioreactors are also used for animal

cell culture.
Animal cells are trapped in gels or on the surface

of special particles known as "microcarriers".

Fluidized bed reactors are one example of

perfusion culture technology used for animal cell

culture.

Comparing fluidised bed and

fixed reactors
Fluidised bed reactors are considerably more efficient
than fixed bed reactors for the following reasons:
1) A high concentration of cells can be immobilized in
the reactor due to the larger surface area for cell
immobilization is available
2) Mass transfer rates are higher due to the larger
surface area and the higher levels of mixing in the
reactor.
3) Fluidised bed reactors do not clog as easily as fixed
bed reactors.

Comparing fluidised bed and

fixed reactors
Fluidised bed reactors are however more difficult

to design than fixed bed reactors.

Design considerations include:
Setting the flow rate to achieve fluidisation
Ensuring that bubble size remains small
during the fermentation.
Prevention of the cells from falling or
"sloughing" off the particles.
Minimising particle damage.

Flocculated cell reactors

In flocculated cell reactors, the cells are trapped in

the reactor due to an induced or natural

flocculation process. In flocculation cells tend to
group together causing them to come out of
solution and to sink towards the base of the reactor.
Flocculated cell reactors are used widely in
anaerobic waste treatment processes.
In these reactors, the methanogenic and other
bacteria form natural flocs. The flocs move due to
the release of methane and carbon dioxide by the
cells.

Flocculated cell
reactors
Large scale anaerobic flocculated cell systems,

known as Upflow Anaerobic Sludge Blanket

processes are widely used in Europe for the
anaerobic digestion of high strength industrial
wastewaters.
The reactors are typically egg-shaped.

Flocculated cell
reactors

Cells form flocs which

gently fall and rise with
gases they produce

Enzyme Reactors
Membrane reactors have been used quite recently as
have applications of whole cell processes.
Retention of the cells within the reactor may be achieved
by membrane separation or by the same
immobilization methods that are used for isolated
enzymes (34).
In principle, the cell itself can be regarded as a form of
native immobilization of enzymes.
Biosensors are a very special form of carrier-fixed
biocatalysts.

When considering the use of soluble or

carrier-fixed enzymes, the following topics
we have to be addressed:
1. Additional costs for support and chemicals
performing the immobilization have to be
balanced against the increase of stability.
2. Loss of activity during the immobilization step.
3. When the catalyst is immobilized only by
adsorption or entrapment without covalent
attachment, its leakage from the carrier
support has to be examined and compared with
the overall deactivation rate.

4. Mass transfer limitations for enzymes on a

support may cause problems when adjusting of
the pH is necessary during the reaction.
5. With soluble enzymes, higher volumetric
activities at high catalyst concentrations are
possible, enabling conversion of poor substrates
at reasonable rates.
6. Whereas membrane reactors can be easily
sterilized before use, this is not possible for
reactors with carrier-fixed enzymes.
To prevent microbial contamination, these
processes are quite often operated at higher
temperatures.

7. When enzymes are to be used

together with organic solvents to
increase reactant or product solubility
or to alter their kinetics, it may
become necessary to immobilize them
on a support
The support will at the same time act as a
water pool to maintain the enzymatic
activity.
In such systems, water-insoluble organic
solvents have less effect on the enzyme

Process design and operational

strategies of immobilized enzyme
reactors
The final decision for a certain reactor
design should be based on an optimization
process
covering
all
relevant
factors
contributing to the overall costs, including
investment, catalyst consumption, or productivity.