Immobilized Enzyme and Cell Systems

• Advantages:
– Enzyme and cell re-utilization
– Simpler product recovery
– Enzyme function/activity may increase
• Disadvantages:
– Leaking
– Diffusion limitiations
– Lack of control of environmental
conditions

1
Immobilized Cells and Enzymes

Product
PACKED BED

Recycle

Feed

Product
FLUIDIZED BED

Recycle

Feed

1
 BIOE 382
Fall 1998
BIOFILMS
Supplementary Notes

1 Introduction
To date we have considered cell cultures in suspension, that is when the cells are
homogeneously distributed in the fermenter broth. This is a desirable situation if
we want the cells as our product and want to continuously remove them from the
bioreactor. However, if the cells are making another product that we want and if this
product is being produced extracellularly then we may not want the cells to leave the
reactor. What would be the advantage of this? This is what we will be considering in
this section. Let’s say that we want to have a high production rate of product from
our bioreactor and to do so requires a high concentration of cells in the reactor vessel.
However, to obtain a high production rate we need to have a high feed rate to the
reactor. If the cells were in suspension and no attempt was made to retain them in the
reactor then the cells would have to be present in the reactor long enough for them to
grow. This means that the residence time in the bioreactor must be longer than the
inverse of the specific growth rate of the bacteria.
1
τ> (1)
µ
We will be deriving this equation later in the course when we develop material
balance equations for chemostats (another word for continuous stirred tank reactors).
The residence time is equal to the volume of the reactor divided by the flow rate of
feed (nutrients and raw materials) into the reactor.
V
τ= (2)
F
Thus, for a particular organism and growth conditions, if the reactor volume was
constant then we would be limited by a certain flow rate. If the flow rate was to
be kept constant then we would be limited to reactors larger than a minimum size.
For example, if we are growing sulphate reducing bacteria whose specific growth rate
µ = 0.06 h−1 . Then,
V
> 16.67 hours
F
If we wanted to treat 0.1 m3 of water per minute our reactor has to be larger than
100 m3. In ground water treatment processes where we have to inevitably treat large
flows of water, the slow rate of growth of these bacteria requires that we build very
large reactors. This may preclude the application of bioremediation technologies to
treat ground water. What happens if we decrease the residence time below 1/µ? Then
the cells do not spend enough time in the reactor to grow and wash out occurs. That
is the cell concentration in the reactor disappears to zero.

1
 The solution to this problem and a way to be able to use smaller bioreactor vessels
to treat larger flow rates is to retain the cells inside the reactor and prevent them from
leaving with the effluent. To do this we must provide a surface on which the cells will
attach and form biofilms or we need to entrap the cells inside a particle that is large
enough to prevent from leaving the reactor. In this section, we will be talking about
the ways in which we can immobilize cells and enzymes inside bioreactors. But first
we will consider all of the advantages and disadvantages to doing so.

2 Advantages and disadvantages of immobilized cells

and enzymes
Advantages

1. To keep an isolated enzyme in the bioreactor

2. To prevent loss of expensive enzymes

3. To simplify separation of the product downstream of the bioreactor

4. Immobilized enzymes may retain their activity for longer.

5. Immobilizing an enzyme near other enzymes participating in a catalytic sequence

may increase efficiency of the reaction.

6. To enable a large throughput of substrate.

7. To achieve higher cell densities

8. Some mammalian cells grow only if attached to surfaces

9. Can be used for biosensors.

10. To control cell morphology and broth rheology.

11. To prevent washout.

Disadvantages

1. There are diffusion limitations.

2. Cell growth and gas evolution may disrupt immobilization.

3. Suitable only where the product is secreted by the cells.

4. Immobilization may be disrupted due to shear stresses.

2
 Techniques for Cell and Enzyme
Immobilization
Unattched cells and enzymes

entrapped
bound

support encapsulated E
E

E
E covalently
bound
to a
support
(enzymes)
adsorption
to a surface

trapped
inside a
membrane,
gel,
or micelle

2
A scanning electron microscope picture of a lava rock
sample. Lava rock is one type of solid support
material that is used for biomass immobilization.

A close-up showing different organisms growing on

the surface:

(See if you can pick out the different cells)

An even closer picture of bacteria cells in a biofilm

Pictures from http://www-scf.usc.edu/~chitwood/biofilm.html

Recipe of a method for immobilization
of cells in K-carrageenan gel

This is a polysaccharide from seaweed

composed of β -D-galactose sulphate and 3,6,
anhydro-α-D-galactose units.

Steps:

1. Make a gel by dissolving at 70-80◦ C in a

saline solution (2-5% concentration).

2. Add the cell suspension, which is at 40-50◦ C.

3. Cool and add 0.1M KCl

Example of an application: For immobilization of

yeast for ethanol production.

3
Calculations for the rates of reactions
in immobilized biocatalysts

1. Describe the concentration profiles in the

biofilm and in solid biocatalysts.

2. Develop equations for the concentration

profile.

3. Recognize and describe the Thiele

Modulus and the effectiveness factor.
4. Use the Thiele Modulus and effectiveness
factor to find the kinetics of immobilized
reactions.

4
SUBSTRATE CONCENTRATION PROFILE IN
A SOLID BIOCATALYST

Spherical
cells Particle
Bulk Boundary
liquid: layer
well
mixed

R
r
CAb
concentration

CAs
substrate

In some cases the substrate

concentration could drop to
zero inside the particle.

distance
 BIOFILMS

- Multilayer growth of cells on a

solid support material
- Occurs in natural and industrial
fermentations
- Formed by polymeric substances
EPS (extracellular polymeric substances)

Biofilm
Solid Support

CAb

CAi

Bulk Fluid
CAs
Assumptions for the Mass Balance:
• Isothermal
• Diffusion only
• Fick’s Law
∂CA
JA = DA,e
∂r
• Homogeneous particle
• Partition coefficient is unity
• Steady State
• Substrate diffuses towards center
 Mass Balance Substrate
Concentration in a Solid
Biocatalyst
Mass transfer resistance is important in
solid particles due to the density of the
immobilizing medium and/or the tortuous
passages. Diffusion can be very slow.
 De The Effective Diffusivity.

This is a measure of the movement of a molecule

inside the pores of a porous compound. We need
De so that we can use Fick’s Law to calculate the
flux of molecules in the pores.

Pore diffusion occurs by three mechanisms:

• Ordinary diffusion
• Knudsen diffusion (small pores, mostly for
gases)

• Surface diffusion

2
 Diffusivity is influenced by the length of the
tortuous path, irregular shape and constrictions in
the path.

DAB θ
De =
τ

DAB is the molecular diffusion.

θ is the volume void fraction.
τ is the tortuosity factor.

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 True and Observed Reaction
rates in solid biocatalysts
The true rate is a function of the
substrate concentration in the biocatalyst
(zero or first order, Monod, Michaelis
Menten). It is different at different
positions in the solid particle. The true
rate is difficult to measure.
The observed rate is the rate at which
the substrate disappears from the bulk
solution
dCA,b
=
dt
The observed rate can be measured. We
will learn how to predict the observed rate
from the reaction kinetics for the intrinsic
(true) rate.
 Mass Balance for Substrate in the
Solid Support

For a porous support or a gel bead: spherical

geometry.
For biofilm: flat plate geometry.
For spherical geometry:
!
d2 CA 2 dCA
DAe 2
r + 2r − r 2 rA = 0 (1)
dr dr

r is the distance from the centre.

rA is the substrate uptake rate.
µmax. S
For microbial growth rA = Y 1 K
XS s +s

It is difficult to solve Eqn. 1 for Monod kinetics

(must be done numerically). Monod kinetics are
between zero and first order. We can solve Eqn. 1
analytically for a zero and first order reaction.

4
 Prediction of the overall
reaction rate

For first order kinetics,

robs. = k1 CA

CA is a function of the distance inside

the biocatalyst (or biofilm). Substitute this
expression (Eqn. 12.11) into the above
equation and integrate over the entire
biocatalyst volume (from r = 0 to
r = R).

1
 The Effectiveness Factor

This is the ratio of the observed rate to

the rate that would occur if
CA = CAs throughout the particle.

rA,obs.
η= ∗
rAs

η is proportional to R, k, DAe and

sometimes CAs .

2
 Thiele Modulus

Dimensionless variable:

For a first order reaction:

s
R k1
φ=
3 DAe

See Table 12.2 for other Thiele moduli.