2 Basic Bioreactor Concepts

2.1 Information for Bioreactor Modelling

Both physical and biological information are required in the design and
interpretation of biological reactor performance, as indicated in Fig. 2.1.
Physical factors that affect the general hydrodynamic environment of the
bioreactor include such parameters as liquid flow pattern and circulation time,
air distribution efficiency and gas holdup volume, oxygen mass transfer rates,
intensity of mixing and the effects of shear. These factors are affected by the
bioreactor geometry and that of the agitator (agitator speed, effect of baffles)
and by physical property effects, such as liquid viscosity and interfacial tension.
Both can have a large effect on gas bubble size and a corresponding effect on
both liquid and gas phase hydrodynamics. The biokinetic input involves such
factors as cell growth rate, cell productivity and substrate uptake rate. Often this
information may come from laboratory data, obtained under conditions which
are often far removed from those actually existing in the large scale bioreactor.
Although shown as separate inputs in Fig. 2.1, there are, in fact, considerable
interactions between the bioreactor hydrodynamic conditions and the cell
biokinetics, morphology and physiology, and one of the arts of modelling is to
make proper allowance for such effects. Thus in the large scale bioreactor,
some cells may suffer local starvation of essential nutrients owing to a
combination of long liquid circulation time and an inadequate rate of nutrient
supply, caused by inadequate mixing or inefficient mass transfer. Agitation and
shear effects can affect cell morphology and hence liquid viscosity, which will
also vary with cell density. This means that the processes of cell growth affect
the bioreactor hydrodynamics in a very complex and interactive manner.
Changes in the cell physiology, such that the cell processes are switched from
production of further biomass to that of a secondary metabolite or product, can
also be affected by selective limitation on the quantity and rate of supply of
some essential nutrient in the medium. This can in turn be influenced by the
bioreactor hydrodynamics and also by the mode of the operation of the
bioreactor.
The overall problem is therefore very complex, but as seen in Figure 2.1,
when all the information is combined successfully in a realistic and well
founded Bioreactor Model, the results obtained can be quite impressive and

Biological Reaction Engineering, Second Edition, I. J. Dunn, E. Heinzle, J. Ingham, J. E. Pfenosil

Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-30759-1
56 2 Basic Bioreactor Concepts

may enable such factors as cell and product production rates, product
selectivities, optimum process control and process optimization to be
determined with some considerable degree of confidence.

Physical Aspects Biokinetics

(flow patterns, residence (order, inhibition,pH,
time, mass transfer) temperature)

Production rate
Selectivity
Control

Figure 2.1. Information for bioreactor modelling.

2.2 Bioreactor Operation

The rates of cell growth and product formation are, in the main, dependent on
the concentration levels of nutrients and products within the bioreactor. The
concentration dependencies of the reaction or production rate are often quite
simple, but may also be very complex. The magnitude of the rates, however,
depend upon the level of concentrations, and it will be seen that concentration
levels within the bioreactor depend very much on its type and mode of
operation. Differing modes of operation for the bioreactor can therefore lead
to differing rates of cell growth, to differing rates of product formation and
hence to substantially differing productivities.
Generally, the various types of bioreactor can be classified as either stirred
tank or tubular and column devices and according to the mode of operation as
batch, semi-continuous or continuous operation.
2.2 Bioreactor Operation 57

2.2.1 Batch Operation

Most industrial bioreactors are operated under batch conditions. In this, the
bioreactor is first charged with medium, inoculated with cells, and the cells are
allowed to grow for a sufficient time, such that the cells achieve the required
cell density or optimum product concentrations. The bioreactor contents are
discharged, and the bioreactor is prepared for a fresh charge of medium.
Operation is thus characterized by three periods of time: the filling period, the
cell growth and cell production period and the final emptying period as
depicted in Fig. 2.2. It is only during the reacting period, that the bioreactor is
productive. During the period of cell growth, strictly speaking, no additional
material is either added to or removed from the bioreactor, apart from minor
adjustments needed for control of pH or foam, small additions of essential
precursors, the removal of samples and, of course, a continuous supply of air
needed for aerobic fermentation. Concentrations of biomass, cell nutrients and
cell products thus change continuously with respect to time, as the various
constituents are either produced or consumed during the time course of the
fermentation, as seen in Fig. 2.3.

Filling Reacting Emptying Cleaning

Figure 2.2. Periods of operation for batch reactors.

concentration
A biomass
ubstrate

product

time

Figure 2.3. Concentration-time profiles during batchwise operation.

58 2 Basic Bioreactor Concepts

During the reaction period, there are changes in substrate and product
concentration with time, and the other time periods are effectively lost as
regards production.
Since there is no flow in or out of the bioreactor, during normal operation,
the biomass and substrate balances both take the form,

(Rate of accumulation within the reactor) = (Rate of production)

This will be expressed in more quantitative terms in Ch. 4.

Batch reactors thus have the following characteristics:

1) Time-variant reaction conditions

2) Discontinuous production
3) Downtime for cleaning and filling

2.2.2 Semicontinuous or Fed Batch Operation

In semi-continuous or fed batch operation, additional substrate is fed into the

bioreactor, thus prolonging operation by providing an additional continuous
supply of nutrients to the cells. No material is removed from the reactor, apart
from normal sampling, and therefore the total quantity of material within the
reactor will increase as a function of time. However if the feed is highly
concentrated, then the reactor volume will not change much and can be
regarded as essentially constant.

Figure 2.4. Fed batch bioreactor configuration.

2.2 Bioreactor Operation 59

Semi-continuous operation shares the same characteristics as pure batch

operation, in that concentration levels generally change with time and that some
downtime occurs during the initial charging and final discharge period at the
end of the process.
The ability to manipulate concentration levels within the bioreactor by an
appropriate controlled feeding strategy confers a high degree of flexibility to
fed batch or semi-continuous operation, since differing concentration levels can
be utilized to manipulate the rates of reaction. In Fig. 2.4, both the volumetric
feeding rate, F, and the feed substrate concentration SQ, may be constant or may
vary with time, giving the possibility of such feeding strategies as:

1. Slow constant feeding, which can be shown to result in linear growth

of the total cell biomass.

2. Exponential feeding to maintain constant substrate concentration and,

resulting in unlimited, exponential cell growth.

3. Feedback control of the feed rate, based on monitoring some key

component concentration.

The important characteristics of fed batch operation are therefore as follows:

1. Extension of batch growth or product production by additional

substrate feeding.

2. Possibility of operating with separate conditions for growth and

production phases.

3. Control possibilities on feeding policies.

4. Development of high biomass and product concentration.

For fed-batch operation, the cell balance follows the same form as for batch
operation, but since additional substrate feeding to the reactor now occurs, the
substrate balance takes the form:
Rate
( <* "| ( Substrate \ ( Substrate >|
accumulation = (f^d [n) _ consumption
V of substrate J \ rate )

Under controlled conditions, in which the substrate concentration is maintained

constant or kept small, the accumulation term in the above equation will also be
small, with the result that the feed rate of substrate into the reactor will balance
the rate of consumption by reaction.
60 2 Basic Bioreactor Concepts

One other balance equation, however, is also necessary, i.e. the total mass
balance,

f Rate of accumulation of ^ ( Mass flow rate of feed ^

=
V mass in the reactor / V to the reactor )

which for constant density conditions reduces to

(Rate of change of volume) = (Volumetric rate of feeding)

Further extensions of fed batch operation are possible, such as the cyclic or
repeated fed batch, which involves changing volume with a filling and
emptying period. The changing reactor concentrations repeat themselves with
each cycle. This operation has similarities with continuous operation and
approaches most closely to continuous operation, when the amount withdrawn
is small and the cycle time is short. The simulation examples FEDBAT, Sec.
8.1.3 and in Sec. 8.3 (VARVOL, PENFERM, PENOXY, ETHFERM, REPFED)
allow detailed investigations of fed batch performance to be made on the
computer.

2.2.3 Continuous Operation

In continuous operation fresh medium is added continuously to the bioreactor,

while at the same time depleted medium is continuously removed. The rates of
addition and removal are such that the volume of the reactor contents is
maintained constant. The depleted material, of course, contains any products
that have been excreted by the cells and, in the case of suspended-cell culture,
also contains effluent cells from the bioreactor.
Continuous reactors are of two main types, as indicated in Fig. 2.5, and these
may be considered either as discrete stages, as in the continuous, stirred-tank
bioreactor, or as differential devices, as represented by the continuous tubular
or column reactor.

Continuous tank bioreactor Continuous tubular bioreactor

Figure 2.5. The two main types of continuous reactors.

2.2 Bioreactor Operation 61

As shown later, these two differing forms of continuous reactor operation have
quite different operational characteristics. Both however are characterized by
the fact that after a short transient period, during which conditions within the
bioreactor change with time, the bioreactor will then achieve a steady state. This
means that operating conditions, both within the bioreactor and at the
bioreactor outlet, then remain constant, as shown in Fig. 2.6.

Startup Steady state

Concentration period

time

Figure 2.6. Startup of a continuous reactor.

Continuous reactors, however, have found little use as biological reactors on a

production scale, although there are a few important examples (Id's single-cell
protein air lift process, wastewater treatment and the isomerization of corn sugar
to fructose syrup). Frequent use is made of continuous reactors in the
laboratory for studying the kinetics of organism growth and for enzyme
reaction kinetics. This is because the resulting form of the balance equation,
leads to an easy method for the determination of reaction rate, as discussed in
Ch. 4.
The behavior of the two differing forms of continuous reactor, are best
characterized by their typical concentration profiles, as shown in Fig. 2.7. In
this case, S is the concentration of any given reactant consumed, and P is the
concentration of any given product.

Tank Tube
So So

Cone. Cone.

distance distance

Figure 2.7. Profiles of substrate and product in steady state continuous tank and tubular
reactors.

As seen, the concentrations in a perfectly mixed tank are uniform, throughout

the whole of the reaction vessel contents and are therefore identical to the
concentration of the effluent stream. In a tubular reactor the reactant
concentration varies continuously, falling from a high value at the inlet to the
62 2 Basic Bioreactor Concepts

lowest concentration at the reactor outlet. The product concentration rises from
inlet to outlet. These differences arise because in the tank reactor the entering
feed is continuously being mixed with the reactor bulk contents and therefore
being diluted by the tank contents. The feed to the tubular reactor, however, is
not subject to mixing and is transformed only by reaction, as material moves
down the reactor.
No real situation will exactly correspond to the above idealized cases of
perfect mixing or zero mixing (plug flow), although the actual behavior of
tanks and tubes tends in the limit towards the corresponding idealized model.
The characteristics of continuous operation are as follows:

1. Steady state after an initial start-up period (usually)

2. No variation of concentrations with time
3. Constant reaction rate
4. Ease of balancing to determine kinetics
5. No down-time for cleaning, filling, etc.

The balance equations at steady state for a well-mixed tank reactor have the
form
0 = (Input) - (Output) + (Production)

since at steady-state the rate of accumulation and therefore the rate of change is
zero.
This equation predicts that the reaction rate causes a depletion of substrate
from the feed condition to the outlet, (the product will increase) and that the
rate of production can be obtained from this simple balance:

(Rate of production) = (Rate of output) - (Rate of input)

For a non well-mixed reactor such as a tubular or column reactor, steady-state

implies the same non-transient conditions, but now concentrations also vary
with position. The same situation also applies to the case of a series of well-
mixed tanks.
The balance form is then:
0 = (Rate of input) - (Rate of output) + (Overall Rate of Production)

Here the overall rate of reaction is obtained by summing or integrating over

every part of the reactor volume.
The concentration characteristics of a tubular reactor, as shown in Fig. 2.7,
are well approximated by a series of tank reactors. Referring to Fig. 2.8, and
moving downstream along the reactor cascade, the substrate concentration
decreases stepwise from tank to tank, while the product concentration increases
in a similar stepwise manner. As the number of tanks in the cascade increases,
so the performance becomes more and more similar to that of a tubular reactor.
In the case of a reaction, whose rate of reaction increases with increasing
2.2 Bioreactor Operation 63

substrate concentration S, the multiple tank configuration or a tubular reactor

would thus have a kinetic advantage over that of a single tank. The same is true,
in the case of product inhibition kinetics, in which the rate would be lowered by
high product concentration, P. Substrate inhibition systems would be run
preferably in single tanks, however, since then the substrate concentration is
always at its lowest value.

Cone.

distance

Figure 2.8. Stirred tanks in series and their concentration profiles.

A calculation of the tank volume or residence time requirement involves the

formulation of the tank balance equations, as before and then the application of
the equations, successively from tank to tank such that the effluent from the
preceding tank is the feed of the next and so on. Tanks-in-series bioreactor
operations are illustrated by the simulation examples TWOSTAGE, STAGED
and DEACTENZ in Sec. 8.4.

2.2.4 Summary and Comparison

The operating characteristics of the various reactor modes are summarized in

Table 2.1.
The important bioreactor operating parameters will depend on the mode of
operation. In batch operation, concentration levels can be varied by adjustment
of the initial values, whereas in continuous and semi-continuous operation, the
concentration levels depend on the feed rate and feed concentration. As
indicated previously, the manner in which the bioreactor is operated can
therefore give rise to different concentration levels and therefore differing
productivities. The consequent concentration profiles depend, of course, on the
reaction kinetics, which express the rate of reaction as a function of the
concentrations of reactants and products.
64 2 Basic Bioreactor Concepts

Table 2.1. Summary of reactor modes.

Mode of operation Advantages Disadvantages

Batch Equipment simple. Suitable Downtime for loading and

for small production. cleaning. Reaction
conditions change with
time.

Continuous Provides high production. Requires flow control.

Better product quality due Culture may be unstable
to constant conditions. over long periods.
Good for kinetic studies.

Fed batch Control of environmental Requires feeding strategy to

conditions, e.g. substrate obtain desired
concentration. concentrations.

Table 2.2 lists the main operating parameters for the three differing modes of
bioreactor operation.

Table 2.2. Operating variables for batch and continuous bioreactors.

Batch Continuous Semicontinuous

Initial medium composition Inlet medium Feed and initial substrate

and inoculum composition composition

Temperature, pressure Temperature, pressure Temperature, pressure

pH if controlled pH if controlled pH if controlled

Reaction time Liquid flow rate Liquid flow rate

(residence time) (residence time)
Aeration rate
Aeration rate Feeding rate and control
Stirring rate program
Stirring rate
Aeration rate

Stirring rate
2.2 Bioreactor Operation 65

The foregoing discussion of the varying characteristics of the different reactor

types and their concentration profiles allows a qualitative comparison of the
volume requirements for the different types of reaction, according to the
particular kinetics. For this it is first necessary to consider the qualitative nature
of the basic forms of kinetic relationship: zero order, first order, product and
substrate inhibition. The detailed quantitative treatment of these kinetic forms is
dealt with in Ch. 3.
The rate of a zero order reaction is independent of concentration. Many
bioreactions at high substrate concentrations follow zero order kinetics and are
therefore insensitive to concentration and to the effects of concentration
gradients. From the kinetic viewpoint, therefore, any reactor type would be
equally suitable.
First order reaction rates are directly proportional to concentration.
Bioreactions at low concentration are generally first order, and this would favor
operation in either a batch or a tubular/column type reactor. This is because
reactant concentrations in such reactors are generally high overall and hence
the overall rates of reaction are also consequently high. Hence the reactor
volume required for a given duty would generally be small. (In the case of a
batch reactor, this of course neglects the time lost for filling, emptying and
cleaning.)
A reaction with substrate inhibition would be best run in a tank at low
substrate concentration, since the concentration would be low throughout the
whole of the tank contents. Conversely, product inhibition would be more
pronounced in tank reactors, since product concentration would be at its
highest. In this case, a tubular type reactor or batch reactor would be preferred.

Table 2.3. Kinetic considerations for reactor choice.

Continuous
Reaction Batch Tank Tanks-in- Continuous Fed Batch
Kinetics Series or Single Tank
Tubular
Zero order OK OK OK Low con-
version
First order Best Best Low con- OK
version only
Substrate Low initial Low tank Best Best
inhibition concentration concentrations

Product Best Best Low con- Low con-

inhibition version only version only

Production OK for temp- Possible Not suitable Best for con-

triggered by erature-shift centration-
shift in shift
environment
66 2 Basic Bioreactor Concepts

Table 2.3 compares the performance of batch tanks, continuous tubular or

tanks-in-series reactors and single continuous tank reactors. As discussed in
Sec. 4.2.1, batch tank concentration-time profiles are exactly analogous to the
steady state concentration-distance profiles obtained in continuous tubular
reactors. In terms of performance, therefore, the batch reactor would be the
same as a tube, when compared on the basis of equal batch time in the tank and
time of passage through the tube. Tanks-in-series reactors, as shown in Fig. 2.8,
involve step wise gradients, which in the limit are very similar to those of
continuous tubular reactors, hence, making their performance similar to that of
a tubular reactor. Owing to the high degree of mixing which leads to a uniform
concentration, the performance of the single continuous stirred tank reactor is
very different to that of the other reactor types. An exact quantitative
comparison can be made using the mass balance equations developed in Ch. 4
for each reactor type.