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Bioprocess Engineering Prof. Dr.

Rainer Buchholz

5. Fermentation Processes

Schematic course of a bioprocess

Substrate Pretreatment / Substrate analysis

pH-Adjustment H 2O
Preparation of the Mixing
Addition of salts, Culture medium
P-, N - sources etc. Cultivation of the
production strain
Sterilisation
Inoculum
Energy
CO2 and other Gases
Aeration Fermentation
Measurement and Energy
control
Separation of Remaining culture broth
Filtration and / or
biomass and
culture broth
Separation Biomass

Product isolation Waste

Product purification Waste


confection

Product Waste elimination

Fig 5 - 1: Schematic flow-sheet of aerobic cultivation

The following parameters for the bioprocess are necessary to define:

1. Flow pattern (batch-reactor, continuous flow reactor, semi flow


reactor)
2. Reactor type (stirred-tank, bubble-column, etc.)
3. Reactor volume
4. Reactor material
5. Reaction conditions (temperature, pressure, concentrations, residence
time, mixing time, degree of dispersion of gas-phase or cells, purity of
educts)
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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Presupposition for the commitment of these parameters is the knowledge of


the quantity of the product, its quality and the raw materials. Its also
important to consider the national laws and environmental boundary
conditions. During process development it is necessary to keep the
production costs under the given conditions described above as low as
possible. But in any case the production costs are strongly influenced by all
these parameters.

The influence of the amount of production and utilisation of equipment


capacity are briefly discussed:
A common relation between the relative increase of investment for bigger
equipment and its capacity is given by the following equation.
x
investment capacity
(5 1)
investment 0 capacity 0

Whereby x = 0.6

The installation of two parallel equipments for the increase of a production


plant never leads to a reduction of the invest amount. A reduction is only
possible in the state of planning while using one bigger plant with a higher
capacity. The estimation of a correct reactor-scale leads to less production
costs and in this way to an increase of profit.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

1 Chlorine-alkali-electrolysis
5 (x = 0.38)
a
2 Ethanol (x = 0.6)
3 3 Ethylene (x = 0.71)
4

3 4 Ethyleneoxide (x = 0.78)
2 5 Low pressure polyethylene
b
2 (x = 0.9)

1 Fermentation plants
a > 200 m3
b < 200 m3
1

Fig 5-2: Relation between the relative investment and the relative plant
capacity

The reduction of costs is increasing by decreasing the degression coefficient


x. In the case of x ~ 1 it makes more sense to increase plant-capacity by
installation of a secondary (parallel) plant. In this case the equipment is
more suitable to react on the request of the market situation.

Another important point is that the capacity of a production plant should


always run at high level. Only in this case the production costs are as low as
possible and the higher investment for the bigger plant is taken into
account.

As in "chemical reaction engineering" the production costs of biochemical


products consist of two types of costs:

1. Fixed costs
2. Variable costs

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Table 5-1 gives a listing of these costs.

Tab. 5-1 Types of production costs of a biochemical product

a) fixed costs (independent of b) variable costs (dependent on


productive capacity used) productive capacity used)
1.) depreciation of plant or rent for 1.) raw material costs
production plant
2.) interests for invested capital 2.) energy costs
3.) taxes depending on company 3.) other costs (water,
property cleaning....)
4.) wages for permanent 4.) wages for additional
production staff production staff
5.) cost for strain culture 5.) distribution costs, transport
costs
6.) costs for environmental
protection ("social costs") not
included in investment costs

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

1
2
6000
Profit
3
3000
Loss
B
S 4

20 40 60 80 100 120
Capacity [%]
Fig 5-3: Production cost as a figure of use of the capacity
1 realised proceed
2 constant + variable costs
3 variable costs
4 constant costs
B break even point; S shut down point

In fig. 2-6 the constant and variable costs are plotted. The constant costs are
independent from the amount of production rate while the variable ones
show a proportional increase with the amount of production rate. The total
production cost is calculated by adding both parts.
The proceeding is also increasing proportional with the sold amount of the
product. The difference between realised proceeding and production cost is
the profit. In this example the plant is running under profitable conditions in
the case of running capacity over 50%. Less running capacity leads to
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Bioprocess Engineering Prof. Dr. Rainer Buchholz

losses. From a running capacity over 35% up to 50% the loss is less than
the constant costs of the production plant, in this case it's more economic
not to shut down the plant. In the case that the loss exceeds the constant
cost it is necessary to shut down the plant.

Modern petrochemical plants need a "break even point" over 70 to 75%. In


biotechnology the "break even point" can vary over a broad range. It
depends on the type of product: while commodities as single cell protein
also need a similar range of the "break even point" like a petrochemical
plant, other products like diagnostics, therapeutics or high value enzymes
are economical by even reaching only a few percent of the running capacity
of the plant.

In general it is important to increase the economy of bioproducts by


minimizing the production costs. Therefore it is necessary to consider the
whole production process (that means all steps like up-stream, fermentation,
down-stream process) and optimise the whole process over all. Optimising
single steps often leads to suboptimal results!

The biological process, which should be optimised, determines some of the


process conditions (e.g. aerobic, anaerobic). Furthermore, the running
conditions (T, p, pH, C), the type and scale of the bioreactor and its running
mode (batch, continuous) have to be according to the biological
requirements.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.1 Operation modes of bioreactors

The operation modes of bioreactors are distinguished in:


1. Batch mode
2. Steady state mode
3. Semi-continuous mode
5.1.1 Batch mode

The batch or so-called charge mode is indicated by the following


characteristics:

The sterile fermentor broth is inoculated by the production strain


(monoseptic mode) and cultivated for a defined reaction time. During this
time a change in biomass and substrate concentration occurs. Due to
effective mixing within the reactor there is no local gradient in concentration
and temperature. The reactor is characterized by its non-stationary
behaviour. As a reactor model the discontinuous ideal mixed vessel can be
used.

reactor concentration profile


in time in space
C C
Ca Ca t=a

Ce Ce t=e
DIK
t z
BSTR

Fig. 5 - 4: Concentration course of a batch reactor

Advantages:
- low investment costs due to relatively low control equipment

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

- high flexibility due to the use of different products or specifications


- high conversion rate by given reaction time
- due to relatively short cultivation time there is a minor risk of infection
and mutation

Disadvantages:
- non-productive dead times for filling and idling, heating and cooling
- high personnel costs for running the non-stationary process
- higher risk for the operating staff coming in contact with the pathogen
organisms or poisonous products
- higher wear on the equipment by numerous sterilizations

The batch process is used, if:


- only small amounts of products are formed
- the reactor should be used for different products
- there is a high risk of infection
- the organisms show the tendency towards mutation
- the product recovery from the biomass is difficult (e.g. break up of the
cells)

5.1.2 Steady state mode

The steady state or so-called continuous mode is indicated by the following


characteristics:
The culture broth is introduced into the reactor by constant feed. The feed
stream can be sterile or contaminated by the production strain. The same
amount of culture broth is taken out of the fermentor. All reactants,
concentrations of the compound, and of the cells, temperature etc. will be
kept constant. In the case of a continuous tube reactor (ideal plug flow

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reactor), the conditions are described as stationary, however not


homogeneous.
In the case of a stirred tank reactor (ideal mixed flow reactor), there is no
local and no time gradient in concentration and temperature due to
effective mixing within the reactor. The reactor is described as being
capable of stationary behaviour and homogeneity. As a reactor model the
continuous ideal mixed vessel can be used.

reactor concentration profile


in time in space
C C
Ca Ce
Ca z=a Ca

KIR Ce z=e Ce
CPFR t z

Fig. 5 - 5: Concentration course of a plug flow reactor

reactor concentration profile


in time in space
C C
Ca Ce
Ca Ca

Ce Ce
KIK t z
CSTR

Fig. 5 - 6: Concentration course of a continuous mixed flow reactor

Advantages:
- high standard of automation
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Bioprocess Engineering Prof. Dr. Rainer Buchholz

- low staff cost


- smaller reaction volumes due to no non-productive times for filling and
idling, heating and cooling
- constant product quality due to constant operation conditions
- lower risk for the operation staff caused by non-manual operations
- lower wear on the equipment by reducing the sterilization cycles

Disadvantages:
- low flexibility due to fixed operation conditions
- demand for a constant quality of the raw materials
- high investment costs for automation and measurement devices
- higher risk of infection and mutation due to the long cultivation times

Up to now the continuous mode is seldom used in biotechnology. Due to


the advantages and disadvantages there is an applicability only for bulk
production and, if microorganisms are used that are stable and show no
mutation.

5.1.3 Semi-continuous mode

The semi-continuous mode is a combination between the batch mode and


the steady state. In this case some components are running under batch
conditions, others (e.g. oxygen) under continuous conditions.
Due to the low solubility of oxygen it is necessary for all aerobe cultivations
to add oxygen continuously. Therefore, all batch reactors for aerobe
cultivations are running in a semi-continuous mode.
Furthermore, during the fermentation process the production of undesired
by-products occurs. These disturbing materials have to be constantly
removed (e.g. CO2 or organic acids).

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.2 Mixing (Ideal Reactor Types)

Most of the reactors used in practice are describable with more or less
complicated mathematical models. One successful application uses a few
basic Ideal Reactor Types, which are combined or expanded to describe the
non ideal reactors.

The flow pattern of the ideal reactor types:

1. BSTR (Batch Stirred Tank Reactor)


In a BSTR one finds ideal mixing conditions realised by intensive stirring.
This reactor type runs under uniform composition everywhere in the
reactor, but of course the composition changes with time. For a batch
reactor the input and output rates are zero!
Therefore the balance equation is given:
dn i
VR ri (5 2)
dt
This reactor works homogeneous but not under steady-state conditions.

2. CSTR (Continuous Stirred Tank Reactor)


This reactor type runs under continuous conditions, but like the BSTR this
reactor type is uniformly mixed. That means same composition everywhere,
within the reactor and at the exit. While this reactor runs under steady-state
conditions the accumulation term becomes zero in Equ. 5 - 2.
dn i
0 (5 3)
dt
While there is no change in composition during time and volume this
reactor type is running under homogeneous and steady-state conditions.

3. CPFR (Continuous Plug Flow Reactor)

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dni
0 (5 4)
dt x const

This reactor type is an idealised long tube without any back-mixing inside,
which means fluid flows with a flat velocity profile. It also runs under steady-
state conditions but the composition changes progressively through the
reactor.

5.2.1 Residence time distribution

The discussed two ideal flow patterns, plug flow, and mixed flow can give
very different behaviour depending on order of reaction rate. But these two
cases are extrema and often not realisable in technical equipments. In the
following part the real mixing in technical equipments will be discussed.

! Residence time and space time


The residence time t of a defined element of the volume leaving the
reactor is the period of this element staying in the reactor. These periods of
each volume element (and even single molecules) differ from each other.
This fact leads to the residence time distribution.
The average residence time t of all volume elements is not easy to
define in general for all cases of mixing conditions. This is the reason to
introduce the space time.

The space time is defined as that time one needs to run the same
volume as reactor volume at starting conditions through the vessel.
VR

a
time (5 5)
V
VR - reactor volume
a
V - feed stream at reactor entrance

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Space time is the relation between reactor volume and volumetric feed flow
(at reactor entrance and under defined conditions). If there is no change in
density of culture medium inside the reactor then space time is equivalent to
the average residence time.

The inverted value of space time is called space velocity s:


1 V 1
s a time (5 6)
VR

This is the relation between volumetric feed flow and the reactor volume.
Batch Stirred Tank Reactor (BSTR)

Equations for calculating the reaction time and the reaction volume:

Residence time t:
A
d A n Ka n K
t n Aa conversion (5 7)
a V rA n Ka

V = const.:
cA
dc A
t rA
(5 8)
c Aa

For 1st order reaction, -rA = k cA, is valid:


1 c Aa
t ln (5 9)
k cA
1 1
fr Aa 0 : t ln (5 10)
k 1 A
A
d A
beiV Va 1 A : t n Aa rA Va 1 A (5 11)
Aa

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Ideal Stirred
Ideal Tube Reactor (KIR)
Tank (KIK)
(plug flow)
(for Aa 0 )

A
d A n Aa
Reactor Volume n Aa
rA A
Aa rA

Space time =

A
d A c Aa
a) generally c Aa rA rA
A
Aa

b) First order
reaction, 1 1 1 A
ln
constant density, k 1 A k 1 A
Aa 0

c) 1 order reaction,
1 1 1 A
a 1 A
V
V 1 ln A 1 A
k 1 A k 1 A
Aa 0

(5-12) to (5-14)

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Ideal Tube Reactor


Ideal Stirred Tank (KIK)
(KIR)
(for Aa 0 )
(plug flow)

Mean
Generally
residence time

A
d A c Aa A
t c Aa a
V t
Aa

rA V rA 1 A 1 A

For 1. order
1 A
reaction,
k 1 A
constant density

(5-15)

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.3 Continuous fermentation

In practice, continuous production methods are used in a variety of


chemical processes. These continuous methods have replaced the non
stationary processes in many cases. Particularly in the production of
commodities there are numerous advantages of using continuous methods:
S higher productivity
S simplier process automation
S homogeneous product composition
In contrast to chemical technology continuous methods are rarely used in
biotechnology. One important application is the biological waste water
treatment.
The most essential reasons that have prevented the use of continuous
methods in biotechnology until now are:
S Problems of sterility
It is difficult to run long time fermentations in a sterile mode.
S Specification of product charges
For the validation of pharmaceutical products, a charge number has
to be assigned to the product necessary.
S Danger of mutation and degeneration of cells
High productivity mutants are often unstable and due to their
degeneration a decrease in productivity occurs.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

S Missing knowledge of process dynamics does not permit


optimal automation
Biological processes are subject to a broader spread, contrary to
chemical reactions, which run under a far reaching reproductiveness
obtaining the same productive quality. The knowledge of the course
of biological reactions is often missing.

5.3.1 Classification of continuous fermentations

A fermentation process can be distinguished by the type of control.

a) External control (Chemostat)


Fermentations with external control are indicated by a constant growth rate.
This growth rate lies below the maximum value due to a controlled influx.
The growth of the cells is kept constant by the concentration of the
nutrients. Each substrate can be used as a limiting factor. In practice such a
system is controlled by keeping the liquid level constant in the fermentor.

b) Internal control (Turbidostat)


The cell concentration is measured (optical density, biomass probes) and its
growth is kept constant by the adjustment of the substrate influx. This type
of control is particularly desirable.

Classification by the used fermentor type


This classification is similar to that of chemical reactors and is based on
physical and operational criteria. The nature of the fermentation and
biological criteria is not taken into account.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

The following two subdivisions can be pointed out:

a) open systems
b) closed systems

Both may be divided into:

1) homogeneous systems
2) heterogeneous systems
3) mixed systems

Concerning the open systems, the newly formed biomass is continuously


diluted out of the fermentor. In the closed systems, the biomass is held
back in the fermentor by either immobilization or back flow. The back flow
as part of the biomass is also possible in open systems.
Homogeneous systems are using well mixed reactor types (ideal stirred
tank characteristic). There are no local gradients in the concentration of the
cells and of the substrate. Those gradients are found in heterogeneous
systems (as a rule, they are desired in this case).

So called single phase systems are fermentation systems which consist of


only one homogeneous liquid phase. Contrarily, multi phase systems
contain a second phase (gas or solid phase). If two or more non mixable
fluids are used, the system is called multi component system.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

homogeneous

cellophane tube reactor stirred tank reactor with


100 % recycled cells

Fig. 5 - 7a: Closed systems (homogeneous)

heterogeneous (single phase)

S
100%
100%

continuous tube reactor divided tank reactor with


with 100 % recycled cells 100 % recycled cells

Fig. 5 - 7b: Closed systems (heterogeneous, single phase)

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

heterogeneous (multi phase)

packed bed reactor

Fig. 5 - 7c: Closed systems (heterogeneous, multi phase)

homogeneous
(single stage)

stirred tank reactor stirred tank reactor with


particular recycled cells

Fig 5 - 8a: Open systems (homogeneous, single stage)

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

homogeneous
(multi stage)
S S Z Z

cascade cascade (multi substrate dosage)

Fig 5 - 8b: Open systems (homogeneous, multi stage)

heterogeneous
single phase multi phase
cells in substrate out

S
cells out substrate in

continuous tube reactor multi phase column

Fig 5 - 8c: Open systems (heterogeneous)

mixed system

stirred tank connected with tube reactor

Fig 5 - 8d: Open systems (mixed)

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.3.2 Theoretical fundamentals of the continuous fermentation


5.3.2.1 Mass balance

Basically, the quantitative description of continuous cultures is the mass


balance of all the involved compounds (microorganisms, substrates and
products).
The construction of mass balance equations are clarified in the following
chapter.

cA cA2
1

Fig. 5 - 9: Balance area

The usual procedure:


1. Definition of the balance area
2. Estimation of the in- and out-flow
3. Determination of the involved compounds
4. Formulation of the balance equation

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

V0 Ve
ci
o V R ci

Fig. 5 - 10: Mass balance

For the general case of the continuous stirred tank fermentor, the mass
balance for the cell density can be formulated as follows:

F [l/h] F
X0 [g/l] X
S0 [g/l] S11

Fig. 5 - 11: Continuous stirred tank fermentor


F flow rate
X0 cell density (in-flow)
X1 cell density (out-flow)
S0 substrate concentration (in-flow)
S1 substrate concentration (out-flow)
(for the CSTR also the concentration inside the fermentor)
V reaction volume

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Balance
dX1 dX
V F X 0 F X1 V 1 (5 17)
dt dt growth
2
dX1 F F dX
X 0 X1 1 (5 18)
dt V V dt growth

Definition:
F flow rate
D dilution rate
V reaction volume
The increase of cell density due to growth is given by:
dX1
X1
dt
Combining these expressions one obtains:
dX1
D X 0 X1 X1 (5 19)
dt
For a stationary case the change of biomass in time is equivalent to zero. If
the in-flow contains no microorganisms (X0 = 0), the following result is
obtained:
D X1 X1 or
(5 20)
D
For a steady state the specific growth rate is equivalent to the dilution rate.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.3.2.2 Mass balance of cell density, product and substrate in


a stirred tank cascade containing n vessels

The next case shows an illustration concerning a mass balance of an


arbitrary stirred tank depicting a cascade.

F [l/h ]

X [g/l]
0

S [g/l] X1 X Xn
0 n-1

p [g /l] S1
0 V V .. S n-1 V Sn
P1
P Pn
n-1

1 2 n

Fig. 5 - 12: Mass balance of a stirred tank cascade

The mass balance for the cell density X inside the nth vessel is given by:
dX n
V F X n 1 F X n V n X n (5 21a)
dt
or, after divided by the volume:
dX n
DX n 1 X n n X n (5 21b)
dt
The mass balance for the substrate S inside the nth vessel is given by:
dSn dS
V F Sn 1 F Sn V n (5 22a)
dt dt growth

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

or, after divided by the volume:


dSn 1
DSn 1 Sn nXn
dt YX / S
(5 22b)
Y
DSn 1 Sn P / X n X n
YP / S
whereby:
X
YX / S cell substrate yield
S
P
YP / S product substrate yield
S
P X
YP / X YX / S
X S
The mass balance for the product P inside the nth vessel is given by:
dPn dP
V F Pn 1 V n F Pn (5 23a)
dt dt production

or:
dPn
DPn 1 Pn YP / X n X n (5 23b)
dt
In the case of the steady state, the left sides of the equations (5 21) to (5
23) are zero. Therefore, the biomass of the nth vessel is given as:
D X n 1
Xn for n 1
D n
D X n 2
X n 1
D n 1

D X n 1 D2 Xn 2
Xn ..........
D n D n D n 1

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

The value for Xn can be obtained by:


D n 1 X1
Xn n
(5 24)
D i
i2

Equation (5 24) allows the estimation of biomass Xn within the nth vessel.
Moreover, the specific growth rate n can be calculated from the biomass
Xn, Xn-1, etc.
As for the boundary condition n=1, meaning the cascade has only one
vessel, the biomass X0 on the right side of equation (5 21) to approach
zero. Because the functional correlation between the specific growth rate
and the substrate concentration, defined by Monod, is valid, the following
equation applies:
S1
1 D max (5 25)
K S S1

Since
S1
1
K S S1

the maximum specific growth rate can never be reached under those
conditions, the conclusion is
D max
so that no wash-out of the cells occur.
The stationary product concentration for the nth vessel can be determined in
an analogue way:
D Pn 1 YP / X n X n
Pn (5 26)
D

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

For the boundary condition n = 1 results in P0 = 0:


YP / X 1 X1
Pn YP / X X1 (5 27)
D
The stationary substrate concentration can be calculated by:
1 YP / X
Sn Sn 1 n Xn
D YP / S

or:
1 1
Sn Sn 1 nXn (5 28)
D YX / S

The substrate concentration for a single vessel is:


YP / X / YP / S
S1 S0 1X1
D
or, for = D
The result for S1 is:
YP / X
S1 S0 X1
YP / S
1
S1 S0 X1 (5 29)
YX / S

5.3.2.3 Criteria for reactor design

For the stationary mode, in the case of which one single vessel is used, Eq.
(5 25) can be transformed into:
KS
S1 (5 30)
max

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Accepting that D = :
D KS
S1
max D

or transformed:
KS
S1 (5 31)
max
1
D
As long as the dilution rate is much smaller than max, the substrate
concentration can be obtained from Eq. (5 31) as:
D KS
S1 (5 32)
max

Introduced to Eq. (5 29), the biomass can be obtained after


transformation as:
D KS
X1 YX / S S0 (5 33)
max

It is evident according to Eq. (5 31) and (5 32), that for D<<max the
concentration of the limiting substrate proportional increase starts at zero. If
D approaches the value of max, the concentration of S1 increases rapidly
and becomes infinite by D = max.
However, this case can not occur in reality, due to the fact that:
S1 S0
That means S1 can never overcome the initial substrate concentration!
The cell concentration is given at small values of D approximately by YX/S
S 0.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

For
D max and S S0 follows X1 0 !
This condition is called wash-out, and it appears when the provided dilution
rate is greater than the specific growth rate.
Conclusion: the residence time of the microorganisms inside the bioreactor
gets shorter than the average generation time.
The relations during continuous fermentations are shown in a clear
representation by the graph X over D, or S over D.

5 X 5

4 8 4

3 6 DX 3

2 4 S0 2

1 2 S 1

0,25 0,50 0,75 1,00


flow rate (h-1)

Fig. 5 13: X/D- or S/D-diagram

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

The diagram represented here contains the following data:


max = 1,0 h-1
YX/S = 0,5
S0 = 10 g/l
KS = 0,2 g/l

Productivity of a continuous culture

Generally, for the productivity of a continuous culture it is found that:


Pr X D
using Eq. (5 33) and (5 31) it follows:
D KS
X D D YX / S S0 (5 34)
max D
To identify the value of D which leads to a maximum of XD, it is necessary
to formulate the expression:
dX D
0
dD
and solve it for the dilution rate (D)Pr,max. This calculation is the result for the
maximum productivity:
KS
D Pr,max max 1
(5 35)
K S S0
Due to the fact that KS << S0, it consequently shows that generally the
maximum of productivity is the yield near the wash-out point.

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

The biomass at the dilution rate for maximum productivity can be obtained
from Eq. (5 33) and (5 31):

KS
X1 YX / S S0 (5 36)
max
1
D
Substitution of D by Eq. (5 35) leads to:



KS
X1 YX / S S0
max
1
KS

max 1

K S S0
KS
KS KS
K S S0
X1 YX / S S0
KS
K S S0

K K S S0
X1 YX / S S0 S KS
KS


X1 YX / S S0 K S K S S0 K S (5 37)

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Graphic methods

The following part shows, how the biomass concentration can be


determined graphically in the case of equilibrium. From the balance
equation (5 - 19) under the boundary condition X0 = 0 and steady state
one obtains:
dX1 dX
0 1 D X1
dt dt growth

or transformed:
dX1

dt growth
X1 (5 38)
D
The growth rate (dX1/dt)growth is obtained directly from the grade of the
growth curve given by the cultivation under batch conditions.

X
d X1
X1
d t

t
Fig. 5 - 14: Growth curve

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Bioprocess Engineering Prof. Dr. Rainer Buchholz

Plotting dX/dt versus X, one obtains a straight line with the gradient:
1 dX
gradient
X dt
which is similar to the dilution rate D under steady state conditions of the
continuous culture.
The point of intersection between the straight line with the grade D and the
growth curve from the batch culture is a stationary operating point of the
continuous culture.

dX
d t Operating point

D
X1
X

Fig. 5 - 15: Operating point of a continuous culture

The point of intersection between the straight line with the grade D and the
growth curve from the batch culture is a stationary operating point of the
continuous culture.

- 104 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

For a multi stage plant follows consistently:


dX 2 dX
0 2 D X 2 X1 (5 39)
dt dt growth

dX 2

dt growth
X 2 X1 (5 40)
D
The positions on the operating points are shown in Fig. 5-16. This graphic
method can only be applied, if the growth curve of the batch culture is
equivalent to those of the continuous culture. This assumption is often not
fulfilled due to the continuous change of the environmental conditions of a
batch culture!
Nevertheless, this method can be consulted to estimate the stable operating
point of a continuous culture approximately from batch data.

Operating point 1

dX
d v
Operating point 2
D

X1 X2
X

Fig. 5 - 16: Operating points of a continuous multi stage plant

- 105 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.4 Balance of a multi stage reactor system


5.4.1 Vessel with cell recycling

FR=(1+" )F
F
S0 X1 F,X2,S1
X0= 0 S1

FX,XX,S1

V, X 1
"F, XX, S1

Fig 5 17: Vessel with cell recycling


F = flow rate (inflow)
FR = flow rate (outflow of the reactor)
F* = flow rate (clearified outflow of the separator)
F*X = flow rate (concentrated outflow of the separator)
= relation of back flow
F = flow rate of the recycled cell suspension

The figure shows that:


FR 1 F (5 41)

and:
F F* FX* (5 42)

- 106 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

Calculating the cell density by applying the mass balance, the result is:
dX1 dX
V X X F 1 FX1 1
dt dt growth

or divided by the volume V:


dX1
X X D 1 DX1 1X1 (5 43)
dt
Introducing the Monod equation:
dX1 S1
X X D 1 DX1 max X1 (5 44)
dt K S S1
Subsequently, the mass balance can be applied for the determination of
substrate as follows:
dS1 S
V FS0 FS1 1 FS1 V 1
dt dt growth

dS1 1
DS0 DS1 1 DS1 1X1 (5 45)
dt Y / S

dS1 1 S1
DS0 DS1 1 DS1 max X1 (5 46)
dt Y / S K S S1

can be obtained from equation (5-43) for the steady state.


1 dX1 X
1 D X D
X1 dt X1

or:
X
D1 1 X (5 47)
X1

- 107 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

For the balance of biomass in the separator it is found:


1 FX1 F*X 2 FX* X X FX X

F* X 2 X X FX* F
1 F F*
X2 XX *

X 1 X1

FX F
From this follows:
XX X
1 F F 2
X1 X1
FX* F

or:
F* X 2
1
XX F X1

X1 FX*

Introducing FX* = F - F*:


F* X 2
1
XX F X1
(5 48)
X1 F*
1
F
By inserting equation (6-48) into equation (6-47), one gets:
F* X 2
1
F X1
D1 1 (5 49)
F*
1
F

- 108 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

For X2/X1 0 this equation is simplified to:




1
D1 1 (5 50)
F*
1
F

1,0
F*
: F
=0

D 0,8

0,6
0,6

0,4 0,8

0,9
0,2
F* 0,95
= 1
F 0,98
0
0 1 2 3
"
Fig. 5-18: Influence of the relation of back flow on /D

Consequently, figure 5 - 18 shows that stationary states are realized in the


bioreactor where the dilution rate D is greater than the specific growth rate
. Those conditions were used specifically in the field of waste water
treatment.
For the condition F*/F = 0 the same situation is found in a single vessel
system without recycled cells. In this case the specific growth rate must be
equal to the dilution rate for stationary behavior.

- 109 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

From equation (5-47) follows for the stationary case:


S1 X
max 1 X D AD (5 51)
K S S1 X

with
X
1 X A
X
The substrate concentration S1 is given by:
AD
S1 K S S1
max

or solved for S1:


K S AD
S1 (5 52)
max AD

For the biomass in the vessel one obtains correspondingly:


YX / S
X1 S0 S1 (5 53)
A
From the equations (5-51) to (5-53) the coherence between biomass X,
substrate concentration S, the productivity XD and the dilution rate can be
calculated. The courses are presented in fig. 5-19. To compare the
conditions without back flow of the cell are also pointed out in this graph.

- 110 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

10
A

X 5 C

0
0 1 2

D
Fig. 5-19: Courses of concentration during a continuous fermentation

These calculations are based on:


max = 1,0 [h-1] = 0,5
YX/S = 0,5 Xx/X = 2
S0 = 10 [g/l] KS = 0,2 [g/l]

Legend of fig. 5 -19:


A: Biomass concentration in the fermentor
B: Productivity
C: Cell concentration in the outlet
D: Cell concentration without back flow
E: Productivity without back flow

- 111 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.4.2 Two stage fermentor without cell recycling

Two stage plants were seldom used in biotechnology. Applications are


known in the waste water treatment and in the case of different substrates
during induced conversions. Under normal conditions the substrate S will
be utilised nearly completely in the first level. That means that the
concentration of the substrate is decreasing to low values and in the case
without substrate addition, only a subordinating growth can be obtained!
Another point of view is to run the first reactor close to the wash out point,
then the utilisation of substrate is low in this vessel.
Third, it is possible to run both vessels under the same growth rate. But it
can easily be shown that under these conditions the productivity is the same
as the yield in one vessel with the equivalent volume.
Advantages of the use of multi stage systems are only found if
- a complex growth media is used
- a certain physiological growth condition is desired.
The case of complex media is given, if two or more substrates are present,
which leads to different growth rates. In such a broth the better usable
substrate can be utilised in the first vessel at relatively high dilution rate, and
the other substrate is converted much slower but nearly complete in the
second vessel as the only substrate (multi-stage waste water treatment).

For this case the following example can be given:


The fermentor broth contains the substrate A and B and the following
assumptions are valid:
YXA/ S YXB/ S K SA K SB Amax Bmax

- 112 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

F F
F
A A A
S0 S1 S2
B B
B S1 S2
S0
V1 X1 V2 X2

A
S0
X S B0
XB
S
XA

D
Fig. 5-20 and 5-21: Two stage facility without back flow of the cells

Assumption: S1A 0 and S0B ~ S1B

The mass balance of the first stage is equivalent to that of a single vessel, for
the second stage it is found for the biomass:
dX 2 B SB2
D 2 X1 D 2 X 2 max X 2 B (5 54)
dt K S SB2
- 113 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

for the substrate:


dSB2 1 SB
D 2 S1B D 2 SB2 Bmax B X 2 B 2 B (5 55)
dt YX / S K S S2

steady state:
X 2 X1 YXB/ S S1B SB2 (5 56)
D 2 X1
X2 (5 57)

D 2 K SB SB2 Bmax SB2

D
X1 YXA/ S S0A K SA A 1 B (5 58)
max S2

- 114 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.4.3 Two stage facility with substrate addition in the second


vessel

An additional substrate in the second vessel also guarantees growth even if


the substrate S0 is utilised in the first vessel.
In the second stage a wash out can never happen, as long as the biomass
from the first stage is fed. That means D2 can even be greater than max.

F2 S0,2

F1
(F1 + F2 )
S0 S1 S2
X1 X2
V1 X1 F1 V2 X2

Fig. 5-22: Two stage facility with additional substrate in the second vessel

Mass balance of the substrate in the second vessel:


dS2 V1 F 1 S2
D1S1 2 S0, 2 D 2S2 max X 2 (5 59)
dt V2 V2 YX / S K
S S 2

with
F1 F F2
D1 andD 2 1
V1 V2

- 115 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.4.4 Two stage facility with additional second substrate in the


second vessel

(Biotransformation)

F2 Z0

F1
(F1 + F2 )
S0 S1 S2
X1 X2
V1 X1 F1 V2 X2 P2
Z2

Fig. 5 - 23: Two stage facility with an additional second substrate in the
second vessel
Legend of fig. 5 - 23:
P = product
Z = convertible substrate
YP/Z = -dP/dZ
YP/Z = -dP/dZ

Numerous reactions for transformation follow the Michaelis Menten kinetic.


In these cases the following attempt is valid:

dZ dP Z
YP / Z k X (5 60)
dt dt KZ Z
For the case that KZ << Z the product formation rate is given:
dP
kX
dt
- 116 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

For the dilution rates D1 and D2 the following equations are valid:
F1 F1 F2
D1 and D 2
V1 V2

Accepting that S1 0, which means that there is no growth in the second


vessel, the next equation directly follows:
X 2 F1 F2 F1 X1
or:
F1 F1
X 2 X1 YX / S S0 (5 61)
F
1 F2 F
1 F2

Due to the fact that the substrate for conversion is added in the second
vessel, the balance of Z is given by:
dZ 2 1 F F2 F
kX 2 1 Z 2 2 Z 0 (5 62)
dt YP / Z V2 V2
For steady state:
F2 V2 1
Z 2 Z 0 kX 2 (5 63)
F1 F2 F1 F2 YP / Z

A complete conversion is obtained providing the concentration of Z at the


exit of the second vessel (Z2) equal to zero. In this case the equation (5-45)
becomes:
1 1 F1
F2 Z 0 V2 kX 2 V2 k YX / SS0 (5 64)
YP / Z YP / Z F1 F2

- 117 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

5.5 Residence time distribution of steady state reactors


Ideal and non-ideal residence time behaviour

Ideal: CPFR, CSTR

Non-ideal: a) dead or stagnant region within the reactor,


b) channel flow (bypass)
c) back flow

Fig. 5-24: Examples for reactor types with non-ideal residence time
distribution

Residence time function


2 extrema: a) plug flow reactor (ideal), CPFR,
b) stirred tank reactor (ideal), CSTR.
Step input at time t = 0 at the entrance of the reactor (t < 0: no tracer; t >
0 with tracer)

- 118 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

Residence time of a volume element:

a) Tube reactor with plug flow, CPFR:


All flow elements leaving the reactor at a given time (t = n) have an
identical residence time.
ci
c ia

1,0

0
J t
Fig. 5-27
c i part of volume elements at reactor exit

c ia with a residence time shorter than t

b) Stirred tank reactor with ideal backmixing, CSTR:


The concentration of ci within the reactor is constant at any place (ideal
mixed flow). The concentration at reactor exit must be identical with the
concentration ci within the reactor. Changing the input flow at time t = 0
from concentration ci to concentration cia leads to a change in concentration
at reactor exit as a function in time.
t
ci
1 e 1 e (5 65)
c ia

t
dimensionless time

Fr t :
ci
1 e 1 0,63212
c ia
- 119 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

ci
c ia
1,0

0
J t
Fig. 5-28

Residence time function

The concentration course ci/cia as a function in t f(t) with cia = 1 gives the
step response curve, the so called Residence time sum function F.

The function F (t) gives that part of exit flow with a residence time shorter
than t within the reactor.
t0 F0
t F 1
The part of exit flow with a given residence time within the reactor could be
determined by differentiating the F-curve at t:
d Ft
E t (5 66)
dt

E(t) is the so called Residence time distribution function.

The internal age distribution function I(t) is defined as:


l t 1 F t (5 67)

That part of exit flow staying a time period between t and (t + dt) within the
reactor is given by:
d Ft Et d t (5 68)
- 120 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

Therefore the relationship between F and E is:


t
Ft Et d t
0

F Et d t 1 (5 69)
0

F0 0

The average residence time t :

The average residence time is given as the average value of residence time
of all flow elements.

t N t N t E t
t 0
0
t E t d t (5 70)
N N 0

N(t) = is the number of elements with a residence time between t and


(t + dt).
= N E(t)
VR
t (5 71)
V

The investigation of residence time distributions is carried out without


change in density and chemical reaction (isothermal conditions), therefore:

V
a; VR
V t space time (5 72)
a
V

It may be suitable for comparing different reactor types with regard to their
characteristic residence time behaviour to use the dimensionless time :
t V t
(5 73)
VR

- 121 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

Residence time functions with :


dt
d
t
F Ft (5 74)
dF dF
E t t E t dimensionless
d dt

Residence time functions (summarise)

Name Normalised Step Exit Age


Function Distribution

Symbol F E

Relation to the other t d Ft


Ft Et d t E t
function dt
0

Experimental method Step marker Pulse marker

Function for ideal tube F 0 fr t E 0 fr t


(IR) F 1 fr t E fr t

Function for ideal t


t
t

F 1 e E e
continuous tank (KIK)

- 122 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

Experimental methods (non chemical) for finding residence time


functions

There are four methods:

a) The step experiment; input: step-function


(Heaviside-function).
b) The pulse experiment; input: pulse function, (Dirac-
function, Delta-function).
c) The periodic input experiment; input: periodic function (Sinus-
function)
d) The random input experiment; input: random function (white
noise)

Fig. 5-29:
- 123 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

Tanks in Series Model

A cascade of stirred tank reactors can be described as a row of N ideal


stirred vessels.
change in reactant concentration
by reaction
Damkhler number Da l
convective mass transport
(5 75)
VR rA
Da l
V c
A

In the case of constant density:

a ; VR ;
V rA
V Da l
a
V cA
(5 76)

space time
For the continuous stirred tank reactor (CSTR):
A V

Da l with V a (5 77)
1 A
For a cascade of n ideal vessels (each of them with the same volume and
with constant density) the following equation is valid:
N
c Ne c e 1 V
a
with V
c1a c a N Da l 1
N
(5 78)
c 1
cascade N 1 e 1
ca N Da l 1
The conversion N N-vessel cascade with the same volume for each vessel is
given for a 1st order reaction at constant densty by:

V rA
N
1
Da l R k N 1 (5 79)
cA
V k i 1
- 124 -
Bioprocess Engineering Prof. Dr. Rainer Buchholz

The space time is defined under these conditions as:


1

N c a N
N i 1 (5 80)
k c e

Residence time sum function for a cascade reactor:


1 1
N 1 N N 2 ... N N 1
2! N 1!

F 1 e
(5 81)

Residence time distribution function for a cascade reactor:


N
E N N 1 e N (5 82)
N 1!

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