3.

Fed-Batch Fermentations

3.1. Fixed volume fed-batch
3.2. Variable volume fed-batch
3.3. Advantages and disadvantages of fed-batch culture
3.4. Equipment
3.4.1. Vessels
3.4.2. Pumps
3.5. Control techniques for fed-batch fermentations
3.6. Modelling of fed-batch reactors
3.6.1. Fixed volume fed-batch
3.6.2. Variable volume fed-batch
3.6.3. Models of possible situations that may occur in fed-batch fermentation
3.7. Parameters used to control fed-batch fermentations
3.7.1. Calorimetry
3.7.2. Specific growth rate
3.7.3. Substrate (carbon or nitrogen source)
3.7.4. By-product concentration
3.7.5. Inductive, enhancer or enrichment components
3.7.6. Respiratory quotient
3.7.7. General feeding mode
3.7.8. Proton production rate
3.7.9. Fluorescence
3.8. Parameters to start and finish the feed, and stop the fed-batch
fermentation
3.9. Preliminary knowledge required to implement fed-batch
3.10. Algorithms for operating a fed-batch reactor at optimum specific
growth rate (model independent and applicable to adapting systems)

3.11. Some examples of fed-batch use in industry




Two basic approaches to the fed-batch fermentation can be used: the constant volume
fed-batch culture - Fixed Volume Fed-Batch - and the Variable Volume Fed-Batch. The
kinetics of the two types of fed-batch culture will be described in section 3.6.

3.1. Fixed volume fed-batch
In this type of fed-batch, the limiting substrate is fed without diluting the culture.
The culture volume can also be maintained practically constant by feeding the growth
limiting substrate in undiluted form, for example, as a very concentrated liquid or gas
(ex. oxygen).
Alternatively, the substrate can be added by dialysis or, in a photosynthetic culture,
radiation can be the growth limiting factor without affecting the culture volume
5
.
A certain type of extended fed-batch - the cyclic fed-batch culture for fixed volume systems -
refers to a periodic withdrawal of a portion of the culture and use of the residual culture
as the starting point for a further fed-batch process. Basically, once the fermentation
reaches a certain stage, (for example, when aerobic conditions cannot be maintained
anymore) the culture is removed and the biomass is diluted to the original volume with
sterile water or medium containing the feed substrate
22
. The dilution decreases the
biomass concentration and result in an increase in the specific growth rate (see
mathematical description in section 3.6). Subsequently, as feeding continues, the growth
rate will decline gradually as biomass increases and approaches the maximum
sustainable in the vessel once more, at which point the culture may be diluted again
26
.
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3.2. Variable volume fed-batch
As the name implies, a variable volume fed-batch is one in which the volume changes
with the fermentation time due to the substrate feed. The way this volume changes it is
dependent on the requirements, limitations and objectives of the operator.
The feed can be provided according to one of the following options:
(i) the same medium used in the batch mode is added;
(ii) a solution of the limiting substrate at the same concentration as that in the initial
medium is added; and
(iii) a very concentrated solution of the limiting substrate is added at a rate less than (i),
(ii) and (iii)
21
.
This type of fed-batch can still be further classified as repeated fed-batch process or cyclic
fed-batch culture, and single fed-batch process.
The former means that once the fermentation reached a certain stage after which is not
effective anymore, a quantity of culture is removed from the vessel and replaced by
fresh nutrient medium. The decrease in volume results in an increase in the specific
growth rate, followed by a gradual decrease as the quasi-steady state is established.
The latter type refers to a type of fed-batch in which supplementary growth medium is
added during the fermentation, but no culture is removed until the end of the batch.
This system presents a disadvantage over the fixed volume fed-batch and the repeated
fed-batch process: much of the fermentor volume is not utilized until the end of the
batch and consequently, the duration of the batch is limited by the fermentor volume
26
.
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3.3. Advantages and disadvantages of the fed-batch
reactors

Fed-batch fermentation is a production technique in between batch and continuous
fermentation
12
. A proper feed rate, with the right component constitution is required
during the process
8
.
Fed-batch offers many advantages over batch and continuous cultures. From the
concept of its implementation it can be easily concluded that under controllable
conditions and with the required knowledge of the microorganism involved in the
fermentation, the feed of the required components for growth and/or other substrates
required for the production of the product can never be depleted and the nutritional
environment can be maintained approximately constant during the course of the batch.
The production of by-products that are generally related to the presence of high
concentrations of substrate can also be avoided by limiting its quantity to the amounts
that are required solely for the production of the biochemical. When high
concentrations of substrate are present, the cells get "overloaded", this is, the oxidative
capacity of the cells is exceeded, and due to the Crabtree effect, products other than the
one of interest are produced, reducing the efficacy of the carbon flux. Moreover, these
by-products prove to even "contaminate" the product of interest, such as ethanol
production in baker's yeast production, and to impair the cell growth reducing the
fermentation time and its related productivity.
Sometimes, controlling the substrate is also important due to catabolic repression. Since
this method usually permits the extension of the operating time, high cell
concentrations can be achieved and thereby, improved productivity [mass of
product/(volume.time)]. This aspect is greatly favored in the production of growth-
associated products
1
.
Additionally, this method allows the replacement of water loss by evaporation and
decrease of the viscosity of the broth such as in the production of dextran and xanthan
gum
13
, by addition of a water-based feed.
As previously mentioned, fed-batch might be the only option for fermentations dealing
with toxic or low solubility substrates.
When dealing with recombinant strains, fed-batch mode can guarantee the presence of
an antibiotic throughout the course of the fermentation, with the intent of keeping the
presence of an antibiotic-marked plasmid. Since the growth can be regulated by the
feed, and knowing that in many cases a high growth rate can decrease the expression of
encoded products in recombinant products, the possibility of having different feeds and
feed modes makes fed-batch an extremely flexible tool for control in these cases
7, 8
.
Because the feed can also be multisubstrate, the fermentation environment can still be
provided with required protease inhibitors that might degrade the product of interest,
metabolites and precursors that increase the productivity of the fermentation
19
.
Finally, in a fed-batch fermentation, no special piece of equipment is required in
addition to that one required by a batch fermentation, even considering the operating
procedures for sterilization and the preventing of contamination
12
.
A cyclic fed-batch culture has an additional advantage: the productive phase of a
process may be extended under controlled conditions. The controlled periodic shifts in
growth rate provide an opportunity to optimize product synthesis, particularly if the
product of interest is a secondary metabolite whose maximum production takes place
during the deceleration in growth
22
.

As a summary of what was described above, fed-batch mode of fermentation has the
following features:
Advantages:
- production of high cell densities due to extension of working time (particularly
important in the production of growth-associated products)
- controlled conditions in the provision of substrates during the fermentation,
particularly regarding the concentration of specific substrates as for ex. the
carbon source
- control over the production of by-products or catabolite repression effects due to
limited provision of substrates solely required for product formation
- the mode of operation can overcome and control deviations in the organism's
growth pattern
1
as found in batch fermentation
- allows the replacement of water loss by evaporation
- alternative mode of operation for fermentations leading with toxic substrates
(cells can only metabolize a certain quantity at a time) or low solubility
compounds
- increase of antibiotic-marked plasmid stability by providing the correspondent
antibiotic during the time span of the fermentation
- no additional special piece of equipment is required as compared with the batch
fermentation mode of operation
Disadvantages:
- it requires previous analysis of the microorganism, its requirements and the
understanding of its physiology with the productivity
- it requires a substantial amount of operator skill
13
for the set-up, definition and
development of the process
- in a cyclic fed-batch culture, care should be taken in the design of the process to
ensure that toxins do not accumulate to inhibitory levels and that nutrients other
than those incorporated into the feed medium become limiting, Also, if many
cycles are run, the accumulation of non-producing or low-producing variants
may result
22
.
- the quantities of the components to control must be above the detection limits of
the available measuring equipment
25


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3.4. Equipment
No special piece of equipment is required over the equipment required for batch
13
.
However, some considerations should be made over the equipment used for a fed-batch
fermentation.
3.4.1. Vessels
The vessels, particularly those used for the acid and base control, must be constructed
from a non-toxic, corrosion-resistant material which is capable of withstanding repeated
sterilization cycles
13
. Figure 3.4.1. Illustrates two methods of assembling vessels for easy
transfer of either inoculum or medium to the fermentor.

Figure 3.4.1. Holding vessels. A. Screw-neck borosilicate glass vessel with medium/inoculum addition
assembly. (a) Stainless steel rod; (b) Silicon tubing; (c) Silicon disc; (d) Hypodermic needle; (e) Air vent; (f)
Screw cap; (g) Magnetic bar. B. Aspirator-type vessel for introducing an inoculum of filamentous fungi
into the fermentor. (a) Cotton-wool plug; (b) Magnetic stirrer bar
13
.


3.4.2. Pumps
There are two types of pumps which are suitable for the aseptic pumping of small
volumes of culture media: the peristaltic pump and the diaphragm-dosing pump. Other
pumps are unsuitable because they are difficult to sterilize and cannot be used for
pumping small volumes
13
.
The peristaltic pump is typically constituted by a main body that comprises both the
drive motor and electrics, and the rotating unit of rollers. This unit of rollers occludes
the tube which, as it recovers to its original size passes to the nest roller until is
expelled, as the unit moves round. The flow rate can be varied by either the speed
setting or by changing the diameter of the tube being used.
The diaphragm-dosing pump consists of a main body and a detachable heat-sterilizable
head. The fluid is sucked in to the pump head. The suction inlet tube then closes and
the pressure discharge tube opens and forces the fluid out. The suction and pressure
forces in the pump head are generated by the reciprocating action of both the
diaphragm plunger and the return spring. For a more accurate description of these
pumps, reference 13 can be consulted.

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3.5. Control techniques for fed-batch fermentation
Adaptive control is the name given to a control system in which the controller learns
about the process by acquiring data from a certain process and keeps on updating a
control model. A parameter estimator monitors the process and estimates the process
dynamics in terms of the parameters of a previously defined mathematical model of the
process. A control design algorithm is then used to generate controller coefficients from
those estimates, and a controller sets up the required control signals to the devices
controlling the process. An extremely important feature of an adaptive controller is the
structure of the model used by the parameter estimator to analyze estimates of process
dynamics. The process can be described by a set of mass balance equations, whose
quantities can be measured directly or indirectly
26
. Figure 3.5.1. describes schematically
the concept.

Figure 3.5.1. Adaptive control: the controller compares the estimates from a mathematical model applied to the
system to the readings obtained from the fermentation process. The controller then sends the signal to the device
controlling the fermentation, for example, by increasing or decreasing a flow rate.
The optimal strategy for the fed-batch fermentation of most organisms is to feed the
growth-limiting substrate at the same rate that the organism utilizes the substrate, this
is, to match the feed rate with demand for the substrate.
Four basic approaches have been used in attempts to balance substrate feed with
demand (listed in order of increasing accuracy and/or complexity):
(i) open-loop control schemes in which feed is added according to historical data or
predicted data;
(ii) indirect control of substrate feed based on non-feed source parameters such as pH,
offgas analysis, dissolved O2 or concentrations of organic products;
(iii) indirect control schemes based on mass balance equations, the values of which are
calculated from data obtained by sensors; and
(iv) direct control schemes based on direct, on-line measurements substrates
9
.
Better and more flexible control may be obtained when there is direct measurement of
substrate or an excreted metabolite in the medium, which can be used to influence
feeding rates to the fermentation. This can be done off-line or semi-on-line, but on-line
measurements are more useful because of
- the shorter analysis required,
- lower personnel requirement and
- a reduced chance of fermentor contamination
25
.
Regardless the type of control, the design is strongly influenced by both mathematical
model availabilities and measurement possibilities
14
.
Control and optimization of bioreactors is strongly influenced by the quality of the sensors
available for crucial response variables
4
. Of primary importance is the ratio of the dynamic
parameters of the sensor to those of the process. When these variables cannot be measured easily
or quickly enough, a mathematical model must be used in some way in place of feedback
information.
When an exact mathematical model is at disposal, an open-loop process control can be
proposed which generally proves to be insufficient
14
. The advantage of a feedback
control is that a response to unforeseen and unexpected conditions during the
fermentation is achieved and the process is controlled within the desired limits
29
.
An indirect feedback control utilizes an observable parameter, such as dissolved
oxygen, pH, respiratory quotient, partial pressure of CO2, culture fluorescence or by-
product formation, which is closely related to the course of microbial fermentation. As
examples of fed-batch systems using this concept, one can mention the pH-stat - a
system in which the feed is provided depending on the pH, - and the DO-stat - a system
in which the feed is provided depending on the reading of the dissolved oxygen
24, 29
.
A direct feedback controller uses the concentration of limiting substrate in the culture
medium as a feedback feed -related parameter for control. A direct feedback control can
have the disadvantage of not being very feasible due to the difficulty associated with
obtaining accurate on-line measurements of substrate concentrations or even by the
absence of on-line sensors for the important compound to control
14
. The advantage of a
feedback control is that a response to unforeseen and unexpected conditions during the
fermentation is achieved and the process is controlled within the desired limits
29
.

A feedback control can be implemented accordingly to not only a single measurement,
but also to obtain a finer control action in a dual-level system. Turner at al.
25
, describes
a control method applied to a fed-batch culture of recombinant Escherichia coli in which
a two-level control was preferred because it provided much greater flexibility and
better control over the substrate concentration in the medium and the production of by-
products
25, 29
.

As compared with the batch fermentation, two more parameters need to be specified to
determine the operating conditions of a fed-batch fermentation: feed and initial feeding
time. These parameters are usually process and/or microorganism specific and the
parameters commonly used to define them are explained in section 3.7.

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3.6. Modeling fed-batch fermentations

3.6.1. Fixed volume fed-batch
The mathematical development that is going to be presented here has the following
assumptions
22
:
o The feed is provided at a constant rate
o The production of mass of biomass per mass of substrate is constant
during the fermentation time and
o A very concentrated feed is being provided to the fermentor in such a way
that the change in volume is negligible.
The equations that describe the system in terms of specific growth rate, biomass and product
concentration (for both growth and non-growth associated products) with time are the following:
Table 3.6.1.1. Mathematical modelling of fixed volume fed-batch.
Parameter Equation Equation #
Specific Growth Rate u = (F . Yx/s) / x (3.6.1.1)
Biomass (as a function of time) x
t
= x
o
+ F . Y
x/s
. t (3.6.1.2)
Product Concentration
(non-growth associated)
P= Pi + qp . xo . t + qp . F . Y x/s . t
2
/2

(3.6.1.3)
Product Concentration
(non-growth associated)
P= Pi + r
p
. t (3.6.1.4)

- x is the biomass [mass biomass/volume]
- x
o
is the biomass in the beginning of the fermentation [mass biomass/volume]
- t is time
- F is the substrate feed rate [mass substrate/(volume.time)] and
- Y x/s is the yield factor [mass biomass/mass substrate]
- u is the specific growth rate [time
-1
]
- P is the product concentration {mass product/volume] and
- qp is the specific production rate of product [mass product/(mass biomass . time)
- rpis the product formation rate [mass product/(volume . time)]
From equations (3.6.1.1) and (3.6.1.2), it can be observed that
(i) the specific growth rate decreases with time because the biomass (in the denominator) is
increasing with time and
(ii) the biomass increases linearly with time.
The product variation with time will depend on its being growth or non-growth associated, this
is, will depend on q
p
(specific product formation defined as the product formation rate divided by
the biomass) being dependent on the specific growth rate or not, respectively.
To obtain the derivations that yielded these equations, please click here.
Figure 3.6.1.1. depicts the typical behavior of a fixed-volume fed-batch culture.


Figure 3.6.1.1. Time profiles for a fixed-volume fed-batch culture. u= specific growth rate, x = biomass
concentration, S(GLS) = growth limiting substrate, SN = any other substrate other than the S(GLS), P(nga)
is the non-growth associated product and P(ga) is the growth associated profile for product
concentration.


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3.6.2.Variable volume fed-batch

In a variable volume fed-batch fermentation, an additional element should be considered: the
feed. Consequently, the volume of the medium in the fermenter varies because there is an inflow
and no outflow. Again, it is going to be considered that the growth of the microorganism is
limited by the concentration of one substrate.
For the mathematical developments that will be presented, the assumptions are
o Specific growth rate is uniquely dependent on the concentration of the limiting
substrate
o The concentration of the limiting substrate in the feed is constant
o The feed is sterile
o The yields are constant during the fermentation time


Table 3.6.2.1. summarizes the equations that apply to this situation. These relations are
the base for all further calculations and specific cases of a variable volume fed-batch
fermentation.
Table 3.6.2.1. Mass balances for the main components for a fed-batch reaction.
Component Mass Balance Equation Equation #
Overall F = dV/dt (3.6.2.1)
Biomass dx/dt = x . (u . V - Kd . V - F) / V (3.6.2.2)
Substrate ds/dt = F . (so - s)/V - u. x/ Yx/s (3.6.2.3)
Product dP/dt = qp . x -P . F / V (3.6.2.4)

- V is the volume of the fermentor
- t is the time
- F is the feed rate [volume/time].
- x is the biomass concentration [mass biomass/volume]
- u is the specific growth rate [time
-1
]
- Kd is the specific death rate [time
-1
]
- s is the substrate concentration in the fermentor [mass substrate/volume]
- so is the substrate concentration in the feed [mass substrate/volume]
- Y x/s is the yield factor [mass biomass/mass substrate]
- P is the product concentration {mass product/volume] and
- qp is the specific production rate of product [mass product/(mass biomass . time)
To obtain the derivations that yielded these equations, please click here.

For a non-growth associated product
In this case it is desirable to have a high cell density. The process can be then divided in
two stages: the first stage of the process would therefore be to grow up a high cell
concentration, followed by a phase where growth is suppressed and only sufficient of
the substrate is supplied for maintenance and product formation the batch feeding phase.
The first stage can be translated by the equations in table 3.6.2.1. For the second stage, u
should be zero and the production formation rate is defined as rp = |cto. x
15
.

s = 0; ds/dt = 0 and rx= 0 &n
bsp; (3.6.2.5)
x = K1 . Kd . exp(-Kd . t)/(1 K1 . K . exp(-Kd . t)) &nb sp; (3.6.2.6)
Where K1 is defined as the ratio xo / ( Kd + K . x) ; (3.6.2.7)
F/V = K . x ; &n
bsp; (3.6.2.8)
where K = { ms + |/ Yp/s} . 1/so &nb
sp; (3.6.2.9)
P= |cto / K . [ 1 - exp (-K . f(t)] (3.6.2.10)

A similar and a much simpler development would be implemented if Kd would be
negligible. In this situation, the solutions for biomass concentration, flow rate, product
concentration and volume variations with time would be given by
x = xo / (1 + K . xo . t) &nbs p; &
nbsp; (3.6.2.11)
F = K . xo. Vi &nb sp; &nb
sp; (3.6.2.12)
(where Vi is the volume in the beginning of the fed-batch phase)
P = |cto . x . t / (1 + K . x . t) &nb
sp; (3.6.2.13)
V = Vi . (1 + K . xo.t) &nb
sp; (3.6.2.14)
Figure 3.6.2.1. depicts the change in volume, product concentration, feed and biomass
concentration with time for a fermentation as described above.

Figure 3.6.2.1. Time profiles for a variable-volume fed-batch culture for a process involving non-growth
associated production. V=volume of the fermentor, P=product concentration, u=specific growth rate,
X=biomass and S(gls)=growth limiting substrate concentration. The feed follows a similar profile to that
one of the specific growth rate.

To obtain the derivations that yielded these equations, please click here.
For a growth associated product
13

Nomenclature:
- Y'p/s is the mass of product formed per fraction of substrate mass
- Y'x/s is the mass of cells formed per fraction of substrate mass
- S is the difference (so- s)
In this case, substrate is provided in such a way that maximizes the specific growth rate,
assuming that the substrate is not growth or product formation inhibitory for that
concentration. The substrate is supplied here not only for maintenance and product
formation but also for biomass production. The product formation is such that rp =
otqo . u. x
15
, being alpha a specific constant of the bioprocess.
For a matter of simplicity, Kd is going to be considered to be approximately zero and
s = constant; ds/dt = 0 ; &n
bsp; (3.6.2.15)
x = K1 . u. exp(-u . t)/( K1 . K . exp (-u . t) - 1) &nbs
p; (3.6.2.16)
K = - { u/ Y'x/s + ms + otqo . u/ Y'p/s} . 1/S (3.6.2.17)
and K1 is defined as the ratio xo / (u + K . x) (3.6.2.18)

For product variation with time
P = otqo . u / K . { 1 - exp (-K . F(t)} &n
bsp; (3.6.2.19)



Figure 3.6.2.2. Time profiles for a variable-volume fed-batch culture for a process involving growth
associated production. V=volume of the fermentor, P=product concentration, u=specific growth rate,
X=biomass and S(gls)=growth limiting substrate concentration.

To obtain the derivations that yielded these equations, please click here.
Microorganisms growing exponentially
29

Another approach to a situation in which the specific growth rate is maintained
constant goes as following.
Nomenclature:
- rs is the consumption rate of substrate (mass substrate/(volume. time)
- xo is the initial concentration of biomass inside the fermentor for time zero [mass
biomass/volume]
- Vo is the initial volume of the fermentor for time zero
- V is the volume of the fermentor for time t

F = u . xo . Vo . exp(u . t)/[(so - s) . Y x/s] &n
bsp; (3.6.2.20)
V = Vo (u + A . xo . exp(u . t) A . xo)/ uwhere
A = u / (s . Y x/s) &nbs p; &
nbsp; (3.6.2.22)
x = u . xo . exp(u . t)/ (u + A . xo . exp(u . t) - A . xo) &nbs
p; (3.6.2.23)

To obtain the derivations that yielded these equations, please click here.

Another alternative would be to maintain the concentration of biomass constant with
time the quasi-steady state
6
. In this case,

V = Vo + F . t &nb
sp; (3.6.2.24)

s = F. t/ (Vo + F . t) . (so - x/ Y x/s) &nbs
p; (3.6.2.25)

Which, for small times, approximates zero and for large times approximates

S = so - x/ Y x/s &nb sp; (3.6.2.26)

The growth rate varies as following
For small times: du/dt = -F
2
/Vo
2 ; &n bsp;
(3.6.2.27)

For large times du/dt = -1/t
2 &nb sp;
(3.6.2.28)

Which shows that the specific growth rate decreases more in the beginning of the
fermentation but decreases less as the time passes by
6
.
To obtain the derivations that yielded these equations, please click here.

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3.6.3. Models of possible situations that may occur in a
fed-batch fermentation

The models that are presented here can be directly used in the equations summarized in
table 3.6.3.1.

Table 3.6.3.1. List of growth models that can be found in biotransformations
1

Model Form
Monod
Constant yield
u = umax s/(K m + s)
Y
x/s
= Y0
Substrate inhibition
Constant yield
u = umax s/(K
m
+ s + s
2
/Ki)
Y
x/s
= Y0
Substrate inhibition
Variable yield
u = umax s (1 -

T. s)/(K m + s + s
2
/Ki)
Y
x/s
= Y0 (1 - T. s)/(1 + R. s + G. s
2
)
Substrate and product inhibition
Inhibitions
Constants yields
u = umax s/(K
m
+ s + s
2
/Ki)
u = umax
o
(1 - P/P m )
q p = alpha. u+ beta
otqo, |cto and Y
p/s

Ki refers to inhibition constants and have the same units as the substrate concentration
(mass of substrate/volume). Tand Rare constants with units that are volume/mass
substrate and G has (volume/substrate)
2
units. These constants are defined by
experimental data analysis
1
.

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3.7. Parameters used to control fed-batch fermentations
All the parameters that are about to be described have as an ultimate aim, to control a
fed-batch fermentation, more specifically the growth rate and/or the flow through the
central carbon metabolism and/or to reduce the overflow of carbon source to metabolic
by-products. The control can be a one-step type in which only one of the parameters is
used or it can be dual-level control in which two parameters are used, yielding a more
"refined" control
25, 29, 4
. For example, some processes require the control of different
parameters at different stages of the fermentation. Such is the example of high density
bacterial fermentation like the production of baker's yeast and penicillin. In these cases,
as an example, two phases can be distinguished: (i) a phase in which the substrate needs
to be controlled so as to avoid by-products formation and (ii) a second stage in which,
due to the high cell density, oxygen transfer is limiting and so,this parameter is the one
to be controlled above a critical value, under which the cellular metabolism changes.
The control constraints switch from specific growth rate to oxygen concentration after
some critical period of time in the fermentation process
4
.
The choice of each parameter is system dependent and the decision should be based on
convenience and experimental data
25
. The mathematical model is used in two ways: first
to estimate the controlling variable and second to calculate the control action
4
.
A very important fact that should be considered is the quality of the sensors available for the
variables that are whished to be controlled. Temperature, speed of agitation, flow rates, pH,
dissolved oxygen, redox potential of broth are examples of commonly controlled variables.
These sensors usually respond quickly enough (faster than the change of the variable itself in the
system) and so, proportional feedback controllers are often suitable for these variables. Of
particular interest, the dead time - time for the sensor to start responding - and the overall time
constant - time required to yield the final reading. The crucial design factor is the ratio of the
first order time constant to the dead time
4
. Cohen-Coon derived the following expression to
determine the proportionality constant of proportional controllers:
K
c
= T
p
/ (K
p
. t
d
) . [1 + t
d
/ (3T
p
)] &nbs p; &
nbsp; 3.7.1.
where
- K
c
is the constant of the proportional controller
- T
p
is the overall time constant
- K
p
is the slope of the response curve, or the process gain
The steady stater error for this controller can be defined by being equal to -1/(1+K
c
K
p
).
Proportional - integral (PI) and proportional - integral - derivative (PID) controllers
14
can also be
used, yielding a more refined control and eliminating the set-point offset usually related with
proportional controllers. Even when these "more effective" controllers are used, some problems
can still occur due to nonlinearities inherent to the process
4
.
To design a feedback controller, a certain parameter to be maintained within certain limits is
analyzed as far as requirements to keep its value within the desired range or level. The process
response curve, an open loop response of the pH to a step change is generated by changing the
value of the parameter by small increments.
Computer technology allows the handling of mathematical models which are solved on-line and
predict and evaluate the future performance.
Because of some difficulties with measurement of some variables, some linear estimation of state
can be used such as the Kalman filter.The Kalman filter uses past measurements for a weighted
least square estimate of the current variable as reflected through the dynamic model
4
. Another
alternative is the use of a Dynamic Matrix Control, which is basically a linear dynamic
mathematical modelof the process that calculates the response resulting from initial conditions,
disturbances, manipulated variable inputs and setpoint changes. The compensation is then
applied in such a way that minimizes the sum of squares of deviations from the setpoint,subject
to constraints on the manipulated variable
4
.
3.7.1. Calorimetry
Calorimetry is an an excellent tool for monitoring and controlling microbial
fermentations
11
. Its main advantage is the generality of this parameter, since microbial
growth is always accompanied by heat production, and the measurements are
performed continuously on-line without introducing any disturbances to the culture.
Moreover, the rate of heat production is stoichiometrically related to the rate of
substrate consumption and product, including biomass formation. In many cases it can
be replaced by exhaust gas analysis, although this approach can not be considered in
anaerobic processes which proceed without formation of gaseous products.
This technique has been proved successful to indirectly determine the substrate and
product concentrations continuously during aerobic batch growth of Saccharomyces
cerevisiae with glucose as the carbon and energy source. In the presence of this substrate,
this yeast shows diauxic growth by initially consuming the glucose with concomitant
production of ethanol and then, once glucose is depleted, using the produced ethanol as
an energy source. Calorimetry can then be used to control the feed rate in such a way
that ethanol formation is avoided.
Another interesting description of a temperature-based controlled reactor follows a
stability criterion that predicts that the range of operation is controlled by the reactant
feed, being the flow rate of the cooling medium the control variable
23
. Although the
study has been performed in a chemical reactor, the concepts can be easily extended to a
biotransformation process.
3.7.2. Specific growth rate
For the production of a growth-associated product, the production of a certain product
is related with the specific growth rate of the producing microorganism. Consequently,
it is of interest to feed the fermentor in such a way that the specific growth rate remains
constant. Such is the case of the production of hepatitis B surface antigen by
Saccharomyces cerevisiae
1, 2
. The yield of the antigen is ten times more than that of the
fed-batch cultivation for the same volume and total substrate added. Care should be
given to the value of the chosen specific growth rate, because cells may not be
"activated" easily
19
, stress proteases can be produced that may degrade the product
19

and also there might be a threshold value of specific growth rate above which there is
production of by-products
29
.


3.7.3. Substrate (carbon and nitrogen source)
Substrate is a particularly important parameter to control due to eventual associated
growth inhibitions and to increase the effectiveness of the carbon flux, by reducing the
amount of by-products formed and the amount of carbon dioxide evolved.
An example of adaptive feedback control of glucose concentration is found in the fed-
batch production of thuringiensin, an exotoxin that shows efficacious control against
flies, beetles, bugs and mites by Bacillus thuringiensis
8
. Glucose concentration was
estimated by using empirical correlation equations between the consumed glucose and
the values of "Oxygen Uptake Rate" (OUR) and "Carbon Dioxide Production Rate"
(CPR). Then, the glucose concentration G(t) in the fermentor was expressed as
G(t) = [{initial mass amount} - {consumed mass} + {mass in
feed}]/V(t) (3.7.3.1.)

G(t) = [Gi . Vi - Gcons(t) + Gf . Va]/V(t) &nb
sp; (3.7.3.2.)

Where

- Gi is the initial glucose concentration in the fermentor
- Vi is the initial working volume
- Gcons(t) is the mass of glucose consumed
- Gf is the glucose concentration of the feed medium
- Va is the added volume of substrate during fed-batch culture and
- V(t) is the volume of the fermentor for time t

The equation for adaptive control of substrate addition is

dG(t)/dt = a . G(t) + b . u(t) &nbs p; &
nbsp; (3.7.3.3.)
Where

- a and b are estimated and
- u(t) is the feeding rate (volume/time)

Now, the model strategy is given by

dG'(t)/dt = A . [G'(t) - Gs] &nb
sp; (3.7.3.4.)

being A a negative constant, G(t) the value of glucose concentration that is estimated
and Gs the setpoint of G(t). An error signal E(t) can be defined as

E(t) = G(t) - G'(t) &n
bsp; ; (3.7.3.5.)

Combining equations 3.7.3.3., 3.7.3.4. and 3.7.3.5., gives

dE(t)/dt = A . E(t) + (a - A) . G(t) + b . u(t) + A . Gs ; (3.7.3.6.)

By making the error signal approach zero,

dE(t)/dt = A . E(t) &nb
sp; (3.7.3.7.)

Then, the feeding rate is determined by combination of equations (3.7.3.6.) and (3.7.3.7.)

u(t) = - 1/b . (a -A) . G(t) - 1/b . A . Gs &nb
sp; (3.7.3.8.)

By using this adaptive type of control, the production of thuringiensin was significantly
improved, with readings that were ten times higher for fed-batch (according to the feed
medium) as compared with batch fermentation
8
.
A predictive and feedback control algorithm can be also set up to form a product or
grow cells such as E. coli
9
. The control in this case will be more refined than that of a
feedback control. Basically, the control scheme can be divided in two: a feed-forward
component that predicts (according to same statistical method and the previous
collected data) the "need" of a certain substrate measurable on line, and a feed-back
controller which corrects for minor errors in the predicted "need". These errors can
occur in exponentially growing microorganisms, in which the predicted value will be
greater than the effective value
9
. The main advantage to this system is that the
investigator does not need to know the metabolic constants for a given organism prior
to growth of that organism in the system, and it can be applied to any substrate that can
be measured on-line, being particularly valuable in the minimization of by-products.
In some other situations such as otqo-amylase production in recombinant Bacillus
brevis, the fermentation kinetics are mainly driven by the nitrogen source, since this
microorganism responds very slowly to glucose depletion and it prefers a nitrogen
source over a carbon nutrient for growth and production of recombinant protein
16
.
However, when a large amount of nitrogen source is fed, the larger the cell mass is
produced but the lower the recombinant gene production is. Therefore, the feed
nitrogenous sources need to be maintained at low levels and can be provided in optimal
manner using L-amino acids concentrations control at various levels on-line
17
.
As a final comment the operator should be aware of the analytical equipment that will
be using so as to guarantee that the readings from the substrate concentrations fall
within the limits of detection of the assay and/or analytical assay. At the same time, the
operator should make sure that the concentrations are low enough to prevent by-
product formation
21
. Another aspect to consider are eventual interferences in the
readings of this due to some other component that might be present in the growth
medium.


3.7.4. By -product concentration
The production of by-products is undesirable because reduces the efficacy of the carbon
flux in a fermentation. The production of these components take place whenever the
substrate is provided in quantities that exceed the oxidative capacity of the cells. This
approach has been used in the fermentation of Saccharomyces cerevisiae, in which acid
production rate is used to provide on-line estimates of the specific growth rate
1
. Also, in
modern fed-batch processes for yeast production, the feed is under strictly control
based on the measurement of traces of ethanol in the exhaust gas of the fermenter
22
.

3.7.5. Inductive, enhancer or enrichment components
In certain fermentations it is of interest to continuously add either an inductive or fast
consumed components and not only a limiting substrate. An example is the continuous
addition of an antibiotic in recombinant microorganims bearing an antibiotic marked
plasmid
29
. Another example is given by the production of gluthathione by high-
gluthathione-accumulating Saccharomyces cerevisiae, the commonly microorganims used
for commercial production. Cysteine was found to be the only amino acid that
enhanced gluthathione formation. However, the growth inhibition occurred and it was
related to the concentration of cysteine. This problem was then resolved by an adequate
addition of cysteine in exponential fed-batch culture without growth inhibition
11
.
Fed-batch proves to be an appropriate mode of fermentation in microorganisms that are
producing heterologous proteins and whose elevated protein expression results in
product degradation by activation of proteases. A general insight on this subject was
the study of a recombinant E. coli for production of chloramphenicol acetyltransferase.
A gradual induction with IPTG and phenylalanine (rate limiting precursor) addition
strategies were able to reduce the physiological burden imposed on the bacterium,
thereby avoiding cellular stress responses and enhancing bioreactor productivity
19
. In
this case, IPTG and phenylalanine were the driving parameters that dominated the feed.
As a final note, the addition of precursors or inducers should take into account if the
product of interest is growth associated or not. For example, the use of a tyrosine-
deficient strain of E. coli in the production of phenylalanine requires a balance feed of
tyrosine that, if not provided in low quantities is used as carbon source with subsequent
production of excessive biomass synthesis at the expense of phenylalanine synthesis.
This limitation on biomass production is possible because the phenylalanine production
was not growth associated
19
.


3.7.6. Respiratory quotient (RQ)
Gas analyzers,especially mass spectrophotometers are relatively fast
4
. Respiratory
quotient, the ratio between the moles of carbon evolved per moles of oxygen consumed,
has been a general method used to determine indirectly the lack of substrate in the
growth medium
4
. It is a fairly rapid method of measurement that is useful because the
gas analyses can be related to crucial process variables. The method is not "universal"to
all bioprocesses since some biosystems can produce by-products that affect the
productivity of the process without affecting the RQ, such as the production of acetic
acid by E.coli
4
.
Usually, the signal is characterized by a sharp rise in dissolved oxygen
13
. The process
response of RQ can be represented very closely by a first order transfer function defined
by RQ/ (feed of substrate). Equation 3.7.1. can be still used but, any steady state offset
for a proportional controller in this case is not desirable. Instead, a PI can be considered.
Based on the concept of respiratory quotient, there are the so called DO -stats, in which
the feed is regulated in accordance with the dissolved oxygen
29
. The analysis of the
dissolved oxygen or carbon dioxide evolution rate can also be used to control or
prevent the production of by-products,
1, 10
. The respiratory quotient is often analyzed
to study the carbon flux, this is, the feed should be conditioned in such a way that it
should prevent excess of carbon dioxide evolution caused by unnecessarily severe
substrate limitation
4,9
.
Care should be given to the mathematical model so that equations are explanatory for certain
substrates limits or ranges for which below or above the system behaves differently due to
different metabolic reactions and,consequently, different metabolism. Suppose that the desirable
point of operation is at RQ=1,corresponding to zero by-product formation (such is the case with
Baker's yeast). However, if the substrate feed rate is further reduced, the RQ will remain at 1 but
suboptimal conditions will occur. Conversely,RQ can be still equal to 1 if that is the
steicheometry of the consumption of the by-product as alternative energy source
4
. The objective
thus is slightly modified to control RQ as near to 1 as possible, but just slightly larger like 1.02
4
.
For most processes,amore reliable control system is required
4
.


3.7.7.General feeding mode
The feeding mode influence a fed-batch fermentation by defining the growth rate of the
microorganisms and the effectiveness of the carbon cycle for product formation and
minimization of by-product formation. Inherently related with the concept of fed-batch,
the feeding mode allows many variances in substrate or other components
constitution
24
and provision modes and consequently, better control over inhibitory
effects of the substrate and/or product. The feed mode can be defined based on an
open-loop, if an exact mathematical model is at disposal (not very common and usually
insufficient)
14
, a feedback control (ex. pH - stat or dissolved oxygen (DO) stat) or in
any other way depending on the specific kinetics of each fermentation and even within
the time frame of the fermentation process. In fact, the feed can be modified accordingly
to the different phases of the microbial growth, as a consequence of physiological
alterations that the cells undergo upon transfer through eventual consecutive stages of
the fed-batch cultivation
10
.
Usually, a fed-batch starts as a batch mode and after a certain biomass concentration or
substrate consumption, the fermentor is fed with the limiting susbtrate solution.
However, that approach does not need to be the absolute rule. Some cases happen in
which the rate of production of a certain product is limited not only by the susbtrate but
also by a primary product, associated with the growth of the microorganism. That is the
case of streptokinase formation. Streptokinase is a vital and effective drug for the
treatment of myocardinal infection, that is currently produced in industry by mainly
natural or mutated strains of streptococci. The specific growth rate is inhibited by the
susbtrate and by lactic acid. A near optimal feed policy based on a chemotaxis
algorithm has been established that defines an initial decreasing feeding phase,
followed by a batch fermentation with no more added substrate in the medium. The
starting point was the data provided by the batch fermentations and the feed was
defined as being a polynomial function of time. By iterative calculations and having the
batch time fermentation or the maximum allowable volume of the fermentor as time
limits, a feed strategy was defined yielding a 12% increase in streptokinase activity over
batch fermentations
16
. This type of approach has been previously suggested also for
ethanol production by Saccharomyces cerevisiae that follows the same kinetics
16
.
Finally, the feed can be continuous, can be provided in pulses
24
, as a shot feeding
11
,
single or multisubstrate
24
, increasing linearly
19
, be exponential
7,19
or constant with time.
The design of the feed solution may follow a conventional approach in which the
nutrients are more concentrated as compared with the growing medium in the
fermentor - or follows a quantitative design in such a way that depletion or
accumulation of nutrients can be avoided or reduced
28
. An optimization problem for a
feed to the production of a non-growth associated product is given by Meszaros
14
.

3.7.8.Proton production
An "unusual" type of controlling process parameters is the proton production to
estimate on-line the specific growth rate in a fed-batch culture and indirectly, the
substrate concentration. In an anaerobic alcohol fermentation, Won et al.
27
defined
specific growth rate (u) as being

u= dln(proton production)/dt ; &n
bsp; (3.7.8.1.)

The measured amount of proton produced during the fermentation was calculated
based on the volume of base added to the fermentor to control the pH at a pre-set value.
The control based on pH usually uses a on-off mode because the magnitude of the acid and alkali
feeds is so low that implemententing a proportional controller is difficult. On -off operation is
similar to proportional control with a very high gain because a small amount of acid/alkali feed
raises the pH above the set point. Flow must be discontinued to await the reaction to bring the
pH back down to the desired value. A variation on the on-off control valve system is frequency
modulation. If the pH is very far from the set point, the valve will be open for a long period of
time and closed for a shorter period of time. The reversal happen when the pH is in the vicinity
of the setpont value. The frequency modulated on-off controller offers more accurate control if
that is needed
4
.

3.7.9. Fluorescence
A linear relationship exists between the culture fluorescence (as a function of the
intracellular NAD(P)H pool) and the dry cell weight concentration up to 30g dry cell
weight/liter
29
. Thus, fluorescence can be used to estimate on-line the biomass
concentration and be a controlling parameter in the feed provision
29
.

| Outline | Top of document |

3.8.Parameters to start and finish the feed, and stop the
fed-batch fermentation

The times at which the feeding should start and finish, as well as the criteria to stop a
fed-batch fermentation is very much dependent on the specific cultivation kinetics and
the operators interest. For example, in substrate limited processes, the feed should start
immediately after all substrate is consumed from the batch phase, otherwise the process
may be difficult to control, for example, because of a lag phase due to previous
starvation
13
. The most commonly criteria to start the feed is the depletion of substrate
24
,
which can be measured by a multitude of techniques, from specific enzymatic assays,
HPLC
25
to indirect methods such as the exhaust gas analysis
7, 9
. Still related with the
amount of substrate in the medium, the operator might not find necessary to reach the
complete depletion but to be below a predetermined set-point (eventually related with
historical data, growth models and known yields)
8,19
.
The fed-batch fermentation should be halted when the production slows down because
of cell death
13
, because the metabolic potential of the culture becomes inadequately low
or because by-product excretion starts at significant levels
10
. Some other criteria can be
an increase in viscosity that implies an increased oxygen demand until the oxygen
limitation is achieved, which is the case for penicillin production
22
.
| Outline | Top of document |

3.9.Preliminary knowledge required to implement fed-
batch

Before starting a fed-batch process, a batch fermentation should be implemented to "get
to know" the fermentation of the microorganism. From a batch fermentation, the
operator should have a knowledge of:
- Best abiotic conditions such as temperature, light, agitation, pH, growth
medium, etc.
- Specific needs of precursors, inducers or other enrichment factors
- The different growth phases and the consumed (substrate) and produced
components (product of interest and by-product)
- The relationship between the biomass and product formation (growth or non-
growth associated product) and the oxygen uptake rates
- Limiting substrate for growth and the relationship between the specific growth
rate and the limiting substrate concentration
- Eventual inhibitions from the substrate and/or product
Now, the operator should define the objective functions and the best parameter to
control the fermentation, considering both accuracy of data and convenience. Also, the
operator should define if the control that wishes to be implemented is based on an
feedback control (direct or indirect) or an open-loop control based on mathematical
models established for the system.
| Outline | Top of document |

3.10.Algorithms for operating a fed-batch reactor at
optimum specific growth rate (model independent and
applicable to adapting systems)
1


The control of a fed-batch fermentation can implicate many difficulties: low accuracy of
on-line measurements of substrate concentrations, limited validity of the feed schedule
under a variety of conditions and prediction of variations due to strain modification or
change in the quality of the nutrient medium. These aspects point to the need of a fed-
batch fermentation strategy which is model independent, identifies the optimal state
on-line, incorporates a negative feedback control into the nutrient feeding system and
contemplates saturation kinetic model, variable yield model, variation in feed substrate
concentration and product inhibited fermentation. The following described algorithms
describe methods for operating a fed-batch fermentor at the maximum possible rate of
fermentation (so that the productivity is maximized). The only requirement is the
establishment of a reliable on-line estimate of the specific-fermentation rate
1
.

3.10.1. Open-loop performance
In an open-loop operation system, a predetermined feed schedule is used
1
. This
approach considers that the system can be exactly translated in a set of mass balance
equations in which the specific growth rate. However, it is easy to assume that due to a
non-identified physiological problem of the cells the specific growth rate can be either
higher or lower than the one that was previously established. If, for example, the
specific growth rate is higher than the pre-set one, and if the substrate is being fed in
such a way that is assumed that all the substrate is being consumed as soon as it enters
the fermentor, then there will be substrate limitation during the course of the
fermentation. Consequently, the open-loop feed policy does not always result in an
optimal operation.

3.10.2. Feed-back control algorithm
This algorithm requires only a reliable on-line estimate of the specific growth rate, that
can be provided by any of the parameters described in sections 3.7.1. to 3.7.9. Since the
objective of the algorithm is to optimize the cell-mass production by controlling the
specific growth rate (u) at an optimum value uopt, the feedback law can be defined:

Fin(t n+1) = Fin (tn) Kc [u opt (tn) -u(tn)] &n
bsp; (3.10.2.1.)

This relation can be used to manipulate the feed flow rate (Fin) to the fermentor, where
Kc is a controller constant which is assumed to be positive. When u is different from
uopt, either S < Sopt or S > Sopt. Then, the positive sign in equation (3.10.2.1.) applies to the
former case and, similarly, the negative sign applies to the latter case. By analysis of
what has just been described, then opt and Sopt need to be identified. Figure
3.10.2.1.describes a flow diagram of the simple control algorithm to find those values by
an initial open-loop period. This period continues until starts to decrease. The
maximum value of obtained during this period is set as uopt and the corresponding
value of S as Sopt.

Figure 3.10.2.1. A flow diagram of the control agorithm
1
.

In situations where it is difficult to obtain on-line measurements or estimates of S, it is
proposed to estimate S as S and Sopt as Sopt during the open-loop period, in which opt
value is identified. Figure 3.10.2.2 shows the flow diagram that includes all the features
that has just been described. This algorithm is model independent and therefore can be
applied to many industrial fermentations which utilize complex media. Moreover, since
the values of opt and

S
opt
are continuously being updated, the control methodology should work
well even when the microbial system undergoes adaptation.


Figure 3.10.2.2. A flow diagram of the control algorithm including estimates of optimum substrate and
specific growth rate
1
.
| Outline | Top of document |

3.11. Some examples of fed-batch use in industry

The use of fed-batch culture by the fermentation industry takes advantage of the fact
that the concentration of the limiting substrate may be maintained at a very low level,
thus
- avoiding repressive effects of high substrate concentration
- controlling the organisms growth rate and consequently controlling the oxygen
demand of the fermentation.
Saccharomyces cerevisiae is industrially produced using the fed-batch technique so as to
maintain the glucose at very low concentrations, maximizing the biomass yield and
minimizing the production of ethanol, the chief by-product
13, 15, 22
.
Hepatitis B surface antigen (HbsAg) used as a vaccine against type B hepatitis has been
purified from human plasma and expressed in recombinant yeast, being now produced
commercially. Again, the production of the recombinant protein is achieved using fed-
batch culture techniques very similar to that developed for Saccharomyces cerevisiae. A
cyclic method is used due to reports of superior productivity
22
.
Penicillin production is an example for the use of fed-batch in the production of a
secondary metabolite. The fermentation is divided in two phases: the rapid-growth
phase during which the culture grows at the maximum specific growth rate, and the
slow-growth phase in which penicillin is produced. During the rapid-growth phase, an
excess of glucose causes an accumulation of acid and a biomass oxygen demand greater
than the aeration capacity of the fermentor, whereas glucose starvation may result in
the organic nitrogen in the medium being used as a carbon source, resulting in a high
pH and inadequate biomass formation. During the production phase, the feed rates
utilized should limit the growth rate and oxygen consumption such that a high rate of
penicillin synthesis is achieved and sufficient dissolved oxygen is available in the
medium
15, 22
.
Some other examples are the production of thiostrepton from Streptomyces laurentii
and the production of cellulase by Trichoderma reesei. The production of thiostrepton
uses pH feedback control and the production of cellulase utilizes carbon dioxide
production as a control factor
15
.

3.6.1. Fixed volume fed-batch (derivations)
The mathematical development that is going to be presented here has the following
assumptions
21
:
o The feed is provided at a constant rate
o The production of mass of biomass per mass of substrate is constant
during the fermentation time and
o A very concentrated feed is being provided to the fermentor in such a way
that the change in volume is negligible.

Consider a batch culture in which the growth of the process organism has depleted the
limiting substrate to a limiting level. If this limiting substrate is fed to the fermentor in
such a way that the volume does not change (as a very concentrated feed, for example),
then
dx/dt = F . Y x/s ( 3.6.1.1.)
where
- x is the biomass [mass biomass/volume]
- t is time
- F is the substrate feed rate [mass substrate/(volume.time)] and
- Y p/x is the yield factor [mass biomass/mass substrate]
But dx/dt = u . x (3.6.1.2)
Where u is the specific growth rate [time
-1
]
Using equation (3.6.1.2) in (3.6.1.1), then u . x = F . Y p/x <=> u = (F . Y x/s ) / x (3.6.1.3)

Considering that (F . Y x/s )/x has as a upper limit umax, then the limiting substrate will be
consumed as soon as it enters the fermentor and ds/dt is approximately zero, being "s" the
concentration of substrate inside the fermentor [mass substrate/volume]. However, because cells
are growing in the fermentor and then biomass is increasing with time, dx/dt is not zero.
Integrating equation (3.6.1.1) between the initial time (t=0) and between time t, and between the
biomass concentration at the onset of the fed-batch culture (x
o
) and the biomass concentration
after operating the fed-batch system after t time (x
t
), equation (3.6.1.4) is obtained.


x
t
= x
o
+ F . Y
x/s
. t (3.6.1.4)

Then, from equations (3.6.1.3) and (3.6.1.4), it can be observed that
(i) the specific growth rate decreases with time because the biomass (in the denominator) is
increasing with time and
(ii) the biomass increases linearly with time.

In terms of a product P, the product balance is

dP/dt = qp . x (3.6.1.5)

where
- P is the product concentration {mass product/volume] and
- qp is the specific production rate of product [mass product/(mass biomass . time)
Note that equations (3.6.1.2) and (3.6.1.5) do not take into consideration the variable
volume because we are treating a fixed-volume fed-batch process.
If equation (3.6.1.4) is substituted into equation (3.6.1.5), then

dP/dt = qp . (xo + F . Y x/s . t) (3.6.1.6)

It is observed that the production of product rises with biomass, if qp is constant (non-
growth associated products). If equation (3.6.1.6) is integrated between the initial time (t
= 0) and time t, and initial product concentration Pi and the concentration P for time t

P= Pi + qp . xo . t + qp . F . Y x/s . t
2
/2

(3.6.1.7)

If fed-batch mode is established from the beginning of the fermentation, then Pi can be
zero or approximated to zero.
If qp is related with u (growth associated product) then the relationship between
product concentration and time will vary according to that relationship. By definition qp
is defined as the ratio of the product production rate over the biomass concentration in
accordance to equation (3.6.1.8)

qp = rp / x (3.6.1.8)

where rp is the product formation rate [product mass/(time * volume)]. Assume that rp
is a constant. Combining equations (3.6.1.4), (3.6.1.6) and (3.6.1.8), it comes to

dp/dt = rp (3.6.1.9)

which reflects a linear relationship between product and time. Figure 3.6.1.1. depicts
these relationships for constant or non-constant qp.
Other relationships between qp and the biomass production can be used in equation
(3.6.1.6) so that, by integration, the concentration of product with time is known.
The same conclusions can be withdrawn if the Ludeking Piret (1959)
20, 12
model type is
used.
For non-growth associated products
rp = |cto . x (3.6.1.10)

Using equation (3.6.1.8), then qp becomes a constant of value |, which confirms
equation (3.6.1.7).

For growth-associated products

rp = otqo . u. x (3.6.1.11)

If this equation is used in combination with equation (3.6.1.3), then

rp = otqo . F . Y x/s (3.6.1.12)

Once again, using equation (3.6.1.8), it can be seen that qp is not a constant and varies
with time, in accordance with the increase of biomass. Further substitution into
equation (3.6.1.5) yields

dP / dt = otqo . F . Y x/s (3.6.1.1 3)

which, in accordance with was already concluded, reflects a linear relationship between
product and biomass.
Now, if the growth is inhibited by the product such that

u = umax
. (1 - P/Pm) (3.6.1.14)


being Pm the maximum product concentration that can be attained without stopping the specific
growth. Then, the relationships defined by equations (3.6.1.1) (3.6.1.4) are maintained.
However, if equation (3.6.1.13) is combined with equations (3.6.1.3) and (3.6.1.4), the following
relationship of P=f(t) becomes

P= Pm . [1 - F . Y x/s /(umax . (x + F . Y
x/s
. t))] (3.6.1.15)

The situation in which the substrate is growth inhibitory is not considered here because it was
assumes previously that the growth rate of the organism is defined by F and Y
x/s
, does not
exceed the umax and the substrate is consumed as soon as it enters the fermentor.

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3.6.2.Variable volume fed-batch

In a variable volume fed-batch fermentation, an additional element should be considered: the
feed. Consequently, the volume of the medium in the fermenter varies because there is an inflow
and no outflow. Again, it is going to be considered that the growth of the microorganism is
limited by the concentration of one substrate
5,21
.
For the mathematical developments that will be presented, the assumptions are
o Specific growth rate is uniquely dependent on the concentration of the limiting
substrate
o The concentration of the limiting substrate in the feed is constant
o The feed is sterile
o The yields are constant during the fermentation time


Considering the overall mass balance

{in} = {out} + {accumulation} (3.6.2.1)
F = 0 + dV/dt <=> F = dV/dt (3.6.2.2)

Where
- V is the volume of the fermentor
- t is the time
- F is the feed rate [volume/time].

Considering now the balance to the biomass

{accumulation} = {in} + {produced}-{lost by cell death} (3.6.2.3)

d(Vx)/dt = F . xo + rx . V - rd . V (3.6.2.4)

Where
- x is the biomass concentration [mass biomass/volume]
- xo is the biomass concentration in the feed [mass biomass/volume] and
- rx is the biomass production rate [mass biomass/(volume. time)] and
- rd is the biomass death rate [mass biomass/(volume. time)].


But the feed is considered to be sterile, then the {in} amount equals zero. If the left hand-
side term derivative is now developed equation (3.6.2.5) is obtained.

V . dx/dt + x . dV/dt = V . dx/dt + x . F = rx . V - rd . V (3.6.2.5)

But rx . V =u . X . V and rd . V = Kd . x . V (3.6.2.6)

Where u is the specific growth rate [time
-1
] and Kd is the specific death rate [time
-1
].

Substituting in (3.6.2.5) and rearranging the equation

dx/dt = x . (u . V - Kd . V - F) / V (3.6.2.7)

For a matter of simplicity, the specific death rate is considered to be much smaller than
the specific growth rate and consequently, it can be neglected.

Considering now the balance to the limiting substrate

{accumulation} = {in} + {consumed} (3.6.2.8)

d(V . s)/dt = F . so - rs . V (3.6.2.9)

Where
- s is the substrate concentration in the fermentor [mass substrate/volume]
- so is the substrate concentration in the feed [mass substrate/volume]
- rs is the consumption rate of substrate [mass substrate/(volume. time)]

It should be noted that the consumption rate of substrate include the specific
consumption used for biomass production, product formation and maintenance of the
cells
14
. Introducing now the concept of yield Yx/s as being the ratio between the mass of
cells produced per mass of substrate consumed.

rs . V = u. X . V/ Yx/s (3.6.2.10)

If the derivative on the left-hand side of equation (3.6.2.8) is now developed

d(V . s)/dt = V . ds/dt + s . dV/dt = V . ds/dt + s . F (3.6.2.11)

Substituting now in equation (3.6.2.9) and rearranging the equation, one gets

ds/dt = F . (so - s)/V - u . x/ Yx/s (3.6.2.12)

Considering a mass balance for the product

{accumulation} = {produced} (3.6.2.13)

d(V . P)/dt = rp . V (3.6.2.14)

Where
- Pis the product concentration [mass product/volume] and
- rp is the product formation rate [mass product/(volume.time)]

Developing the derivative in the left-hand side of equation (3.6.2.14) as made
previously, and knowing that

rp = qp . x (3.6.2.15)

being qp the specific product formation [mass product/(volume. time)], equation
(3.6.2.14) becomes

dP/dt = qp . x P. F / V (3.6.2.16)

Table 3.6.2.1. summarizes the equations that have just been developed. These relations
are the base for all further calculations and specific cases of a variable volume fed-batch
fermentation.
Table 3.6.2.1. Mass balances for the main components for a fed-batch reaction.
Component Mass Balance Equation Equation #
Overall F = dV/dt (3.6.2.2)
Biomass dx/dt = x . (u . V - Kd . V - F) / V (3.6.2.7)
Substrate ds/dt = F . (so - s)/V - u. x/ Yx/s (3.6.2.12)
Product dP/dt = qp . x -P . F / V (3.6.2.16)

|Back to section 3.6 - modelling fixed-volume fed-batch fermentations | Top of the document |

For a non-growth associated product
12

Nomenclature:
- V is the volume of the fermentor
- t is the time
- F is the feed rate [volume/time].
- x is the biomass concentration [mass biomass/volume]
- u is the specific growth rate [time
-1
]
- Kd is the specific death rate [time
-1
]
- s is the substrate concentration in the fermentor [mass substrate/volume]
- so is the substrate concentration in the feed [mass substrate/volume]
- Y x/s is the yield factor [mass biomass/mass substrate]
- ms is the substrate consumption rate for cell maintenance [mass
substrate/(volume.time)]
- Y'p/s is the mass of product formed per fraction of substrate mass
- P is the product concentration {mass product/volume] and
- qp is the specific production rate of product [mass product/(mass biomass . time)
- rs' is the substrate consumption rate for maintenance and product formation
[mass of substrate/(volume.time)]
In this case it is desirable to have a high cell density. The process can be then divided in
two stages: the first stage of the process would therefore be to grow up a high cell
concentration, followed by a phase where growth is suppressed and only sufficient of
the substrate is supplied for maintenance and product formation the batch feeding
phase. The first stage can be translated by the equations in table 3.6.2.1. For the second
stage, u should be zero, rp = beta . x
14
being beta a specific constant of the bioprocess,
and hence:

s = 0; ds/dt = 0 and rx= 0 (3.6.2.17)
Applying these relations to equations (3.6.2.2), (3.6.2.7), (3.6.2.12) and (3.6.2.16), the
following system of equations is achieved:
- Biomass: dx/dt = -x . (Kd . V + F) / V (3.6.2.18)
- Substrate: 0 = F . so / V - rs' (3.6.2.19)
(where rs' is the substrate consumption rate for maintenance and product
formation)
rs' = ms . x + rp / Yp/s (3.6.2.20)
- Product: dP/dt = |cto . x

- P. F / V (3.6.2.21)
o Overall: dV/dt = F (3.6.2.22)
To obtain the optimum operating system, so will be fixed and F is allowed to vary.
Equation (3.6.2.19) can be rearranged to give

0 = - K . so . x + F . so /V (3.6.2.23)

where K = { ms + |/ Yp/s} . 1/so (3.6.2.24)

From equation (3.6.2.23),

F / V = K . x (3.6.2.25)

Which substituting in equation (3.6.2.18) gives

dx/dt = - x . (Kd + K . x) (3.6.2.26)

By integration, between t = 0 and time t, and xo (the initial cell concentration when the
batch feeding phase begins) and x (the correspondent cell concentration in the
fermentor for time t) gives

x = K1 . Kd . exp (-Kd . t) / (1 - K1 . K . exp (-Kd . t)) (3.6.2.27)

Where K1 is defined as the ratio xo/( Kd + K . x) (3.6.2.28)

The variation of volume with time is given by combination of equations (3.6.2.27) and
(3.6.2.22). The variation of the product concentration with time is again given by a
combination of equations (3.6.2.27) and (3.6.2.21). Mathematically, the resulting
expression is more difficult to solve although this can be circumvented if
t
F(t) = I[ x dt (3.6.2.29)
0
t
where I[ is the integral.
0
Then

P= |cto / K . [ 1 - exp (-K . f(t)] (3.6.2.30)

A similar and a much simpler development would be implemented if Kd would be
negligible. In this situation, the solutions for biomass concentration, flow rate and
product concentration variations with time would be given by

x = xo / (1 + K . xo . t) (3.6.2.31)
F = K . xo. Vi (3.6.2.32)
(where Vi is the volume in the beginning of the fed-batch phase)
P = |cto . x . t / (1 + K . x . t) (3.6.2.33)
V = Vi . (1 + K . xo.t) (3.6.2.34)

|Back to section 3.6 - modelling fixed-volume fed-batch fermentations | Top of the document |

For a growth associated product
12

Nomenclature:
- V is the volume of the fermentor
- t is the time
- F is the feed rate [volume/time].
- x is the biomass concentration [mass biomass/volume]
- u is the specific growth rate [time
-1
]
- Kd is the specific death rate [time
-1
]
- s is the substrate concentration in the fermentor [mass substrate/volume]
- so is the substrate concentration in the feed [mass substrate/volume]
- Y x/s is the yield factor [mass biomass/mass substrate]
- ms is the substrate consumption rate for cell maintenance [mass
substrate/(volume.time)]
- Y'p/s is the mass of product formed per fraction of substrate mass
- P is the product concentration {mass product/volume] and
- qp is the specific production rate of product [mass product/(mass biomass . time)
- rs' is the substrate consumption rate for maintenance and product formation
[mass of substrate/(volume.time)]rx is the cell growth rate [mass
substrate/(volume. time)]
- Y'x/s is the mass of cells formed per fraction of substrate mass
In this case, substrate is provided in such a way that maximizes the specific growth rate,
assuming that the substrate is not growth or product formation inhibitory for that
concentration. The substrate is supplied here not only for maintenance and product
formation but also for biomass production. The product formation is such that rp =
otqo . u. x
14
, being alpha a specific constant of the bioprocess.
For a matter of simplicity, Kd is going to be considered to be approximately zero and

s = constant; ds/dt = 0 (3.6.2.34)


Applying these relations to equations (3.6.2.2), (3.6.2.7), (3.6.2.12) and (3.6.2.16), the
following system of equations is achieved:
o Biomass: dx/dt = x . (u . V- F) / V (3.6.2.35)
o Substrate: 0 = F . (so- s) /V - r's (3.6.2.36)
(where r's is the substrate consumption rate for growth, maintenance and product
formation)
r's = rx / Y'x/s+ ms . x + rp / Y'p/s ( = . x/ Yx/s ) (3.6.2.37)
o Product: dp/dt = otqo . u. x

- p . F / V (3.6.2.38)
o Overall: dV/dt = F (3.6.2.39)
Once again, to obtain the optimum operating system, so will be fixed and F is allowed to
vary. Moreover, the specific growth rate is a constant and s is a constant also, hence (so
- s) is a constant that will be named S. Equation (3.6.2.36) can be rearranged to give

0 = F . S /V + K . so . x (3.6.2.40)

in which now

K = - { / Y'x/s + ms + otqo . u/ Y'p/s} . 1/S (3.6.2.41)

However, for K to be a constant, u needs to be constant also. Considering that is the
case, then, the solutions for biomass concentration, flow rate and product concentration
variations with time would be given by

x = K1 . u. exp(-u . t)/( K1 . K . exp (-u . t) - 1) (3.6.2.42)

Where K1 is defined as the ratio xo / (u + K . x) (3.6.2.43)

Again, the variation of the volume with time is given by the combination of equations
(3.6.2.39) and (3.6.2.40), using F(t) and by direct integration. For product variation with
time

P = otqo . u / K . { 1 - exp (-K . F(t)} (3.6.2.44)

Microorganisms growing exponentially
28

Another approach to a situation in which the specific growth rate is maintained
constant goes as following. Taking equation (3.6.2.9)

d(V . s)/dt = F . so rs . V (3.6.2.9)

Y x/s = rx . V / (rs .V) = u . X . V/(rs . V) (3.6.2.45)

If the growth is exponential

d(x . V)/dt = u. x . V (3.6.2.46)

Integrating equation (3.6.2.46) gives

X . V = xo . Vo . exp (u . t) (3.6.2.47)

Where
- rs is the consumption rate of substrate (mass substrate/(volume. time)
- xo is the initial concentration of biomass inside the fermentor for time zero [mass
biomass/volume]
- x is the concentration of biomass for time t [biomass/volume]
- Vo is the initial volume of the fermentor for time zero
- V is the volume of the fermentor for time t

Using equation (3.6.2.47) in equation (3.6.2.45), gives

rx . V = u. xo . Vo .exp(u . t) / Y x/s (3.6.2.48)

If now the derivative in the left-hand side of equation (3.6.2.9) is developed

d(V . s)/dt = V . ds/dt + s . dV/dt (3.6.2.49)

But since the specific growth rate is constant, which implies constant substrate
concentration in the medium, then ds/dt equals zero. Equation (3.6.2.49) is resumed to

d(V . s)/dt = s . dV/dt = s . F (3.6.2.49)

being the last relation allowed by equation (3.6.2.2). Finally, substituting equations
(3.6.2.48) and (3.6.2.49) in (3.6.2.9), gives

F = u . xo . Vo . exp(u . t)/[(so - s) . Y x/s] (3.6.2.50)


Which basically reflects the need of an exponential feed to maintain a constant specific
growth rate in a culture growing exponentially and, consequently the substrate
concentration. To determine how the biomass is increasing with time, take

F = u . x . V/ (s . Y x/s) = u . xo . Vo . exp (u . t) / (s . Y x/s) = dV/dt (3.6.2.51)

By integration of this equation, V comes as a function of time

V = Vo (u + A . xo . exp(u . t) A . xo)/ uwhere
A = u/(s . Y x/s) (3.6.2.52)

Consequently, by substituting equation (3.6.2.52) in equation (3.6.2.47)

X = u . xo . exp(u . t)/ (u + A . xo . exp(u . t) - A . xo) (3.6.2.53)


|Back to section 3.6 - modelling fixed-volume fed-batch fermentations | Top of the document |


Another alternative would be to maintain the concentration of biomass constant with
time the quasi-steady state. In this case,

dx/dt = 0 (3.6.2.54)

Right from equation (3.6.2.7) and assuming Kd negligible, = F/V, which means that
the specific growth rate will decrease with time since the volume is increasing
simultaneously. Both the substrate concentration and the growth rate are changing with
time. In this case, to solve the system of equations, the feed should be defined. Consider
a constant feed F (volume/time). Then, equation (3.6.2.2) can be directly integrated and

V = Vo + F . t (3.6.2.55)

Substituting in equation (3.6.2.12), and by integration, it comes

s = F. t/ (Vo + F . t) . (so - x/ Y x/s) (3.6.2.56)

Which, for small times, approximates zero and for large times approximates

S = so - x/ Y x/s (3.6.2.57)

To determine how the growth rate varies, then

du/dt = d(F/V)/dt =d[F/( Vo + F.t)]/dt = -F
2
/V
2
= -F
2
/( Vo + F.t)
2
(3.6.2.58)

For small times: d/dt = -F
2
/Vo
2
(3.6.2.59)

For large times d/dt = -1/t
2
(3.6.2.60)

Which shows that the specific growth rate decreases more in the beginning of the
fermentation but decreases less as the time passes by
5
.

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