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1.1 A microorganism: a microscopic biochemical factory

A single cell is considered as a microscopic biochemical factory. Materials

such as carbon, nitrogen, oxygen and others are brought into the cell and
converted within the cell via the hundreds of reactions to the various
constituents of the cell as well as to biochemical products which may be
retained or transported back into the environment outside the cell . 1 metabolic
activities inside the cell are regulated at various levels both inside and outside
the cell. Moreover, biological activity of the cell is extremely sensitive to the
environment it is exposed to. Because of this multi - level complex regulation,
by an engineering point of view, it is of utmost importance to understand the
nutritional and environmental factors affecting cell metabolism.

1.2 Bioreactor as a controlled environment for the growth of microorganisms

An engineer is always interested in consistently producing large quantities of

product of interest over long periods of time. The best way to achieve this goal
wants to be to grow the cells in a bioreactor where the cellular activity can be
controlled efficiently. The three basic modes of bioreactor operation are batch,
fed-batch and continuous. The control issues related to each of these will be
discussed in later sections.

1.3 Importance of process control in bioreactors

Development of methods for monitoring and control of commercial

bioprocessing including reliable and robust real-time sensors has been listed on
outreaching priority for federal investment in a report published in July 95 from
the Biotechnology Research Subcommittee (BRS) of the Committee on
Fundamental Science of the The National Science and Technology Council.
The best performance of the upstream processing can not be achieved without
the knowledge of the state of the system and on control algorithms that can
optimize the process. Controls of Bioprocesses is challenging, particularly in
batch and fed-batch bioreactors, due to high degree of nonlinearity (meaning
that nonlinear differential equations are required for mathematical modeling),
and their potential for instability when they involve high-yield mutant or
recombinant microorganism. These problems are further complicated by the
scarcity of on-line real-time sensors and realistic models that capture the
intricate complexities of biological systems.

2. Parameter characterizing a bioreactor

The presence of the living microorganisms inside the bioreactor makes it more
complicated than the conventional chemical reactor. As already indicated, it is
extremely important to gather the knowledge about the state of the bioreactor
prior to design and implementation of any control system for the reactor. The
complete state of the biochemical reactor can be assessed by knowing the
following parameters: physical parameters, chemical (extracellular) parameters,
biochemical (intracellular), and biological parameters. 3 The following
subsections will list the various parameters in each category. The common
methods for the determination of these parameters are discussed in section 9.2.

2.1 Physical parameters

The following are the important physical parameters for the operation of a
bioreactor: agitation power, agitation speed, broth volume, color, expanded
broth volume (density), foaming, gas flow rate, gas humidity, heat generation
rate, heat transfer rate, liquid feed rate, liquid level, mass, osmotic pressure,
pressure, shear rate, tip speed, temperature, turnover time, and viscosity. 3 Many
of these parameters have important implications in the control of bioreactors.

2.2 Chemical Parameters

The following list gives the different parameters that define the chemical
environment inside the reactor: amino acids, carbon dioxide (gas), cation level,
conductivity, inhibitor, intermediate (s), ionic strength, Malliard reaction
products, nitrogen (free and total) , nutrient composition, oxygen, pH,
phosphorous, precursor, product, redox and substrate. 3

2.3 Biochemical (intracellular) parameters

Biochemical parameters are the intracellular parameters that indicate the

metabolic state of the cell at any given time during the cell growth. These
include amino acids, ATP / ADP / AMP, carbohydrates, cell mass composition,
enzymes, intermediates, NAD / NADH, nucleic acids , total protein, and

2.4 Biological parameters

Biological parameters characterize the bioreactor in terms of what is happening

inside the reactor at the cellular level. The list includes age / age distribution,
aggregation, contamination, degeneration, doubling time, genetic instability,
morphology, mutation, size / size distribution, total cell count and viable cell
3. Control Strategies for Bioprocesses

All the bioreactors used oxygen at the present time use control strategies for
three basic environmental factors: pH, temperature and dissolved. Invariably,
these control implementations are achieved through regulation of flow rate of
acid / base, flow rate of fluid through the cooling coil, and agitation
respectively. Needless to say, these three parameters are extremely important
for optimal cellular activity. But they alone do not guarantee the maximum
productivity, which is the objective for most of the industrial fermentations.
This paper will explore the control strategies which are used to accomplish this

Before attempting to understand the details of the control strategies used for
bioprocess, one should be familiar with the common features in the field of
controls. One of the feature integrated into any control system is control
algorithm. The control algorithm is that part of the control system that takes the
available measurements and level of process understanding and decides on the
best way to influence the process with the available manipulated variable to
achieve The desired objective. 4

A control system can not be implemented unless the process under

consideration is understood. An efficient way of understanding the process is a
mathematical model of the process. A good process model is an invaluable tool
to deveolp a control algorithm. It is not implied that controllers can not control
poorly understood processes, indeed, that is often their function. However, an
expensive, and time - consuming trial and error adjustment of the control
algorithm is required in that case. 4

A common approach to obtain a simple, empirical model for controller design

is to make small step changes in the inputs and observe the dynamic behavior
of the outputs. One can then obtain a linear time - invariant process model in a
straightforward fashion. A more fundamental approach is to formulate mass
and energy balances for different components, resulting in a set of nonlinear
ordinary differential equations. The latter approach has an advantage that the
nonlinear model may better represent the process over a significant range of
state values, whereas the linear empirical model resulted from the former
approach may not be reliable for process states away from the state at which
model is identified. This is particularly important in case of batch and fed -
batch fermentations in which the process state changes significantly during
operation. The disadvantage of the latter approach is that the available
controllers - design tools are less developed for the nonlinear models. 4
There are three modes of bioreactor operation: batch, fed-batch and
continuous. As discussed in subsequent sections of this paper, each of these
modes presents different challenges to the control algorithm.

4. Bioreactor dynamics

4.1 Bioreactor as a multivariable system with nonlinear dynamics

Prior to discussing specific control applications, some general features of

bioreactors relevant to control applications should be mentioned. The two main
characteristics that are important to know before designing a control system for
a bioreactor are:

Multivariable system: As one would anticipate, control of a bioreactor will

involve a system involving many variables.

Nonlinear dynamics: The control of a bioreactor is complicated by the fact that

nonsteady-state behavior is nonlinear. This has several consequences.
Hysteresis is often observed. For example, a step increase in reactor feed rate
in case of CSTBR (continuously stirred tank bioreactor) will result in a
transient that will be different than when the corresponding equivalent step
decrease in feed rate back to initial conditions is made. Moreover, multiple
steady states are often observed for identical feed conditions, and in certain
cases, exotic dynamics like limit cycles, oscillatory transients, long time lags
may be exhibited. 1

The reasons for the above mentioned behaviors are ultimately related to the
complexities of living cells. Finally, many of the important variables which are
desirable for monitoring and control are only measurable with Large time lags
or not measurable at all This gives scope for accurate mathematical models and
/ or state estimation techniques. Fortunately, simple models and single input -
single output feedback loops are available and work well in many cases.

4.2 Cell growth modeling in a batch reactor

The simplest way to model cell growth will be to consider in unstructured,

unsegregated model for cell growth. For this kind of model,

r x = dX / dt = mX (1)

where, r x = rate of cell generation (g / l-hr)

X = cell concentration (g / l)
m = specific growth rate (hr -1)

The most commonly used expression that relates the specific growth rate of the
cell to the substrate concentration is Monod's equation, which is given as

m = m max S / (K s + S) (2)
where, m = specific growth rate (hr -1)
m max = maximum specific growth (hr -1)
S = substrate concentration (g / l)
K S = saturation constant for substrate (g / l)

Figure 1 depicts the dependence of? on S according to Monod's equation. One

should note that Monod's equation is empirical and does not have any
mechanistic basis. 5 The equation is only valid for an exponentially growing
culture under condition of balanced growth. The equation does not fair well in
transient conditions. Despite its simplicity and no fundamental basis, it works

Figure 1 Monod's growth curve

surprisingly well in a large number of steady state and dynamic situations.
This characteristic has important implications in control of bioreactors.

4.3 Continuous bioreactor dynamics

For a continuously fed bioreactor, the cells are continuously supplied substrate
at growth limiting level, and hence they remain in the exponential phase. Since
the cells remain in the exponential phase, Monod's equation can be applied. A
cell balance on the reactor can be written as

FX - FX f + V (dX / dt) = r x (3)

where F = volumetric flow rate (l / hr)
X = cell concentration inside the reactor and in the outlet stream (g / l)
F X = cell concentration in the feed (g / l)
V = reactor volume (l)
r x = rate of cell generation (g / l-hr)

For a sterile feed (X f = 0)) and noting that the reaction rate can be written in
terms of the specific growth rate (r x = mX, equation (3) can be reduced to

dX / dt = (m - D) X (4)
where D = dilution rate = F / V (hr -1)

A balance on the substrate yields the following equation

FS - FS f + V (dS / dt) = r s V (5)

where F = volumetric flow rate (l / hr)
S = cell concentration inside the bioreactor and in the outlet stream (g / l)
S f = substrate concentration in the feed (g / l)
V = reactor volume (l)
r s = rate of substrate consumption (g / l-hr)

A yield parameters (Y x / s) is defined that relates the amount of cell mass

produced per amount of substrate consumed, and is mathematically represented

Y x / s = mass of cells produced / mass of substrate consumed x = r / r s (6)

Combining equations (1), (5), and (6) yields

dS / dt = D (S f - S) - mX / Y x / s (7)
The CSTBR (continuous stirred tank bioreactor) is now completely described
by equations (4) and (7) with m given by equation (2). At steady state (with
fixed Sf and D), the following are the values for m (specific growth rate), S
(substrate concentration) and cell concentration (X)

m = D (8)

DK S = S / (m max - D) (9)
X = Y x / s (S f - S) (10)

There are a few characteristics of an open-loop CSTBR that are conceptually

different from that of a chemical reactor which are important to know before
any control system for a bioreactor can be designed. Figure 2 shows that D
must be less than m max for a realistic value of S to be achieved. The same
conclusion can

Figure 2 Relationship between dilution rate and specific growth rate for a
steady state CSTBR

be derived by looking at the steady state solution of equation (4). The two
solutions are equation (8) and
X = 0 (11)

The corresponding substrate concentration is

S = S f (12)

Equation (11) and (12) define a situation called washout. This situation is
encountered whenever the value of dilution rate equals or exceeds m max. A
rigorous discussion of washout would point to the fact that whenever m (S f), ie,
m f evaluated at S is less than? Max, then the critical dilution rate for washout
will occur at D = m (S f), and not at D = m max. 1 The control algorithm should be
completely aware of this unproductive state.

For the given set of equations, numerical solution is required since the system
is described by two coupled nonlinear differential equations, ie, equations (4)
and (7). Linear control theory can be applied only in a limited sense, ie, only
near the steady state when the system model is linearized. 1

Start up is an important consideration as well. The general procedure in the

start up avoiding washout would be to initiate cell growth in a batch mode until
the exponential phase is reached. At this point, the sterile feed would be started
with a dilution rate such that D <m (Sf). A non washout steady state would be
reached after a transient phase. 1

4.4 Multiplicity and stability of steady states in a continuous bioreactor

Though the control loop of a CSTBR is simple, the system is complicated by

the presence of multiple steady states and the stability considerations of these
steady states. The following discussion will highlight these problems.

As already implied, the control design of a biological reactor described by

equation (4) and (7) should take into account the nonlinear nature of these
differential equations. Multiple critical points are common with nonlinear
systems. This has been shown earlier in the discussion of washout. A
systematic approach to an efficient control design will involve
1. calculation of the number of steady states
2. characterization of the nature of the steady states with respect to their
3. design of appropriate control loops based on the results from step 1 and step

4.4.1 Calculation of multiple steady states

Once the governing equations describing the system are in place, the steady
states are found by replacing all time derivatives by zero. This can be done by
inspection and algebric solution. For high order or complex models, a
nonlinear root finding technique should be employed. 1

4.4.2 Stability of a steady-state

A steady state is stable if, for initial conditions near the steady state, all
transients converge to it. If the transients diverge, steady state is called
unstable. The diverging transients always end at some other stable state.
Stability analysis of a steady state would involve Whether the steady state
under consideration is stable or not and the information about state - to - state
transitions in case of unstable steady states. 1

The information about the local stability and dynamics of the steady states is
accomplished through a linear stability analysis. It should be borne in mind that
the results of the linear stability analysis are good only near the steady state.
For general (nonlocal) behavior and information about state - to - state
transitions, generation of the phase plane is suitable. 1

4.5 Proportoinal control of a CSTBR with Monod's kinetics

Before designing the closed - loop continuous bioreactor, one should

understand the open - loop CSTBR fully since the scope of closed - loop
CSTBR will be given only by the knowledge about the open - loop CSTBR.
Linear stability analysis and phase plane analysis for open - loop CSTBR and
closed - loop CSTBR are detailed below.

4.5.1 Stability analysis of open - loop CSTBR Linear stability analysis for open - loop CSTBR

As already discussed, for an open - loop CSTBR with Monod's kinetics, there
exist two steady states, ie, a nontrivial steady state (defined by equation (8), (9),
and (10)), washout and steady-state (defined by equation (11) and (12)). The
Jacobian J for the system defined by equation (4), and (7) with m given by
equation (2) is
where X '= dX / dt
S '= dS / dt

Substituting the values for X 'and S' from equations (4) and (7) yields the value
of Jacobian for the system as

For the nontrivial steady state, stability is guaranteed if the following equations
are satisfied

Trace J <0 (15)

Det J> 0 (16)

Which yields

-D - m'X / Y x / s <0 (! 7)
Xm'm / Y x / s> 0 (18)
In case of Monod's equation, m '> 0 for all p. Hence, nontrivial state is always
stable. For the washout state, the conditions for stability are derived from a
similar procedure

m (S f) - D <0 (19)
(m (S f) - D) (D)> 0 (20)

Equations (19) and (20) indicate that D must be greater than m (S f) for the
washout steady state to be stable. Thus, any dilution rate which gives any
realistic solution (X> 0, and S> 0) will result in washout being unstable. The
same conclusion can be derived by the analysis phase plane which is also
discussed in the next subsection. Phase plane analysis for open - loop CSTBR

Construction of the phase plane for open - loop CSTBR be achieved through
integrating equations (5) and (8), selecting several time points, plotting the
values of S and X at each point, then repeating for new initial conditions or
sketched directly from the results of the linear analysis. As shown in Figure 3,
all-state initial conditions result in the achievement of the desired steady. 1

The motive for controlling this reactor would be to maintain a closed - loop
system such that washout could be avoided regardless of flow fluctuations. An
easy-state approach to achieve this would be to measure the cell concentration
and manipulate the flow rate to force the reactor to nontrivial steady. This can
Figure 3 Open loop phase plane for bioreactor with Monod's kinetics (S R is
the feed substrate concentration and alpha and beta are the steady-state cell and
substrate concentrations)

easily be accomplished with a simple proportional controller Whose stability

analysis is discussed in next subsection.

4.5.2 Stability analysis of closed - loop bioreactor

The governing equation for the proportional controller which manipulates the
flow rate as a response to changing cell concentration inside the reactor is given

D = D ss + K c (X - X sp) (21)
Where, D = dilution rate that is manipulated by the controller (hr -1)
D ss = dilution rate corresponding to the nontrivial steady state for X = X sp in
open loop CSTBR (hr -1)
K c = controller gain (l / g-hr)
X = cell concentration in the reactor (g / l)
X sp = controller set point and the desired cell concentration in the reactor (g /

Substituting the value of D from equation (21) into equations (4) and (7) shows
that X = 0, S = S f is no longer a steady-state solution. A rigorous analysis for
this system will show that the worst case for this system as X approaches 0
corresponds to D = 0 This is equivalent to saying that the CSTBR wants to
approach the behavior of a batch reactor.

The conditions for the stability of the system under consideration according to
linear stability analysis are

K c - D ss - m '/ Yx / s <0 (22)

K c + m '/ Y x / s> 0 (23)

Any positive value of K c is sufficient to satisfy equations (22) and (23), and
hence guarantee stability. This state is not surprising keeping in mind the
stability of nontrivial steady in the open - loop CSTBR. It seems fair to expect
the closed - loop phase plane similar to open - loop phase plane for reasonable
values of K c.

The whole discussion can be summarized as follows. Since the nontrivial state
is always stable for realistic D values, there is little incentive for closed - loop
operation other than to prevent washout from large flow disturbances. The
incentive for closed - loop operation increases significantly if the growth
kinetics are more complex, eg, substrate inhibited growth kinetics. This is
discussed in the next section.

4.6 Control of a continuous bioreactor with substrate inhibition kinetics

Though Monod's kinetics makes a nice model for substrate - limited cases, it
does not approximate the real cases very well since all the biological systems
are inhibited by high substrate concentration. Hence, understanding these kind
of reactors are important. The dynamics of the CSTBR with substrate inhibition
kinetics leads to an interesting control problem. Interested reader may find
detailed analysis by Dibiasio for an open - loop as well as closed - loop CSTBR
with substrate. 1

A number of reports have been published regarding control issues related to

continuous bioreactors.

4.7 Fed batch reactor dynamics

Though CSTBR is an excellent tool to study bacterial metabolism, it is not

extensively used in biotechnology - industry. The most widely used mode for
fermentation industrial production of biochemicals is fed - batch fermentation.
The state-fed - batch system is an interesting system to study since it does not
have a true steady. In this case, evaluation of the state variables will locate the
position of the system on a trajectory through the operational cycle. Since these
state variables can not be measured online, the estimation of state becomes an
important element of optimization and control of the reactor. There are
numerous reports about the theoretical and experimental issues related to the
fed - batch fermentation.

4.8 Bifurcation analysis

A convenient method of classifying the various types of possible dynamic

behaviors that can be exhibited by a fermentation model is provided by
bifurcation analysis. This theory has a large literature, and the interested reader
is directed to Razon and Schmitz.

Feedback control is an action by which PID controllers as well as the

controllers based on advanced control strategies implement their control action.
Feedback control is an action by which PID controllers as well as the
controllers based on advanced control strategies implement their control action.
In feedback control system, the controlled variable is measured and compared
to the setpoint. The feedback control system, the controlled variable is
measured and compared to the setpoint. Subsequently, an error signal is
generated by subtracting the setpoint from the value of the controlled variable.
Subsequently, variable an error signal is generated by subtracting the value
from the setpoint of the controlled. Then the controller calculates the the
appropriate corrective action, to be implemented by the manipulated variable,
by using the value of the error signal. 16 Then the controller calculates the the
appropriate corrective action, to be implemented by the manipulated variable,
by using the value of the error signal. 16