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- Slides 77 Bioprocess
- Effect of Substrate Concentration on α -Amylase
- Salivary Amylase
- kinetics
- Experiment 2 - Batch Fermentation of E Coli in Bio Reactor
- bioreactor
- Effect of Temperature on Enzyme Activity
- Enzyme Kinetics Lab Report
- Immobilized Enzymes
- 6085547-r05222301bioprocessengineering
- 01 Enzyme Kinetics
- Crystallization Equipment
- Bio Reactor Engineering Chapt 1-EDITED- Stdt
- Experiment 2_lowry x
- Chapter 3
- 95507914-Report
- Experiment 1final
- Doran - Bioprocess Engineering Principles - Fluid Flow and Mixing
- Scale-up Strategies in Stirred and Aerated Bio Reactor
- Enzyme Kinetics Principles and Methods

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INTRODUCTION

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.

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.

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.

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.

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.

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

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

vitamins.

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

count.

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

goal.

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

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

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

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:

involve a system involving many variables.

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.

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

r x = dX / dt = mX (1)

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)

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

surprisingly well in a large number of steady state and dynamic situations.

This characteristic has important implications in control of bioreactors.

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

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)

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)

produced per amount of substrate consumed, and is mathematically represented

as

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)

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)

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 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

the presence of multiple steady states and the stability considerations of these

steady states. The following discussion will highlight these problems.

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

stability

3. design of appropriate control loops based on the results from step 1 and step

2

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

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

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.

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

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.

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)

analysis is discussed in next subsection.

The governing equation for the proportional controller which manipulates the

flow rate as a response to changing cell concentration inside the reactor is given

by

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 /

l)

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 + 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.

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

continuous bioreactors.

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.

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.

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

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