Biological Reaction Engineering

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

Dynamic Modelling Fundamentals
with Simulation Examples
Second, Completely Revised Edition
Biological Reaction Engineering. Second Edition. \. J. Dunn. E. Heinzle, J. Ingham, J- E. Pfenosil

Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheitn
ISBN: 3-527-30759-1
Irving J. Dunn, Elmar Heinzle, John Ingham, Jifi E. Pf enosil
Biological
Reaction Engineering
Dynamic Modelling Fundamentals
with Simulation Examples
Second, Completely Revised Edition
WILEYVCH
WILEY-VCH GmbH & Co. KGaA
Dr. Irving J. Dunn

ETH Zurich
Department of Chemical Engineering
CH-8092 Zurich
Switzerland
Professor Dr. Elmar Heinzle
University of Saarland
Department of Technical Biochemistry
P.O. Box 15 11 50
D-66041 Saarbrucken
Germany
Dr. John Ingham
University of Bradford
Department of Chemical Engeering
Bradford BD7 1DP
United Kingdom
Dr.JiriE.Prenosil
ETH Zurich
Department of Chemical Engineering
CH-8092 Zurich
Switzerland
This book was carefully produced. Nevertheless,

authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements,
data, illustrations, procedural details or other
items may inadvertently be inaccurate.
First Edition 1992

Second, Completely Revised Edition 2003
Library of Congress Card No.: Applied for.

British Library Cataloguing-in-Publication Data:
A catalogue record for this book is available from
the British Library.
Bibliographic information published by Die Deutsche Bibliothek. Die Deutsche Bibliothek lists this
publication in the Deutsche Nationalbibliografie;
detailed bibliographic data is available in the
Internet at <http://dnb.ddb.de>.
2003 WILE Y-VCH Verlag

GmbH & Co. KGaA, Weinheim
All rights reserved (including those of translation
into other languages). No part of this book may be
reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or
translated into a machine language without written permission from the publishers. Registered
names, trademarks, etc. used in this book, even
when not specifically marked as such, are not to
be considered unprotected by law.
Printing: Strauss Offsetdruck, Morlenbach
Bookbinding: GroBbuchbinderei J. Schaffer
GmbH & Co. KG, Griinstadt
Printed in the Federal Republic of Germany.
Printed on acid-free paper.
ISBN 3-527-30759-1
Table of Contents
TABLE OF CONTENTS
PREFACE
V
XI
PART I PRINCIPLES OF BIOREACTOR MODELLING
NOMENCLATURE FOR PART I
MODELLING PRINCIPLES
1.1
FUNDAMENTALS OF MODELLING
9
7.7.7
Use of Models for Understanding, Design and Optimization of Bioreactors 9
1.1.2
General Aspects of the Modelling Approach
10
1.1.3
General Modelling Procedure.....
72
1.1.4
Simulation Tools
75
7.7.5
Teaching Applications
75
1.2
DEVELOPMENT AND MEANING OF DYNAMC DIFFEREOTTAL BALANCES
16
1.3
FORMULATION OF BALANCE EQUATIONS
..21
7.5.7
Types of Mass Balance Equations
27
1.3.2
Balancing Procedure
23
1.3.2.1 Case A. Continuous Stirred Tank Bioreactor
24
1.3.2.2 CaseB. Tubular Reactor
24
1.3.2.3 Case C. River with Eddy Current
25
1.3.3
Total Mass Balances
33
1.3.4
Component Balances for Reacting Systems
34
1.3.4.1 Case A. Constant Volume Continuous Stirred Tank Reactor
35
1.3.4.2 Case B. Semi-continuous Reactor with Volume Change
37
1.3.4.3 Case C. Steady-State Oxygen Balancing in Fermentation
38
1.3.4.4 Case D. Inert Gas Balance to Calculate Flow Rates
39
7.5.5
Stoichiometry, Elemental Balancing and the Yield Coefficient Concept.. 40
1.3.5.1 Simple Stoichiometry
40
1.3.5.2 Elemental Balancing
42
L3.5.3 Mass Yield Coefficients
44
VI
Table of Contents
1.3.5.4
1.3.6
1.3.6.1
1.3.6.2
1.3.7
1.3.6.3
Energy Yield Coefficients

Equilibrium Relationships
General Considerations
Case A. Calculation of pH with an Ion Charge Balance
Energy Balancing for Bioreactors.....
Case B. Determining Heat Transfer Area or Cooling Water Temperature
BASIC BIOREACTOR CONCEPTS

2.1
INFORMATION FOR BIOREACTOR MODELLING...
2.2
BIOREACTOR OPERATION
2.2.7
Batch Operation
2.2.2
Semicontinuous or Fed Batch Operation.....
2.2.3
Continuous Operation
2.2.4
Summary and Comparison
45
46
46
47
49
52
55
BIOLOGICAL KINETICS
.....55
.....56
57
....58
60
63
67
3.1
ENZYME KINETICS
68
3.1.1
Michaelis-Menten Equation
68
3.1.2
Other Enzyme Kinetic Models
73
3.1.3
Deactivation
76
3.1.4
Sterilization
76
3.2
SIMPLE MICROBIAL KINETICS
77
3.2.1
Basic Growth Kinetics
77
3.2.2
Substrate Inhibition of Growth
80
3.2.3
Product Inhibition
81
3.2.4
Other Expressions for Specific Growth Rate
81
3.2.5
Substrate Uptake Kinetics
83
3.2.6
Product Formation
85
3.2.7
Interacting Microorganisms
....86
3.2.7.1 Case A. Modelling of Mutualism Kinetics.....
88
3.2.7.2 Case B. Kinetics of Anaerobic Degradation
89
3.3
STRUCTURED KINETIC MODELS
..........91
3.3.1
Case Studies
93
3.3.1.1 Case C. Modelling Synthesis of Poly-B-hydroxybutyric Acid (PHB)
93
3.3.1.2 Case D. Modelling of Sustained Oscillations in Continuous Culture
94
3.3.1.3 Case E. Growth and Product Formation of an Oxygen-Sensitive Bacillussubtilis Culture
97
4
BIOREACTOR MODELLING
4.1
GENERAL BALANCES FOR TANK-TYPE BIOLOGICAL REACTORS
4.1.1
The Batch Fermenter.
4.1.2
The Chemostat
4.1.3
The Fed Batch Fermenter
4.1.4
Biomass Productivity
4.1.5
Case Studies
101
101
103
104
1 06
109
109
Table of Contents
4.1.5.1 Case A. Continuous Fermentation with Biomass Recycle
4.1.5.2 Case B. Enzymatic Tanks-in-series Bioreactor System
4.2
MODELLING TUBULAR PLUG FLOW BIOREACTORS
4.2.1
Steady-State Balancing
4.2.2
Unsteady-State Balancing for Tubular Bioreactors
5
MASS TRANSFER
VII
110
112
113
113
775
117
5.1
MASS TRANSFER IN BIOLOGICAL REACTORS
117
5.7.7
Gas Absorption with Bioreaction in the Liquid Phase
777
5.1.2
Liquid-Liquid Extraction with Bioreaction in One Phase
778
5.1.3
Surface Biocatalysis
778
5.7.4
Diffusion and Reaction in Porous Biocatalyst
779
5.2
INTERPHASEGAS-LIQUID MASS TRANSFER
119
5.3
GENERAL OXYGEN BALANCES FOR GAS-LIQUID TRANSFER
123
5.3.1
Application of Oxygen Balances
725
5.3.1.1 Case A. Steady-State Gas Balance for the Biological Uptake Rate
125
5.3.1.2 Case B. Determination of KLa Using the Sulfite Oxidation Reaction
126
5.3.1.3 Case C. Determination of Kjâ by a Dynamic Method
126
5.3.1.4 Case D. Determination of Oxygen Uptake Rates by a Dynamic Method
128
5.3.1.5 Case E. Steady-State Liquid Balancing to Determine Oxygen Uptake Rate.. 129
5.3.1.6 Case F. Steady-State Deoxygenated Feed Method for KJJI
130
5.3.1.7 Case G. Biological Oxidation in an Aerated Tank
131
5.3.1.8 Case H. Modelling Nitrification in a Fluidized Bed Biofilm Reactor
133
5.4
MODELS FOR OXYGEN TRANSFER IN LARGE SCALE BIOREACTORS
137
5.4.1
Case Studies for Large Scale Bioreactors
7 39
5.4.1.1 Case A.Model for Oxygen Gradients in a Bubble Column Bioreactor
139
5.4.1.2 Case B.Model for a Multiple Impeller Fermenter
140
6
DIFFUSION AND BIOLOGICAL REACTION IN IMMOBILIZED

BIOCATALYST SYSTEMS
145
6.1
EXTERNAL MASS TRANSFER
6.2
INTERNAL DIFFUSION AND REACTION WITHIN BIOCATALYSTS
.....
6.2.1
Derivation of Finite Difference Model for Diffusion-Reaction Systems.
6.2.2
Dimensionless Parameters from Diffusion-Reaction Models
6.2.5
The Effectiveness Factor Concept.
6.2.4
Case Studies for Diffusion with Biological Reaction
6.2.4.1 Case A. Estimation of Oxygen Diffusion Effects in a Biofilm
6.2.4.2 Case B. Complex Diffusion-Reaction Processes (Biofilm Nitrification)....
AUTOMATIC BIOPROCESS CONTROL FUNDAMENTALS

7.1
7.2
ELEMENTS OF FEEDBACK CONTROL

TYPES OF CONTROLLER ACTION
7.2.7
On-OffControl
7.2.2
Proportional (P) Controller
7.2.3
Proportional-Integral (PI) Controller
146
149
151
754
755
757
157
157
161
161
163
163
764
765
VIII
Table of Contents
7.2.4
Proportional-Derivative (PD) Controller
7.2.5
Proportional-Integral-Derivative (PID) Controller
7.3
CONTROLLER TUNING
7.3.1
Trial and Error Method
7.3:2
Ziegler-Nichols Method.
7.3.3
Cohen-Coon Controller Settings
7.3.4
Ultimate Gain Method
7.4
ADVANCED CONTROL STRATEGIES
7.4.1
Cascade Control
7.4.2
Feed Forward Control
7.4.3
Adaptive Control
7.4.4
Sampled-Data Control Systems
7.5
CONCEPTS FOR BIOPROCESS CONTROL
7.5.7
Selection of a Control Strategy
7.5.2
Methods of Designing and Testing the Strategy
REFERENCES
REFERENCES CITED IN PART I
RECOMMENDED TEXTBOOKS AND REFERENCES FOR FURTHER READING
PART II
166
167
169
769
769
170
777
172
772
173
774
774
175
776
7 78
181
181
184
DYNAMIC BIOPROCESS SIMULATION EXAMPLES AND

THE BERKELEY MADONNA SIMULATION LANGUAGE. 191
SIMULATION EXAMPLES OF BIOLOGICAL REACTION

PROCESSES USING BERKELEY MADONNA
193
8.1
INTRODUCTORY EXAMPLES
193
8.7.7
Batch Fermentation (BATFERM)
793
8.7.2
ChemostatFermentation (CHEMO)
799
8.1.3
Fed Batch Fermentation (FEDBAT)
204
8.2
BATCH REACTORS
209
8.2.7
Kinetics of Enzyme Action (MMKINET)
209
8.2.2
Lineweaver-Burk Plot (LINEWEAV)
.....272
8.2.3
Oligosaccharide Production in Enzymatic Lactose Hydrolysis (OLIGO) 215
8.2.4
Structured Model for PHB Production (PHB)
....279
8.3
FED BATCH REACTORS
224
8.3.1
Variable Volume Fermentation (VARVOL and VARVOLD)
224
8.3.2
Penicillin Fermentation Using Elemental Balancing (PENFERM)
230
8.3.3
Ethanol Fed Batch Diauxic Fermentation (ETHFERM)
240
8.3.4
Repeated Fed Batch Culture (REPFED)
245
8.3.5
Repeated Medium Replacement Culture (REPLCUL)
249
8.3.6
Penicillin Production in a Fed Batch Fermenter (PENOXY)
253
8.4
CONTINUOUS REACTORS
257
8.4.7
Steady-State Chemostat (CHEMOSTA)
257
8.4.2
Continuous Culture with Inhibitory Substrate (CONINHIB)
267
8.4.3
Nitrification in Activated Sludge Process (ACTNITR)
267
Table of Contents
IX
8.4.4
Tubular Enzyme Reactor (ENZTUBE)
272
8.4.5
Dual Substrate Limitation (DUAL)
275
8.4.6
Dichloromethane in a Biofllm Fluidized Sand Bed (DCMDEG)
280
8.4.7
Two-Stage Chemostat with Additional Stream (TWOSTAGE)
286
8.4.8
Two Stage Culture with Product Inhibition (STAGED)
290
8.4.9
Fluidized Bed Recycle Reactor (FBR)
295
8.4.10
Nitrification in a Fluidized Bed Reactor (NITBED)...
299
8.4.11
Continuous Enzymatic Reactor (ENZCON)
305
8.4.12
Reactor Cascade with Deactivating Enzyme (DEACTENZ)
308
8.4.13
Production ofPHB in a Two-Tank Reactor Process (PHBTWO)
314
8.5
OXYGEN UPTAKE SYSTEMS
318
8.5.1
Aeration of a Tank Reactor for Enzymatic Oxidation (OXENZ)
318
8.5.2
Gas and Liquid Oxygen Dynamics in a Continuous Fermenter (INHIB) 321
8.5.3
Batch Nitrification with Oxygen Transfer (NITRIF)
327
8.5.4
Oxygen Uptake and Aeration Dynamics (OXDYN)
331
8.5.5
Oxygen Electrode for Kia (KLADYN, KLAFIT and ELECTFIT)
335
8.5.6
Biofiltration Column with Two Inhibitory Substrates (BIOFILTDYN). 342
8.5.7
Optical Sensing in Microtiter Plates (TITERDYN and T1TERB1O)
349
8.6
CONTROLLED REACTORS
354
8.6.1
Feedback Control of a Water Heater (TEMPCONT)
354
8.6.2
Temperature Control of Fermentation (FERMTEMP)
358
8.6.3
Turbidostat Response (TURBCON)
363
8.6.4
Control of a Continuous Bioreactor, Inhibitory Substrate (CONTCON)367
8.7
DIFFUSION SYSTEMS
....371
8.7.1
Double Substrate Biofilm Reaction (BIOFILM)
377
8.7.2
Steady-State Split Boundary Solution (ENZSPLIT)....
377
8.7.3
Dynamic Porous Diffusion and Reaction (ENZDYN)....
383
8.7.4
Oxygen Diffusion in Animal Cells (CELLDIFF)
388
8.7.5
Biofilm in a Nitrification Column System (NITBEDFILM)
393
8.8
MULTI-ORGANISM SYSTEMS
..400
8.8.1
Two Bacteria with Opposite Substrate Preferences (COMMENSA)
400
8.8.2
Competitive Assimilation and Commensalism (COMPASM)
406
8.8.3
Stability of Recombinant Microorganisms (PLASMID)
411
8.8.4
Predator-Prey Population Dynamics (MIXPOP)
417
8.8.5
Competition Between Organisms (TWOONE)
422
8.8.6
Competition between Two Microorganisms in a Biofilm (FILMPOP). 425
8.8.7
Model for Anaerobic Reactor Activity Measurement (ANAEMEAS).... 433
8.8.8
Oscillations in Continuous Yeast Culture (YEASTOSC)
441
8.8.9
Mammalian Cell Cycle Control (MAMMCELLCYCLE)
445
8.9
MEMBRANE AND CELL RETENTION REACTORS
451
8.9.1
Cell Retention Membrane Reactor (MEMINH)
451
8.9.2
Fermentation with Pervaporation (SUBTILIS)
455
8.9.3
Two Stage Fermentor With Cell Recycle (LACMEMRECYC)
464
8.9.4
Hollow Fiber Enzyme Reactor for Lactose Hydrolysis (LACREACT). 470
8.9.5
Animal Cells in a Fluidized Bed Reactor (ANIMALIMMOB)
477
Table of Contents
APPENDIX: USING THE BERKELEY MADONNA LANGUAGE.. 483

9.1
9.2
A SHORT GUIDE TO BERKELEY MADONNA

SCREENSHOT GUIDE TO BERKELEY MADONNA
483
488
10 ALPHABETICAL LIST OF EXAMPLES
497
11 INDEX
499
Preface
Our goal in this textbook is to teach, through modelling and simulation, the
quantitative description of bioreaction processes to scientists and engineers. In
working through the many simulation examples, you, the reader, will learn to
apply mass and energy balances to describe a variety of dynamic bioreactor
systems. For your efforts, you will be rewarded with a greater understanding of
biological rate processes. The many example applications will help you to gain
confidence in modelling, and you will find that the simulation language used,
Berkeley Madonna, is a powerful tool for developing your own simulation
models. Your new abilities will be valuable for designing experiments, for
extracting kinetic data from experiments, in designing and optimizing
biological reaction systems, and for developing bioreactor control strategies.
This book is based on part of our successful course, "Biological Reaction
Engineering", which has been held annually in the Swiss mountain resort of
Braunwald for the past twenty five years and which is now known, throughout
European biotechnology circles as the "Braunwald Course". More details can
be found at our website www.braunwaldcourse.ch. Modelling is often
unfamiliar to biologists and chemists, who nevertheless need modelling
techniques in their work. The general field of biochemical reaction engineering
is one that requires a very close interdisciplinary interaction between applied
microbiologists, biochemists, biochemical engineers, engineers and managers; a
large degree of collaboration and mutual understanding is therefore important.
Professional microbiologists and biochemists often lack the formal training
needed to analyze laboratory kinetic data in its most meaningful sense, and
they may sometimes experience difficulty in participating in engineering
design decisions and in communicating with engineers. These are just the very
types of activity required in the multi-disciplinary field of biotechnology.
Chemical engineering's greatest strength is its well-developed modelling
concepts, based on mass and energy balances, combined with rate processes.
Biochemical engineering is a discipline closely related to conventional
chemical engineering, in that it attempts to apply physical principles to the
solution of biological problems. This approach may be applied to the
measurement and interpretation of laboratory kinetic data or as well to the
design of large-scale fermentation, enzymatic or waste treatment processes. The
necessary interdisciplinary cooperation requires the biological scientists and
chemical engineers involved to have at least a partial understanding of each
other's field. The purpose of this book is to provide the mathematical tools
necessary for the quantitative analysis of biological kinetics and other
biological process phenomena. More generally, the mathematical modelling
XII
Preface
methods presented here are intended to lead to a greater understanding of how

the biological reaction systems are influenced by process situations.
Engineering science depends heavily on the use of applied theory,
quantitative correlations and mathematics, and it is often difficult for all of us
(not only the biological scientist) to surmount the mathematical barrier, which
is posed by engineering. A mistake, often made, is to confuse "mathematics"
with the engineering modelling approach. In modelling an attempt is made to
analyze a real and possibly very complex situation into a simplified and
understandable physical analog. This physical model may contain many
subsystems, all of which still make physical sense, but which now can be
formulated as mathematical equations. These equations can be handled
automatically by the computer. Thus the engineer and the biologist are freed
from the difficulties of mathematical solution and can tackle complex problems
that were impossible before. Models, however, still have to be formulated and
one of the most important tools of the biochemical engineer, in this operation,
is the use of material balance equations. Though it may not be easy for the
microbiologist to fully appreciate the importance of differential equations, mass
balance equations are not so difficult to understand, since the first law of
conservation, namely that matter can neither be created nor destroyed, is
fundamental to all science. Mass balances, when combined with kinetic rate
equations, to form simple mathematical models, can be used with very great
effect as a means of planning, conducting and analyzing experiments. Models
are especially important as a means of obtaining a better understanding of
process phenomena. A rational approach to experimentation and design
requires a considerable knowledge of the system, which can really only be
achieved by means of a mathematical model. This book attempts to
demonstrate this by way of the many detailed examples.
The contribution made to biotechnology by the biochemical engineering
modelling approach is especially important because the basic procedure can be
developed from a few fundamental principles. An aim of this book is to
demonstrate that you do not have to be an engineer to learn modelling and
simulation. The basic concepts of the material balance, combined with
biological and enzyme kinetics, are easily applied to describe the behavior of
well-stirred tank and tubular fermenters, mixed culture dynamics, interphase
gas-liquid mass transfer and internal biofilm diffusional limitations, as
demonstrated in the computer examples supplied with this book. Such models,
when solved interactively by computer simulation, become much more
understandable to non-engineers.
The Berkeley Madonna simulation language, used for the examples in this
book, is especially suitable because of its sophisticated computing power,
interactive facility and ease of programming. The use of this digital simulation
programming language makes it possible for the reader, student and teacher to
experiment directly with the model, in the classroom or at the desk. In this way
it is possible to immediately determine the influence of changing various
Preface
XIII
operating parameters on the bioreactor performance - a real learning

experience.
The simulation examples serve to enforce the learning process in a very
effective manner and also provide hands-on confidence in the use of a
simulation language. The readers can program their own examples, by
formulating new mass balance equations or by modifying an existing example
to a new set of circumstances. Thus by working directly at the computer, the
no-longer-passive reader is able to experiment directly on the bioreaction
system in a very interactive way by changing parameters and learning about
their probable influence in a real situation. Because of the speed of solution, a
true degree of interaction is possible with Berkeley Madonna, allowing
parameters to be changed easily. Plotting the variables in any configuration is
easy during a run, and the results from multiple runs can be plotted together
for comparison. Other useful features include data fitting and optimization.
In our experience, digital simulation has proven itself to be absolutely the
most effective way of introducing and reinforcing new concepts that involve
multiple interactions. The thinking process is ultimately stimulated to the point
of solid understanding.
Organization of the Book

The book is divided into two parts: a presentation of the background theory in
Part I and the computer simulation exercises in Part II. The function of the text
in Part I is to provide the basic theory required to fully understand and to make
full use of the computer examples and simulation exercises. Numerous case
studies provide illustration to the theory. Part II constitutes the main part of this
book, where the simulation examples provide an excellent instructional and
self-learning tool. Each of the more than fifty examples is self-contained,
including a model description, model equations, exercises, computer program
listing, nomenclature and references. The exercises range from simple
parameter-changing investigations to suggestions for writing a new program.
The combined book thus represents a synthesis of basic theory and computerbased simulation examples.
Quite apart from the educational value of the text, the introduction and use
of the Berkeley Madonna software provides the reader with the considerable
practical advantage of a differential equation solution package. In the appendix
a screenshot guide is found concerning the use of the software.
Part I: "Principles of Bioreactor Modelling" covers the basic theory
necessary for understanding the computer simulation examples. This section
presents the basic concepts of mass balancing, and their combination with
kinetic relationships, to establish simple biological reactor models, carefully
presented in a way that should be understandable to biologists. In fact,
engineers may also find this rigorous presentation of balancing to be valuable.
XIV
Preface
In order to achieve this aim, the main emphasis of the text is placed on an
understanding of the physical meaning and significance of each term in the
model equations. The aim in presenting the relevant theory is thus not to be
exhaustive, but simply to provide a basic introduction to the theory required for
a proper understanding of the modelling methodology.
Chapter 1 deals with the basic concepts of modelling, the basic principles,
development and significance of differential balances and the formulation of
mass and energy balance relationships. Emphasis is given to physical
understanding. The text is accompanied by example cases to illustrate the
application of the material.
Chapter 2 serves to introduce the varied operational characteristics of the
various types of bioreactors and their differing modes of operation, with the
aim of giving a qualitative insight into the quantitative behavior of the
computer simulation examples.
Chapter 3 provides an introduction to enzyme and microbial kinetics. A
particular feature of the kinetic treatment is the emphasis on the use of more
complex structured models. Such models require much more consideration to
be given to the biology of the system during the modelling procedure, but
despite their added complexity can nevertheless also be solved with relative
ease. They serve as a reminder that biological reactions are really infinitely
complex.
Chapter 4 is used to derive general mass balance equations, covering all types
of fermentation tank reactors. These generalized equations are then simplified
to show their application to the differing modes of stirred tank bioreactor
operation, discussed previously and which are illustrated by the simulation
examples.
Chapter 5 explains the basic theory of interfacial mass transfer as applied to
fermentation systems and shows how equations for rates of mass transfer can be
combined with mass balances, for both liquid and gas phases. A particular
extension of this approach is the combination of transfer rate and material
balance equations to models of increased geometrical complexity, as
represented by large-scale air-lift and multiple-impeller fermenters.
Chapter 6 treats the cases of external diffusion to a solid surface and internal
diffusion combined with biochemical reaction, with practical application to
immobilized biocatalyst and biofilm systems. Emphasized here is the
conceptual ease of handling a complex reaction in a solid biocatalyst matrix.
The resulting sets of tractable differential-difference equations are solved by
simulation techniques in several examples.
Chapter 7 describes the importance of control and summarizes control
strategies used for bioreaction processes. Here the fundamentals of feedback
control systems and their characteristic responses are discussed. This material
forms the basis for performing the many recommended control exercises in the
simulation examples. It also will allow the reader-simulator to develop his or
her own control models and simulation programs.
Preface
XV
Part II, "Dynamic Bioprocess Simulation Examples and the Berkeley Madonna
simulation language" comprises Chapter 8, with the computer simulation
examples, and Chapter 9, which gives the instructions for using Madonna, Each
example in Chapter 8 includes a description of its physical system, the model
equations, that were developed in Part I, and a list of suggested exercises. The
programs are found on the CD-ROM. These example exercises can be carried
out in order to explore the model system in detail, and it is suggested that work
on the computer exercises be done in close reference to the model equations
and their physical meaning, as described in the text. The exercises, however, are
provided simply as an idea for what might be done and are by no means
mandatory or restrictive. Working through a particular example will often
suggest an interesting variation, such as a control loop, which can then be
programmed and inserted. The examples cover a wide range of application and
can easily be extended by reference to the literature. They are robust and are
well tested by a variety of undergraduate and graduate students and by also the
350 participants, or so, who have previously attended the Braunwald course. In
tackling the exercises, we hope you will soon come to share our conviction that,
besides being very useful, computer simulation is also fun to do.
For the second edition, the text was thoroughly revised and some of our
earlier, less relevant material was omitted. On the other hand, a number of new
examples resulting mainly from the authors' latest research and teaching work
were added. There was also an opportunity in this new edition to eliminate
most of the past errors and to avoid new ones as much as possible. Most
importantly, the examples have been rewritten in Berkeley Madonna, which all
of our reader-simulators will greatly appreciate.
Our book has a number of special characteristics. It will be obvious, in
reading it through, that we concentrate only on those topics of biological
reaction engineering that lend themselves to modelling and simulation and do
not attempt to cover the area completely. Our own research work is used to
illustrate theoretical points and from it many simulation examples are drawn. A
list of suggested books for supplementary reading is found at the end of
Chapter 6, together with the list of cited references. The diversity of the
simulation examples made it necessary to use separate nomenclature for each.
The symbols used in Chapters 1 - 6 are defined at the end of Part I. The
authors' four nationalities and three mother tongues, made it difficult to settle
on American or British spelling. Somehow we like "modelling" better than
"modeling".
We are confident that the book will be useful to all life scientists wishing to
obtain an understanding of biochemical engineering and also to those chemical
and biochemical engineers wanting to sharpen their modelling skills and
wishing to gain a better understanding of biochemical process phenomena. We
hope that teachers with an interest in modelling will find this to be a useful
textbook for undergraduate and graduate biochemical engineering and
biotechnological courses.
XVI
Preface
Acknowledgements
A major acknowledgement should be made to the excellent pioneering texts of
R. G. E. Franks (1967 and 1972) and also of W. L. Luyben (1973), for
inspiring our interest in digital simulation.
We are especially grateful to our students and to the past-participants of the
Braunwald course, for their assistance in the continuing development of the
course and of the material presented in this book. Continual stimulus and
assistance has also been given by our doctoral candidates, especially at the
Chemical Engineering Department, ETH-Zurich, as noted throughout the
references.
We are grateful to and have great respect for the developers of Berkeley
Madonna and hope that this new version of the book will be useful in drawing
attention to this wonderful simulation language.
Part I
Principles of
Bioreactor Modelling
Biological Reaction Engineering, Second Edition, I. J. Dunn, E. Heinzle, J. Ingham, J. E. Pfenosil

Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-30759-1
Nomenclature for Part I
Symbols
A
A
a
a
b
b
B
c
C
CP
CPR
D
D
d
d
DO
E
E
ES
f
f
F
G
G
hi
H
AH
I
I
j
K
K
KD
Units
Area
Magnitude of controller input signal
Specific area
Constant in Logistic Equation
Constant in Luedeking-Piret relation
Constant in Logistic Equation
Magnitude of controller output signal
Fraction carbon converted to biomass
Concentration
Heat capacity
Carbon dioxide production rate
Diffusivity
Dilution rate
Differential operator and diameter
Fraction carbon converted to product
Dissolved oxygen
Enzyme concentration
Ethanol
Enzyme-substrate concentration
Fraction carbon converted to CO2
Frequency in the ultimate gain method
Flow rate
Gas flow rate
Intracellular storage product
Partial molar enthalpy
Henry's Law constant
Enthalpy change
Inhibiting component concentration
Cell compartment masses
Mass flux
Mass transfer coefficient
Constant in Cohen-Coon method
Acid-base dissociation constant
m2
various
m2/m3
1/h
1/h
m3/kg h
various
kg/m3, kmol/m3
kJ/kg K, kJ/mol K
mol/h
m2/h
1/h
-, m
g/m3, 9 air sat.

g/m3
kg/m3
g/m3
1/h
m3/h and m3/s
m3
kg/m3
kJ/mol
bar m3/kg
kJ/mol or kJ/kg
kg/m3
kg/m3
kg/m2h, mol/m2h
1/h
various
Biological Reaction Engineering, Second Edition. I. J. Dunn, E. Heinzle, J. Ingham, J. E. Pfenosil

ISBN: 3-527-30759-1
Nomenclature
k
Kca
kGa
KI
KLa
kLa
KM
Kp
KW
KS
L
M
m
N
N
n
n
OTR
OUR
P
P
P
R
R
R
r
fi
r
i/j
RQ
rx
S
S
s
T
T
T
L
t
Tr
U
V
V
v
max
Constant
Gas-liquid mass transfer coefficient
Gas film mass transfer coefficient
Inhibition constant
Gas-liquid mass transfer coefficient
Liquid film mass transfer coefficient
Michaelis-Menten constant
Proportional controller gain constant
Dissociation constant of water
Monod saturation coefficient
Length
Mass
Maintenance coefficient
Mass flux
Molar flow rate
Number of mols
Reaction order
Oxygen transfer rate
Oxygen uptake rate
Pressure
Product concentration
Output control signal
Total transfer rate
Specific rate
Ideal gas constant
Recycle flow rate
Residual active biomass
Reaction rate
Reaction rate of component i
Reaction rate of component i to j
Respiration quotient
Growth rate
Concentration of substrate
Slope of process reaction curve
Stoichiometric coefficient
Temperature
Enzyme activity
Time lag
Time
Transfer rate
Heat transfer coefficient
Volume
Flow velocity
Maximum reaction rate
Wastage stream flow rate
various
1/h
1/h
kg/m3, kmol/m3
1/h
1/h
kg/m3, kmol/m3
various
kg/m3
m
kg or mol
1/h
kg/m2 h
mol/h
_
mol/h and kg/h
mol/h and kg/h
bar
kg/m3 and g/m3
various
kg/h and mol/h
kg/kg biomass h
bar m3/ K mol
m3/h
kg/m3
kg/m3h, kmol/m3h
kg i/m3h
kg /m3h, kmol/m3h
mol CO2/mol 2
kg biomass/m3 h
kg/m3, kmol/m3
various
CorK
kg/m3
h, min. or s
h, min and s
mol/m3 h
kJ/m2 C h
m3
m/h
kmol/m3 h
m3/h
Nomenclature
w
X
Y
Yi
y
Z
Mass fraction
Biomass concentration
Yield coefficient
Yield of i from j
Mol fraction in gas
Length variable
kg/m3
kg/kg
kg i/kg j,mol i/mol j
m
Greek
8
a
8
A
O
Z
T
T
T
Controller error
Partial differential operator
Concentration difference quantity
Difference operator
Thiele Modulus
Effectiveness factor
Specific growth rate
Maximum growth rate
Stoichiometric coefficient
Density
Summation operator
Residence time
Controller time constant
Electrode time constant
various
kg/m3
1/h
1/h
kg/m3
h and s
s
s
Indices
*
Refers to equilibrium concentration

Refers to initial, inlet, external, and zero order
1
Refers to time ti, outlet, component 1, tank 1, and first order
2
Refers to tank 2, time t2 and component 2
1,2,..., n
Refers to stream, volume elements and stages
A
Refers to component A, anions and bulk
ARefers to anions
Refers to ambient
a
Ac
Refers to acetoin and acetoin formation
aer
Refers to aerobic
agit
Refers to agitation
anaer
Refers to anaerobic
Refers to apparent
app
ATP/S,Ac Refers to ATP yield from reaction glucose --> Ac
ATP/S,CO2 Refers to ATP yield from glucose oxidation
ATP/NADH Refers to ATP produced from NADH
ATP/X
Refers to consumption rate ATP > biomass
Nomenclature
avg
B
Bu
CO2
d
D
D
E
E
f
G
H+
i
I
I
inert
K
K+
L
m
m
max
n
NH4
NC>2
NOs
O and O2
P
PA
PB
Q
Q/O2
Q/S
R
r
r,S
s
S
SL
Sn
tot
X
X/i
X/S
Refers to average
Refers to component B, base, backmixing, and surface position
Refers to butanediol
Refers to carbon dioxide
Refers to deactivation and death
Refers to derivative control
Refers to D-value in sterilization
Refers to electrode
Refers to energy by complete oxidation
Refers to final
Refers to gas and to cellular compartment
Refers to hydrogen ions
Refers to component i and to interface
Refers to inhibitor
Refers to integral control
Refers to inert component
Refers to cellular compartment
Refers to cations
Refers to liquid
Refers to maximum
Refers to metabolite
Refers to maximum
Refers to tank number
Refers to ammonium
Refers to nitrite
Refers to nitrate
Refer to oxygen
Refers to product
Refers to product A
Refers to product B
Refers to heat
Refers to heat-oxygen ratio
Refers to heat-substrate ratio
Refers to recycle stream
Refers to reactor
Refers to reaction of substrate
Refers to settler
Refers to substrate and surface
Refers to liquid film at solid interface
Refers to substrate n
Refers to total
Refers to biomass
Refers to biomass-component i ratio
Refers to biomass-substrate ratio
Nomenclature
Refers to difference between cations and ions

Bar above symbol refers to dimensionless variable
Modelling Principles
1.1
Fundamentals of Modelling
1.1.1
Use of Models for Understanding, Design and

Optimization of Bioreactors
An investigation of bioreactor performance might conventionally be carried

out in an almost entirely empirical manner. In this approach, the bioreactor
behavior would be studied under practically all combinations of possible
conditions of operation and the results then expressed as a series of
correlations, from which the resulting performance might hopefully be
estimated for any given set of new operating conditions. This empirical
procedure can be carried out in a very routine way and requires relatively little
thought concerning the actual detail of the process. While this might seem to
be rather convenient, the procedure has actually many disadvantages, since very
little real understanding of the process would be obtained. Also very many
experiments would be required in order to obtain correlations that would cover
every process eventuality.
Compared to this, the modelling approach attempts to describe both actual
and probable bioreactor performance, by means of well-established theory,
which when described in mathematical terms, represents a working model for
the process. In carrying out a modelling exercise, the modeller is forced to
consider the nature of all the important parameters of the process, their effect
on the process and how each parameter can be defined in quantitative terms,
i.e., the modeller must identify the important variables and their separate
effects, which, in practice, may have a very highly interactive combined effect
on the overall process. Thus the very act of modelling is one that forces a
better understanding of the process, since all the relevant theory must be
critically assessed. In addition, the task of formulating theory into terms of
mathematical equations is also a very positive factor that forces a clear
formulation of basic concepts.
Once formulated, the model can be solved and the behavior predicted by the
model compared with experimental data. Any differences in performance may
ISBN: 3-527-30759-1
10
1 Modelling Principles
then be used to further redefine or refine the model until good agreement is
obtained. Once the model is established it can then be used, with reasonable
confidence, to predict performance under differing process conditions, and it
can also be used for such purposes as process design, optimization and control.
An input of plant or experimental data is, of course, required in order to
establish or validate the model, but the quantity of experimental data required,
as compared to that of the empirical approach is considerably reduced. Apart
from this, the major advantage obtained, however, is the increased
understanding of the process that one obtains simply by carrying out the
modelling exercise.
These ideas are summarized below.
Empirical Approach: Measure productivity for all combinations of reactor
operating conditions, and make correlations.
- Advantage:
Little thought is necessary
- Disadvantage: Many experiments are required.
Modelling Approach: Establish a model, and design experiments to determine
the model parameters. Compare the model behavior with the experimental
measurements. Use the model for rational design, control and optimization.
- Advantage:
Fewer experiments are required, and greater understanding is
obtained.
- Disadvantage: Some strenuous thinking may be necessary.
1.1.2
General Aspects of the Modelling Approach
An essential stage in the development of any model, is the formulation of the

appropriate mass and energy balance equations (Russell and Denn, 1972). To
these must be added appropriate kinetic equations for rates of cell growth,
substrate consumption and product formation, equations representing rates of
heat and mass transfer and equations representing system property changes,
equilibrium relationships, and process control (Blanch and Dunn, 1973). The
combination of these relationships provides a basis for the quantitative
description of the process and comprises the basic mathematical model. The
resulting model can range from a very simple case of relatively few equations
to models of very great complexity.
Simple models are often very useful, since they can be used to determine the
numerical values for many important process parameters. For example, a
model based on a simple Monod kinetics can be used to determine basic
parameter values such as the specific growth rate (JLI), saturation constant (Ks),
biomass yield coefficient (Yx/s) and maintenance coefficient (m). This basic
kinetic data can be supplemented by additional kinetic factors, such as oxygen
transfer rate (OTR), carbon dioxide production rate (CPR), respiration quotient
(RQ) based on off-gas analysis and related quantities, such as specific oxygen
1.1 Fundamentals of Modelling
11
uptake rate (qo2)> specific carbon dioxide production rate (qco2)> may also be
derived and used to provide a complete kinetic description of, say, a simple
batch fermentation.
For complex fermentations, involving product formation, the specific
product production rate (qp) is often correlated as a complex function of
fermentation conditions, e.g., stirrer speed, air flow rate, pH, dissolved oxygen
content and substrate concentration. In other cases, simple kinetic models can
also be used to describe the functional dependence of productivity on cell
density and cell growth rate.
A more detailed "structured kinetic model" may be required to give an
adequate description of the process, since cell composition may change in
response to changes in the local environment within the bioreactor. The greater
the complexity of the model, however, the greater is then the difficulty in
identifying the numerical values for the increased number of model parameters,
and one of the skills of modelling is to derive the simplest possible model that
is capable of a realistic representation of the process.
A basic use of a process model is thus to analyze experimental data and to
use this to characterize the process, by assigning numerical values to the
important process variables. The model can then also be solved with
appropriate numerical data values and the model predictions compared with
actual practical results. This procedure is known as simulation and may be
used to confirm that the model and the appropriate parameter values are
"correct". Simulations, however, can also be used in a predictive manner to test
probable behavior under varying conditions; this leads on to the use of models
for process optimization and their use in advanced control strategies.
The application of a combined modelling and simulation approach leads to
the following advantages:
1. Modelling improves understanding, and it is through understanding that
progress is made. In formulating a mathematical model, the modeller is
forced to consider the complex cause-and-effect sequences of the
process in detail, together with all the complex inter-relationships that
may be involved in the process. The comparison of a model prediction
with actual behavior usually leads to an increased understanding of the
process, simply by having to consider the ways in which the model might
be in error. The results of a simulation can also often suggest reasons as
to why certain observed, and apparently inexplicable, phenomena occur
in practice.
2. Models help in experimental design. It is important that experiments be
designed in such a way that the model can be properly tested. Often the
model itself will suggest the need for data for certain parameters, which
might otherwise be neglected, and hence the need for a particular type of
experiment to provide the required data. Conversely, sensitivity tests on
the model may indicate that certain parameters may have a negligible
12
effect and hence that these effects therefore can be neglected both from
the model and from the experimental program.
3.
Models may be used predictively for design and control. Once the
model has been established, it should be capable of predicting
performance under differing sets of process conditions. Mathematical
models can also be used for the design of relatively sophisticated control
algorithms, and the model, itself, can often form an integral part of the
control algorithm. Both mathematical and knowledge based models can
be used in designing and optimizing new processes.
4.
Models may be used in training and education. Many important aspects

of bioreactor operation can be simulated by the use of very simple
models. These include such concepts as linear growth, double substrate
limitation, changeover from batch to fed-batch operation dynamics, fedbatch feeding strategies, aeration dynamics, measurement probe
dynamics, cell retention systems, microbial interactions, biofilm diffusion
and bioreactor control. Such effects are very easily demonstrated by
computer, as shown in the accompanying simulation examples, but are
often difficult and expensive to demonstrate in practice.
5. Models may be used for process optimization. Optimization usually

involves considering the influence of two or more variables, often one
directly related to profits and one related to costs. For example, the
objective might be to run a reactor to produce product at a maximum
rate, while leaving a minimum amount of unreacted substrate.
1.1.3
General Modelling Procedure
One of the more important features of modelling is the frequent need to

reassess both the basic theory (physical model) and the mathematical equations,
representing the physical model (mathematical model) in order to achieve the
required degree of agreement, between the model prediction and actual plant
performance (experimental data).
As shown in Fig. 1.1, the following stages in the modelling procedure can be
identified:
(i) The first stage involves the proper definition of the problem and hence the
goals and objectives of the study. These may include process analysis,
improvement, optimization, design and control, and it is important that the aims
of the modelling procedure are properly defined. All the relevant theory must
then be assessed in combination with any practical experience with the process,
13
Physical Model
LJt
Revise ideas
and equations
New
experiments
Mathematical Model
Solution: C = f(t)
Experimental Data
NO
Comparison
OK?
YES
Use for design,

optimization and control
Figure 1.1. Information flow diagram for model building.
and perhaps alternative physical models for the process need to be developed
and examined. At this stage, it is often helpful to start with the simplest possible
conception of the process and to introduce complexities as the development
proceeds, rather than trying to formulate the full model with all its complexities
at the beginning of the modelling procedure.
(ii) The available theory must then be formulated in mathematical terms.
Most bioreactor operations involve quite a large number of variables (cell,
substrate and product concentrations, rates of growth, consumption and
production) and many of these vary as functions of time (batch, fed-batch
operation). For these reasons the resulting mathematical relationships often
consist of quite large sets of differential equations. The thick arrow in Fig. 1.1
designates both the importance and the difficulty of this mathematical
formulation.
(iii) Having developed a model, the model equations must then be solved.
Mathematical models of biological systems are usually quite complex and
highly non-linear and are such that the mathematical complexity of the
14
equations is usually sufficient to prohibit the use of an analytical means of

solution. Numerical methods of solution must therefore be employed, with the
method preferred in this text being that of digital simulation. With this
method, the solution of very complex models is accomplished with relative ease,
since digital simulation provides a very easy and a very direct method of
solution.
Digital simulation languages are designed specially for the solution of sets
of simultaneous differential equations using numerical integration. Many fast
and efficient numerical integration routines are now available and are
implemented within the structure of the languages, such that many digital
simulation languages are able to offer a choice of integration routine. Sorting
algorithms within the structure of the language enable very simple programs to
be written, having an almost one-to-one correspondence with the way in which
the basic model equations were originally formulated. The resulting simulation
programs are therefore very easy to understand and also to write. A further
major advantage is a convenient output of results, in both tabulated and
graphical form, that can be obtained via very simple program commands.
(iv) The validity of the computer prediction must be checked and steps (i) to
(iii) will often need to be revised at frequent intervals during the modelling
procedure. The validity of the model depends on the correct choice of the
available theory (physical and mathematical model), the ability to identify the
model parameters correctly and the accuracy of the numerical solution method.
In many cases, owing to the complexity and very interactive nature of
biological processes, the system will not be fully understood, thus leaving large
areas of uncertainty in the model. Also the relevant theory may be very
difficult to apply. In such cases, it is then often very necessary to make rather
gross simplifying assumptions, which may subsequently be eliminated or
improved as a better understanding is subsequently obtained. Care and
judgement must also be used such that the model does not become over
complex and so that it is not defined in terms of too many immeasurable
parameters. Often a lack of agreement between the model and practice can be
caused by an incorrect choice of parameter values. This can even lead to quite
different trends being observed in the variation of particular parameters during
the simulation.
It should be noted, however, that often the results of a simulation model do
not have to give an exact fit to the experimental data, and often it is sufficient to
simply have a qualitative agreement. Thus a very useful qualitative
understanding of the process and its natural cause-and-effect relationships is
obtained.
15
1.1.4 Simulation Tools

Many different digital simulation software packages are available on the market
for PC and Mac application. Modern tools are numerically powerful, highly
interactive and allow sophisticated types of graphical and numerical output.
Most packages also allow optimisation and parameter estimation. BERKELEY
MADONNA is very user-friendly and very fast. We have chosen it for use in
this book, and details can be found in the Appendix. With it data fitting and
optimisation can be done very easily. MODELMAKER is also a more recent,
powerful and easy to use program, which also allows optimisation and
parameter estimation.
ACSL-OPTIMIZE has quite a long history of
application in the control field, and also for chemical reaction engineering.
MATLAB-SIMULINK is a popular and powerful software for dynamic
simulation and includes many powerful algorithms for non-linear optimisation,
which can also be applied for parameter estimation.
1.1.5
Teaching Applications
For effective teaching, the introduction of computer simulation methods into

modelling courses can be achieved in various ways, and the method chosen will
depend largely on how much time can be devoted, both inside and outside the
classroom. The most time-consuming method for the student is to assign
modelling problems to be solved outside the classroom on any available
computer. If scheduling time allows, computer laboratory sessions are
effective, with the student working either alone or in groups of up to three on
each monitor or computer. This requires the availability of many computers,
but has the advantage that pre-programmed examples, as found in this text, can
be used to emphasize particular points related to a previous theoretical
presentation. This method has been found to be particularly effective when
used for short, continuing-education, professional courses. By use of the
computer examples the student may vary parameters interactively and make
program alterations, as well as working through the suggested exercises at his or
her own pace. Demonstration of a particular simulation problem via a single
personal computer and video projector is also an effective way of conveying
the basic ideas in a short period of time, since students can still be very active in
suggesting parametric changes and in anticipating the results. The best
approach is probably to combine all three methods.
16
1.2
Development and Meaning of Dynamic

Differential Balances
As indicated in Section 1.1, many models for biological systems are expressed
in terms of sets of differential equations, which arise mainly as a result of the
predominantly time-dependent nature of the process phenomena concerned.
For many people and especially for many students in the life sciences, the
mention of differential equations can cause substantial difficulty. This section
is therefore intended, hopefully, to bring the question of differential equations
into perspective. The differential equations arise in the model formulation,
simply by having to express rates of change of material, due to flow effects or
chemical and biological reaction effects. The method for solution of the
differential equations will be handled automatically by the computer. It is
hoped that much of the difficulty can be overcome by considering the
following case. In this section a simple example, based on the filling of a tank
of water, is used to develop the derivation of a mass balance equation from the
basic physical model and thereby to give meaning to the terms in the equations.
Following the detailed derivation, a short-cut method based on rates is given to
derive the dynamic balance equations.
Consider a tank into which water is flowing at a constant rate F (m3/s), as
shown in Fig. 1.2. At any time t, the volume of water in the tank is V (m3) and
the density of water is p (kg/m3).
Figure 1.2. Tank of water being filled by stream with flow rate F.
During the time interval At (s), a mass of water p F At (kg) flows into the tank.
As long as no water leaves the tank, the mass of water in the tank will increase
by a quantity p F At, causing a corresponding increase in volume, AV.
Equating the accumulation of mass in the tank to the mass that entered the tank
during the time interval A t gives,
pAV = p F A t
Since p is constant,
At
-F
h
-
1.2 Development and Meaning of Dynamic Differential Equations
17
Applying this to very small differential time intervals (At > dt) and replacing
the A signs by the differential operator "d", gives the following simple first
order differential equation, to describe the tank filling operation,
dV
dT = F
What do we know about the solution of this equation? That is, how does the
volume change with time or in model terms, how does the dependent variable,
V, change with respect to the independent variable, t? To answer this, we can
rearrange the equation and integrate it between appropriate limits to give,
or for constant F,
= F Jtf l l dt= F(ti-to)

o
Integration is equivalent to summing all the contributions, such that the total
change of volume is equal to the total volume of water added to the tank,
IV = IF At
For the case of constant F, it is clear that the analytical solution to the
differential equation is,
V = F t + constant
In this case, as shown in Fig. 1.3, the constant of integration is the initial volume
of water in the tank, VQ, at time t = 0.
dt
Vo
Figure 1.3. Volume change with time for constant flow rate.
18
Note that the slope in the variation of V with respect to t, dV/dt, is constant, and
that from the differential equation it can be seen that the slope is equal to F.
Suppose F is not constant but varies linearly with time.
F = Fo-kt
The above model equation applies also to this situation.
Solving the model equation to obtain the functional dependence of V with
respect to t,
JdV = |F dt = J(F0 - k t)dt = FO Jdt - k Jt dt
Integrating analytically,
V = FO t -
kt"
+ constant
The solution is,
v = FO t - kt'
+ V0
."t
Figure 1.4. Variation of F and V for the tank-filling problem.
Note that the dependent variable starts at the initial condition, (Vo), and that the
slope is always F. When F becomes zero, the slope of the curve relating V and t
also becomes zero. In other words, the volume in the tank remains constant
and does not change any further as long as the value of F remains zero.
Derivation of a Balance Equation Using Rates
A differential balance can best be derived directly in terms of rates of change.
For the above example, the balance can then be expressed as:
/The rate of accumulation^
V of mass within the tank )
/The flow rate of mass^

\ entering the tank )
1.2 Development and Meaning of Dynamic Differential Equations
19
Thus, the rate of accumulation of mass within the tank can be written directly as
dM/dt where the mass M is equal to p V. The rate of mass entering the tank is
given by p F, where both sides of the equation have units of kg/h.
=
PF
and
d(oV)
= pF
Thus this approach leads directly to a differential equation model, which is the
desired form for dynamic simulation. Note that both terms in the above
relationship are expressed in mass quantities per unit time or kg/h.
At constant density, the equation again reduces to,
dV
dT = F
which is to be solved for the initial condition, that at time t = 0, V=Vo and for a
variation in flow rate, given by,
F = F0-kt
which is valid until F = 0.
These two equations, plus the initial condition, form the mathematical
representation or the mathematical model of the physical model, represented
by the tank filling with an entering flow of water. Thus this approach leads
directly to a differential equation model, which is the desired form for
simulation. This approach can be applied not only to the total mass but also to
the mass of any component.
We have seen an analytical solution to this model, but it is also interesting to
consider how a computer solution can be obtained by a numerical integration
of the model equations. This is important since analytical integration is seldom
possible in the case of real complex problems.
Computer Solution
The numerical integration can in principle be performed using the relations:
dV
20
where t - to represents a very small time interval and V - VQ is the resulting

change in volume of the water in the tank. As before, the flow is assumed to
decrease with time according to F = FQ - k t.
This integration procedure is equivalent to the following steps:
1) Setting the integration time interval.
2) Assigning a value to the inlet water flow rate at the initial value, time
t = to3) The term involving the water flow rate, F-kt, is equal to the derivative
value, (dV/dt), at time t = to.
4) Knowing the initial value of V and the slope dV/dt, enables a new value of
V to be calculated over the small interval of time, equivalent to the
integration time interval or integration step length.
5) At the end of the integration time interval, the value of V will have
changed to a new value, representing the change of V with respect to time
from its original value. The new value of V can thus be calculated.
6) Using the new value of V, a new value for the rate of change of V with
respect to time, (dV/dt), at the end of the integration time interval can
now be calculated.
7) Knowing the value of V and the value of dV/dt at the end of the
integration time interval, a new value of V can be estimated over a further
step forward in time or integration time interval.
8) The entire procedure, as represented by steps (2) to (7) in Fig. 1.5 below,
is then repeated with the calculation moving forward with respect to time,
until the value of F reaches zero. At this point the volume no longer
increases, and the resulting steady-state value of V is obtained, including
all the intermediate values of V and F, which were determined during the
course of the calculation.
Figure 1.5. Graphical portrayal of numerical integration, showing slopes and approximated
values of V at each time interval.
1.3 Formulation of Balance Equations
21
Using such a numerical integration procedure, the computer can thus be used
to generate data concerning the time variations of both F and V. In practice,
more complex numerical procedures are employed in digital simulation
languages to give improved accuracy and speed of solution than illustrated by
the above simplified integration technique.
1.3
Formulation of Balance Equations
1.3.1
Types of Mass Balance Equations
Steady-State Balances
One of the basic principles of modelling is that of the conservation of mass,
which for a steady-state flow process can be expressed by the statement,
Rate of mass flow^

into the system J
f Rate of mass flow^j

^ out of the system J
Dynamic Total Mass Balances

Many bioreactor applications are, however, such that conditions are in fact
changing with respect to time. Under these circumstances, a steady-state mass
balance is inappropriate and must be replaced by a dynamic or unsteady-state
mass balance, which can be expressed as:
Rate of accumulation of ^
mass in the system J
( Rate of ^
^mass flow inj
( Rate of ^
^mass flow out J
Here the rate of accumulation term represents the rate of change in the total
mass of the system, with respect to time, and at steady-state this is equal to zero.
Thus the steady-state mass balance represented earlier is seen to be a
simplification of the more general dynamic balance, involving the rate of
accumulation.
At steady-state:
22
(
Rate of
^
= 0 = (Mass flow in) - (Mass flow out)
I accumulation of mass .
hence, when a steady-state is reached:
(Mass flow in) = (Mass flow out)
Component Balances
The previous discussion has been in terms of the total mass of the system, but
most fluid streams, encountered in practice, contain more than one chemical or
biological species. Provided no chemical change occurs, the generalized
dynamic equation for the conservation of mass can also be applied to each
component. Thus for any particular component:
Rate of
accumulation of mass
of component
in the system
Mass flow of A
the component _
into the system J
Mass flow of >

the component out
of the system ,
Component Balances with Reaction

Where chemical or biological reactions occur, this can be taken into account by
the addition of a further reaction rate term into the generalized component
balance. Thus in the case of material produced by the reaction:
' Mass flow ^
^ Mass flow ^
Rate of ^
accumulation
of the
of the
=
of mass
component - component +
of component
into
out of
^the system,
^the system,
^ in the system,
'
Rate of
production
of the
component
by the reaction>
and in the case of material consumed by the reaction:

Rate of '
accumulation
of mass
=
of component
în the system,
Mass flow
' Mass flow
of the
of the
component - component into
out of
^the system^
v the system,
Rate of
consumption
of the
component
by the reaction
23
Elemental Balances
The principle of the mass balance can also be extended to the atomic level and
applied to particular elements. Thus in the case of bioreactor operation, the
general mass balance equation can also be applied to the four main elements,
carbon, hydrogen, oxygen and nitrogen and also to other elements if relevant
to the particular problem. Thus for the case of carbon:
' Rate of accumulation^! (Mass flowrate of "\ ( Mass flow rate of \
of carbon in
=
carbon into
the carbon out
the system
J ^ the system J V of the system )
Note the elemental balances do not involve reaction terms since the elements do
not change by reaction.
The computer example PENFERM, is based on the use of elemental mass
balance equations for C, H, O and N which, when combined with other
empirical rate data, provide a working model for a penicillin production
process.
While the principle of the mass balance is very simple, its application can
often be quite difficult. It is important therefore to have a clear understanding
of both the nature of the system (physical model), which is to be modelled by
means of the mass balance equations, and also of the methodology of
modelling.
1.3.2
Balancing Procedure
The methodology described below outlines six steps, I through VI, to establish
the model balances. The first task is to define the system by choosing the
balance or control region. This is done using the following procedure:
I.
Choose the balance region such that the variables are

constant or change little within the system. Draw
boundaries around the balance region
The balance region may be a reactor, a reactor region, a single phase within a
reactor, a single cell, or a region within a cell, but will always be based on a
region of assumed constant composition. Generally the modelling exercises will
involve some prior simplification. Often the system being modelled is usually
considered to be composed of either systems of tanks (stagewise or lumped
24
parameter systems) or systems of tubes (differential systems), or even

combinations of tanks and tubes, as used in Case C, Sec. 1.3.2.3.
1.3.2.1
Case A. Continuous Stirred Tank Bioreactor
AO
Total mass = pV
Mass of A = C VA
Balance region
Figure 1.6. The balance region around the continuous reactor.
If the tank is well-mixed, the concentrations and density of the tank contents
are uniform throughout. This means that the outlet stream properties are
identical with the tank properties, in this case CA and p. The balance region can
therefore be taken around the whole tank.
The total mass in the system is given by the product of the volume of the
tank contents V (m3) multiplied by the density p (kg/m3), thus Vp (kg). The
mass of any component A in the tank is given as the product of V times the
concentration of A, CA (kg of A/m3 or kmol of A /m3), thus V CA (kg or kmol).
1.3.2.2
Case B. Tubular Reactor

Balance region
'AO
A1
Figure 1.7. The tubular reactor concentration gradients.
In the case of tubular reactors, the concentrations of the products and reactants
will vary continuously along the length of the reactor, even when the reactor is
operating at steady-state. This variation can be regarded as being equivalent to
25
that of the time of passage of material as it flows along the reactor and is
equivalent to the time available for reaction to occur. Under steady-state
conditions the concentration at any position along the reactor will be constant
with respect to time, though not with position. This type of behavior can be
approximated by choosing the balance regions sufficiently small so that the
concentration of any component within a region can be assumed to be
approximately uniform. Thus in this case, many uniform property subsystems
(well-stirred tanks or increments of different volume but of uniform
concentration) comprise the total reactor volume.
13.2.3
Case C. River with Eddy Current
For this example, the combined principles of both the stirred tank and
differential tubular modelling approaches need to be applied. As shown in Fig.
1.8 the main flow along the river is very analogous to that of a column or
tubular process, whereas the eddy region can be approximated by the behavior
of a well-mixed tank. The interaction between the main flow of the river and
the eddy, with flow into the eddy from the river and flow out from the eddy
back into the river's main flow, must be included in any realistic model.
The real-life and rather complex behavior of the eddying flow of the river,
might thus be represented, by a series of many well-mixed subsystems (or
tanks) representing the main flow of the river. This interacts at some particular
stage of the river with a single well-mixed tank, representing the turbulent eddy.
In modelling this system by means of mass balance equations, it would be
necessary to draw boundary regions around each of the individual subsystems
representing the main river flow, sections 1 to 8 in Fig. 1.9, and also around the
tank system representing the eddy. This would lead to a very minimum of nine
River
Eddy
Figure 1.8. A complex river flow system.
26
component balance equations being required. The resulting model could be

used, for example, to describe the flow of a pollutant down the river in rather
simple terms.
1
>
',
i
2
2
',
i
3
3
4
4
,
i
>k
V
5
5
,
i
66
,
i
7
7
,
i
>
8
8
fc
Flow interaction
fitSliM^B
:i
iK&^'ffK<&y^
Figure 1.9. A multi-tank model for the complex river flow system.
//.
Identify
system
the transport streams which flow across the

boundaries
Having defined the balance regions, the next task is to identify all the relevant
inputs and outputs to the system. These may be well-defined physical flow
rates (convective streams), diffusive fluxes, and also interphase transfer rates.
It is important to assume a direction of transfer and to specify this by means of
an arrow. This direction might reverse itself, but will be accomodated by a
reversal in sign.
Out by diffusion
Convective
flow out
Convective flow
in
In by diffusion
Figure 1.10. Balance region showing convective and diffusive flows in and out.
///.
Write the mass balance in word form
This is an important step because it helps to ensure that the resulting

mathematical equation will have an understandable physical meaning. Just
27
starting off by writing down equations is often liable to lead to fundamental

errors, at least on the part of the beginner. All balance equations have a basic
logic as expressed by the generalized statement of the component balance
given below, and it is very important that the mathematical equations should
retain this. Thus:
' Rate of
accumulation
of mass
of component
the system )
f Mass flow ^\
of the
component
into
\the system^
f Mass flow ^
of the
component
out of
^the system^
Rate of
production
of the
component by
\ the reaction /
This can be abbreviated as,

(Accumulation)
(In) -
(Out) + (Production)
IV. Express each balance term in mathematical form with

measurable variables
A. Rate of Accumulation Term
This is given by the derivative of the mass of the system, or the mass of some
component within the system, with respect to time. Hence:
(Rate of accumulation of mass of component i within the system) =
dMi
where M is in kg or mol and time is in h, min or s.

Volume, concentration and, in the case of gaseous systems, partial pressure
are usually the measured variables. Thus for any component i
dMj
dt
_ d(CjV)
dt
where, Q is the concentration of i (kmol/m3 or kg/m3), and pi is the partial

pressure of i within the gas phase system. In the case of gases, the Ideal Gas
Law can be used to relate concentration to partial pressure and mol fraction.
Thus,
piV= niRT
where R is in units compatible with p, V, n and T.
In terms of concentration,
28
_ = ni
c
i T
Pi
yiP
RT = "RT
where yi is the mol fraction of the component in the gas phase and p is the total
pressure.
The accumulation term for the gas phase can be written as,
/piV
dMj _ d(CjV) _ d(QV) _ . d
_
For the total mass of the system:
dM _ d(p V)
dt = dt
with units
m3
B. Convective Flow Terms

Total mass flow rates are given by the product of volumetric flow multiplied by
density. Component mass flows are given by the product of volumetric flow
rates times concentration.
Mass \
(VolumeJ
kg
s
m 3 kg
- s m3
Total mass flow = F p

Component mass flow MI = F Q
A stream leaving a well-mixed region, such as a well stirred tank, has the same
properties as the system volume as a whole, since for perfect mixing the
contents of the tank will have uniform properties, identical to the properties of
the fluid leaving at the outlet. Thus, the concentrations of component i both
within the tank and in the tank effluent are equal to Qi, as shown in Fig. 1.11.
29
lilf
Figure 1.11. Convective flow terms for a well-mixed tank bioreactor.
C. Diffusion of Components
As shown in Fig. 1.12, diffusional flow contributions can be expressed by
analogy to Pick's Law for molecular diffusion
Ji = -i dZ
where jt is the flux of any component i flowing across an interface (kmol/m2 h

or kg/m2 h) and dQ/dZ (kmol/m) is the concentration gradient as shown in
Fig. 1.12.
Figure 1.12. Diffusion flux j j driven by concentration gradient (Qo - Cji) / AZ through
surface area A.
In accordance with Pick's Law, diffusive flow always occurs in the direction of
decreasing concentration and at a rate proportional to the concentration
gradient. Under true conditions of molecular diffusion, the constant of
proportionality is equal to the molecular diffusivity for the system, Dj (m2/h).
For other cases, such as diffusion in porous matrices and turbulent diffusion, an
30
effective diffusivity value is used, which must be determined experimentally.

The concentration gradient may have to be approximated in finite difference
terms (Finite differencing techniques are described in more detail in Sec. 6.2).
Calculating the mass rate requires the area, through which diffusive transfer
occurs.
( Massrate
of
(^component i
( Diffusivity YConcentrationY
Area
^
(
of
gradient
perpendicular =-DJ
(^component ij\^
ofi
J^ to transport J
kg
sm 2
2
m
_ m 2 kg
2 _ kg
" s m4 m " T
D. Interphase Transport
Interphase mass transport also represents a possible flow into or out of the
system.
In bioreactor modelling applications, this is most frequently
represented by the case of oxygen transfer from air to the liquid medium,
followed by oxygen taken up by the cells during respiration. In this case, the
transfer of oxygen occurs across the gas liquid interface, which exists between
the surface of the air bubbles and the surrounding liquid medium, as shown in
Fig. 1.13.
Figure 1.13. Transfer of oxygen across a gas-liquid interface of specific area "a" into a liquid
phase of volume V.
Other applications may involve the supply of oxygen to the bioreactor by

transfer from the air, across a membrane and then into the bulk liquid. Where
there is interfacial transfer from one phase to another, the component balance
equations will need appropriate modification to take this into account. Thus, an
oxygen balance for the well-mixed gas phase, with transfer from the gas to the
liquid, can be written as,
31
Rate of
\
Rate
\
/Mass flow\ /Massflow\ / of interfacial
accumulation
of the
of the
of the
mass transfer
oxygen
oxygen
=
- from the gas
mass of oxygen
into the
from the
in the gas
phase into
Vgas
phase
)
^
gas phase> V the liquid s
V phase system J
This form of transfer rate equation will be examined in much more detail in
Chapter 5. Suffice it to say here that the rate of transfer can be expressed in the
form shown below:
Rate Of \ ( Mass ^ Ârea peA /Concentration^ /SystemA
Uass transferJ= tra"sPrtf ^ volume ) ^driving force ) VvolumeJ
V coefficient.
f
= KaACV
where, a is a specific area for mass transfer, A/V (m2/m3), A is the total
interfacial area for mass transfer (m2), V is the liquid phase volume (m3), AC is
the concentration driving force (kmol/m3 or kg/m3) and, K is the overall mass
transfer coefficient (1/s). Mass transfer rate expressions are usually expressed
in terms of kmol/s, and can be converted to mass flows (kg/s), if desired.
The units of the terms in the equation (with appropriate mass quantity units)
are:
kg
1 kg
mj
Production Rate
This term in the component balance equation allows for the production or
consumption of material bv
by reaction and is incorporated into the component
balance equation. Thus,
Rate of >\
accumulation
of mass
of component
the system )
/ Massflow\
of the
component
into
\the system /
/ Massflow\
of the
component
out of
the system /
Rate of
production
of the
component by
v the reaction /
Chemical production rates are often expressed on a molar basis and, as in the
case of the interfacial mass transfer rate expressions, can be easily converted to
mass flow quantities (kg/s). The production rate can then be expressed as
32
f Mass rate \
production of
^component Ay
/^Reaction rate\
= rA V = ^ per volume ) (Volume of system)
kg
__
kg
m3
s m3
Equivalent molar quantities may also be used. The quantity r^ is positive when
A is formed as product, and TA is negative when a reactant A is consumed.
The growth rate for cells can be expressed in the same manner, using the
symbol rx- Thus,
/ Mass rate of
^
Vbiomass production^
/^Growth rate^
= rx V = ^per vo lume) (Volume of system)
kg
kg
mj
s m3
The consumption rate of substrate, r$, is often directly related to the cell growth
rate by means of a constant yield coefficient YX/S, which has the units of kg
biomass produced per kg substrate consumed. Thus,
( Ma< rate \
U>nsumptionJ
kg
s m3
V.
growth rate V
1
\
er
M> volume ABiomass-substrate yield) (Volume)
kg biomass kg substrate
kg biomass
s m3
Introduce other relationships and balances such that the

number of equations equals the number of dependent
variables
The system mass balance equations are often the most important elements of
any modelling exercise, but are themselves rarely sufficient to completely
formulate the model. Other relationships are therefore needed to supplement
the material balance relations, both to complete the model in terms of other
important aspects of behavior and to satisfy the mathematical rigor of the
modelling, such that the number of unknown variables must be equal to the
number of defining equations.
33
Examples of this type of relationships which are not based on balances, but
which nevertheless form an important part of any model are:
Reaction rates as functions of concentration, temperature, pH

Stoichiometric or yield relationships for reaction rates
Ideal gas law behavior
Physical property correlations as functions of concentration
Pressure variations as a function of flow rate
Dynamics of measurement instruments as a function of the instrument
response time
Equilibrium relationships (e.g., Henry's law)
Controller equations
Correlations of mass transfer coefficient, gas holdup volume, and
interfacial area, as functions of system physical properties and degree of
agitation or flow velocity
How these and other relationships are incorporated within the development of
particular modelling instances are shown later in the cases given throughout the
text and in the simulation examples.
VI. For additional insight with complex problems, draw

an information flow diagram
Information flow diagrams can be useful in understanding complex
interactions (Franks, 1966). They help to identify missing relationships and
provide a graphical aid to a full understanding of the interactive nature of
system. An example is given in the simulation example BATFERM.
1.3.3
Total Mass Balances
In this section the application of the total mass balance principles will be
presented. Consider some arbitrary balance region, as shown in Fig. 1.14 by
the shaded area. Mass accumulates within the system at a rate dM/dt, owing to
the competing effects of a convective flow input (mass flow rate in) and an
output stream (mass flow rate out).
34
Mass flow rate out
Mass flow rate In
Figure 1.14. Balancing the total mass of an arbitrary system.

The total mass balance is expressed by,
dM
( Mass flow \
fMass flow out\
U .he system) - Uhe system J
= Mass rate in - Mass rate out
or in terms of volumetric flow rates, F, densities, (p), and volume, V

d(p V) system
3t
= F
oPo-FiPi
When densities are equal, as in the case of water flowing in and out of a tank,
dV
dT = F O-FI
The steady-state condition of constant volume in the tank (dV/dt = 0) occurs

when the volumetric flow in, FQ, is exactly balanced by the volumetric flow out,
FI. Total mass balances therefore are mostly important for those bioreactor
modelling situations in which volumes are subject to change.
1.3.4
Component Balances for Reacting Systems
Each chemical species can be described with a component balance around an

arbitrary, well-mixed, balance region, as shown in Fig. 1.15.
35

Species i
outflow
Species i
inflow
Figure 1.15. Component balancing for species i.
Thus for any species i, involved in the system, the component mass balance is
given by:
/
Rate of
\
accumulation
of mass
of component i
i in tnp cvct<=m
'Mass flow of^

component i
into
v the system ^
/^MassflowoA f
Rate of N
component i
production of
out of
component!
^ the system ) \ by reaction
Expressed in terms of volume, volumetric flow rate and concentration, this is

equivalent to:
~ = (F 0 C i0 )-(F 1 C il )+(r i V)
with units of mass/time:
,3*6.
m~
m^
m3
m-
1.3.4.1
Case A.
ill 3
Constant Volume Continuous Stirred Tank

Reactor
A constant volume, continuous, tank reactor with reaction A > 2B is

considered here, as shown in Fig. 1.16.
36
FQ
C AO
1 C A 1 C B1
C BO
>f
'.; '.??: 5: ' ' :; f'.<$ 'XI ' ;:?-'?:;':- ''M
I!
i
l
l
ilili
;|i
Figure 1.16. Continuous stirred tank reactor with reaction A > 2B.
Component A is converted to component B in a 1 to 2 molar ratio.

The component balances for A and B are:
d(VC A1 )
dt
= F0 CAO -
d(VC B i)
dt
F0CBo -
Here it is convenient to use molar masses, such that each term has the units of
kmol/h.
Under constant volume conditions:
d(VCA) = VdC A
d(VCB) = VdC B
and in addition FQ = FI. Thus the two model equations, then simplify to give:
dCAi
- CAI) +
and
dCBi
~dT~
F
V ( c BO~C B i)
In these two balances there are four unknowns CAI, Q*i, rAl an(^ rB l 8
kinetics are assumed to be first order, as often found in biological systems at
low concentration. Then:
r
Al = According to the molar stoichiometry,
37
rfil = -2r M = + 2 k C A !
Together with the kinetic relations there are 4 equations and 4 unknowns, thus
satisfying the conditions necessary for the model solution. With the initial
conditions, CAI and CBI at time t = 0, specified, the solution to these two
simultaneous equations, combined with the two kinetic relations, will give the
resulting changes of concentrations CAI and CBI as functions of time. The
simulation example ENZCON, is similar to the situation of Case A.
1.3.4.2
Case B.
Semi-continuous Reactor with Volume

Change
The chemical reaction and reaction rate data are the same as in the preceding
example, but now the reactor has no effluent stream. The operation of the
reactor is therefore semi-continuous.
AO
2B
Figure 1.17. A semi-continuous reactor example.
The kinetics are as before:

t ^
rA = -kC A
moles
T
m s
In terms of moles the stoichiometry gives,

rB = - 2 rA = + 2 k CA
The component balances with no flow of material leaving the reactor are now:
= FC AO + r A V
d(V C )
at
= IB v
B
38
The number of unknowns is now five and the number of equations is four, so
that an additional defining relationship is required for solution. Note that V
must remain within the differential, because the volume of the reactor contents
is now also a variable and must be determined by a total mass balance.
Assuming constant density p, this gives the defining equation as:
dt
= FF
With initial conditions for the initial molar quantities of A and B, (VGA,
and the initial volume of the contents, V, at time t = 0 specified, the resulting
system of equations can be solved to obtain the time varying quantities VCA(t),
VCs(t), V(t) and hence also concentrations CA and CB as functions of time.
Similar variable volume situations are found in examples FEDBAT, and
VARVOL.
1.3.4.3 Case C.
Steady-State Oxygen Balancing in

Fermentation
Calculation of the oxygen uptake rate, OUR, by means of a steady-state oxygen

balance is an important application of component balancing for fermentation.
In the reactor of Fig. 1.1.8, the entering air stream flow rate, oxygen
concentration, temperature and pressure conditions are shown by the subscript
0 and the exit conditions by the subscript 1.
Gas
>
i>yi> T i> PI
Air
F
0'yO'T0'P0
Figure 1.18. Entering air and exit gas during the continuous aeration of a bioreactor.
Writing a balance around the combined gas and liquid phases in the reactor
gives,
f Rate of accum-^j_ f Flowrateî ( Flowrate"\ /^Rate of O2 uptake A
( ulationofO 2 J~ [ofO 2 in J~(ofO 2 out J~ I by the cells
J
39
At steady-state, the accumulation terms for both phases are zero and
Flow of O2 in - Flow of C>2 out = Rate of C>2 uptake.
For gaseous systems, the quantities are often expressed in terms of molar
quantities.
Often only the inlet air flow rate FQ and the mol fraction of 62 in the outlet
gas, yi9 are measured. It is often assumed that the total molar flow rate of gas is
constant. This is a valid assumption as long as the number of carbon dioxide
mols produced is nearly equal to the number of oxygen mols consumed or if
the amounts of oxygen consumed are very small, relative to the total flow of
gas.
Converting to molar quantities, using the Ideal Gas Law,
pV = nRT
or in flow terms:
pF = N R T
where N is the molar flow rate, R is the gas constant and F is the volumetric flow
rate. Thus, for the inlet gas flow:
P
where NO is molar flow rate of the oxygen entering. Note that the pressure, po,
and temperature, TO, are measured at the point of flow measurement.
Assuming NO = NI, then measurement of NO gives enough information to
calculate oxygen uptake rate, OUR, from the steady-state balance. Thus,
0 = yo NO - yi NI - ro2 VL
OUR = ro2 VL = yo NO - yi NI
If NO is not equal to NI, then this equation will give large errors in oxygen
uptake rate, and NI must be measured, or determined indirectly by an inert
balance. This is explained in the Sec. 1.3.4.4 below.
1.3.4.4
Case D.
Inert Gas Balance to Calculate Flow Rates
Differences in the inlet and outlet gas flow rates of a tank fermenter can be
calculated by measuring one gas flow rate and the mol fraction of an inert gas
in the gas stream. Since inert gases, such as nitrogen or argon, are not
consumed or produced within the system (rinert = 0), their mass rates must
40
therefore be equal at the inlet and outlet streams of the reactor, assuming
steady-state conditions apply. Then for nitrogen
/Molar flow of\
V nitrogen in )
/Molar flow of\

^ nitrogen out /
NI yi
and in terms of mol fractions,

NO YO inert
inert
From this balance, calculation of NI can be made on the basis of a combination

of measurements of NO and the inert gas partial pressures (yinertX at both inlet
and outlet conditions.
N,1 =
y i inert
Since the inlet mol fraction for nitrogen in air is known, the outlet mol fraction,
yi inert' must be measured. This is often done by difference, having measured
the mol fraction of oxygen and carbon dioxide concentration in the exit gas.
1.3.5
Stoichiometry, Elemental Balancing and the

Yield Coefficient Concept
Stoichiometry is the basis for any quantitative treatment of chemical and

biochemical reactions. In biochemical processes it is a necessary basis for
building kinetic models.
1.3.5.1
Simple Stoichiometry
The Stoichiometry of chemical reactions is used to relate the relative quantities

of the different materials which react with one another and also the relative
quantities of product that are formed. Most chemical and biochemical reactions
are relatively simple in terms of their molar relationship or Stoichiometry. For
single reactions stoichiometric coefficients are clearly defined and may usually
easily be determined. Some examples are given below:
C3H4O3 + NADH + H+ < C3H6O3 + NAD+
Pyruvic Acid
Lactic Acid
41
This relation indicates that 1 mol of pyruvic acid reacts with 1 mol of NADH to
produce 1 mol of lactic acid.
Another example of stoichiometry is that of the oxidative decarboxylation of
pyruvic acid to yield acetyl-CoA
C3H4O3 + CoA-SH + NAD+ -> CH3CO-S-CoA + CO2 + NADH + H+
Pyruvic Acid
Acetyl-CoA
Stoichiometry relations also describe more complex pathways and can be
written with exact molar relationships, like the pentose-phosphate pathway
below.
Glucose + 12 NADP+ + ATP + 7 H2O -> 6 CO2 + 12 (NADPH + H+) +
+ ADP + Pi
where 1 mol of glucose reacted, consumes 7 mol of water and produces 6 mol
of carbon dioxide. Here the molar quantities of NADPH and ATP produced
and consumed, respectively, are shown.
For many complex biological reactions, however, not all the elementary
reactions and their contributions to the overall observed reaction stoichiometry
are known (Roels, 1983; Bailey and Ollis, 1986; Moser, 1988).
Thus the case of a general fermentation is usually approximated by an
overall reaction equation, where
Substrate + Nitrogen source + O2 -> Product + CO2 + H2O
v N H3(t)NH 3 + v02(t)O2
>
VC02(t) CO2 + VH2o(0 H2O
where the i-th product, such as metabolites or biomass, is given by a general

formula.
In the case above, the generalized elemental formulae are used for substrate,
biomass and products, but the nitrogen source is given simply as ammonia. The
stoichiometric coefficients, v, for each component are taken relative to that of
substrate and their coefficients may vary as a function of time as a result of
changing fermentation conditions.
Some indication as to the relative
magnitudes of the stoichiometric coefficients can be obtained from elemental
balancing techniques, but in general the problem is so complex that other
concepts, such as the more approximate yield coefficient concept, are used to
relate the relative proportions of materials undergoing conversion during the
fermentation.
42
1.3.5.2
Elemental Balancing
The technique of elemental balancing can be represented as follows:

Taking the general case of
CHmOi + a NH3 + b 02 >
[substrate]
c CHpOnNq + d CHrOsNt + e H2O + f CO2

[biomass]
[product]
where c, d and f are the fractions of carbon converted to biomass, product and
CO2, respectively.
Elemental balances for C, H, O and N give
C
H
O
N
1
m+3 a
l+2b
a
= c +d +f
= c p + dr + 2e
=cn + ds + e + 2f
=cq+dt
In this general problem there are too many unknowns for the solution method
to be taken further, since the elemental balances provide only four equations
and hence can be solved for only four unknowns. Assuming that the elemental
formulae for substrate, biomass and product and hence 1, m, n, p, q, r, s and t are
defined, there still remain six unknown stoichiometric coefficients a, b, c, d, e
and f and only four elemental balance equations. Thus the elemental balances
need supplementation by other measurable quantities such as substrate, oxygen
and ammonia consumption rates (assuming controlled pH conditions), and
carbon dioxide or biomass production rates, such that the condition is satisfied
that the number of unknowns is equal to the number of defining equations. In
principle the problem then becomes solvable. In practice, there can be
considerable difficulties and inaccuracies involved, although the technique of
elemental balancing can still provide useful data. The application of so-called
macroscopic principles (Roels, 1980, 1982 and 1983; Heijnen and Roels, 1981)
introduces a more strict systematic system of analysis. This is depicted in Fig.
1.19.
43
<P2
C32
04
Substrate
H
b2 Oc2 Nd2
N Source
C34 Hb4 Oc4 Nd4
Figure 1.19. Flow inputs into a system.
The system is represented here in terms of the various flow inputs, where f is the
corresponding flow vector
() = <]>
<1>
<E>
<5
The steady state balance for the system is then represented by: <|) E = 0
where E is the elemental composition matrix
E = a 4 b4 c 4 d 4 O4
0 0 2 0 O5
1 0 2 0 O6
0 2 1 0 <D7
(C) (H) (O) (N)
The combination of 7 unknown quantities and 4 elemental balance equations)

leave 3 quantities are independent. Thus assuming fluxes <E>1 (biomass), O2
(substrate) and <J>3 (product) are known, the unknown fluxes 04, 05, Og and
<I>7 can be obtained by methods of linear algebra and which are detailed by
Roels (1983).
44
In more complex cases with growth and product formation, more

information is needed. The introduction of the concept of the degree of
reduction is useful. For organic compounds this is defined as the number of
equivalent available electrons per gram atom C, that would be transferred to
CC>2, H2O and NH3 upon oxidation. Taking charge numbers: C = 4, H = 1 , O =
-2, and N = -3, reductance degrees (y) can be defined for
substrate (S)
biomass (X)
product (P)
ys = 4 + m - 2 1
yx = 4 + p - 2 n - 3 q
yp = 4 + r - 2 s - 3 t
The reductances for NHs, fÔ and CC>2 are of course zero.
Often the elemental composition of the substrate is not known and then the
reductance method may be supplemented by the following regularities, which
apply to a wide variety of organic molecules.
Qo2 = 27 J per g equivalent of available electrons transferred to oxygen
Yx = 4.29 g equivalent of available electrons per equivalent 1 g atom C in
biomass
GX = 0.462 g carbon / g dry biomass
1.3.5.3
Mass Yield Coefficients
Yield coefficients are biological variables, which are used to relate the ratio
between various consumption and production rates of mass and energy. They
are typically assumed to be time-independent and are calculated on an overall
basis. This concept should not be confused with the overall yield of a reaction
or a process. The biomass yield coefficient on substrate (Yx/s) is defined as:
Y
v
x/S = rs
In batch systems, reaction rates are equal to accumulation rates, and therefore
/dX\
Y
IdTj
dX
X/S = - TdST = - dS"

IdTj
After integration from time 0 to time t the integral value is obtained:
..
* x/s
45
amount of biomass produced

total amount of substrate consumed
X(t)-X(t=0)
S(t=0)-S(t)
For a steady-state continuous system the mass balances give

rs
SQ-S!
where index 0 and 1 indicate feed and effluent values, respectively.

In the literature, yield coefficients for biomass with respect to nutrients are
most often used (e.g. Dekkers, 1983; Mou and Cooney, 1983; Roels, 1983;
Moser, 1988). In many cases this is very useful because the biomass
composition is quite uniform, and often product selectivity does not change
very much during an experiment involving exponential growth and associated
production. Some useful typical values are given in Table 1.1.
1.3.5.4
Energy Yield Coefficients
Energy yield coefficients may be defined similarly to mass yield coefficients.

In terms of oxygen uptake,
TO
amount of heat released
Yq/02 = =
7
TQ2
amount or oxygen consumed
In terms of carbon substrate consumed,
v
- rQ rs
amount of heat released

amount of carbon source consumed
46
Table 1.1. Typical mass and energy yield values (Roels, 1983; Atkinson and
Mavituna, 1991).
Type of yield coefficient
Dimension
Value
c-mol / c-mol
c-mol / c-mol
c-mol / mol
c-mol / mol
kJ / mol
kJ / mol
kJ / c-mol
kJ / c-mol
0.4-0.7
0.1-0.2
1-2
0.35
380-490
460
325-500
120-190
X/S,aer
Yx/S,anaer
Yx/02 (Glucose)
YX/ATP
Y
Q/O2
YQ/C02
YQ/x,aer (Glucose)
Yq/x,anaer
Note: The molecular weight of biomass is taken here as 24.6 g/C-mol

The yield coefficients are usually determined as a result of a large number of
elementary biochemical reactions and it can easily be understood that their
values might vary depending on environmental and operating conditions.
A detailed description of some of these dependencies is given in the literature.
Despite this inconsistency, measured yield coefficients are often very useful for
practical purposes of process description and modelling.
1.3.6
1.3.6.1
Equilibrium Relationships
General Considerations
In many biological systems, processes with large ranges of time constants have
to be described. Usually it is important to start with a simplification of a system,
focusing on the most important time constant or rate. For example, if the
growth of an organism is to be modelled with a time constant of the order of
hours, it is very useful to ignore all aspects of biological evolution with time
constants of years. Also fast equilibrium reactions or conformational changes
of proteins having time constants below milliseconds should be ignored. Fast
reactions can, however, be very important when considering allosteric activation
or deactivation of proteins or simply pH-changes during biochemical reactions.
47
pH changes can have dramatic effects on the enzyme and microbial activity but
can also strongly influence absorption and desorption of carbon dioxide.
A typical equilibrium reactions is the dissociation of a receptor-protein ligand
complex, RL, into the free protein, L, and the receptor protein, P
This reaction is characterized by the corresponding dissociation equilibrium

constant KD
CLP
k_j
In most cases such relationships can be used to express the concentration of all
concentrations in explicit form using, e.g. a protein balance.
r1 _i_ c*
C Ptot
~ ^P ~r ^LP
^
KD
- ^ptot Cr + K
The total concentration, Cptot, is then included in a material balance equation

and the concentrations of the free receptor and the receptor-ligand complex are
determined by the equilibrium relationship. This is also true for a simple acidbase equilibrium relationship.
In more complex cases with interactions of various receptors or with a buffer
system containing several components, it is not possible to express the
concentrations in explicit forms and a non-linear algebraic equation has to be
solved during the simulation. The implementation of such problems into
BerkeleyMadonna is shown below with the example of pH calculation
1.3.6.2
Case A.
Calculation of pH with an Ion Charge

Balance.
Modelling systems with variable pH requires modelling of acid-base equilibria,

whose reactions are almost instantaneous. Production of acids or bases causes a
variation of pH, which depends on the buffer capacity of the system. pH also
influences the biological kinetics. It has been shown that only the undissociated
acid forms are kinetically important substrates in anaerobic systems. The
48
concentration of these species is a function of the pH as can be seen in the

equilibrium equation
Base' + H+
Acid
with dissociation constant
CBase- H+
CAcid
where CAcid is the concentration of the undissociated acid and CBase" is the
concentration of the corresponding base (salt).
An ion charge balance can be written
(cations * charge)
= (anions * charge)
In the pH range of interest (usually around pH = 7) all strong acids and strong
bases are completely dissociated. Moderately strong acids and bases exist in
both the dissociated and non-dissociated forms,
In the usual pH range the sum of the cations are much larger than the H+
ions.
ICK+CH+
where ]CK+ is the total cation concentration.
Negative ions originate mainly from strong acids (e.g. Cl% SO42') but also
arise from weak acids (Ac", Pr, Bu~, HCO3'). The concentration of CO32' is
always much smaller than that of
The ion balance reduces to
KBj
KW
Btot,i+ K+ =
V1
KAi
where KAI are the acid dissociation constants (e.g. KAC); KBI are the base
dissociation constants (e.g. KNHS); KW is the dissociation constant of water;
Cfitot,i are the total concentrations of base i; CAtot,i are the total concentrations
of acid i and EC An" is the sum of the anions.
The pH can be estimated from the above equation for any situation by
solving the resulting non-linear implicit algebraic equation, provided the total
concentrations of the weak acids, CAtot,i> weak bases, CBtot,i cations of strong
bases, CK+, and anions of strong acids, CAIT> are known.
49
It is convenient to use only the difference between cations and anions
After neglecting any ammonia buffering effect, it is useful to rearrange the

above equations in the form,
The example ANAMEAS, Sec. 8.8.6 includes this ion balance for pH
calculation. This equation represents an algebraic loop in a dynamic simulation
which is solved by iteration at each time interval until 8 approaches zero. This is
accomplished with the root-finding feature of Berkeley Madonna.
If there is pH control, then strong base or acid would be usually added. The
addition of strong alkali for pH control would cause an increase in CK+
which in accordance with the above equation would result in a decrease of CH+.
An alternative approach, which avoids an algebraic loop, is to treat the
instantaneous equilibrium reactions as reactions with finite forward and
backward rates. These rates must be adjusted with their kinetic constants to
maintain the equilibrium for the particular system; that is, these rates must be
very fast compared with the other rates of the model. This approach replaces
the algebraic loop iteration with a stiff er and larger set of differential equations.
This could be an advantage in some cases.
1.3.7
Energy Balancing for Bioreactors
Energy balances are needed whenever temperature changes are important, as

caused by reaction heating effects or by cooling and heating for temperature
control. For example, such a balance is needed when the heat of fermentation
causes a variation in bioreactor temperature. Energy balances are written
following the same set of rules as given above for mass balances in Sec. 1.3.
Thus the general form is as follows:
Accumu-^
lation
rate of
Ênergy ,
Rate of^ 'Rate of ^ 'Rate of"

energy
energy
energy
out by
out by
in by
^flow , flow
<transfer>
'Rate of >
energy
generated
^by reactiony
'Rate of >
energy
added by
âgitation ^
50
The above balance in word form is now applied to the measurable energy
quantities of the continuous reactor shown in Fig. 1.20.
AL H
agit
\
M)'
PO
P1
' '
U,A,T S
l!
Figure 1.20. A continuous tank fermenter showing only the energy-related variables.
An exact derivation of the energy balance was given by Aris (1989) as,
- h n ) ) + U A (Ta - TI) + rQ V + AHagit

agi
"dT =
where ni is the number of moles of component i, cpi are the partial molar heat
capacities and hi are the partial molar enthalpies. In this equation the rate of
heat production, TQ, takes place at temperature TI. If the heat capacities, cpi, are
independent of temperature, the enthalpies at TI can be expressed in terms of
heat capacities as
hn = hio + Cpi (TI -TO)
and with
S
2>i0
1=1
- 2X
1=1
= vp
Thus with these simplifications,

Vpc p
F0 p cp (T0 - TI) + U A (Ta - TI) +rQ V + AHagi
The units of each term of the equation are energy per time (kJ/h or kcal/h).
51
Accumulation Term
Densities and heat capacities of liquids can be taken as essentially constant.
dT
VpcPdF
has units:
m3 (kg/m3) (J/kg K) K
s
_
~
kJ
s
Here (p cp T) is an energy "concentration" term and has the units,

/jnass\ / energy \
_ /energy \
Vvolumey Vmass degree^ VaegreeJ - Vyolume,/
Thus the accumulation term has the units of energy/time (e.g. J/s)
Flow Terms
The flow term is F p CP (T0 - TI)
with the units, /energy

i^nn^J\ /volume\
\ctisr) = /energy \
This term actually describes heating of the stream entering the system with TO
to the reaction temperature TI. It is important to note here that this term is
exactly the same for a continuous reactor as for a fed-batch system.
Heat Transfer Term
The important quantities in this term are the heat transfer area A, the
temperature driving force or difference (Ta-Ti), where Ta is the temperature of
the heating or cooling source, and the overall heat transfer coefficient, U. The
heat transfer coefficient, U, has units of energy/time area degree, e.g. J/s m2 C.
The units for U A AT are thus,
(heat transfer rate) = U A (Ta - TI)
energy
~B55~
energy
area time degree (area) (degree)
The sign of the temperature difference determines the direction of heat flow.
Here if T a > TI heat flows into the reactor.
Reaction Heat Term
The term rq V gives the rate of heat released by the bioreaction and has the
units of
52
energy
_ energy
volume time ( volume ) - time
The rate term TQ can alternatively be written in various ways as follows:
In terms of substrate uptake and a substrate-related heat yield,
rq = rs YQ/S
In terms of oxygen uptake and an oxygen-related heat yield,

rq = ro2YQ/Q2
In terms of a heat of reaction per mol of substrate and a substrate uptake rate,
rQ = AHr,s rs
Here rs is the substrate uptake rate and AHr?s is the heat of reaction for the
substrate, for example J/mol or kcal/kg. The rs AHr>s term therefore has
dimensions of (energy/time volume) and is equal to TQ.
Other Heat Terms
The heat of agitation may be the most important heat effect for slow growing
cultures, particularly with viscous cultures. Other terms, such as heat losses
from the reactor due to evaporation, can also be important.
1.3.6.3
Case B.
Determining Heat Transfer Area or Cooling

Water Temperature
For aerobic fermentation, the heats of reaction per unit volume of reactor are
usually directly related to the oxygen uptake rate, ro2Thus for a constant-volume batch reaction with no agitation heat effects, the
general energy balance is
/Accumulation rate^
V
of energy
)
/Energy out^
~ ~ \ by transfer J
/Energy generated\
V by reaction
)
where YQ/Q2 often has a value near 460 kJ/mol 2, as given in Table 1.1.
53
If T is constant (dT/dt = 0):

UA(Ti-Ta) = r 0 2YQ/o 2 V
(heat transfer rate) = (rate of heat release)
Using this steady-state energy balance, it is possible to calculate the cooling
water temperature (Ta) for a given oxygen uptake rate and cooling device.
Thus,
_-ro 2
UA
Alternatively this same relation can be used in other ways:
1) To calculate the additional heat transfer area required for a known
increase in cooling water temperature.
2) To calculate the biomass concentration allowable for a given cooling
system, knowing the specific oxygen uptake rate (kg O2 / kg biomass h).
3) To calculate the cooling area required for a continuous fermenter with
known volume inlet, temperature, flow rate and biomass production rate.
Basic Bioreactor Concepts
2.1 Information for Bioreactor Modelling

Both physical and biological information are required in the design and
interpretation of biological reactor performance, as indicated in Fig. 2.1.
Physical factors that affect the general hydrodynamic environment of the
bioreactor include such parameters as liquid flow pattern and circulation time,
air distribution efficiency and gas holdup volume, oxygen mass transfer rates,
intensity of mixing and the effects of shear. These factors are affected by the
bioreactor geometry and that of the agitator (agitator speed, effect of baffles)
and by physical property effects, such as liquid viscosity and interfacial tension.
Both can have a large effect on gas bubble size and a corresponding effect on
both liquid and gas phase hydrodynamics. The biokinetic input involves such
factors as cell growth rate, cell productivity and substrate uptake rate. Often this
information may come from laboratory data, obtained under conditions which
are often far removed from those actually existing in the large scale bioreactor.
Although shown as separate inputs in Fig. 2.1, there are, in fact, considerable
interactions between the bioreactor hydrodynamic conditions and the cell
biokinetics, morphology and physiology, and one of the arts of modelling is to
make proper allowance for such effects. Thus in the large scale bioreactor,
some cells may suffer local starvation of essential nutrients owing to a
combination of long liquid circulation time and an inadequate rate of nutrient
supply, caused by inadequate mixing or inefficient mass transfer. Agitation and
shear effects can affect cell morphology and hence liquid viscosity, which will
also vary with cell density. This means that the processes of cell growth affect
the bioreactor hydrodynamics in a very complex and interactive manner.
Changes in the cell physiology, such that the cell processes are switched from
production of further biomass to that of a secondary metabolite or product, can
also be affected by selective limitation on the quantity and rate of supply of
some essential nutrient in the medium. This can in turn be influenced by the
bioreactor hydrodynamics and also by the mode of the operation of the
bioreactor.
The overall problem is therefore very complex, but as seen in Figure 2.1,
when all the information is combined successfully in a realistic and well
founded Bioreactor Model, the results obtained can be quite impressive and
ISBN: 3-527-30759-1
56
2 Basic Bioreactor Concepts
may enable such factors as cell and product production rates, product
selectivities, optimum process control and process optimization to be
determined with some considerable degree of confidence.
Physical Aspects
(flow patterns, residence
time, mass transfer)
Biokinetics
(order, inhibition,pH,
temperature)
Production rate
Selectivity
Control
Figure 2.1. Information for bioreactor modelling.
2.2
Bioreactor Operation
The rates of cell growth and product formation are, in the main, dependent on
the concentration levels of nutrients and products within the bioreactor. The
concentration dependencies of the reaction or production rate are often quite
simple, but may also be very complex. The magnitude of the rates, however,
depend upon the level of concentrations, and it will be seen that concentration
levels within the bioreactor depend very much on its type and mode of
operation. Differing modes of operation for the bioreactor can therefore lead
to differing rates of cell growth, to differing rates of product formation and
hence to substantially differing productivities.
Generally, the various types of bioreactor can be classified as either stirred
tank or tubular and column devices and according to the mode of operation as
batch, semi-continuous or continuous operation.
57
2.2 Bioreactor Operation
2.2.1 Batch Operation

Most industrial bioreactors are operated under batch conditions. In this, the
bioreactor is first charged with medium, inoculated with cells, and the cells are
allowed to grow for a sufficient time, such that the cells achieve the required
cell density or optimum product concentrations. The bioreactor contents are
discharged, and the bioreactor is prepared for a fresh charge of medium.
Operation is thus characterized by three periods of time: the filling period, the
cell growth and cell production period and the final emptying period as
depicted in Fig. 2.2. It is only during the reacting period, that the bioreactor is
productive. During the period of cell growth, strictly speaking, no additional
material is either added to or removed from the bioreactor, apart from minor
adjustments needed for control of pH or foam, small additions of essential
precursors, the removal of samples and, of course, a continuous supply of air
needed for aerobic fermentation. Concentrations of biomass, cell nutrients and
cell products thus change continuously with respect to time, as the various
constituents are either produced or consumed during the time course of the
fermentation, as seen in Fig. 2.3.
Filling
Reacting
Emptying
Cleaning
Figure 2.2. Periods of operation for batch reactors.
concentration
ubstrate
biomass
product
time
Figure 2.3. Concentration-time profiles during batchwise operation.
58
During the reaction period, there are changes in substrate and product
concentration with time, and the other time periods are effectively lost as
regards production.
Since there is no flow in or out of the bioreactor, during normal operation,
the biomass and substrate balances both take the form,
(Rate of accumulation within the reactor) = (Rate of production)
This will be expressed in more quantitative terms in Ch. 4.
Batch reactors thus have the following characteristics:
1)
2)
3)
Time-variant reaction conditions

Discontinuous production
Downtime for cleaning and filling
2.2.2 Semicontinuous or Fed Batch Operation

In semi-continuous or fed batch operation, additional substrate is fed into the
bioreactor, thus prolonging operation by providing an additional continuous
supply of nutrients to the cells. No material is removed from the reactor, apart
from normal sampling, and therefore the total quantity of material within the
reactor will increase as a function of time. However if the feed is highly
concentrated, then the reactor volume will not change much and can be
regarded as essentially constant.
Figure 2.4. Fed batch bioreactor configuration.
59
Semi-continuous operation shares the same characteristics as pure batch

operation, in that concentration levels generally change with time and that some
downtime occurs during the initial charging and final discharge period at the
end of the process.
The ability to manipulate concentration levels within the bioreactor by an
appropriate controlled feeding strategy confers a high degree of flexibility to
fed batch or semi-continuous operation, since differing concentration levels can
be utilized to manipulate the rates of reaction. In Fig. 2.4, both the volumetric
feeding rate, F, and the feed substrate concentration SQ, may be constant or may
vary with time, giving the possibility of such feeding strategies as:
1.
Slow constant feeding, which can be shown to result in linear growth

of the total cell biomass.
2.
Exponential feeding to maintain constant substrate concentration and,

resulting in unlimited, exponential cell growth.
3.
Feedback control of the feed rate, based on monitoring some key

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

1.
Extension of batch growth or product production by additional

substrate feeding.
2.
Possibility of operating with separate conditions for growth and

production phases.
3.
Control possibilities on feeding policies.
4.
Development of high biomass and product concentration.
For fed-batch operation, the cell balance follows the same form as for batch
operation, but since additional substrate feeding to the reactor now occurs, the
substrate balance takes the form:
Rate
<* "|
accumulation
V of substrate J
( Substrate \ ( Substrate >|

(f^d
[n) _ consumption
\ rate
)
Under controlled conditions, in which the substrate concentration is maintained

constant or kept small, the accumulation term in the above equation will also be
small, with the result that the feed rate of substrate into the reactor will balance
the rate of consumption by reaction.
60
One other balance equation, however, is also necessary, i.e. the total mass
balance,
f Rate of accumulation of ^
V mass in the reactor
/
( Mass flow rate of feed ^

V
to the reactor
)
which for constant density conditions reduces to

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

In continuous operation fresh medium is added continuously to the bioreactor,
while at the same time depleted medium is continuously removed. The rates of
addition and removal are such that the volume of the reactor contents is
maintained constant. The depleted material, of course, contains any products
that have been excreted by the cells and, in the case of suspended-cell culture,
also contains effluent cells from the bioreactor.
Continuous reactors are of two main types, as indicated in Fig. 2.5, and these
may be considered either as discrete stages, as in the continuous, stirred-tank
bioreactor, or as differential devices, as represented by the continuous tubular
or column reactor.
Continuous tank bioreactor
Continuous tubular bioreactor
Figure 2.5. The two main types of continuous reactors.
61
As shown later, these two differing forms of continuous reactor operation have
quite different operational characteristics. Both however are characterized by
the fact that after a short transient period, during which conditions within the
bioreactor change with time, the bioreactor will then achieve a steady state. This
means that operating conditions, both within the bioreactor and at the
bioreactor outlet, then remain constant, as shown in Fig. 2.6.
Concentration
Startup
period
Steady state
time
Figure 2.6. Startup of a continuous reactor.
Continuous reactors, however, have found little use as biological reactors on a

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

the whole of the reaction vessel contents and are therefore identical to the
concentration of the effluent stream. In a tubular reactor the reactant
concentration varies continuously, falling from a high value at the inlet to the
62
lowest concentration at the reactor outlet. The product concentration rises from
inlet to outlet. These differences arise because in the tank reactor the entering
feed is continuously being mixed with the reactor bulk contents and therefore
being diluted by the tank contents. The feed to the tubular reactor, however, is
not subject to mixing and is transformed only by reaction, as material moves
down the reactor.
No real situation will exactly correspond to the above idealized cases of
perfect mixing or zero mixing (plug flow), although the actual behavior of
tanks and tubes tends in the limit towards the corresponding idealized model.
The characteristics of continuous operation are as follows:
1.
2.
3.
4.
5.
Steady state after an initial start-up period (usually)

No variation of concentrations with time
Constant reaction rate
Ease of balancing to determine kinetics
No down-time for cleaning, filling, etc.
The balance equations at steady state for a well-mixed tank reactor have the
form
0 = (Input) - (Output) + (Production)
since at steady-state the rate of accumulation and therefore the rate of change is
zero.
This equation predicts that the reaction rate causes a depletion of substrate
from the feed condition to the outlet, (the product will increase) and that the
rate of production can be obtained from this simple balance:
(Rate of production) = (Rate of output) - (Rate of input)
For a non well-mixed reactor such as a tubular or column reactor, steady-state
implies the same non-transient conditions, but now concentrations also vary
with position. The same situation also applies to the case of a series of wellmixed tanks.
The balance form is then:
0 = (Rate of input) - (Rate of output) + (Overall Rate of Production)
Here the overall rate of reaction is obtained by summing or integrating over
every part of the reactor volume.
The concentration characteristics of a tubular reactor, as shown in Fig. 2.7,
are well approximated by a series of tank reactors. Referring to Fig. 2.8, and
moving downstream along the reactor cascade, the substrate concentration
decreases stepwise from tank to tank, while the product concentration increases
in a similar stepwise manner. As the number of tanks in the cascade increases,
so the performance becomes more and more similar to that of a tubular reactor.
In the case of a reaction, whose rate of reaction increases with increasing
63
substrate concentration S, the multiple tank configuration or a tubular reactor

would thus have a kinetic advantage over that of a single tank. The same is true,
in the case of product inhibition kinetics, in which the rate would be lowered by
high product concentration, P. Substrate inhibition systems would be run
preferably in single tanks, however, since then the substrate concentration is
always at its lowest value.
Cone.
distance
Figure 2.8. Stirred tanks in series and their concentration profiles.
A calculation of the tank volume or residence time requirement involves the

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

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

Mode of operation Advantages
Disadvantages
Batch
Equipment simple. Suitable Downtime for loading and

for small production.
cleaning. Reaction
conditions change with
time.
Continuous
Provides high production.

Better product quality due
to constant conditions.
Good for kinetic studies.
Requires flow control.

Culture may be unstable
over long periods.
Fed batch
Control of environmental
conditions, e.g. substrate
concentration.
Requires feeding strategy to

obtain desired
concentrations.
Table 2.2 lists the main operating parameters for the three differing modes of
bioreactor operation.
Table 2.2. Operating variables for batch and continuous bioreactors.
Batch
Continuous
Semicontinuous
Initial medium composition Inlet medium
and inoculum
composition
Feed and initial substrate

composition
Temperature, pressure
Temperature, pressure Temperature, pressure
pH if controlled
pH if controlled
pH if controlled
Reaction time
Liquid flow rate

(residence time)
Liquid flow rate

(residence time)
Aeration rate
Feeding rate and control

program
Aeration rate
Stirring rate
Stirring rate
Aeration rate
Stirring rate
65
The foregoing discussion of the varying characteristics of the different reactor

types and their concentration profiles allows a qualitative comparison of the
volume requirements for the different types of reaction, according to the
particular kinetics. For this it is first necessary to consider the qualitative nature
of the basic forms of kinetic relationship: zero order, first order, product and
substrate inhibition. The detailed quantitative treatment of these kinetic forms is
dealt with in Ch. 3.
The rate of a zero order reaction is independent of concentration. Many
bioreactions at high substrate concentrations follow zero order kinetics and are
therefore insensitive to concentration and to the effects of concentration
gradients. From the kinetic viewpoint, therefore, any reactor type would be
equally suitable.
First order reaction rates are directly proportional to concentration.
Bioreactions at low concentration are generally first order, and this would favor
operation in either a batch or a tubular/column type reactor. This is because
reactant concentrations in such reactors are generally high overall and hence
the overall rates of reaction are also consequently high. Hence the reactor
volume required for a given duty would generally be small. (In the case of a
batch reactor, this of course neglects the time lost for filling, emptying and
cleaning.)
A reaction with substrate inhibition would be best run in a tank at low
substrate concentration, since the concentration would be low throughout the
whole of the tank contents. Conversely, product inhibition would be more
pronounced in tank reactors, since product concentration would be at its
highest. In this case, a tubular type reactor or batch reactor would be preferred.
Table 2.3. Kinetic considerations for reactor choice.
Continuous
Reaction
Batch Tank
Tanks-inContinuous
Kinetics
Series or
Single Tank
Tubular
Zero order
OK
OK
OK
First order
Best
Best
Substrate
inhibition
Low initial
concentration
Low tank
concentrations
Product
inhibition
Best
Best
Production
triggered by
shift in
environment
OK for temp- Possible

erature-shift
Low conversion only

Best
Fed Batch
Low conversion
OK
Best
Low conversion only
Low conversion only
Not suitable
Best for concentrationshift
66
Table 2.3 compares the performance of batch tanks, continuous tubular or

tanks-in-series reactors and single continuous tank reactors. As discussed in
Sec. 4.2.1, batch tank concentration-time profiles are exactly analogous to the
steady state concentration-distance profiles obtained in continuous tubular
reactors. In terms of performance, therefore, the batch reactor would be the
same as a tube, when compared on the basis of equal batch time in the tank and
time of passage through the tube. Tanks-in-series reactors, as shown in Fig. 2.8,
involve step wise gradients, which in the limit are very similar to those of
continuous tubular reactors, hence, making their performance similar to that of
a tubular reactor. Owing to the high degree of mixing which leads to a uniform
concentration, the performance of the single continuous stirred tank reactor is
very different to that of the other reactor types. An exact quantitative
comparison can be made using the mass balance equations developed in Ch. 4
for each reactor type.
Biological Kinetics
As explained in Sec. 2.1, a realistic bioprocess model will usually require the
input of kinetic rate data. In the case of even simple chemical reactions, this
data has to be obtained by laboratory experiment. Since biochemical reactions
are controlled by enzymes, it is appropriate to start with a consideration of
simple enzyme kinetics (Sec. 3.1), In the case of modeling the behavior of
enzyme reactors, knowledge of the enzyme reaction kinetics is most important.
The sheer complexity of the biological reactions, occurring in a living cell,
seem to imply an almost impossible task in obtaining meaningful rate data for
biological modelling applications. Fortunately this is not the case and, as
shown in Section 3.2, a quite reasonable overall description of cell growth rate
data is possible, based on an overall empirical relationship, the Monod
Equation, which has been found to give a good fit to many general
observations of cell growth. This overall view, based on the net result of many
simultaneously occurring and highly interacting biochemical reactions, of
course represents an incredible oversimplification of the actual situation.
Fortunately it seems to work in many instances and can also be easily modified
to allow for the uptake of substrate by the cells and to include such additional
effects, as substrate limitation, multiple substrate limitation and product
inhibition. It is interesting, that the basic enzyme rate equation, or MichaelisMenten equation, based on the theory indicated in Sec. 3.1, is of the same basic
form as the empirically-based Monod equation for the growth of microorganisms.
When used in this manner, the cell kinetics are completely devoid of any
mechanistic interpretation and constitute what is known as an unstructured
kinetic model (Fig.3.1 A). In other cases, it may be necessary to look in some
detail at individual cell processes and reactions, in order to obtain a more
realistic description, thus leading on to the use of structured kinetic models
(Fig. 3.IB) as described in Sec. 3.3. In a most simple case, the cells are
composed of a catalytic part comprising proteins, RNA, DNA and other cellular
compounds and of a storage part, e.g. poly-hydroxy-alkanoic acids (PHAs) or
inclusion bodies of recombinant proteins. A most simple segregated model
considers different stages of cells and therefore a distribution of cell stages in a
culture without structuring the cell composition (Fig. 3.1C). In the most
realistic, but most complex situation the model is structured and segregated
ISBN: 3-527-30759-1
68
3 Biological Kinetics
(Fig. 3.ID). For the purpose of this book, the differences of these models can
be best described by their different balance regions.
Non-structured
Structured
3
CO
t
o
Figure 3.1. Types of kinetic models for cells. Balance regions: A Total cell biomass, B Cell
parts, C biomass parts, D Biomass and cell parts.
3.1
3.1.1
Enzyme Kinetics
Michaelis-Menten Equation
The rate of reaction catalyzed by a soluble enzyme can usually be described by

the Michaelis-Menten kinetic equation. This equation can be derived from the
accepted Briggs-Haldane mechanism for a simple enzyme reaction, which is
very similar to that for conventional chemical heterogeneous catalysis.
Thus,
3.1 Enzyme Kinetics
69
>
S 4- E
<
ES
ES
- P + E
where E is enzyme, S is substrate, P is product and ES is an enzyme-substrate

complex.
For a batch reaction, the balances for S and ES are written in terms of the
mechanism as,
JQ
= - ki S E + k_i (ES)
dt
dt
= ki S E - (k_i + ki) (ES)
with initial conditions at t = 0:
(ES) = 0
S = S0
The concentration changes for a batch reactor are shown qualitatively in Fig.
3.2. While the enzyme concentration is usually much lower than that of the
substrate, most of the enzyme is present during the reaction in the form of the
enzyme-substrate complex, ES.
Analytical solution is then possible by assuming a quasi-steady state for the
enzyme-substrate complex, ES,
d(ES)
-= 0
dt
This assumption is valid for E SQ.
Using the total enzyme mass balance,
EO = E + (ES)
the above equations can be solved for the unknown concentrations E and ES to
give,
and
ki S E
0 ~ k_i .+ k2
E = E
k.i + k2
k_i + k2
= E
70
1
Q)
O
-^
time
Figure 3.2. Concentration changes of the reaction species for a simple enzymatic reaction
taking place in a batch reactor.
Substituting for E and ES the substrate balance becomes,
dS _
dt ~
k2SEo
giving the Michaelis-Menten equation,
where the parameters in terms of the mechanistic model are for the maximum
reaction rate (kmol/m3min):
v max = k 2 E 0
and for the Michaelis-Menten constant (kmol/m3):

KM -
k-i + k2
ki
The Michaelis-Menten equation exhibits three distinct regions for the reaction
rate. At very low and very high substrate concentrations the rs versus S curve is
essentially linear, as seen in Fig. 3.3.
71
3.1 Enzyme Kinetics
vm
rS
Michaehs-Menten region
vm/2""
KM
10 KM
5 KM
15 KM
Figure 3.3. Reaction rate versus substrate concentration for the Michaelis-Menten equation.
The low concentration region can be approximated by first-order kinetics. The

Michaelis-Menten equation becomes for S KM,
_ VmaxS
For high substrate concentration (S KM) the relation approaches zero-order,

r
S = v max
and the rate of reaction is thus independent of substrate concentration and is

constant at the maximum value.
In the intermediate substrate concentration range, 0.1 KM < S < 10 KM, the
full Michaelis-Menten equation must be used to guarantee an accuracy for rs
greater than 10 %. The parameters vmax and KM can be determined from
experimental data, either graphically following a linearization of the MichaelisMenten equation or, better, numerically by using nonlinear parameter
estimation techniques.
Graphical determination of KM and vmax is based on rearrangement of the
Michaelis-Menten equation into a linear form,
Inversion and rearrangement give,
72
1
rs
KM I
v
max S
max
A graphical representation of this equation is called the Lineweaver-Burk

diagram (Fig. 3.3) from which the kinetic parameters vmax and KM may be
determined.
Figure 3.3. Lineweaver-Burk double-reciprocal plot.
Typical values of the enzyme kinetic constants are given in Table 3.1. It is
interesting to note that the rate of formation of the enzyme-substrate complex
can be extremely fast, with the constant ki approaching 1 x 1010 L/mol s. This
is the maximum value for a rate constant of a reaction that is limited by
diffusion of a small substrate molecule in aqueous solution.
Table 3.1. Typical values of the constants of the Michaelis-Menten equation.
Constant
Value range
KM
105 - 109 L/mol s

10- 104 1/s
1 - 106 1/s
10-6 10-l mol/L
3.1 Enzyme Kinetics
73
The simulation example MMKINET enables a computer study of the basic

characteristics of the Michaelis-Menten equation to be carried out, and
LINEWEAV simulates the study of the Lineweaver-Burk plot for a batch
enzyme reaction.
3.1.2
Other Enzyme Kinetic Models
The reaction mechanism of enzyme catalysis can be very complex, resulting in

complicated kinetic equations that are treated in specialized textbooks, as given
in the reference section. Some of the more readily used forms of the modified
Michaelis-Menten kinetics are presented here.
Double Michaelis-Menten Kinetics
This refers to the case when two substrates are involved in the reaction:
v
max Si 82
s - (KMi + Si) (KM2 + S2)
Inhibition
Inhibition occurs when a substance, inhibitor (I), reduces the rate of an
enzyme-catalyzed reaction, usually by the inhibitor binding to the enzyme
active site. Three simple types of reversible inhibition kinetics are given in Tab.
3.2.
Table 3.2. Enzymatic inhibition kinetics.
Mechanism
Inhibition
Rate equation, rs
v
maxS
competitive
KM (1 + I/KI) + S
E+SES-P
c
c
El ESI
non-competitive
(i + I/KI) (KM + S)
ES +1 => ESI
uncompetitive
vmax
74
Usually, the substance I is the substrate or the product, and the reaction kinetics
are known as substrate or product inhibition, respectively.
Allosteric Kinetics
A simple model to describe allosteric inhibition is given, in which the enzyme
can bind to more than one substrate molecule. Thus:
nS + E
ESn
nP + E
when n is the number of substrate molecules.

The resulting kinetic expression is referred to as Hill kinetics,
Vmax S n
s
" K M n + Sn
As shown in Fig. 3.5, for values of n > 1 an S-shaped function results. KM is

the substrate concentration with r$ = vmax/2. The simulation example PHB
employs this kinetic form.
Temperature and pH Influence
Rates of biological reactions, including growth rates, exhibit a maximum when
plotted versus temperature or pH. The maximum point is referred to as the
temperature optimum or pH optimum for the system. The term temperature
optimum must be used with caution because the curve is a result of two
temperature dependent processes, the enzyme catalysed reaction and the
enzyme deactivation reaction, respectively.
75
3.1 Enzyme Kinetics
Figure 3.5. Michaelis-Menten and Hill kinetics: v max = 1; Km = 5; n = 1, 2, 3, 5.
At temperatures well below the optimum the enzyme deactivation may be

neglected and the temperature influence on the reaction rate described by the
Arrhenius equation. At higher temperatures both enzymatic reaction and
enzyme deactivation rate equations must be solved together with their respective
kinetic constants expressed in terms of the Arrhenius equation.
Most enzymes exhibit a distinct pH optimum. This can be explained by
dissociation of acidic and basic groups of the enzyme, especially of its active
center. The following equation is a useful description of this.
r =
S,H+CH+CH/ K I,H
76
3.1.3
Deactivation
Biocatalysts in reactors usually undergo irreversible conformational changes

generally known as denaturation or deactivation. This often causes an
exponential decrease of activity with time and can be described by a first-order
reaction rate process:
rd = -kdE
Considering that for a batch reactor, dE/dt = r^, the integrated form can be
written as
E = E 0 e- kdt
Substitution in the Michaelis-Menten equation yields
=
MOJL
kdt
KM+S
This equation suggests an exponential decrease of reaction rate regardless of
substrate concentration. The simulation example DEACTENZ, Sec. 8.4.12
illustrates this.
Engineering models for the kinetics of deactivation are given by Prenosil et
al. (1987).
3.1.4
Sterilization
Similar to enzyme deactivation, sterilization kinetics can be viewed as a process

of inactivation or the removal of viable organisms or cells from the system.
Inactivation can be achieved by using heat, radiation or chemicals. It is a
statistical process, with the rate of killing being usually proportional to the
number of the organisms at any time. Therefore it can be described again by
first-order kinetics:
rd = - k d X
where
For a batch reactor,
dX
= - k d X =-k d =k 0 e- E a / R T X
which upon integration gives,
3.1 Enzyme Kinetics
77
_ ,,-kdt
where XQ is the initial live biomass concentration, X is the viable biomass after
the treatment time t, and kd is the specific deactivation constant (1/s).
The sterilization time will depend on the initial level of contamination. For
this purpose the D-value is defined as the treatment time required to reduce the
population by a factor of ten. This time is related to the rate constant by
2.3
3.2
3.2.1
Simple Microbial Kinetics

Basic Growth Kinetics
Under ideal conditions for growth, when a batch fermentation is carried out, it
can be observed experimentally that the quantity of biomass, and therefore also
the concentration, increases exponentially with respect to time. This phenomena
can be explained by the fact that all cells have the same probability to multiply.
Thus the overall rate of biomass formation is proportional to the biomass itself
This leads to an autocatalytic reaction, which is described by a first order rate
expression as
where rx is the rate of cell growth (kg cell/m3 s), X is the cell concentration
(kg cell/m3) and k is a kinetic growth constant (1/s). For a batch system, this is
equivalent to,
where dX/dt is the rate of change of cell concentration with respect to time
(kg cell/m3 s). The analytical solution of this simple, first-order differential
equation is of the form
In X = k t + In X0
or,
= ekt
where XQ is the initial cell concentration at time t = 0.
78
Plotting experimental growth data in the form of the natural logarithm of cell
concentration versus time will often yield a straight line over a large portion of
the curve, as shown below in Fig. 3.6.
InS
Limitation \Stationary
InX
Death
Exponential
Lag
time
Figure 3.6. Biomass and substrate concentrations during batch growth.
In the range from ti to t2 the logarithmic curve is linear, and this is the region
of exponential growth. Three other regions can be identified: between t = 0
and ti, there exists a period of cell adaptation or lag phase, and before t2 there
is a region where the growth is limited by the lack of a particular substance,
which is known as the limiting substrate.
The slope of the linear part of the curve between ti and t2 is the growth rate
per unit mass of cells or specific growth rate and is given the symbol \i.
=
1 dX
X" "dT
= specific growth rate = |i
In many processes cells begin to die (after ts), because of lack of nutrients,
toxic effects or cell aging. This process can typically be described by a first
order decay,
rd = - kd X
where rd is the death rate and k^ is the specific death rate, with the same
dimensions as the specific growth rate. This expression is identical with
sterilization kinetics, Sec. 3.1.4.
The exponential and limiting regions can be described by a single relation,
that sets JLI equal to a function of substrate concentration. It is observed
experimentally that |a is at a maximum when the particular limiting substrate
concentration S is large, and for low concentration ja is proportional to S. Over
the whole range from low to high S, |i is described by the following Monod
equation.
79
3.2 Simple Microbial Kinetics
Thus |i varies with S in the same fashion as does the enzymatic rate of
Michaelis-Menten kinetics.
Again, this is a two-parameter equation involving two constants, the
maximum specific growth rate |im and the saturation constant KS. It is best
considered to be an empirical relation, but since it has the same form as the
Michaelis-Menten enzyme kinetics equation, it is sometimes taken to be related
to a limiting enzymatic step. Although very simple, it often describes
experimental data for growth rates very well. The form of this relation is shown
in Fig. 3.7.
M
Monod Relation
Figure 3.7. Specific growth rate versus limiting substrate concentration according to the
Monod relation.
The important properties of this relationship are as follows:
S - 0,
S = KS,
Urn
Jl = ~
The first introductory simulations in Sec. 8.1 are based on Monod kinetics.
When two substrates can be limiting, it is often the case that a double Monod
type relationship can be used, as given in Sec. 3.2.4 and as shown by the
simulation examples NITRIF, Sec. 8.5.3, and BIOFILM, Sec. 8.7.1.
80
3.2.2
Substrate Inhibition of Growth
Many substrates can be utilized by organisms at low concentration, but at high

concentrations they can also act as toxic growth inhibitors. The |i versus S
curve may then appear in the form shown in Fig. 3.7, and can be described by
the relation:
_
|imS
^ " (KS + s + s2/KO
whose shape depends on the values for KS and KI.

This is a modified Monod relation to allow for the inhibitory effects of high
substrate concentration. As shown in Fig. 3.8, the inhibition term (S2/Ki),
which is small in magnitude at low values of S, increases dramatically at high
values of S and causes a decrease in \i. Note that high values of KI correspond
to a decreasing effect of substrate inhibition. It is seen that larger values of KS
shift the left side of the curves towards the right, while increasing values of KI
raise the right side of the curves. Thus a wide range of shapes can be achieved
by varying the three parameters, but a maximum value of (I is always obtained
at some intermediate value of S.
1.0 -,
0.8 -
0.6 -
0.0
Figure 3.7. Substrate inhibition kinetics for various values of KS and Kj. The parameters used
are as follows: For all curves |im = 1.0 1/h. Curve A: KS = 1 and KI = 10, Curve B: KS = 0.1 and
KI = 10; Curve C: KS = 1 and KI = 20; Curve D: KS = 0.1 and KI = 20. The units of KS, KI and S
are g/m^.
81
The substrate inhibition kinetic curve has the following characteristics, which
result from the kinetic equation:
1) When S = Ks
_
- - 2 + Ks/Kj
2) When S = KI
- 2 + Ks/Ki
3) The maximum occurs at S = (Ks Ki)-5 and
M- =
3.2.3
Mm
2 (Ks/Ki)-5 + 1
Product Inhibition
When the formation of product inhibits the rate of cell growth, the basic Monod
equation can be modified, by the addition of a product inhibition term P/Kj.
Thus,
3.2.4
Other Expressions for Specific Growth Rate
The Modified Monod Form

MmS
M- - KS S0 + S
shows the influence of initial concentration, which is sometimes observed if
other components are limiting.
The Teisser Equation
ILL = |Li m (l-e-S/k)
relates |Li to S exponentially.
82
The Contois Equation
- KX + S
expresses the effective saturation constant as being proportional to the biomass
concentration X. At high X, |i is inversely proportional to X. This is
sometimes used to represent a diffusion limitation in flocculating or
immobilized biomass.
The Logistic Equation
M, = ( a - b X )
encompasses exponential growth and the levelling off to zero growth rate at
high X. For a batch fermentation the biomass balance is,
= aX-bX2
Thus when X is small, growth is exponential and given by
dX
When X is large,
At steady state or zero growth rate,
0 = a X - b X2
and thus
X = a/b
Multiple-Substrate Monod kinetics
can be used to describe the influence of many substrates, which for two
substrates takes the form,
Si ^ f S2 A
l + SiJ ^2 + 827
In this way either substrate may be limiting under conditions when the other
substrate is in excess. Note that the multiplicative effect gives for S\ = K\ and
83
82 = K2, ILL = |LLm/4. An example of such kinetics is the simultaneous requirement

of glucose and oxygen by aerobically growing organisms.
Double-Monod kinetics
can also be written for two substrates as parallel reactions, according to
lSi
k2S2 V
This form gives an additive, fractional contribution for each substrate. Thus for
Si = KI and 82 = K2, the result is |Li = |Lim/2. For the case Si = 0 and 82 large,
then ji = |iim k2/(ki+k2). Each substrate thus allows a different maximal growth
rate. If both Si and 82 are large then |ii = |iim. Note that the flexibility of this
kinetic form requires twice as many kinetic parameters as the simpler double
Monod kinetics. An example of this kinetics is the parallel use of alternative
substrates, such as various types of sugars.
Diauxic Monod Growth can be modelled for two substrates by the relation
K 2 + S2 + S / K !
in this way the consumption of substrate 82 will be inhibited until Si is
exhausted, for suitably low values of Kj. Diauxic growth can be observed in
many organisms. An example is E. Coli, where the uptake of lactose is
repressed in the presence of glucose. The simulation example SUBTILIS, Sec.
8.9.2 uses this kinetic form.
3.2.5
Substrate Uptake Kinetics
The rate of uptake of substrate by micro-organisms is generally considered to

be either related to that of growth or to that required for cell maintenance. This
can be expressed as:
~rx
v
rs =
?5 " m X
where rs is the rate of substrate uptake by the cells (kg substrate/m3 s). As
explained in Section 1.3.5, YX/S is the stoichiometric factor or yield coefficient
and relates mass cells/mass substrate.
84
The maintenance factor, m, gives the (mass substrate/mass cells time)

required for non-growth functions. The total substrate utilization for cell
maintenance is, of course, taken to be proportional to the total quantity of cells,
and therefore for a batch reactor it is proportional to cell concentration, X.
Often the uptake and production rates of substances are expressed in terms
of the particular quantities related to unit mass of cells and are then known as
specific cell quantities. Thus:
For the specific growth rate (1/h)
rx
= X
For the specific substrate uptake rate (kg S/kg biomass h),
rs
qs = x
For the specific oxygen uptake rate (kg C>2/kg biomass h),
r
O2
qo2 =
For the specific carbon dioxide uptake rate (kg CCVkg biomass h),
qco2 = "IT"
For the specific product production rate (kg P/kg biomass h),
qp =
Note that qx = rx/X = |JL is the specific biomass production rate.

Specific rate quantities may take simple or complicated forms, for example, for
the case of substrate:
rs = y^" - m X
then,
qs =
where |i is also a function of S.
85
By necessity, in wastewater treatment systems the substrate concentration, S, is

taken often as total dissolved organic carbon, rather than considering a specific
substance, such as glucose. The biomass concentration, X, also must be related
to the total of all microbial species present. Naturally a gross simplification of
such a complex system results.
In wastewater treatment systems, biomass growth is immeasurably slow,
whereas the substrate uptake can usually be measured fairly accurately. Under
such circumstances it is then more useful to base the kinetics on the more
measurable rate and to express r$ as a separate rate equation, which is
independent of rxFor example, this can be done using an expression, analogous in form to that
of the Monod equation,
VmS
where the constant vm is proportional to the quantity of biomass in the system

and is the maximum rate of substrate consumption, observed at high S.
3.2.6
Product Formation
The kinetics of product formation can be exceedingly complex. Product is

sometimes formed during growth and sometimes after all growth has stopped.
A useful equation for the rate of product formation, flexible enough to find
frequent application, is that of the Luedeking-Piret model:
X
where rp is the rate of product formation (kg product/m3 s). YX/P is a yield
factor (kg cell produced/kg product produced), relating the growth and product
stoichiometry in the "growth associated" term of the Luedeking-Piret equation,
and b is a non-growth related term and is important for cultures which produce
product independent of growth. Often both coefficients of the above equation
(YX/P and b) are not constant but are functions of substrate or product
concentration.
When little is known about the detailed kinetics of product formation, a more
general expression rp = qp X is used, where qp will usually vary with culture
conditions and concentrations.
86
3.2.7
Interacting Microorganisms
Multiple-organism populations will involve interactions in which one species of

organisms will exert some influence on another. Such interactions and their
models are described below. Considering two microbial species, A and B, three
types of interactions on each other are possible; a positive beneficial effect (+),
a negative detrimental effect (-), or a neutral effect (0). The resulting
interaction possibilities are given in Table 3.3.
The different types of interactions can also be described by a graphical
representation. Thus the growth kinetics can be described by a solid arrow
connecting the substrate to the product, where the organism involved is shown
above the arrow. A solid arrow from one substrate symbol to the same symbol
in another growth path indicates that the product from one organism acts as a
substrate for another. Substrate or product inhibition is indicated by a dashed
arrow connecting the component to the inhibited organism. The symbols +, or 0 at the right hand side of the diagram indicate whether or not the organism
involved has benefited by the interaction. This is made clear by the examples
below:
Table 3.3. Definition of microbial interaction types.
Interaction-type
Neutralism
Commensalism
Mutualism
Predator-prey
Predator-prey
Amensalism
Amensalism
Competition
A
0
0
Organisms
B
0
+
0
0
Predator-Prey Kinetics
Organism A consumes substrate S, and organism B consumes organism A.
The simulation example MIXPOP demonstrates this type of system.
87
Commensalism
Organism A uses substrate 82 to produce product P; organism B uses substrate
Si to produce product 82, which benefits organism A since product 82 acts as
its substrate.
A
81
The following processes with the compound 82, shown in brackets, involve this
form of commensalism:
nitrification (NC>2~ )
anaerobic digestion (organic acids)
methanogenation (H2, 62) as found in simulation example ANAMEAS,
Sec. 8.8.7.
Commensalism with Product Removed
Organism A utilizes a substrate 82, which inhibits the growth of B on substrate
Si.
s 2 - - -> p 2
This effect may be found in the removal of toxic wastes in mixed cultures with
multiple carbon sources. An example is found in ANAMEAS in which the
hydrogen substrate of the methanogens (A) inhibits the acetogenic organisms
(B).
Mutualism with Product Removed
Organism A utilizes substrates 82 to produce product P. Organism B utilizes
substrate S\ to produce 82, which inhibits organism B.
An example of this would be found in anaerobic digestion for hydrogen gas

(see ANAMEAS, Sec. 8.8.7)
88
Mutualism with Products Used Mutually as Substrates

Both organisms benefit from each other's products.
3.2.7.1
Case A. Modelling of Mutualism Kinetics
Organism A utilizes the product from organism B, which also helps B because
PB inhibits its growth.
An example of kinetic modelling is presented for this case, in which the growth
of the two organisms, A and B, takes place in a batch reactor with initial
substrate concentrations SIQ and 820- The growth rate is expressed by Monodtype kinetics and constant yield factors are used to express the substrate uptake
and product formation rates.
Substrate Si balance,
dt
_
-
dt
_
-
Substrate 82 balance.
Product PB balance,
dPB
IT
A PB
Species A balance,
dXA
"3f" =
Species B balance,
BS2
89
dXB =
~3T
^B XB
The kinetics are given by Monod-type relations, with a double form of Monod
equation employed for species A and a product inhibition term employed for
B,
Si
PB
HA - MmA KI + Si
B = K2 + S2 + (PB/KI)
Other examples of interacting microorganism effects are given in the
simulation examples ACTNITR (neutralism), Sec. 8.4.3, COMPASM
(competitive assimilation and commensalism), Sec. 8.8.2, MIXPOP (predatorprey population dynamics), Sec. 8.8.4 and TWOONE (competition between
organisms), Sec. 8.8.5.
3.2.7.2
Case B. Kinetics of Anaerobic Degradation
Anaerobic degradation is a very complex multi-substrate, multi-organism

process, as is depicted in Fig. 3.9. It is shown here how its modelling can be
approached using Monod-type kinetics. This problem is of interest because
overloading of an anaerobic reactor causes accumulation of intermediates
(organic acids, hydrogen) and consequent inhibition of the final methanogenic
step (Gujer and Zehnder, 1983).
In a first step, polymer materials (carbohydrates, proteins or lipids) are
hydrolyzed to yield the monomer compounds (amino acids, sugars and fatty
acids). In a second acidogenic step, these compounds are fermented to organic
acids (mainly acetic, propionic and butyric acid) and hydrogen. In the third
acetogenic step, organic acids with more than three atoms of carbon per
molecule are converted to acetic acid and hydrogen. In a last methanogenic
step, the intermediates, acetic acid and hydrogen and carbon dioxide are
converted to methane. Five different groups of organisms accomplish these
reactions as shown in Fig. 3.9.
According to thermodynamic calculations (Archer, 1983) the oxidation of
propionic to acetic acid should only be possible at a hydrogen partial pressure
f PH2 < 10~4 bar. From a series of observations it seems evident that
disturbances in the methanogenic step, which is generally considered to be the
most sensitive one, lead to the accumulation of H2- Additionally the state of an
anaerobic reactor may be characterized by its volatile fatty acid levels based on
the CH4/CO2 ratio and the total gas production rate.
90
(0
'55
CO
g0)
TJ
o>
"5
W
'(0
0)
0)
D)
O
I
O
Figure 3.9. Reaction scheme of anaerobic degradation. The symbols are: Poly - polymer
material (proteins, fats, hydrocarbons, etc.); XJJY - Biomass hydrolyzing Poly; Mono monomeric materials from hydrolysis of Poly; XAG ~acid generating biomass; HPr - propionic
acid; Pr" - propionate; Xpr - biomass growing on propionate; HBu - butyric acid; Bu" - butyrate;
XBU - biomass growing on butyrate; HAc - acetic acid; Ac" - acetate; XAC - biomass growing on
acetate; XH - biomass growing on hydrogen and carbon dioxide. Dashed arrows indicate gaseous
compounds transfer to the liquid phase. T - gaseous compounds transferred to liquid gas phase.
The respective reaction rates rj for the production of biomass Xi, for the
consumption of substrate Si and for the formation of product Pi in each step
are:
rxi = Hi Xi - kdi Xi
3.3 Structured Kinetic Models
91
Hi Xj
rpi =
where k^ is the specific death rate, including maintenance metabolism, and the
specific growth rates take the Monod form,
Mimax Si
W - KSi + Si
or its modified form in the case of substrate inhibition by acetate,

Mimax Si
These kinetics can then be combined with the mass balances as discussed in Ch.
4 for each component, Si and Pi, and for the biomass balances for each
organism type, Xi
Following this approach a model was established (Denac et al., 1988) and
combined with particular control algorithms to simulate a continuous anaerobic
digestor with feed rate control. This included a gas phase balance,
thermodynamic equilibrium constraints and acid-base equilibria using an ion
charge balance (Sec. 1.3.6.2). The simulations were used to adjust the control
parameters, which were employed on laboratory reactors (Heinzle et al., 1992).
The simulation example ANAEMEAS, Sec. 8.8.7, gives details concerning this
model.
33
Structured Kinetic Models
In many cases the characterization of biological activity by simply the total

biomass concentration is insufficient for a true model representation
(Fredrickson et al., 1970; Roels, 1983; Moser, 1988). A variation in the
biomass activity per unit biomass concentration may be caused by:
Loss of plasmids (Imanaka and Aiba, 1981). See also the simulation
example PLASMID.
Induction and repression of genes.
Variation of RNA content of the cells (Harder and Roels, 1982;
Furukawaet al., 1983).
Variation of enzyme content of the cells.
Post-translational modification of proteins, e.g. phosporylation.
Signaling networks.
92
Membrane transport.
Accumulation of storage materials (Heinzle and Lafferty, 1980; Heinzle
et al., 1983). See example PHB, Sec. 8.2.4.
Morphological changes, e.g., branching of filamentous organisms,
volume to surface ratio of yeast cells (Fig. 3.10), Furukawa et al., 1983.
Such variations in biomass activity and composition require a more complex

description of the cellular metabolism and a more structured approach to the
modelling of cell kinetics. Structured models are based on a compartmental
description of the cell mass as shown in Fig. 3.1.
In general it is very difficult experimentally to obtain sufficient mechanistic
knowledge about the cell metabolism for the development of a "realistic"
structured model. Parameter estimation may be very difficult, and the
application of complex numerical methods may easily lead to physically
meaningless results. Often the verification of even simple unstructured models
is not possible owing to experimental difficulties. This problem becomes much
more significant with increasing complexity of the model. For this reason,
structured models are seldom used for design or control. Structured models
may be useful to model transient behavior of a biological system. Such models
also may be required if a wide range of changes of environmental conditions
have to be described with one model and one set of parameters (Moes et al.,
1985 and 1986).
Changes of cellular composition as functions of steady-state growth rate are
well known. For example, in long-term experiments the cellular composition
of lipids, carbohydrates, protein, RNA and DNA in Baker's Yeast were found to
change as a function of dissolved oxygen and dilution rate (Furukawa et al.,
1983). In this work the yeast shape and specific area also changed with
dissolved oxygen concentration (Fig. 3.10).
L/d; S/V x 10
2.0
[1/m]
L/d
1.5
S/V
1.0
0.01
0.1
1.0
D0[g/m3]
Figure 3.10. Dissolved oxygen concentration, DO, influenced the shape of yeast in
continuous culture as given by the ratios of length/diameter (L/d) and surface/volume (S/V).
93
In what follows three cases involving structured kinetics models will be

discussed briefly. Case C describes a simple, two-compartment model with
storage material. Case D gives an example of a complex, structured threecompartment model. Here the biomass contains storage material and an
enzyme that degrades the storage material. In this example the problem of
model discrimination is discussed briefly. Case E describes the application of
ATP balancing, a method of linking stoichiometry of various pathways in
complex models.
3.3.1
3.3.1.1
Case Studies
Case C. Modelling Growth and Synthesis of Poly-Bhydroxybutyric Acid (PHB)
Fig. 3.11 represents the process of cell growth and the synthesis of intracellular
product PHB. Residual biomass (R) is the difference between total cell dry
mass (X) and product PHB (P). Synthesis of PHB occurs with a single limiting
substrate NH4+ (S) and constant dissolved gas concentrations of H2, C>2 and
CO2 (So). Mass flows are indicated by solid lines, and regulatory mechanisms
are symbolized by dashed lines.
inhibiting
inhibiting
Figure 3.11. Schematic diagram of growth and synthesis of the intracellular product PHB (P)
with constant concentrations of dissolved gases H2, O2, and CO2 (SG) X is the total biomass
(X=R+P).
94
It can be seen that the catalytically active biomass, R, is produced from both S
and SQ. During exponential growth the PHB content is constant, and thus the
rate of intracellular product formation is proportional to the rate of formation
of the residual biomass. On this basis, the basic mass balance equations for a
batch process can be formulated as shown in the simulation example PHB, Sec.
8.2.4. This model was used successfully in describing experimental batch
growth and the PHB product formation (Heinzle and Lafferty, 1980), as shown
in Fig. 3.12.
S [g/L]
X,P,R [g/L]
P/X [-]
3 -
2 -
1 -
Figure 3.12. Comparison of simulation results from the structured PHB model with
experimental data (Heinzle and Lafferty, 1980).
3.3.1.2
Case D.
Modelling of Sustained Oscillations in

Continuous Baker's Yeast Culture
Oscillations of continuous culture of baker's yeast have been observed by a

number of researchers. An example of such sustained oscillations is shown in
Fig. 3.13 (Heinzle et al., 1983).
95
15
10
10
0.5
X [g/L]
'1 DO [mg/L]
0.05
0.0
0.5
E [g/L]
S [mg/L]
0
100
2.0
50
1.5
RQ
q X [mmol/h L]
1.0
0
10
20
t[h]
Figure 3.13. Oscillating profiles from a continuous culture of S. cerevisiae. (Heinzle et al.,
1983). Symbols used are X (total biomass), DO (dissolved oxygen), E (ethanol), S (glucose),
QCO2 and QO2 (specific gas reaction rates), and RQ (respiratory quotient).
One possible mechanism to explain the observed results is that a storage

material (G) having the same oxidation state as glucose (S) must cyclically be
produced and consumed. This storage material was identified as trehalose and
glycogen. Under conditions of high glucose uptake rate and high degradation
Figure 3.14. Structured model to describe Baker's Yeast oscillations.
96
rate of G, ethanol, E, is accumulated in the medium and can be later oxidized

to yield biomass, R. From this and additional information on the activity of the
enzyme T, which catalyzes the degradation of G, the reaction scheme shown in
Fig. 3.14 was developed and used as a basis for model formulation.
Here R is the biomass, not including the intracellular storage material, G. The
enzyme, T, is not considered to contribute significantly to the total biomass and
was therefore neglected in the total biomass balance. The detailed kinetic model
for continuous culture is given in the simulation example YEASTOSC, Sec.
8.8.8.
Many parameters could be determined from independent experiments. Some
were taken from the literature, and some, especially those describing the
enzyme T activity had to be based on simulation results. The model leads to
oscillations (Fig. 3.15), whose existence and dependency on operating
conditions agree qualitatively with experimental observations.
(g/L)
10
Time (h)
15
Time (h)
D=0.05 h'1
I ' ' ' ' I

10
15
Time (h)
Figure 3.15. Simulation of the Baker's yeast model (simulation example YEASTOSC, Sec.
8.8.8, showing oscillations of all the components, Q.
97
3.3.1.3 Case E. Growth and Product Formation of an OxygenSensitive Bacillus subtilis Culture
This example shows how knowledge of the biochemical pathways, when
combined with experimental data, can lead to model development. In this
research an oxygen-sensitive culture was to be used for mixing studies, and it
was important to establish the kinetic model (Moes et al, 1985, 1986) in order
to describe the batch profiles as shown in Fig. 3.17.
Since it was not possible to describe the growth behavior by simple Monodtype models, an ATP balance was used to establish the available energy for
biomass synthesis. This was possible because the biochemical pathways (Fig.
3.18) for the fermentation and their associated chemical energy production and
consumption steps were known.
Gl
Ac, Bu (g/L)
X (9/L)
- 3
10
- 2
- 1
Figure 3.17. Growth and product formation of Bacillus subtilis at constant DO. Gl - glucose,
X - biomass, Ac - acetoin, Bu - butanediol.
The formulation starts with a steady state ATP balance, which assumes that all
energy-producing steps are balanced by those that consume energy. The form
of this balance is as fpllows:
i A ' I' U
dt
= 0 = [ qs/co2 YATF/s,co2 + qs/Ac YATP/S,AC +

u - qBu/Ac) YATP/BU - qATp/x ]X
98
In this equation q$/GO2 is the rate at which sugar S is converted to CC>2 by

respirative growth. The rate of the sugar conversion to acetoin is qs/Ac- The
rates of conversion of acetoin to butanediol and the reverse reaction are given
by qAc/Bu, and QBu/Ac- Energy is required for growth, and the ATP
consumption rate is qATP/X- The corresponding ATP yields Y convert these
rates to ATP equivalents. The nomenclature at the end of this example defines
all symbols in detail.
Here knowledge of the yields of ATP for each step is important. Rate
expressions for the reaction pathways, as given in Fig. 3.18, were thus
established for each step in terms of the participating reactants. Batch mass
balances for all species were then written in terms of the individual rates of
production and consumption of each relevant component.
NADH
Cells
Glucose
>*
6CO 2
2Pyruvate
NADH
ATP
Acetoin ^
^ NAD+
NADH
ADP
>
Butanediol
NAD"1"
^ ATP
Figure 3.18. Biochemical pathways for the production of acetoin and butanediol.
Using the steady-state approximation that the ATP level does not vary
significantly, allows setting the condition that dATP/dt = 0. The steady state
ATP balance is then solved for qATP/X, which is the rate of ATP available for
growth. The required yields can be calculated from the reactions given in Fig.
3.17. In these calculations it was assumed that at high oxygen concentration 1
mol NADH was converted to 3 mol ATP in the respiratory chain. At low
oxygen concentrations, the conversion equivalent was assumed to be a function
of oxygen as determined by parameter estimation, based on the experimental
data.
The glucose substrate balance can be written in terms of the rates at which
substrate was consumed for complete oxidation (qs/GO2) fr biomass
(qATP/X YX/ATP / YX/S) and for product formation (qs/Ac)'
dS
,.
qATP/x YX/ATP
99
It was observed experimentally that the reaction stoichiometry for the

formation of metabolites was almost constant and was given by Yp/S = 0.57
mol (Ac + Bu)/mol glucose. Thus, another balance can be written for the
substrate:
_ qS/Ac X
dt
Y
P/S
These two equations were used to determine dS/dt and qs/GO2The biomass balance is,
dX
qATP/x YX/ATP X
The metabolite balances are,

dAc
"dt" = ( qS/Ac ~ qAc/Bu + qBu/Ac )
and,
dBu
~dt~
( Âc/flu ~ qBu/Ac ) X
In the above balances, all specific rate terms, q, are in the units
(mol/g biomass h). All concentrations (ATP, S, Ac, Bu) are in mol/L units
except X (g/L). All yield coefficients Y are in mol/mol units except when
involving biomass, e.g. YX/S is in units of g/mol.
Empirical Monod-type kinetic relationships, not given here, were established
to calculate the rate of glucose to acetoin, qs/Ac an(i the reversible acetoin to
butanediol rates, qAc/Bu and qBu/Ac as a function of reactant concentrations for
glucose, S, for acetoin, Ac, for butanediol, Bu, and for dissolved oxygen, DO.
Additional empirical kinetic terms were needed to fit the following
experimental observations:
1)
2)
3)
4)
5)
Growth stopped when S approached zero.

qs/Ac was limited by C>2
qBu/Ac was promoted by high C>2
qAc/Bu was promoted by low C>2
The influence of DO with S = 0 was negligible.
The many kinetic parameters were determined partly by direct experiments and
partly by fitting the data using a parameter estimation computer program. The
influence of oxygen was determined using data from experiments at controlled
oxygen conditions and determining the best values of the oxygen sensitive rates
by parameter estimation procedures. A simple graphical procedure then
allowed determination of the appropriate constants.
100
The quantities YATP/NADH and YX/ATP are linearly dependent on each other and
could therefore not be determined from experimental data. The maximum
value of YATP/NADH was arbitrarily fixed at the maximum theoretical value of
3.0, which has a direct influence on the estimation of YX/ATP (here 5.7 g/mol).
Good agreement of the batch curves with the model at constant DO was
achieved as shown in Fig. 3.18. From these results it is seen that the model
predicts the metabolite and biomass profiles. The model was quite versatile and
reasonably accurate, considering the large differences in biomass formation at
high DO (X = 3.4 g/L) and low DO (X = 2.5 g/L), as well as the variation of the
butanediol formation at high DO (Bu = 0.2 g/L) and low DO (Bu = 2 g/L),
X(g/L)
GI(g/L)
t(h)
Figure 3.19. Comparison of simulation results with the Bacillus subtilis fermentation (Moes
et al., 1986). X=biomass, Ac=acetoin, Bu=butanediol, Gl=glucose.
The simulation example SUBTILIS, Sec. 8.9.2, employs a more conventional

approach to the kinetics but also makes use of the biochemical pathways.
Bioreactor Modelling
4.1
General Balances for Tank-Type Biological

Reactors
Fermentation systems obey the same fundamental mass and energy balance
relationships as do chemical reaction systems, but special difficulties arise in
biological reactor modelling, owing to uncertainties in the kinetic rate
expression and the reaction stoichiometry. In what follows, material balance
equations are derived for the total mass, the mass of substrate and the cell mass
for the case of the stirred tank bioreactor system as shown in Fig. 4.1.
' X0! F0
1f
F1
Figure 4.1. The variables for a tank fermenter.
In this generalized case, feed enters the reactor at a volumetric flow rate FQ, with
cell concentration, XQ, and substrate concentration, SQ. The vessel contents,
which are well-mixed are defined by volume V, substrate concentration Si and
cell concentration X\. These concentrations are identical to those of the outlet
stream, which has a volumetric flow rate FI.

ISBN: 3-527-30759-1
102
4 Bioreactor Modelling
General Balance Form

As shown previously, the general balance form can be derived by setting:
(Rate of accumulation) = (Input rate) - (Output rate) + (Production rate)
and can be applied to the whole volume of the tank contents.
Expressing the balance in equation form, gives:
Total mass balance:
d(Vp)
= P(FO-FI)
Substrate balance:
d(VSi)
gj = F o S o - F i S i + r s V
Organism balance:
d(V Xi)
J J t = FO XQ - FI X j + rx V
where the units are: V (m3), p (kg/m3), F (m3/s), S (kg/m3), X (kg/m3) with rs
and rx (kg/m3 s).
The rate expressions can be simply:
= KS + S!
and using a constant yield coefficient,
-*x
fs =
Yxl
but other forms of rate equation may equally apply.

The above generalized forms of equations can be simplified to fit particular
cases of bioreactor operation.
4.1 General Balances for Tank-Type Biological Reactors
4.1.1
103
The Batch Fermenter
A batch fermenter is shown schematically in Fig. 4.2.
Jiiilii
Figure 4.2. The batch fermenter and variables.
Starting from an inoculum (X at t = 0) and an initial quantity of limiting

substrate, S at t = 0, the biomass will grow, perhaps after a short lag phase, and
consume substrate. As the substrate becomes exhausted, the growth rate will
slow and become zero when substrate is completely depleted. The above
general balances describe the particular case of a batch fermentation if V is
constant and F = 0. Thus,
Total balance:
Substrate balance:
dSi
V^r = r s V
Organism balance:
V
T = rxV
Suitable rate expressions for r$ and rx and the specification of the initial
conditions would complete the batch fermenter model, which describes the
exponential and limiting growth phases but not the lag phase. The simulation
example BATFERM, Sec. 8.1.1, demonstrates use of this model.
104
4.1.2 The Chemostat

A chemostat, as shown in Fig. 4.3, normally operates with sterile feed (Xo = 0)
at constant volume steady state conditions, meaning that dV/dt = 0,
d(VSi)/dt = 0, d(VXi)/dt = 0. In addition constant density conditions can be
taken to apply
UN
Figure 4.3. The chemostat and its variables.
The total mass balance simplifies to,
o = FQThe dynamic component balances are then,

Substrate balance
VdSi
= F(S 0 -Si)
Cell balance
VdXi
= F(X0-Xi)
where F is the volumetric flow through the system.

These dynamic equations are used in the simulation example CHEMO, Sec.
8.1.2.
At steady state, dSi/dt = 0 and dXi/dt = 0, Hence for the substrate balance:
0 = F (S0 - Si) + rs V
105
Chemostats normally operate with sterile feed, XQ = 0, and hence for the cell
balance,
0 = - F X! + rx V
Inserting the Monod-type rate expressions gives:
For the cell balance,
FXi
= rx = Ji X!
hence
F
H = v =
Here D is the dilution rate and is equal to 1/T, where i = V/F and is equal to the
tank mean residence time.
For the substrate balance,
]
from which:
Xi = YX/S (So-Si)
Thus the specific growth rate in a chemostat is controlled by the feed flowrate,
since [I is equal to D at steady state conditions.
Since |Li, the specific growth rate, is a function of the substrate concentration,
and |Li is also determined by dilution rate, the flow rate F then also determines
the outlet substrate concentration Si. The last equation is, of course, also
simply a statement that the quantity of cells produced is proportional to the
quantity of substrate consumed, as related by the yield factor YX/SThe curves in Fig. 4.4 represent solutions to the steady-state chemostat
model as obtained from the simulation example CHEMOSTA, Sec. 8.4.1, with
KS = 1.0.
The variables Xi, Si, as well as the productivity DXi are plotted versus D.
Thus as flow rate is increased, D also increases and causes the steady state value
of Si to increase and the corresponding value of Xi to decrease. It is seen
when D nears |im, Xi becomes zero and Si rises to the inlet value SQ. This
corresponds to a complete removal of the cells by flow out of the tank; a
phenomenon known as "washout". Fig. 4.4 shows washout occurring for D well
below |jm, which is caused by the large value of KS. . When D is nearly zero
(low flow rates) then Si approaches zero, and Xi approaches YSo The
productivity of biomass, DXi, (kg X/m3 h) passes through a maximum rather
close to the washout region.
106
X1,S1,DX1
10.0 T
5.0 -
0.25
1.0
D (1/h)
Figure 4.4. Variation of the steady state variables in a chemostat with Monod kinetics as a
function of dilution rate.
Chemostat applications are largely found in research laboratories. Microbial

physiology studies can be made conveniently, since the cells can be controlled
by the flow rate to grow at a particular value of specific growth rate \i. Kinetic
studies can be made by measuring the concentration of the limiting substrate
for a range of |u (=D) values, permitting the kinetics, |j = f(Si), to be
determined. The yield coefficient can be determined by steady state
measurements of substrate, biomass and product. The influence of any
substrate in the culture can be tested by adding it to the medium at various
concentrations.
4.1.3
The Fed Batch Fermenter
As shown in Fig 4.5 the outlet is zero for a fed batch fermenter, and the inlet
flow, FO, may be variable. As a result the reactor volume will change with time.
107
S.F,
o1 ' o
Figure 4.5. The fed batch fermenter and its variables.
The balance equations then become for constant density,

dT
T7
0
d(VSi)
= F0 S0 + rs V
dt
d(V XQ
= rxV
dt
Expanding the differential terms, which are products of the two variables V
and Si and V and Xi, respectively, and substituting for dV/dt = FQ gives:
VdSi
= F 0 (S 0 -Si)
The above equations are identical to those for a constant volume chemostat
reactor. It can be shown by simulation that a quasi-steady state can be reached
where dXi/dt = 0 and fi = F/V (Dunn and Mor, 1975) as seen in the Fig. 4.7.
Since V increases, therefore n must decrease, and thus the reactor moves
through a series of changing steady states for which |a = D, during which Si
and p decrease, and Xi remains constant. This is analogous to a constant
volume reactor with slowly decreasing F. These phenomena are demonstrated
by the simulation examples FEDBAT, Sec. 8.1.3, and VARVOL, Sec. 8.3.1.
108
A fed batch fermenter, in which the inlet feed rate is very low, will not exhibit
a large increase in volume and will not reach a quasi-steady state, unless X is
very high. Assuming V to be approximately constant, the general equations
can be integrated analytically for the case of |j = constant, giving an
exponential increase in X. The constant |u condition is maintained by constant
Si, which can be obtained via exponential feeding. Another phenomenon can
be proven from these equations for the case of constant feed rate and
essentially constant V; this is the linear growth situation, where X increases
linearly with time. As shown in Fig. 4.6, the slope of the curve is related to the
feed rate and the yield coefficient. If V changes as a consequence of dilute
feed, then the total quantity of biomass (VX) will increase linearly.
FS
0 Y X/S
Figure 4.6. Linear growth under conditions of feed limitation with constant volume.
Figure 4.7. Repeated fed batch operation in terms of dimensionless variables for substrate
inhibition kinetics. Two cycles of operation are shown. The dimensionless variables are
defined in the simulation example VARVOL, Sec. 8.3.1.
109
Other important types of operation described by the general balances are

cyclic fed batch (Keller and Dunn, 1978). An example of cyclic fed batch
operation in which the quasi-steady state can be observed is shown in Fig. 4.7.
Similar results can be obtained from the simulation example REPFED.
4.1.4
Biomass Productivity
The specific biomass production rate for a chemostat, DXi, (kg biomass/m3 h)
can be calculated by applying the above model equations. Thus,
D X i = DY x /s(So-Si) = DYx/s
The conditions for the maximum value of DXi as shown in Fig. 4.4, can be
obtained by setting
d(DXi)
dD
or by running the simulation example CHEMOSTA, Sec. 8.4.1.

The production rate for a batch culture can be calculated by dividing the
biomass concentration by the time for the culture. Thus, the batch biomass
production rate is equal to Xf/tf.
Comparing the continuous biomass production rate to the batch biomass
production rate, it is found that the continuous fermenter will have a 2 - 3 times
larger maximum biomass production rate. This is because the batch
fermentation starts at the inoculum value of X and has correspondingly low
initial growth rates.
Biomass production rates for fed batch fermenters are calculated by taking
the total mass produced over the time of operation, or (VX)f - (VX)o/tf . For
cyclic operation the biomass production has been shown to depend on the
starting and final volume ratio (Keller and Dunn, 1978). This and more
complex questions regarding product productivity for particular kinetics can be
answered by making suitable changes in the simulation example REPFED, Sec.
8.3.4. The productivity of a repeated fed batch as compared to chemostat
operation will depend on the operating variables, as well as the kinetics (Dunn
et al., 1979).
110
4.1.5
Case Studies
4.1.5.1 Case A.
Continuous Fermentation with Biomass

Recycle
The retention of active biomass is a means of improving the reactor

productivity or efficiency for substrate uptake. The biomass separation could
be performed by any suitable process (flotation, sedimentation, membrane
filtration, or centrifugation). The cell recycle stream has a volumetric flow rate
R and a biomass recycle concentration XR, where X R > X I and Xi is the
biomass concentration in the stream leaving the fermenter.
Consider the operation of a biological reactor with cell recycle as shown in
Fig. 4.8.
S^X^F+R
Cell recycle
Figure 4.8. A bioreactor with cell separation and recycle.
The mass balances around the entire system are as follows: Biomass
accumulates both within the reactor of volume Vr and also within the separation
unit with volume Vs. Assuming that biomass leaves only in the wastage stream
and that growth occurs only in the reactor, the balance is then
dXi
where W is the wastage flow rate.

At steady state,
0 = -
=0-WXR
111
Thus, the wastage rate of biomass must equal its production rate, otherwise Xi
will change. Wastage rate is an important control parameter in wastewater
treatment, where the separator is usually a sedimentation tank.
For the substrate, which is consumed only in the reactor section,
dSi
dSR
At steady state,
0 = F So FI Si W Sj + r$ Vr
Here it is seen that the uptake rate is equal to the difference between the inlet
and outlet mass flows. The efficiency of the continuous biomass separation
determines XI/XR, which controls the recycle ratio, R/(F+R).
Considering the fermentation tank only, the balances are as follows:
For the biomass,
At steady state,
0 =RXR-(F + R)X!+rxVr
Cell separation and recycle lead to high cell concentrations in the reactor,
which, when neglecting the contribution by growth, would be XR/(F+R). Since
the rates are proportional to Xi, an increase in reactor efficiency is obtained.
Assuming, rx = |n Xi gives,
MX,
where, XR > Xi , and D = F/V is the nominal dilution rate. This equation means
that the specific growth rate is decreased from the chemostat value, D. This is
due to the reduction in substrate concentration, Si, which is caused by the
higher biomass concentration, resulting from the cell recycle. Washout is
impossible due to the complete biomass retention, and for this reason flow rates
greater than in a chemostat are possible.
The substrate balance gives
JQ
Vr == F S0 + R Si - (F + R) Si + rs V
dt
which shows that at steady state
112
This would also be the case without cell recycle, since the substrate is assumed
to pass unchanged in concentration through the separator.
The above equation can be written as
=
where the right hand side of this equation is the F/M (food/biomass) ratio. This
gives a theoretical basis to the F/M concept, which is well known for the control
of waste treatment plants. The simulation example ACTNITR, Sec. 8.4.3,
enables the main operating characteristics of cell recycle systems to be studied.
A related simulation example MEMINH, Sec. 8.9.1, considers the retention of
enzyme using a membrane, and SUBTILIS, Sec. 8.9.2, involves the retention of
biomass.
4.1.5.2
Case B.
Enzymatic Tanks-in-series Bioreactor

System
A three tanks-in-series system is used for an enzymatic reaction, as shown in

Fig. 4.9.
illi
11
: :';: y~: :
81
w>
Vv
S2
>
1
1
1
1
S;^;:-);'::;HaKS
>
illi
Figure 4.9. Tanks-in-series bioreactor.
For the first tank,
dSi
jp = F(S 0 -Si)
dividing by
SQ-SI
where i\ = Vi/F and is the mean residence time of the liquid in tank 1.
The balances for tanks 2 and 3 have the same form except for the subscripts:
4.2 Modelling Tubular Plug Flow Bioreacrors
113
For known flow rate, F, and known tank volumes, there are six unknowns in
these three equations. Note that different tank sizes may be accounted for by
differing values for the tank residence times TI, 12 and 13.
If the kinetic terms r$ are only dependent on S, then the above equations can
be solved without any further balances. It is often the case that enzymatic rate
equations of the form given below can be used for each tank n = 1, 2 and 3:
This gives now six equations and six unknowns, and the problem is solvable by
simulation methods.
If the situation is more complex, such that r$ depends on other components,
for example P, in the case of product inhibition or biomass X, then additional
balance equations for these components must be included in the model. When
combined with equations for the complete kinetics description (rates as a
function of all the influential concentrations), the model can be solved to obtain
the dynamics of the system and also the final steady state values. It can be
shown that a three tanks-in-series reactor system will provide a good
approximation to the performance of a corresponding tubular reactor, except
for very high conversions.
4.2
4.2.1
Modelling Tubular Plug Flow Bioreactors

Steady-State Balancing
The tubular reactor can be modelled for steady state conditions by considering
the flow as a series of fluid elements or disks of liquid, which behave as a batch
reactor during their time of passage through the reactor. This can be
understood by considering a pulse of unreacting tracer in Fig. 4.10 that passes
from entrance to exit unchanged without mixing.
114
tracer pulse
response
Figure 4.10. Plug flow idealization of the tubular reactor with no axial mixing.
A reaction will cause a steady state axial concentration profile as shown in Fig.
4.11. Thus at steady state, the concentrations vary with distance in a manner
which is analogous to the time-varying concentrations that occur in a batch
reactor.
Concentration
Figure 4.11. Axial profiles of steady-state concentrations in a tubular reactor.
This means that steady-state tubular reactor behavior can be modelled by direct
analogy to that of a simple batch reactor. Thus using the batch reactor
substrate balance (p = constant),
dS
dF = fS
The flow velocity, v, for the liquid is defined as,
v
dZ
= dF
or,
dt =
dZ
where v = F/A and F is the volumetric flow rate through the tube with crosssectional area A. Thus substituting for dt,
dS_
dZ
rs_
v
4.2 Modelling Tubular Plug Flow Bioreacrors
115
This is the steady state tubular reactor design equation.

With the kinetics model, rs = f(S), the equation can be integrated from the
inlet, at position Z = 0, to the outlet, at Z = L, to obtain the steady state
concentration profile of S. Additional component balances would be required
for more complex kinetics. This is demonstrated by the simulation example
for a tubular enzyme reactor ENZTUBE, Sec. 8.4.4.
4.2.2
Unsteady-State Balancing for Tubular

Bioreactors
If dynamic information is needed for tubular or column systems, then changes

with respect to both length and time must be considered. In order to achieve
this, the reactor can be considered by dividing the volume of the reactor into N
finite-differenced axial segments (Fig. 4.12), and treating each segment
effectively as a separate stirred tank.
)))))>
Figure 4.12. Finite-differencing the tubular reactor.
Figure 4.13. Balancing the difference segment n for the tubular reactor.
Formulating the substrate balance for S over a single segment n of volume AV

= A AZ:
f Accumulation^ ( input \
{ output \
^production rate\
V rate of S ) = Vrate of S) ~ \ rate of S) + ^ of S by growth)
The balances have the same form. Thus for segment n,
AV
dSn
"dT = S n-l F n-l - Sn Fn + rSn AV
Constant density gives F n _i = Fn = F. Dividing by AV,
116
dSn
dt
A(SF)
+ rsn
AV
Setting AV AdZ and AS > dS gives the partial differential equation, which
describes changes in time and distance, as,
38
at
1 d(SF)
-- x
A az
J_
When the volumetric flow, F, is constant,
At steady state,
as
_F as + rs
at ~ ~ A az
and
V
+ fS
dZ
which is the steady state equation derived earlier in Section 4.2.1.

To model the dynamics by simulation methods, the partial differential
equation must be written in difference form as,
AV ^- = F (Sn.j - Sn) + rsn AV
or
dt
AV/F
where AV/F = x , the residence time of the liquid in a single segment.
Mass Transfer
5.1
Mass Transfer in Biological Reactors
Multiphase reaction systems usually involve the transport of material between

two or more phases. Usually one of the reactants is transferred from one phase
into a second phase, in which the reaction takes place. The following cases are
examples of biological systems.
5.1.1 Gas Absorption with Bioreaction in the Liquid

Phase
The gas phase is dispersed as gas bubbles within the liquid phase. Mass transfer
occurs across the gas-liquid interface, out of the gas into the liquid, where the
reaction occurs. The typical example is aeration of the bioreactor broth and
the supply of oxygen to the cells as shown in Fig. 5.1.
Figure 5.1. Absorption of oxygen from an air bubble to the liquid medium.

ISBN: 3-527-30759-1
118
5.1.2
5 Mass Transfer
Liquid-Liquid Extraction with Bioreaction in

One Phase
An immiscible liquid phase is dispersed in a continuous liquid phase. Mass

transfer of a reactant takes place across the liquid-liquid interface, shown here
(Fig. 5.2) from the continuous phase into the dispersed phase, where reaction
occurs. An example might be the transfer of a substrate in an oil phase to an
enzyme in the droplet aqueous phase.
Figure 5.2. Liquid-liquid extraction plus reaction.
5.1.3
Surface Biocatalysis
In this case, a liquid phase is in contact with solid biocatalyst. Substrates A and
B diffuse from the liquid to the reaction sites on the surface of the solid, where
reaction occurs. The product C must similarly be transferred away from the
solid reaction surface, as shown in Fig. 5.3. Examples are found with
immobilized enzyme and cell systems. In Sec. 6.1 the modelling aspects of this
type of system are considered in detail.
5.2 Interface Gas-Liquid Mass Transfer
119
Figure 5.3. Reaction of two substrates on a solid biocatalyst surface.
5.1.4
Diffusion and Reaction in Porous Biocatalyst
Here a porous biocatalyst sphere is suspended in a liquid medium. Substrates

diffuse into the porous internal structure of the biocatalyst support and react.
Similarly, the products must diffuse away from the reaction sites within the
solid to the outer surface, where they are then transported into the liquid.
Detailed modelling of this process is treated in Ch. 6.
Figure 5.4. Reaction within a solid biocatalyst.
5.2
Interphase Gas-Liquid Mass Transfer
Concentration gradients are the driving forces for mass transfer. Actual
concentration gradients (Fig. 5.5) in the very near vicinity of the gas-liquid
120
5 Mass Transfer
interface, under mass transfer conditions, are very complex. They result from
an interaction between the mass transfer process and the local fluid
hydrodynamics, which change gradually from stagnant flow, close to the
interface, to perhaps fully-developed turbulence within each of the bulk phases.
According to the Two-Film Theory, the actual concentration profiles, as
represented in Fig. 5.5 can be approximated by linear gradients, as shown in
Fig. 5.6.
A thin film of fluid is assumed to exist at either side of the interface. Away
from these films, each fluid is assumed to be in fully developed turbulent flow.
There is therefore no resistance to mass transfer within the bulk phases, and the
concentrations, CG and CL, are uniform throughout each relevant phase. At the
phase interface itself, it is assumed there is no resistance to mass transfer, and
the interfacial concentrations, CGI and CLI, are therefore in local equilibrium
with each other. All the resistance to mass transfer must, therefore, occur within
the films. In each film, the flow of fluid is assumed to be stagnant, and mass
transfer is assumed to occur only by molecular diffusion and therefore to be
Interface
Gas
Figure 5.5. Concentration gradients at a gas-liquid interface.
described by Pick's law, which says that the flux JA (mol/s m2) for the molecular
diffusion of some component A is given by,
dZ
121
5.2 Interface Gas-Liquid Mass Transfer
Gas
Interface
Liquid
Figure 5.6. Concentration gradients according to the Two-Film Theory.
where D is the molecular diffusion coefficient (m2/s) and dC/dZ is the steady
state concentration gradient (mol/m3). Thus applying the same concept to mass
transfer across the two films,
JA = DG
where DG and DL are the effective diffusivities of each film, and ZG and ZL are
the respective thicknesses of the two films.
The above equations can be expressed in terms of mass transfer coefficients
kc and kL (m2/s) for the gas and liquid films,
JA = k G (C G -C G i) = k L (C Li -C L )
The total rate of mass transfer, Q (mol/s), is given by,
Q = JAA = jA(aV)
where "A" is the total interfacial area available for mass transfer, and "a" is
defined as the specific area for mass transfer or interfacial area per unit liquid
volume (m2/m3). Thus for the total rate of mass transfer:
In terms of the total interfacial area A,
Q = k G A(C G -C G i ) = k L A(C L i -C L )
In terms of a and VL,
Q = ko a (Co - CGi) VL = k L a(C L i -C L )V L
122
5 Mass Transfer
Since the mass transfer coefficient, k, and the specific interfacial area, a, depend
on the same hydrodynamic conditions and system physical properties, they are
frequently combined and referred to as a "ka value" or more properly a mass
transfer capacity coefficient.
In the above theory, the interfacial concentrations CGI and CLI cannot be
measured, and are therefore of relatively little use, even if the values of the film
coefficients are known. For this reason, by analogy to the film equations,
overall mass transfer rate equations are defined, based on overall coefficients of
mass transfer, KG and KL, and overall concentration driving force terms, where:
Q = K G A(C G -C G *) = K L A(C L *-C L )
Here, CG* and CL* are the respective equilibrium concentrations, corresponding
to the bulk phase concentrations, CL and CG, respectively, as shown in Fig. 5.6.
Equilibrium relationships for gas-liquid systems, at low concentrations of
component A usually obey Henry's law, which is a linear relation between gas
partial pressure, PA, and equilibrium liquid phase concentration, CLA*:
PA=
where HA (bar m3/kg) is the Henry's law constant for component A in the
medium. Henry's law is generally accurate for gases with low solubility, such as
the solubility of oxygen in water or in fermentation media. Thus from this
relation, as shown in Fig. 5.7, the corresponding equilibrium concentrations can
be easily established.
CLC*
Figure 5.7. Equilibrium concentrations based on Henry's law.
For gases of low solubility, e.g., oxygen and carbon dioxide in water, the
concentration gradient through the gas film is very small, as compared to that
within the liquid film, as illustrated in Fig. 5.6. This results from the relatively
5.3 General Oxygen Balances for Gas-Liquid Transfer
123
low resistance to mass transfer in the gas film, as compared to the much greater
resistance to mass transfer in the liquid film. The main resistance to mass
transfer is predominantly within the liquid film. This causes a large change in
concentration (Cy - CL), since the resistance is almost entirely on the liquid
side of the interface.
At the interface, the liquid concentration, Cy, is in equilibrium with that of
the gas, CGI, and since CGI is very close in magnitude to the bulk gas
concentration, CLI must then be very nearly in equilibrium with the bulk gas
phase concentration, CG- This is known as liquid film control and corresponds
to the situation where the overall resistance to mass transfer resides almost
entirely within the liquid phase. The overall mass transfer capacity coefficient
is KLa. Hence the overall mass transfer rate equation used for slightly soluble
gases in terms of the specific area is
Q = KLa (C L *-C L )V L
where CL* is in equilibrium with CG, as given by Henry's law,
C G = HCL*,
Mass transfer coefficients in fermentation are therefore generally spoken of as
KL values or Kâ values for the case of mass transfer capacity coefficients.
5.3
General Oxygen Balances for Gas-Liquid

Transfer
In order to characterize aeration efficiency, to predict dissolved oxygen

concentration, or to follow the biological activity it is necessary to develop
models, which include expressions for the rate of oxygen transfer and the rate
of oxygen uptake by the cells. Well-mixed phase regions, in which the oxygen
concentration can be assumed uniform, can be described by simple balancing
methods. Situations in which spatial variations occur require more complex
models, as described in Sec. 5.4. The following generalized oxygen balance
equations are derived for well-mixed phases, using the well-mixed tank
concept. In the situation in Fig. 5.8, both the liquid and gas phases are defined
by distinct well-mixed regions and by the total volumes of each phase, VL and
VG.
124
5 Mass Transfer
Gas
CQO GO
Figure 5.8. The balance regions for well-mixed gas and liquid phases in a continuous reactor.
For the gas phase the oxygen balance can be developed as follows:
Rate of
accumulation
of oxygen
in gas
( Rate of ^
>
Flow of
f Flow of \
transfer
oxygen in
_ oxygen out
_ of oxygen
Vin exit stream/
; inlet gas streamy
v from gas ,
Thus, for the gas phase,

dCGi
K L a(C L i*- C L i)V L
where VQ represents the volume of gas in the dispersed phase, or the gas
holdup.
For the liquid phase,
Rate of
^
accumulation
of oxygen
^ in liquid
/Flow of
( Flow of
oxygen
oxygen
in inlet
out in
liquid
exit
V stream J
' Rate of
consumption
of oxygen
in liquid
Rate of oxygen consumption = -rO2 = -qo2
Rate of
transfer
of oxygen
^ from gas
125
Thus for the liquid phase,

dCLi
- LiC L i + K L a(C L 1 *-C L i)V L -
The above equations include accumulation, convective flow, interphase transfer

and biological oxygen uptake terms. Here CLI* is the equilibrium solubility of
oxygen corresponding to the gas phase concentration, CGI , and is calculated
by Henry's law, according to the relationship:
Typical units are as follows: CG and CL (kg/m3); G and L (m3/s); Kâ (1/s);
VG and VL (m3); qO2 (kg/kg s); X (kg/m3).
In the next sections, the general equations, given above, will be applied to
important special situations.
5.3.1
5.3.1.1
Application of Oxygen Balances
Case A. Steady-State Gas Balance to Determine the

Biological Uptake Rate
The convective terms in the generalized liquid balance equation can usually be
neglected, owing to the low solubilities of oxygen in water (about 8 g/m3). This
gives the steady state liquid balance, dCL/dt=0, relation as:
K L a(C L i*- CLI) = qo2Xi
Thus at steady-state, the oxygen transfer rate is effectively equal to the oxygen
uptake rate. Even during batch fermentations this is approximately true.
Substituting this relationship into the steady state gas balance gives,
0 = GO CGO - GI CGI - qo2 X VL

The above equation can also be derived from a steady state balance around the
entire two-phase system. It shows that the biological oxygen uptake rate can be
calculated from knowledge of the gas flow rates and the gas concentrations.
126
5 Mass Transfer
This application is very important in fermentation technology, since it permits

the on-line monitoring of the rate of fermentation, by gas balancing methods
(Heinzle and Dunn, 1991, Ingham and Dunn, 1991).
5.3.1.2
Case B. Determination of Ki,a Using the Sulfite

Oxidation Reaction
If a chemical reaction, classically the oxidation of sodium sulfite, is used to take

up the oxygen from solution, then the term qo2 X VL in the liquid phase
balance may be replaced by the chemical reaction term, ro2 VL- At steadystate,
K L a(C L i*-C L i) = r02
Usually ro2 is obtained by taking samples and titrating for the fractional
conversion of sulfite, which can be related by stoichiometry to the oxygen
consumption.
Since the chemical reaction causes the liquid dissolved
concentration CLI to fall essentially to zero and with CLI* calculated from the
oxygen concentration in the exit gas, the value of the overall mass transfer
capacity coefficient, Kâ, can be estimated. An improved method uses the gas
balance instead of titration to obtain ro2 in the manner outlined above for
qO2 X and also provides a check on the sulfite measurements. The sulfite
method is useful for comparing aeration systems, but the values are difficult to
relate to actual fermentation conditions owing to the very different physical
conditions (coalescence, aeration rates) (Ruchti et al., 1985).
5.3.1.3
Case C. Determination of Ki,a by a Dynamic Method
If water is initially deoxygenated and is then re-aerated, the concentration of

the dissolved oxygen will increase with time, from zero to effectively 100% air
saturation at the end of the experiment. The exact form of the response curve
obtained depends on the values of KLa, the driving force, (CLI* - CLI), and the
measurement dynamics of the dissolved oxygen electrode. The liquid balance,
for the unsteady state batch aeration condition, gives:
=
K L a(CLi*-C L i)V L
127
The classical dynamic KLa method assumes that Kâ and CLI* are constant.
Under these conditions, the differential equation can be integrated analytically
to give the relationship:
CL* ^
= K L at
r *
CL - rCL\}
Plotting the natural logarithmic concentration function on the left side of the
equation versus time, should, in principle, give a straight-line relationship, with
â as the slope.
Usually deoxygenation is accomplished with nitrogen, so that initially the gas
phase consists of nitrogen, which is gradually displaced and mixed with air.
Under these conditions, CLI* is nt constant, and a gas balance must be
employed to calculate the variation in CGI versus t. Since the liquid phase
concentration, CLI, is measured by means of a membrane covered oxygen
electrode, the dynamics of measurement method usually cannot be neglected.
The dynamics of the measurement electrode can be described, approximately,
by a first-order lag equation,
dCE
where TE represents the electrode time constant, and CE is the measurement

signal.
The fractional response of the electrode for a step change in CL would
appear as shown in Fig. 5.9.
time
Figure 5.9. Response of electrode for a step change in CE from zero to 100 % saturation
according to a first-order lag model.
Note that TE corresponds to the time for the electrode to reach 63 % of the final
response. The overall process dynamics involves thus the gas phase, the liquid
128
5 Mass Transfer
phase and the electrode response. The three responses might appear as shown
below:
time
Figure 5.10. Response of the gas, the liquid and the electrode measurement during a dynamic
KLa experiment.
The values of three individual time constants determine the process response.
These are TG = VG/G, (representing the dynamics of the gas phase), 1/KLa
(representing the dynamics of the liquid phase mass transfer process), and IE
(representing the measurement dynamics). This is illustrated in the simulation
example KLADYN, Sec. 8.5.5.
5.3.1.4
Case D. Determination of Oxygen Uptake Rates by a

Dynamic Method
Low oxygen uptake rates, as exist in slow growing systems (plant and animal
cell cultures, aerobic sewage treatment processes, etc.), cannot easily be
measured by a gas balance method, since the measured difference between inlet
and outlet oxygen gas phase concentrations is usually very small. Due to the
low solubility of oxygen in the liquid media, quite small oxygen uptake rates
will cause measurably large changes in the dissolved oxygen concentration.
Thus it is possible to measure qo2 X either by taking a sample and placing it in
a small chamber or by turning off the reactor air supply, according to the
liquid balance equation
dCLi
129
Dissolved oxygen concentration decreases linearly and is equal to qo2 X as

shown in Fig. 5.11.
Figure 5.11. Oxygen uptake rate determined by a dynamic method.
When the time required for an appreciable decrease in dissolved oxygen is

large, as compared to the electrode time constant, the method is quite accurate
and no correction for the electrode measurement dynamics is required (Mona
et al., 1979). If the response is too fast the sample can be diluted. This method
is illustrated by the simulation example OXDYN, Sec. 8.5.4. A similar
simulation example, ANAMEAS, Sec. 8.8.7, illustrates dynamic measurements
in anaerobic systems.
5.3.1.5
Case E. Steady-State Liquid Balancing to Determine

Oxygen Uptake Rate
If the biomass is immobilized or retained by membranes within the reactor,

oxygen can be supplied to the cells by means of a circulating liquid supply,
which is aerated in a separate unit, external to the reactor.
130
5 Mass Transfer
CL1
CLO
Figure 5.12 Oxygen uptake rate determined by a steady-state liquid balance.
It then becomes possible to determine the oxygen uptake rate, simply by

measuring the liquid flow rate and the difference in dissolved oxygen in the
liquid inlet and outlet flow streams, according to the following steady-state
liquid phase balance equation:
0 = L(CLo - CLI) - qo2 Xi VL
Thus the rate of oxygen supply via the liquid is equal to the rate of oxygen
uptake by the cells. This method provides a very sensitive way of measuring
low oxygen uptake rates (Keller et al., 1992, Tanaka et al., 1982). The casestudy H in Sec. 5.3.1.8 is an example of this use for an experimental reactor.
The simulation example FBR, Sec. 8.4.9, also demonstrates this method.
5.3.1.6
Case F.
KLa
Steady-State Deoxygenated Feed Method for
Feeding a deoxygenated liquid continuously to an aerated tank (Fig. 5.11)

allows the oxygen transfer rate to be determined by difference measurement.
Thus the liquid phase balance becomes
0 = L (CLO - CLI) + KLa (CLi* - C L i)V L
Knowing the flow rate L, the oxygen liquid concentrations CLO and CLI and the
outlet oxygen in the gas phase (to determine CLI*) permits the calculation of
131
KLa. Another variation of this would be to gas with oxygen-enriched air or

with nitrogen, which would avoid the difficulty of producing a continuous
source of deoxygenated liquid. A similar steady state method has been
employed to obtain steady oxygen concentration profiles in column (Meister et
al., 1980), and tubular bioreactors (Ziegler et al., 1977). A suitable steady state
model for the tubular reactor then allows calculating the unknown Kâ by
parameter estimation (Shioya et al., 1978).
Deoxygenated
C
liquid
LO
/"VX
"N^
-^^^
r-^^-~\_/~\^^x
nM&^/m^M:WM.
Xiiiiiiiiiiji ^^^SRSSSS
^^îO^wiBii
O
Wiiiiilmiiiiiii
Air
Figure 5.12. Steady-state dissolved oxygen difference measurement for Kjâ.
5.3.1.7
Case G. Biological Oxidation in an Aerated Tank
A batch reactor liquid is aerated with a continuous flow of air to support a

biological reaction, as shown in Fig. 5.13.
air
air
Figure 5.13. A batch bioreactor with continuous aeration.
132
5 Mass Transfer
The biological reaction in the liquid phase is first-order in oxygen

concentration. Since oxygen is relatively insoluble (approximately 8 g/m3
saturation for air-water) the transfer rate is important to maintain a high
dissolved oxygen concentration CL. The batch oxygen balance for the liquid
phase is then:
f
Rate of
\
accumulation of
V 02 in liquid )
dCL
,^
f
(Transfer
rate of f N\
l02 mto the hqmdj
/ Uptake rate of
^
by the cells
= K L a(CL*-C L )VL - k C L V L
A steady-state can be reached for which the mass transfer rate is equal to the
oxygen uptake rate by reaction:
0 = KLa (CL* - CL) - k CL
giving for CL
KLaCL*
CL = K L a
Using this equation, the reaction rate constant, k, can be determined if CL is

measured and Kâ is known or measured. The equilibrium value, CL*, can be
calculated from the gas phase concentration, and if there is little oxygen
depletion it can be calculated from the inlet gas conditions.
If the oxygen depletion in the gas phase is appreciable, then the mole
fraction of oxygen in the exit may not be the same as in the inlet, and a gas
phase balance must be applied to determine CL*:
Figure 5.14. Inlet and outlet oxygen mole fractions and total gas molar flow rates.
133
From the ideal gas law as shown before, assuming a well-mixed gas phase in
steady state, N = (p / RT) F, where NO is the molar flow rate of air and F is the
air volumetric flow rate.
0
/ Rate of O2 \
/ Rate of O2 \
/ Transfer rate of \
V in by flow ) ~ Vout byflow) ~ \ C>2 to the liquid )
Using the nomenclature in Fig 5.13,
0 = yoN0-yiNi-KLa(CL*-CL)VL
where
Assuming NO = NI, these equations can be solved to obtain yi and CLSolving for CL gives,
k~" CL*
CL =
or for the apparent reaction rate,
k
re = -
k~ CL
Thus it is possible to distinguish between two different regimes for this system,
transfer control and reaction control:
1)
2)
3)
Reaction rate control applies for low values of k/KLa, when re approaches
k CL*, and CL approaches CL*
Diffusion control applies for high values of k/KLa, when re approaches
KL& CL* and CL approaches 0.
If KLa = k, then rc = - (k/2) CL*, and CL approaches (1/2) CL*.
5.3.1.8
Case H. Modelling Nitrification in a Fluidized Bed

Biofilm Reactor
Nitrification is a two-step microbiological process, in which the ammonium ion

is oxidized to nitrite ion and further to nitrate ion as shown:
NH4+ - NO2- - NO3-
134
5 Mass Transfer
This reaction is important in waste water treatment because of the toxicity of

ammonia and its large oxygen demand. Several known organisms can gain
energy from either of the two oxidation steps, but most commonly
Nitrosomonas and Nitrobacter are responsible for steps (1) and (2),
respectively. These organisms grow very slowly, obtaining their carbon from
dissolved carbonate. Due to the very slow grow rates, it is of interest to retain
the biomass within the reactor.
One possibility considered here is to
immobilize the biomass as a natural biofilm on a fluidized bed of sand (Tanaka
and Dunn, 1982).
The stoichiometric relations for the reaction steps (1) and (2) are:
, 3
,
NH4+ + j O2 -> NO2" + H2O + 2 H+
O2 -> NO3"
Summing the above steps (1) and (2) gives

NH4+ + 2O 2 The reactor of volume, Vr, consisted of a conical sand bed column, which was
fluidized by the liquid recycle stream flowing up through the bed. The recycle
stream was oxygenated in a separate, baffled, tank contactor of volume VT, with
turbine impeller and air or oxygen sparging. The reactor and oxygenator were
thus separate parts of a recycle loop configuration. This could be operated
batchwise or with a continuous feed and effluent stream flow to and from the
system. When operating at high recycle rates, the whole system acted
effectively as one well-mixed tank system.
The reactor-oxygenator recycle loop can be analyzed as a total system or
broken down into its individual components as shown in Fig. 5.14. These
include liquid phase balance over the reactor and combined phase, liquid phase
and gas phase balances over the oxygenator and over the total system.
135
Figure 5.15. Mass balancing regions for the fluidized bed reactor nitrification system.
The mass balances to be considered are those for oxygen and the nitrogencontaining reactants and products. The oxygen balance taken over the total
system can be simplified by neglecting the accumulation terms and the liquid
flow terms, that will be small compared to the gas rates and the consumption by
reaction, owing to the relatively low solubility of oxygen in the liquid medium.
Thus the oxygen balance becomes,
0 =
Here Vr is the volume of the reactor column.

The nitrogen (N) components, NH4+, NCV, and NOs', in the liquid phase
can be balanced around the total system by considering the accumulation, flow,
and reaction terms for each of the N-containing components. For the total
system each component equation has the form,
VdCp
= F(C N i-C N 2 )
dt
When the reactor is operated as a batch system, F = 0, and when used as a
continuous steady state reactor, dCN2/dt = 0. This equation can be used in
column systems for very low single-pass conversion, when the differences in
local reaction rate at the reactor inlet and outlet are not large. Although the
reactions actually occur in the solid phase, because of the high solid-liquid
interfacial area, the system is treated here as being quasi-homogeneous.
The gas-liquid interfacial mass transfer area will often be small enough to be
important for the overall process, and it is therefore useful to consider the gas
136
5 Mass Transfer
and liquid phases as separate balance regions. The absorption tank can be
described by the oxygen balances for the liquid phase:
0 = F R (C L4 -C L3 ) + K L a(C L *-C L3 )V T
and for the gas phase:
0 = G(CGi - CG2) - KLa (CL* - CL3) VT
The liquid phase oxygen balance for the total system is
0 = KL
where ro2 is the oxygen uptake rate by the reaction. These equations, which
assume ideally mixed phases, are useful in designing the gas absorber
according to the required oxygen transfer coefficient.
Balancing the oxygen around the reactor gives
0 =
Since CL4 at the reactor outlet is usually very low, then,

FR CL3 = - ro2 Vr
which says that the oxygen uptake rate by reaction must be equal to the supply
rate from the oxygenation tank. This is the condition of reaction-rate
limitation by the oxygen transfer in the absorber.
From the stoichiometry, the relationships between the molar reaction rates
(rNH4> rO2 rH* r2,NO2 an^ fNO3) can be found. Thus, for example, the first
nitrification step gives
TNH4 =
2
T r l , O 2 = ~ri,NO2
and the total rate for 02 is given by the sum of the rates for steps (1) and (2).
r
O2 = r l,O2 + r2,O2
From the measured concentration dependency of these rates, the reaction

kinetics of the individual steps can be determined. The dependency of these
rates on the individual concentrations can then be used to establish the reaction
kinetic model. This model is the basis of the simulation example NITRIF, Sec.
8.5.3. A similar type of recycle, fluidized-bed reactor is the theme of
simulation examples FBR, Sec. 8.4.9 and DCMDEG, Sec. 8.4.6.
5.4 Models for Oxygen Transfer in Large Scale Bioreactors
5.4
137
Models for Oxygen Transfer in Large Scale

Bioreactors
Large-scale industrial fermenters can generally be expected to exhibit

deviations from the two idealized flow conditions of perfect mixing or perfect
plug flow. Thus the assumption of completely mixed gas or liquid phases may
not be valid. Little experimental information is available on concentration
inhomogeneities or concentration gradients within large bioreactors. Residence
time distribution information, from which a physical and mathematical model
could be established, is also generally not available.
Convection currents within the liquid phase of a bioreactor are usually caused
by the mechanical energy inputs of agitation and aeration. It is often
reasonable to assume that slowly changing quantities, such as biomass
concentration, substrate concentration, pH and temperature are uniform within
the whole mass of bioreactor liquid. Oxygen must be considered, however, as a
rapidly changing substrate, owing to its low solubility in fermentation media. It
is therefore necessary to consider that differences in oxygen transfer and
uptake rates will create oxygen concentration gradients throughout the reactor.
Buoyancy forces carry the gas from the lower gas inlet point up to the top
liquid surface. In the absence of mechanical agitation, the gas phase might
move from the bottom to the top of the reactor in an approximate plug flow
manner, with very little backmixing. If the stirring power supplied to the
fermenter, however, is sufficient to create liquid velocities, that are greater than
the free rise velocity of a bubble (about 26 cm/sec) then the bubbles will
circulate around the fermenter, before eventually escaping. Very high power
inputs can cause the smaller bubbles to circulate many times within the vessel
and spend an appreciable time before reaching the surface. Under such
conditions, if no bubble coalescence occurs, the gas phase would contain a
fraction of small bubbles, depleted of oxygen but with a large surface area.
Obviously any well-mixed phase assumption becomes difficult to justify.
The gas phase flow conditions in large scale industrial fermenters usually lie
somewhere between the extreme cases of idealized plug flow and perfect
mixing. Experimental residence-time distribution information, obtained by
helium tracer techniques under actual operating conditions, are then necessary
to characterize the gas phase flow. Unfortunately very little experimentation on
industrial scale equipment has been reported.
Hydrostatic pressure gradients in tall fermenters will cause large differences
in the oxygen solubility, CL*, with regard to the depth position in the tank. In a
10m tall reactor, the oxygen solubility for a given gas composition will be twice
that at the bottom of the tank as compared to the top surface, since the total
pressure is effectively doubled. This is seen by Henry's law which can be written
as:
138
5 Mass Transfer
d* = yH2p
where yo2 is the mole fraction of oxygen in the air and p is the total pressure at
some point in the tank.
The possibility that oxygen gas compositions, dissolved oxygen
concentrations, oxygen solubilities, gas holdup volumes, bubble sizes and other
transfer parameters can vary with depth in a tall fermenter introduces a much
greater degree of complexity to the problem of modelling the reactor. This
makes it difficult to obtain data on oxygen mass transfer coefficients.
Although it is impossible to give specific recommendations that apply to any
particular situation, a further discussion of possible models and their
underlying assumptions may help to define the problem. Incorporated into the
more complex models, discussed below, are such factors as gas and liquid phase
flow pattern, gas composition gradients and the effects of hydrostatic pressure.
Great caution and wisdom must be exercised to avoid creating a model that is
too complex to verify by experimentation. Experienced engineers will say
"Keep it simple!" and "Avoid too much model!".
All large scale reactors, whether multi-impeller tanks or column fermenters,
will display some axial dissolved oxygen concentration gradients. The most
general method for modelling is to represent the reactor using balances in a
series of sections or stages. Mass balances in multi-stage process are easy to
formulate, since both the liquid and gas phases may be assumed to be wellmixed, for any given stage of the cascade.
Figure 5.16. A single gas-liquid stage with backmixing of the liquid phase.
The formulation of the mass balances for a single stage, as shown in Fig.
5.16, follows closely that described previously, except that now the reactor is
made up of many stages which are interconnected by the flows of gas and
liquid between stages and by diffusive mass transfer mechanisms.
139
5.4.1.
Case Studies
5.4.1.1
Case A. Model for Oxygen Gradients in a Bubble

Column Bioreactor
The application of the stagewise modelling approach is shown below, where a

bubble column reactor is modelled as a five-stage reactor system. The reactor
will be assumed to operate cocurrently, as would be also the case for the riser of
an airlift bioreactor.
Exit Gas
Exit Liquid
A CQB A CLS
Gas
Gas Feed
ill
Liquid Feed
GO
Figure 5.17. Stagewise model of a bubble column bioreactor.
L'
LO
140
5 Mass Transfer
The oxygen balance equations for the gas and liquid phases of each stage are
as follows:
if
= FG(C G n-l-C Gn ) - K L a(C Ln *-C Ln )V L
f\(~^
VL-dT = F L(C L n-i-C Ln ) + K L a(C L n*-C L n)VL - rn VL

where,
r<~Ln * - 1
r c * C
H r
Gn or CLn
02
-77rl
and
rn = Qo2m |
For simplification only oxygen is assumed limiting and growth is not

considered; however the biomass concentration is contained in the maximum
oxygen uptake Qo2m- The dynamics of the oxygen transfer and uptake
processes are obtained by solving these differential equations simultaneously
for each stage. The resulting solution then gives CLH and CGn > for each stage
as functions of time and also yields the resulting final steady state values.
Note that the biomass concentrations Xn are assumed constant, otherwise
biomass balance and growth kinetics equations would have to be added to the
model. Using simulation methods, other effects, such as the effect of
hydrostatic pressure on CG or on bubble size could be included. The
simulation example DCMDEG, Sec. 8.4.6, demonstrates some aspects of the
stagewise modelling approach.
5.4.1.2
Case B. Model for a Multiple Impeller Fermenter
Mixing in a tank reactor is complex, and it would be necessary to consider

liquid flow in both directions. It is generally assumed, however, that the
intensity of mixing is such that no radial variations occur. Fig. 5.16 represents
a multiple impeller reactor with well-mixed liquid zones in the region of each
impeller. The reactor can be described approximately by means of a threestage model. Mixing of the liquid in a direction which is directly opposite to
that of the main flow liquid (here upwards) can be incorporated into the model,
by the assumption of a backmixing stream, with flow rate FB- This backmixing
stream accounts for a flow interaction between the mixing zones and for
deviations from ideal stage mixing. To determine FB, a tracer experiment
141
would need to be performed to obtain the necessary information regarding the

degree of backmixing actually existing in the reactor.
Exit Gas
Exit Liquid
FG3 A
A C L3
\_^
Gas
L2
L3
G2
Gas
'LI
G1
FL+FB
Gas
G- C GO
\\>cu>
Liquid Feed
Inlet Gas
Figure 5.18. Stagewise approximation for stirrer regions in multi-stirrer tank.
To model this system, the liquid-phase impeller zones are assumed to be wellmixed, and the plug-flow gas is described by a series of well-mixed phases,
together with an arithmetic-mean, concentration-driving-force approximation.
Here the flow rates and mass transfer coefficients are assumed constant.
Stage 1:
dCLi
VL -ar
= FLCLO + FBCL2 - (FL + FB) CLI +
+ K L a(CLi*-C L i)VL + riV L
V
dCGl
"""'
xx-<
-CGI) -KLa(CLi -CLI)VL

f\
-rr-
//~1
/"I
\A 7
where the plug flow nature of the gas is partially accounted for by
142
5 Mass Transfer
and
CLI
= - Qo2r
Stage 2:
dCL2
VL -gf = (FL+ FB) CLI + FBCL3 - (FL+ FB) CL2 - FB CL2
K L a(C L 2*-C L 2)V L
= FG (CGI - CG2) - KLa (CL2* - CL2)VL

where
CL2. .
CL2
n
r2 = -Qo2mK 0 + CL2
and
Stage 3:
dCL3
K L a(C L 3*-CL3)V L + r 3 V L
V
- CG3) - KLa (CL3*- CL3) VL
where
and
CL3. .
CL3
= -QO2n K0 + CL3
The above equations describe the dynamic oxygen concentrations in the multiimpeller continuous bioreactor. Note that the liquid phase balances for the two
end stages 1 and 3 differ from that of the intermediate stage 2, owing to the
143
absence of any backmixing flow contribution exterior to the column. A batch

reactor would be described by setting the liquid flow, FL, equal to zero. Since
the biomass balance and growth kinetics are not included here, the solution
would be valid at only one time during the fermentation, corresponding to the
assumed value of Qo2m> which is proportional to the value of X existing at that
time. Variations in X are, however, easily incorporated into the model by
adding cell and substrate balance equations.
Diffusion and Biological Reaction in

Immobilized Biocatalyst Systems
The retention and immobilization of enzymes and cells usually requires the
presence of an additional solid carrier phase or flocculant cell mass. As
illustrated in Fig 6.1, in order to reach a reaction site, substrate S must first be
transported by convection from the bulk liquid to the exterior stagnant film
(point A). Then transport by diffusion must occur through the film (from A to
B) to the surface of the carrier (point B), where surface reaction can take place.
If further reaction sites are available within the carrier matrix, an additional
internal diffusion path (from B to C) is then also required. Similarly product P,
formed within the carrier matrix, must diffuse out of the matrix towards the
surface, and then away from the surface via the external mass transfer laminar
film to the bulk liquid.
Concentration
Diffusion film
Bulk
liquid
Solid
carrier
Figure 6.1. Concentration profiles for a biocatalyst immobilized on a solid carrier.
The stagnant film and the immobilization matrix constitute mass transfer
resistances which may slow the overall reaction rate, since reaction cannot
proceed at a rate greater than the rate at which substrate is supplied by the
mechanism of diffusion. The diffusional mass transfer process via the external
film is referred to as external mass transfer. Since the reaction site may often
be located within a gel, a porous solid, biofilm or biofloc, the transfer of
substrate or substrates from the exterior surface of the biocatalyst to reaction
sites, located within the internal structure of the carrier, is also usually
necessary. This process is therefore referred to as internal mass transfer or
intraparticle transfer. In what follows, external transport and internal transport
ISBN: 3-527-30759-1
146
6 Diffusion and Biological Reaction in Immobilized Biocatalyst Systems
are considered separately, although, of course, the two effects can exert a
combined effect in reducing the effective reaction rate, compared to that which
would be obtained if there were no diffusional limitations.
6.1
External Mass Transfer
Fig. 6.2 illustrates the substrate concentration profile, existing in the very near
region of an immobilized biocatalyst surface, supported on a non-porous
carrier. Also shown is the idealized concentration profile, as represented by the
film theory. As previously discussed in Sec. 5.2, the "film theory" assumes the
presence of a stagnant layer of liquid to exist at the solid-liquid interface. This
stagnant region is termed the diffusion film or Nernst-diffusion film and
constitutes the external resistance to mass transfer. It thus determines the rate of
supply of substrate to the surface, for subsequent reaction.
Substrate cone.
SA A
Figure 6.2. External diffusion model of substrate transport to a reactive enzyme immobilized
on a solid surface.
The rate of supply of substrate to the surface is defined by mass transfer

considerations, such that the mass flux to the catalyst surface is given by,
JS = ks L (SA-S B )
147
6.1 External Mass Transfer
where, js is the mass flux (mol/m2 s), ksL is the mass transfer coefficient (m/s)
and SA, SB are the substrate concentrations for the bulk and surface conditions
[mol/m3], respectively.
The steady-state balance can be written for the transport-reaction process,
(Rate of supply by diffusion)
= (Surface reaction rate)
SL (SA - SB) = ks SB = rapp
In the following treatment, the surface reaction is assumed to be first-order,

such as found for a biocatalytic reaction with Michaelis-Menten kinetics and
S KM- The apparent reaction rate per unit surface area, r app (mol/m2 s), is
equal to the rate of both processes.
Solving the equation, for the surface concentration, SB,
SB =
-SA
and hence
= ksSB=U^
l k S+ k SL
Two extreme conditions can be identified:

1) For ks/ksL 1, SB approaches zero, and the reaction is completely mass
transfer controlled, with rapp =
2) For ks/ksL .! SB approaches SA, and the reaction is kinetically
controlled, with an apparent rate equal to that defined by the reaction
kinetics, with rapp = ks SAThe intermediate situation is given by the full equation, for which the apparent
reaction rate is influenced by both the true kinetic rate constant ks and by the
diffusional mass transfer coefficient ksLFor a zero-order reaction:
k S L(S A -S B ) = ks
where ks is now a zero-order kinetic rate constant. The concentration at the
reaction surface SB is thus,
SB = S A -ks/k S L
148
which indicates that the ratio of the magnitudes of the kinetic rate constant to
that of the mass transfer coefficient determines SB- If the reaction is zeroorder, the overall order of reaction rate is not influenced by diffusional
considerations, but the effective rate will still be reduced, owing to the lowered
concentration SBFor Michaelis-Menten kinetics, which encompass the range between effective
zero and first order reaction kinetics, the relation between rate of supply and
the rate of reaction becomes,
kSL (SA - SB) = : > - = r app
After rearrangement, the resulting quadratic equation can be solved for SB, with
the solution indicating that, in general, the magnitudes of all the coefficients
can influence the overall reaction rate and also that the external transfer can
change the overall observed reaction kinetics. Thus they no longer follow the
Michaelis-Menten form with respect to bulk concentration, and the apparent
kinetics can differ substantially from the intrinsic true reaction kinetics. Under
these conditions, it is no longer correct to equate the Michaelis-Menten
constant, KM, to the substrate concentration at which the observed reaction rate
is equal to the half of the maximum observed rate. This can be most easily
seen from the above equation; when SB is low, the effective surface rate reduces
to the form, rapp = vm SB/KM- The overall reaction rate then becomes,
=
app
(v m /K M )S A
(v m /K M k S L ) + l
showing that the apparent rate of reaction depends on the magnitude of the
mass transfer coefficient
It is only possible to measure true reaction kinetics, by operating
experiments in a truly kinetic regime, such that any influence of the external
diffusional mass transfer is negligible. This can be achieved by ensuring that
the ratio of vm/ksL is sufficiently low. Under these conditions,
which are the intrinsic Michaelis-Menten kinetics.

The ratio can be made low by increasing the mass transfer coefficient, k$L,
and by increasing the mass transfer rate enhancing parameters, such as flow
velocity and stirring speed. Conversely those factors affecting the maximum
reaction rate, vm, should be decreased, for example enzyme loading and
temperature.
The regimes of possible external mass transfer influence on the observed
kinetics are summarized in Table 6.1, together with the important parameters.
6.1 External Mass Transfer
149
Table 6.1. Characteristics of overall reaction rate influenced by external

transfer.
Regime of operation
Reaction parameter having an influence

on overall rate
Transfer control
Temperature (slight influence due to viscosity and

diffusion rate). Stirring speed in tank. Flow in packed
and fluidized beds. S in bulk liquid.
Kinetics control
S in bulk liquid. Enzyme loading on surface.

Temperature.
Intermediate regime
All of above.
6.2
Internal Diffusion and Reaction within

Biocatalysts
Reactions with enzymes and whole cells entrapped or immobilized in a porous

solid matrix will be subjected to a mass transfer influence. Example systems
are whole cells immobilized in alginate, enzymes adsorbed on ion-exchange
resins, or naturally occurring biological films on surfaces or flocculated
biomass. In the case of a biological film attached to an impermeable solid, the
substrate can enter from only one surface, as shown in Fig. 6.1, through the
diffusion layer A-B and into the biocatalyst B-C.
In the case of an alginate bead, a biofloc or its two-dimensional
approximation, substrate can enter from opposing directions, as shown below in
Fig 6.3. In this case, the diffusion will result in a symmetrical, steady state
concentration profile. The case of complete penetration of substrate through
the biofloc is shown by the solid line. Whereas an incomplete penetration, as
shown by the dashed line, results in the center of the film being completely
ineffective, in terms of reaction capability. Note that in this case, the effects of
external diffusion are neglected.
The uptake of substrates within solid material requires transport by a
diffusional process.
The driving force for diffusion is a gradient in
concentration, and the diffusional flux is given by Pick's law,
150
jA = -D A
dCA
dZ
with j having units of kg/m2 s.
'AO
'AO
Diffusion
Diffusion
'AO
Figure 6.3. Internal concentration profiles in a symmetrical rectangular biocatalyst matrix.
If a reaction occurs within the matrix, a concentration gradient will be

established as a result of the simultaneous diffusion and reaction processes.
The reaction rate at each position, being usually a function of concentration,
will vary, and the overall or apparent reaction rate per unit volume of matrix
(kg/s m3), rapp, will be determined by the transfer rate at the surface (kg/s),
/Apparent rate\
Vin bulk liquid)
/Rate of substrate^ /Net rate of reaction^

\entering matrix ) = v within matrix )
Vr a p p = (j|z=o)A = r a v g A L
where the units of each term are kg/s. Here ravg represents an average value in
the matrix, which will increase with higher internal substrate concentrations.
Regarding the influence of diffusion for a particular situation, it is possible
to arrive at some quantitative guidelines without considering any mathematical
details. Obviously the concentration profiles are caused by a competition
between reaction and diffusion. The ratio of the maximum intrinsic reaction
rate (not influenced by transfer) to maximum diffusion rate provides a useful
dimensionless parameter,
6.2 Internal Diffusion and Reaction within Biocatalyst

r
max A L
r(C 0 ) A L
JA
D (Co/L) A
151
maximum reaction rate

maximum diffusion rate
For first order reaction, r = k CQ, this dimensionless group becomes k L2/D,
and for zero order reaction, r = k, it is k L2/D CQ.
Therefore for any kinetic form of equation, the distance coordinate or length
of diffusion path, L, plays an important role since the ratio of maximum
diffusion rate to maximum reaction rate varies according to L2. The higher the
value of this ratio, the greater in magnitude are the substrate gradients. With
this qualitative feeling for diffusion-reaction phenomena, more quantitative
aspects can be considered.
6.2.1
Derivation of Finite Difference Model for

Diffusion-Reaction Systems
Diffusion with biological reaction can be treated by mathematical modelling,

and from this it is possible to develop equations describing changes of
concentration, with respect to both time and position. The same technique of
finite differencing is used as in the modelling of the dynamic behavior of
tubular bioreactors, Sec. 4.2.2.
Consider the case where the substrate varies from a concentration SQ in the
bulk liquid to some concentration, at the position L (a wall or the center of a
symmetrical particle). At the center by symmetry or at a wall, owing to the
absence of diffusion into the wall, the concentration gradient must be zero.
The actual continuous concentration profile, through the slab, may be
approximated by a series of increments, as indicated in Fig. 6.4 and by a series
of biocatalyst matrix elements as shown in Fig. 6.5.
Liquid
Figure 6.4. Finite differencing a solid, showing concentration gradient approximation.
152
In
jn-1
n-1
>
,
I n+1
n+1
Figure 6.5. Series of volume elements connected with diffusion fluxes.
A magnified view of element n is shown in Fig. 6.6, where the flux, jn, depends
on the local concentration gradient, and the reaction rate, rsn, depends on the
local concentration in element n.
Jn-1
AZ
Figure 6.6. A single element n of volume AV and thickness AZ, showing the diffusion fluxes.
A component mass balance is written for each segment and for each
component as
/Accumulation^
V
rate
)
A AZ
/Diffusion^ /Diffusion\ / Production \

\ rate in ) ~ \ rate out ) + \rate by reaction/
dSn
~dT = Jn-1 A - jn A + rSn A AZ
Using Pick's law in the difference form,
(Sn-i ~ Sn)
s
and similarly for Jn gives,

n-l ~ Sn)
+rSnAAZ
153
Dividing by A AZ,
dSn
T
(S n ,j -2S n + S n+ i)
s
+ rsn
The equivalent partial differential equation is,
as
a2s
+ rs
The finite-differenced forms of the model equations, however, are especially

suitable for simulation programming. Thus, N equations are obtained, one
substrate balance equation for each element, and these are solved
simultaneously. Note that the boundary conditions, for elements 1 and N, must
be described separately.
For the above case, the boundary conditions are dS/dZ = 0 at Z = L and
S = SQ at Z = 0. Thus the equations for the first and last elements must be
written accordingly, as shown in simulation example BIOFILM, Sec. 8.7.1.
Note also that it would be also possible, in principle, to include external
diffusion effects, by formulating a boundary condition, balance for the first
element as:
/Accumulation^
V
rate
/
/External trans- A /Diffusion^ f Production \

V port rate in ) ~ \ rate out ) + Vrate by reaction/
where the external transport rate through area A is,

Q = k S L (So-Si)A
where SR is the bulk reactor concentration.
Coupling the Biocatalyst Matrix to the Reactor Liquid.

The biocatalyst diffusion model can be combined with a well-mixed tank
model, as shown in Fig. 6.7. The bulk liquid-phase component balances take
the form:
^= |(S F -SO)-Jsa| z = 0
where a=A/V,
,
A dS ,
Js a| z=o = - DS v dZ I z=o
where,
154
dS dZ I z=o =
The direction of the positive diffusion is into the biofilm, since dS/dZ is
negative and (SR - Si) /AZ is positive. Here SR corresponds to So in Fig. 6.4,
and Si is the concentration in the first element. The boundary condition is
S = SR at Z = 0.
F,SF
F,SO
Figure 6.7. Coupling the biofilm model to the continuous tank model.
In this way it is possible to simulate immobilized biocatalyst performance in a

single tank or in a column by using a tanks-in-series model, The simulation
example BIOFILM, Sec. 8.7.1, demonstrates this approach.
6.2.2
Dimensionless Parameters from DiffusionReaction Models
There are several advantages of formulating model equations in dimensionless

form. The number of variables in the model is reduced, thus reducing the
number of experiments or simulations required to investigate all combinations.
It is also possible, on the basis of the numerical values of the parameters, to
access the relative importance of certain terms. Finally, the dimensionless form
makes the solution much more generalized because the units of the individual
quantities are no longer important.
The governing dimensionless parameters can be obtained by re-examining
the defining model equations and arranging them such that the variables range
155
only between the values of zero and unity. Thus new dimensionless variables,
S=S/S 0 , Z = Z/L and dimensionless time, f = t / ( L I D ) , can be defined.
Substituting these new variables into the diffusion-reaction, partial differential
equation, for the case of a first order biochemical reaction, gives,
as
S
(L /D8)at
as
or
_
at - az2 " DS
_
s
Thus the solution depends only on the value of [lq L2/DsL which is a
dimensionless diffusion-reaction parameter.
For zero-order reaction the
equation becomes,
as a2s2
at " az
where ko L2/Ds SQ is the governing parameter.
It is seen that the dimensionless parameters in the model have the same form
and significance as was derived from the qualitative reasoning presented earlier.
For heterogeneous reaction systems this dimensionless group is known as the
Damkohler Number, and its square root is called the Thiele Modulus.
In the above equations, all the terms, excepting that of the reaction term, have
dimensionless parameters of unity. On this basis, it can be said that if the
reaction parameter for a first order reaction, [ki L2/DsL has a value of 1.0 or
greater, then the reaction will have a large effect on the solution, that is, on the
concentration gradients. Similarly, ko L2/D$ SQ will govern a zero order
reaction. Such "order of magnitude analysis" is important for physical
understanding and also to obtain information from differential equations
without having to actually develop an analytical solution.
Dimensionless formulation of equations is also explained in the simulation
example VARVOL, Sec. 8.3.1, and KLADYN, Sec. 8.5.5.
6.2.3
The Effectiveness Factor Concept
The relative influence of diffusion on biochemical reaction rate, can be

expressed by means of an effectiveness factor, T|, where,
156
T|
actual apparent rate

rate at bulk liquid concentration
Solutions of the above diffusion reaction model are available in the literature
for simple reaction orders (Satterfield and Sherwood, 1963). In Fig. 6.8 the
values of t| for zero, first and second reaction order have been plotted against
the Thiele Modulus ,<f>, where,
O = '\/L 2 kSo n - 1 /D
This figure shows that a zero order reaction is not influenced by concentration
gradients until the substrate falls to zero in the matrix, corresponding to O > \2
. The other reaction-types are influenced by low concentrations, as the curves
for T| indicate. It is seen, for example, that for a first order reaction a value of
O = l corresponds to t| = 0.8.
1.0
0.8
0.6
0.4
0.2
Incomplete penetration at O >V~2~
A
A = zero-order
B = first-order
C = second-order
0 = L(kSo n ' 1 /D) 172

Figure 6.8. Effectiveness factor TJ versus the dimensionless reaction/diffusion parameter
(Thiele Modulus O ) for reactions in a flat film with diffusion from one side (after Satterfield and
Sherwood, 1963).
157
6.2.4
Case Studies for Diffusion with Biological

Reaction
6.2.4.1
Case A.
Estimation of Oxygen Diffusion Effects in a

Biofilm
For a biofilm or floe, whose oxygen uptake might be taken as a constant (zero
order), the corresponding group would be [L2 qo2 Xbi0fiim/D Co2L where qo2
Xbiofiim corresponds to the rate constant k and the oxygen concentration in the
outside liquid phase is Co2- Note that Xbiofiim is the biomass per unit of
biofilm volume and is not easy to measure. Substituting values obtained from
an aerobic biofilm nitrification experiment gives,
2
0
L2 qQ2 X
= D C02
(0.01 mm2) (80 mg O2/L min)

(0.1 mm2/min) (8 mg O2/L)
For this order-of-magnitude analysis, the value of 1.0 can be used to separate
the regions of reaction and diffusion dominance. Thus it is seen if L = 0.1 mm,
then the dimensionless group will have a value of 1.0, and it could therefore be
expected that a film or floe thickness greater than 0.1 mm would be oxygen
limited. From the exact solution, as seen in Fig. 6.8, gradients would appear for
a zero order reaction at a value of <&2 = 2.0, instead of 1.0. This example
shows how the Thiele Modulus can be used to make useful estimates for
diffusion reaction problems, providing rate and diffusion data are available.
6.2.4.2
Case B.
Complex Diffusion-Reaction Processes

(Biofilm Nitrification)
Nitrification reactions, considering only the substrate conversion reactions and

ignoring the slow organism growth processes, the reactions can be written as,
NH4+ + 3/2O 2 ->
N02- + 1/2 02 -> N03
158
The oxygen requirements for the first and second steps can be related to the
nitrogen content of NH4+ and NC>2~. These values are si = 3.5 mg 62 / mg
NH4+ - N and 82 = 1.1 mg 62 / mg NCV - N. The low yields and low growth
rates make it unnecessary to consider growth requirements and kinetics. In
previous work (Tanaka and Dunn, 1982) the intrinsic substrate uptake kinetics
for the two steps were shown to have a double Monod form for the first step,
rNH4 = v m i
. K NH4
and for the second step,

rN02 = vm2
where v m i and vm2 represent the maximum rates for a particular biomass
concentration and the chemical symbols represent concentrations.
Considering the diffusion phenomena in the biofilm to be represented by
one-dimensional diffusion with quasi-homogeneous reaction, differential
balance equations can be written for all reactants and products to describe the
concentration profile in the film. Proceeding as described in Sec. 6.2.1, a
component mass balance is written for segment n and for each component:
(Accumulation^ _ (Diffusion^ _ (Diffusion^
^
rate
) ~ \ rate *n ) ~ \ rate out J
( Production ^
Vrate ^ reactin J
and the equivalent partial differential equation is obtained by letting AZ

approach zero as
3S
32S
Applying this to each component gives the following balances:

For NH4+,
a2NH4+
3NH4+
= DNH4
For NO2",
aNQ2~3r
= D
NO2
^2
-wo
- TNH4
+ rNH4 - TNO2
159
For NO3%
32N03
3N03
For O2,
3202
" s l rNH4 ~
The stoichiometric oxygen requirements for the first and second reaction steps
are given by si and s2. The boundary conditions used represent the bulk liquid
phase or reactor concentrations and the zero gradient at the biofilm- solid
interface, as discussed earlier.
These equations can be written as differential-difference equations using the
finite-differencing technique (Sec. 6.2.1). Thus for each of N increments, four
component balances will be needed. Three simulation examples, BIOFILM,
ENZDYN, CELLDIFF in Sec. 8.7, demonstrate this approach.
This system was also analyzed in terms of dimensionless variables. A
comparison of the resulting dimensionless NH4+ and O2 balances reveals that,
when the second reaction is neglected, the equations are identical if
DNH4 = Do2 and if,
Q2R
where the subscript R refers to the concentrations in the bulk reactor liquid.
Under these conditions, to a good approximation, the penetration distances
of O2 and NH4+ would be the same. The ratio O2R/NH4+R, which can be varied
according to the reactor operating conditions, can thus be used as a criterion to
evaluate whether NtLj."1" or O2 might be penetration-limiting. The O2R/NH4+R
criterion indicates which component can be limiting, O2 if the ratio is less than
3.5 or NH4+ if the ratio is greater than 3.5. These conditions are not sufficient
for limitation, but indicate which component would be limiting. Simulation
results from a model that was developed using finite-differencing demonstrates
this phenomenon. The profiles in Fig. 6.9, are for the case O2R/NH4+R = 0.07.
160
NH 4 ,NO3,N0 2 (mg/L)
100
0 2 (mg/L)
20
80
60
NO
10
40
20
Figure 6.9. Steady state profiles for constant bulk concentrations showing incomplete
oxygen penetration.
Coupling the liquid and biofilm for a batch nitrification reactor as explained in
Fig. 6.7, gave the results in Fig. 6.10. Here the influence of oxygen limitation
caused the oxygen in the midpoint of the film to rise as the nitrogen substrates
were successively consumed.
NH4
0 2 [mg/L]
NO 3, NO2" (mg/L)
100
80
80
64
60
48
40
32
20
16
12
10
2
0
120
t (min)
Figure 6.10. Simulated biofilm nitrification profiles in a batch reactor. The N-component
concentrations are in the bulk liquid. O2 is in the midpoint of the biofilm and indicates
limitation during the first 60 minutes.
Automatic Bioprocess Control

Fundamentals
The purpose of automatic process control is to maintain time-dependent

changes of the relevant process variables (deviations, errors), within prescribed
limits and without a direct action of an operator. Process control may be
considered as a corrective action involving three steps:
1.
2.
3.
Measuring the variable to be controlled (controlled variable)

Comparing the measurement with the desired value (set point)
Adjusting some other variable (manipulated variable) that has a direct
effect on the controlled variable, until the set point is reached.
A number of advantages or reasons for process control may be listed, which

include uniform and higher quality products, safety, increase of productivity,
minimization of waste, optimization, freeing the labor force from drudgery and
danger, and decrease of labor costs.
Obviously, process control is highly dynamic in nature, and therefore its
modelling requires the solution of sets of differential equations, and it is
therefore highly suited to solution by digital simulation. A brief introduction to
the basic principles of process control required for solution of simple
simulation examples is given here.
7.1
Elements of Feedback Control
The simple temperature control of a fermenter shown in Fig. 7.1 illustrates the
essential idea of any automatic control system that the process and the
controller form a closed loop, which usually functions in a feedback fashion.

ISBN: 3-527-30759-1
162
7 Automatic Bioprocess Control Fundamentals
Controller
mechanism
Desired
value
I Measured
I value
I
Thermocouple
Fermenter
Figure 7.1. Simple feedback control system.
The components of such a control system can be best understood using a

generalized block diagram (Fig. 7.2). They are the process itself, the
measuring element (thermometer), the controller (including a comparator),
the final control element (automatic control valve) and the transmission lines.
The information on the measured variable, temperature, taken from the system
is used to manipulate the flow rate of the cooling water in order to keep the
temperature at the desired constant value, or set point.
Controller
mechanism
Load
Actuator
1 Com)jarator Controller
_
i'
$~
Desired+c
value | *A/
1
1
Measured
value
1
K
1
1
Process
-*U
Measuring
element
k
A
A
A
A
A
A
A
A
ÂÂÂAAÂ
Figure 7.2. Block diagram of the feedback control system in Fig. 7.1.
Controlled
variable
7.2 Types of Controller Action
163
Similar temperature control systems are given for a simple water heater,
TEMPCONT, in Sec. 8.6.1 and for a batch fermenter, FERMTEMP, in Sec.
8.6.2
7.2
7.2.1
Types of Controller Action

On-Off Control
The most common and simplest type of control is an on-off or two position
action, sometimes called discontinuous control (Fig. 7.3). An example is a
contact thermometer, which closes or opens the heater circuit. The controller
changes the value of the controller output, or the manipulated variable, from
one extreme to the other, when the controlled variable moves above or below
the set point. This leads to oscillations that could become very fast, depending
on the speed of response of the process. The real on-off controller has
therefore a built in feature called a differential gap or a dead zone. It is a small
interval on either side of the set point, within which the controller does not
respond. When the controlled variable moves outside the dead zone, the
manipulated variable goes on or off. This is illustrated in Fig. 7.3. Such shifts
from the set point are known as offset. Such a controller is simple and
inexpensive, but the oscillatory nature of the action and the offset make it very
imperfect.
The usefulness of this type of control was demonstrated for a biological,
sequential batch process by Hediger and Prenosil, (1985). More sophisticated
function control modes consider the magnitude and time behavior of the
control error. Three principal functional modes of control generally employed
for process control are proportional (P), integral (I) and derivative (D) control.
164
100
Manipulated
variable
0
Control
variable
Xl___4/_
.Set point
Differential gap
Figure 7.3. On-Off controller with differential gap or dead zone.
7.2.2
Proportional (P) Controller
The produced output signal P is proportional to the detected error, e, according

to
P = PO + Kp 8
where Kp is the proportional gain, and P0 is the controller output for zero error.
An example of a level control is shown in Fig. 7.4. The action of this type of
controller is shown in Fig. 7.5 and 7.6.
Figure 7.4. Response of proportional-mode controller to sinusoidal error input.
165
Systems with proportional control often exhibit pronounced oscillations and

for sustained changes in load the controlled variable attains a new equilibrium,
steady-state position, or control point. The difference between this point and
the set point is the offset (Fig. 7.5) Integral and derivative modes are used
mostly in combination with the basic proportional control mode. The
simulation Example INHIB, Sec. 8.5.2 includes the application of this simple
control mode in the recommended exercises.
F + AF
Setpoint
Figure 7.5. An example of proportional-mode level controller illustrating offset.
7.2.3
Proportional-Integral (PI) Controller
Pi-controller, sometimes called automatic reset, produces an output signal

related to the error by
Kpe+
P =
or for e = constant
dP
for t=Ti
dP
dF = K P
TI
166
where i\ is the integral time constant or reset time. This is the time required to
enable the controller to repeat the initial proportional output action (Fig. 7.6).
The integral part of the control mode eliminates the offset and it is especially
useful for correction of very small errors because the controller output P will
continue to change as long as an error persists. This can be understood by
considering a constant error, which would cause P to increase linearly (Fig. 7.6)
at a rate proportional to the error. This type of controller is found most often
and the simulation examples TEMPCONT, FERMTEMP, TURBCON and
CONTCON in Sec. 8.6 demonstrate the use of this control mode. The
examples also show the ease by which the programming of the PI controller
equations is made using the simulation language.
2Kp
Controlled
output
PI action
Kp
P action
Error
signal
Figure 7.6. Response of a proportional-integral controller to a unit step change in error.
7.2.4
Proportional-Derivative (PD) Controller
A controller with derivative function projects the error in the immediate future
and the controller output is proportional to the current rate of the error change.
The output signal varies only if the error is changing.
167
d
P = P0 + Kp e + Kp TD gj-
where TD is the derivative time constant. A PD-controller output is compared

with pure proportional mode in Fig. 7.7.
P (alone)
t
Figure 7.7. Response of a PD-Controller to a constant rate of decrease in error. Comparison
with P and PID modes.
The drawbacks of the derivative control mode standing alone are that a constant
error (e ^ 0) gives no response at all, since de/dt = 0, and therefore an
unnecessarily large response might occur as a result of small but fast error
changes.
7.2.5
Proportional-Integral-Derivative (PID)
Controller
In industrial practice it is common to combine all three modes, sometimes

termed as Proportional-Reset-Rate-Control. The action is proportional to the
error (P) and its change (D) and continues if residual error is present (I):
168
KD r
de
P = P0 + Kp e + ^ J dt+ Kp TD -gjTI
This combination gives the best control using conventional feedback

equipment. It retains the specific advantages of all three modes: proportional
correction (P), offset elimination (I) and stabilizing, quick-acting character,
which is especially suitable to overcome lag presence (D). The action of a PIDcontroller as a response to a ramp function is shown in Fig, 7.7. The
performance of the different feedback control modes can also be seen in Fig.
7.8.
Controlled
variable
/ Uncontrolled
response
Figure 7.7. Response of controlled variable to a step change in error using different control
modes.
The selection of the best mode of control depends largely on the process
characteristics. Further information can be found in the recommended texts
listed in the reference section. Simulation methods are often used for testing
control methods.
7.3 Controller Tuning
7.3
169
Controller Tuning
The purpose of controller tuning is to choose the controller constants, such that
the desired performance is obtained.
This usually means that the control
variables should be restored in an optimal way, following either a change in the
set point or as a result of an input disturbance to the system.
Thus the
controller constants can be set by experimentation. A rational basis for such
experimental tuning is given in what follows. Other methods for tuning
combine process dynamic experimentation with theoretically-based control
methods; some of the standard methods are also described below. The
simulation example TEMPCONT, Sec. 8.6.1, provides exercises for controller
tuning using the methods explained below.
7.3.1
Trial and Error Method
Controllers can be adjusted by changing the values of gain Kp, reset time i\ and
derivative time ID- By experimentation, either on the real system or by
simulation, the controller can be set by trial and error. Each time a disturbance
is made the response is noted. The following procedure might be used to test
the control with small set point or load changes:
1. Starting with a small value, Kp can be increased until the response is
unstable and oscillatory. This value is called the ultimate gain KPQ.
2. K p is then reduced by about 1/2.
3. Integral action is brought in with high i\ values; they are reduced by
factors of 2 until the response is oscillatory, and tj is set at 2 times this
value.
4. Include derivative action, increase ID until noise develops and set ID at
1/2 this value.
5. Increase Kp in small steps to achieve the best results.
7.3.2
Ziegler-Nichols Method
This method is an empirical open-loop tuning technique, obtained by

uncoupling the controller. It is based on the characteristic curve of the process
response to a step change in manipulated variable, equal to A. This response is
called a process reaction curve, whose magnitude is B. The two parameters
170
important for this method are given by the normalized slope of the tangent
through the inflection point, S = slope/A and by its intersection with the time
axis (lag time TL), as determined graphically in Fig. 7.9. The actual tuning
relations which are based on empirical criteria for the "best" closed-loop
response are given in Table 7.1.
Manipulated
Variable
X
Slope
Time
Figure 7.9. Process reaction curve as a response to a step change in manipulated variable.
7.3.3
Cohen-Coon Controller Settings
Cohen and Coon observed that the response of most uncontrolled (controller
disconnected) processes to a step change in the manipulated variable was a
sigmoidal shape curve. This can be modelled approximately by a first order
system with time lag TL, as given by the intersection of the tangent through the
inflection point with the time axis. The theoretical values of the controller
settings obtained by the analysis of this system are summarized in Table 7.1
The model parameters for a step change A to be used with Table 7.1 are
calculated as follows:
K = B/A
T = B/S
where B is from Fig. 7.9 and S is the slope at the inflection point/A.
7.3 Controller Tuning
171
Table 7.1. Controller Settings Based on Process Responses

Controller
Kp
Ziegler-Nichols
1/TLS
0.9/TL S
1.2/TLS
P
PI
PID
3.33TL
2TL
TL/2
Co/ten-Coon
P
TL \
PI
4
30 + 3TL/T
TL \
32 + 6TL/T
Ultimate Gain
PI
PID
7.3.4
0.5 Kpo
0.45 Kp0
0.6 Kpo
l/1.2fp0
l/2fpo
l/8fpO
Ultimate Gain Method
The previous tuning transient response methods are sensitive to disturbances

because they rely on open-loop experiments. Several closed loop methods
were developed to eliminate this drawback. One of them is the empirical tuning
method, ultimate gain or continuous-cycling method. The ultimate gain, Kpo,
is the gain which brings the system with the proportional control mode to
sustained oscillations (stability limits) of the frequency fpo, where l/f p o is called
the ultimate period. It is determined experimentally by increasing Kp from low
172
values in small increments until continuous cycling begins. The controller

settings are then calculated from Kpo and f p o according to the tuning rules
given in Table 7.1.
While this method is very simple it can be quite time consuming in terms of
number of trials required and if the process dynamics are slow. In addition, it
may be hazardous to experimentally force the system into unstable operation.
Simulation methods can be very useful if a suitable model is available as was
shown by Heinzle et al. (1992) for a one and two stage anaerobic system, using
a kinetic model similar to the simulation example, ANAMEAS, Sec. 8.8.7.
7.4
7.4.1
Advanced Control Strategies

Cascade Control
In control situations with more then one measured variable but only one
manipulated variable, it is advantageous to use control loops for each measured
variable in a master - slave relationship. In this, the output of the primary
controller is usually used as a set point for the slave or secondary loop.
This may be relevant for some wastewater treatment plants where the high
concentration of some substrate may be toxic for the microorganisms. For
example, the simulation Example TURBCON, Sec. 8.6.3, could be easily
adapted to this situation if the substrate concentration were subject to significant
changes, as shown schematically in Fig. 7.10.
173
7.4 Advanced Control Strategies
|
1
lilllll
:
';S:'|||::||:|-
XI
. .. N
/6oncentrationV/Concentriilonl
Controller
)H f errand
x.
,s
\ transmitter
/
^
Biomass
controller
V^
illlll
ill lit:
J_
[ Turbidometer
Figure 7.10. Cascade control of a fermenter with toxic substrate.
The simulation example TURBCON, Sec. 8.6.3. could be modified similarly by

adding biomass as a measured variable. An interested reader may try to
implement the cascade control strategy in these simulation programs.
7.4.2
Feed Forward Control
Feedback control may never be perfect as it only reacts to the disturbances

which are measured in the system output. The feedforward method tries to
eliminate this drawback by another approach. Rather than using the process
output as the measured variable, this is taken as the measured inlet disturbance
and its effect on the process is anticipated by means of a process model. Thus
action is taken on the manipulated variable by the model, which relates the
measured variable at the inlet, the manipulated variable and the process output.
The success of this control strategy depends largely on the accuracy of the
model prediction. For this reason sometimes an additional feedback loop is
used.
Many of the continuous process simulation examples in Ch. 8 could be
altered in this fashion. It would be interesting to program an example using
simple kinetics for the feedforward control and to describe the "actual" system
with more complex kinetics. The discrepancies between the "simple" model
174
prediction and the more complex "actual" process kinetics could then be taken
care of by a feedback control loop.
7.4.3
Adaptive Control
This control system can automatically modify its behavior according to the
changes in the system dynamics and disturbances. Especially systems with
nonlinear and unsteady characteristics call for use of this control strategy.
There are a number of actual adaptive control systems. Programmed or
scheduled adaptive control uses an auxiliary measured variable to identify
different process phases for which the control parameters can be either
programmed or scheduled. The "best" values of these parameters for each
process state must be known a priori. Sometimes adaptive controllers are used
to optimize two or more process outputs, by measuring these and fitting the
data with empirical functions, as employed on anaerobic treatment process, by
Ryhiner, et al. (1992).
7.4.4
Sampled-Data Control Systems
When discontinuous measurements are involved the control system is referred

to as sampled-data. Concentration measurements by chromatography would
represent such a case.
Controlled
variable
Figure 7.11. Sampled control strategy.
Here a special consideration must be given to the sampling interval T (Fig.

7.11). In general the sampling time will be short enough if the sampling
7.5 Concepts for Bioprocess Control
175
frequency is equal to 2 times the highest frequency of interest or T is equal to

0.5 times the minimum period of oscillation. When the sampling time satisfies
the above criteria, the system will behave as if it were continuous. Details of this
and other advanced control topics are given in specialized process control
textbooks, some of which are listed in the reference list.
7.5
Concepts for Bioprocess Control
Bioprocess control consists of establishing a strategy for the management of the

biocatalyst environment. In a natural environment microorganisms and cells of
higher organisms very rarely produce large amounts of products.
In
biotechnological processes organisms are usually kept in a completely
"unnatural" environment. Control is therefore often necessary to induce them
to produce substances in economically important amounts.
Process optimization is closely linked with control. The objectives of
optimization and control may be to maximize productivity, final concentration,
yield or to minimize effluent concentration and energy costs. Although the
criteria for optimal processes differ widely, all bioprocesses need control and
automation to run under optimal conditions. The selection of control variables
strongly depends on the process and the final goal to be achieved.
Information about the dependency of biological rates, yields and selectivities
on environmental conditions is usually required, as given in Ch. 3.
All
biological reactions have distinct temperature and pH optima, and all respond
to substrate concentrations. Therefore it is common to control these variables.
Heat is produced in all biological reactions and therefore temperature
control is necessary. In large-scale production, heat removal capacity may be
the limiting factor. It is important to maintain the temperature at an optimum
level. This is the theme of the simulation example, FERMTEMP, Sec. 8.6.2.
As discussed in Ch. 3, bioreaction rates usually follow the Arrhenius' Law below
the optimal temperature, which means that the growth rates can be expected to
increase exponentially with increasing temperature. Above the optimum
temperature, further temperature increases usually cause a dramatic decrease in
activity, mainly due to inactivation of enzymes. Also, temperature shocks may
be important since enzyme formation may often be induced by a shock at the
end of the exponential growth phase.
The variable pH has certain similarities with temperature because there
usually exists an optimum for biological activity; it can be relatively easily
measured and is often controlled. The biological rates also exhibit a maximum
at the optimal pH value, which is usually in the neutral pH 7 region. Again
control is often required since in almost all biological reactions acids (e.g.
lactic, pyruvic acid) or bases (e.g. NH3) are either produced or consumed.
176
Biological rates usually depend on substrate concentration (e.g.: sugar,

mineral salts, oxygen, precursor, etc.) though in many cases kinetics are of
zero-order type above certain concentration levels. In the latter case control is
not important, since high concentrations will guarantee maximum rates. The
situation is more complex if process selectivity changes with substrate
concentration. The most well-studied process of this kind is Baker's Yeast
production, where high glucose concentration (> 100 mg L"1) and oxygen
limitation causes undesired ethanol formation. Substrates (e.g. mineral salts,
components of wastewater), precursors (e.g. in antibiotica production or
transformation processes) or products (e.g. ethanol) may be inhibiting or even
toxic at higher concentration levels. In such processes it is necessary to control
the concentration within certain limits.
7.5.1
Selection of a Control Strategy
The first step in controlling a process is to choose a control strategy. Simple

examples are the set-point control of constant temperature, pH, substrate and
precursor concentration. Table 7.2 gives examples of methods and strategies
for control of biological reactors. Much of the difficulty in control lies in
finding a suitable sensor. Calculated values using indirect measurements can be
very useful, e.g. measuring oxygen uptake to control substrate level. Often
variables such as pH or dissolved oxygen (DO) control can be used to
indirectly keep substrate concentration constant
Table 7.2. Examples of methods and strategies for the control of bioprocesses
(Heinzle and Saner, 1991).
Process
Method
Controlled
Manipulated
and strategy
variable(s)
variable(s)
Baker's yeast
production
Discrete
control
RQ
(Gas analysis)
Glucose
feed rate
Baker's yeast
production
Feedback
control
RQ
(Gas analysis)
Glucose
feed rate
Baker's yeast
production
Two point
control
DO
(electrode)
Feed rate,
agitation speed
aeration rate
177
Table 7.2. (Continued).

Process
Method
and strategy
Ethanol
PID
production
Controlled
Manipulated
variable(s)
variable(s)
Sucrose cone, by Feed rate
enzyme thermister
Wastewater
treatment
Adaptive
control
DO
(electrode)
Aeration rate
Diverse
fermentations
Various
DO
(electrode)
Stirring speed,
gas composition,
aeration rate,
pressure
Bacillus subtilis
at low DO
Cascade
DO
(electrode)
Gas flow,
valve setting
Penicillin
production
Set-points
(growth and
production)
Growth rate
(CO2 rate)
Feed rate
a-Amylase by
Bacillus amyloliquefaciens
Feed profile
Feed rate
(off-line)
Feed rate
Fed-batch
penicillin
Feed profile
Substrate and
biomass cone.
Feed rate
Baker's yeast
Feed profile
RQ
Feed rate
Cephalosporin C Profiled pH
pH, temp.
production
and temperature (electrode,
control
thermister)
Alkali feed rate,

cooling water rate
Recombinant
E. coli
Growth rate
(pH)
Glucose and alkali

rate
Phenol uptake
(O2 uptake)
Flow rate
Conventional
with added
glucose
Phenol oxidation Adaptivequesting
For constant value or set-point control usually constant control parameters are
used. Because of non-linearities or varying process dynamics (e.g. exponential
178
growth phase followed by production phase) control parameters of a linear

controller may be inadequate to control the process. It is therefore necessary to
adapt control parameters (e.g. proportional gain) according to the process
requirements. Minimization of an objective function can be used to guide the
adaptive tuning of the controller. A rather simple method uses a somewhat
empirical adjustment mechanism, which is driven by a secondary measurement.
This was used for DO control by Heinzle et al. (1986), in which the oxygen
uptake rate measurement was used to adjust the controller gain.
In addition to constant value control, optimal profile control may be applied.
The predefined optimal profile is then followed, which may be calculated from
off-line simulation and optimization. An example is found in the exponential
feeding profiles that can be calculated from the models for fed batch
fermenters in Ch 4.
If no suitable dynamic model is available and the process changes in
unpredictable ways, then on-line adaptive optimizing control may be useful.
This would however require measurements of the key inputs and outputs of the
process. An example is the optimization of a continuous anaerobic process by
Ryhiner et al. (1989) in which the methane and organic acids output rates were
correlated with the input flow rate. The optimization involved a compromise
between high methane rates and low organic acid concentration.
7.5.2
Methods of Designing and Testing the

Strategy
Selection of a control strategy and its parameters (e.g. for a PID controller)
may be difficult, since the process and controller dynamics are often not well
understood. If possible, it is useful to use dynamic models to select a control
strategy, and to use it for testing and tuning. An example with anaerobic
digestion is given by Heinzle et al. (1992). In Fig. 7.12 are shown the results of
a simulation and a corresponding experiment for the control of the propionic
acid concentration by manipulation of the feed flow rate.
179
Propionic acid [mg/l]

Feed flow [ml/min]
1
I
0
6(
Propionic acid [mg/l]

Feed flow [ml/min]
5(
4(
o
CD
O
3( -
"E
V2
20-
1(
2
Time [h]
Figure 7.12. Control of anaerobic digestion of whey wastewater. Simulation (A) and
experiment (B) of control after step change (Heinzle et al. (1992). Here the controlled variable
was the propionic acid concentration, and the manipulated variable was the feed flow rate.
References
References Cited in Part I
Archer, D.B. (1983) The Microbiological Basis of Process Control in

Methanogenic Fermentation of Soluble Wastes. Enzyme Microb. Technol. 5,
570-577.
Aris, R. (1989)
Boston.
Elementary Chemical Reactor Analysis. Butterworths,
Atkinson, B. and Mavituna, F. (1991) Biochemical Engineering and

Biotechnology Handbook. 2nd. Ed., Stockton Press, New York.
Bailey, J.E. and Ollis, D.F. (1986) Biochemical Engineering Fundamentals.
2nd. Ed., McGraw-Hill, N.Y.
Blanch, H.W. and Dunn, I.J. (1973) Modelling and Simulation in Biochemical
Engineering. Adv. Biochem. Eng, 3, 127-165.
Dekkers, R.M. (1983) State Estimation of a Fed-batch Baker's Yeast
Fermentation., in: Modelling and Control of Biotechnological Processes, (Ed.:
A.Halme) Pergamon Press, Oxford, 73.
Denac, M., Miguel, A., Dunn, I.J. (1988) Modeling Dynamic Experiments on
the Anaerobic Degradation of Molasses Wastewater. Biotechnol. Bioeng. 31, 110.
Dunn, I.J. and Mor, J.R. (1975) Variable Volume Continuous Cultivation.
Biotechnol. Bioeng. 17, 1805-1822.
Dunn, I.J., Shioya, S. and Keller, R. (1979) Analysis of Fed Batch
Fermentation Processes. Annals N.Y. Acad. ScL 326, 127-139.
ISBN: 3-527-30759-1
References
181
Franks, R.G.E. (1966) Mathematical Modeling in Chemical Engineering. Wiley,

New York.
Franks, R.G.E. (1973) Modeling and Simulation in Chemical Engineering.
Wiley, New York.
Fredrickson, A.G., Megee, R.D., Tsuchita, HM. (1970) Mathematical Models
for Fermentation Processes. Adv. Appl. Microbiol. 13, 419-465.
Furukawa, K., Heinzle, E., and Dunn, IJ. (1983) Influence of Oxygen on the
Growth of Saccharomyces cerevisiae in Continuous Culture. Biotechnol.
Bioeng. 25, 2293-2317.
Gujer, W., Zehnder, A.J.B. (1983) Conversion Processes in Anaerobic
Digestion. Water Sci. Technol. 15, 127-167.
Harder, A., Roels, J.A. (1982) Application of Simple Structured Models in
Bioengineering. Adv. Biochem. Eng./Biotechnol. 21, 56-107.
Hediger, T. and Prenosil, I.E. (1985) Microprocessor Automated Sequential
Batch Process, Biotechnol. Progr.l, 216-225.
Heinzle, E. and Lafferty, R.M. (1980) Continuous Mass Spectrometric
Measurement of Dissolved H2, O2, and CO2 during Chemolitho- autotrophic
Growth of Alcaligenes eutrophus strain H 16. Eur. J. Appl. Microbiol.
Biotechnol. 11, 8-16.
Heinzle, E., Furukawa, K., Dunn, I.J., and Bourne, J.R. (1983) Experimental
Methods for On-line Mass Spectrometry in Fermentation Technology.
Bio/Technology 1, 14-16.
Heinzle E. and Dunn I. J. (1991) Methods and Instruments in Fermentation
Gas Analysis, in Biotechnology, Vol 4, (Ed.: H.-J.Rehm and R.Reed). VCH,
Weinheim, 27-74.
Heinzle, E. and Saner, U. (1991) Methodology for Process Control in Research
and Development, Ed. Pons, M.-N., in Bioprocess Monitoring and Control.,
Hanser, Munich. 223-304.
Heinzle, E., Dunn, IJ. and Ryhiner, G. (1992) Modelling and Control for
Anaerobic Wastewater Treatment, Adv. Biochem. Eng., Springer Verlag.

ISBN: 3-527-30759-1
182
References
Imanaka, T., Aiba, S. (1981) A Perspective on the Application of Genetic

Engineering: Stability of Recombinant Plasmid. Ann. N. Y. Acad. Sci. 369, 114.
Ingham, J., and Dunn, I.J. (1991) "Bioreactor Off-Gas Analysis", in "Bioreactors
in Biotechnology", Ed. A. Scragg, Ellis-Horwood, Chichester, 195-220.
Keller, R. and Dunn, I.J. (1978), Computer Simulation of the Biomass
Production Rate of Cyclic Fed Batch Continuous Culture, J. Appl. Chem.
Biotechnol. 28, 508-514.
Keller, R. and Dunn, I.J. (1978) Fed Batch Microbial Culture: Models, Errors,
and Applications. J. Appl. Chem. Biotechnol. 28, 508-514.
Keller, J., Dunn, I. J. and Heinzle, E. (1991). A Fluidized Bed Reactor for
Animal Cell Culture, in preparation, Biotechnol. Bioeng.
Luyben, W.L. (1973) Process Modeling, Simulation, and Control for Chemical
Engineers. McGraw-Hill.
Meister, D., Post, T., Dunn, I.J. and Bourne, J.R. (1979) Design and
Characterization of a Multistage, Mechanically Stirred Column Absorber.
Chem. Eng. Sci., 34, 1376.
Moes, J., Griot, M., Keller, J., Heinzle, E., Dunn, I.J., and Bourne, J.R. (1985) A
Microbial Culture with Oxygen-sensitive Product Distribution as a Tool for
Characterizing Bioreactor Oxygen Transport. Biotechnol. Bioeng. 27, 482489.
Moes, J., Griot, M., Heinzle, E., Dunn, I.J., and Bourne, J.R. (1986) A Microbial
Culture as an Oxygen Sensor for Reactor Mixing Effects. Ann. N. Y. Acad. Sci.
469, 482-489.
Mona, R., Dunn, I.J. and Bourne, J.R. (1979) Activated Sludge Process
Dynamics with Continuous TOC and Oxygen Uptake Measurements.
Biotechnol. Bioeng. 21, 1561-1577.
Moser, A. (1988) Bioprocess Technology, Springer, N.Y.
Mou, D.G. and Cooney, C.L. (1983) Growth Monitoring and Control through
Computer-aided On-line Mass Balancing in a Fed-batch Penicillin
Fermentation. Biotechnol. Bioeng. 25, 225-255.
References
183
Prenosil, J., Dunn, I.J., and Heinzle, E. (1987) Biocatalyst Reaction Engineering,
in: Biotechnology Vol.7a, (Ed.: HJ.Rehm and R.Reed) VCH, Weinheim, 489545.
Roels, J.A. (1983) Energetics and Kinetics in Biotechnology. Elsevier
Biomedical Press, Amsterdam.
Ruchti, G., Dunn, I.J., Bourne, J.R. and v. Stockar, U. (1985) Practical
Guidelines for Determining Oxygen Transfer Coefficients with the Sulfite
Oxidation Method. Chem. Eng. J. 30, 29-38.
Russell, T.W.F., Denn, M.M. (1972). Introduction to Chemical Engineering
Analysis. Wiley, New York.
Ryhiner, G., Dunn, IJ, Heinzle, E, Rohani S., (1992) Adaptive On-line Optimal
Control of Bioreactors: Application to Anaerobic Degradation, J. BiotechnoL.
22, 89-106.
Ryhiner, G., Heinzle, E, Dunn, IJ. (1991) Modelling of Anaerobic Degradation
and Its Application to Control Design: Case Whey, in: Dechema Biotechnology
Conferences Vol.3, (Ed.: Behrens, D. and Driesel, A.J.), 469-474.
Saner, U., Bonvin, D., Heinzle, E. (1990) Application of Factor Analysis for
Elaboration of Stoichiometry and its On-line Application in Complex Medium
Fermentation of B. subtilis, in: Dechema Biotechnology Conferences Vol.3,
(Ed.: Behrens, D. and Driesel, A.J.), 775-778.
Satterfield, C.N. and Sherwood, T.K. (1963) The Role of Diffusion in Catalysis.
Addison-Wesley, N.Y.
Shioya, S., Dang, N.D.P. and Dunn, IJ. (1978). Bubble Column Fermenter
Modeling: A Comparison for Pressure Effects. Chem. Eng. Sci., 33, 1025 1030.
Tanaka, H. and Dunn, IJ. (1982). Kinetics of Biofilm Nitrification. BiotechnoL
Bioeng., 24, 669 - 689.
Tanaka, H., Uzman, S., Dunn, IJ. (1981). Kinetics of Nitrification Using a
Fluidized Sand Bed Bioreactor with Attached Growth. BiotechnoL Bioeng., 23,
1683 - 1702.
Ziegler, H., Meister, D., Dunn, IJ., Blanch, H.W., Russell, T.W.F. (1977). The
Tubular Loop Fermenter: Oxygen Transfer, Growth Kinetics and Design.
BiotechnoL Bioeng. 19, 507.
184
References
Recommended Textbooks and References for Further Reading
Biochemical
Engineering
Aiba, S., Humphrey, A.E. and Millis, N.F. (1973) Biochemical Engineering.
Academic Press, N.Y.
Atkinson, B. and Mavituna, F. (1991) Biochemical Engineering and
Biotechnology Handbook., 2nd. Ed., Stockton Press, New York.
Bailey, I.E. and Ollis, D.F. (1986) Biochemical Engineering Fundamentals.
2nd. Ed., McGraw-Hill, N.Y.
Blanch, H.W., Clark, D.S. (1996) Biochemical Engineering; Marcel Dekker,
N.Y.
Bu'lock, J. and Kristiansen Eds (1987) Basic Biotechnology Acad. Press,
London 1987.
Doran, P. M. (1995) Bioprocess Engineering Principles; Academic Press
Limited: London.
Glick, B.R., Pasternak, J.J. (1995) Molekulare Biotechnolgie; Spektrum,
Heidelberg.
Grady, C. P. L. and Lim H. C. (1980) Biological Wastewater Treatment, Marcel
Dekker.
Hastings, A. (1997) Population biology. Concepts and models; Springer, N.Y.
Heinrich, R., Schuster, S. (1996) The Regulation of Cellular Systems; Chapman
& Hall, New York.
Klefenz, H. (2002) Industrial Pharmaceutical Biotechnology; Wiley-VCH.
Ladisch, M.R. (2001) Bioseparations Engineering: Principles, Practice and
Economics. Wiley, New York.
Lee, J. M. (1992) Biochemical Engineering. Prentice Hall.
Moo-Young, M., Ed. (1985) Comprehensive Biotechnology, Vols. 1-4
Pergamon Press, Oxford.
References
185
Moser, A. (1988) Bioprocess Technology.Springer, N.Y.

Nielsen, J., Villadsen, J. (1994) Bioreaction Engineering Principles. Plenum
Press.
Pirt, S. John (1975) Microbe and Cell Cultivation. Blackwell Scientific Publ.,
Oxford.
Rehm H.J. and Reed R. Eds (1988) Fundamentals of Biochemical Engineering,
in Biotechnology, Vol. 2, VCH, Weinheim,
Roels, J.A. (1983) Energetics and Kinetics in Biotechnology. Elsevier
Biomedical Press, Amsterdam.
Schiigerl, K., Bellgardt, H. (Eds.) (2000) Bioreaction Engineering. Springer,
Berlin, Heidelberg.
Schiigerl, K. (1987) Bioreaction Engineering. Wiley, New York.
Schiigerl, K. (1994) Solvent Extraction in Biotechnology : Recovery of
Primary and Secondary Metabolites. Springer, Berlin.
Shuler, M. L., Kargi, F. (2002) Bioprocess Engineering. Basic Concepts;
Prentice-Hall.
Spier, R.E., Griffiths, J.B. (1985) Animal Cell Biotechnology. Vol. 1-3,
Academic Press.
Wang, D.I.C., Cooney, Ch.L., Demain, A.L., Dunnill, P., Humphrey, A.E., Lilly,
M.D. (1979) Fermentation and Enzyme Technology. Wiley, New York.
Wingard, L.B., Jr., Katschalski-Katzir and L. Goldstein Eds (1976-1983)
Applied Biochemistry and Bioengineering, Vols. 1-4 Acad. Press, London.
Bioreactor Design and Modelling

Asenjo, J.A., Merchuk, J.C. (1995) Bioreactor system design; Marcel Dekker,
N.Y.
Hannon, B., Ruth, B. (1997) Modeling Dynamic Biological Systems; SpringerVerlag, New York.
186
References
Scragg, A.H. (1991). Bioreactors in Biotechnology. Ellis Horwood.

Sinclair, C.G., Kristiansen, B., Bu'Lock, J.D. (1987) Fermentation Kinetics and
Modelling. Open University Press, Milton Keynes.
Schugerl, K. (1987) Bioreaction Engineering.Vol.1, John Wiley, Chichester.
Subramanian, G. (1998) Bioseparation and Bioprocessing. A Handbook
Volume II: Processing, Quality and Characterization, Economics, Safety and
Hygiene, Wiley-VCH, Weinheim.
van't Riet, K., Tramper, J. (1991) Basic Bioreactor Design. M. Dekker, New
York.
Vieth, W.R. (1994) Bioprocess Engineering, J. Wiley & Sons, N.Y.
Webb, C., Black, G.M., and Atkinson, B. (1986) Process Engineering Aspects of
Immobilized Cell Systems.Pergamon Press Ltd., Oxford.
Enzyme Engineering and Kinetics

Bisswanger, H. (2002) Enzyme Kinetics. Principles and Methods; Wiley-VCH.
Buchholz, K., Kasche, V. (1996) Biokatalysatoren und Enzymtechnologie,
VCH.Weinheim.
Cornish-Bowden, A. (1979) Fundamentals of enzyme kinetics, Butterworth,
London.
Drauz, K., Waldmann, H. (1995) Enzyme Catalysis in Organic Synthesis.
Volume I. VCH Weinheim.
Drauz, K., Waldmann, H. (1995) Enzyme Catalysis in Organic Synthesis.
Volume II. VCH, Weinheim.
Fessner, W.-D. (1999) Biocatalysis - From Discovery to Application, Springer,
Berlin.
Godfrey, T., West, S. (1996) Industrial enzymology, Macmillan Press, London.
Hayashi, K.and Sakamoto, N. (1986) Dynamic Analysis of Enzyme Systems,
Japan Sci. Soc. Press, Tokyo, Springer Verlag, Berlin.
References
187
Kennedy J. F., Ed. (1987) Enzyme Technology, in Biotechnology Vol. 7a,

VCH, Weinheim,
Liese, A., Seelbach, K., Wandrey, C. (2000) Industrial Biocatalysis, Wiley-VCH.
Scheper, T. (1997) Advances in Biochemical Engineering Biotechnology. New
Enzymes for Organic Synthesis, Springer.
Segel, I.H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady-state Enzyme Systems. Wiley, New York.
Metabolic Engineering
Lee, Papoutsakis (1999) Metabolic Engineering, Marcel Dekker: New York,
Basel.
Stephanopoulos, G. N., Aristidou, A. A., Nielsen, J. (1998) Metabolic
Engineering. Principles and Methodologies, Academic Press: USA.
Chemical Reaction Engineering

Aris, R. (1989) Elementary Chemical Reactor Analysis. Butterworth Publ.,
Stoneham.
Fogler, H. S. (1992) Elements of Chemical Reaction Engineering, PrenticeHall.
Hagen, J. (1993) Chemische Reaktionstechnik, VCH: Weinheim.
Ingham, J., Dunn, I.J., Heinzle, E., Prenosil, I.E. (2000) Chemical Engineering
Dynamics: An Introduction to Modelling and Computer Simulation, VCH
Verlagsgesellschaft mbH: Weinheim, Germany,.
Levenspiel, O. (1999) Chemical Reaction Engineering. John Wiley & Sons,
New York.
Massart, D. L., Vandeginste, B. G. M., Buydens, L. M. C., de Jong, S., Lewi, P.
J., Smeyers-Verbeke, J. (1997) Handbook of Chemometrics and Qualimetrics:
Part A, Elsevier: Amsterdam.
188
References
Richardson, J. F., Peacock, D. G. (1994) Coulson & Richardson's Chemical

Engineering. Volume 3: Chemical & Biochemical Reactors & Process Control,
Pergamon, Trowbridge.
Satterfield, C.N. and Sherwood, T.K. (1963) The Role of Diffusion in Catalysis,
Addison-Wesley, New York.
Modelling and Simulation

Basmadjian, D. (1999) The Art of Modeling in Science and Engineering,
Chapman & Hall/CRC: Boca Raton.
Deaton, M. L., Winebrake, J. J. (1999) Dynamic Modelling of Environmental
Systems, Springer: New York.
Dodson, C. T. J., Gonzalez, E. A. (1995) Experiments in Mathematics using
Maple, Springer-Verlag: Berlin,
Franks, R.G.E. (1966) Mathematical Modeling in Chemical Engineering. Wiley,
New York.
Franks, R.G.E. (1972) Modeling and Simulation in Chemical Engineering.
Wiley, New York.
Russell, T.W.F., Denn, M.M. (1972). Introduction to Chemical Engineering
Analysis. Wiley, New York.
Ruth, M., Hannon, B. (1997) Modeling Dynamic Economic Systems, Springer
Verlag, New York.
Dynamics and Control

Astrom, K.J. and Wittenmark, B. (1989). Adaptive Control. Addison-Wesley,
Reading.
Coughanowr D. R. and Koppel L. B. (1965) Process System Analysis and
Control. McGraw-Hill, New York.
References
189
Fish, N. M., Fox, R.L, and Thornhill, N.F. (1989) Computer applications in
fermentation technology: Modelling and control of biotechnological processes.
Elsevier, London.
Halme, A. (1983) Modelling and Control of Biotechnical Processes. Pergamon
Press, Oxford.
Johnson, A. (1986) Modelling and Control of Biotechnological Processes.
Pergamon Press, Oxford.
Luyben W. L. (1973) Process Modeling, Simulation, and Control for Chemical
Engineers, McGraw Hill, New York.
Pons, M.-N., Ed.(1991) Bioprocess Monitoring and Control. Hanser, Munich.
Snape, J. B., Dunn, I. J., Ingham, J., Prenosil, J. E. (1995) Dynamics of
Environmentel Bioprocesses, VCH Verlagsgesellschaft mbH, Weinheim,.
Stephanopoulos, G. (1984) Chemical Process Control: An Introduction to
Theory and Practice, Prentice Hall.
Weber. W. J., Jr., DiGiano, F. A. (1996) Process Dynamics in Environmental
Systems, Wiley.
Part II Dynamic Bioprocess

Simulation Examples
and the Berkeley
Madonna Simulation
Language

ISBN: 3-527-30759-1
Simulation Examples of Biological

Reaction Processes Using Berkeley
Madonna
8.1
Introductory Examples
8.1.1
Batch Fermentation (BATFERM)
System
The system is represented in Fig. 1, and the important variables are biological
dry mass or cell concentration, X, substrate concentration, S, and product
concentration, P. The reactor volume V is well-mixed, and growth is assumed
to follow kinetics described by the Monod equation, based on one limiting
substrate. Substrate consumption is related to cell growth by a constant yield
factor YX/S- Product formation is the result of both growth and non-growth
associated rates of production, where either term may be set to zero as required.
The lag and decline phases of cell growth are not included in the model.
Figure 1. Stirred batch fermenter with model variables.

ISBN: 3-527-30759-1
194
8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna
Model
Mass Balances:
(Rate of accumulation)
= (Rate of production)
For cells
VTT = r xv
or
dX
For substrate
V
dS
dF
or
dS
dF = rS
For product
dP
V-3T =
or
dP
dF = r?
Kinetics:
rx = f i X
with the Monod relation, constant yield relation, and product formation
kinetics:
tx/s
rP = (ki + k2 |^) X
where ki is the non-growth associated coefficient, and k2 is the coefficient

associated with growth.
If the number of equations is equal to the number of unknowns, the model is
complete and the solution can be obtained. The easiest way to demonstrate this
is via an information flow diagram, as shown below in Fig. 2.
195
8.1 Introductory Examples

x
Biomass
Balance
4_
So
T*
Growth
Rate
PO
Substrate
Balance
4_
X
^
Monod
Kinetics
f'x
Substrate
Rate
Product
Balance
A
4
s
^
Product
Rate
-^M
,-_
Figure 2. Information flow diagram of the batch fermenter model equations,
It is seen in that all the variables required for the solution of any one equation
block are obtained as the products of other blocks. The information flow
diagram thus emphasizes the complex inter-relationship involved in even this
very simple problem. Solution begins with the initial conditions XQ, SQ and PQ
at time t=0. The specific growth rate |i is calculated, enabling rs, rx and rp to
be calculated, and hence the initial gradients dX/dt, dS/dt and dP/dt. At this time
the integration routine takes over to estimate revised values of X, S and P over
the first integration step length. The procedure is repeated for succeeding step
lengths until the entire X, S and P concentration time profiles have been
calculated up to the required final time.
Program
The following Berkeley Madonna program solves the above fermentation
problem:
{BATPERM}
{Batch
growth
{Constants}
UM=0 . 3
KS = 0 . 1
Kl=0.03
K2=0.08
Y=0. 8
with
product
;kg/m3
;kgP/kgX h
;kgP/kgX h
;kg X/kg S
formation}
196
X0=0.01
SO = 10
P0=0
/Initial
;Initial
;Initial
biomass inoculum, kg/m3

substrate cone., kg/m3
product conc.,kg/m3
{Initial Conditions}
INIT X=XO
INIT S=SO
INIT P=PO
(Mass
X'=
S'=
P' =
Balances}
RX
RS
RP
;BIOMASS BALANCE
;SUBSTRATE BALANCE
;PRODUCT BALANCE
{Kinetics}
RX = U*X
U = UM*S/(KS + S)
RS = -RX/Y
RP= (K1 + K2*U) *X
Limit
; BIOMASS RATE EQUATION, kg/m3 h

;MONOD EQUATION, 1/h
; SUBSTRATE RATE EQUATION, kg/m3 h
/PRODUCT RATE EQUATION, g/m3 h
S>=0.0
The semicolon or curly brackets are used for comments.

INIT specifies the initial conditions. XQ, SQ and PQ are used here for the initial
conditions, or the values at time=0. The form X' designates the time derivative
or d/dt(X) can be used. Most models are conveniently structured in terms of
mass balances and kinetics. Any result quantity on the left of the equal sign is
stored for further calculations or for use in graphing. Usually concentration
versus time is of interest, but rates versus concentrations make very useful plots
for understanding the kinetics. The five integration methods require specifying
time intervals, such as DT, DTMIN and DTMAX. This requires a bit of
experience. Care must be taken to see that the same results are obtained by two
different methods or for at least two different DT values.
As is seen in the Appendix, Berkeley Madonna provides many possibilities to
change the parameters and graph new runs. These include the following:
changing parameters with the parameter window and making overlay plots;
changing parameters with sliders; using the Batch Runs facility.
197
Nomenclature
Symbols
k ] and k2
KS
P
r
S
V
X
Product formation constants

Saturation constant
Reaction rate
Substrate concentration
Reactor volume
Yield coefficient
1/h and kg/kg

kg/m 3
mg/m3
kg/m 3 h and kg/m 3 h
kg/m 3
m3
kg/m 3
kg/kg
1/h
Indices
Refers
Refers
Refers
Refers
Refers
Refers
2
m
P
S
X
to non-growth association rate

to growth-association rate
to maximum
to product
to substrate
to biomass
Exercises
1.
Vary KS, Mm separately and observe the effects in the graphs. It is

useful to zoom in on regions of importance by using the zoom tool
in the tool bar.
2.
Vary the product kinetics constants (Kj and K2>, and observe the
effects. Observe the P versus time curve when S reaches zero.
3.
Plot the rates versus the concentrations.
198
Results
The plots of X, S and P versus T in Fig. 3 show that when substrate is depleted,
the growth stops, and the product continues to increase, but only linearly. The
results of Fig. 4 are obtained by varying the product formation rate constants,
ki in three runs using a slider, which is defined in the Parameter Menu.
Run 1:1500 steps in 0 seconds
.10
Figure 3. Plots of X, S and P versus time during batch growth and production.
Run 3: 1500 steps in 0.0333 seconds
^.......^
-10
"l"""'"*"'.^_
/
**
-S:2
P:2
-S:3
P:3
/*
-'7^'^
5
15
.5
..
\
20
cn
-4
.**&'*' \
0
/ /'
\ /s' \,"~'"
-rp^ fm
vtf*EV
9
-8
25
3()
TIME
Figure 4. Plots of P and S versus time created by varying the product formation rate constant
199
8.1.2
Chemostat Fermentation (CHEMO)
System
A continuous fermenter, as shown in Fig. 1, is referred to as a chemostat. At
steady state the specific growth rate becomes equal to the dilution rate, |a = D.
Operation is possible at flow rates (F) which give dilution rates (D = F/V) below
the maximum specific growth rate (|um). Washout of the organisms will occur
when D > (a. The start-up, steady state and washout phenomena can be
investigated by dynamic simulation.
S,X
D,S F
Figure 1. Chemostat with model variables.
Model
The program BATFERM may be easily modified to allow for chemostat
operation with sterile feed by modifying the mass balance relationships to
include the inlet and exit flow terms. The corresponding equations are then:
For cells
dX
.= - D X + rx
200
For substrate
dS
3f
= D (SF - S) + rs
For product
dP
df
= -DP
+ rp
where D is the dilution rate and Sp the concentration of the limiting substrate in
the feed.
The same kinetic expressions as in BATFERM will be applied here.
Program
Note the conditional statement for D which allows a batch startup.
{CHEMO}
(Chemostat startup and steady state.

batch reactor until time=tstart}
{Constants}
UM=0.3
KS = 0.1
Kl = 0.03
K2=0.08
Y = 0.8
X0 = 0.01
S0=10
P0=0
SF = 10
Dl = 0.25
t start = 5
;
;
;
;
;
;
;
;
;
;
;
Startup
1/h
kg/m3
kgP/kgX h
kgP/kgX
kg X/kg S
Initial biomass inoculum, kg/m3
Initial substrate cone., kg/m3
Initial product conc.,kg/m3
Feed cone. ,kg/m3
Dilution rate, 1/h
Start time for the feed
(Initial Conditions}
Init X=XO
Init S=SO
Init P=PO
{Mass Balances}
X'=-D*X+RX
; BIOMASS BALANCE EQUATION
S =D* (SF-S) +RS
; SUBSTRATE BALANCE EQUATION
P'=-D*P+RP
; PRODUCT BALANCE EQUATION
as
201
{Kinetics}
RX = U*X
U = U M * S / ( K S + S)
RS=-RX/Y
RP= (K1 + K2*U) *X
; BIOMASS RATE EQUATION, kg/m3 h

; MONOD EQUATION, 1/h
; SUBSTRATE RATE EQUATION, kg/m3 h
;PRODUCT RATE EQUATION, kg/m3 h
{Conditional equation for

D=if time>=tstart then Dl
Prod=D*X
D}
else
/Productivity
0
for
biomass,
kg/m3
Nomenclature
Symbols
D
ki and
KS
P
r
Dilution rate
Product formation constants
Saturation constant
Reaction rate
S
X
Y
Yield coefficient
Time lag constant
ILL
1
Indices
F
MONOD
P
S
X
Refers to feed
Refers to Monod kinetics
Refers to product
Refers to substrate
Refers to biomass
1/h
1/h and kg/kg
kg/m3
mg/m3
kg/m3h and
kg/m3
kg/m3
kg/m3
kg/kg
1/h
202
Exercises
1.
Increase D interactively to obtain washout.
2.
Note the steady state values of X and S; calculate Y from these.
3.
Change SF. Does this alter S at steady state? Why?
4.
Calculate S at steady state from D. Verify by simulation.
5.
Change the program to account for biomass in the feed.
6. Operate initially as a batch reactor with D = 0, and switch to

chemostat operation with D < |jm. Does this reduce the time to reach
steady state? Is the exact time of switchover important?
7. Include maintenance requirements to the substrate uptake kinetics
using RS = - ( U / Y + M ) * X . Remember to add a value of the
maintenance coefficient M to the constants. Investigate the influence of
the value of M on the steady state biomass concentration.
8. Using a Parameter Plot, obtain steady state values of X and S for a
range of Dl.
9. Rapidly-changing dynamic fermentations do not follow
instantaneous Monod kinetics. Modify the model and the program with
a dynamic lag on jo, such that d|j /dt= (nMonod - l-O/t- Compare the
response to step changes in D for suitable values of the time lag constant
t.
Results
The graphical output in Fig. 2 shows three startups of the fermenter under
initially batch growth conditions, using three values for D l . The break in the
concentration-time dependency as feeding starts is quite apparent, and the new
transient then continues up to the eventual steady state chemostat operating
condition or washout in the case of one run. For the results of Fig. 3 the
program was changed by adding the line PROD = X*D, and the final, steady
state value of production rate was plotted versus Dl for twenty runs, using the
Parameter Plot feature of Madonna.
203

10
Figure 2. Startups of the chemostat after initial batch growth for 3 values of Dl.
Figure 3. Productivity in a chemostat. Steady states are shown for 20 runs using the Parameter
Plot.
204
8.1.3
Fed Batch Fermentation (FEDBAT)
System
In this case the model equations allow for the continuous feeding of sterile
substrate, the absence of outflow from the fermenter and the increase in volume
(accumulation of total mass) in the fermenter, schematically as shown in Fig. 1.
Simulation of fed batch fermenters can be used to demonstrate the important
characteristics of quasi-steady state, linear growth, and use of alternative feed
strategies.
F,SF
V
X
s
p
Figure 1. Fed batch fermenter with model variables.
Model
For fed batch operation, the equations become as follows:
Total balance
dV
dT =
For cells
For substrate
205
For product
where F is the volumetric feed rate, Sp is the feed concentration and V is the
volume of the fermenter contents at time t. Thus the mass quantities, VX, VS,
and VP are calculated and are divided by the volume at each time interval to
obtain the concentration terms required for the kinetic relationships. The
kinetics are taken to be the same as in BATFERM.
Program
The "IF" statement in the program causes the continuous feed to start when time
reaches tfeed, at which point batch operation stops and the fedbatch starts.
(FEDBAT)
{Fermentation
{Flow rate is
time=tfeed.}
with
batch
initially
start
zero
up}
and
is turned
on at
{ Constants}
UM=0.3
; 1/h
KS = 0 . 1
; kg/m3
; kgP/kgX h
Kl = 0.03
K2 = 0.08
; kgP/kgX
; kg X/kg S
Y = 0.8
; Initial biomass inoculum, kg/m3
X0 = 0 .01
S0 = 10
; Initial substrate cone., kg/m3
P0 = 0
; Initial product conc.,kg/m3
; Feed conc.,kg/m3
SF = 10
; Feed flow rate, m3/h
Pl-1. 5
tfeed=22.5
; Start time for the feed
{Initial Conditions}
init V=l
init VX=V*XO
init VS=V*SO
init VP=V*PO
206
{Mass balances,
d/dt(V)=F
d/dt(VX)=RX*V
d/dt(VS)=F*SF+RS*V
kg/h}
d/dt(VP)=RP*V
{kg/h}
{Calculation
of
concentrations}
X=VX/V
S=VS/V
P=VP/V
{Kinetics}
RX=U*X
U=UM*S/(KS+S)
RS=-RX/Y
RP=(K1+K2*U)*X
D=F/V
{nominal
dilution
{Turning the feed on at time = tfeed}

F=if time>=tfeed then Fl else 0 {batch
rate,
start
1/h}
up}
Nomenclature
Symbols
D
F
KS
ki, k2
M
P
r
S
X
V
Y
|i
T
Dilution rate
Flow rate
Saturation constant
Constants in product kinetics
Maintenance coefficient
Reaction rate
Reactor volume
Yield coefficient
Time delay constant
1/h
m3/h
kg/m3
1/h and kg/kg
kg/kg h
kg/m3
kg/m3 h
kg/m3
kg/m3
m3
kg/kg
1/h
h
207
Indices
F
P
S
X
Refers
Refers
Refers
Refers
to feed
to product
to substrate
to biomass
Exercises
Results
Operation begins under initial batch conditions, and feeding of substrate is
started at tfeed=22.5 h. In Fig. 2, the break in the batch growth transient, as
semi-batch feeding starts is very apparent, with the transient continuing to an
apparent "quasi" steady state operating condition. Under these conditions the
biomass concentration becomes constant, while the substrate concentration (not
shown) is below the KS value and decreases very slowly. As seen in the zoom
of Fig. 3, the values of D (= F/V) also decrease since V increases due to the
incoming feed, and D eventually becomes equal to p when S falls below K$.
The total biomass is determined by the yield coefficient times the total amount
of substrate that has been consumed, which is approximately equal to the
amount in the reactor initially plus the amount added during the feeding
period. During the quasi-steady state, the total biomass will increase linearly
with time if, as in this case, the feeding flow rate is constant. This is a "linear
208
growth" situation in which the growth rate is limited by the feeding rate. In
Fig. 3 the values of X, S, and P are plotted versus T for a switch from batch
(F = 0) to fed batch (F = 5) at time T = 20 h. The product production rate
depends linearly on biomass concentration, and thus even when ja becomes very
low, P will continue to increase linearly in mg/m3 amounts.
TIME= 34.13 X = 12.34
.^
10-.-
10
20
30
40
50
60
70
80
90
100
Figure 2. Transients during the fedbatch fermentation.

0.4.
0.35-
-~, I
V
0.3-
0.25.
3
Q 0.2-
CO
0.15-
-J.
T
0.10.05027
28
29
30
31
32
TIME
Figure 3. Zooming in on the quasi-steady state.
33
34
35
36
37
8.2 Batch Reactors
8.2
8.2.1
209
Batch Reactors
Kinetics of Enzyme Action (MMKINET)
System
The intermediate enzyme-substrate complex is the basis for the simplest form
of enzymatic catalysis (Fig. 1):
E +S
ES
*-
E +P
k2
Figure 1. Mechanistic model for enzymatic reaction.
Model
The equations for substrate, enzyme-substrate complex and product in a batch
reactor are:
- = ki E S - k 2 ES
dt
dFS
^ = ki E S - (k2 + k3) ES
dt
Using the steady state approximation for the change of active complex,
dt
the Michaelis-Menten equation is obtained.

_dS _
~ dt ~
M +S
210
where vmax = k3 E0 and KM = (k2+k3)/ki.
Program
The program with the detailed mechanism is on the CD-ROM.
Nomenclature
Symbols
E
ES
k
KM
P
S
Vmax
mol/m3
Enzyme-substrate complex concentration mol/m3
Reaction rate constants
various
mol/m3
mol/m3
mol/m3
Maximum velocity
mol/m3 h
Indices
0
1
2
3
S
Mm
Exercises
Refers to initial values

Refers to reaction 1
Refers to substrate
Refers to Michaelis-Menten
211
8.2 Batch Reactors
Results
Figs. 2 and 3 give the results of the full model and the Michaelis-Menten
simplification, respectively
Run 1:119 steps in 0.0167 seconds
0.009.
0.0080.007-
Lx""""^"~
\
f'
\ /
0.006
^0.005-
0.7
^..]
...
8:1
0.6
ES:1
--
0.5
P:1
riL
t ~m
f \
LU
0.0040.003-
*i*
0.001 - i
0.4
0.3
0.002
-0.8
:.
tn
.0.9
fc
*v.
0.2
\. i
0.1
" %^
""""%-,
^*"'..._
.0
10
20
30
40
50
TIME
Figure 2. Results from the full model
70
80
90
100
a
*
to
212
\
\
10
20
30
40
50
60
70
80
90
100
TIME
Figure 3. Results from the Michaelis-Menten simplification.
8.2.2
Lineweaver-Burk Plot (LINEWEAV)
System
This program simulates the batch uptake of substrate using Michaelis-Menten
kinetics, of the form,
r
s = K^TS-
The inverse rate is plotted versus the inverse concentration (Fig. 1).
Comparison of this plot with the concentration-time plot together with the Km
value, demonstrates the importance of data in the Km region and the difficulty
of obtaining this in a batch reactor. It is useful to make specially-scaled graphs
in the KM region.
213
8.2 Batch Reactors
Figure 1. Lineweaver-Burk plot to determine vm and
Model
The model is that of a batch reactor with Michaelis-Menten kinetics.
dS
dF = ~ r s
Program
To make the Lineweaver-Burk plot, the inverse values of S and rs are calculated
in the program on the CD-ROM.
Nomenclature
Symbols
KM
r
S
Reaction rate
kg/m3
kg/m3
kg/m3
214
Si
V
Inverse substrate concentration

Reaction velocity or rate
Inverse reaction velocity or rate
m3/kg
kg/m3 h
m3 h/kg
Indices
0
m
S
Refers to feed
Refers to maximum
Refers to substrate
Exercises
Results
The results are shown in Fig. 2 (rates and concentrations versus time) for a
range of Michaelis-Menten constants KM and in Fig. 3 the corresponding
Lineweaver-Burk plots.
215
8.2 Batch Reactors
'0.5
0.45
.0.4
0.35
.0.3
.0.25
0.2
-0.15
0.1
0.05
140
160
Figure 2. Rate and concentration plots for KM = 0.2, 0.5, 1.0 and 2.0 (bottom to top curves).
Figure 3. Lineweaver-Burk plots for KM = 0.2, 0.5, 1.0 and 2.0 (bottom to top curves).
8.2.3
Oligosaccharide Production in Enzymatic

Lactose Hydrolysis (OLIGO)
System
Some enzyme catalyzed reactions are very complex. For this reason their
rigorous modelling leads to complex kinetic equations with a large number of
constants. Such models are unwieldy and are usually not suitable for practical
216
purposes. One approach to simplify them is to neglect formation of enzymesubstrate complexes altogether and to deal only with overall reactions of the
react ants to products.
An example of such a reaction is the enzymatic lactose hydrolysis, a
complex process involving a multitude of sequential reactions leading to higher
saccharide (oligosaccharides) intermediates. The mechanistic model is rather
complex even when only trisaccharides are considered (Fig. 1).
La + E
Ga E + La
GaE + H2O
^-
LaE
**
E + Tr
E + Ga
Ga + GI + E
Figure 1. Complex and simplified models for the enzymatic hydrolysis of lactose, where the
symbols are La for lactose, Ga for galactose, Gl for glucose, Tr for trisaccharide and E for
enzyme.
Neglecting the enzyme complexes, however, gives a simplified model (Fig. 2)

requiring only three constants:
1L.a
a
fî
Ga T. V3II
to
^ Tr
i
La -i- Ga
fcaCl
Figure 2. Simplified model for the enzymatic hydrolysis of lactose.
The simulation of this model is easy, and the constants can be adjusted to
achieve good agreement with experimental data.
Model
This simple batch reactor model is equivalent to the Michaelis-Menten product
inhibition model.
217
8.2 Batch Reactors
dLa
-gjdGa
- K! La - KI La Ga + K2 Tr
= Kj La - KI La Ga + K2 Tr
dTr
= KI La Ga - K2 Tr
Initial conditions: Lao =150 mmol/m3, Gao = 0, Trg = 0

Range of the kinetic constants: KI = 0.02 - 0.06 miir1, KI = 0.02 - 0.1
L/mmol min, K2 = 1 - 50 min"1.
Program
It was found that K2 must be two orders of magnitude greater than KI in order
to bring the simulation into agreement with the experimental data. The
program is on the CD-ROM.
Nomenclature
Symbols
Ga
Gl
K2
La
Tr
Galactose concentration
Glucose concentration
Reaction rate constant (La > Ga + Gl)
Reaction rate constant (La + Ga -> Tri)
Reaction rate constant (Tri -> La + Ga)
Lactose concentration
Trisaccharide concentration
Indices
0
Refers to initial concentration
mmol/L
mmol/L
1/min
L/(mmol min)
1/min
mmol/L
mmol/L
218
Exercises
Results
The outputs in Figs. 3 and 4 show the influence of KI, KI and Lao on the sugar
concentration profiles.
100.
r100
90.
80.
70
60.
. 50.
40
30
20
10
0
20
100
TIME
Figure 3. Sugar concentrations with Kr = 0.04, K{ = 0.05, La0 = 100.
180
200
219
8.2 Batch Reactors

160
80
'
80
100
120
140
160
180
200
Figure 4. Sugar concentrations with Kj = 0.06, KI = 0.1 Lao = 160.
Reference
Prenosil, J. E., Stuker, E. and Bourne, J. R. (1987) "Formation of
Oligosaccharides during an Enzymatic Lactose Hydrolysis Process", Parts I and
II: Biotechnol. Bioeng. 30, 1019-1031.
8.2.4
Structured Model for PHB Production (PHB)
System
Heinzle and Lafferty (1980) have presented a structured model to describe the
batch culture of Alcaligenes eutrophus under chemolithoautotrophic growth
conditions, as discussed in Case C, Sec. 3.3.1. Growth and storage of PHB are
described as functions of limiting substrate S (NH4+), residual biomass R and
product P (PHB) concentrations.
220
Figure 1. Structured kinetic model for PHB synthesis.
Model
In the model seen in Fig. 1 the whole cell dry mass (X) consists of two main
parts, namely PHB (P) and residual biomass (R), where R is calculated as the
difference between the total cell dry weight and the concentration of PHB (R =
X - P). R can be considered as the catalytically active biomass, including
proteins and nucleic acids. With constant concentrations of the dissolved gases,
two distinct phases can be recognized: growth and storage. During the growth
phase there is sufficient NH4+ to permit protein synthesis. When the limiting
substrate NH4+ (S) is exhausted, the protein synthesis ceases, and the
production rate of PHB is increased. During the storage phase only PHB is
produced. The limiting substrate NH4+ (S) is essential to produce R and limits
its synthesis at low concentrations.
For the batch process,
dR
dF
= r
R = MR
where TR is the rate of synthesis of R and (j is the specific rate of synthesis of R,

where
S
(S/Ks,2)n
+ S) + ^m,2 ! + (S/KS,2)n
where n is the empirical Hill coefficient (see Sec. 3.1.2), having a value of 4 in
this example.
This is based on the postulate that there are two different mechanisms for the
assimilation of NH4+ in procaryotes. This formulation is not a mechanistic one,
8.2 Batch Reactors
221
since in reality the enzyme system, using energy to assimilate NH4+, is

repressed by high concentrations of NH4+.
For the substrate
dS
1
dF = rs = -YR/S **
The rate of synthesis of P(rp) is assumed to be the sum of a growth associated

term (rpj) and a biomass associated term (rp,2) and is given by,
dP
df
= rp = r P j + rP,2
where r P j = YP/R rR
The non-growth associated term of the synthesis of P(rp,2) is assumed to be a
function of the limiting substrate S, of the residual biomass R and of the
product P. When the PHB content in the cells is high, the rate of synthesis of P
is decreased, which can be formally described as an inhibition.
Program
The program is found on the CD-ROM.
Nomenclature
Symbols
KI
KS
n
P
R
rp
TR
rs
Inhibition constant, for (NH^SC^

Saturation constant
Hill Coefficient
Product concentration (PHB)
Residual biomass concentration
Rate of synthesis of PHB
Rate of synthesis of R
Rate of substrate uptake
kg/m3
kg/m3
kg/m3
kg/m3
kg/m3
kg/m3
kg/(m3 h)
222
X
YP/R
YR/S
Limiting substrate concentration

NHj as (NH4)2S04
Yield coefficient
Yield coefficient,
Specific rate of synthesis of R (rR/R)
Specific rate of synthesis of P (rp/P)
Indices
1
2
m
Exercises
Refers to maximum
kg/m3
kg/m3
kg/kg
kg/kg
1/h
1/h
223
8.2 Batch Reactors
Results
4-1
-16
3.5-
3-
-.^
1.5-
y
/ "
y "
1-
^^
*"^
0.50-
-10
"."T.-'V/T
I
J
'""
ISi$%
M*
/
*"'"
12
T
'"U--b
of
-*'
2.5-
14
^"~'"~*" "
'. .'
-8 a.
-'
'*"
-6
f'
-2
_j__ *%
10
_n
15
20
25
30
35
40
TIME
Figure 2* Profiles of residual biomass concentration R, substrate S and product P in the batch
fermentation.
Run 4: 41 6 steps in 0.01 67 seconds
35- ...
5
'"'V^
4.5
^.^.-
30-
-4
'v
25-
\
\
20-
a
15-
.._.. 3:3(2,3)
~- P:3(2.3)
/
1
-*.
P:4 (5)
3.5
3
-2.5 (/)
-2
"""-,
10-
"\ ""
.*""r-'"
-1.5
\"' /î''^'
-1
-0.5
^^";:^
0.
10
15
-n
20
25
30
35
40
TIME
Figure 3. PHB formation at two different initial substrate concentrations.
References
Heinzle, E., and Lafferty, R. M. (1980) Continuous Mass Spectrometric
Measurement of Dissolved H2, O2, and CC>2 during Chemolitho-autotrophic
224
Growth of Alcaligenes eutrophus strain H16. Eur. J. Appl. Microbiol.

BiotechnoL 11, 8.1
8.3
Fed Batch Reactors
8.3.1
Variable Volume Fermentation (VARVOL and

VARVOLD)
System
Semi-continuous or fed batch cultivation of micro-organisms is common in the
fermentation industries. The fed batch fermenter mode is shown in Fig. 1 and
was also presented in the example FEDBAT. In this procedure a substrate feed
stream is added continuously to the reactor. After the tank is full or the
biomass concentration is too high, the medium can be partially emptied, and
the filling process repeated. Since the variables, volume, substrate and biomass
concentration change with time, simulation techniques are useful in analyzing
this operation. This example demonstrates the use of dimensionless equations.
Figure 1. Filling and emptying sequences in a fed batch fermenter.
Model
The balances are as follows:
Volume,
dv
dT = FO
Substrate,
224
Growth of Alcaligenes eutrophus strain H16. Eur. J. Appl. Microbiol.

BiotechnoL 11, 8.1
8.3
Fed Batch Reactors
8.3.1
Variable Volume Fermentation (VARVOL and

VARVOLD)
System
Semi-continuous or fed batch cultivation of micro-organisms is common in the
fermentation industries. The fed batch fermenter mode is shown in Fig. 1 and
was also presented in the example FEDBAT. In this procedure a substrate feed
stream is added continuously to the reactor. After the tank is full or the
biomass concentration is too high, the medium can be partially emptied, and
the filling process repeated. Since the variables, volume, substrate and biomass
concentration change with time, simulation techniques are useful in analyzing
this operation. This example demonstrates the use of dimensionless equations.
Figure 1. Filling and emptying sequences in a fed batch fermenter.
Model
The balances are as follows:
Volume,
dv
dT = FO
Substrate,
ISBN: 3-527-30759-1
8.3 Fed Batch Reactors
225
- = F0S0
Biomass,
d(VX)
dt
= rx
The kinetics are
rx = MX
|LimS
** - (Ks + S)
and
rx
rs = -Y
The dilution rate is defined as
In order to simplify the equations and to present the results more generally, the
model is written in dimensionless form. Defining the dimensionless variables:
v
V
X
X< =
F =
- _ JL
Mm
tft' = t
226
Expanding the derivatives gives

d(V S) = V dS + S dV
and
d(V X) = V dX + X dV
Substituting, the dimensionless balances now become:

Volume
dV'
Biomass
dX'
dt'
Substrate
dS'
dt
= (l-S)D-jiX
The Monod equation is:

KS +S
In Fig. 2 a computer solution shows the approach to and attainment of the

quasi-steady state of the dimensionless fed-batch model.
227
Quasi- steady
Figure 2. Dynamic simulation results for a fed batch culture.
Programs
The program VARVOL is based on the model equations with normal
dimensions. The program VARVOLD is based on the dimensionless equations
as derived above. Both are on the CD-ROM.
Nomenclature
Symbols
D
F
KS
r
S
V
Dilution rate
Flow rate
Saturation constant
Reaction rate
Reactor volume
1/h
m3/h
kg/m3
kg/m3 h
kg/m3
m3
228
X
Y
Yield coefficient
Indices
0
f
m
S
X
Refers
Refers
Refers
Refers
Refers
Refers
to feed and initial values

to final
to maximum
to substrate
to biomass
to dimensionless variables
Dimensionless Variables
S'
V
X1
t'
Exercises
Dimensionless flow rate

Dimensionless saturation constant
Dimensionless substrate concentration
Dimensionless volume
Dimensionless biomass concentration
Dimensionless time
Dimensionless specific growth rate
kg/m3
kg/kg
1/h
229
Results
During the quasi-steady state, \l becomes equal to D, and this requires that S
must decrease steadily in order to maintain the quasi-steady state as the volume
increases (Fig. 3). Increasing flow rates from 0.01 to 1.0 causes a delay in the
onset of linear growth and causes the final biomass levels to be higher (Fig. 4).
4.5
Figure 3. Fed batch concentration and growth rate profiles, showing quasi-steady state.
2.5
10
C/>
230
Figure 4. Influence of flow rate on growth. Flow rate increase from 0.01 to 1.0.
References
Dunn, I.J., and Mor, J.R. (1975) Variable Volume Continuous Cultivation.
Biotechnol. Bioeng. 17, 1805.
Keller, R., and Dunn, I.J. (1978) Computer Simulation of the Biomass
Production Rate of Cyclic Fed Batch Continuous Culture. J. AppL Chem.
Biotechnol. 28, 784.
8.3.2
Penicillin Fermentation Using Elemental

Balancing (PENFERM)
System
This example is based on the publication of Heijnen et al. (1979), and
encompasses all the principles of elemental balancing, rate equation
formulation, material balancing and computer simulation. A fed batch process
for the production of penicillin as shown in Fig. 1 is considered with
continuous feeding of glucose. Ammonia, sulfuric acid and o-phosphoric acid
are the sources of nitrogen, sulfur and phosphorous respectively.
Ophosphoric acid is sufficiently present in the medium and is not fed. Oxygen
and carbon dioxide are exchanged by the organism. The product of the
hydrolysis of penicillin, penicilloic acid, is also considered, thus taking the slow
hydrolysis of penicillin-G during the process into account.
231
Glucose
Carbon dioxide
Oxygen
Precursor
Phenylacetic acid
Sulfuric acid
Ammonia
Figure 1. Streams in and out of the penicillin fed batch reactor.
Table 1. lists the components and their conversion rate designation.
232
Table 1. Component properties and rate designations.

Compound
Chemical formula
Mol wt. Enthalpy Conversion
(Daltons) (kcal/mol) rate (mol/h)
Glucose
180
- 303
C6H1206
Rl
Mycelium
24.52
28.1
R2
CHi.64Oo.52No. 16
So.0046P<).0054
C16H1804N2S
Penicillin
334
- 115
R3
C16H2005N2S
R4
352
- 183
Penicilloic acid
Oxygen
02
32
0
R5
CO2
Carbon Dioxide
44
-94
R6
NH3
Ammonia
17
- 19
R8
H2SO4
R9
Sulfuric Acid
98
- 194
Phosphoric Acid H3PO3
98
- 319
RIO
Phenylacetic Acid C8H802
136
-69
RH
H2O
Water
18
-68
Rl2
Model
a) Elemental Balancing
Knowing the composition of all chemical substances and the biomass mycelium
(Table 1) allows the following steady state balances of the elements in terms of
mol/h:
For carbon
6 RI + R6 + 16 R3 + 8 RH + 16 RH + R2 = 0
For oxygen
6 RI + 2 R5 + 2 R6 + R12 + 4 R3 + 4 R9 + 4 R10 + 2 RH + 5 R4 + 0.52 R2 = 0

For nitrogen
0.16R 2 + 2 R 3 + 2R 4 + R8 = 0
For sulfur
0.00 46 R2 + R3 + R4 + R9 = 0
233
For hydrogen
12 RI + 1.64 R2 + 18 R3 + 20 R4 + 3 R8 + 2 R9 + 3 RIO + 8 R n + 2 R12 = 0
For phosphorus
0.0054 R2 + RIO = 0
A steady state enthalpy balance gives the following
- 303 RI - 28.1 R2 - 115 R3 - 183 R4 - 94 R6 - 19 R8 - 194 R9 -
- 3 1 9 R i o - 6 9 R n -68Ri 2 + rH = 0
where TH is the rate of heat of production (kcal/h).
A total of 12 unknowns (Ri through R6, Rg through Ri 2 and TH) are involved
with a total of 7 equations (6 elemental balances and one heat balance). The
five additional equations are provided by five reaction kinetic relationships.
The remaining rates can be expressed in terms of these basic kinetic equations.
From the carbon balance
- R6 = 6 RI + R2 + 16 R3 + 16 R4 + 8 RH
From the nitrogen balance

- R 8 = 0.16R2 + 2R 3 + 2R 4
From the sulfur balance
- R9 = 0.0046 R2 + R3 + R4
From the phosphor balance
- R i o = 0.0054 R2
From the hydrogen and nitrogen balances
- R5 = -6 RI - 1.044 R2 - 18.5 R3 - 18.5 R4 - 9 R n
From the enthalpy balance

rH = - 669 RI - 110.1 R2 - 1961 R3 - 1961 R4 - 955 RH
234
To complete the model, equations for glucose uptake rate (-Ri), biomass
formation rate (R2), rate of penicillin formation (Rs), precursor consumption
rate (-Rn), and rate of penicillin hydrolysis (R4) must be known. Note that the
reaction rates are defined with respect to total broth weight, since the process is
the fed-batch type and broth weight is variable with respect to time.
b) Formulation of the Kinetic Equations

Substrate (Glucose) Uptake Rate:
A MONOD type equation for the uptake of sugar by P. Chrysogenum is used.
-QlCiM2
Biomass Formation Rate:
A linear relationship between the glucose consumption rate and growth rate of
biomass is assumed. Hence,
1
- Rl = y^ ^2 + m M2
where Y2 is the maximum growth yield and m is the maintenance rate factor
(mol glucose/mol mycelial biomass h).
Some sugar is used in the formation of the product. Hence,
- Rl = Yj R2 + m M2 + YJ (R3 + R*)
where 3 is the conversion yield for glucose to penicillin (mol penicillin/mol

glucose).
The total rate of biomass formation equals the net rate of formation,
corrected for the amount transformed to penicilloic acid. Therefore,
R2 = - Y 2 R i - Y 2 m M 2 - yf (Rs + 4)
Precursor Conversion Rate
It is assumed that the precursor is only used for penicillin synthesis. Thus
- R l l = R 3 + R4
where - RH is the precursor consumption rate.
235
Rate of Penicillin Synthesis

The specific rate of penicillin synthesis is assumed not to be a function of
specific growth rate. So that
R 3 = Q3 M2 - R4
where Q3 is the maximum specific rate of penicillin synthesis (mol/mol h),

Equation for the Rate of Penicillin Hydrolysis
The hydrolysis of penicillin takes place by a first-order reaction.
R 4 = K 3 M3
c) Balance Equations
Total Mass Balance
The individual feed rates of glucose, sulfuric acid and ammonia are adjusted to
equal their molar consumption rates. Water lost by evaporation is neglected.
The change in mass due to gas uptake and production is neglected. The mass
flow rates are calculated from the molecular weights, the uptake rates and the
mass ratio compositions.
Feed rate of glucose stream (kg/h)
F
l =
180
500
F
2.78
where F = mol glucose /h.

Feed rate of NH3 stream (kg/h)
F8 = R 8 25Q
= T4JT
Feed rate of [2804 stream (kg/h)

18
F9 - R95QO
R9
~ 2.55
The total mass in reactor G (kg/h) changes with time according to

dG
"dT = Tn
R9
235"
Component Balances
Expressed in mol/h the dynamic balances are,
Rg
TTTT
236
Glucose
~3T = R l + F
Biomass
dM2
Penicillin
~ar = R2
dM3
Penicilloic acid
dM4
The concentrations in mol/kg are as follows:
M2
Tr
M3
M4
c4 =
where the masses MI, M2, M3 and M4 are in mol units.
d) Metabolism Relations
The various metabolic relationships are given from
Specific growth rate for cells
R2
* = Ml
R
Re
Q = R7
Oxygen uptake rate

OUR = -R 5
CO2 production rate

CPR = R6
Fraction of N2 in mycelium
237
R2
f 2 = 0.16 R|
N2 fraction in penicillin
f3 = 1-F 2
Fraction of sulfur used for mycelium
R2
f 4 = 0.046 R|
Sulfur fraction used for penicillin
f5 = 1 - F4
Fraction of glucose for cell growth
=
R2
Fraction of glucose for penicillin

R3 + R4
S3 = - Y3 R!
Fraction of glucose for maintenance
M2
g4 = -M R^-
Program
The Madonna program covers a fermentation time of 200 h starting from the
initial conditions of 5500 mol glucose, 4000 mol biomass, 0 mol penicillin and
0.001 mol penicilloic acid in an initial broth weight of IxlO 5 kg. The program
is on the CD-ROM.
Nomenclature
Symbols
a, b
C
CPR
F
Flow rate variables

Component concentration
Feed rate
various
mol/kg
mol/h
kg/h
238
h
fl
f5
G
82
g3
g4
Kl
K3
M
m
OUR
Q
R
RQ
rq
Y
Fraction of nitrogen in mycelium

Nitrogen fraction in penicillin
Fraction of sulfur used for mycelium
Fraction of sulfur used for penicillin
Mass in reactor
Fraction of glucose for cell growth
Fraction of glucose for penicillin
Fraction glucose for maintenance
Saturation constant
Hydrolysis rate constant
Mass of individual components
Maintenance rate factor
Oxygen uptake rate
Maximum specific rates
Conversion
Heat production rate
Respiratory quotient
Yield coefficient
Indices
0
1
2
3
4
5
6
8
9
10
11
12
Exercises
initial
glucose
biomass
penicillin
penicilloic acid
oxygen
carbon dioxide
ammonia
sulfuric acid
phosphoric acid
phenylacetic acid
water
kg
mol/kg
1/h
mol
mol/(mol h)
mol/h
mol/(mol h)
mol/h
kcal/h
1/h
239
Results
The results of Fig. 2 show the substrate MI to pass through a maximum, while
the penicillin M2 develops linearly, for this constant feeding situation.
Increasing the feeding linearly with time (F = 500 + 5* time) gave the results in
Fig. 3, where it is seen that maintenance accounts for about 70 % of glucose
consumption at the end of the fermentation.
20
40
60
80
100
120
140
160
180
200
TIME
Figure 2. Penicillin fed batch fermentation with total masses of glucose (M]) and biomass
(M2).
240
0.9-,
0.80.7-
0.6-
r- 0.40.30.20.1-
020
60
80
100
120
TIME
Figure 3. Linear increase of feeding with time F = 500 + 5*T.
Reference
Heijnen, J., Roels, J. A., and Stouthamer, A.H. (1979). Application of Balancing
Methods in Modeling the Penicillin Fermentation. Biotechnol. and Bioeng., 21,
2175-2201.
8.3.3
Ethanol Fed Batch Diauxic Fermentation

(ETHFERM)
System
Yeast exhibits diauxic behavior with respect to the glucose and ethanol in the
medium as alternative substrates. In addition, the glucose effect, when glucose
levels are high, will cause fermentation, instead of respirative oxidation, to take
place, such that the biomass yields are much reduced (Fig. 1). In this example
the constant a designates the fraction of respiring biomass and (1 - a) the
fraction of biomass that ferments. The rates of the process are controlled by
three enzymes.
24 1
^^
C02 + X
Glucose
^^^*-
Ethanol + X
Figure 1. Pathways of aerobic ethanol fermentation.
Model
The rates of the processes are as follows:
Respirative oxidation on glucose,
R, =
Glu+K sl
Fermentation to ethanol,
R2 = K2 (1 - a) X
Glu + KS2
Conversion of ethanol to biomass,
Enzyme activation for the transformation of ethanol to biomass is assumed to

involve an initial concentration of starting enzyme EQ, which is converted to enzyme 2 and which catalyzes growth on ethanol through an intermediate
enzyme EI.
Thus, the production rate of enzyme EI is inhibited strongly by glucose,
R4 = - -rXEo
K S4 +Glu 3
and the production rate of enzyme 2 controlling the conversion of biomass to

ethanol depends on EI,
R5 = K 5 X E i
The mass balances for the biomass, substrates and enzymes are those for a fed
batch with variable volume.
242
For the total mass balance with constant density,
dt
The component balances are written by separating the accumulation term,
noting that
d(VC) _ VdC
CdV _ VdC
- +
- dt
dt
dt
dt
Thus,
dt
V
f
ô
dt
E0Q
V
f -*-.-*
Program
Note that the program on the CD-ROM is formulated in terms of C-mol for the
biomass. This is defined as the formula weight written in terms of one C atom,
thus for yeast CHL667Oo.5No.i67-
243
Nomenclature
Symbols
C
E
EtOU
Glu
K
Q
R
V
X
Y
a
Component concentration
Ethanol concentration
Substrate feed concentration
Rate constants
Feed flow rate
Reaction rate
Reactor volume
Yield coefficient
Fraction of respiring biomass
Indices
0
1
2
3
4
5
Exercises
Refers
Refers
Refers
Refers
Refers
Refers
to feed
to reaction
to reaction
to reaction
to reaction
to reaction
1
2
3
4
5
mol/m3
mol/m3
mol/m3
mol/m3
various
m3/h
mol/m3 h
m3
C-mol/m3
mol/mol
244
Results
Seen in Fig. 3 are the simulation results giving the concentrations (glucose,
ethanol and biomass) during the fed batch process. In Fig. 4 the maximum in
ethanol concentration as a function of feedrate is given from a Parameter Plot.
30
25
60
Figure 3. Batch yeast fermentation.
245

30
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Figure 4. Influence of flowrate on the maximum ethanol concentration.
Reference
This example was contributed by C. Niklasson, Dept. of Chemical Reaction
Engineering, Chalmers University of Technology, S - 41296 Goteborg,
Sweden.
8.3.4
Repeated Fed Batch Culture (REPFED)
System
A single cycle of a repeated fed batch fermentation is shown in Fig. 1. In this
operation a substrate is added continuously to the reactor. After the tank is full,
the culture is partially emptied, and the filling process is repeated to start the
next fed batch. The operating variables are initial volume, final volume,
substrate feed concentration and flow rates of filling and emptying.
246
Figure 1. One cycle of a repeated fed batch.
Model
The equations are the same as given in the example FEDBAT (Section 8.1.3),
where the balances for substrate and biomass are written in terms of masses,
instead of concentrations. The only difference is that an outlet stream is
considered here to empty the fermenter at the end of the production period.
Program
Since in a Madonna program, the initial conditions cannot be reset, an outlet
stream is added. The inlet and outlet streams are controlled by conditional
statements as shown below. The full program is on the CD-ROM.
{Statements to switch the feed and emptying streams)
Fin=if time> = 10 then Flin else 0
{batch start up}
Flin= if time> = 33 then 0.5 else if time> = 32 then 0
else if time> = 21 then 0.5 else if time> = 20 then 0
else 0.5
Fout= if time>=33 then 0 else if time>=32 then 5.39
else if time> = 21 then 0 else if time> = 20 then 5.39
else 0
247
Nomenclature
Symbols
D
F
Kl and K2
KS
Dilution rate
Flow rate
Product kinetic constants
Saturation constant
P
S
X
V
V
0
VX
VS
Y
|i
Reactor volume
Initial volume of liquid
Biomass in reactor
Substrate in reactor
Yield coefficient
Indices
S
X
0 (zero)
initial
in
out
Exercises
Refers
Refers
Refers
Refers
Refers
Refers
to substrate
to biomass
to initial and inlet values
to initial values
to inlet
to exit
1/h
m3/h
various
kg/m3
g/m3
kg/m3
kg/m3
m3
m3
kg
kg
kg/kg
1/h
248
Results
Shown below are results of a simulation with three filling cycles.
60 -|
.-80
A
pr^Ti
/- 70
| "vsi|
, go
/ I
/ I
/"(
/
.50
/ I
'
J
-40
f
I /
\
/
I /
i /-V
/
I/
I / ^\
\l
XV
,*
\
-10
50-
40-
30-
20-
10-
--*'"-'
0-
10
15
20
25
30
35
L
40
45
-Q
50
TIME
Figure 2. Masses of substrate and biomass during filling and emptying cycles.
249
10
Figure 3. Concentrations of product, substrate and biomass during filling and emptying
cycles. The volume is also shown.
References
Dunn, I.J., Mor, J.R., (1975) Variable Volume Continuous Cultivation
Biotechnol. Bioeng. 17, 1805.
Keller, R., Dunn, I.J. (1978) Computer Simulation of the Biomass Production
Rate of Cyclic Fed Batch Continuous Culture J. AppL Chem. Biotechnol. 28,
784.
8.3.5
Repeated Medium Replacement Culture

(REPLCUL)
System
Slow-growing animal and plant cell cultures require certain growth factors and
hormones which begin to limit growth after a period of time. To avoid this,
part of the entire culture is replaced with fresh medium. A single cycle of
repeated replacement culture is shown in Fig. 1. In this procedure part of the
medium volume (with cells) is removed after a certain replacement time and
replaced with fresh medium. Each cycle operates as a constant volume batch in
250
which the concentration of substrate decreases, while that of biomass increases.

The operating variables are replacement volume, replacement time, and
substrate concentration in the replacement medium. The initial conditions for
each cycle are determined by the final values in the previous cycle and the
replacement volume and concentration.
Replacement
VX
Final Conditions
VS
Initial Conditions
Figure 1. One cycle for medium replacement culture.
Model
The equations are those of batch culture, where for convenience the total
masses are used.
dVS = r
"dT
sV
dvx
Monod kinetics is used.

The effective starting conditions for each batch can be calculated using the
final conditions of the previous cycle from the volume replaced, VR? and the
total volume, V, by the equations,
* VR
f=
VX = (1 - ) VXF
251
VS= ( l - f ) V S F + V R S 0
where f is the volume fraction replaced.
Program
The program as shown on the CD-ROM makes use of the PULSE function to
vary the biomass and substrate concentrations corresponding to the
replacement of a fraction F of the culture medium. The time for each batch is
the value of INTERVAL.
Nomenclature
Symbols
D
f
KS
X
V
v
vx
vs
VR
Y
Dilution rate
Fraction of volume replaced
Saturation constant
Reactor volume
Initial volume of liquid
Biomass in reactor
Substrate in reactor
Volume replaced
Yield coefficient
Indices
F
S
X
0
Refers
Refers
Refers
Refers
to final values at end of the cycle

to substrate
to biomass
to initial and inlet values
1/h
g/m3
g/m3
g/m3
m3
m3
kg
kg
m3
1/h
252
Exercises
Results
Fig. 2 shows how the biomass increases, until after six cycles the time profiles
become almost identical.
TIME= 19.29 X = 1.26
10
20
30
40
50
TIME
60
70
90
100
Figure 2. Oscillations of biomass and substrate concentrations with replacement cycles for
Interval 10 and F=0.8
253
8.3.6
Penicillin Production in a Fed Batch

Fermenter (PENOXY)
A fed batch process is considered for the production of penicillin, as described

by Muttzall (1), The original model was altered to include oxygen transfer and
the influence of oxygen on the growth kinetics.
Figure I. Fed batch reactor showing nomenclature.
Model
As explained in the example FEDBAT the balances are:
Total mass
dt
Biomass:
d(MassX)
= Vr-X
dt
Substrate:
d(MassS)
= FSf+Vrs
dt
Product:
254

d(MassP)
dt
_.
= V fp
p
Dissolved oxygen, neglecting the content of the inlet stream is calculated from
d(MassO)
= K L a*(O sat -0) + Vr 0
dt
The influence of biomass concentration
approximated here by
on
the
oxygen
transfer is
KX+X
The concentrations are calculated from
_MassX
V
1\. """""""""""""^ ,
MassS
,
V
MassP
,
V
MassO
V
The growth kinetics take into account the oxygen influence
o
The substrate uptake kinetics includes that amount used for growth, for product
and for maintenance
J*o ~
Jft o _/V
V
Y
XS
V"
Y
PS
Product production involves two terms whose constants are turned on and off
according to the value of |ii, as seen in the program.
Oxygen uptake includes growth and maintenance
=-TT
Y
xo
255
Program
The program is on the CD-ROM.
Nomenclature
Symbols
F
KLa
Ko
KS
KX
Mass
mo
ms
Osat
Sf
V
'max
YPS
YXO
YXS
H-max
Exercises
Feed flowrate
Oxygen transfer coeff.
Monod constant for oxygen
Monod constant for glucose
Constant for biomass effect on
Component mass
Maintenance coeff. for oxygen
Maintenance coeff. for glucose
Saturation for oxygen
Feed cone, of glucose
Volume
Maximum volume
Yield product to substrate
Yield biomass to oxygen
Yield biomass to substrate
max.specific growth rate
m3/h
1/h
kg/m 3
kg/m 3
kg/m3
kg
kg O/kg X h
kg S/kg X h
kg/m3
kg/m3
m3
m3
kg/kg
kg/kg
kg/kg
1/h
256
II!
References
K. Mutzall, "Modellierung von Bioprozesses", Behr's Verlag, 1994.
Program and model developed by Reto Mueller, ETH Zurich.
Results
0.008
20
40
60
80
Figure 2. Dynamics of the fed batch reactor.
100
120
140
160
180
200
257
8.4 Continuous Reactors

-0.008
120
0.007
100
! I
i I
80
0.006
0.005
: I
Li I
I
- 60
40
0.004 O
-0.003
-0.002
20
-0.001
0
0
20
40
60
80
100
120
140
160
180
200
TIME
Figure 3. Influence of initial KLa value from 100 to 160 h"^ on the S and O profiles.
8.4
8.4.1
Continuous Reactors
System
The steady state operation of a continuous fermentation having constant
volume, constant flow rate and sterile feed is considered here (Fig. 1).
257
-0.008
120
-0.007
100
1I
80
\
. ll
'-, 1
- 60
\\
40
-0.006
I
I
. !i
i1
-0.005
-0.004 O
v i
-0.003
|\ i
I i '\ *
\ \! \l
!
\\ \
20
-0.002
-0.001
-0
20
40
60
80
100
120
140
160
180
200
TIME
Figure 3. Influence of initial KLa value from 100 to 160 h"^ on the S and O profiles.
8.4
8.4.1
Continuous Reactors
System
The steady state operation of a continuous fermentation having constant
volume, constant flow rate and sterile feed is considered here (Fig. 1).

ISBN: 3-527-30759-1
258
D,S
Figure 1. Chemostat fermenter with model variables.
Model
The dynamic balance equations may be modified to apply only to the steady
state by setting the time derivatives equal to zero. The corresponding equations
are then:
For biomass,
0 = - D X + rx
For substrate,
0 = D (S0 - S) + rs
Growth kinetics,
rx = ^ X
Substituting into the biomass balance gives

\i = D
where S is determined by the kinetics
\i = f(S)
The Monod relation results in,

S =
The substrate balance gives,

X = Y(S 0 -S)
259
The productivity of the reactor for biomass is X D.

The above equations represent the steady state model for a chemostat with
Monod kinetics. Using them it is possible to calculate the values of S and X,
which result from a particular value of D, and to investigate the influence of the
kinetic parameters.
Program
In Madonna programs, time can be used as a variable which will increase from
the starting time. Here it is renamed D. Thus equations will be solved for
increasing values of the dilution rate. Fortunately X and S can be explicitly
solved for in this problem. If not, the ROOT FINDER facility of Madonna can
be used. The program is found on the CD-ROM.
Nomenclature
The nomenclature is the same as the example CHEMO, Sec. 8.1.2.
Exercises
260
Results
The steady state curves of X, S, and XD versus D are given Fig. 2. The results
in Fig. 3 were obtained by varying K$ in each run. An interesting effect can be
observed on the position of the washout point.
-4
10 9-
3.5
87-
6v-
*S**~\ !I
\1 i
Y.-I
m ^r
~s;i
f*r
mm
\!
-2.5
S"
"
-3
4-
1.5
3-
//
2-
/{
0.5
1-
0-
0.1
0.2
0.3
0.4
0.5
0.6
Figure 2. Steady state curves of X, S and XD versus D.
0.7
0.8
0.9
261
Figure 3. Runs obtained by varying KS from 0.2 to 1.0.
8.4.2
Continuous Culture with Inhibitory Substrate

(CONINHIB)
System
Inhibitory substrates at high concentrations reduce the specific growth rate
below that predicted by the Monod equation. The inhibition function may be
expressed empirically as
where KI is the inhibition constant (kg/m3).

If substrate concentrations are low, the term S2/Kj is lower in magnitude than
KS and S, and the inhibition function reduces to the Monod equation. In batch
cultures the term S2/Kj may be significant during the early stages of growth,
even for higher values of K[. The inhibition function passes through a
maximum at Smax = (Kg Ki)-5. A continuous inhibition culture will often lead
262
to two possible steady states, as defined by the steady state condition JLI = D and
as in shown Fig. 1.
D=
Figure 1. Possible steady states for a chemostat with inhibition kinetics.
One of these steady states (A) can be shown to be stable and the other (B) to be
unstable. Thus, only state A and the washout state (S = SQ) are possible.
Model
A model of a chemostat with its variables is represented schematically in Fig. 2.
F,S 0
+-
Figure 2. Model variables.
F,S,X
263
Cell material balance,

VdX
jj- = ^ i V X - F X
or,
where D is the dilution rate = F/V.

Substrate material balance,
VdS
= F (S 0 -S)
^VX
-~^f
or,
dS
dT
MX
= D (S0 - S) -
where Y is the yield factor.
Program
When the system equations are solved dynamically, one of two distinct steady
state solutions are obtained, the stable condition A and the washout condition.
The initial substrate and organism concentrations in the reactor will determine
the result. This is best represented as a phase-plane plot X versus S. All results
indicate washout of the culture when the initial cell concentration is too low;
higher initial substrate concentrations increases the likelihood of washout.
Nomenclature
Symbols
D
KI
KS
Dilution rate
Inhibition constant
Saturation constant
1/h
kg/m3
kg/m3
264
S
Smax
X
Y
Maximum in S for inhibition function
Yield coefficient
Indices
0
I
m
Exercises
Refers to inlet
Refers to initial value
Refers to maximum
kg/m3
kg/m3
kg/m3
kg/kg
1/h
265
Results
1-1-2
10
15
20
TIME
25
30
35
40
Figure 3. Time course of X, S and U.
5 i
4.5 -
4
3.5
3
e/> 2.5
2
1.5
1
0.5
0
0.5
2.5
Figure 4. Phase-plane plot of X versus with varying ST from 0 to 5 kg/m3 using Batch Runs
with overlay.
266
Run 9: 2000 steps in 0 seconds
5-
X,
4.5-
4 -
^^^>
*"
^ *t %%
3.5-
* * C^1 v
\
"|
f/ // // /;^',--^,
. *x
2
1.5-
0.5-
"%
\ ^ x
i ! l
</) 2 . 5 -
3:2 (0.5)
3:3 (0.5857)
_ - - 3:4(0.6714)
3:5(0.7571)
3:6(0.8429)
3:7(0.9286)
3:8(1.014)
3:9(1.1)
t**
j* S ^
f f / t / ,f
rf'^T
:' / /
* / Jf*'^
* \ f 1 C & (
"^ -s^^-
"^"^>*i'^' ** -^
^^^*^^$^S? **
^^*^^ **
0.5
Figure 5. Phase plane plot of influence of the initial biomass Xi from 0.5 to 1.1 for
Steady states upper left and lower right.
= 0.0.
Figure 6. Influence on the inhibition function made by varying KI between 1 and 3.
Reference
Edwards, V.H, Ko, R.C. and Balogh, S.A. (1972). Dynamics and Control of
Continuous Microbial Propagators Subject to Substrate Inhibition Biotechnol.
and Bioeng. 14, 939-974.
267
8.4.3
Nitrification in Activated Sludge Process

(ACTNITR)
System
Nitrification is the process of ammonia oxidation by specialized organisms,
called nitrifiers. Their growth rate is much slower than that of the heterotrophic
organisms which oxidize organic carbon, and they can be washed out of the
reactors by the sludge wastage stream (Fs). In an activated sludge system
(Fig. 1) when the organic load (F So/V) is high, then the high biomass growth
rates require high waste rates. Nitrification will not be possible under these
conditions because the concentration of nitrifiers (Ni) will become very low.
O,F O
2, F4
Reieto*
2, F3
Figure 1. Configuration and streams for the activated sludge system.
Model
The dynamic balance equations can be written for all components around the
reactor and around the settler. The settler is simplified as a well-mixed system
with the effluent streams reflecting the cell separation.
Organic substrate balance for the reactor:
= F 0 So + F 2 S 2 -
268
Ammonia substrate balance in the reactor:

= FQ AQ + F2 A2 - FI A
R2Vt
Reactor balance for the heterotrophic organisms:

pj7
= p2 O2 FI QI + RI Vj_
Reactor balance for the nitrifying organisms:

l
= F 2 N 2 - F i N i + R2Vi
di
Organic substrate balance in the settler:

V 2 dS 2
j^
= F i S i - F3S2 - F4S2
Ammonia substrate balance for the settler:

V 2 dA 2
3t
= F A
I I -
- F4A2
Balance for heterotrophic organisms in the settler:

V 2 d0 2
dt
= Fl l - F3 02
Balance for nitrifying organisms in the settler:
V2 dN2
34 = F i N i - F 3 N 2
The equations for the flow rates are given below.
Recycle flowrate:
F2 = F 0 R
where R is the recycle factor.
Reactor outlet flow:
Flow of settled sludge:
FI = F2 + F0 = F O R + FO
269
where C is the concentration factor for the settler.

Flow of exit substrate:
F4 = FI - F3>
Flow of exit sludge wastage:
F5 = F3 - ?2.
Note that C and R must be chosen so that F5 is positive.

Monod-type equations are used for the growth rates of the two organisms.
l^2max
2 = ^Ni =
Program
The program is given on the CD-ROM.
Nomenclature
Symbols
A
C
F
Fo-5
KI
K2
N
O
R
Ammonia substrate concentration

kg/m3
Concentrating factor for settler
Flow rate
m3/h
Flow rates, referring to the figure
m3/h
Saturation constant of heterotrophs
kg/m3
Saturation constant of nitrifying organisms
kg/m3
Concentration of nitrifiers
kg/m3
Concentration of heterotrophs
kg/m3
Recycle factor
-
270
Rl
R2
V
Y
Hi
Growth rate of heterotrophics

kg/m3h
Growth rate of nitrifying organisms
kg/m3h
Organic substrate concentration
kg/m3
Volumes
m3
Yield coefficients
kg/kg
Specific growth rate of heterotrophs
1/h
Specific growth rate of nitrifying organisms
1/h
Indices
Flow and concentration indices referring to Fig. 1 are as follows:
0
Refers to feed and initial values
1
Refers to reactor and organic oxidation
2
Refers to settler and ammonia oxidation
3
Refers to recycle
4
Refers to settler effluent
5
Refers to sludge wastage
m
Refers to maximum
Exercises
271
Results
The results in Fig. 2 demonstrate the influence of flow rate on the effluent
organics 82- The ammonia in the effluent A2 is seen, in Fig. 3, to respond
similarly to FQ, but for a very high value of FQ = 1000 m3/h the nitrification
ceases, and A2 becomes the same as the inlet value AQ. This corresponds to
washout of the nitrifiers, which would be seen by plotting NI versus time.
0.9 ,
0.8 -I
,/"
M if
If J
II /
It
0.3- rr
J
II
"
0-1 JM
02.
" .
6
10
12
82:1(20)
82:2(180)
82:3(340)
82:4(500)
14
16
18
20
TIME
Figure 2. Transient of S2 at various flow rates F0 (20 to 500m3/h, bottom to top).

0.1 -I
0.090.08-
3 0.06 -|-_005 J*
1
A2:1(20)
A2:2(180)
-_A2:3(340)
I
A2:4(5QO)
S.
0.040.03-
0.02J
0
10
TIME
12
14
16
18
20
Figure 3. Ammonia in the effluent (A2) at various flow rates F0 (5 to lOOOm^/h, bottom to
top).
272
8.4.4
Tubular Enzyme Reactor (ENZTUBE)
System
A tubular, packed-bed, immobilized-enzyme reactor is to be investigated by
simulation. The flow is assumed to be ideal plug flow. The distribution of the
enzyme is not uniform and varies linearly from the inlet to higher values at the
outlet, as shown in Fig. 1.
Enzyme
concentration
Enzyme distribution
Distance along reactor, Z

Figure 1. Distribution of enzyme along the tubular reactor.
Model
The equations for steady state operation are given below.
Substrate balance,
dS
dZ
1
~v
Kinetics,
The linear flow velocity is increased by the presence of the solid enzyme carrier
particles according to
273
V7
L
F
Ae
~
The reaction velocity depends on the enzyme concentration,

vm = KE
and the linear distribution of enzyme distribution given by,

E = E0 + mZ
Program
The model is solved by renaming the independent variable, TIME, to be the
reactor length coordinate Z. The program is given on the CD-ROM.
Nomenclature
Symbols
A
F
K
KM
m
r
S
vm
vz
Z
e
E
Reactor tube cross section

Flow rate
Rate constant
Enzyme distribution constant
Reaction rate
Maximum reaction velocity
Linear flow velocity
Reactor length
Void volume fraction of packing
m2
m3/h
1/h
kg/m3
kg/m3 m
kg/m3 h
kg/m3
kg/m3h
m/h
m
kg/m3
274
Indices
0
S
Refers to inlet
Refers to substrate
Exercises
Results
Flow rate is the primary operating variable, along with enzyme loading and
inlet concentration. In Fig. 2 the influence of F is seen in the steady-state, axial,
substrate profile.
275
10
12
14
16
18
20
Figure 2. Substrate profile under the influence of F (1 to 10 m^/h, bottom to top).
8.4.5
Dual Substrate Limitation (DUAL)
System
In defined-nutrient growth media, one substrate can usually be made to be
limiting by adjusting its concentration relative to those of the other medium
components. In general, however, more than one substrate may limit the cell
growth rate. In this case the yield coefficients for the various components,
Yxsi> may vary depending upon the growth regime. This situation was
discussed by Egli et al. (1989), who examined results at steady state with dual
nutrient limitation. The present mathematical model simulates the transient
behaviour of such a dual (Si -carbon, 82 -nitrogen) nutrient-limited system
when carried out in a chemostat. The model assumes that the yield coefficients
are each a function of the ratio 81/82, i.e. the ratio of the carbon-nitrogen
substrate concentrations in the vessel. The original paper took the carbonnitrogen ratio in the feed stream as the controlling parameter. Here the
concentrations in the reactor are assumed to be controlling.
Model
Assuming a perfectly mixed, constant volume continuous-flow stirred-tank
reactor, the mass balance equations for the cells and for the two limiting
substrates are as follows:
276
= D
(SlFeed - Si) -
/O
O \
I ^__i^^_ I i i "V
(o2pee(j o2) lv YQO J ]LlA
where D = F/V.
The specific growth rate is modelled as
Si
The yield coefficients are assumed to vary with the carbon-nitrogen ratio in the
reactor.
Si
RATIO = ^
The yield coefficients are varied according to RATIO using the following logic:
Y X Sl=Yi m i n
and
and
YXS2 = Y2min
YXS2 = Y2max
if
if
RATIO < B i
RATIO > B2
where,
_
Y2min
Y 2max
Bi = \r
1 - and Bo = 1v~,T"
Imax
- 1mm
The boundaries of the three growth regimes in Fig. 1 are defined by the
quantities BI and B2.
277
C limitation
N limitation
Double limitation
10
XSi
0.8
r
XS1
B2
S2
Figure 1. Limitation regions for carbon and nitrogen showing influence on yield.
The yield coefficients for biomass on nitrogen and carbon take maximum or
minimum values when only one substrate is limiting and vary linearly with
opposing tendencies in the double-limitation region.
Program
Note that the programing of this example is rather more complicated than usual
owing to the need to allow for the logical conditions of carbon limitation,
nitrogen limitation or both substrates together causing limitation. A partial
listing is seen below and the full program is on the CD-ROM.
(CALCULATION
OF
YIELD
VALUES)
YXSl=if (RATIO < Bl) then YlMAX else ( if (RATIO >

B2) then Y1MIN else (Y1MAX+(RATIO-B1)/(B2B1)*(Y1MIN-Y1MAX))
)
YXS2 = if (RATIO < Bl) then Y2MIN else ( if (RATIO >
B2) then Y2MAX else
(Y2MIN+(RATIO-B1)/(B2Bl)*(Y2MAX-Y2MIN))
)
278
Nomenclature
Symbols
Bi
B2
Cc
Cn
D
F
KS
R
Si
S2
X
Y
H
Ratio of Y 2min /Yi max

Ratio of Y 2max /Yi min
Carbon source concentration
Nitrogen source concentration
Dilution rate
Volumetric feed rate
Affinity constant
Reaction rates
Carbon source concentration
Nitrogen source concentration
Yield coefficient
Indices
1
2
Exercises
Refers to carbon source

Refers to nitrogen source
_
-
kg/m3
kg/m3
1/h
m3/h
kg/m3
kg/m3 h
kg/m3
kg/m3
kg/m3
kg/kg
1/h
279
Results
The startup of a continuous culture is shown in Fig. 2. Note that the nitrogen
level 82 in the reactor drops to a low level after 15 h and causes a change in the
yield coefficients. The influence of dilution rate on the system was investigated
by varying D from 0 to 1.5 as shown in Fig. 3.
3-c
Figure 2. Startup of a continuous culture.
X 1.5
Figure 3. Variation of D from 0.1 to 1.5 (top to bottom).
280
Reference
Egli, Th., Schmidt, Ch. R. (1989). "On Dual-Nutrient-Limited Growth of
Microbes, with Special Reference to Carbon and Nitrogen Substrates", in
Proceed. Microb. Phys. Working Party of Eur. Fed Biotech. Eds. Th. Egli, G.
Hamer and M. Snozzi, Hartung-Goree, Konstanz, 45-53.
This example was developed by S. Mason, ETH-Zurich.
8.4.6
Dichloromethane in a Biofilm Fluidized Sand

Bed (DCMDEG)
System
The process involves the removal of dichloromethane (DCM) from a gas stream
and the subsequent degradation by microbial action. The reactor consists of
biofilm sand bed column with circulation to an aeration tank, into which the
substrate and oxygen enters in the gas phase, or the substrate can be fed in a
liquid stream, as shown in Fig. 1. The column is approximated by a series of six
stirred tanks. The reaction is treated with homogeneous, double saturation
kinetics with dichloromethane (DCM) inhibition. The oxidation of one mole of
DCM produces 2 moles of HC1, making a hydrogen ion balance for pH important. The yield with respect to oxygen is 4.3 mg DCM/mg 62. In practice,
care must be taken to prevent stripping of DCM to the air stream.
281
SR6>
SRin C jn , pH jn
Figure 1. Schematic of fluidized bed column with external aeration vessel.
Model
The model does not include a gas balance on the aeration tank, since it is
assumed that the gas phase dynamics are comparatively fast and hence an
equilibrium with the inlet concentration of oxygen and DCM may be assumed.
The biomass is assumed to grow slowly, and growth rates are therefore also not
modelled. The model for pH changes does not include buffering effects.
For the inlet section 1 at the bottom of the column the balances are as follows:
O2 balance,
dCQ1 ^
dt
282
DCM balance,
dC
Srl _
dt
Srin~ C Srl
H ion balance,
i -CHI
dCHi
2r S i
84900
Here T is the residence time of the liquid in one section of the column. The
constant 84,900 converts grams to moles and includes the stoichiometry.
pHi = -0.434 log |Cm|
Evaluation of rates for the inlet section 1:
maxCSrl
-01
KI )
For the aeration tank the 62 and DCM balances are:

K L a 0 2(Co2eq-Coin)
dC
R
V
-~ = (CSr6 - CSrin )
at
DCM (Cs2eq -
Tr- (CSFO ~ CSrin
Program
The program constants describe DCM entering the reactor in the gas stream.
The DCM concentration in the liquid feed is set to zero. The program is on the
CD-ROM.
Nomenclature
oin
srin
CSFO
CSG
F
KI
KLa
KS
pHn
R
VR
VT
Vmax
YSO
Exercises
11
H+ ion concentration in section n

kg mol/m3
Inlet dissolved oxygen concentration
g/m3
Oxygen saturation constant
g/m3
DCM saturation constant
g/m3
DCM inlet concentration
g/m3
DCM concentration in section n
g/m3
Oxygen concentration in section n liquid g/m3
DCM concentration in feed
g/m3
DCM gas concentration
g/m3
Feed rate
m3/h
Inhibition constant
g/m3
Transfer coefficients for DCM and 2 1/h
Saturation constants
g/m3
pH in n section n
pH units
Recirculation rate
m3/h
Oxygen uptake rate in section n
g/m3 h
Substrate uptake rate in section n
g/m3 h
Reactor volume
m3
Volume of aeration tank
m3
Maximum degradation rate
g/m3 h
Yield coefficient for DCM/oxygen
Liquid residence time in one section
h
283
284
Results
The concentrations in the stream leaving the top of the column (CSr6) during
startup of the fluidized bed are shown in Fig. 2 for four values of F (0.5 to 10)
The change of the pH for one flow rate (F = 0.5) is shown in Fig. 3.
285
0.05
0.1
0.15
0.2
0.25
0.3
0.4
0.35
0.5
0.45
TIME
Figure 2. Fluidized bed startup for four values of F (0.5 to 10, bottom to top).

3.5
3
2.5
CSR6:1
... PH6:1
2
1.5
1
0.5
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
TIME
Figure 3. Change of carbon substrate and pH in the top section 6 during startup.
Reference
D. Niemann Ph.D. Dissertation 10025, ETH, 1993.
286
8.4.7
Two-Stage Chemostat with Additional Stream

(TWOSTAGE)
System
Two chemostats are arranged in series (Fig. 1) with the intention that the first
operates at a relatively high rate of cell growth, while the second operates at low
growth rate, but high cell density, for secondary metabolite production.
Additional substrate may be fed to the second stage.
, 810
X1.S!
Hi
US
Figure 1. Two-stage chemostat with two feed streams.
Model
The balance equations are written for each component in each reactor.
Stage 1 with sterile feed,
= F[S O -S!] -
287
Stage 2 with additional substrate feed and an input of cells and substrate from
Stage 1,
V2
- [F + Fi]X2
V2 ^2.= F [Si - S2] + F! [Sio - S2] dt
Yv
KS + S2
Productivity for biomass:

First stage,
Prodi =
V,
Both stages,
Prod2 =
Program
Nomenclature
Symbols
F
KS
Prod
S
V
X
Y

Saturation constant
Productivity for biomass
Reactor volume
Yield coefficient
m3/h
kg/m3
kg/m3 h
kg/m3
3
kg/m3
kg/kg
1/h
288
Indices
0
1
2
10
m
Refers
Refers
Refers
Refers
Refers
to tank 1 inlet
to tank 1 and inlet of tank 2
to tank 2 and outlet of system
to separate feed for tank 2
to maximum
Exercises
Results
The results in Fig. 2 give biomass concentrations and productivities for both
tanks during a startup with a constant feed stream to the first tank (F = 0.5). In
Fig. 3 the influence on X2 of feed to the second tank Fl (0 to 1.0) with
constant F is shown.
289

T5
35
40
Figure 2. Biomass (Xj X2) and productivities for both tanks (F = 0.5).
5
4.5
4
3.5
32.5
2
1.5
1
0.5
0
10
15
20
25
30
35
40
TIME
Figure 3. Influence on X2 of feed to the second tank (Ft = 0 to 1.0, curves right to left).
290
8.4.8
Two Stage Culture with Product Inhibition

(STAGED)
System
Products may inhibit growth rates. Under such conditions a multi-staged
continuous reactor as shown in Fig. 1 will have kinetic advantages over a single
stage. This is because product concentrations will be lower and consequently
the rates in the first tank will be higher as compared with a single tank. This
effect may be conveniently investigated by simulation. Batch cultures can be
expected to have similar kinetic advantages for product inhibition situations.
Figure 1. Two-stage chemostat with product inhibition.
Model
The inhibition function is expressed empirically as
When product concentrations are low, the equation reduces to the Monod
equation.
The product kinetics are according to Luedeking and Piret, with dependence
on both growing and non-growing biomass,
ISBN: 3-527-30759-1
291
rpn = (On + (3n \ln) Xn
In addition, the non-growth term, an, is assumed to be inhibited according to,
an - a"Q
~ 1i-r+rPn
When product concentrations are low, a = ano.
Kinetics for growth:
Kinetics for substrate consumption (neglecting consumption for product):
_ _rxn
where Y is the yield factor.
Mass balances:
Stage 1,
j- = F[So-Si] +r S iVi
jp = F[P 0 -Pi] + rp^j
Stage 2 with additional substrate feed FI,
dX2
V 2 -gjT- = F Xj - [F + F!]X2 + rX2V2
dS2
V2 -gj- = F [Si - S2] + FI [Sio - S2] + rS2V2
dP2
- =FPl- [F + Fi]P2 + rp2V2
Productivity for product:

First stage,
292
Prodi =
Both stages,
Program
Nomenclature
Symbols
F
KI
KS
P
Prod
r
S
V
X
Y
a
OC0

Inhibition constant
Saturation constant
Productivity for product
Reaction rate
Reactor volume
Yield coefficient
Non-growth product rate term
Non-growth term with no inhibition
Growth dependent product yield
Maximal specific growth rate
Indices
n
0
1
Refers to tank n
Refers to tank 1 inlet
Refers to tank 1 and inlet of tank 2
m3/h
kg/m3
kg/m3
kg/m3 h
kg/m3 h
kg/m3
m3
kg/m3
kg/kg
kg P/kg X h
kg P/kg X h
kg/kg
1/h
1/h
2
10
293
Refers to tank 2 and system of outflow

Refers to inlet concentration of tank 2
Exercises
Results
The startup and approach to steady state for the two stages is shown in Fig. 2.
The influence of the inhibition can be tested by varying KI from 0.1 to 10.0, as
shown in Fig. 3. The higher the KI the lower is the degree of inhibition and the
greater is the product concentration P2-
294
r 10
3.5
3
2.5
,
1.5
1
0.5
0
10
15
TIME
Figure 2. Startup and approach to steady state for the two stages.
1.4.,
1.3
1.2.
1.1
I
1
0.9.
0.8
0.7 J
10
12
14
16
18
20
22
24
26
TIME
Figure 3. Product concentration P at various values of KI (1 to 5), curves bottom to top.
Reference
Herbert, D. (1961). A Theoretical Analysis of Continuous Culture Systems.
Soc. Chem. Ind. Monograph No. 12, London, 2L
295
8.4.9
Fluidized Bed Recycle Reactor (FBR)
System
A fluidized bed column reactor can be described as 3 tanks-in-series (Fig. 1).
Substrate, at concentration SQ, enters the circulation loop at flow rate F. The
flow rate through the reactor due to circulation is FR. Oxygen is absorbed in a
well-mixed tank of volume VT. The reaction rate for substrate (r$) depends on
both S and dissolved oxygen (CL)- The rate of oxygen uptake (ro) is related to
S by a yield coefficient (Yos)- The gas phase is not included in the model,
except via the saturation concentration (CLS)- The oxygen uptake rate of
reactor can be determined by the difference in CL inlet and outlet values.
? So , Fn
Fluidized
Bed
F,S
Figure 1. Biofilm fluidized bed with external aeration.
296
Model
The model balance equations are developed by considering the individual tank
stages and the absorber separately. The gas phase in the absorber is assumed to
be air.
Substrate balances:
For the absorption tank
dS
FR
dF =
For each stage n

dSn
FR
-3T = -^(Sn-!-Sn)- rsn
Oxygen balances:
For the absorption tank
r
= ^(C L 3-C L )+K L a(C L s-C L )

VT
For each stage

dCLn
~dT"
FR
V
(CLn
-! ~ C Ln) ~ rOn
Kinetics for stage n:
V Tm
K n +Sn K 0 +C Ln
Program
297
Nomenclature
Symbols
CL
CLS
F
FR
KLa
Ks
Ko
r
S
V
VT
^m
x
Y
T
Dissolved oxygen concentration

Saturation oxygen concentration
Feed flow rate
Recycle flow rate
Transfer coefficient
Saturation constant
Saturation constant for oxygen
Reaction rate
Reactor volume of one stage
Volume of absorber tank
Maximum velocity
Yield coefficient
Inverse liquid residence time
Indices
0
l,2,3,n
m
O
S
T
X
Exercises
Refers to feed
Refer to the stage numbers
Refers to maximum
Refers to oxygen
Refers to substrate
Refers to aeration tank
Refers to biomass
g/m3
g/m3
m3/h
m3/h
1/h
kg/m3
g/m3
kg/m3 h
kg/m3
m3
m3
kg/m3 h
kg/m3
kg/kg and g/kg
1/h
298
Results
Note from the results below that the steady state for oxygen is reached rather
quickly, compared to that of substrate.
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Figure 2. Oxygen concentrations in fluidized bed reactor. Top of column is the lower curve.
299
35
tf
Figure 3. Substrate concentrations from the run as in Fig. 2.
8.4.10
Nitrification in a Fluidized Bed Reactor

(NITBED)
System
Nitrification is an important process for wastewater treatment. It involves
the sequential oxidation of NFLt"1" to NO2~ and NC>3~ that proceeds
according to the following reaction sequence:
NH4+ + 1 02 -> N02- + H20 +2H+
NO2~ +
O2 - NO3~
The overall reaction is thus

NH4+ + 2O2
NO3- + H2O + 2H+
Both steps are influenced by dissolved oxygen and the corresponding nitrogen
substrate concentration. Owing to the relatively slow growth rates of nitrifiers,
treatment processes benefit greatly from biomass retention.
300
In this example, a fluidised biofilm sand bed reactor for nitrification, as

investigated by Tanaka et al. (1981), is modelled as three tanks-in-series with a
recycle loop (Fig. 1). With continuous operation, ammonium ion is fed to the
reactor, and the products nitrite and nitrate exit in the effluent. The bed
expands in volume because of the constant circulation flow of liquid upwards
through the bed. Oxygen is supplied external to the bed in a well-mixed gasliquid absorber.
Model
The model balance equations are developed by considering, separately, the
individual tank stages and the absorber. Component balances are required for all
components in each section of the reactor column and in the absorber, where the
feed and effluent streams are located. Although the reaction actually proceeds
in the biofilm phase, a homogeneous model with apparent kinetics is employed
rather than a biofilm model, as in the example NITBEDFILM.
03.
Fluidized
bed
Figure 1. Biofilm fluidised-bed recycle loop reactor for nitrification.
In the absorber, oxygen is transferred from the air to the liquid phase. The
nitrogen compounds are referred to as Si, 82, and 83, respectively. Dissolved
301
oxygen is referred to as O. Additional subscripts, as seen in Fig. 1, identify the

feed (F), recycle (R) and the flows to and from the tanks 1, 2 and 3, each with
volume V, and the absorption tank with volume VAThe fluidised bed reactor is modelled by considering the component
balances for the three nitrogen components (i) and also for dissolved oxygen.
For each stage n, the component balance equations have the form
Similarly for the absorption tank, the balance for the nitrogen-containing
components include the input and output of the additional feed and effluent
streams, giving
The oxygen balance in the absorption tank must account for mass transfer from
the air, but neglects the low rates of oxygen supply and removal of the feed and
effluent streams. This gives
For the first and second biological nitrification rate steps, the reaction kinetics
for any stage n were found to be described by
v
r
r2n
ml Sin
Qn
K + S K + O
m2 S2n
n
S
K
2+ 2n O2+n
The oxygen uptake rate is related to the above reaction rates by means of the
constant yield coefficients, YI and 2, according to
ron = - H n Y i -r 2 n Y 2
The reaction stoichiometry provides the yield coefficient for the first step
302
YI = 3.5 mg O2/(mg NNH4)

and for the second step
Y2 = LI mg O2/(mg NNO2)
Program
Nomenclature
Symbols
F
FR
Kjâ
K
KI
K2
O
Os and O*
OUR
r
S
V
VA
Feed and effluent flow rate

Recycle flow rate
Saturation constant for ammonia
Oxygen solubility, saturation cone.
Oxygen uptake rate
Reaction rate
Volume of one reactor stage
L/h
L/h
h
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L h
mg/L
L
L
vm
Y
Maximum velocity
Yield coefficient
mg/L h
mg/mg
Indices
1,2,3
1,2,3
A
F
ij
m
Refer to ammonia, nitrite and nitrate, resp.

Refer to stage numbers
Refers to absorption tank
Refers to feed
Refers to substrate i in stage j
Refers to maximum
Ol and O2
S1,S2
S and *
Exercises
Refer to oxygen in first and second reactions

Refer to substrates ammonia and nitrite
Refer to saturation value for oxygen
303
304
Results

6
10
15
20
25
30
35
40
45
50
Figure 2. Dynamic startup of continuous operation showing oxygen concentrations and

nitrogen compounds at the top of the column.

280
P 2.5
270
260
250
1^240.
,230<
220
M I
210
200
0.5
190
180
15
20
25
30
35
40
KLA
Figure 3. Parametric run of continuous operation showing oxygen and ammonia in the effluent
versus
305
8.4.11
Continuous Enzymatic Reactor (ENZCON)
System
This example, schematically shown in Fig. 1 involves a continuous, constant
volume, enzymatic reactor with product inhibition in which soluble enzyme is
fed to the reactor.
EO.FE
I S 1f P 1 F1
Figure 1. Continuous enzymatic reactor with enzyme feed.
Model
The mass balance equations are formulated by noting the two separate feed
streams and the fact that the enzyme does not react but is conserved.
Total flow:
FS + FE =
Mass balances:
dSi
= FsSo-FiS1+rsV
306
r = -FiP1+rpV
Kinetics with product inhibition:

r
S - - v mK M + S + (P/Ki)
vm = EI K2
rP = - 2 r s
Program
Nomenclature
Symbols
E
F
KI
KM
K2
P
r
S
V
vm
Flow rate
Inhibition constant
Saturation constant
Rate constant
Reaction rate
Reactor volume
Maximum rate
Indices
0
Refers to inlet values
kg/m3
m3/h
kg/m3
1/h
kg/m3
kg/(m3 h)
kg/m3
m3
kg /(m3 h)
307
1
E
P
S
Refers
Refers
Refers
Refers
to reactor and outlet values

to enzyme
to product
to substrate
Exercises
Results
Variations in the flows FE (Fig. 2) or Fs (Fig. 3) cause the product levels to
change.
308
Run 3: 8004 steps in 0.1 83

SeC ndS
1.8
1.6
--
0.8
.0.6
0.4
tS
P1:2(0.2)
P1:3(0.3)
,-'"
0.2
10
20
30
40
TIME
50
60
70
80
Figure 2. Performance for three values of FE.
Run 3: 8004 steps in 0.233

seconds
r4.5
3.5
x--'
3
2.5
.2
-1.5
r *>"*
-^ - **" ~ ""
jft
. P1:2(1.5)
.. P1:3(2)
<*/r
0
10
20
30
40
50
60
70
80
TIME
Figure 3. Performance for three values of Fs.
8.4.12
Reactor Cascade with Deactivating Enzyme

(DEACTENZ)
System
Biocatalysts usually deactivate during their use, and this has to be considered in
the bioreactor design. One of the methods to keep productivity fluctuations
low, and hence to efficiently utilize the biocatalyst, is to use a series of reactors
with biocatalyst batches having different times-on-stream in each reactor. In
309
this example a series of three stirred tanks of a constant equal volume with
biocatalyst deactivating by first order reaction kinetics is investigated (Fig. 1).
After a time period ILAG? ê biocatalyst from the tank with longest time-onstream (first tank in the cascade) is discarded and replaced by a fresh batch.
The streams are switched over so that tank 1 becomes tank 3, the last reactor in
the series. Other tanks are switched over correspondingly. This is equivalent to
replacing the used enzyme with fresh enzyme in tank 3 and moving the used
enzyme upstream from tank 3 to tank 2 to tank 1, which is easier to simulate.
(3-galactosidase was taken as an example of the biocatalyst. This obeys
Michaelis-Menten kinetics with competitive product inhibition, and the kinetic
constants were determined with considerable accuracy. The same constants are
used also in this substrate inhibition model.
F,S 0
F,Si
F,S 2
F,S 3
Figure 1. Tanks in series reactor with immobilized enzyme.
Model
Using the stoichiometry, S > P, the mass balances for the ith tank (i = 1, 2, 3)
with the volume V can be written
Substrate
Product
dt
Enzyme (active)
= F(P M -Pi)
310
=r Ei v
where rate of substrate consumption
(competitive)
is given by product
inhibition
s
i
Si = ' v max b i -7-Z~^\
inh
According to the molar stoichiometry

R P i = -R Si
The rate of enzyme deactivation is assumed to be:
rEi = - kD EI
For each batch of enzyme in tank i

dE
-i
This equation can be applied by changing the initial conditions for each tank
when the enzyme is moved from tank to tank. Thus the final value in tank n
becomes the initial condition in tank i-1. The initial conditions can also be
calculated by analytical integration of the enzyme deactivation equation at
times corresponding to the respective ages of the biocatalysts in the respective
reactors (multiples of TLAG)- Fresh enzyme with the activity EQ is in the third
tank. The other tanks start with the following enzyme activities:
EI = E0 e C- (3 - i) ko TLAG]
Program
In the program on the CD-ROM note that the cost calculation at the end of the
program is included only as a comment but could be incorporated into the
program with the corresponding values for the constants.
311
Nomenclature
Symbols
COST
E
ECOST
F
ICOST
kD
Kinh
Km
OCOST
P
RC
m
rs
S
T
t
TDOWN
TLAG
v
max
Specific product costs

Enzyme cost
Flow rate
Investment cost
Deactivation constant
Inhibition constant
Michaelis - Menten constant
Operating cost
Total amount of product
Reactor refill cost
Reaction rate of deactivation
Reaction rate of substrate
Residence time
Time
Down time
Time-on-stream difference
Maximum specific reaction rate
Indices
0
i
Exercises
Refers to initial, feed

Refers to reactor number
$/kg
kg/m3
$/kg
m3/h
$/kg
1/h
mol/m3
mol/m3
$/kg
mol
$/kg
kg/(m3 h)
mol/(m3 h)
mol/m3
h
h
h
h
mol/kg h
312
Results
The results from DEACTENZ show an exponential decrease of the biocatalyst
activity (Fig. 2), which causes dynamic changes in the substrate and product
concentrations (Fig. 3) in all three reactors.
313
0.5-,\
0.45-
-4000
\
\
%
0.4-
i**
*.
0.35-
"%
03
a '
.0.25-
"*
s
-'
-.
0.1-
^ -"
0
100
200
-3000
^
-2500 3
I
-2000 Q_
_- Totalproduct:1
-1500 p
0.050-
-3500
. E2:1
*"">cr
^r "'"'"-'"*. |
j+
_/
" . -i..
0.15-
s ***
"''
0.2-
r*
j**
-1000
i...

300
400
500
.500
-0
600
700
800
900
1000
TIME
Figure 2. Exponential biocatalyst deactivation and total product during one run.

120
140-i
120-
100
10080
l
C/l
^
cn
a-
60
40
40-
20
20
0
100
200
300
400
500
600
700
800
TIME
Figure 3. Dynamic changes in the substrate and product concentrations.
900
1000
314
References
Prenosil, I.E., Peter, J., Bourne, J.R. (1980). Hydrolytische Spaltung des
Milchzuckers der Molke durch immobilisierte Enzyme im Festbett-Reaktor.
Verfahrenstechnik 14, 392.
Prenosil, J.E. (1981). Optimaler Betrieb fur einen Festbett- und einen FliessbettReaktor mit desaktivierendem Katalysator. Chimia 35, 226 .
Prenosil, J.E., Hediger, T. (1986). An Improved Model for CapillaryMembrane, Fixed-Enzyme Reactors. In Membranes and Membrane Processes,
Plenum, N. Y., 515.
8.4.13
Continuous Production of PHB in a Two-Tank

Reactor Process (PHBTWO)
System
This example considers a two-stage process for the production of PHB, a
biopolymer. The kinetics of this fermentation is presented in the example PHB.
The structured kinetic model involves a Luedeking-Piret-type expression and
also an inhibition by the product. From this it might be expected that two stages
would be better than one, and it is the goal of this example to optimize the
process. The volume ratio and the feed rate are the obvious design and
operating parameters.
Sfeed,
> 82, F0
Figure 1. Configuration of the two-tank system.
315
Model
The details of the structured model will not be repeated here (See PHB). The
biomass consists of a synthesis part R and the intracellular product P. The
biomass growth rate of R is proportional to the specific growth rate, which is
given by a two-part expression
S
(S/Ks,2)n
(KS,i + S) -*
The synthesis rate of PHB is given by a two-part expression
The term -kiP represents a product inhibition.

The model requires component balances for P, R and S for both tanks, as seen
in the program. The relative reactor volumes are determined by the parameter
Vrat. The volumetric productivities are calculated to compare the results.
Program
The program is found on the CD-ROM
Nomenclature
Symbols
FO
KI
KS
n
P
PROD
R
rp
TR
Feed flow rate

Inhibition constant, for (NH4>2SO4
Saturation constant
Hill Coefficient
Product concentration (PHB)
Productivity
Residual biomass concentration
Rate of synthesis of PHB
Rate of synthesis of R
m3/h
kg/m3
kg/m3
kg/m3
kg/(m3h)
kg/m3
kg/m3
kg/m3
316
Sfeed
Vi and V2
X
YP/R
YR/S
MP
Rate of substrate uptake

Limiting substrate cone. NH4+ as
(NH4)2S04
Feed concentration
Reactor volumes
Yield coefficient
Yield coefficient,
Specific rate of synthesis of R (rR/R)
Specific rate of synthesis of P (rp/P)
Indices
1
2
m
Exercises
Refers to reaction 1 and tank 1

Refers to reaction 2 and tank 2
Refers to maximum
kg/(m3 h)
kg/m3
kg/m3
m3
kg/m3
kg/kg
kg/kg
1/h
1/h
317
Results
90
100
Figure 2. A run showing the dynamic approach to steady state for X, S, P in both tanks.
0.2
Figure 3. Here with FO set at the optimum value of 1.24, the influence of VRAT is investigated
giving a value for the maximum in PROD corresponding to the OPTIMIZE results. VRAT is seen
not to be very important. Thus equal-sized tanks are adequate.
318
8.5
8.5.1
Oxygen Uptake Systems

Aeration of a Tank Reactor for Enzymatic
Oxidation (OXENZ)
System
The influence of gassing rate and stirrer speed on an enzymatic, aerated reactor,
as shown in Fig. 1, is to be investigated. The outlet gas is assumed to be
essentially air, which eliminates the need for a gas balance for the well-mixed
gas phase.
gas
Hi
Ill
+ 02
ii
air
Figure 1. Schematic of the enzymatic oxidation batch reactor.
Model
The reaction kinetics are described by a double Monod relation:
_S
CL
The batch mass balances lead to:

ISBN: 3-527-30759-1
8.5 Oxygen Uptake Systems
319
dS
dF
dCL
= r
= K L a(C L * -CL) - r s Y o /s
dP
dF
~ r s Y P/s
KLa varies with stirring speed (N) and aeration rate (G) according to:
KLa = kN 3 G- 5
where k = 4.78 x 10-13 with N in 1/h, G in m3/h and KLa in 1/h.
Program
Nomenclature
Symbols
CL
CLS>CL*
G
KCL
â
KS
k
N
P
r
S
vm
Y

Aeration rate
Saturation constant
Constant in Kâ correlation
Stirring rate
Growth rate
Maximum degradation rate
Yield coefficient
g/m3
g/m3
m3/h
kg/m3
1/h
kg/m3
complex
1/h
kg/m3
kg/(m3 h)
kg/m3
g/(m3 h)
kg/kg
320
Indices
0
o
p
s
Refers
Refers
Refers
Refers
to feed
to oxygen
to product
to substrate
Exercises
Results
The results in Fig. 2 show the influence of stirrer speed N on the dissolved
oxygen level. Variations from 30,000 to 5,000 1/h were made with the Batch
Run facility. Runs to obtain the results in Fig. 3 were made by varying the gas
flow rate G from 25 to 5 m3/h (curves top to bottom).
321

8
7s"
--'/
_..-....._..-..._
7 -
*
*
.***
6 5
CL1 (3e+4)
CL:2 (2.1676+4)
-- CL:3 (1.3336+4)
-^_CL:4 (5000)
*
1
/
4
2 -
-"-''
0
C
TIME
Figure 2. Influence of stirrer speed on dissolved oxygen levels.

<j 6.5
5
TIME
Figure 3. Influence of the gas flow rate on dissolved oxygen levels.
8.5.2
Gas and Liquid Oxygen Dynamics in a

Continuous Fermenter (INHIB)
System
Cell growth is limited by the oxygen mass transfer rate, and hence by the
dissolved oxygen concentration. It is also inhibited by an inhibitory substrate S.
Liquid phase balances for X, S and 62 in the liquid phase are therefore used,
together with a gas phase oxygen balance to determine the rate of O2 supply.
322
To avoid washout of cells, it is important that the reactor should never enter the
range of inhibitory behavior. Schematic representation of a continuous aerated
fermenter is given in Fig. 1.
feed F, S 1
gas G, CG2
liquid F,S 2 , X
air, G, CG1
Figure 1. Schematic of the continuous fermentation with oxygen transfer.
Model
The liquid phase mass balances are as follows:
For biomass,
dX
rxVL
For substrate,
dS2
For oxygen,
dCL2
The kinetics are as follows:
L2
KS+S2+(S22/KI)K0+CL2
rx = |a X
323
rs =
The balance for oxygen in the gas phase is:
The oxygen equilibrium relates the concentration in the gas phase to the liquid
phase saturation concentration,
CL2* = MC G2
The gas holdup fraction is,
VG = eV L
Proportional control of the feed rate, based on exit substrate concentration, can
be added with,
F = Fo + KpE
with E = S2set ~ $2- Here the sign must be adjusted depending on the substrate
region above or below the maximum kinetic rate.
Program
Nomenclature
Symbols
CL
CLS
E
F
G
KI

Control error
Flow rate
Gassing rate
Inhibition constant
mg/L
nig/L
g/L
L/h
L/h
g/L
324
KLa
Ko
KP
KS
M
OUR
r
S
V
X
Yx/s
Yo
8
H
Oxygen saturation constant
Proportional control constant
Saturation constant
Equilibrium coefficient
Oxygen uptake rate
Reaction rate
Reactor volume
Yield coefficient
Mole fraction of oxygen in outlet gas
gas/liquid volume ratio
Indices
0
1
2
G
I
L
m
O
P
S
X
Exercises
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
to feed
to inlet
to outlet
to gas
to inhibitor
to liquid
to maximum
to oxygen
to product
to substrate
to biomass
to equilibrium
1/h
mg/L
L/h/g/L
g/L
mg/h
g/Lh
g/L
L
g/L
g/g
1/h
325
326
Results
8-
0.1
^
7.
0.09
L
S2:1 (1)
.. CL2:1 (1)
S2:2 (4)
6-
S2:3 (7)
5-
a'
4-
CL2:3(7)
82:4(10)
-CL2:4(10)
|N ^^
% *%- N**
-0.08
-0.07
-0.06
-0.05
0.04
-0.03
3" -^ .^^
0.
1.
i)
-0.02
.**-*uT-ZZ_H.r. r 0.01
"-""'
0
K)
TIME
Figure 2. Dissolved oxygen versus time at various feed rates.

.70
DJ
5
TIME
Figure 3. Influence of the control on the reactor. The setpoint 82 is 5.0 kg/m^.
8.5.3
327
Batch Nitrification with Oxygen Transfer

(NITRIF)
System
Nitrification in a biofilm fluidized bed is to be modelled.
oxidation of NtLj.* to NC>2~ and NC>3" proceeds according to:
The sequential
NH4+ + 02 -> N02O2 -> NO3The two steps are shown schematically in Fig. 1.
Ammonium ion -^
Nitrite ion -> Nitrate ion
Figure 1. Reaction sequence for nitrification.
The stoichiometry is for the first step YI = 3.5 g O2/ (g NPfy-N) and for the
second step Y2 = 1.1 g O2/(g NO2-N).
Model
Neglecting the details of the biofilm diffusion, the apparent kinetics of this
biofilm process can be approximately described with homogeneous kinetics
that follow a double Monod limitation:
Si
CL
*
I + S| KOI +C
= vm2
The batch balances are as follows:

ForNH 4 + (Si):
S2
K 2 +S 2
CL
328
~dT = - f i
For NO2~ (S2):

dS2
dt
= ri - r2
For NO3- (S3):

dS3
~3T = r2
For oxygen (CL):

dCL
= - Y i r i - Y 2 r 2 + K L a(C L *-C L )
Program
Nomenclature
Symbols
CL
CLS
K
KLa
r
Si
S2
S3
Vm

Oxygen transfer coefficient
Reaction rate
Concentration of NH4+ - N
Concentration of NC>2~ - N
Concentration of NO3~ - N
Maximum degradation rates
Yield coefficients
Indices
0
1,2,3
O
Refers to feed
Refer to reaction steps
Refers to oxygen
Refers to substrate
g/m3
g/m3
g/m3
1/h
kg/m3
g/m3
g/m3
g/m3
g/m 3 h
g/g
Exercises
329
330
Results
100
+*
90
80
70
CO
X *'
...X'-''
60
$ 50
5) 40
30
20
10
0
1.5
TIME
Figure 2. NH4+, NO2~ and NC>3" and dissolved oxygen in a batch nitrification with KLa = 40 h"1/
2.5
Figure 3. NH 4 + and dissolved oxygen in batch nitrification using three values of KLa from 20
8.5.4
331
Oxygen Uptake and Aeration Dynamics

(OXDYN)
System
The aeration of a batch culture (with essentially constant biomass X) is stopped
and the dissolved oxygen (CL) is allowed to fall zero before re-aerating. The
slope of the CL curve is the oxygen uptake rate and it is approximated by the
slope of the electrode response curve CE curve. The dynamics of the electrode
are known, and it is desired to investigate the lag effects as shown in Fig. 1.
Model
The following equations represent the model:
Oxygen uptake rate,
OUR = q 0 2X
Specific OUR,
qo2m CL
102 - KQ + CL
Oxygen balance,
dCL
*
T = K L a(C L *-C L ) -OUR
332
C (mg/L)
20
40
60
80
time(s)
Figure 1. Typical response of the batch oxygen uptake and reaeration experiment.
Measurement dynamics for the liquid film may be important with a viscous
culture,
dCp
CL - Cp
TF
and for the electrode lag,
dt
Program
Experimental data, in the file OXDYNDATA, and the program are found on
the CD-ROM.
Nomenclature
Symbols
C
KLa
Oxygen concentrations
g/m3
1/h
333
KO
OUR
Q
X
T

Oxygen uptake rate
Specific oxygen uptake rate
Time constants
Indices
E
F
L
m
02,0
S
*
Exercises
Refers to electrode
Refers to liquid film
Refers to liquid
Refers to maximum
Refer to oxygen
Refers to saturation
Refers to saturation
g/m3
g/m3s
g/kgs
kg/m3
334
g Simulation Examples of Biological Reaction Processes Using Berkeley Madonna
Results
The results shown in Fig. 2 demonstrate the effect of changing Kâ. Runs
varying the electrode time constant TE gave the results of Fig. 3.
Run 2:10004 steps in 0.183 second:

8
70
Figure 2. Aeration turned on at low CL for two KLa values.
80
90
100
335
10
20
30
40
50
TIME
Figure 3. Variation of the electrode time constant, TE from 1 to 25.
8.5.5
Dynamic Oxygen Electrode Method for KLa

(KLADYN, KLAFIT and ELECTFIT)
System
A simple and effective means of measuring the oxygen transfer coefficient
(â) in an air- water tank contacting system involves first degassing the batch
water phase with nitrogen (Ruchti et al., 1981). Then the air flow is started and
the increasing dissolved oxygen concentration is measured by means of an
oxygen electrode.
336
VQ.CQ
Hii
G> CGO
Figure 1. Aerated tank with oxygen electrode.
As shown below, the influence of three quite distinct dynamic processes play a
role in the overall measured oxygen concentration response curve. These are
the processes of the dilution of nitrogen gas with air, the gas-liquid transfer and
the electrode response characteristic, respectively. Whether all of these processes
need to be taken into account when calculating Kâ can be determined by
examining the mathematical model and carrying out simulations.
Measurement
CF
Gas phase
Liquid phase
Electrode
diffusion film
Electrode
Figure 2. Representation of the process dynamics.
Model
The model relationships include the mass balance equations for the gas and
liquid phases and equations representing the measurement dynamics.
Oxygen Balances
The oxygen balance for the well-mixed flowing gas phase is described by
VG
dCG_ _
= G (CGO - CG) - KLa (CL* - CL) VL
dt
337
where VG/V = TG , and Kâ is based on the liquid volume.

The oxygen balance for the well-mixed batch liquid phase, is
dt
= K L a(C L *-C L )V L
The equilibrium oxygen concentration CL* is given by the combination of

Henry's law and the Ideal Gas Law equation where
RT
r
CL * = r
CG
and CL* is the oxygen concentration in equilibrium with the gas concentration,
CG- The above equations can be solved in this form as in simulation example
KLAFIT. It is also useful to solve the equations in dimensionless form.
Oxygen Electrode Dynamic Model
The response of the usual membrane-covered electrodes can be described by
an empirical second-order lag equation. This consists of two first-order lag
equations to represent the diffusion of oxygen through the liquid film on the
surface of the electrode membrane and secondly the response of the membrane
and electrolyte:
dC F _ C L -C F
dt
TF
and
dCg
Cp ~Cg
dt "
TE
Tp and TG are the time constants for the film and electrode lags, respectively. In
non- viscous water phases Tp can be expected to be very small, and the first lag
equation can, in fact, be ignored.
Dimensionless model equations
Defining dimensionless variables as
C =
CG
CGO
CL =
C GO (RT/H)
the component balance equations then become
TG
338
dt'
'
VG H
and
Initial conditions corresponding to the experimental method are,

t1 = 0 ;
C' L =C' G =0
In dimensionless form the electrode dynamic equations are

C
=
L-CF
dt
and
dC
E
dt'
C
=
F-CB
TE/^G
where Cp is the dimensionless diffusion film concentration.
C GO (RT/H)
and CE is the dimensionless electrode output
CE
C GO (RT/H)
As shown by Dang et al. (1977), solving the model equations by Laplace
transformation gives
1
,RTVL
= =
V
-. + 1) TO + TE + TF
KLa + ( H
G
where oc is the area above the CE versus t response curve, as shown in Fig. 3.
339
1.0
CE'
Time (s)
Figure 3. Determination of the area a above the CE' versus time response curve.
Program
The program KLADYN can be used to investigate the influence of the various
experimental parameters on the method, and is formulated in dimensionless
form. The same model, but with dimensions, is used in program KLAFIT and
is particularly useful for determining Kâ in fitting experimental data of CE
versus time. A set of experimental data in the text file KLADATA can be used
to experiment with the data fitting features of Madonna. All are on the CDROM.
The program ELECTFIT is used to determine the electrode time constant in
the first-order lag model. The experiment involves bringing CE to zero by first
purging oxygen from the water with nitrogen and then subjecting the electrode
to a step change by plunging it into fully aerated water. The value of the
electrode time constant, TE can be obtained by fitting the model to the set of
experimental data in the file, ELECTDATA.
The value found in this
experiment can then be used as a constant in KLAFIT.
Nomenclature
Symbols
Oxygen concentration
g/m3
340
G
H
KLa
RT/H
t
V
Gas flow rate

m3/s
Henry coefficient
Pa m3/mol
1/s
(Gas constant)(Abs. temp.)/Henry coeff. Time
s
Reactor volume
m3
Area above Cn'-time (s) curve
s
Time constant
s
Indices
E
F
G
L
Exercises
Refers to electrode
Refers to film
Refers to gas phase
Refers to liquid
Prime denotes dimensionless variables
341
Results
20
40
60
80
100
120
140
TIME
Figure 4. Response of CG, CL and CE versus Ttime from KLAFIT.
160
180
200
342
TIME
Figure 5. A fit of experimental data (open circles) as dimensionless CE versus time (s) to
determine KLA using KLAFIT, which gives 0.136 1/s.
References
Dang, N.D.P., Karrer, D.A. and Dunn, IJ. (1977).Oxygen Transfer Coefficients
by Dynamic Model Moment Analysis, Biotechnol. Bioeng. 19, 853.
Ruchti, G. Dunn, IJ. and Bourne J.R. (1981). Comparison of Dynamic Oxygen
Electrode Methods for the Measurement of KLa, Biotechnol. Bioeng., 13, 277.
8.5.6
Biofiltration Column for Removing Two

Inhibitory Substrates (BIOFILTDYN)
System
Biofiltration is a process for treating contaminated air streams. Moist air is
passed thrpugh a packed column, in which the pollutants in the contaminated
air are adsorbed onto the wetted packing. There in the biofilm solid phase the
resident population of organisms oxidizes the pollutants.
343
L, S
G, C1T6
Tank6
Gas
Transfer
TriT6
wifsp#K
iS:Tl;niiiS?'
Illllpll
G.Q1T5
*
Tanks
Gas
I
I
I
I
Tank4
illlii
llllll
Transfer
tlwilffi
I
l
l
l
l
l
l
l
Gas
fankS
'"';:"H? H'MK'SK
Transfer
i
illlll
Illlllll
Transfer
Gas
Tank 2
Transfer
: : ' : . ; : - : ^ v \ r : : ^ -^ ;.;ji: ' ;' ';':'
Gas
Tank1
Gas
^îiiriS
i;|tii^|;i;||
I
Transfer
^
TriT1
^îinS^p:
OliiulllI
L, S1T1
Figure 1. Biofiltration countercurrent column.
Such columns can be run with a liquid phase flow (bio-trickling filter) or only
with moist packing (biofilter). The work of Deshusses et al. (1995) investigated
the removal of two ketones, methyl isobutyl ketone (MIBK) and methyl ethyl
ketone (MEK), in such a biofilter. The kinetics of this multi-substrate system is
especially interesting since both substances exhibit mutual inhibitory effects on
their rates of degradation.
344
Model
The model requires stagewise mass balances in the gas and liquid phases for
both components. Transfer takes place from the gas to liquid phases with
reaction in the liquid phase. The symbols used for the concentrations of
substrates 1 and 2 in the n th tank are for the liquid phase Sixn and S2Tn and
for the gas phase CiTn and C2Tn- The reaction rates are RiTn and R2Tn and the
transfer rates are designated TriTn and Tr2TnG, C1Tn, C2Tn
G,C1Tn-1,C2Tn-1
L, S1Tn+1, S2Tn+1
L, S1Tn, S2Tn
m
nth
Figure 2. Single n
stage for the biofiltration countercurrent column.
Referring to above figure, the mass balances for a single tank can be written as:
Gas phase
ÔL~(G(C2Tn.1-C2Tn)-Tr2Tn
Liquid phase
-^ = ^- (L(SlTn+l - SlTn ) +TrlTn -rlTn VS )
345
1Q
n =
^~ (L(S2Tn+l - S2Tn ) + Tr2Tn -r2Tn VS )
Here the reaction is assumed to occur in a solid phase of volume Vs.
Vs = (1 - EG - e L ) Ê.
For the transfer terms

Vc
2Tn = K L a ( S 2EQn - S2Tn)~
Tr
For the gas-liquid equilibria:
For the reaction rate terms the following equations are used to describe the
mutual inhibition. Note that oxygen is assumed to be in excess.
For substrate 1 (MEK) in tank n:
v
r
lTn = "
mi SlTn
For substrate 2 (MIBK) in tank n:

V
r
2Tn V
m2 S2Tn
|1+
i i SlTn |
Program
The program developed by M. Waldner, ETH, is given on the CD-ROM.
346
Nomenclature
Symbols
C
G
Ki
KLa
Km
L
M
N
r
S
Tr
VC
VG
VL
vm
VS
z
e
Concentration in gas phase

Gas flow rate
Inhibition constant
Mass transfer coefficient
Monod coefficient
Liquid flowrate
Partition coefficient
Number of tanks
Reaction rate
Concentration in liquid phase
Transfer rate
Volume of column
Volume of gas phase
Volume of liquid phase
Maximum reaction velocity
Volume of solid phase
Length or height
Volume fraction
kg/m3
m3/s
kg/m3
1/s
kg/m3
m3/s
kg/m3s
kg/m3
kg/s
m3
m3
m3
kg/m3s
m3
m
Indices
Eq
G
in
L
M
n
Tn
1
2
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
to equilibrium value
to gas
to inlet
to liquid
to maximum
to nth stage
to nth tank
to methyl ethyl ketone (MEK)
to methyl isobutyl ketone (MIBK)
347
Exercises
Reference
Deshusses, M. A, Hamer, G. and Dunn, I. J. (1995) Part I, Behavior of Biofilters
for Waste Air Biotreatment: Part I, Dynamic Model Development and Part II,
Experimental Evaluation of a Dynamic Model, Environ. Sci. Technol. 29,
1048-1068.
348
Results
1.2
0.0025-
0.8 ^
0.002-
0.6
. 0.0015-
0.001 -
&-
,0.4 W
H
'0.2 (0
5e+5
1e+6 1.5e+6 2e+6 2.5e+6 3e+6 3.5e+6 4e+6 4.5e+6 5e+6

TIME
Figure 3. Dynamic startup of the column.
0.1
5e-5
1e-4
1.5e-4
2e-4
2.5e-4
3e-4
3.5e-4
4e-4
4.5e-4
Figure 4. Influence of gas flowrate on the steady state fraction removed.
5e-4
349
8.5.7
Optical Sensing of Dissolved Oxygen in

Microtiter Plates (TITERDYN and
TITERBIO)
System
Measurement of dissolved oxygen in microtiter plates is of potential interest for
the screening of oxygen-consuming enzymes (e.g., oxidases), aerobic cell
activities, and biological degradation of pollutants, and for toxicity tests. John et
al. developed microtiter plates with the integrated optical sensing of dissolved
oxygen by immobilization of two fluorophores at the bottom of 96-well
polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to
dissolved oxygen concentration, whereas the oxygen-insensitive one served as
an internal reference. As modelled in TITERDYN, oxygen transfer coefficients
were determined by a dynamic method in a commercial microtiter plate reader
with an integrated shaker. For this purpose, the dissolved oxygen was initially
depleted by the addition of sodium dithionite and, by oxygen transfer from air,
it increased again after complete oxidation of the dithionite. Available
commercial readers have an intermittent operation. After a certain period of
shaking, the plate is moved around to measure dissolved oxygen concentration.
During this period the plate moves more slowly and oxygen transfer rate is
reduced. This may lead to oxygen depletion during the measurement process.
It is essential to know the size of the errors that are introduced by this
intermittent procedure. This is evaluated by the simulation example TITERBIO.
Microtiter plate
Filter
Light
Figure 1. Microtiter well showing light path and sensor layer.
350
Model
Experiments involved measuring the oxygen uptake rate by removing the

oxygen from the liquid using a chemical reaction (oxidation of sodium
dithionite). Oxygen is depleted immediately after the addition of dithionite.
After the consumption of the dithionite the oxygen transfer increased the
oxygen in the liquid. The following model was used to evaluate the KLa value.
dCT
dt
where CL is the dissolved oxygen concentration, CL* is the saturation value and
OUR is the oxygen uptake rate in mM/min.
The experiment starts with high values of dissolved oxygen concentration,
CL After addition of dithionite OUR increases as calculated by
OUR
= ko CL CD
As oxidation proceeds the dithionite concentration changes according to

dC D ^ 2 f
dt "" 3
In order to account for some time delay of the sensor a first order equation was
used
dC E ^C L -C E
dt
TE
The time constant TE was estimated to be about 1 s.
In further experiments this method was also applied to simulate a microbial
cultivation in the wells of a microtiter plate. In this case the OUR value was
taken to be a constant value as measured in a larger fermentation vessel. KLa
varied periodically simulating the high value during shaking and the lower
value during the measurement period. The questions of interest are how much
the measured OUR or KLa would differ from the actual one provided KLa or
OUR were already known.
351
Program
Two separate programs are given on the CDROM: TITERDYN for the chemical
oxidation with re-aeration and TITERBIO for the biological oxidation and reaeration dynamics during a cultivation in a microplate reader. For the program
TITERDYN there is experimental data on the file TITERDYNDATA available
to allow fitting the value of KLa. In TITERBIO KLa during measurement is a
fraction of KLa during shaking and is determined by the parameter kmax. KLa
during measurement is defined as,
KLameasure=KLashaking*(kmax-l)/kmax.
In the original model settings, kmax has a value of 2. The larger kmax, the
larger the error of KLa or OUR estimation.
Nomenclature
The program TITERDYN uses minutes and TITERBIO uses seconds.
Additional symbols are defined in the programs.
Symbols
CD
CL
ko
â
KQ
OUR
Q
TE
Dithionite concentration
Oxygen concentration
Rate constant for dithionite reaction
Oxygen uptake rate
Specific oxygen uptake rate
Time constant for measurement
Indices
E
D
L
S and *
Refers to electrode
Refers to dithionite
Refers to liquid
Refer to saturation
mM
mM
1/min mM
1/s
mM
mM/s
mM/ s
s
352
Exercises
353
Results
1:1019 steps in 0.0167 seconds
'0.1
100
200
300
400
500
600
700
800
900
1000
TIME
Figure 2e Dynamics of biological uptake and reaeration. Program TITERBIO.

it
Figure 3.
Data fitting
Duration=0.48.
using
TTTERDYN, yielding
'VtV'tj " *
KLa=0.201,
Calcfact=102
and
Reference
John, G.T., Klimant, I., Wittmann, C., Heinzle, E. (2003). Integrated Optical
Sensing of Dissolved Oxygen in Microtiter Plates - A Novel Tool for Microbial
Cultivation, Biotechnol. Bioeng., 81, 829-836.
354
8.6
Controlled Reactors
8.6.1
Feedback Control of a Water Heater

(TEMPCONT)
System
A simple feedback control system involving a stirred tank, temperature
measurement, controller and manipulated heater is shown in Fig. 1.
T 0 ,F
IT*
F,T R
ip
Figure 1. Feedback control of a simple continuous water heater.
Model
The energy balance for the tank is
dTR
where Q is the delayed heat input from the heater represented by a first order
lag
dt
TQ
The measurement of temperature is also delayed by a sensor lag given by

ISBN: 3-527-30759-1
8.6 Controlled Reactors
355
u*sens .... *R ~ *sens

dt
Tsens
A proportional-integral feedback controller can be modelled by
where the control error is given by
Program
Random disturbances in flowrate or feed temperature can be generated using
the RANDOM function in Madonna, as explained in the HELP on the CDROM.
Nomenclature
Symbols
cp
f
F
Kp
Q
T
V
8
p
TD
TI
TQ
Specific heat
Frequency of oscillations
Flow rate
Proportional control constant
Heat input
Temperature
Reactor volume
Error
Density
Differential control constant
Integral control constant
Time constant for heater
Time constant for measurement
kJ/(kg C)
1/h
m3/h
kJ/(h C)
kJ/h
C
m3
C
kg/m 3
h
h
h
h
356
Indices
C
R
sens
set
0
Exercises
Refers
Refers
Refers
Refers
Refers
to controller
to reactor
to sensor
to setpoint
to inlet or initial
357
Results

7000
6000
3000
1000
70
Figure 2. Approach to steady state for a setpoint of 80C.
80
90
100
358

-1.2e+4
1e+4
8000
100.
6000
4000
80
2000
60
0
-2000
-4000
20
40
80
100
120
140
160
180
200
TIME
Figure 3. Response to a step change in the inlet temperature TO at 120 h. The controller
constant Kp was set higher than in the run of Fig. 2.
8.6.2
Temperature Control of Fermentation

(FERMTEMP)
System
Heat effects in fermentation can be important, especially on a large scale.
Shown in Fig. 1 is a batch fermentation process, during which the cooling water
flowrate is controlled by a feedback controller. The rate of heat generation is
related to rate of substrate uptake by a constant yield factor YQS. The cooling
coil is modelled as a well-mixed system.
359
Water
Figure 1. Feedback control of the temperature in during a fermentation.
Model
The batch fermentation model is given by,
dX
df =
dS
dt
-H
= Y
^ = Ks+S
The energy balance equation for the reactor is,
dT
dtR _
~
rQ
UA
(TR-Tc)
VpCp
360
where,
TQ = ^XY Q S /Y
For the well-mixed cooling coil, the energy balance equation is:
dTr
(Tcin TC) +
UA
(TR ~ Tc)
The controller is a proportional-integral type

F = F0 + K P 8 +
=
Program
As seen on the CD-ROM and below, the control equations can be written in
terms of the error and its integral.
{CONTROL EQUATIONS FOR
d/dt(EInt)=E
F=FO+KP*E+(KP/TI)*EInt
limit F> = 0
E=TR-TSET
FLOWRATE}
Nomenclature
Symbols
Cp
F
Kp
KS
UA
r
V
Heat capacity
Flow rate
Controller constant
Saturation coefficient
Reactor transfer-area constant
Rate of heat production and transfer
Reactor volume
kcal/(kg C)
m3/h
m3/(h C)
kg/m3
kcal/kg
kcal/(m3 h)
m3
361
X
Y
YQS
P
T
s
e
H
Yield coefficient
Heat yield for substrate
Density
Time constant of controller
Temperature error
Indices
C
m
Q
R
S
O
P
set
Exercises
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
Refers
to coolant and cooling

to integral control
to maximum
to heat
to reactor
to substrate
to normal value and inlet value
to proportional
to setpoint (desired value)
kg/m3
kg/kg
kcal/kg
kg/m3
h
kg/m3
C
1/h
362
Results
TIME= 3.452
S = 18.25
TR= 24.6
Figure 2. Cooling flow starts when TR > Tset (25 C); after batch growth finishes at time=4.6 h
the reactor cools. Here Kp=0.6 and TI = 0.6.
363

'0.2
100
150
200
250
300
350
400
450
500
Figure 3. With Parameter Plot, the integral of the error and minimum water temperature versus
Kp for a fixed value of Tj=9.
8.6.3
Turbidostat Response (TURBCON)
System
Although not so widely used as the chemostatic type of operation of
continuous culture, the turbidostat may offer advantages for the investigation of
particular problems. As shown in Fig. 1, the flow rate of the incoming substrate
is controlled by the biomass concentration (more correctly, the turbidity) in the
vessel. In practice, this control is usually on-off or proportional, but more
sophisticated control would be simple to implement.
364
F,S0
Feed pump
X,S
Turbidometer
Figure 1. Feedback control of the biomass concentration using a turbidostat.
Model
For the well-mixed tank with Monod growth:
dS
M_X
dt" = F(So-S) - -y~

dX
FX
dT = -IT
Considering product production with Luedeking-Piret kinetics:
dP
FP
dT = -"V" +
The turbidometer control is modelled by:

KPP ,.
F = F0 + K P e + fedt
s
365
8 = (X-X S et)
Program
Nomenclature
Symbols
A
B
F
Fo
Kp
KS
P
S
V
X
Y
e
Growth-associated constant
Nongrowth-associated constant
Flow rate
Normal feed flow rate
Proportional controller constant
Saturation constant
Reactor volume
Yield coefficient
Error
Integral control time constant
Indices
m
P
S and set
0
Refers
Refers
Refers
Refers
to maximum
to proportional control
to setpoint
to inlet stream
1/h
m3/h
m3/h
m6/h kg
kg/m3
kg/m3
kg/m3
m3
kg/m3
kg/kg
kg/m3
1/h
366
Exercises
Results

1-5
,3.5
5
TIME
Figure 2. Startup and response of the controlled reactor.
c/>
367

-5
3.5-
3.
45
4
2.5-
-3.5
2"
_X:1
-3
X"
"
1.5.
F:
C^
'
-2.5
,m
1
n e
VJ.O
0-
-2
i T...
-1.5
...^. ._..._. ._..._,,....
-1
' "
8
10
TIME
12
14
16
18
20
Figure 3. Response of the controlled reactor to a step change in X se t o
8.6.4
Control of a Continuous Bioreactor with

Inhibitory Substrate (CONTCON)
System
The continuous fermenter is equipped with feedback control based on substrate
measurement, as shown in Fig. 1. This type of controlled fermenter has been
referred to as an auxostat.
368
F,S,
Feed
pump
X,S
' Substrate
measurement
Controller
Figure 1. Flow diagram of a feedback loop to control substrate concentration.
Model
The biomass and substrate mass balances are the same as in the previous
TURBCON model.
Kinetics:
Biomass balance,
V =
dt
VX-FX
or,
f
where D is the dilution rate (= F/V). Thus steady-state behaviour, where dX/dt
= 0, is represented by the conditions that |u = D.
Substrate mass balance,
or,
dt
f "><*>--f
369
where Y is the yield factor for biomass from substrate. Also from this equation
at steady state, since (j = D and dS/dt = 0, the steady-state cell concentration is
given by
X = Y(S 0 -S)
A continuous inhibition culture will often lead to two possible steady states, as
defined by the steady-state condition (a = D, as shown in Fig. 2.
Control equations:
Sset- S
Kp r
F = F0 + KP e + I edt
>
Program
When the system equations are solved dynamically, one of two distinct steadystate solutions is obtained, i.e., the reactor passes through an initial transient but
then ends up under steady-state conditions either at the stable operating
condition, or at the washout condition, for which X=0. The initial
concentrations for the reactor will influence the final steady state obtained. A PI
controller has been added to the program, and it can be used to control a
substrate setpoint below Smax. The controller can be turned on setting by
Kp>0. The control constants Kp, and the time delay tp can be adjusted by the
use of sliders to obtain the best results. Appropriate values of control constants
might be found in the range 0.1 to 10 for Kp and 0.1 to 10 for TJ. Note that
the control does not pass Smax even though the setpoint may be above Smax.
Another feature of the controller is a time delay function to remove chatter.
The program comments on the CD-ROM should be consulted for full details.
Nomenclature
Symbols
D
Dilution rate
1/h
370
F
KI
KP
KS
S
V
X
Y
H(U)
T
I
TF
Flow rate
Inhibition constant
Controller constant
Saturation constant
Volume
Yield coefficient
Specific growth rate coefficient
Controller time constant
Time constant controller delay
Indices
0
I
m, max
Exercises
Refers to inlet
Refers to initial value
Refers to maximum
m3/h
kg/m3
kg/m3
m 6 /kgh
kg/m3
m3
kg/m3
kg/kg
1/h
h
h
371
8.7 Diffusion Systems
Results
r 0.3
0.25
0.2
1.5
0.15
TIME
Figure 2. A control simulation of the process with the setpoint below Sn
References
Edwards, V. H, Ko, R. C. and Balogh, S. A. (1972) Dynamics and Control of
Continuous Microbial Propagators Subject to Substrate Inhibition, Biotechnol.
Bioeng. 14, 939-974.
Fraleigh, S. P., Bungay, H. R. and Clesceri, L. S. (1989) Continuous Culture,
Feedback Control and Auxostats. Trends in Biotechnology, 7, 159-164.
8.7
8.7.1
Diffusion Systems
Double Substrate Biofilm Reaction
(BIOFILM)
System
A biocatalyst is immobilized inside a solid matrix (gel or porous solid) through
which substrates diffuse and react. As shown in Fig. 1, for simulation purposes
371
Results
r0.3
0.25
0.2
1.5
0.15
TIME
Figure 2. A control simulation of the process with the setpoint below Sn
References
Edwards, V. H, Ko, R. C. and Balogh, S. A. (1972) Dynamics and Control of
Continuous Microbial Propagators Subject to Substrate Inhibition, Biotechnol.
Bioeng. 14, 939-974.
Fraleigh, S. P., Bungay, H. R. and Clesceri, L. S. (1989) Continuous Culture,
Feedback Control and Auxostats. Trends in Biotechnology, 7, 159-164.
8.7
8.7.1
Diffusion Systems
Double Substrate Biofilm Reaction
(BIOFILM)
System
A biocatalyst is immobilized inside a solid matrix (gel or porous solid) through
which substrates diffuse and react. As shown in Fig. 1, for simulation purposes
ISBN: 3-527-30759-1
372
the matrix is divided into segments, and the diffusion flux, j, from segment to
segment, is expressed in terms of the concentration difference driving force.
Solid Biocatalyst Matrix with N Segments
Liquid
j n-1
n-1
^_ n
^
jn
^ n+1
Figure 1. Finite-differencing of the concentration profiles within the immobilized biocatalyst

into segments 1 to N.
Model
A multicomponent reaction whose reactants and products diffuse to and from
the reaction site, for example into an immobilized enzyme or biofilm, can be
described by diffusion-reaction equations. The original problem in terms of
non-linear partial differential equations, is described by a large number of
time-dependent differential-difference equations by discretizing the length
variable.
A component mass balance is written for each segment and for each
component:
[Accumulation |
^
rate
J
(Diffusion^ _ (Diffusion^
^ rate in J
^ rate out J
^Production rate^
v by reaction }
dSn
' F = J n - l A - j n A +r S n A A Z
Using Pick's law,

n-1 =
and dividing by A AZ,
S n -l ~ Sn
AZ
373
dSn
"dT = s
(Sn.i -2S n + S n +i)

AZ2
+ rs
Thus N dynamic equations are obtained for each component at each

one for each element. The boundary conditions are for the above case
0 at Z = L and S = So at Z = 0. The equations for the first and last
must be written accordingly.
The kinetics used here consider carbon-substrate inhibition and
limitation. Thus,
S
O
position,
dS/dZ =
elements
oxygen
At steady state, the overall reaction rate or consumption rate of substrate can be
calculated from the gradient at the outer surface. To find the resulting change
of bulk concentration, the liquid phase can be coupled with suitable mass
balances. For a well-mixed, continuous-flow, liquid the resulting balance
equation would be
dS0
F
So)
- a DS
SQ-SI
^Z
For oxygen transferred from the gas phase:

dO0
= K L a(Os-0 Q ) - a Do
AZ
Program
As seen below, the program on the CD-ROM uses the array-vector form which
permits plotting the values at time=Stoptime versus the distance index. Also the
number of finite-difference elements N can be varied.
a/at (s[i. .N-i] >=DS* (s[i-u )/(Z*Z)+RS[i]
a/at (o[i. .N-I] )=DO* (o[i-u

2*0[i]+0[i+l])/(Z*Z)
374
Nomenclature
Symbols
a
A
C
D
F
j
K
KLa
O
0S
R,r
S
V
vm
Yos
Specific area perpendicular to the flux 1/m

Area perpendicular to diffusion flux m2
Concentration
g/m3
Diffusion coefficients
m2/h
Volumetric flow rate
m3/h
Diffusion
flux
g/ (m2 h)
g/m3
1/h
g/m3
Saturation concentration for oxygen
g/m3
Reaction rate
g/ (m3 h)
Substrate concentration of carbon source g/m3
Volume of tank
m3
Maximum reaction rate
g/ (m3 h)
Yield for oxygen uptake
Length of element
m
Indices
0
1 - 10
I
O
S
n
Feed
Exercises
Refers to bulk liquid

Refer to sections 1-10
Refers to inhibition
Refers to oxygen
Refers to carbon source
Refers to section n
Refers to feed
375
376
Results
.1
Figure 2. Oxygen and substrate time profiles for a step change in KLA.
1.6-1
1.4-
10
Figure 3. Oxygen and substrate distance profiles at the end of the run in Fig. 2.
377
Figure 4. Dynamic response of oxygen and substrate mid-points caused by a step change in
KLA (as Fig. 2) followed at 3 h by a step reduction in Sfeed.
8,7.2
Steady-State Split Boundary Solution

(ENZSPLIT)
System
A rectangular slab of porous solid supports an enzyme. For reaction, substrate
S must diffuse through the porous lattice to the reaction site, and, as shown in
Fig. 1, it does so from both sides of the slab. Owing to the decreasing
concentration gradient within the solid, the overall rate is generally lower than
that at the exterior surface. The magnitude of this gradient determines the
effectiveness of the catalyst.
378
Biocatalyst Matrix
Substrate diffusion
diffusion
X = L -*
X = 0 ^
X=L
Figure 1. Symmetrical concentration gradients for substrate and product.
Model
Under steady state conditions:
f Rate of diffusion of
Uubstrate into the slab
Rate at which reactant \

îs consumed by reaction^
dX
A quasi-homogeneous form for the reaction term is assumed.

The boundary conditions are given by:
At X = L:
S = S0 , P = P0
At X = 0:
dX
dX
379
The external concentration is known, and the concentration profile throughout

the slab is symmetrical.
The reaction rate is expressed by the Michaelis-Menten equation with
product inhibition
kES
=
KM(I+P/KI)+S
where k, KM and K\ are kinetic constants and E and P are the enzyme and
product concentrations. At steady state, the rate of diffusion of substrate into
the slab is balanced by the rate of diffusion of product out of the slab.
Assuming the simple stoichiometry S > P
dS
SdX
dP
-PdX
which on integration gives

P = (So-S)
DS
where P is assumed zero at the exterior surface.

Defining dimensionless variables
S<
=^ '
' =^
and X' =
gives
d^'
dX'2
L2R'
DSS0 ~
where,
R' =
kES'
(K M /S 0 )(1 +(S 0 F/K I ))
and,
P = (1 - S')
with boundary conditions at

X' = 1
X' = 0
S' = 1
dS'/dX' = 0
The catalyst effectiveness may be determined from

DS Sp (dSVdX')x=l
^
L2R0
=
380
where RQ is the reaction rate determined at surface conditions,

kES0
~K M (i +P O /K I ) + SO
Program
The dimensionless model equations are used in the program on the CD-ROM.
Since only two boundary conditions are known, i.e., S at X = L and dSVdX' at
X' = 0, the problem is of a split-boundary type and therefore requires a trial
and error method of solution. Since the gradients are symmetrical, as shown in
Fig. 1, only one-half of the slab must be considered. Thus starting at the midpoint of the slab at X1 = 0, where dSVdX' = 0, an initial value for S1 is assumed
(SGUESS). After integrating twice, the computed value of S is compared with
the known value of SQ at X' = 1. A revised guess for S' at Xf = 1 is then made.
This is repeated until convergence is achieved.
Nomenclature
Symbols
D
E
K
k
L
P
R
S
X
Diffusion coefficient
Kinetic constant
Reaction rate constant
Distance from slab center to surface
Reaction rate
Length variable
Effectiveness factor
Indices
I
M
Refers to Michaelis-Menten
m2/h
mol / m3
kmol / m3
1/h
m
kmol / m3
kmol /(m3 h)
kmol / m3
m
381

P
S
!
0
GUESS
Exercises
Refers
Refers
Refers
Refers
Refers
to product
to substrate
to dimensionless variables
to bulk concentration
to assumed value
382
Results
Run 5:1000 steps in 0

seconds
0.2
0.5
0.3
0.6
0.7
Figure 2. Substrate profiles generated by manual slider iterations.
Run 5:1000 steps in 0

seconds
1.86
1.84
Q.
1.82
1.8
1.78
1.76
1.74
0.1
0.2
0.3
0.4
0.5
Figure 3. Product profiles for the runs in Fig. 2.
0.6
0.7
0.8
0.9
8.7.3
383
Dynamic Porous Diffusion and Reaction

(ENZDYN)
System
This example involves the same diffusion-reaction situation as the previous
example ENZSPLIT, except that here a dynamic solution is obtained by finite
differencing. The substrate concentration profile in the porous biocatalyst is
shown in Fig. 1.
Model
With complex kinetics a steady state split boundary problem of the type of
Example ENZSPLIT may not converge satisfactorily, and the problem may be
reformulated in the more natural dynamical form. Expressed in dynamic
terms, the model relations become,
3S
dt
ap
a2p
where at the center

dX~dX
+R
384
Center
Outside
S2
S3
S4
Figure 1. Finite-differencing for ENZDYN.
Using finite differencing techniques (refer to Sec. 6.2.1), these relations may be
expressed in semi-dimensionless form for any given element n by
dS'n
W
dP'n
ar
n+l - 2S'n + S'n.f

_= 2iL(*2
AX'2
L I
)
f
'P
r
R'n
Sl
P'
n+l OP
^r n _i_
"* r
n+ A
'
2
AX'
R'n
I
+ S
where
D S S 0 1-S
L 2 R n AX'
and
S'n = Sn/SI; Fn = Pn/Si andAX' = AX/L
Sj is the external substrate concentration and AX is the length of the finite
difference element. Boundary conditions are given by the external
concentrations Sj and PI and at the slab center by setting SN+I = SN and PN+I =
PN.
385
Catalyst effectiveness may be determined according to two different

methods:
(a) the effectiveness factor based on the ratio of actual rate to maximum rate
(here for eight segments).
R
R
Ri + R29 + Ra3 + RA4 + RS5 + R*
-J
6 + 77 + a8.
R
(b) an estimate of the slope of the substrate concentration at the solid surface
_
o
D OQ
I
c)
1~ O1
^"L^RO AX'
Where the rate at the bulk conditions is
kES 0
+ P 0 /K I ) + S0
The same constant values are used as in Example ENZSPLIT.
Program
The numerical results of example ENZSPLIT and should be essentially the
same as the steady state of ENZDYN. Both programs are on the CD-ROM.
Nomenclature
The nomenclature is the same as ENZSPLIT with additional symbols and
indices:
Symbols
AX
r|2
Increment of length
Effectiveness factor based on rates
m
-
386
111
K2
Effectiveness factor based on flux

Same as KM
Indices
refers to segment n
Exercises
References
Blanch, H.W., Dunn, I.J. (1973) Modelling and Simulation in Biochemical
Engineering in Advances in Biochemical Engineering, Eds. T.K. Ghose, A.
Fiechter, N. Blakebrough, 3, Springer.
Goldman, R., Goldstein, L. and Katchalski, Ch.L (1971) in Biochemical Aspects
of Reactions on Solid Supports, Ed. G.P. Stark, Academic Press.
387
Results
1
0.9
* 0.8
B?' 0.7
0.6
81:1
,.. 82:1
.. 83:1
. 84:1
- 85:1
_ _S6:1
, 87:1
_S8:1
tf"
tfO.3
W0.2
</)
0.1
0
30
50
TIME
80
Figure 2. Substrate concentrations in porous enzyme catalyst during dynamic solution.
Run 1:1005 steps in 0.0833

seconds
P5:1
-- P6:1
-ST-J7U.
_ -P8:1
, P3:1
-P4:1
10
20
30
50
TIME
60
Figure 3. Product concentrations in porous enzyme catalyst.
70
80
90
100
388
8.7.4
Oxygen Diffusion in Animal Cells

(CELLDIFF)
System
This example treats a diffusion-reaction process in a spherical biocatalyst bead.
The original problem stems from a model of oxygen diffusion and reaction in
clumps of animal cells by Keller (1991), but the modelling method also applies
to bioflocs and biofilms, which are subject to potential oxygen limitation.
Sphere
Oxygen
Product
I/I
AV
Substrate
Rp
Rp
Figure 1. The finite differencing of the spherical bead geometry.
Diffusion and reaction takes place within a spherical bead of volume = 4/37cRp3
and area =47iRp2. It is of interest to find the penetration distance of oxygen for
given specific activities and bead diameters. As shown, the system is modelled
by dividing the bead into shell-like segments of equal thickness. The problem
is equivalent to dividing a rectangular solid into segments, except that here the
volumes and areas are a function of the radial position. Thus each shell has a
volume of 4/3 n (rn3 - rn_i3). The outside area of the nth shell segment is 4n rn2
and its inside area is 4n r n _i 2 .
389
in
Figure 2. The diffusion fluxes entering and leaving the spherical shell with outside radius rn
and inside radius r n _j.
Model
Here the single limiting substrate S is taken to be oxygen.
The oxygen balance for any element of volume AV is given by
The diffusion fluxes are
Ar
S
jn-l =
n~ S n-l
Ar
Substitution gives
dSn
dt "'
3D
The balance for the central increment 1 (solid sphere not a shell) is
4
Since ri = Ar, this becomes
3 dS
. ,,
= J.
22
4
4
-
390
dS
3D
S /q
o N , p
I f - ^ S 2- S l) +R sl
r
The reaction rate is expressed by a Monod-type equation

RSn = - (
where X is the biomass concentration (cell number/m3) in the bead, OUR is the
specific oxygen uptake rate (mol/cell s) and Sn is the oxygen concentration
(mol/m3) in shell n.
Program
As shown below, segments are programmed using the array-vector facility of
Madonna, numbered from the outside to the center. The effectiveness factor,
expressing the ratio of the reaction rate to its maximum, is calculated in the
program, part of which is shown below. The number of elements N is called
Array in the program, which is on the CD-ROM.
{Shells 2 to Array-1}
a/at (S [2. . (Array-1) ] )=3*D*( ((r[i]**2)*(S[i-l]/(deltar*( (r[i]**3)-(r[i+l]**3))
Nomenclature
Symbols
D
KS
OURmax
r
Rp
Diffusion coefficient
Saturation constant in Monod equation
Maximum specific oxygen uptake rate
Radius at any position
Outside radius of bead
m2/s
mol/m3
mol/cell s
m
m
RS
S
X
Ar (Deltar)
391
Reaction rate in the Monod equation

Oxygen substrate concentration
Increment length, r/N
Indices
1
2
n
P
S
Exercises
Refers
Refers
Refers
Refers
Refers
to
to
to
to
to
segment 1
segment 2
segment n
particle
substrate
mol/s m3
mol/m3
cells/m3
m
392
Results
10
20
30
40
50
60
70
80
90
100
Figure 3. Profiles of oxygen concentrations versus time for each shell.
Figure 4. Doubling the bead radius causes oxygen deficiency inside the bead (lower curve) as
these radial profiles show.
Reference
Keller, J. (1991) PhD Dissertation No. 9373, ETH-Zurich.
8.7.5
393
Immobilized Biofilm in a Nitrification

Column System (NITBEDFILM)
Nitrification is the sequential oxidation of NH4+ to NO2~ and NO3" which

proceeds according to the following reaction sequence:
NH4+ +1 O2 -> NO2- + H2O +2H+
NO2- + \ O2 -> NO3The overall reaction is thus

NH4+ + 202 -> N0 3 -+H 2 0
Both steps are influenced by dissolved oxygen and the corresponding substrate
concentration and are catalyzed by two different organism species. Since their
growth rates are very low, nitrification as a wastewater treatment process benefits
greatly from biomass retention.
In this example, a biofilm column reactor for nitrification is modelled as three
tanks-in-series with a recycle loop (Fig. 1). Oxygen is supplied only in an
external contactor and circulates to the reaction column in dissolved form.
This is similar to the example NITBED. However, in this case the reaction takes
place within an immobilized biofilm, similar to the single tank example
BIOFILM.
394
O3, Si3
Fluidized
bed
SiA
Figure 1. Biofilm column reactor with recycle loop for nitrification.
Model
The column reactor is assumed to be described by three tanks. The model
balance equations for the liquid phase are developed by considering both the
individual tank stages and the absorber. Component balances are required for
all components in each section of the reactor column and in the absorber,
where the feed and effluent streams are located. For the solid biofilm phase,
where the reaction takes place, the concentrations change both with distance
and time. Therefore, a descretization of the length variable is required as
developed for the example BIOFILM.
395

To tank n+1
t
S2ntO]
S3ntO]
On[0]
From tank n-1

Figure 2. Schematic of a single tank in the column.
Because of the complexity involving four components in two phases and four
regions care must be taken with the nomenclature. The nitrogen compounds are
referred to as Si, 82, and 83, respectively. Dissolved oxygen is referred to as O.
Referring to the above figure, a single tank n is shown with the four
components. [0] refers to the liquid phase in contact with the solid. Transfer to
the biofilm is by diffusion to the first section, denoted [1].
Figure 3. Schematic of a single section i of biofilm in tank n.
Further diffusion brings substrate to all the biofilm sections, as shown above, for
a single substrate in section i. The reactions occur in these sections.
In the absorber, oxygen is transferred from the air to the liquid phase.
Additional subscripts, as seen in Fig. 1, identify the feed (F), recycle (R) and the
flows to and from the tanks 1, 2 and 3, each with volume V, and the absorption
tank with volume VAThe fluidised bed reactor is modelled by considering the component balances
for the three nitrogen components (i) and also for dissolved oxygen. For each
stage n, the liquid phase component balance equations have the form
dSin[0]
dt
396
-J0[0]
For the absorption tank, the balance for the nitrogen containing components
include the input and output of the additional feed and effluent streams, giving
ds
iA
-
_(S/QiF -SQiA )\
The oxygen balance in the absorption tank must account for mass transfer from
the air, but neglects the low rates of oxygen supply and removal by the
convective streams. This gives
For the first and second biological nitrification rate steps, the reaction kinetics
for any stage n are given by
v
ml Slni
ni
Kl+ S lni K O i + O ni
V
rs2n
m2 S2ni
2+ S 2ni
02+ni
The oxygen uptake rate is related to the above reaction rates by means of the
constant yield coefficients, YI and Y2, according to
i -r S 2niY2
The reaction stoichiometry provides the yield coefficient for the first step
YI = 3.5 mg 02/(mg N-NNH4)
and for the second step
Y2 = 1.1 mg O2/(mg N-NO2)
397
Nomenclature
Symbols
A
F
FR
KLa
K
KI
K2
L
N
0
Os and O*
OUR
r
S
V
VA
vm
Y
Specific area of film

Feed and effluent flow rate
Recycle flow rate
Biofilm thickness
Number of biofilm segments
Oxygen solubility, saturation cone.
Oxygen uptake rate
Reaction rate per volume of biofilm
Volume of one reactor stage
Maximum velocity
Yield coefficient
1/m
m3/h
m3/h
h
g/m 3
g/m 3
g/m 3
m
g/m 3
g/m 3
g/ m3 h
g/ m3 h
g/m 3
m3
m3
mg/L h
mg/mg
Indices
1,2,3
1,2,3
A
F
jn[I]
m
Ol and O2
S1,S2
S and *
Refer to ammonia, nitrite and nitrate, resp.

Refer to stage numbers
Refers to absorption tank
Refers to feed
Refers to substrate j in stage n in segment i
Refers to maximum
Refer to oxygen in first and second reactions
Refer to substrates ammonia and nitrite
Refer to saturation value for oxygen
398
Exercises
Program
399
Results
'4.5
*C
3
A
.3.5 g
4
I 60 '
j,g 5 0 -
13
40-
2.5
.2
a?
3
S
1.5
520.
55 io-
o60
50
70
80
90
100
TIME
Figure 4. Time profiles of the nitrogen component bulk concentrations in the first tank and
the oxygen bulk concentrations in the three tanks.
4.5
4
3.5
0.5
0
0
10
20
30
40
50
60
70
80
90
100
TIME
Figure 5. Time profiles of the oxygen concentrations within the 10 segments of biofilm in
the first tank.
400
8.8
8.8.1
Multi-Organism Systems
Two Bacteria with Opposite Substrate
Preferences (COMMENSA)
System
Considered here (Fig. 1) is the batch growth of a two-organism culture on two
substrates, in which both species can utilize both substrates (Kim et al., 1988),
but where the organisms have opposing substrate preferences. The two
bacterial species involved are: Klebsiella oxytoca (XA) and Pseudomonas
aeruginosa (XB). The two substrates are glucose (Y), which is preferred by K.
oxytoca, and citrate (Z), which is preferred by P. aeruginosa.
XA
XB
Figure 1. Organism XA prefers substrate Y, and organism Xg prefers substrate Z.

The assumptions are as follows:
- The overall individual growth rate of each species at any time is the sum of
the rate of growth on glucose plus the rate on citrate.
- The specific growth rate on each substrate depends on the concentration
level of some key enzyme responsible for the rate-controlling step E.
- The key enzyme for the preferred substrate is assumed to be constitutive.
- The production of the key enzyme for the secondary substrate is subject to
induction and repression by the preferred substrate.
- An inhibitor I is produced from the growth of K. oxytoca on glucose and
inhibits the growth of P. aeruginosa on citrate. The inhibitor is thus a
growth-associated product.

ISBN: 3-527-30759-1
8.8 Multi-Organism Systems
401
- The total rate of substrate consumption is the sum of the rates of

consumption by each organism plus the rate of consumption of substrate for
the production of inhibitor.
- The oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER)
involve the sum of the individual contributions from each organism.
- The dissolved oxygen tension in percentage air saturation (DOT) is obtained
using a steady state oxygen balance.
Model
The growth rates, jny, for each organism are the sums of the growth rates on
glucose and citrate. The subscripts i and j have the following meaning: i refers
to the organisms (K. oxytoca - A and P. aeruginosa = B) and j refers to the
substrate (glucose = Y and citrate = Z). The levels of the key enzymes are
denoted by E.
The biomass balances for the batch system are
dXA
(MAY + MAZ) XA
dXB
I
The specific growth rate equations for the two organisms on each substrate are
given by:
A*maxAYSYEAY
K
SAY + S Y
MmaxAZSZEAZ
K
SAZ +S Z
A*maxBZSZEBZ I
M-BZ - K--
S
"FT
SBZ+ Z V I +
The substrate balances are given by:
402
dS
Y =
SAY
I )
" YSAZ
XA
SBY
dSz
dt
- - YSBZ
XB
Inhibitor (I) production is growth associated to organism A, and its decay is

proportional to the cell concentration. The balance for the inhibitor is
dl
gj- = oc|u AY X A -pX A
The balances for the key enzymes, which control the growth on secondary
substrates are given by:
Sz
KRAZ
- -*^--rt\iu
kpAZf\z^
EAZ
T-T
"
and
dE
BY
- "
SY
--Y
KRBY
KbY
where the consecutive terms in the above equations represent induction,

repression, and dilution due to cell division, respectively. Here the enzyme
levels E are normalized with respect to the maximum levels (See reference).
Because growth on the preferred substrates is constitutive, EAy and EBZ are
equal to 1 .
The oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and
dissolved oxygen tension (DOT) are given by:
OUR =
OAY
OAZ
OBY
OBZ
CER =
Y
CAY
CBY
CAZ
CBZ
403
DOT = 100 1-
OUR
K L aC 0
The cell mass fractions are given by:

FA=-
X A + XB
FB = 1 - F A
Program
Nomenclature
Symbols
CER
DOT
F
I
K
KLaC0*
OUR
S
X
Y
E
m
a
P
Carbon dioxide evolution rate

Dissolved oxygen tension
Cell mass fractions
Inhibitor concentration
Saturation and inhibitions constants
Oxygen transfer rate
Oxygen uptake rate (normalized)
Biomass concentrations
Yield coefficients
Level of key enzyme
Specific maintenance rates
Yield constant for inhibitor
Inhibitor consumption rate constant
kg/m3 h
kg/m3
kg/m3
kg/m3 h
kg/m3 h
kg/m3
kg/m3
kg/kg
kg/kg h
kg/kg
kg/kg h
1/h
404
Indices
A
B
C
I
M
O
P
R
S
Y
Z
Exercises
Refers to K. oxytoca
Refers to P. aeruginosa
Refers to carbon dioxide
Refers to inhibitor
Refers to maximum
Refers to oxygen
Refers to dilution due to cell division
Refers to repression
Refers to substrate
Refers to glucose
Refers to citrate
405
Results
The graphical results in Fig. 2 show the dynamic changes in biomass fractions
FA and FB for two values of a: 0.007 kg/kg and 0.0007 kg/kg .
1
0.9.
0.8
0.7
0.6
s-0.45
0.3
0.2
0.1
0
4
TIME
Figure 2. Dynamic changes in biomass fractions FA and FB for a = 0.007 and 0.0007.
Reference
Kim, S. U., Kim, D. C, Dhurjati, P. (1988). Mathematical Modeling for Mixed
Culture Growth of Two Bacterial Populations with Opposite Substrate
Preferences. Biotechnol. Bioeng., 31, 144-159.
This example was developed from the original paper by J. Lang, ETH-Zurich.
406
8.8.2
Competitive Assimilation and Commensalism

(COMPASM)
System
The interactions between two microbial species (Ma and Mb) in a mixed
continuous culture are considered (Miura et al., 1980). The population
dynamics of the two microbes, is described by competitive assimilation of
substrate Si and commensalism, with the participation of growth factor Ga that
is excreted by microbe Ma and required by microbe Mb for growth. Mb also
consumes a second substrate 82 from the medium. These interactions are
represented in the Fig. 1.
,G
Figure 1. Interaction of two organisms and two substrates in continuous culture.
Model
For the chemostat shown above the unsteady-state material balances are as
follows:
Dilution rate:
407
Organism Ma:
dXa
j- = ftia-D)Xa
Organism Mb:
dXb
-gj- = (jib - D) Xb
Substrates S\ and 82:
ar = - "a
dS2
M-b Xb + D (S2
-ar = - ~YT"
"S2)
The yields for organism Mb on the two substrates are assumed here, for
simplicity, to have the same values, Yb.
The growth factor balance is
dGaa
"P
11
Jib Xb
T-X f*
The mass balance for the growth factor, Ga, is formulated by assuming a
formation rate, Pa |ia Xa, and consumption rate, (|Lib Xb)/Ybg. Here Xa and Xb
are the concentrations of microorganisms Ma and Mb, respectively. The
constants Pa and Ybg are the biological yield constants for the formation and
consumption of Ga, respectively. The specific growth rates of microbes Ma and
M b are expressed by:
Organism Ma:
Si
KSa+Si
Organism Mb:
Si
Ga
82
K Sb 2 + S2 Kg + Ga
408
where K$a and K$bi are the saturation constants of Ma and Mb for substrate Si,
Ksb2 is the saturation constant of Mb for substrate 82, and Kg is the saturation
constant of Mb for growth factor Ga. Setting \imb2 = 0 corresponds to the
consumption of only one substrate Si.
A rigorous stability analysis of the system has been carried out by Miura et.
al. (1980). This involves linearizing the mass balances by Taylor's method in
the vicinity of the steady state solution and determining the characteristic
eigenvalues of the resultant matrix. The following relationship for co-existence
of the two microbes can be derived for the case of a single substrate.
Sio > KSa D Oima - D) +
^sm ma D(K Sbl -K Sa )

Y F
a aCmmbKSa - m ma K Sbl)
Also, a critical dilution rate, where the maximum dilution rates of the two
organisms cross-over can be written:
.
Cnt
_
"
Sa
Sbl
Four particular cases depending on the values of the maximum specific growth
rate and saturation constants of both microbes can be simulated for the single
substrate case (|imb2 = 0).
1- M-ma > l^mbli Ksa > K$bi:
2. |ima > Mmbi; Ksa < KSbi:
3. |ima < |imbi; Ksa > Ksbi:
K$a >
Coexistence below a certain value of D

No coexistence range
Coexistence with wider range of stable focus
Coexistence at higher D and wider range of
Program
409
Nomenclature
Symbols
D
F
G
K
P
S
V
X
Y
Dilution rate
Feed rate
Growth factor concentration
Yield constant
Reactor volume
Yield coefficient
1/h
m3/h
g/m3
g/m3
g/m3
m3
g/m3
g/g
1/h
Indices
0
1
2
a
b
bl
b2
g
m
Exercises
Refers to feed
Refers to substrate 1
Refers to substrate 2
Refers to organism a
Refers to organism b
Refers to organism b growing on substrate 1
Refers to organism b growing on substrate 2
Refers to growth factor
Refers to maximum
410
Results
A simulation is given in Fig. 2 for the parameters as given in the program with
feed flow rate F = 0.24 m3/h, and the feed concentrations SIQ = 500 mg/L and
820 = 1500 mg/L. The oscillating concentrations are given for Xa and S\
versus time. It is seen that this solution is stable and homes into a steady state,
corresponding to case 1.

300
250
200
150 CD
100
300
Figure 2. Competition and commensalism of two organisms (F = 0.24 m3/h, S10 = 500 mg/L
and S2o = 1500 mg/L), showing the biomass concentrations.
411
Reference
Miura, Y., Tanaka, H., Okazaki, M. (1980). Stability Analysis of Commensal
and Mutual Relations with Competitive Assimilation in Continuous Mixed
Culture. Biotechnol. Bioeng., 22, 929.
Example developed from the original paper by S. Ramaswami, ETH-Zurich.
8.8.3
Stability of Recombinant Microorganisms

(PLASMID)
System
In genetic engineering, microorganisms are used as host cells to produce
important biochemicals by inserting a small portion of extra-chromosomal
DNA (on plasmids) into the cell. These plasmids carry the genetic instructions
to produce the desired product and tend to lose their engineered properties
during cell division because of non-uniform plasmid distribution. The
engineered or recombinant strain usually grows more slowly than the wild-type,
nonplasmid-bearing strain, so that engineered strain may be lost through
extinction. By exploiting the difference in the adaptation times of wild and
engineered strains, a possibility exists of maintaining a plasmid-bearing
population in continuous culture by cycling the substrate feed concentration or
the dilution rate. This dynamic problem is adapted from Stephens and
Lyberatos (1987 and 1988), based on the concept of plasmid stability from
Aiba and Imanaka (1981).
The Monod model assumes a balanced growth in which all cellular
components change in the same proportion at all times but does not account
for dynamic effects. Dynamic first order lag relations are added to account for
the response of the organisms to rapid changes in the medium. It is assumed
that the time constants for the two strains are different and that the responses to
changing concentrations are therefore different. As a consequence, the strain
with the smallest time constant has the advantage when the concentration of the
limiting substrate is oscillating. The simulation model based on Fig. 1 is used
to predict the stability in the competition between wild (Xi) and engineered
(X2) strains in continuous culture.
412
S.XLX2
Figure 1. Competitive cultures x j and X2 in a continuous system.
Model
The following dimensionless parameters are defined: s = S/K, P = 0,1/0.2, t = treai
2, Rmd = torn/Ok, Dd = D/a2, xi = Xi/Y K, x2 = X2 A" K.
The mass balances in dimensionless form are:
= (ill (zi) xi - D xi + p [12 (Z2) X2
dx2
2 - D X2
ds
g^ = D (SQ - s) - (Lli (s) Xi - |I2 (s) X2
where the time delayed specific growth rates are,
"SIT
Z2)
413
and the growth rates are
In the above, z\ and Z2 represents the time-delayed substrate concentrations,

and the specific growth rates of xi and X2 are taken as functions of z.
Thus:
dz
= P(S-ZI)
where p = ai/a2, and cxi and a2 are the adaptability factors or inverse time
constants. The effect of (3 is to delay the substrate for growth in the wild and
engineered organisms according to their first order time constants. For p > 1 the
wild type is delayed with a shorter time constant. At high values of oci and OC2,
the model describes an undelayed, instantaneous Monod growth model. It is
assumed, that (li^ > H2m .
The probability factor p represents the probability (or fraction) of
conversion to the wild strain during growth of the engineered strain. Thus the
growth rate of the engineered strain is multiplied by [1 - p].
Program
In the program on the CD-ROM, the square-wave input for SQ is generated by
the Madonna Conditional Operator, using the parameter MARK (ratio of the
time during which the function has the value 1 to the time of the complete
period) and PER (period).
{Square wave feeding generated by conditional
operator}
SO=IF(Time/PER-INT(Time/PER))<=MARK THEN SI ELSE
414
Nomenclature
Symbols
D
K
MARK
PER
R
S
s
X
X
Y
P
r
t
X
z
a
P
H
Dilution rate
1/h
Saturation constant
kg/m3
Ratio controlling step function
h
Time period
Biomass ratio
kg/m3
Substrate concentration, dimensionless
kg/m3
Biomass concentration, dimensionless
Yield coefficient
kg/kg
Probability factor
Growth rate
kg/m3 h
Dimensionless time
Biomass concentration, dimensionless

Delayed substrate concentration, dimensionless Adaptability factors
1/h
Ratio of adaptability factors
1/h
Indices
0
1
2
d
i
m
real
Refers to inlet stream

Refers to wild strain
Refers to engineered strain
Refers to dimensionless
Refers to 1 or 2
Refers to maximum
Refers to real time
415
Exercises
The stability problem can be studied by the variation of several parameters.
Results
The output in Fig. 2 gives the substrate oscillations created by the square wave
feed concentrations, showing the engineered organism X2 being washed out. A
similar situation for sine wave feeding is given in Fig. 3.
416
100
250
200
150
TIME
Figure 2. Square wave substrate feeding, causing X2 to wash out.
100
150
200
TIME
Figure 3. Sine wave feeding. Similar to Fig. 2 but with longer period and a higher b value.
References
Aiba, S., Imanaka, T. (1981) in Annals of the New York Acad. of Sciences, 369,
1-15.
417
Stephens, M.L., Lyberatos, G. (1988) Biotechnol. and Bioeng., 31, 464-469.

Stephens, M.L., Lyberatos, G. (1987) Biotechnol. and Bioeng., 29, 672-678.
Example developed by N. Mol, ETH-Zurich.
8.8.4
Predator-Prey Population Dynamics

(MIXPOP)
System
The growth of a predator-prey mixed culture in a chemostat can be described
with a reaction kinetics formulation. In this growth process, the dissolved
substrate S is consumed by organism Xi (the mouse), while species X2 (the
monster) preys on organism Xi, as shown in Fig. 1.
Figure 1. Monster attacks mouse while it unsuspectingly feeds on S.
Model
The model involves the chemostat balances for each species with the
corresponding kinetics. The variables are given in Fig. 2.
418
Sl,X-|,X2
^*
S0 D
1
II
Figure 2. Chemostat predator-prey reactor.
Substrate balance,
dS
= D(So-Si) Species 1 (prey) balance,
^2X2
Species 2 (predator) balance,
dX2
- DX 2
where D is the dilution rate,
~"v
The kinetics are given by Monod relations,
Program
419
Nomenclature
Symbols
D
F
K
S
V
X
Y
Dilution rate
Flow rate
Saturation rate constant
Reactor volume
Yield constants
Indices
0
1
2
m
Exercises
Refers
Refers
Refers
Refers
to feed stream
to prey
to predator
to maximum
1/h
m3/h
kg/m3
kg/m3
m3
kg/m3
kg/kg
1/h
420
Results
Stable steady states for the system are shown in Fig. 3 for |LLmi = 0.5 and
|Lim2 = 0.11. Oscillations in the biomass populations are achieved by setting the
specific growth rates nearly equal (\im\ = 0.5 and |Lim2 = 0.49) as shown in
Fig. 4 and also by the phase plane plot of Fig. 5.
3.5
3
2.5
2
1.5
0.5
50
100
150
200
250
TIME
300
Figure 3. Stable steady state (|iml = 0.5 and [im2 = 0.11).
350
400
450
500
421
450
Figure 4. Oscillatory state (|iml = 0.5 and p,m2 = 0.49).
Figure 5. Phase plane plot of oscillations.
422
8.8.5
Competition Between Organisms (TWOONE)
System
Consider organism A and organism B with their respective specific growth rates,
(HA and ILLB, which both grow independently on substrate S. Assume:
S/(KSA + S)
S/(KSB + S)
Depending on the values of |LIM and K$, these two functions may occur in two
different forms, as shown in Fig. 1.
M
inter
inter
Figure 1. Comparison of growth rate curves for the competitive chemostat growth.
It is clear that the curves B and A will cross each other if (IMB < MMA and KSB
< KSA- In Fig. 1, the situation on the left indicates that B will grow fastest at
any value of S. For this case, in chemostat cultures with dilution rate DI, after
an initial start up period, a substrate concentration S i will be reached at which
(LIB = DI and for which |LIA < DI. Organism A will then be washed out, and
only organism B will remain in the reactor.
The situation in the right of Fig. 1 shows (IB crossing (IA- The point of
intersection can be found easily by simple algebra where:
423
Wnter =
Solving for S at the intersection,
Sinter
For this case a chemostat can theoretically operate stably at D = Jiinter such
that both A and B will coexist in the reactor. This however is an unrealistic
metastable condition, and with D < Hunter* A will wash out. With D > Hunter A
will grow faster, causing B to be washed out.
Model
The equations for the operation of chemostat with this competitive situation are,
d*A
dXB
jp = 0 - D XB + JIB XB
dT = D(S0 - S) In addition, the Monod relations, |IA = f(S) and |LLB = f(S), are required.
Solution of these equations will simulate the approach to steady state of A and
B competing for a single substrate.
Program
424
Nomenclature
Symbols
D
F
KS
S
V
X
Y
Dilution rate
Flow rate
Substrate concentrations
Reactor volume
Biomass concentrations
Yield coefficient
Specific growth rates
1/h
m3/h
kg/m3
kg/m3
m3
kg/m3
kg/kg
1/h
Indices
A
B
M
0
inter
Exercises
Refers
Refers
Refers
Refers
Refers
to organism A
to organism B
to maximum
to inlet stream
to the intersection of the ju versus S curves
425
Results
4.5
4
3.5
3
!
2.5 '
2
1.5
1
0.5
9
10
Figure 2. Organism A and B competing for substrate.
8.8.6
Competition between Two Microorganisms for

an Inhibitory Substrate in a Biofilm
(FILMPOP)
System
Wastewater with toxic chemicals is often treated directly at the source with
specialized microbial cultures in small-scale biofilm reactors. A model may
help to understand, optimize, and control such reactors. In a paper by Soda et
al. a simple biofilm model was developed to simulate the competition between
two microorganisms for a common inhibitory substrate. In the model the
following assumptions were made: (i) the biofilm has a uniform thickness and
is composed of 5 segments, (ii) each microorganism A and B utilizes a
common substrate, and the growth rates are expressed by Haldane kinetics with
a spatial limitation term but is otherwise independent of the other
microorganism and (iii) the diffusion of the substrate, movement of the
microorganisms, and continuous loss of the biomass by shearing are expressed
by Pick's law-type equations.
425
Results
4.5
4
3.5
3
!
2.5 '
2
1.5
1
0.5
9
10
Figure 2. Organism A and B competing for substrate.
8.8.6
Competition between Two Microorganisms for

an Inhibitory Substrate in a Biofilm
(FILMPOP)
System
Wastewater with toxic chemicals is often treated directly at the source with
specialized microbial cultures in small-scale biofilm reactors. A model may
help to understand, optimize, and control such reactors. In a paper by Soda et
al. a simple biofilm model was developed to simulate the competition between
two microorganisms for a common inhibitory substrate. In the model the
following assumptions were made: (i) the biofilm has a uniform thickness and
is composed of 5 segments, (ii) each microorganism A and B utilizes a
common substrate, and the growth rates are expressed by Haldane kinetics with
a spatial limitation term but is otherwise independent of the other
microorganism and (iii) the diffusion of the substrate, movement of the
microorganisms, and continuous loss of the biomass by shearing are expressed
by Pick's law-type equations.

ISBN: 3-527-30759-1
426
Sf
S0
Wall
Figure 1. Schematic of the continuous reactor with biofilm, showing the descretization into
five layers.
Model
Fig. 1 illustrates an idealized flat biofilm with a uniform thickness Lf (m). The
biofilm is divided into N segments for simulation purposes and each has a
thickness of AZ = Lf/N (m). Wastewater containing the substrate is fed to the
reactor at a constant feed rate and a concentration Sf (mg/L). The bulk liquid in
the reactor is mixed throughout the tank and the substrate diffuses into the
biofilm. The substrate is transported from the bulk liquid having a
concentration S[0] (mg/L) to the surface of the biofilm having a concentration
S[l] (mg/L). A diffusion layer of a thickness Lj (m) is used to represent the
external mass transport resistance.
Using the same approach as in the example BIOFILM, the mass balances in
the bulk liquid for the substrate and microorganisms A and B with a continuous
flow are simply described as following:
dS[0]
dt
Wll
v f
427
_,
'
S[0]-S[1]
" DZ
m [01X fC] mB[0]XB[0]

YA
YB
,
XA[0]-XA[1] ,
}
- -DXA|0] - aDXA
(mALO] - bA JXA[0]
/\/1
where S is substrate concentration (mg/L). XA and XB are biomass of

microorganisms A and B (mg/L), respectively. Each number in the brackets
refers to the bulk liquid or a segment illustrated in Fig. 1 . DX, b, Y, and |Li are
diffusion coefficient of microorganisms (m2/day), biomass decay coefficient
(day1), yield coefficient (-), and net specific growth rate (day1). Subscripts A
and B refer to microorganisms A and B. D, Ds, a, and t are dilution rate (day"1),
diffusion coefficient of substrate (m2/day), specific area perpendicular to the
flux (nr1), and time (day), respectively.
Reactions within the biofilm are described by diffusion reaction equations. The
mass balances of the surface segment are described as following:
s[0]-sm
dt
LjAZ
dX A [l]
dt
AZ2
XA[0]-XA[1]
XA[1]-XA[2]
- -- DXA -
LZXZ,
at ~
dX B [l]
=: \j v"-p
XB
XB[0]-XB[1]
L,AZ
XB[1]-XB[2]
n
LJ YT3
^
XB
AZ2
Component mass balances are written for each segment (i = 2, .., N-l), where:
428
dS[i]
dt
5>[i-l]-2S[i]+S[i+]L]
AZ2
DS
MA[i]XA[i]
/iB[i]XB[i]
YA
YB
dXA[i]
X A [i-l]-2X A [i] + XA[i
t~DXA
2
dXB[i]
^t-DXB
XB[i-l]-2XB[i] + XB[i
The mass balances of the boundary segment on the support wall are described
by the following equations:
dS[N] = p S[N-1]-S[N]
S
dt
AZ2
The "diffusion" coefficients of microorganisms, DXA and DXB represent

displacement by cell division and by shearing off at the film boundary
contacting the bulk liquid.
Growth Kinetics Of Microorganisms

The inhibitory influence of high substrate concentration was described by the
Haldane kinetics. The two types of microorganisms compete for substrate, but
in the biofilm they also have to compete for the limited space available.
Therefore, growth of the microorganisms was described by the Haldane kinetics
with a spatial limitation term which was originally proposed as cell inhibition
kinetics by Han and Levenspiel (1988).
..
429
m_
K IA
[i]=
K IB
where KI? Ks, and (im are inhibition constant (mg/L), half saturation constant
(mg/L), and maximum specific growth rate (day" ). Xm (mg/L) is the maximum
capacity of total biomass of microorganisms A and B in a segment.
The formulation of the spatial limitation term used here is the most simple
one possible with non-restricted growth at zero biomass concentration and zero
growth at maximal biomass concentration Xm.
Applying the above model it was found (Soda et al., 1999) that the
qualitative behavior of the biofilm reactor is characterized by 5 regions,
depending on the operating conditions, the substrate concentration in the feed
and the dilution rate. In region I, both microorganisms are washed out of the
biofilm reactor. In region II, microorganism B is washed out, and in region III,
microorganism A is washed out of the biofilm. In region IV, both
microorganisms coexist with one another. In region V, both microorganisms
coexist with a sustained oscillatory behavior. Convergence to regions I-V
depends on the initial conditions. In regions II-V, washout of either or both
microorganisms is also observed when the initial conditions are too far away.
Nomenclature
Symbols
a
b
D
DS
Dx
K!
KS
specific area perpendicular to the flux,

related to bulk liquid volume
biomass decay rate
dilution rate
diffusion coefficient of substrate
diffusion coefficient of microorganisms
inhibition constant
saturation constant
m"
day"
day"
m /day
m /day
mg/L
mg/L
430
LI
Lf
N
S[i
Sf
Y
AZ
thickness of diffusion layer

thickness of biofilm
number of segments in biofilm
substrate concentration in element i
substrate concentration in feed
biomass
spatial capacity of total biomass
of microorganisms A and B in a segment
yield coefficient
thickness of each segment
maximum specific growth rate
Indices
A
B
refers to microorganism A
refers to microorganism B
Numbers in brackets
0
1-5
Exercises
refers to bulk liquid

refer to segments 1-5
m
m
mg/L
mg/L
mg/L
mg/L
m
"
day
431
References
Soda, S, Heinzle, E,, Fujita, M. (1999) "Modeling and simulation of
competition of two microorganisms for a single inhibitory substrate in a
biofilm reactor." Biotechnol. Bioeng., 66, 258-264.
Han, K. and Levenspiel, O. 1988. "Extended Monod kinetics for substrate,
product, and cell inhibition." Biotechnol. Bioeng. 32: 430-437.
Program
Shown below is a portion of the program. The full program is on the CD-ROM.
{BALANCES FOR BIOFILM IN 10 SEGMENTS}
d/dt (S[2. .nslabs-1] )=DS* (S[i-l] 2*S[i]+S[l+l])/ (Z*Z)-UA[i] *XA[i] /YA-UB[i] *XB[i] /YB
d/dt (XA[2. .nslabs-1] ) =DSA* (XA[i-l]2*XA[i] +XA[i+l] )/(Z*Z)+(UA[i] -kdA) *XA[i]
d/dt ( X B [ 2 . . n s l a b s - 1 ] ) =DSB* ( X B [ i - l ] ) / ( Z * Z ) + ( U B [ i ] -kdB) * X B [ i ]
2*XB[i]
432
Results
/ \
/-
^^'
sr^^"
'
0.035
-0.03
\,
2 40-

m
/
-0.025
\
I
x 20
0.02
XAmid:1
. . XBmid:1
Smid:1
(A
-0.015
i
\
-0.01
g.
-0.005
---. '--.
10
20
30
40
50
60
70
80
90
100
TIME
Figure 2. Results corresponding to Case 2: UmB=0.4, KSB=0.1, KIB=10.

0.05
0.045
0.04
0.035
0.03
0.025
(A
0.02
0.015
0.01
0.005
10
20
30
40
50
60
70
80
90
100
Figure 3. Results corresponding to Case 4: UmB=1.8, KSB=0.01, KIB=0.01.
8.8.7
433
Model for Anaerobic Reactor Activity

Measurement (ANAEMEAS)
System
As already discussed in Chapter 3, anaerobic processes can be described by
multi-substrate, multi-organism kinetics. As shown in Table 1, organic acids
are formed from monomeric and polymeric substrates contained in wastewater.
These are then converted into hydrogen, CO2 and acetic acid. In a last step,
acetic acid and H2 with CO2 form methane.
Table 1. Stoichiometry of Anaerobic Reactions.
Step 1:
Hydrolysis (example: carbohydrate-hexoses)

(C6Hi2O6)n + n H2O -> nC 6 Hi 2 O 6
Step 2:
Acid production (example glucose)

C6Hi2O6
->
CH3(CH2)2COOH + 2 H2 +
C6Hi2O6 + 2 H2
->
2 CH3CH2 COOH +
2H 2 O H 2CH3COOH + 4 H2 + 2 CO2
Step 3:
Step 4:
Acetic acid production

CH3(CH2)2COOH + 2 H2O
->
2 CH3COOH +
CH3CH2COOH + 2 H20 -> CH3COOH + 3 H2 + CO2
2 CO2
2 H2O
2 H2
Methane production
CH3COOH -> CH4 + CO2
4 H2 + C02 -> CH4 + 2 H20
In order to evaluate the activity of an anaerobic reactor and to evaluate the

correctness of the reactions in Table 1, an off-line measurement system has
been designed. This involves a small batch reactor coupled to a mass
spectrometer. A sample of biomass with medium is taken from the larger
continuous anaerobic reactor and put into the small batch reactor. Dissolved
gases are stripped by helium, all gas bubbles are removed and substrate is
added to start the reaction. The accumulation of organic acids, CO2, H2 and
CtLj. is measured. pH is adjusted according to total acid concentration and
buffer capacity. Biomass concentration is constant throughout the experiment.
434
The model was developed to aid the design of the measurement system and in
the interpretation of the data.
Model
A five organism model with lumped hydrolysis and acid-generating bacteria
was established. Substrate, intermediate and product balances of the batch
reactor are
dSi
"dT = IrSi
where rsi are the rates of consumption and synthesis of S[.
The respective reaction rates rj for the consumption of substrate Si and for
the formation of product Pj in each step are those from the reactions in Table 1
as follows:
and the specific growth rates take the Monod form,

M-imax Si
Mi = KSi + Si
or modified in the case of substrate inhibition for acetate,

M^maxi Si
The individual equations for each substrate Si are given in the program,
Thermodynamic equilibrium constraints on the Step 3 reactions (Table 1)
are also included in the model.
435
Reaction Equilibrium
In the acetogenic step (Step 3 reaction in Table 1), acetic acid, hydrogen and
carbon dioxide are produced from propionic and butyric acid.
The
thermodynamic equilibria for these reactions are incorporated by estimating
the chemical equilibrium limits for butyric acid:
CH3(CH2)2COO- + 2 H2O 2 CH3COO- + 2 H2 + H+
AGo = 48.3 kJ
From this the equilibrium constant is KBU = 2.02 x 10~3 (mol4 nr12) given by
KBU
"
For propionic acid similarly,

CH3CH2COO- + 2 H2O
2 CH3COO- + 3 H2 + CO2 AG^ = 76.1 kJ
and a equilibrium constant of Kpro = 1.35 x 10~12 (mol4 nr12).

4 CEAc2 CEH23 CEC02
Pro 3 CEPro
The factor 4/3 is necessary because concentrations here are given in C-mol.
An empirical approach was chosen to slow the reactions down on
approaching the equilibrium, and they were not allowed to proceed to the right
side when the equilibrium condition was reached. Using the actual
concentrations, the parameters KBU* and Kpro* were estimated.
*
KBU
C A c 2 C H2 2 CH +
~~
CBu
_ 4 CAc2 CH23 CCQ2

3C Pro
The ratio of these values to the true equilibrium constant, K*BU/KBU, and
K*pro/Kpro will be greater than unity if the equilibrium has not yet been
reached. Using these ratios with the empirical S-shaped curve of Fig. 1, the
factor FEQ was determined and was used to modify the growth rates. This
somewhat arbitrary function starts from FEQ = 0 at K*/K < 1 and rises to FEQ
= 1 at K*/K > 2. The factor FEQ causes the reaction to the right to stop when
436
the equilibrium is reached, but there is no reverse reaction when concentrations

of acetate, hydrogen, CO2 and H+ exceed the equilibrium values caused by
other reactions. The reaction proceeds irreversibly further away from the
equilibrium (K*/K > 2).
1.2
1.00.8.
a
uj
u_
0.60.40.2.
0.
-0.2K*/K
Figure 1. Equilibrium factors (FEQ) to slow the growth rates near equilibrium.
The kinetics of biomass growth butyric acid, and propionic acid were modified
by these empirical equilibrium factors, FEQ, according to
i =FEQi
Ion Charge Balance to Estimate pH

As discussed in Sec. 1.3.7, in calculating the pH an ion charge balance can be
written to account for the acid-base dissociation buffer effects. The ion balance
represents an implicit non-linear equation in the dynamic model and must be
solved by iteration for each time interval, such that 8 = 0 in the equation
I + CH+
Thus CH+ is varied iteratively until 8 becomes essentially zero. This numerical
solution is not always trivial using conventional methods for non-linear
437
algebraic equations (e.g., Newton-Raphson, and Regula falsi). Fortunately this

type of equation can be handled conveniently by the root finding feature of
BerkeleyMadonna, as shown in the program on the CD-ROM and below.
If base is added to control pH, an additional balance for cations of strong
bases (K+, Na+, ...) and anions of strong acids (Cl% SC>42~, ....) becomes
necessary as follows:
dCz
=
titr Qtitr
Program
The program nomenclature is rather extensive and is defined within the
program. The Berkeley Madonna ROOTS feature is used to calculate the pH,
as shown below. The full program is on the CD.
(PH>
GUESS CHPLUS = le-4
ROOTS CHPLUS =
KW/CHPLUS+KdBu/(KdBu+CHPLUS)*BU/4+KdPr/(KdPr+CHPLUS)
*Pr/3
+KdAc/(KdAc+CHPLUS)*Ac/2
+KdC/(KdC+CHPLUS)*Cg
+KdBuf/(KdBuf+CHPLUS)*BUFFER-lonen-CHPLUS
LIMIT CHPLUS >= 0
LIMIT CHPLUS <= 1000
pH=-loglO(chplus)+3
Nomenclature
The nomenclature of the program is partially defined within the program.
Symbols
C
Cons
F
FEQ
Ftitr
Concentration
Consumption rate
Stoichiometric coefficients
Equilibrium factor
Titration flow rate
C-mol/m3
C-mol /m3h
(-)
(-)
m3/h
438
KD
Ks
S
P
Pro
X
YP/S
YX/S
1^
Dissociation constant
Inhibition constant
Saturation constant
Product Concentration
Production rate
Yield coefficient, product from substrate
Yield coefficient, biomass from substrate
Specific rate of biomass synthesis
mol /m3
C-mol /m3
C-mol/m3
C-mol /m3
C-mol /m3
C-mol /m3 h
C-mol /m3
C-mol /C-mol
C-mol /C-mol
1/h
Indices
Ac
Bu
Buf
d
Hy
i
in
Mo
Pr
titr
Tot
Refers to acetic acid

Refers to butanediol
Refers to buffer
Refers to death rate
Refers to hydrogen gas
Refers to reaction i
Refers to initial
Refers to whey substrate
Refers to propionic acid
Refers to titration
Refers to total
Refers to difference between cations and ions
Results
The first of the three graphs in Fig. 2 shows dynamic profiles of substrate whey
(Mo), CH4 (CH), dissolved CO2 (CO) and dissolved hydrogen (Hy). The whey
is almost instantaneously consumed. Hy reaches a maximum very soon and is
then quickly reduced to almost zero. CH4 is produced with varying rates. CC>2
reaches a maximum, which is partly caused by pH changes and by
consumption by hydrogen-consuming organisms. The peaks in the CC>2 curve
originate from numerical inaccuracies in the stiff system. In the second graph,
Fig. 3, the total concentration of volatile acids acetate (Ac), propionate (Pr) and
butyrate (Bu) are given. The thermodynamic inhibition of acetogenesis is
clearly seen in the early phase of the experiment. Ac reaches a maximum much
later than Pr and Bu, since it is produced from these two acids. In the third
graph, Fig. 4, the pH versus time profile is given, exhibiting an early decrease,
followed by almost constant pH during the rest of the simulation.
439
0.06
-0.3
0.05
-0.25
0.04
-0.2
' 0.03
0.15
0.02
-0.1
0.05
0.01
0.005
0.025
0.03
Figure 2. Dynamic profiles of substrate whey (Mo), CH4 (CH), dissolved CO2 (CO) and
dissolved hydrogen (Hy). Zoomed to show the early period.
0.3
0.25
r 0.2
0.15 -
-0.05
0.35
Figure 3. Total concentration of volatile acids acetate (Ac), propionate (Pr, lower curve) and
butyrate (Bu). The whey (Mo) peak is hardly visible at T = 0.
440
6.18
-0.2
6.16
0.18
6.14
0.16
6.12
.0.14
6.1
.0.12
0.1
: 6.08
0.08
6.06
0.06
6.04
6.02
..'
0.04
0.02
5.98
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Figure 4. Variation of pH and acetate with time.
Exercise
References
Heinzle, E., Dunn, I.J. and Ryhiner, G. (1993) "Modelling and Control for
Anaerobic Wastewater Treatment." Adv. Biochem. Eng. 48, 79-114.
Yamada, N., Heinzle, E. and Dunn, I.J. (1991) "Kinetic Studies on
Methanogenic Cultures Using Mass Spectrometry." in: Biochemical
Engineering - Stuttgart (Eds. Reuss, M., Chmiel, H., Gilles).
441
Ryhiner, G. Heinzle, E., Dunn, I.J. (1992) "Modelling and Simulation of

Anaerobic Waste water Treatment and Its Application to Control Design: Case
Whey," Biotechnol. Progr. 9, 332-343.
8.8.8
Oscillations in Continuous Yeast Culture

(YEASTOSC)
System
Oscillations in continuous cultures of baker's yeast have often been observed.
An example of measurements is shown in Chapter 3, whose oscillations were
modelled by the reaction scheme in Fig. 1.
Figure 1. Pathways of proposed model for yeast culture oscillations.
Model
The balance equations for continuous culture with dilution rate D are as
follows:
dR
dE
= - D E + [ rGE(R,G,E) + rSE(S) - rEX(E) ] R

- = D (SF - S) - [ rSE(S) + rSG(S,E) ] R
442
dG
dT
= - D G + [ rSG(S,E) - rGE(R,G,E) ] R
= - D T + [ rTi(R,G,E) - rT2(R,G,E) ] R
The species in parenthesis indicate the dependencies of the rates. The kinetic
expressions used in the balance equations are as follows:
GEm E
/KGX\n
l /
S
Ks + S
TEX =
ME)
1 + ( KG/G + KET/E )n
rT2 =
Many of the parameters were determined independently from experiments,
some were taken from the literature, and some, especially those describing the
enzyme activity (T), had to be chosen during simulations. This model leads to
oscillations whose existence and dependency on operating conditions
qualitatively agree with experimental results. Also the directions in the phase
plane plot agree with the experiments.
443
Nomenclature
Symbols
D
E
G
K
n
R
r
S
sig
T
X
Dilution rate
Ethanol concentration
Storage material
Growth rate constants
Empirical exponent in rate model
Residual biomass without G
Growth rates
Rate constants. Example: sigGEm = TGEM
Indices
E
G
m
S
T
X
Exercise
Refers
Refers
Refers
Refers
Refers
Refers
to ethanol
to storage material
to maximum
to glucose
to enzyme
to biomass
1/h
kg/m3
kg/m3
kg/m3
g/m3
kg/m3 h
kg/m3
various
g/m3
kg/m3
1/h
444
Results
The influence of dilution rate is given below in plots from simulations: Fig. 2
with D = 0.05 and Fig. 3 with D = 0.1. In Fig. 4 the phase plane from the run
of Fig. 2 is shown.
-0.45
35-
0.4
30-
0.35
250.3
20-
0.25
(
0.2
15-
-0.15
10
0.1
50-
M..JL.JL...
20
40
0.05
-0
60
100
TIME
120
140
160
180
200
Figure 2. Biomass and substrate oscillations for D = 0.05.

.2
1.8
-1.6
1.4
1.2
-1
0.8
.0.6
.0.4
0.2
20
40
60
80
100
TIME
120
Figure 3. Biomass and substrate oscillations for D = 0.1.
140
160
180
200
</>
445
0.09-|
0.080.070.060.05-
i
0.040.030.020.01 -
012
16
18
20
22
24
26
28
30
Figure 4. Phase plane giving S versus X from the run of Fig. 3. Zoomed in for detail.
Reference
Heinzle, E., Dunn, I.J., Furukawa, K. and Tanner, R.D. (1983). Modelling of
sustained oscillations observed in continuous culture of Saccharomyces
cerevisiae. in Modelling and Control of Biotechnical Processes (ed. A.Halme),
Pergamon Press, London, p.57.
8.8.9
Mammalian Cell Cycle Control

(Mammcellcycle)
System
Modeling of mammalian cell cycle control is of great importance for
understanding development and tumor biology. Hatzimanikatis et al. (1999)
presented a model in the literature using simplified molecular mechanisms as
depicted in Fig. 1.
446
cycE+cdk2
CycE:cdk2-P+1 f>
Rb+E2F
cycE:cdk2-P:1
Rb-P+E2F
Figure 1.
Schematic representation of the molecular mechanism of components and
interactions believed to be most important in controlling the Gl-S transition. cycE- cyclin E;
cdk2 - cyclin dependent kinase 2; Rb - pRb, a pocket protein; E2F - a transcription factor; P phosphate.
Model
For this reaction scheme the dynamic mass balances become
= V2 -Vl
dt ~
dK
dKr
dt
447
dRE
dt
^=v 6 > r -v 6 , f
The symbols are defined as follows:
V are reaction rates.
C is the cyclin E concentration.
K is the cdk2 concentration.
KP is the phosphorylated cyclin E-cdk2 complex concentration.
K P I is the concentration of cyclin E-cdk2 phosphorylated complex bound to
inhibitor.
R is the concentration of the hypophosphorylated form of pRb.
RP is the concentration of the hyper-phosphorylated form of pRb.
RE is the concentration of the hypo-phosphorylated form of pRb that binds
to E2F.
E is the E2F concentration.
I is the concentration of the cyclin E-cdk2 complex inhibitor.
The subscipts "f' and "r" denote the forward and the reverse step,
respectively, of the reversible reactions.
The assumption of near equilibrium operation of reversible reactions (V5 and
V6) and of invariant total amounts of cdk2, pRb, E2F and inhibitor gave the
following dimensionless equations, consisting of 3 differential and 6 algebraic
equations.
=v
vd
dT~ s
y\
dk
drp
di
-= Va3 VA
4
k + k p + k p j =1
r + rp + r^ = 1
448
=1
i + A,kPI=l
re
0I= JPJL
kpi
Where c is the dimensionless concentration of cyclin, k is the dimensionless

concentration of cdk2 and rp is dimensionless concentration of the hyperphosphorylated form of pRb. All details about the kinetic equations and transposing
them into dimensionless form are given in the paper of Hatzimanikatis et al.
(1999).
Nomenclature
Dimensionless symbols as used in the program are listed here.
c
e
i
k
kP
kP,I
r
rE
rP
g
s
1
t
Cyclin E concentration
E2F concentration
Concentration of cyclin E-c
ckd2 concentration
Phosphorylated cyclin E-cdk2 complex
concentration
Concentration of phosphorylated cyclin
E-cdk2 complex bound to inhibitor
Concentration of hypophosphorylated form
of pRb
Concentration of hypophosphorylated form
of pRb that binds to E2F
Concentration of hyperphosphorylated form
of pRb
Ratio of total concentrations of cdk2 and cyclin E
Ratio of total concentrations of pRb and E2F
Ratio of total concentrations of cdk2 and inhibitor
Dimensionless time
449
Exercises
Program
Results
0.5
Figure 2. Profiles of concentrations k, rp and c versus time as obtained from the rate constants
in the program.
450
1
0.9
0.8
0.7
0.6
"o.5
0.4
0.3
0.2
0.1
0.32
0.33
0.34^
0.35
0.36
0.37
0.38
0.39
0.4
0.41
0.42
Figure 3. Phase plane plot of k and rp versus c.
Reference
V. Hatzimanikatis, K. H. Lee, and J. E. Bailey. (1999) "A mathematical
description of regulation of the Gl-S transition of the mammalian cell cycle".
Biotechnol. Bioeng., 65, 631-637.
8.9
8.9.1
Membrane and Cell Retention Reactors
Cell Retention Membrane Reactor (MEMINH)
System
Consider a reactor whose outlet stream passes through a membrane that retains
only the biomass as seen in Fig. 1.
The growth is assumed to follow substrate inhibition kinetics with constant
yields. The oxygen transfer rate influences the growth at high cell density
according to a Monod function for oxygen.
F,S 0
Gas
\"
Membrane modi
module
illiiil
Air
Figure 1. Biocatalyst retention on a continuous reactor.

ISBN: 3-527-30759-1
452
Model
The reactor-membrane system is modelled as a well-mixed tank, except that
biomass is retained in the system batchwise. The balance region is chosen to
include both the reactor and the membrane separator, but the separator volume
is neglected.
Biomass balance:
dX
dT = rx
Substrate balance:
dS
-r s
Oxygen balance (neglecting the oxygen transported by flow):
dCL
-gj- =K L a(CLS-C L )-ro
Kinetics:
S
rx
__
~ ^m
CL
x
s = rx YS/X + MS X
= r
x YQ/X + MO X
where the maintenance coefficients are related by
MS
M0
Program
YS/X
8.9 Membrane and Cell Retention Reactors
453
Nomenclature
Symbols
CL
CLS
F
KI
KLa
Ko
KS
M
r
S
V
X
Y
ILL

Flow rate
Inhibition constant
Saturation constant
Maintenance coefficients
Reaction rate
Reactor volume
Yield coefficient
Indices
0
1
m
O
S
X
Exercises
Refers
Refers
Refers
Refers
Refers
Refers
to feed
to reaction 1
to maximum
to oxygen
to substrate
to biomass
g/m3
g/m3
m3/h
g/m3
1/h
g/m3
kg/m3
kg/(kgh), g/(kgh)
kg/(m3 h)
kg/m3
m3
kg/m3
kg/kg
1/h
454
Results
Oxygen transfer has a pronounced influence on performance as seen in Fig. 2
for variations of Kâ. The dissolved oxygen may reach values below KQ as
shown in Fig. 3 for Kâ values from 0.5 to 5.
20
Figure 2. Influence of oxygen transfer coefficient (KLa = 0.1 to 1.0).
455
\\.
0
10
12
14
16
18
20
TIME
Figure 3. Profiles of dissolved oxygen influenced by KLB (0.5 to 5, curves bottom to top).
8.9.2
Fermentation with Pervaporation (SUBTILIS)
System
The metabolic pathways for the production of acetoin and butanediol are well
known, as shown in Fig. 1. A kinetic model for a Bacillus subtilis strain has
been established from continuous culture experiments using an approach
involving overall stoichiometric relationships and energetic considerations. The
influence on the culture of product removal by pervaporation was investigated
by simulation methods.
Knowledge of these pathways allowed the following overall equations to be
written:
Respiration (reaction RI):
C6Hi2O6
> 6CO 2 + 6H 2 O
Formation of biomass (reaction R2):

1.2 NH3 > 6 CHi.8O0.5No.2 + 0.3 O2 + 2.4 H2O
456
Formation of acetoin (reaction

C 6 Hi 2 0 6 +10 2 > C4H802 + 2 C02 + 2 H2O
Conversion of acetoin to butanediol (reaction R4):
C4H8O2 + H2O -> C4HioO2 + 0.5 O2
Complex Compounds
Biomass
Substrate
Sugars
Carbon Dioxide
(Total Oxidation)
Pyruvate
Lactate
Acetoin
i
+ Carbon
Dioxide
Butanediol
Figure 1. Reaction scheme for the acetoin - butanediol formation.
The continuous reactor was coupled to a pervaporation membrane module and

was influenced by the membrane performance, owing to the removal of
products and the retention of biomass. Since only the volatile products and
water can pass through the membrane, a purge stream was needed to remove
biomass and salts. The recycle between the reactor and membrane module,
shown in Fig. 2, was high enough to provide complete mixing.
Model
Growth
The sugar (S) and dissolved oxygen effects were described in terms of a double
Monod function and the product inhibition by a simple inhibition kinetic term.
Diauxic effects were observed, but the diauxic components were unknown, and
for simplification only one diauxic switchover was assumed. The preferred
457
component is referred to as Ci, and the second component is called C2- An

empirical kinetic relation was devised such that the utilization of C2 was
inhibited by the presence of Ci in amounts greater than a repression constant
kRpi. The formulation then involves considering the growth as the sum of two
terms,
where,
and
C2
Membrane
Recycle
Feed
*pl
Pervaporation Module
Condenser
Bioreactor
Permeate
Fpe
ACpe , Bu
Pump
Figure 2. Bioreactor and membrane pervaporator, showing process variables.

Each product was assumed to inhibit separately,
1
Bu_cc Bu
and
The inhibition constants, kinh,Ac and kinh,BU> an<l the exponents,

were estimated from shake flask experiments.
The complete expression for specific growth rate was thus,
DO
i
1+ [_^_]Ac 1+ |
and
i
Bu_iaBu
458
Formation of Acetoin and Butanediol

The products were assumed to be formed when the respiration was not
sufficient to cover the energy requirement for growth. While the distribution of
the two products was assumed to be dependent on oxygen, through a rapid
equilibrium, according to an S-shaped empirical function described by
fAcBu,max (DO)2
Ac
Bu
To avoid an algebraic loop, the kinetics for the rate of butanediol production
was assumed to be dependent on the deviation from equilibrium,
TBU = qX4 X = kAcBu (Ac - Bu fAcBu)
The constant kAcBu was set high enough to ensure equilibrium conditions.
The specific reaction rate q^3 for product formation was obtained as,
~ YR2/R1 qX,l - YR2/R4
Reaction Rates
The specific reaction rates for each component were obtained from the specific
reaction rates q^ and the stoichiometric coefficients, Vy (component j in
reaction i) as follows,
i
The volumetric rates rj [mol L"1 Ir1] for components X, S, Ac, Bu, 62 and CC>2
were related to the specific rates as,
rj = qj X
The specific rate q^2 of reaction R2 was obtained from \\ as
459
V 2 , X MG X
where V2,x is the stoichiometric coefficient, and MGx is the C-mol mass of
biomass.
The rate for the respiration reaction, qî, was found to be influenced by the
dissolved oxygen, and was described as follows:
qxi =
The yield coefficients for the complex components GI and C2 , were assumed
to be 1 g of each component for 1 g biomass, and the molecular weights of the
components were assumed to be the same as for the biomass.
The initial
amounts of these components in the molasses medium were adjusted in the
simulation. The corresponding rates were proportioned according to the
growth rates as
rri = - rv
and
rr2 =
Pervaporation Model
The mass transfer in the pervaporation module was described as an equilibrium
process using constant enrichment factors. Thus the concentrations of product
in the reactor, Ac and Bu, were related to the concentration in the permeate,
Acpe and Bupe as,
Acpe = PAC Ac
and Bupe = PRU Bu
Dynamic
Reactor Mass Balances
Considering volume-specific flow rates,

_ FO
_
Fpu _
D = y- , Dpu = -^r > Dpe =
where D, Dpu, Dpe were the flows for the feed, purge, and permeate, respectively
intr 1 .
460
The mass balances were as follows:

Total mass
D = Dpu + Dpe
Biomass
dx
"3T = rx - X Dpu
Sugar
dS
df = S0 D - S Dpu - rs
Acetoin
dAc
~dT = rAc ~
Ac D
dBu
~dt~ =
Bu D U
pu ~ Acpe Dpe
Butanediol
rBu
" BuPe DPe
Complex components
dCi
~dT = Ci>0 D - Ci Dpu - rCi
where Q = 1 and 2.
Oxygen transfer from the gas phase and the oxygen uptake rate determine the
DO in percent saturation as,
dDO
-HT~
= KLa (100 - DO)
m
100
^r OUR
2.34xlO"4
Here the equation is in terms of percent saturation using the DO saturation

value at 30 C of 2.34 x 10''4 mol/ L.
Nomenclature
Symbols
ACPE
Ac
ATP
Bu
Ci
CPR
D
DO
Acetoin concentration in permeate

Acetoin
Adenosintriphosphate
Butanediol
Complex components, where i = 1 or 2
Dilution rate
g/L
g/L, mol/L
g/L, mol/L
g/L
mol/L h
1/h
% saturation
461
UcBu
lAcBu.max
F
FM/P
kAcBu
kci
KLa
KS
Kprod
MGX
OUR
P
rpm
Ri
RQ
S
vvm
V
X
Yi/j
Acetoin/butanediol ratio
Max. Ac/Bu ratio
Flow rate
Permeate / purge flow rate
Kinetic const. Ac/Bu
Monod-const., growth on component i
Monod-const., growth depending on DO
Inhibition constant product i
Gas -liquid transfer coefficient for O2
Monod-const. for respiration depending on
DO
Repression const., complex substrate 1 on
component 2
Monod-const., Growth on substrate
Empirical const, for Ac/Bu-equilibrium
Mol mass for biomass (1 C-mol) = 24.6
Oxygen uptake rate
Product, sum Acetoin + Butanediol
Specific rate of component j
Specific rate reaction i, where i =lto 4
Formation or uptake rates, components j
(Ac, Bu, O2, CO2, X and S)
Stirrer speed
Chemical reaction i, where i = Ito 4
Respiration quotient qcO2/(lO2
Substrate concentration (e.g., sugar)
Gas rate per volume liquid
Reaction volume
Yield coefficient (i formed/j used)
L/h
mol/g(acetoin) h
g/L
% saturation
g/L
1/h
% saturation
g/L
g/L
g/mol
mol/L h
g/L, mol/L
mol/g(biomass) h
mol/g(biomass) h
mol/L h
1/min
g/L, mol/L
1/min
L
g/L, mol/L
Greek symbols
ai
pi
\i
R,max
Vi j
Exponent inhibition kinetic product i (Ac or Bu)

Enrichment factor of pervaporation membrane for
component i (Ac or Bu)
Maximum growth rate for component i
Stoichiometric coeff. of component j in reaction i
1/h
1/h
462
Indices
Ac
Bu
CO2
i
j
inh
02
pe
pu
P
Ri
S
X
0
Ai
Acetoin
Butanediol
Component i (unknown components 1 or 2)
Carbon dioxide
Refers to reactions, i (1 to 4)
Refers to components j (Ac, Bu, C>2, CC>2, X and S)
Oxygen uptake
Permeate
Purge
Product
Chemical reaction i (1 to 4)
Sugar
Biomass
Feed or initial concentration
Refers to reactions 1 to 4
Reference
Dettwiler, B. Dunn I. J., Heinzle E., and Prenosil J. E. "A Simulation Model
for the Continuous Production of Acetoin and Butanediol Using B. subtilis with
Integrated Product Separation by Pervaporation" Biotechnol Bioeng. 41, 791
(1993).
Program
Results
The results of Fig. 3 show the influence of the FM/P ratio, which corresponds
to the membrane area per unit reactor volume, on the biomass concentrations.
463
Here the enrichment factor was kept constant (pAc = 2.0), corresponding to the
values found for one of the membranes. In a second set of runs the enrichment
factor was varied for constant FM/P ratio = 1.0.
110
100
60
50
6
10
12
14
16
18
20
Figure 3. Influence of permeate/purge flow ratio on the biomass concentration.

BETAAC=2, FMP=0.4, 1.0, 2.0).
(D=l,

0.25
0.15
..p.-'*""
j- *~ "*"
0.05
Figure 4. Influence of the enrichment factor on the acetoin and butanediol in the permeate.
(D=l, FMP=1 BETAAC=1, 3, 5).
464
8.9.3
Two-Stage Fermentor With Cell Recycle For

Continuous Production Of Lactic Acid
(LACMEMRECYC)
System
This example is based on a paper (1) in which a two stage fermentor-membrane
system for the continuous production of lactic acid was modelled. Membrane
retention of the active biomass can be expected to increase the productivity, but
the biomass concentration must be controlled in each reactor with a bleed
stream. The kinetics for this process, in which glucose is converted to lactic acid
by the bacteria Streptrococcus faecalls is described in the literature (2). Since
the rates are inhibited by product, it can be expected that a multistage system
will be advantageous.
(1-B2XB1F1+I?)
X 2 ,S 2 ,P 2
Figure 1. Two-stage membrane fermenter system for lactic acid production
465
Model
The model assumes completely-mixed stages and complete cell separation. The
operating parameters are feed flow rates or dilution rates and the bleed stream
fractions. The bleed stream from the first fermenter is led to the second
fermenter. The model equations are developed neglecting the volume of the
lines and separators. Note that the retentate streams are returned to their
respective reactors and can be considered as part of the well-mixed system.
The kinetics is given by a product inhibition
S
ii ii
KLP
and a death rate of the bacteria caused by product is also included.
The specific glucose uptake rate is given by

1
Ys
=+
qs
M-
The lactic acid production rate is given by
Mass Balances for the First Stage

Defining the dilution rate as DI=FI/VI
For the total cells
~dT~~
For the living cells
466
For the glucose
For the lactic acid

l
dt
fl
Mass Balances for the Second Stage

The dilution rate for the second stage depends on the bleed ratio from the first
stage and the ratio of feed rates.
For the total cell mass

=
BjDjXn - (8! + f )D!B2X2 +
For the active cells
^ = IBAXJ - I(B! + 00^2X2 + (ji2 - k d2 )x

For the glucose
fD!Sf2 +
BAS1 - ( 8 ! +f)D 1 S 2 -q 2 X 2
For the lactic acid product

1
Clt
UC
(JC
1 .
>
2+V2X2
(JC
Productivity of Lactic Acid

It is assumed that all streams containing product can be recovered from all of
the streams. The productivities are calculated as follows:
From the first stage
fti=D1(P1-Pfl)
467
From the second stage

Pr 2 =-D 1 [(B 1 +f)P 2 -B 1 P 1 -fP f2 ]
VAi
The total productivity

Pr = -i-D 1 [(l-B 1 )P 1 +(B 1 +f)P 2 -P f ,-fP f2 ]
JL ~t~ UC
Substrate Conversion
From stage 1
From stage 2
Xco =1
82
(B 1+ f)S 2
--
B^+fSfz
Total glucose conversion

_
Xg I
(l-B 1 )S 1 +(B 1 +f)S 2
--
f 1 + fSf 2
Program
In the simulation product-free feed streams are assumed with flow rates between
10-30 kg m 3 . The program is on the CD-ROM.
Nomenclature
a
B
D
f
F
kd
Death constant
Bleed ratio
Dilution rate
Flowrate ratio, F2/F1
Volumetric
flowrate
Specific death rate
Basis specific death rate
m 3 kg"1
s'1
m3 s'1
s"1
s"1
468
KP
KS
P
Pr
qs
s
t
V
*s
xt
a
YP,YS
8P,8s
H
V
Exercises
Product inhibition constant

Saturation constant
Lactic acid concentration
Productivity
Specific substrate uptake
Time
Reactor volume
Substrate conversion
Concentration of living cells
Total cell concentration
Reactor volume ratio
Kinetic constants
Kinetic constants
Spec, product formation rate
kg nr3
kg nr3
kg nr3
kg nrV1
kg kg-V1
kg nr3
s
m3
kgnr 3
kgm~ 3
kg kg-1
kg kg-1
s-1
kg kg'1 s-1
469
References
A. Nishiwaki and I. J. Dunn, "Performance of a two stage fermentor with cell
recyle for continous production of lactic acid", Bioprocess Engineering 21;
299-305, 1999.
H. Ohara, K. Hiyama, T. Yoshida, "Kinetics of growth and lactic acid
production in continuous and batch culture" Appl.Microbiol.Biotechnol. 38,
403-407, 1992.
Results
-30
^Xt1:1
...Xt2:1
20-
25
... S2:1
20
P2:1
rsj 15X
gioH
10 CO
10
15
20
30
35
1
40
TIME
Figure 2. Results showing the steady state for biomass substrate and product in both stages.
470
160
-1
140
0.9
| 120-I
B
X
1
flf
0.8
100
-0.7 rf
80
0.6 tfT
X
60
-0.5
40
A..
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.4
0.5
B1
Figure 3. Results showing that the productivity changes slightly with bleed ratio but that the
biomass concentration and the substrate conversion depend highly on bleed ratio.
8.9.4
Tubular Hollow Fiber Enzyme Reactor

Module for Lactose Hydrolysis (LACREACT)
System
This tubular reactor- radial diffusion model assumes a series of nine well-mixed
tanks to describe a single hollow fiber module. Flow of lactose substrate passes
axially through the inner region of the fiber lumen. By diffusion the substrate
is transported radially outward from the lumen through the membrane and into
the cylindrical porous support surrounding the membrane. The reaction takes
place in this support region, where the immobilized enzyme is located. The
products of hydrolysis are glucose and galactose, which diffuse back toward the
liquid phase in the lumen. The parameter N can be used to adjust the number
of shells required. The module is assumed to consist of a large number of
identical fibers*
471
Model
The model is developed by finite-differencing both the axial and radial
directions. Thus there are axial stages in series, here nine plus the recycle tank,
and there are multiple cylindrical sections of porous support in the radial
direction. As depicted in the figures below, there is a convective liquid flow
from each stage to another. Diffusional flows carry substrate and product
radially from one cylindrical section of the porous support to another. Figures
1, 2 and 3 give the geometrical details.
FRfiber
LAfiber
LAtank
FRfiber
Figure 1. Hollow-fiber module showing only a single fiber as modelled by nine stages.
472
Figure 2. Details of a single well-mixed axial section.
Figure 3. Details of a finite-differencing of the porous support in the radial direction.
Fig. 3 shows a cross-section of the hollow fiber membrane showing the inner
hollow fiber region (white) and the outer porous support (shaded). The finitedifference shell of volume V2 (white) is shown with diffusion fluxes of lactate
JLAI entering and leaving JLAI- It is important to account for the radial
variation of volumes and diffusional areas. Note that the segments are
numbered from outside to inside, 1 to N.
For each tank the component balances account for the accumulation, the flow
in and out and the diffusion in or out from segment N of the porous
membrane. For lactose in tank 1
LA
lumenl _
H
ai
473
FR
v
v
fiber */ T A
x , JlAltN]* Aj[N]
T A
v^^tank ' L/Mumenl ) +
77
v
lumen
lumen
For the external recycle tank, the flow leaving all of the fibers enters it and also
the feed stream enters it. The total flow rate leaving must equal the sum of these
rates. Thus for lactate
Number * FRfiher
tank
Ffeed / A
T
T
. LAtank ) + -Jeed_(LA
feed - LA tank )
V
tank
Taking the component balances for each segment in the enzyme zone account
for accumulation, diffusion in and reaction.
Thus for lactose in the enzyme regions of the first tank
where Aj is the area available to diffusion and TLAI!^] is the reaction rate for
lactose in the ith enzyme segment of the first tank. Note that the above
equations do not include the balance for segment 1. The wall condition
requires that this balance contains only the rate of diffusion from segment 1 to
segment 2, as seen in the program.
The reaction rate is assumed, as confirmed by experiment, to have the form of
*!__
LAt[i]
Here the units are mole lactose per cm3 of porous support volume per second.
Program
Repeated here for the first tank section are the lactose balances, fluxes and rates
as given in the program on the CD-ROM.
JLA1[1. . (N-l) ]=-DLA* ( L A l E i + 1 ] -LAl[i] ) /DR
Flux of lactose flowing between segments i+1 and i

JLAl E N ] =DLA* ( L A I [N] -LAlumenl) /DR
Flux of lactose into inner lumen section
474
RATELA1 [1. . N] =-vmax*LAl [i] / (LAI [i]

+Km* (1+ (GA1 [i] / K i n h i b ) ) )
Reaction rate for lactose,mole/s cm3
d/dt (LAlumenl) = (FRf iber/Vlumen) * (Latank-LAlumenl )

+ ( JLA1[N] *AJ[N] ) /Vlumen
Dynamic balance for lactose in the inner lumen volume of the first tank
d/dt (LAI [2 . .N] )-(l/V[i] ) * (JLAl[i-l] *AJ[i-l] JLA1 [ i ] * AJ [ i ] ) +RATELA1 [ i ]
Dynamic balance for lactose in the segment 2 to N of the first tank
d/dt ( LAI [1] )=-(l/V[l] )*(JLA1[1] *AJ[1] )+RATELAl[l]
Dynamic lactose balance for the segment 1 (wall condition) of the first tank.
d / d t ( L A t a n k ) = ( Number * F R f i b e r / V t a n k ) * (LAlumen9L A t a n k ) + ( F f e e d / V t a n k ) * (LAf e e d - L A t a n k )
Dynamic lactose balance for the circulation tank
The geometry of the fiber is programmed such that the lumen radius and the
total fiber radius are given. The number of porous segments can be varied.
Nomenclature
Additional symbols for the geometrical factors are defined in the program on
the CD-ROM
Symbols
DGA
DGL
DLA
EO
FR
GAfeed
GLfeeci
Ki n hib
Galactose diffusivity in porous

membrane
Glucose diffusivity in porous
membrane
Lactose diffusivity in porous
membrane
Enzyme loading
Recycle flow
Galacose in feed
Glucose in feed
Kinetic inhibition constant
cm2/h
cm2/h
cm2/h
mg E in each fiber
cm3
mole/cm3
mole/cm3
mol/cm3
475
LA, GA, GL
Number
V
Kinetic constant
Kinetic constant for vmax
Length of fiber,
Lactose, galactose and glucose
Number of fibers
Volumes
Maximum rate
Indices
fiber
lumen
tank
Exercises
Refers
Refers
Refers
Refers
to lumen
to lumen
to tanks or segments
to recycle tank
mol/cm3
mol/mgE h cm3
cm
mol/cm3
cm
mol/cm3 h
476
Results
100
200
400
500
600
TIME
Figure 4. Approach to steady state for the tank concentrations.

9.4e-5
9.393e-5
Figure 5. Radial concentration gradients for the lactose and glucose. The left axis corresponds
to the outside of the fiber.
477
8.9.5
Immobilized Animal Cells in a Fluidi/ed Bed

Reactor (ANIMALIMMOB)
System
This example is based on experiments with immobilized BHK cells in a
fluidized bed of solid or porous carriers. The fluidized bed itself has an
expanded volume of 700 mL, The complete reactor system contains a volume
of 3.5 L. As seen in the figure below, the arrangement of the electrodes at the
inlet and outlet of the reactor allows an accurate difference measurement of the
oxygen uptake rate. Oxygen transfer takes place only in the conditioning
vessel, while oxygen consumption is only in the fluidized bed column, where
the cells are located.
GAS
OUTLET
GAS INLET
FILTER
AIR
OXYGEN
CARBON
OXIDE
OXYGEN
j (MEASUREMENT
CHAMBER
SAMPLE
PORTM .
VESSEL
THERMOSTAT
Figure 1. Fluidized bed for culturing animal cells on solid carriers.
The recirculation reactor is modelled by taking into account the separate

aeration tank and the geometry of the column reactor. It is assumed that the
reactor is not well mixed, but is described by a tanks-in-series model for the
column with immobilized cells and a separate well-mixed aeration tank reactor,
as shown below.
478
j3, FR
Spent medium
Figure 2. Schematic of the model structure, where Ci refers to any component concentration.
For the tanks-in-series description of the column, 3 (or more) tanks in series are
used. The mass balance for one component in tank 2 is then
Here V is the volume of one tank and r is the reaction rate of the component.
The circulation flowrate is FR. This balance equation form would apply to all
components, but not for the biomass since it is immobilized.
The kinetic model assumes the following:
1. Growth of cells is linked to the consumption of glucose and Yx/s = 0.28g
biomass produced per g glucose consumed.
2. Lactate is produced in proportion to the glucose uptake rate with
YiacG=2.0.
3. Oxygen, glutamine, lactate and glucose concentrations influence the rates.
4. Multiple Monod kinetics can be applied.
5. The medium is in contact with air and the solubility of oxygen is 8 mg/L.
From the data in the dissertation of Keller (1) can be calculated the yield of
lactate with respect to glucose, giving Yiacc = 2.0 mmol lactate/mmole glucose
or 1.1 g sodium lactate/g glucose. This can be used to calculate the production
rate of lactate. Also calculated from the dissertation is YgiutG=:36mg
glutamine/g glucose.
Thus the glucose uptake rate becomes
K lac
"OX
w
sox
K sglut
4-C lac
479
Note that the last term in the equation models an inhibition by lactate.
The growth rates are based on the specific substrate uptake rates. Other specific
rates are related by yield coefficients and biomass concentration.
For example for growth rate,
rx = qG X Yxg
Data of experimental values:

Maximal volume of the expanded fluidized bed; 0.6 L
Entire reactor system volume; 3.5 L
Medium throughput; 6.5 L/day
Feed glucose concentration; 3.9 g/L
Glucose consumption rate; 4.7 mmol/h
Oxygen uptake rate; 3.7 mmol/h
Max. cell density: nonporous carriers; 2*10 cells/ml expanded
bed volume
porous carriers; 4*10 cells/ml expanded bed volume
Ratio of inoculum cell number; approx. 5 % of final cell
number
Total biomass (porous carrier); 6.24 g (Approx. 2*10 cells per
g biomass)
Oxygen transfer coeff.; KLa 2.15 1/hr
Program
Nomenclature
Cgiutf
CgiutF
Cox
Coxsat
F
FR
GUR
Feed cone, for glucose

Feed glutamine concentration
Feed glutamine concentration
Dissolved oxygen
Saturation for oxygen
Flowrate
Circulation flowrate
Glucose uptake rate
g/L
mg/L
mg/L
mg/L
mg/L
L/h
L/h
mmol/h
480
KLa
Klac
K sg
KSglut
KSOX
OUR
qCmax
QOxmax
V
V4
X
YoiutG
YLacG
YoxG
YXG
Exercises
Mass transfer coeff.

Inhibition constant for lactate
Saturation constant, for glucose
Saturation constant for glutamine
Oxygen uptake rate
Glucose uptake rate max
Oxygen uptake rate max
Volume of 3 tanks in the column
Aeration tank volume
Glutamine uptake rate
Yield coefficient glucose
Yield coefficient glucose
Yield coefficient, biomass to glucose
1/h
g/L
g/L
mg/L
mg/L
mmo 1/h
g/h g cells
mg/g cells h
L
L
g/L
mg glut./g glue.
g lactate/g
mg oxygen/g
g/g
481
References
Keller, J. Dissertation No. 9373, ETH, 1991.
Keller, J., Dunn, I. J. and Heinzle E. "Improved Performance of the Fluidized
Bed Reactor for the Cultivation of Animal Cells" in Production of Biologicals
from Animal Cells in Culture, Ed. Spier, Griffiths, Meignier, ButterworthHeinemann, 10th ESACT Meeting, 513-515 (1991).
Keller, J. and Dunn, I. J. "A Fluidized Bed Reactor for the Cultivation of
Animal Cells", In: Advances in Bioprocess Engineering, Eds. E. Galindo and O.
T. Ramirez, Kluver, pp 115-122 (1994).
Results
11 '
10'
6 O
O
9'
8
?
7
6
5
iliii X[3]:1
4
3
20
40
80
TIME
100
140
160
Figure 3. Batch run showing the difference in dissolved oxygen between the tank sections.
482
20
60
80
100
120
140
160
Figure 4. In this run with F=0.05 L/h first oxygen limitation develops and later glucose
limitation. The immobilized biomass in the three sections increases at different rates due to the
glucose gradient in the column.
Appendix: Using the Berkeley

Madonna Language
9.1
A Short Guide to Berkeley Madonna
Computer Requirements
Two Berkeley Madonna versions are supplied with this book on a CD, one for
PC with Windows and one for the Power Macintosh. More information with
downloads can be found on the following website:
http://www.berkeleymadonna.com
Installation from CD
The files are compressed on the CD in the same form as they are available on
Internet. Information on registering Madonna is contained in the files.
Registration is optional since all the examples in the book can be run with the
unregistered version. Registration makes available a detailed manual and is
necessary for anyone who wants to develop his or her own programs.
Running Programs
To our knowledge, Madonna is by far the easiest simulation software to use, as
can be seen on the Screenshot Guide in this Appendix. Running an example
typically involves the following steps:
Start Berkeley Madonna and open a prepared program file.
ISBN: 3-527-30759-1
484
9 Appendix: Using the Berkeley Madonna Language
Adjust the font and size to suit.

Go to Model/Equations on the menu and study the equations and program
logic.
Go to Parameters/Parameter Window on the menu and see how the values
are set. They may be different than on the program. Those with a * can
be reset to the original values. Also, if necessary, here the integration
method and its parameters (DT, Stoptime, DTmax, Dtmin, Tolerance, etc.)
values can be changed.
Decide which plot might be interesting, based on the discussion in the text.
Go to Graph/New Window and then Graph/Choose Variables to select data
for each axis. All calculated results on the left side of the equations are
available and can be selected.
Run the program by clicking on Run.
Adjust the graph by setting the legend with the legend button. Perhaps put
one of the variables on the right side of the graph with Graph/Choose
Variables.
Possibly select the range of the axes with Graph/Axis Settings. Choose
colors or line types with the buttons.
Decide on further runs. It is most common to want to compare runs for
different values of the parameters. This is usually done with Parameters
/Batch Runs and also with Parameters/Define Sliders. If the overlay button
is set then more than one set of runs can be graphed on top of the first set.
Sometimes more than one parameter needs to be set; this is best done with
changes done in the Parameters/Parameter Window, with an overlay graph
if desired.
As seen at the end of the Screenshot Guide, Parameter Plot runs are very
useful to display the steady-state values as a function of the values of one
parameter. For this, one needs to be sure that the Stoptime is sufficient to reach
steady-state for all the runs.
When running a program with arrays, as found in the finite-differenced
examples, the X axis can be set with [i] and the Y axes with the variables of
interest. The resulting graph is a plot of the variable values at the Stoptime in
all of the array sections. For equal-sized segments, this is the equivalent of a
plot of the variables versus distance. If the steady-state has been reached then
9.1 A Short Guide to Berkeley MADONNA
485
the graph gives the steady-state profile with distance. More on running
programs is found in Sec. 2 of the Appendix.
Special Programming Tips

Berkeley Madonna, like all programming languages, has certain functions and
characteristics that are worth noting and that do not appear elsewhere in this
book.
Editing text
The very convenient built-in editor is usually satisfactory. Also the program
can be written with a word processor and saved as a text-only file with the suffix
".mmd". Madonna can then open it.
Finding programming errors. Look at a table output of the variables
Sometimes programs do not run because of errors in the program that cause
integration problems. Some hint as to the location of the error can often be
found by making an output table of all the calculated variables. This is done
by going to Graph/New Window and then Graph/Choose Variables and
selecting all the variables. Then the program is run and the table button is
chosen. Inspection of all the values in the table during the first one or two time
intervals will usually lead to an isolation of the problem for those values that are
marked in red with NAN (not a number). Also, values going negative can be
found easily here and often indicate an integration error. Sometimes this can
be overcome with a limit function of the form, limit X>=0.
Is a bracket missing?
Madonna tests for bracket pairs, and a missing bracket will be indicated.
Setting the axes. Watch the range of values.
Remember that each Y axis can have only one range of values. This means that
you must choose the ranges so that similarly sized values are located on the
same axis.
Are there bugs or imperfections in Madonna?
Yes, there are some that we are aware of. You may find some or you may have
some special wishes for improvements. The Madonna developers in Berkeley,
California would be glad to receive your suggestions. See the homepage to
contact them
Making a pulse input to a process.
This can be done it two ways: To turn a stream on and off either use the preprogrammed PULSE function or use an IF-THEN-ELSE statement.
486
Making a more complex conditional control of a program.

In general the IF-THEN-ELSE conditional statement form is used, combined
with logical expressions as found in the HELP. This can involve a switching
from one equation to another within this statement. Another way is to use flags
or constants that take values of 0 or 1 and are multiplied by terms in the
equations to achieve the desired results. Nesting of multiple IF statements is
possible:
V = I F ( D i s k < l AND P > 1 . 9 )
THEN
0.85*KV*P/SQRT(TR+273)
ELSE IF (Disk<l AND P< = 1.9 AND P>1.1)
THEN KV*P/SQRT(TR+273)*SQRT(1+(1/P)*(1/P))
Parameter estimation to fit parameters to data.

For fitting sets of data to one or more parameters the data can be imported as a
text file and fitted by going to Parameters/Curve Fit. The
Edit/Preferences/Graph Window provides the possibility of having the data as
open circles. The required data format can be found in the file KLADATA.
Optimisation of a variable.
There is optimization available under Parameters/Optimize, but if it is
something simple with one or two parameters, then sliders can also be
effectively used. If the value of a maximum is sought as a function of a single
parameter value, then the Parameter Plot for maximum value can be used.
Finding the influence of two parameters on the steady state?
A Parameter Plot choosing the "final" value can be used to find the influence
of one variable on the steady state. The second parameter can be changed in
the Parameter Window and additional parametric runs made and plotted with an
overlay plot. Thus it is possible to obtain a sort of contour plot with a series of
curves for values of the second parameter. Unfortunately, a contour plot is not
yet possible.
Nice looking results are not always correct.
A warning! It is possible to obtain results from a program that at first glance
seems OK. Always make sure that the same results are obtained when DT is
reduced by a factor of 10 or when a different integration method is used.
Plotting all the variables may reveal oscillations that indicate integration errors.
These may not be detectable on plots of a few variables.
Setting the integration method and its parameters?
It is recommended to choose the automatic step-size method AUTO and to set
equal values of DT and DTMAX. Run the integration once and reduce both
parameters by one-half and run again. If the results are good, try to improve
the speed by increasing both parameters. Finally it should be possible to set
9.1 A Short Guide to Berkeley MADONNA
487
DTMAX higher than DT, but sometimes the resulting curves are not smooth if
DTMAX is too high. In most cases, good results are obtained with AUTO and
DT set to about 1/1000 of the smallest time constant.
If no success is found with AUTO, then try STIFF and adjust by the same
procedure. Oscillations can sometimes be seen by zooming in on a graph; often
these are a sign of integration problems. Sometimes some variables look OK
but others oscillate, so look at all of them if problems arise. Unfortunately
there is not a perfect recipe, but fortunately Madonna is very fast so the trialand-error method usually works out.
Checking results by mass balance
For continuous processes, checking the steady-state results is very useful.
Algebraic equations for this can be added to the program, such that both sides
became equal at steady state. For batch systems, all the initial mass must equal
all the final mass, not always in mols but in kg. Expressed in mols the
stoichiometry must be satisfied.
What is a "Floating point exception"?
This error message comes up when something does not calculate correctly, such
as dividing by zero. This is a common error that occurs when equations
contain a variable in the denominator that is initially zero. Often it is possible
to add a very small number to it, so that the denominator is never exactly zero.
These cases can usually be located by outputting a table of all the variables.
Plotting variables with distance and time.
Stagewise and finite-differenced models involve changes with time and
distance. When the model is written in array form the variable can be plotted as
a function of the array index. This is done by choosing an index variable for
the Y axis and the [ ] symbol for the X-axis. The last value calculated is used in
the plot, which means that if the steady-state has been reached then it is a
steady-state profile with distance. An example is given in the"Screenshot
Guide" in Sec. 2 of the Appendix and in the example CELLDIFF.
Notation for the differential.
In this book the differential form d/dt(x) is usual. However the x' form has the
advantage that it appears in the Choose Variables menu and can be plotted. It
can also be used directly in another equation.
Writing your own plug-in functions or integration methods.
Information on using C or C++ for this can be obtained by making contact
through the BerkeleyMadonna homepage.
488
9.2
Screenshot Guide to Berkeley Madonna
This guide is intended as a supplementary introduction to Berkeley Madonna,

Version 8.0.1.
(CHEMQSTATST
(FILE/CHEMCr1)
{Constants}
UM=OJKS=OJK1
8F*10 D1=0.25
Stoptime=80
{Conditional equation for 0}
!nrfX=1
lnt P=0
{Mass Balances}
; 81OMASS BALANCE EQUATION
; SUBSTRATE QÂNGE EQUATION
; PRODUCT BALANCE EQUATION
9.2 Screenshot Guide to Berkeley MADONNA
489
Figure 1. The example CHEMO has been opened and the Menu (From left: File, Edit, Flowchart
active only for flowchart programs, Model, Compute, Graph, Parameters, Window and Help) and
Graph Buttons (From left: Run, Lock, Overlay, Table, Legend, Parameters, Colors, Dashed Lines,
Data Points, Grid, Value Output and Zoom).
Figure 2. The Berkeley Madonna menus are shown above.
490
9 Appendix: Using the Berkeley MADONNA Language
UP AND QPEFIAT1GW
3T6RTTIME
iTOPTIMI
Kl=Q.03K2=O.OBY=fl.B
BF=10 D1=OJS
MTOUT
LJM
0,02
0
0.3
KS
Kl
0.1
0,03
DT
{Conditional equation for D}

D=iftime=tstirttnenDl
o.oa
0.8
10
01
toil 8=10
initP=G
HITX
NITS
N1TP
{Mass Balances}
EQ
BALANCE
0,34
5
1
10
Figure 3. The Model/Equations was chosen. Seen here is also the Parameter Window.
Figure 4. If a new graph is chosen under Graph/New Window then the data must be selected
under Graph/Choose Variables.
491
4J-,
4-
-9,5
3,5
8.5
-8
2.5
2-
-7.5
7
1.5
f-
-8.5
0,5-
5.5
20
40
80
SO
108
120
140
ISO
130
200
TIME
Figure 5. A graph window for variables on the left and right-side Y axis with Legend Button
selected.
Run 4:10000 steps m 0.167 seconds

..........................10
7.
6-
-7
5-
X 4-
01
-4
3-
-3
4-2
1-
20
40
80
100
120
140
180
!80
200
Figure 6. An Overlay Graph for three values of Dl as selected in the Parameter Window.
492
Figure 7. Part of the window to define the Sliders.
Figure 8. A graph of two slider runs, showing the Parameters Menu pulled down.
493
Figure 9. The Batch Runs window for 5 values of SF.
Figure 10. A Parametric Plot was chosen for 40 runs changing values of Dl to give the
final, steady-state values. The Data Button was pressed to give the points for each run.
494
Figure 11. The Optimize Window, with the value of Dl being selected to minimize the
expression -D*X. The value found was 0.27, corresponding to the Parameter Plot results.
Run 11 KOODOO steps ir, 2.53 seconds
Figure 12. Two Parameter Plots overlaid showing the effect of reducing Y from 0.8 to 0.6.
495
Figure 13. A program written in an array form allows plotting all the values versus time by
choosing the variable vector, here S[ ] versus TIME for the program CELLDIFF.
Figure 14. From the same program as Fig. 13, radial profiles of three runs are plotted in an
overlay plot. The [i] values can be selected in the Choose Variables. Here the parameter
Radius has been changed to demonstrate the large influence of diffusion length.
496
Figure 15. Here the program file KLAFTT is run and fitted to data in the text-file KLADATA.
The data consists of two columns: time and CE at equal intervals as seen by the open circles on
the plot. Note that the fit variable is CE and the parameter varied to minimize the difference in
least squares is KLA.
10
Alphabetical List of Examples
ACTNITR
267
ANEAMEAS
433
ANIMALIMMOB
476
BATFERM
193
BIOFILM
372
BIOFILTDYN
342
CELLDIFF
388
CHEMO
799
CHEMOSTA
258
COMMENSA
400
COMPASM
406
CONINHIB
261
CONTCON
367
DCMDEG
280
DEACTENZ
308
DUAL
275
ELECTFIT
335
ENZCON
305
ENZDYN
383
ENZSPLIT
377
ENZTUBE
272
ETHFERM
240
FBR
295
FEDBAT
204
FERMTEMP
358
FILMPOP
425
INHIB
327
KLADYN
335
KLAFIT
335
LACMEMRECYC
464
LACREACT
470
LINEWEAV
272
MAMMCELLCYCLE 445
MEMINH
450
MIXPOP
477
MMKINET
209
NITBED
299
NITBEDFILM
393
NITRIF
OLIGO
OXDYN
OXENZ
PENFERM
PENOXY
PHB
PHBTWO
PLASMID
REPFED
REPLCUL
STAGED
SUBTILIS
TEMPCONT
TITERBIO
TITERDYN
TURBCON
TWOONE
TWOSTAGE
VARVOL
VARVOLD
YEASTOSC
327
275
337
378
230
253
279
374
477
245
249
290
455
354
349
349
363
422
286
224
224
447

ISBN: 3-527-30759-1
11
Index
Absorption, 117
Absorption tank, 136
Accumulation, 125
Accumulation terms, 135
Acetogenic step, 89
Acid-base equilibria, 47
Acidogenic step, 89
Active, 110
Adaptive Control, 174
Adaptive tuning, 178
Aerated tank, 130
Aerated tank with oxygen electrode, 336
Aeration, 117, 137
Aeration efficiency, 123
Aeration rates, 126
Aeration systems, 126
Aerobic sewage treatment, 128
Agitation, 137
Air or oxygen sparging, 134
Air saturation, 126
Air supply, 128
Airlift bioreactor, 139
Algebraic loop, 49
Alginate, 149
Alginate bead, 149
Allosteric kinetics, 74
Ammonia, 134
Ammonium, 300
Ammonium ion, 133
Anaerobic degradation, 89
Analogous, 114
Analogy, 114
Analytical solution, 19
Analytically, 108
Animal cell culture, 128
Apparent reaction rate, 133
Approximation, 113, 141
Aqueous phase, 118
Arithmetic-mean, 141
Automatic process control, 161
Automatic reset, 165
Auxiliary variable, 174
Axial, 114
Axial profiles, 114
Axial segments, 115
Backmixing, 137
Backmixing flow contribution, 142
Backmixing stream, 140
Balance region, 23
Balances, 101
Batch, 57, 64
Batch aeration, 126
Batch fermentation, 11, 103
Batch reactor periods, 57
Batchwise, 134
Bed, 134
Biocatalysis, 118
Biocatalyst diffusion model, 153
Biocatalytic reaction, 147
Biofilm, 154
Biofilm, 134, 145
Biofilm nitrification, 160

ISBN: 3-527-30759-1
500
Biofilm Reactor, 133

Biofilter, 343
Biofloc, 145
Biofloc, 149
Biological activity, 123
Biological film, 149
Biological floes and films, 388
Biological oxygen uptake, 125
Biological reaction, 145
Biological reactors, 101
Biological systems, 117
Biomass, 10, 103
Biomass recycle, 110
Biomass retention, 111, 299, 393
Biomass separation, 111
Bioprocess control, 175
Bioreaction, 117
Bioreactor, 112
Bioreactor modelling, 101
Bio-trickling filter, 343
Boundary conditions, 153
Briggs-Haldane mechanism, 68
Broth, 117
Bubble, 117, 137
Bubble coalescence, 137
Bubble column, 139
Bubble size, 140
Bulk liquid, 145
Bulk reactor concentration, 153
Buoyancy, 137
Carbon, 134
Carbon dioxide, 122
Carbon dioxide production rate (CPR), 10
Carbonate, 134
Carrier, 145
Carrier matrix, 145
Cascade, 138
Cascade control, 172
Cell concentration, 101
Cell productivity, 55
Cell recycle, 110
Index
Cells, 117
Centrifugation, 110
Chemical reaction, 126
Chemostats, 104
Circulating liquid supply, 129
Circulation time, 55
Closed-loop response, 170
Coalescence, 126
Cocurrently, 139
Cohen-Coon controller settings, 170
Column systems, 135
Commensalism, 87
Comparator, 162
Competition, 150
Competitive, 74
Completely mixed gas or liquid phases, 137
Complex diffusion-reaction processes, 157
Complex kinetics, 115
Complex models, 123, 138
Complexity, 138
Component, 122
Component Balances, 22
Components, 113
Computer Solution, 19
Concentration driving force, 122
Concentration gradient approximation, 151
Concentration gradients, 119, 137
Concentration inhomogeneities, 137
Concentration profile, 114
Conical sand bed, 134
Continuous, 64, 109, 118
Continuous Baker's Yeast Culture, 94
Continuous feed and effluent stream, 134
Continuous Operation, 60
Continuous phase, 118
Continuous-cycling, 171
Contois Equation, 82
Control, 111
Control point, 165
Control region, 23
Control strategy, 176
Index
Controlled variable, 161

Controller, 162
Controller action, 163
Controller equations, 33
Controller output, 166
Controller tuning, 169
Convection currents, 137
Convective flow, 26, 125
Convective streams, 26
Conversions, 113
Cross-sectional area, 114
Cyclic fed batch, 109
Damkohler number, 155
Data fitting, 339
Datafile, 341
Dead zone, 163
Death rate, 78
Degree of backmixing, 140
Density, 104
Deoxygenated, 126
Deoxygenated feed method, 130
Depth, 137
Derivative control, 163
Deviations, 161
Deviations from ideal stage mixing, 140
Difference form, 116
Difference segment, 115
Differential, 107
Differential control constant, 355
Differential equation, 127
Differential equations, 140
Differential gap, 163
Differential-difference equations, 159
Diffusion, 118, 119, 145
Diffusion and reaction, 388
Diffusion control, 133
Diffusion film, 146, 336
Diffusion layer, 149
Diffusion path, 145
Diffusion rate, 149
Diffusional flux, 26, 149, 389
501
Diffusional limitation, 146
Diffusional mass transfer, 145
Diffusional mass transfer coefficient, 147
Diffusion-reaction parameter, 155
Diffusion-reaction phenomena, 151
Diffusion-reaction systems, 151
Diffusive mass transfer, 138
Digital simulation, 14
Digital simulation languages, 14
Dilution rate, 105
Dimensionless, 108
Dimensionless form, 391
Dimensionless group, 151
Dimensionless parameter, 150, 154
Dimensionless variables, 155, 159, 337
Discontinuous control, 163
Disks of liquid, 113
Dispersed, 117, 118
Dispersed phase, 118
Dissociation equilibrium constant, 47
Dissolved oxygen concentration, 123, 335
Dissolved oxygen electrode, 126
Distance, 116
Distance coordinate, 151
Double Michaelis-Menten Kinetics, 73
Double-Monod kinetics, 83, 158
Driving forces, 119
Droplet, 118
D-value, 77
Dynamic, 115
Dynamic component balances, 104
Dynamic kla, 126-127
Dynamic Method, 335
Dynamic simulation, 49
Dynamics of measurement, 127
Dynamics of the liquid phase, 128
Effective diffusivity, 30, 121
Effective rate, 148
Effective reaction rate, 146
Effectiveness Factor, 155, 391
Efficiency, 110
502
Electrode measurement dynamics, 127, 129
Electrode membrane, 337
Electrode response characteristic, 336
Electrode time constant, 127, 129
Elemental balances, 23
Energy balances, 49
Entrance, 113
Enzymatic, 112
Enzyme, 112, 118
Enzyme loading, 148, 149
Enzyme reactor, 115
Enzyme-substrate complex, 69
Equations, 113
Equilibrium, 10, 122
Equilibrium oxygen concentration, 337
Equilibrium relationships, 46
Equilibrium value, 132
Errors, 161
Exit, 113
Experimental reactor, 130
Exponential, 108
Exponential and limiting growth phases, 103
External film, 145
External mass transfer, 145
External transport rate, 153
Extraction, 118
Fed Batch, 58, 64
Feed Forward Control, 173
Feedback, 161
Fermentation, 101
Fermentation media, 137
Pick's Law, 29, 120
Film coefficients, 122
Final, 109
Final control element, 162
Finite difference, 30
Finite difference Model, 151
Finite differencing, 388
Finite differencing technique, 159
Finite-differencing, 115, 153
First order, 65
Index
First order lag equation, 127, 337

First order lag model, 127
First-order, 132
First-order time lag, 354
Flocculant cell mass, 145
Flotation, 110
Flow interaction, 140
Flow velocity, 114, 148
Fluid, 120
Fluid elements, 113
Fluidized bed, 133, 299, 149
Flux, 120
Food/biomass ratio, 112
Fractional conversion, 126
Fractional response, 127
Free rise velocity, 137
Functional modes of control, 163
Gas, 117
Gas absorber, 136
Gas Absorption, 117
Gas and liquid films, 121
Gas balance, 125
Gas balance method, 128, 126
Gas bubbles, 117
Gas concentrations, 125
Gas flow rates, 125
Gas holdup, 55, 124
Gas inlet, 137
Gas phase, 117
Gas-liquid, 117, 120
Gas-liquid systems, 122
Gas-liquid transfer, 336
Gel, 145
Growth, 110
Growth rate, 32, 103
Heat of agitation, 52
Heat of fermentation, 49
Heat Transfer, 51
Henry coefficient, 340
Henry's law, 33, 122, 337
Henry's law constant, 122
Index
Heterogeneous reaction systems, 155

Hill Kinetics, 74
Hydrostatic pressure, 137, 140
Ideal Gas Law, 39
Ideal gas law, 133, 337
Idealized flow conditions, 137
Idealized plug flow, 137
Ideally mixed, 136
Immiscible, 118
Immobilization, 145
Immobilization matrix, 145
Immobilized, 129
Immobilized biocatalyst systems, 145
Immobilized enzyme and cell systems, 118
Impermeable solid, 149
Incomplete oxygen penetration, 160
Increments, 151
Industrial fermenters, 137
Information flow diagram, 33
Inhibition, 73
Inhibitory Substrate, 367
Initial conditions, 103
Initial value, 20
Inlet, 106
Inoculum, 103
Input rate, 102
Integral, 163
Integral control constant, 355
Integral time constant, 166
Integrated, 108, 115
Integration procedure, 20
Integration step length, 20
Integration time interval, 20
Intensity of mixing, 55
Intensity of mixing, 140
Interconnected, 138
Interface, 117, 118, 120
Interfacial concentrations, 122
Internal mass transfer, 145
Internal structure, 119
Interphase, 119
503
Interphase transfer, 26, 125
Intraparticle transfer, 145
Intrinsic reaction rate, 150
Ion charge balance, 47
Ion exchange resins, 149
Kinetic, 106
Kinetic control, 147
Kinetic model, 136
Kinetic rate constant, 147
Kinetic regime, 148
Kinetic relationship, 65
Kinetics control, 149
Kla, 335
Lag phase, 103
Lag time, 170
Laplace transformation, 338
Large bioreactors, 137
Large scale, 137
Length, 115
Length of diffusion path, 151
Limiting, 140, 159
Limiting substrate, 103, 106
Limiting substrate concentration, 78
Linear gradients, 120
Linear growth, 108
Lineweaver-Burk diagram, 72
Liquid, 117
Liquid balance, 125
Liquid balance equation, 128
Liquid film, 337
Liquid film control, 123
Liquid flow terms, 135
Liquid medium, 117
Liquid phase, 117
Liquid recycle stream, 134
Liquid surface, 137
Liquid velocities, 137
Liquid-liquid, 118
Liquid-phase impeller zones, 141
Logistic Equation, 82
Luedeking-Piret model, 85
504
Maintenance coefficient, 10
Maintenance factor, 84
Mammalian Cell Cycle Control, 445
Manipulated variable, 161
Mass balance equation, 16
Mass Transfer, 117, 119
Mass transfer capacity coefficient, 122, 123
Mass transfer coefficients, 121
Mass transfer control, 147
Mass transfer resistance, 145
Material balance equations, 101
Mathematical, 150
Mathematical model, 12, 137
Mathematical modelling, 151
Matrix elements, 151
Maximum, 109
Maximum observed rate, 148
Maximum rates, 158
Maximum reaction rate, 70
Measurement dynamics, 126, 128, 336
Measurement signal, 127
Measurements, 106
Measuring element, 162
Mechanical agitation, 137
Mechanical energy, 137
Medium, 106
Membrane, 112, 127
Membrane filtration, 110
Methanogenic step, 89
Michaelis-Menten constant, 70, 148
Michaelis-Menten kinetics, 148
Microbial interaction, 86
Microbial physiology, 106
Microbiological, 133
Mixing, 113
Mixing zones, 140
Mode of control, 168
Model, 109
Modelling, 113, 388
Molar flow rate of air, 133
Molar reaction rates, 136
Index
Molecular diffusion, 29
Molecular diffusion, 120
Molecular diffusion coefficient, 121
Monod equation, 67
Monod kinetics, 10
Monod-type equation, 390
Monod-type rate expressions, 105
Multiphase reaction, 117
Multiple impeller, 140
Multiple-organism populations, 86
Multiple-substrate Monod kinetics, 82
Multi-stage, 138
Mutual inhibition, 343
Mutualism, 87
Natural logarithmic, 127
Nernst-diffusion film, 146
Nitrate, 300
Nitrate ion, 133
Nitrification, 133, 299
Nitrification reactions, 157
Nitrite, 300
Nitrite ion, 133
Nitrobacter, 134
Nitrogen, 127
Nitrosomonas, 134
Non-competitive, 74
Non-porous carrier, 146
Numerical solution, 19
Objective function, 178
Offset, 163
Oil phase, 118
One-dimensional diffusion, 158
On-line adaptive optimizing control, 178
On-line monitoring, 126
On-Off Control, 163
Open-loop tuning technique, 169
Operation, 110
Order of magnitude analysis, 155
Organism balance, 102
Oscillations, 355
Oscillations of continuous culture, 94
Index
Outlet, 106
Output rate, 102
Overall mass transfer capacity coeff., 123
Overall mass transfer rate equation, 123
Overall order of reaction, 148
Overall rate of reaction, 62
Overall resistance to mass transfer, 123
Oxidation steps, 134
Oxygen, 117, 122, 388
Oxygen balances, 123
Oxygen depletion, 132
Oxygen diffusion effects, 157
Oxygen electrode, 335
Oxygen electrode dynamic, 337
Oxygen gas phase concentrations, 128
Oxygen gradients, 139
Oxygen limitation, 388
Oxygen requirements, 158
Oxygen transfer, 125
Oxygen transfer coefficient, 136, 335
Oxygen transfer rate (OTR), 10
Oxygen uptake rate, 38, 125, 128, 136, 390
OUR, 38
Oxygen-enriched air, 131
Oxygen-sensitive culture, 97
Packed, 149
Parameter, 111
Parameter estimation, 131
Partial differential equation, 116, 153
Partial pressure, 122
Penetration, 149
Penetration distance, 159, 388
Penetration-limiting, 159
Perfect mixing, 137
Perfect plug flow, 137
Performance, 113
pH control, 49
Phase interface, 120
Phases, 117
Physical model, 12, 137
Physical properties, 122
505
Plant cell culture, 128
Plug flow, 113, 141
Poly-6-hydroxybutyric Acid (PHB), 93
Porous, 119
Porous biocatalyst, 119
Porous solid, 145
Power inputs, 137
Predator-Prey Kinetics, 86
Pressure, 137
Process control, 10, 56, 161
Process dynamics, 127
Process optimization, 12, 56
Process reaction curve, 169
Process response, 128
Product, 118
Product inhibition, 113
Product inhibition kinetics, 63
Production rate, 102
Productivity, 105, 110
Programmed, 174
Proportional, 143, 163
Proportional control constant, 355
Proportional-Derivative (PD) Controller, 166
Proportional-Integral controller, 355
Proportional-Integral-Derivative
Controller, 167
Proportlonal-Reset-Rate-Control, 167
Pulse, 113
Quadratic equation, 148
Quasi-homogeneous, 135
Quasi-homogeneous reaction, 158
Quasi-steady state, 107
Radial variations, 140
Rate expressions, 103
Rate of accumulation, 21, 102
Rate of fermentation, 126
Rate of mass transfer, 121
Rate of oxygen transfer, 123
Rate of oxygen uptake, 123
Rate of substrate uptake, 83
Rate of supply, 148
506
Reactants, 117
Reaction, 114, 117, 118
Reaction capability, 149
Reaction control, 133
Reaction Heat, 51
Reaction parameter, 155
Reaction rate constant, 132
Reaction rate control, 133
Reaction site, 118, 145
Reaction surface, 147
Reaction-rate limitation, 136
Reactor, 101, 138
Reactor cascade, 62
Reactor column, 135
Reactor efficiency, 111
Reactor modes, 63
Reactor operating conditions, 159
Reactor outlet, 136
Re-aerated, 126
Recycle loop, 134
Recycle loop configuration, 134
Recycle rates, 134
Recycle ratio, 111
Regimes, 133
Research, 106
Reset time, 166
Residence time, 105, 116
Residence time distribution, 137
Residual error, 167
Resistance to mass transfer, 120, 123
Respiration quotient (RQ), 10
Response, 127
Response curve, 126
Retention, 110, 145
Riser, 139
Sample, 128
Sampled data control, 174
Sampling frequency, 175
Sampling interval, 174
Sand, 134
Saturation, 127
Index
Saturation constant, 10
Scheduled adaptive control, 174
Second-order response lag, 337
Sections, 138
Sedimentation, 110, 111
Semi-Continuous Reactor, 314, 349
Separation, 110
Separator, 111
Series of tank reactors, 62
Set point, 161
Shear, 55
Simulation, 107
Simulation example, 104
Simulation methods, 140
Simulation programming, 153
Simulation programs, 14
Simulation results, 159
Simulation software, 15
Simultaneous diffusion and reaction, 150
Single stage, 138
Single-pass conversion, 135
Slab, 151
Slope, 18, 127
Solid, 118
Solid biocatalyst, 119
Solid carrier, 145
Solid phase, 135
Solid-liquid interfacial area, 135
Solubility, 122
Solution, 143
Spatial variations, 123
Specific area for mass transfer, 121
Specific carbon dioxide production rate, 11
Specific carbon dioxide uptake rate, 84
Specific death rate, 78
Specific growth rate, 10, 78, 105
Specific interfacial area, 122
Specific oxygen, 11
Specific oxygen uptake rate, 84
Specific product production rate, 84
Specific substrate uptake rate, 84
507
Index
Spherical bead, 388

Spherical shell, 389
Stages, 138
Stagewise model, 139
Stagewise modelling, 139, 140
Stagnant, 120
Stagnant film, 145
Stagnant flow, 120
Starting, 109
Startup, 61
Startup period, 61, 62
Steady state, 104
Steady state conditions, 104
Steady state tubular reactor design, 115
Steady state values, 140
Steady-state, 105
Steady-state balances, 21
Steady-state position, 165
Step change, 127
Sterile, 104
Stirring power, 137
Stirring speed, 148
Stirring speed, 149
Stoichiometric coefficients, 40
Stoichiometric oxygen requirements, 159
Stoichiometric relations, 134
Stoichiometry, 40, 126
Structured kinetic model, 11
Substrate balance, 102
Substrate concentration, 101
Substrate gradients, 151
Substrate inhibition, 65, 108
Substrate uptake rate, 55
Sulfite method, 126
Sulfite oxidation, 126
Support, 119
Surface, 118
Surface concentration, 147
Surface reaction, 145
Sustained oscillations, 94
Symmetry, 151
System, 110
Tank, 101
Tank sizes, 113
Tanks-in-series, 112
Teisser Equation, 81
Temperature, 148
Temperature measurement, 354
Theoretical basis, 112
Thiele modulus, 155
Thin film, 120
Time, 109
Time constant for heater, 355
Time constant for measurement, 355
Time constant measurement, 340
Time constants, 128
Time constants for transfer, 340
Time-varying, 114
Titration, 126
Total interfacial area, 121
Total mass balance, 102
Total system, 134
Toxicity, 134
Tracer, 113
Tracer experiment, 140
Tracer techniques, 137
Transfer control, 133, 149
Transfer parameters, 138
Transmission lines, 162
Transport of material, 117
Transport streams, 26
Transport-reaction process, 147
Trial and error method, 169
Tubular, 113
Tubular reactor, 62, 113
Turbine impeller, 134
Turbulence, 120
Turbulent flow, 120
Two position action, 163
Two-Film Theory, 120
Ultimate gain, 169
Ultimate period, 171
508
Uncompetitive, 74
Uptake rate, 111
Variable, 106
Viscosity, 149
Volumetric flow rate, 101
Washout, 105
Wastage, 110
Waste water, 111
Water, 122
Index
Well-mixed, 24, 101, 123
Well-mixed gas phase, 133
Well-mixed liquid zones, 140
Well-mixed tank, 25
Whole cells, 149
Yield coefficient, 10, 32, 102
Zero order, 65, 147, 148
Zero-order kinetics, 391
Ziegler-Nichols Method, 169

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I. J. Dunn, E. Heinzle, J. Ingham, J. E.

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

Irving J. Dunn, Elmar Heinzle, John Ingham, Jifi E. Pf enosil

Dr. Irving J. Dunn

This book was carefully produced. Nevertheless,

First Edition 1992

Library of Congress Card No.: Applied for.

2003 WILE Y-VCH Verlag

PART I PRINCIPLES OF BIOREACTOR MODELLING

NOMENCLATURE FOR PART I

Energy Yield Coefficients

BASIC BIOREACTOR CONCEPTS

DIFFUSION AND BIOLOGICAL REACTION IN IMMOBILIZED

AUTOMATIC BIOPROCESS CONTROL FUNDAMENTALS

ELEMENTS OF FEEDBACK CONTROL

DYNAMIC BIOPROCESS SIMULATION EXAMPLES AND

SIMULATION EXAMPLES OF BIOLOGICAL REACTION

APPENDIX: USING THE BERKELEY MADONNA LANGUAGE.. 483

A SHORT GUIDE TO BERKELEY MADONNA

10 ALPHABETICAL LIST OF EXAMPLES

methods presented here are intended to lead to a greater understanding of how

operating parameters on the bioreactor performance - a real learning

Organization of the Book

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

Nomenclature for Part I

g/m3, 9 air sat.

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

Refers to equilibrium concentration

Refers to difference between cations and ions

Use of Models for Understanding, Design and

An investigation of bioreactor performance might conventionally be carried

General Aspects of the Modelling Approach

An essential stage in the development of any model, is the formulation of the

1.1 Fundamentals of Modelling

Models may be used in training and education. Many important aspects

5. Models may be used for process optimization. Optimization usually

General Modelling Procedure

One of the more important features of modelling is the frequent need to

1.1 Fundamentals of Modelling

Use for design,

Figure 1.1. Information flow diagram for model building.

equations is usually sufficient to prohibit the use of an analytical means of

1.1 Fundamentals of Modelling

1.1.4 Simulation Tools

For effective teaching, the introduction of computer simulation methods into

Development and Meaning of Dynamic

1.2 Development and Meaning of Dynamic Differential Equations

= F Jtf l l dt= F(ti-to)

The solution is,

/The flow rate of mass^

1.2 Development and Meaning of Dynamic Differential Equations

where t - to represents a very small time interval and V - VQ is the resulting

1.3 Formulation of Balance Equations

Formulation of Balance Equations

Types of Mass Balance Equations

Rate of mass flow^

f Rate of mass flow^j

Dynamic Total Mass Balances

Mass flow of >

Component Balances with Reaction

and in the case of material consumed by the reaction:

1.3 Formulation of Balance Equations