Documentos de Académico
Documentos de Profesional
Documentos de Cultura
A. H a r d e r
G i s t - B r o c a d e s R e s e a r c h a n d D e v e l o p m e n t , P.O. B o x 1, 2600 H A Delft, N e t h e r l a n d s
J. A. R o e l s
v a k g r o e p A l g e m e n e e n T e c h n i s c h e Biologic, J u l i a n a l a a n 67, 2628 B C D e l f t ,
Netherlands
t Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 Brief Survey of Microbiological and Biochemical Principles Relevant to the Construction
of Structured Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 Corpuscular Description and its Relation to the Continuum Approach . . . . . . . . . . . . . . . . .
4 Construction of Structured Continuum Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Relaxation Times and their Relevance to the Construction of Structured Models . . . . . . . . .
5.1 The Concept of Relaxation Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 A Model Describing the Dynamics of Product Formation Based on the Relaxation
Time Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 Models of Primary Metabolism in Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1 Two-Compartment Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 A Three-Compartment Model of Biomass Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 Models for the Synthesis of Enzymes Subject to Genetic Control . . . . . . . . . . . . . . . . . . . . . . .
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2 Repressor/Inducer Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3 Catabolite Repression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4 Enzyme Synthesis Mediated by mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5 A Model for Diauxic Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9 Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Mathematical models are an important tool to any engineering discipline. The mathematical treatment
of the processes encountered in bioengineering is complicated by special problems caused by the
complexity of living systems and the segregated nature of microbial life. It is especially this last
mentioned feature which can result in errors if the continuum approach commonly used in
engineering is adopted.
The present paper reviews and updates the theory of the construction of structured continuum
models, which become significant in applications where the common unstructured approach, e.g.
Monod's model, fails. This particularly applies to transient situations in batch, fed batch or continuous
culture.
Emphasize is placed on the need for structured models, which are as simple as possible. A guide
to judging the necessary degree of complexity is provided using the time constant concept, which
is based on judging the time scales on which the various regulatory mechanisms are operative.
The significance of structured models to the description of primary metabolism is described with
special reference to growth energetics.
As a second important range of applications, the dynamics of extracellular and intracellular
enzyme synthesis, is discussed, both from the viewpoint of product formation and diauxy in growth
on mixed substrates.
The need for experimental verification and the potentialities of continuous culture, especially in
transient situations, in that respect are indicated to be the main subjects in which research effort needs
to be invested.
56
A. Harder, J, A. ?~oels
1 Introduction
An important aspect of the methodology of present-day physics is the construction
of mathematical models of aspects of the behavior of a real system. Scientific
progress is made possible by testing the implications of the models, for example by
carefully planned experiments. This generally results in a cyclic process in which the
old model is rejected and a new one postulated (Fig. 1).
It should be emphasized that a model can only represent some of the properties
of a system under consideration. Little would be gained if the behavior of the system
was modelled in all its intricacies, as this would result in a model scarcely more easy
to handle and understand than the real system it represents.
The assessment of which aspects of the system require consideration is essential in
the construction of a workable model and should be guided by the application one
has in mind and the possibility of experimental verification of the model.
A model will hence always be based on assumptions concerning the principles of
the system's behavior. These should be clearly stipulated because their range of
validity will have important consequences for the validity of the conclusions derived
from the model. A more thorough discussion of aspects of the philosophy of modelling
can be found in literature 1-41
TRANSLATIONINTO
A MATHEMATICAL
"']
MODEL
SOLVINGTHE
EQUATIONS
DETERMINATIONOF' ,,,'1
PARAMETERSRNSIVITY
TESTINGTHE MODEL
NO
YES
57
Two broad groups of approaches to the description of systems exist. These are the
continuum approach and the corpuscular method 5). In a corpuscular model, it is
recognized that, probably for all real systems, typical behavior is caused by the concerted actions of objects. The system is inhomogeneous (discontinuous) if length scales
typical of the objects' sizes are considered. For example, the smallest amount of a
chemical substance still having the properties of that substance is a molecule;
likewise, a single bacterium is the smallest quantity having the properties of a bacterial
:species. In view of our present-day image of matter, the corpuscular approach must
be considered the most realistic method of description. Nevertheless, in the engineering
sciences most problems commonly encountered are treated in terms of the aforementioned continuum approach. In a continuum description, the corpuscular nature
of reality is ignored and the system is considered to be continuous in space. Variables
characteristic of this approach are temperature, pressure and concentrations of
substances and organisms. The continuum approach is preferred because these models
are conceptually simpler and can more readily be treated mathematically.
Classical microbiology considers the basic unit of all functioning organisms to be
the cell. Hence, for the description of microbial systems the corpuscular approach
seems to be particularly suitable. This approach has been pursued by Ramkrishna 6)
and by Fredrickson et al. 7~ who apply the term segregated models to this class of
approaches. Despite the fact that organisms are corpuscular in nature, the continuum
approach, earlier termed distributive 4~, is most commonly encountered in the
description of bioengineering systems. It can be shown 3,4.7) that in some instances,
continuum models can be formally derived from a corpuscular description, and both
treatments become equivalent in these cases. In other situations, direct equivalence
cannot be shown and the continuum treatment must be handled with care. A basic
understanding of the corpuscular method is therefore worthwhile and a brief outline
will be given in Sect. 3. After this short excursion into the corpuscular approach, the
continuum approach will be exclusively used.
A second point relevant to models of bioengineering systems is the distinction
between the deterministic and the probablistic approaches 3,4~. The difference between
both approaches rests in the nature of the predictions about the future behavior of
the system that the model allows. In a deterministic approach, the knowledge of the
state vector of the system 3~(a vector composed of all variables necessary to specify the
state of the system at a given moment in time) allows an exact prediction of the future
behavior during an arbitrary time interval. With the probablistic approach, it is only
possible to specify a probability that the state vector will be in a given region of state
space (state space is a coordinate system of the dimensionality of the state vector.
Each point in state space corresponds to a single value of the state vector). The
predictions generally become less and less accurate with increasing time. The
probablistic "behavior" of a system is caused less by the nature of the system than by
the nature of the observer and his observations. A probablistic approach is often
used if the observer is unable to obtain sufficient information about the state of an
object and its subsequent behavior to allow deterministic predictions (for example,
if not all mechanisms are known, or certain important state variables cannot be
measured). Experience shows that the necessity of a probablistic approach increases
if the number of individual objects in the system is low. Thus, the behavior of a large
number of organisms growing in a bioreactor can be adequately described" by a deter-
58
A. Harder, J. A. Roels
ministic model, but the behavior of small numbers of organisms (e.g. in the last stages
of sterilization) calls for a probablistic approach 8~.
In engineering studies deterministic models are almost exclusively used. This
preference is due to the nature of the predictions and the simpler mathematical structure
of these models. Hence, the principles of the probablistic approach will only be
indicated briefly (Sect. 3), attention will be focussed on deterministic models.
A further relevant distinction is the classification into structured and unstructured
models 4~. In unstructured models the state of the organisms in the culture is assumed
to be sufficiently specified by the total number of organisms or the dry weight of
biomass present. However, in a structured model the organism is described in greater
detail, and for example the concentrations of DNA, RNA and protein per unit dry
matter are also specified. Unstructured models are mathematically more tractable and
more easily verified experimentally. Thus, they are therefore to be preferred in all
applications where their accuracy of description of a system is suited to the desired
application. The Monod equation 9) for the substrate limited growth of microorganisms is an example of a successful unstructured model. Originally, it was empirically derived from results on the batch culture of microorganisms. Herbert ~0) introduced a term accounting for endogeneous metabolism, extending it to apply to
growth in the chemostat. More recent work 3.1~) has investigated its application to
fed-batch culture. In general, unstructured models can be considered a good
approximation in two distinct cases. These cases arise when the composition of the
organisms is not relevant to the aspects of the system the model describes, or when it is
independent of time, i.e. in balanced growth 41
Both conditions are fulfilled in chemostat theory, where the outcome of the modelling exercise can be shown to be insensitive to the details of the kinetic assumptions
used. Furthermore, at steady state, the composition of the organisms does not change.
The unstructured approach also assumes composition to be equal at differing dilution
rates, but this is not validated by experimental evidence ~2,~3)
In short, although unstructured models can often be advantageously applied to
the description of a system's behavior there are a large number of applications in
which these models fail to be adequate. This applies when the biomass composition
changes drastically, like in some stages of fed-batch processes and the early stages
of batch growth (lag phenomena), and in situations where a specific constituent (e.g.
protein and RNA content in SCP production) must be modelled. In those cases a
structured approach is necessary. A large number of compositional variables can be
attributed to biomass. If this is performed to the extreme, very complex models
result TM~5). However, these models contain so many parameters that they become
too unwieldy for useful applications in bioengineering. A class of potentially useful
models are formed by a simple extension of the unstructured approach, in which the
amount and the properties of the biomass are specified by two or three variables.
These are the so-called two- or three-compartment models. They combine a better
description of the system's behavior with moderate mathematical complexity and
a sufficiently low number of parameters to permit experimental verification.
Examples of such models are appearing more frequently in the literature. However,
some conceptual difficulties are inherent to the formulation of such models. These,
if not carefully considered, may lead to models which are structurally wrong 16).
The objective of this review is to show some applications of simple structured models
59
6O
A. Harder, J. A. Roels
RNA in d r y m o s s ( % )
40-
(1
30-
20
tx :
10-
o
0
0.5
x
1.0
1,5
Azotobacter chroococcum
Aerobacter aerogenes
o
+
Bacillus megaterlum
Canclida ut]lis
Salmonella
Escher[chio
typhlmurium
Ix ( h -1 )
coli
rates, more RNA is present than is required by the constant efficiency hypothesis ~3)
This unused protein synthesis capacity was shown to be mobilized quickly in transient
states following a sudden increase of the specific growth rate 29)
More drastic changes in the cellular composition are known to follow alterations
in the type of the nutrient supplied. The amounts of the various enzymes produced
by the cell are regulated to meet requirements. The operon model postulated by Jacob
and Monod 3o) explains these phenomena from the existence of controls concerning
the rate of transcription of the codons present on the genetic material. The rate of
transcription of a codon onto messenger g N A is controlled by regulatory genes. The
cell produces a repressor protein which, in the active form, binds to the operator and
blocks transcription. An effector, often derived from the substrate of the enzymic
sequence the operon codes for, interacts allosterically with the repressor protein,
either binding to or releasing the operator, depending on whether the effector is an
anti-inducer or an inducer, respectively. Thus, this mechanism allows the organism to
change its enzymic constitution to suit the demands posed by nutritional changes in the
environment.
In recent years 3~), it has been recognized that a second important control of the
transcription of codons exists. Efficient transcription to m-RNA is postulated to
only take place if a complex of c-AMP (cyclic AMP) and CAP (catabolite activator
protein) is bound to a promotor gene on the DNA. Certain catabotites, such as
glucose, apparently reduce the c-AMP concentration and inhibit the expression of
the codon (positive control, catabolite repression).
The genetic control mechanisms mentioned are relevant to the description of lag
phase phenomena, diauxy and product formation (intracellular and extracellular
enzymes). The time constants of these mechanisms are larger than those mentioned
under b).
61
(1)
62
A. Harder, J. A. Roels
Equation (1) shows the relationship between the number of organisms, dN(o~), having
a state vector in the state space volume element, 1~ dei, the number or organisms per
i
(2)
The first moment is the average value of o for all organisms present in the
culture.
Another important quantity is the second moment, (co2), of the probability-density
function:
( 62) = ~oflqJ(c) d o
(3)
o 2 =
J" ( o -
(o)) 2 ~(o) do
C41
<o~> 2
(5)
Now a function of the property o, fro), is considered, its average value for all
objects in the culture is given by:
(f(o)) = ~ f(o) q~(o) &0
(61
To illustrate the application of Eq. (6) the following example is considered: A culture
or organisms performs an enzymatic reaction due to the action of an enzyme E. The
amount of enzyme per organism is e. The probability-density function for e is W(e).
The number of organisms is assumed to be sufficiently large and a Michaelis-Menten
type Eq. (37) is assumed to apply to each cell. Then the rate of enzymatic reaction
per cell, RE, can be written as:
RE _
keC~
K M + C~
(7~
63
The average rate of reaction per cell for all organisms in the culture is given by"
f
(RE) =
keC~
K M- + C~
W(e) d e
(8)
(e)
(9)
K M 4- C s
The overall rate of reaction, rE, for all organisms in the culture is obtained if the righthand side of Eq. (9) is multiplied by N t
kC,
rE -- KM + C~ N,(e)
(10)
The product Nt(e) is the amount of the enzyme per unit volume of the culture; it
hence is a continuum variable which will be indicated by C E. Thus, Eq. (10) can be
written as:
kCs
rE -- K M+ C~-~CE
( 11 )
(12)
(R) =
co
(13)
For the general case, the integral at the right-hand side of Eq. (13) cannot be
simplified further. Straightforward evaluation is possible if the following conditions
hold:
64
A. Harder, J. A. Roels
a) R(to, y) can be factored out with respect to the individual elements of the
organism's state vector:
R(o, y) = k~T(y) I ] O)i
(14)
y(~o) = H %()
(15)
If Eqs. (15) and (14) are combined with Eq. (13), also considering Eq. (2) for the
restrictive case to which both conditions mentioned under a and b apply, it follows that
( R ) = k~T(y) 1-[ (ml)
(16)
in which the (toi) are the average values of each of the individual properties of
the cells.
Using Eq. (16), the total rate of reaction in the culture is now given by:
rE = k~T(y) 1~ (%) N,
(17)
If one cell has a mass W, and the mass fractions of the various compounds in the cell
are given by %, it follows:
r E = k~T(y) l~ wiW . - t C X
(18)
In Eq. (18), n is the dimensionality of the state vector to and C Xis the concentration
of biomass dry matter.
Equation (18) shows that in the correct approach to structured continuum models,
the extracellular and intracellular concentrations should be treated differently. Special
precautions are not necessary for the a-biotic concentrations (vector y); they can be
expressed as concentrations per unit of culture volume. The biotic concentrations
(i.e. the concentrations of cellular compounds) are, however, best expressed as mass
fractions of the cellular mass, the so-called intrinsic concentrations 16) Finally, the
rate equation (18) is shown to be first order with respect to the total biomass
concentration, Cx, a feature, which is intuitively correct 3)
It is also possible to construct a correct rate equation using biotic concentrations
expressed per unit of culture volume, when the general form of the rate equation
becomes:
r E = kCT(y) [ I xiW ,-1 C~1-,
xi in Eq. (19) is the biotic concentration of compound i expressed per unit volume.
(19)
65
(20)
The structure of this equation is based on the mass-action law rate equations
fundamental to most approaches to chemical kinetics 3s~. It represents, however, an
incorrect approach to bioengineering kinetics when reactions between cellular
constituents are also considered. This is obvious from a comparison of Eqs. (20)
and (19). This difficulty was first pointed out by Fredrickson ~6~ who dealt with
examples of such errors in the literature 39,40). These errors have however also
appeared in the recent literature 4 ~
The problems resulting from the use of equations similar to Eq. (20) were
illustrated by Roels and Kossen 3~ by referring to the model of Williams 39)
There is another problem associated with the use of the continuum approach
which must be discussed. The averaging process according to Eq. (13) only leads to
meaningful deterministic values if the number of objects considered is sufficiently
large. In general, the order of magnitude of the variance of a sample of N objects is
equal to the ensemble variance divided by N. In view of this, if the number of organisms
considered becomes less then 102--10`*3), the deterministic continuum approach
should be handled with caution.
An important problem involves the application of mass-action law considerations
at the level of the bacterial cell where, in many cases, there are only a few molecules per
individual cell. Examples of such problems have been discussed for Michaelis-Menten
kinetics 4.2) and for the operon model 43~ These exercises clearly show that in such
cases, a mass action law approach to kinetics may lead to errors. In the present
review, however, these problems will be ignored.
(21)
The a-biotic state vector, y, contains the concentrations of k compounds which are
not part of the intact biomass. The biotic state vector, x, contains the concentrations
of n compounds which are part of the biomass.
Components present in both the biotic and a-biotic phases are identified by distinct
numbers in both state vectors. The compounds specified by the state vector x, are
assumed to account for all biomass dry matter which, however, does not necessarily
imply the specification of the concentration of each component of the biomass
66
A. Harder, J. A. Roels
separately. The elements of x may also refer to groups of compounds. Under this
condition, the following relationship holds:
C~ = L xi
(22)
i=l
(23)
(24)
In this equation r A is the vector of the net rates of the production of each compound
in the reaction pattern in the system. @ is the vector of the rates of transport of these
compounds to the system. The reaction pattern inside the system is now characterized
by the vector r of the m independent reactions taking place in the system ,s). The net
rate of formation of each compound is now given by:
r A = rot
(25)
Equation (25) defines the stoichiometry matrix, at, an m x p matrix (p is the dimensionality of vector C). In this matrix, the element Qtij gives the amount of compound j
produced in the i-th reaction.
Expressions analogous to Eqs. (24) and (25) may now be formulated for the rates
of change of the a-biotic and biotic state vectors:
= r % + @y
(26)
= r atx + @x
(27)
67
In Eqs. (26) and (27), ay and ~ are the stoichiometric matrices for a-biotic and
biotic compounds; ~y and O~ are the vectors of rates of transport for a-biotic and
biotic compounds.
Equation (26) can be used to describe the dynamics of the a-biotic state vector.
The balance equation for the biotic state vector poses special problems.
Firstly, an equation for ~ must be formulated. As the state vector x refers to intact
cells, transport of compounds to or from the system can only take place as intact cells.
This excludes the possibility of removal or addition of cells of a composition other
than the population mean. Thus, the following equation holds:
O. = q~w
(28)
(29)
(30)
i=l
i= 1
(32)
If Eq. (32) is substituted into Eq. (30), the following equation for the dynamics of w
is obtained after rearrangement:
= (r=~ -- wrO/C,
(33)
68
A. Harder, J. A. Roels
Equations (26), (32) and (33), together with a set of constitutive equations for the
rates of reaction r and constitutive equations for ~x and ~y, form a complete structured
continuum model in which the biotic compounds are treated in terms of intrinsic
concentrations. Equation (33) shows that in the state equation for the intrinsic biotic
state vector w a term --wrx appears. This accounts for the dilution of the biotic
compounds by the increase in the total amount of biomass. Omission of this term in
the formulation of an equation for the biotic state vector dynamics is another
important source of errors in structured continuum models (see article of Fredricks o n 16)).
The approach to structured continuum models developed in this section will be
applied to some examples in the following sections.
69
The time constant concept provides a direct route to the choice of the degree of
complexity required for the description of the behavior of a system. In principle, the
behavior of a culture of organisms is described by a vast number of relaxation
times resulting from, amongst others, the various regulatory mechanisms discussed in
Sect. 2. These mechanisms generally have largely different relaxation times, a highly
speculative picture of which is given in Fig. 3. A description of the system can
be simplified by basing an approach on a comparison of the relaxation times of
the internal processes and those characterizing the relevant changes in external conditions.
If the changes in environmental conditions are slow compared with the rate of
adaptation of a given mechanism, i.e. if the relaxation time of the latter is much smaller,
the dynamics of that mechanism may be ignored. In the case mentioned, the organism
will be at steady state compared to that mechanism and external variables suffice to
describe the state of the organism. An additional relaxation time associated with
the dynamics of adaptation of the given mechanism is not needed. The model can be
simplified by the so-called pseudo-steady-state hypothesis with respect to the mechanism under consideration. A totally different situation occurs when the relaxation
times of the changes in the environment are small with respect to those of the
cell's adaptational mechanism, i.e. if the internal state adjusts very slowly. The
mechanism can then be totally ignored and the state of the organism with respect
to that mechanism will be characterized by the initial state throughout the process.
The description of the behavior of the system can now be simplified by deleting that
mechanism. In order to clarify the nature of both types of simplification vital to the
construction of workable models, some examples will now be dealt with.
a) In the kinetic description of enzymatic reactions, the Michealis-Menten equation
is often used 3 7 ) :
kWEfs
rs = - C~
K M-k C s
(34)
The state of the organism is described by the mass fraction of the enzyme in the
biomass, w E.
I
~ss
ACTION
LAW
101
Io
102 103
104
105
10 6
10
,I,
ALLOSTERI C CONTROLS
RELAXATION
(SECONDS)
,I
CHANGES IN ENZYMIC
CONCENTRATIONS
m-RNA
CONTROL
SELECTION WITHIN A
POPULATION OF ONE OR
MORE SPECIES
EVOLUTIONARy
CHANGES
Fig. 3. Various internal mechanisms and order of magnitude of their relaxation times
TIME
70
A. Harder, J. A. Rods
The derivation of Eq. (34) is based on the following kinetic scheme:
E+S~ES~E+P
(35)
The enzyme is assumed to associate with the substrate to form an intermediate, ES;
this intermediate subsequently dissociates to yield free enzyme and the product. A
detailed solution of the dynamics according to Eq. 35 would require a description in
terms of w E and WEs, the mass fraction of enzyme and enzyme-substrate complex.
Equation (34) is, however, obtained if the relaxation time of the adjustment of the ES
concentration is very small, compared with the other time constants 53, 54)
b) A general approach to the bioenergetics of microbial growth has recently been
developed. This is based upon the pseudo-steady state hypothesis with respect to the
energy metabolism intermediates, ATP and NADH. These have very small time
constants for the adaptation of their concentrations 55, 56).
c) The steady-state behavior of a continuous culture can be adequately described by
the unstructured Monod model lO~.When a continuous culture reaches a steady state,
the relaxation times of the changes in environmental conditions have become
"infinite" and the pseudo-steady-state hypothesis is justified with respect to all
adaptational mechanisms. It may, however, take a long time (approximately 3 times
the largest relaxation time) for all processes to reach their steady-state values. The
phenomenon of selection in continuous culture is an example. This may cause changes
in the steady state of a continuous culture on a time scale which is large compared
with that of other mechanisms. This is a known problem in continuous culture 32~as
well as an effective tool in the selection of organisms with desirable properties from
a mixed culture of organisms 57, 58~. The Monod equation cannot be as successfully
applied to the description of the transient behavior of pure and mixed cultures 49, s9,6o~
Alter a transient shift, the relaxation times for the changes in experimental conditions
are smaller. The pseudo-steady-state hypothesis is then valid with respect to a more
restricted class of internal processes, i.e. those having a relaxation time smaller by a
factor 3 than that of the smallest environmental relaxation time.
Although the application of the relaxation time concept to the simplification of
the description of a system could be further discussed, we will, however, limit
ourselves to indicating its application in a number of examples to be treated in the
next section.
As already pointed out, the transfer function approach, roughly an analogue of the
treatment in terms of relaxation times, has been advocated for the application to
bioengineering systems 49-5~. It is our opinion that such an approach provides a
valuable tool in the identification of the number and the order of magnitude of the
relaxation times necessary for a n adequate description of a system. It should,
however, be borne in mind that the transfer function approach basically only
applies to linear systems. In other words, it only holds in the region around a given
initial state where the system can be sufficiently well described by a linearized set of
differential equations. This severely limits any application to bioengineering where the~
systems are strongly non-linear. Although the same holds, in principle, for the time
constant concept, it may be more easily understood in terms of mechanisms and more
readily adapted to provide a realistic depiction of bioengineering systems. An attempt
to show this will be undertaken in Sect. 5.2. Both approaches are basically "black
71
rs - Ks +
Cx
(36)
(37)
72
A. Harder, J. A. Roels
In Eq. (37), Q is the product formation activity function. The rational behind
Eq. (37) is the assumption that part of the energy flux through the organism is
directed toward product formation, the fraction of the total flux being determined
by the activity function Q.
It is assumed that the fraction of energy directed to product formation remains
small, compared with the total rate of substrate uptake. (This assumption is easily
avoided, but it results in less complicated equations which adequately represent the
general features of the more complex case). The specific growth rate, p, can, for this
case, be calculated from the Herbert/Pirt 10, 70~ equation:
tl = Y~x qK~
. . .+. . C~
C~
m~Y~x
(38)
(39)
Equation (39) provides a relationship between the specific rate of product formation,
%, the activity function, Q, and the specific growth rate la.
In the equation developed above, it is assumed that the substrate is only used as an
energy source. The carbon requirement for growth is assumed to come from
pre-supplied monomers. Again, this assumption can be easily avoided by involving
slightly more complex mathematics. An interesting direct application of Eq. (39) is
obtained if the product formation is directly related to energy generation (for example,
in the anaerobic formation of alcohol, lactic acid, etc.). In this case Q is a constant
which is directly obtained from stoichiometric considerations, and the familiar Luedeking-Piret 63) equation results from a rearrangement of Eq. 39:
%=
Q la+Qm~
~-~
(40)
In this simple case, Eq. (40) suffices to describe continuous cultures as well as fedbatch or batch cultures, provided that relaxation times of primary metabolism adaption are small compared with those of the changes in external conditions. However,
there are cases in which Q is regulated in response to environmental changes in a
manner not directly related to the specific growth rate. In these instances, a definite
relationship between Q and Ix in a steady state can still be assumed to exist:
Q* = f(~t)
(41)
where Q* is the value of Q in a steady state, for example after a sufficiently long period
of continuous culture growth. It is assumed to be an arbitrary function of p, f(p).
From Eqs. (41) and (39) it follows:
Q~
Y~q*
p + msY~x
(42)
73
where q* is the steady-state value of the specific rate of product formation at a specific
growth rate g. Eqs. (42) and (41) allow the determination of Q*(g) from continuous
culture experiments. In order to extend the theory to dynamical situations an equation
is needed for the rate of adaptation of Q to changes in environmental conditions. Such
an equation can be obtained using the following reasoning:
a) The activity function is assumed to be equal or proportional to an identifiable
substance in the cell, i.e. the dynamics of Q can be described by the intrinsic
balance equation derived in Sect. 4:
1
O = ~
(rQ - r,Q)
(43)
(44)
b) When not in a steady state, control mechanisms, which adapt the specific rate of
Q synthesis, are assumed to operate. The difference between the actual rate of Q synthesis and the steady-state rate is assumed to depend on the difference between Q and the
value of Q* consistent with a steady-state at the environmental conditions existing
at the moment considered:
qQ = q~ + g ( Q - Q*)
(45)
Now the function g(Q - Q*) is approximated by a Taylor series expansion 71)
around Q*, truncated as a first approximation after the second term:
g(Q - Q*) = g(O) +
(Q - Q*)
(46)
O =Q*
From the definition of the function g(Q - Q*) it is clear that g(O) equals zero.
Furthermore, from the condition that the steady state must be stable it follows:
Q=Q, =
(48)
(49)
74
A. Harder, J, A. Roels
When introducing constant K, the further assumption that the first derivative of g
with respect to Q, evaluated at Q = Q*, does not depend on Q* is made.
Combining Eqs. (43), (44) and (48) for the rate of the change of Q it results:
()=--(K
(50)
+ p)(Q-Q*)
Equation (50) can now be applied, for example, to a shift in continuous culture,
showing the relaxation time for the adaptation to a new steady state to be equal to
1/(p -t- K). If K is large, a new steady state will be reached almost instantaneously.
Then, the organism will not show any lag in its adaptation to a new steady state.
Alternatively, if K is small, the time constant for adaptation will be equal to l/p, i.e.
dilution through growth will control the adaptational process.
The model presented above has been numerically simulated for a situation where
decreases exponentially. The organism was considered to be fully adapted to the initial
growth rate. The value of K was chosen to vary between 10 -3 times and 100 times
the specific growth rate decay constant. The steady-state relationship for Q* and la
was assumed to be:
Q* = 0.5p
(51)
c 0.5
0
0.4
~~
y-state
relationship
/ /
0.2
t)
~,
o.1
change of J~
I
75
76
A. H a r d e r , J. A. R o e l s
=o
E.
~o
-=
..
.= ._ = "=
=.-=
W.
~_~ ~
~.
=-0
Z~.E
..0
-=
.u
-= =~Z 8
._=
~ o
=
<
Z
<
Z
<
Z
<.~S
=='~=
Zo
"~
~<8~
~D
~g
= ~ ~
"t=
0
.>_,
0
<
'8
,=
a
0
77
..~
~-~
o~
~ ~ o~
~ ~'~,,
o
~~
~
~
.-o
"'~
o.~
~ .
-" ~
~ r.. o~ ~
~= ~ o
~Z
~ . ~
. , ~
~o~
< , ~
I=
e,I) I~
"0
~s:~
"~
~o
[..
78
A. Harder, J. A. Roels
essentially based on that of Williams 39,721, and on its modification and extension by
Roels and Kossen 3), Roels s4) and Harder 44).
A culture in which an organism is growing on one single source of carbon and
energy is considered. The organism is assumed to consist of two compartments
(Fig. 5). The G compartment is thought to contain the enzymes which convert the
substrate to the various building blocks for the macromolecules of the cell. The
remainder of the biomass, the K compartment, is storage material, genetic material,
RNA and the pools of the various precursors. The G compartment is assumed to be
synthetized from the K compartment under the catalytic action of the K compartment.
The latter compartment is thought to be synthetized from substrate under the
catalytic action of the G compartment. This is a great simplification of the
complexity of the cellular processes, but is significantly closer to reality than the
unstructured approach.
For a description of the state of the culture as a function of time, the approach
advocated in Sect. 4 can be used without problems when the kinetics and the
stoichiometry of the processes involved are defined:
a) The conversion of the substrate to the K compartment. The rate will be defined by
rsK. The stoichiometry is defined by the yield factor Y~K, the amount of K compartment material synthetized per unit of substrate used. Bearing in mind that this synthesis
occurs from the substrate under the catalytic action of the G compartment, the
following generalized relationship is proposed:
rsK = fl(fs) f2(WG)C x
(52)
qs, maxCs
WG
- K~+C~ Ko+w~
C~
(53)
rSK
K-compart~
rKG.~compartme,nt
L ...............................................
79
Eq. (53) assumes a Monod-type relationship for the dependence ofrsK on the substrate
concentration, C~, and the weight fraction of the G compartment in the biomass.
b) The transformation of the K compartment into the G compartment.
The rate is defined as rKo and the stoichiometry by the yield constant Y~o, i.e. the
amount of G compartment produced per unit K compartment consumed. The
following relationship is proposed for the rate of transformation of the K to the G
compartment:
rKo = f3(w~) Cx
(54)
Equation (54) expresses the assumption that the transformation of the K to the G
compartment is governed only by composition of the biomass (f.e. wo) and is not a
direct function of the substrate concentration.
c) Turnover of the compartments of the biomass.
In the present example, the G compartment will be assumed to be subject to turnover.
The turnover process is assumed to be modelled by
roK = mGw~C x
(55)
(56)
80
A. Harder, J. A. Roels
Table 2, State equations of the two-compartment model. The following Equations are obtained by
substitution of the rates and flows into Eqs. (26), (32) and (33)
dEs
dt
qs, maxCs
WG
K, + C~ Kc + wG
C,+@~
2.1
--
dC~
qs, m~C~
wG
= YsK
Cx + (Y~G -- 1) f3(WG) C x + qbx
dt
K~+C~ K o + w G
2.2
dWG
2.3
WG
- - = --YsK q~" maxfs
wG + f~(wG){wo + Ylca(1 - wa)} - mGw~
dt
K~+C~ K o + w a
The Equations contain the transport contributions <p~and q~, which depend on the mode of operation
and are given by:
batch
~ = 0,
~ = 0
continuous culture with feed-back of
fraction (1 -- wo) of the biomass
~ = D(Csi - - C s ) ,
~x = -- woDC,
leaving the system
fed batch
~ = F(t),
qb~ = 0
state implies the existence o f a quadratic relationship between the rate o f synthesis
o f the c o m p o u n d and the dilution rate. This is a direct consequence o f the dilution o f
aft intracellular c o m p o u n d due to growth ~6}. This is applicable to the development
o f an equation for fa(wG). The following balance equation can be formulated:
(waC:x) = (qno - - mawo) Cx + ~xwaCx
(57)
(58)
F o r a steady state, wo and (~, equal zero and ~x/Cx equals - - I t ; hence, it follows:
q*a = (It + m G) wB
(59)
q~o=~(wo)
( m G - wSo
w
136j
(60)
81
Now the function f3(w6) introduced in Eq. (54) is identified for a steady state as
being proportional to q*c.
The extrapolation which is now made is based on an assumption implicit in each
of the two compartment models mentioned earlier. It is assumed that the fraction
of the biomass composed of G or K compartments is sufficient to specify the
activity of the biomass, i.e. the two values C x and w G provide sufficient information
to rigorously define the amount and activity of the biomass. This clarifies the
relationship with the unstructured approach. In an unstructured model, the biomass
concentration alone is considered sufficient to specify the activities of the biomass.
The next alternative, a two-compartment model, specifies one compositional variable.
Under the assumptions presented above, Eq. (60) can be generalized to hold even
if the system is not in a steady state:
qKc = YKGh(w~) = ~
1 (wc) 2 + ( m ~ -
-~-c] wc
(61)
The reasoning presented above has resulted in a model of the two compartment
type having minimal complexity. Although highly simplified, it may provide a useful
alternative to unstructured models in situations where these models fail.
The procedure outlined in Sect. 5.2 could also have been applied, resulting in a
different approach in which the adaptation rate is also assumed to be directly
influenced by environmental conditions. This is a more complex, but more flexible
approach.
The necessity for a model of such simplicity, while sufficient knowledge is available
for the construction of a model of much greater realism may not be obvious.
Two factors should be considered:
a) A number of regulatory mechanisms at the level of energy generation and
consumption operate with such small relaxation times that a pseudo-steady-state
hypothesis with respect to these mechanisms is justified. Hence, the introduction of
these details seems to be unnecessary.
b) A minimum of complexity is desirable because a complex model often proves
very difficult to verify and may fit experimental results without having any
relationship with the behavior of the organism a). Only after obtaining experimental
evidence that the simple model should be rejected because of unsufficient fit of the data
or unrealistic parameter values, should additional complexity be introduced. An
additional "hidden" variable must be specified. Careful study however, of the
biochemistry of RNA and protein synthesis 13,81-84), may result in model structures
of greater realism and a complexity similar to that treated in this section.
To give an impression of the typical features of the model presented above, an
analysis of the continuous culture growth using the two-compartment model
presented above was performed. The model exhibits all features of the classical
chemostat theory 26) but also allows for the description of alterations in biomass
composition with growth rate changes (Fig. 6). For a steady-state culture, the
advantages of the two-compartment model over the unstructured approach are not
readily apparent. The differences become clearer in transient situations such as
wash-out from continuous culture. Figure 7 shows the results of a simulation of
wash-out for cells pre-grown at two different dilution rates. The present model
82
A. Harder, J. A. Rods
c:
201
20
10
10
'
- 0
Wk
qSK
1.5
1.0
0.5
0.5
I
I_--
I--
0.5
1.0
1.5 0
0.5
1.0
1.5
Dilution rate D (hq}
Dilution rate D (h-1)
reveals that the cells pre-grown at the lowest growth rate exhibit a more rapid
wash-out.
Although the model is primarily designed to apply to pure cultures, its application
to mixed cultures certainly merits investigation. As was pointed out in Sect. 2 (see
Fig. 2), there exists a tendency for organisms of different origin to have the same
steady-state R N A content at the same specific growth rate. Hence, a compartmental
approach describing a mixed culture in terms of average RNA content could be an
interesting approach to the study of the dynamics of mixed cultures 44).
The two-compartment model exhibits many features observed in batch and
continuous culture experiments..The method is certainly promising as an approach
to the modelling of microbial growth in situations where the relaxation times of the
changes in environmental conditions are of the order of magnitude of the relaxation
time of one of the internal adaptational mechanisms, e.g. in batch or fed batch growth
or during transients in continuous culture. The particular model presented, however,
must be considered as a preliminary proposal because many of the kinetic
assumptions do not rest on solid biochemical facts about the internal regulation of
the ccU. Furthermore, there are difficulties in identifying the compositional nature
of the K and G compartments in terms of structural compounds of the cell. It is
clear that a more thorough study of known regulation phenomena and an empirical
study of transient situations, for example in continuous culture, is needed. In this
respect, the chemostat is a valuable research tool. It is gratifying to note that the study
83
1.0'
0.5
0
Time
of shift
Fig. 7. Simulation of the wash-out curves for biomass from continuous culture. Plot of the biomass
concentration relative to the initial biomass concentration, Cx/C~0 against time for a shift of the
dilution rate from 0.1 to 1.5 (solid line) and a shift
from a dilution rate of 0.9 to 1.5 (dotted line)
t
2
3
l.
Time efter shift (h)
84
A. Harder, J. A. Roeis
TURN OVER
TURN OVER
,I
r ~ = .-7- rR
(62)
where r R is the rate of the R compartment synthesis, Y*R the yield factor for the R
compartment with respect to the substrate (kg R compartment per kg substrate)
and rsR the rate of substrate consumption for the R compartment synthesis. For the K
and G compartments, the situation is more complicated. It is best described by the
assumption that pools of precursors for both compartments are contained in the R
compartment. These pools are supplied by the transformation of substrate to K and
G compartment precursors and by depolymerization of macromolecules to their
precursors. Precursors are drawn from the pools for the synthesis of K and G
compartment. If a pseudo-steady-state hypothesis is applied to the pools (i.e. if the
relaxation times for their adaptation are small), the rates of substrate consumption
due to K and G compartment synthesis are given by:
1
-
rsK-
- -
YsK
rK
r~G = . 7 - r C
YsG
(63)
(64)
85
where rsK and rso are the rates of substrate consumption due to the synthesis of the
compartments K and G; YsK and Yso are their yield factors and r~: and r o the net
rates of synthesis of K and G compartments. The latter rates are obtained from the
total rates (rK)t and (ro) t through correction for turnover:
r K = (rK)t -- mKwKCx
(65)
r o = (ro), -- mowoC ~
(66)
YATP
rR
(67)
(68)
(69)
In these equations, the YATp-Values are the ATP-yields (kg per mol ATP) for the
various compartments. As can be seen, a turnover contribution appears in the rates
of ATP consumption for the synthesis of K and G compartments. Finally, the
contributions in Eqs. (67)--(69) are converted into substrate consumption by the
introduction of the stoichiometry constant czc. This is the amount of ATP (moles)
produced per kg of substrate catabolized. The total rate of substrate consumption
for energy generation is as follows:
r s = - -
0~
P, R
rR + y---~Tp, K rK + y~xp , G rG +
1
1
mGWGCxI
YATP,K mKWKCx+ TAre, G
(70)
86
A. Harder, J. A. Roels
Table 3. Equations for the rate ofsubstrate and ATP consumption on the basis of a three-compartment
approach
Rate of substrate consumption:
I + - -
~ ~ wry-,,
LY,T,,~
Cx
t'
I +~cYATe.R
-Ec
mGWGt
'tl
<,
3.1
YATP,R -- [)
'.YATP.K
FATP= ~
WG( YATP.R
\YATP.G
mGWG)
f mgw g
+C~ - - +
~YATP,K
3.2
~ /
extension of the equations proposed by Herbert 1o) and Pirt 7o) and Stouthamer and
Bettenhausen 93), respectively, for the unstructured approach:
1
r~ = .-z7-- r~ + msC~
1
_ _
+ mArpCx
rATe = (YArP)max
(71)
(72)
87
Y~a
88.9*
222**
0.9
Y~K
0.78
YsG
YATP.a
YArV.K
YATP,O
mK
mo
0.78
81.0x 10 -3
26.8 10 -3
25.6 x 10 - s
0.03
0.03
* P/O = 1
** P/O = 3
Table 5. Calculated values o f Y~, m,, (YATP)mx and mATP for microorganisms o f various compositional
characteristics
Organism
Y~x
(YAlrp)max
P/O
1
P/O
3
0.59
0.69
0.59
x103
m s x 103
mA.rp
P/O
1
P/O
3
26.5
13
!.I
0.69
26.7
12
1.1
0.68
0.78
43.4
0.4
0.65
0.74
35.0
0.7
0.63
0.73
32.0
0.8
Aerobacter aerogenes
(w G = 0.67, w t = 0.31)
Aerobacter aerogenes
(w G = 0.80, w K = 0.16)
Candida utilis
(w G = 0.31, w t = 0A0)
Activated sludge
(w G = 0.49, w K = 0.13)
Activated sludge
(w o = 0.62, w K = 0.10)
carbohydrate content of the organism (e.g. yeast). The maintenance factors reveal
the reverse tendency, being lower for a high carbohydrate content. This phenomenon
may also partly account for the abnormally low maintenance coefficients reported for
activated sludge 44,98~ and axenic cultures 9s). The systematic increase of the
fraction of storage carbohydrate with decreasing growth rate will result in a low
estimate of the maintenance factor if determined by the conventional double
reciprocal plot of yield factor against growth rate.
In the publication of Harder 4,,j, a modified version of the model structure
presented above is used. The uptake of substrate is assumed to take place by
conversion to the R compartment which is subsequently converted to K and G
compartments. Furthermore, the technique described in Sect. 6.1 was used to
model the rates of synthesis of K and G compartments, i.e. the continuous culture
steady-state relationships for the rates of synthesis of these compartments, which follow
directly from the steady-state mass fractions, are assumed to be adequate even when
the organism is not in a steady state.
Furthermore, Harder argues that Monod's equation is unfit to describe the uptake
of substrate by a mixed population, a view which is supported by some authors 99-101)
He proposes a n-th order power law equation for the substrate consumption. Harder
shows that the model fits the results of his continuous culture experiments and is in
fair agreement with some preliminary transient experiments although no attempts
88
A. Harder, J. A. Roels
have been made to adjust the constants of the model such that they optimally fit
the experimental curves. Although the validity of the various assumptions and the
kinetic equations to be used are still uncertain, the method presented appears to be
of future value as an alternative to unstructured models.
89
//
GLUCOSE
/-'71 TlllOGALACTOSIDE
TRANSACETYLASE
/---41 LACTOSE PEP.KEASE
c~roPL~xxc
XE~RANE
GALACTOSIDASE
ADEI~LAT~
CYCLASE~
POSITIVE
-
m I~A
//
REI~ESSOR
+ INDUCER
CHRO~,OSC~t,
DNA
Fig. 9. Schematicrepresentationof the lac operon; its negative and positivecontrol units
is also the base of the operon models of several authors 34.,l l l -114,121), the interaction
between the cytoplasmic repressor (R), the operator (O) and an effector (E) can
be formulated in terms of chemical equilibria.
The ligand-repressor-operator interaction can be described by the following reaction
scheme 115)~
O+R
+
K2
.-~OR
+
nE
nE
":'41"
K41l'~
0 + R E . ~ ORE.
The number of binding sites for the effector on the repressor is given by n. If the
effector (E) is an inducer, the RE, complex results and if it is an anti-inducer, the
ORE. complex is formed.
The equilibrium constants K,, K2, K3, and K 4 are given by
w~E,
K I - w*t..,*~"
R~VVE!
(73)
90
A . H a r d e r , J. A . R o e l s
K2 = - -
(74)
WoW R
W *ORE n
K3
w* *w**"
OR 't,
(75)
El
W *OREn
K4
(76)
,---~WoWREn
(%%=w~+w*RE n
+w%+w*
ORE n
(77)
(w~), = w~ + w * + w*ORE n
(78)
(79)
* t, (Wo)
* t and (W *E)t are the total numbers of moles of R,
In the balance equations, (w~)
O and E, respectively, in the pseudo-steady state. In wild-type E. coli cells, there
are 10-20 times more repressor molecules than there are operators los~. In this case,
w*R and w*ORE n can be neglected in Eq. (77). A more complicated theory is obtained
91
Equation (79) can be simplified by the assumption that the intracellular concentration of effector molecules is in sufficient excess over repressor molecules so
that w*REn + w*
OREn <~ w* and the concentration of non-bound effector is in good
approximation equal to the total effector concentration in the biomass. The total
concentration of intracellular effector molecules is often assumed to be proportional
to the medium effector concentration, C~ al0,m) Also proportionality with respect
to the rate of uptake of the substrate of the inducible enzyme has been postulated 119).
Now an equation can be derived for the fraction of operators which is repressorfree. In fact, this reasoning is analogous to the concept of "the probability of
binding" 12o). By a combination of Eqs. (73), (74), (77--79), the fraction Q1, for the
case of E being an inducer, is given by:
wg
I + K,(w~)"
Ql = - =
(w~)t
1 + Kl(W*)n + K2(w~) t
(80)
1 + K~(w*)"
Q2 =
1 + K,(w~)" + KiK3(w~)"(w~),
(81)
92
A. Harder, J. A. Roels
It has been shown that the CAP-cyclic AMP complex stimulates the fl-galactosidase
synthesis by binding at the promoter site (P) of the lac-operon and initiating the
transcription by RNA potymerase (see Fig. 9)138,139) This antagonistic effect of
catabolite repression by cyclic AMP ('positive control') is thought to be a general
regulatory mechanism in bacteria. However, also catabolite repression in E. coli
mutants, lacking cyclic AMP has recently been observed, suggesting that cyclic AMP
cannot be the unique regulator 140,141} The catabolite modulator factor (CMF),
possibly acting via the CAP protein, might be the regulating effector in these
mutants. Despite this biochemical and genetic information, the mechanism of positive
control by a CAP-cyclic AMP complex has only recently been modelled a4,113,114,t,'2)
Van Dedem and Moo-Young 1t9) modelled catabolite repression by formulating an
equation for the inhibiting effect of the intracellular ATP level on the synthesis of an
inducible permease for the transport of the secondary carbon source. Toda 112)
assumed two different operator genes one of which is repressed by a complex of an
assumed apo-repressor and a co-repressor (e.g. a metabolite of glucose) and the other
operator is repressed by a second molecule which differs from the first. In both
models no allowance has been made for the possibility of positive control by a CAPcyclic AMP complex. The following chemical equilibria are assumed:
K5
(82)
K6
P + CAP - (cAMP)m ~
P - CAP - (cAMP)m
(83)
(84)
WCAp(WcAMP)
Wp .- CAP- (cAMP)m
g 6 =
(85)
W*"
pWCAP - (cAMP)m
In Eqs. (84) and (85), the intracellular concentrations of CAP, cyclic AMP, P
and the complexes at equilibrium are given in terms of moles per unit of cellular dry
mass.
Balances on the amounts of CAP and P result in:
W*(CAP)t ~--- W~A P + W*CAP- (cAMP)ra
(86)
W~p),
(87)
93
(cAMP)m
W~P)t
K 5K6(Wc*AMP)m W~CAP)t
*
*
m
1 + Ks(WcAMP
)m' + KsK 6W *(CAP}t(WcAMP)
(88)
Here, the assumption has been made that the operon shifts from the "closed
complex" of RNA polymerase and lac promoter (see Fig. 9) to a tight "open complex"
positively influenced and initiated by the binding of CAP-cyclic AMP complex to
the promoter site of the operon 133,139,~43,149).The operon model of Gondo et al. 142)
assumes an important equilibrium to exist between the "closed complex" and the
"open" one, independent of the presence of CAP and cyclic AMP. It is the "open"
complex, free from CAP-cAMP complex, which starts transcription of mRNA at
a basal rate. However, the large excess of CAP over bacterial operons 136,137,14.3)and
otherwise basal levels of cyclic AMP at high concentrations of glucose 150,~51)makes
it more probable that a basal level of the CAP-cyclic AMP complex in catabolite
repressed cells determines the basal level of the "open complex" and subsequently
the basal rate of the de novo synthesis of inducible enzymes. In order to account for
the catabolite repression of the intracellular cyclic AMP levels in microorganisms,
experimental evidence suggests an inverse proportionality with respect to the concentration of a catabolite like glucose in the culture medium 132, t51,153) At present,
little seems to be known about the exact mechanism of the interaction between the
concentration of the catabolite and the intracellular concentration of cyclic AMP.
Therefore, a completely arbitrary relationship between cyclic AMP and a catabolite
has been assumed 34).
Keat
W~AUP-- K~at + Cs
(89)
where W'AMP is the molar concentration of cyclic AMP in the dry mass and
Kea t is a constant.
(90)
94
A. Harder, J. A. Roels
or for an anti-inducer:
rmRNA = qmRNA,maxf(p.)Q2Q3Cx
(91)
where rmSNAis the rate of the m-RNA synthesis, qraRNA. . . . the maximum rate of the
mRNA synthesis per unit biomass dry weight and f(la) a function which defines the
dependence of the specific rate of the m-RNA synthesis on the specific growth rate.
QI and Q2 are the fractions of free operators and Q3 is the fraction of occupied promoters. Equations (90) and (91) for the rate of the m-RNA synthesis are valid only if Q1
(or Q2) and Qa are statistically independent, i.e. the probability of the promoter
being occupied does not depend on the state of the operator. The growth-rate
dependent terms for the transcription of mRNA in Eqs. (90) and (91) have been
found to be proportional to la in the case of extracellular hydrolase production 154,15s)
For this case, the function f0t) can be formulated as:
f(la)=
(0~rnRNA~I"+ W~mRNA)0)
(92)
(0~raRNA~max + W(mRNA)0)
where w*(mRNA}
0 is the mass fraction of mRNA at zero specific growth rate and (~mRNA
a proportionality constant for the growth rate. Substituting Eq. (92) into Eq. (90) or
(91) we obtain:
rmRNA ~ qmRNA
QmRNA~ + W*
tmRNA)0 Qt (or Q2) QaCx
~mRNA~max+ W~mRNA)0
(93)
A second process which has to be modelled is the decay of mRNA which depends
on the stability of the messenger. Yagil 117~distinguishes proteins formed on
1) preexisting (latent) stable mRNA, 2) stable mRNA, 3) unstable mRNA,
4) mRNA with a fixed life time.
In general, mRNAs from bacterial operons coding for endogenous enzymes
have a very short half-life, i.e. a few minutes 104.156,157) However, Terui and coworkers 124,126,127)demonstrated the mRNAs for a variety of extracellular hydrolases
such as a-amylase, glucoamylase, acid protease and CMCase from Gram-positive
bacteria and fungi to be very long-lived. Recently, it has been reported 127) that in
Aspergillus niger, mRNAs associated with membrane-bound polysomes are more
stable compared to cytoplasmic mRNAs. The existence of membrane-bound
mRNA is growth rate dependent (0 % in growing cells and 50 % in non-growing
mycelia). The rate of mRNA degradation has been found to be either exponential 158,169,160) or linear 124,126,127) For simplicity, the degradation of mRNA is
assumed to be a first-order process 34,111,154}.
r'mRNA = kaWmRNACx
(94)
where YmRNA
'
i S the rate of mRNA degradation, kd the first-order degradation rate constant and Wr,RNA the fraction of mRNA in dry mass. Now it is assumed that the
de novo synthesis of an inducible enzyme is regulated at the transcriptional level.
95
To save energy and metabolites, this type of metabolite control may be expected to be
more common and hence translation of mRNA follows automatically after
transcription of DNA. The specific rate of translation will be proportional to the
intrinsic mRNA concentration:
rE ~" kEWmRNACx
(95)
r E = kdE'~dECx
(96)
where r~ is the rate of degradation of the enzyme, WE the mass fraction of the enzyme
in dry mass and kdE the kinetic constant of degradation.
The equations developed in this section allow the formulation of models for the
synthesis of intracellular and extracellular enzymes which are subject to negative
and positive controls. An example will be given in the next section.
(97)
The secondary substrate S2 is an inducer of the second enzyme, the first substrate
S~ is a catabolite repressor of the enzyme. The rate of uptake of S2 is expressed by a
Monod type equation:
rs 2 --
qE, raaxWECs 2
Ks 2 + Cs 2 C~
.(98)
96
A. Harder, J. A. Roels
w h e r e qE, max is the maximum specific substrate uptake rate per unit amount of
enzyme and w E the mass fraction of the inducible enzyme in the biomass. The growth
rate of biomass is obtained from an extended version of the linear equation for
substrate consumption Io,70) (see also Sect. 6.2):
(99)
rx
where Ysl, and Ys2x are the yield factors for the first and second substrate,
respectively.
The equations presented above in combination with the equations for the
dynamics of enzyme synthesis developed in Sect. 7.4 have been shown to allow the
formulation of a model for the phenomenon of diauxic growth 34). For the details
of the development of the model the original literature should be consulted.
The model was simulated for batch growth on two substrates using arbitrary parameter values. The results for biomass growth and the consecutive depletion of the
two substrates are shown in Fig. 10. The curves obtained compare well with experimental results, e.g. with those of Monod 9). These kinds of models may certainly be
useful for the description of growth on mixed substrates or for the construction of
models for the production of enzymes.
A number of models concerning the modelling of genetically regulated enzyme
synthesis have been reported. In Table 6 these models are evaluated against the
picture developed in this review. Aspect of the models like wether or not positive
and/or negative controls are taken into account and the way in which translation is
modelled are analyzed. Furthermore, special features are recorded.
As pointed out in Sect. 3, the safest way to model intracetlular processes is in
terms of intrinsic concentrations. Hence, concentrations per unit mass are used in
this section. Only a limited number of publications employ intrinsic concentrations for
intracellular compounds. This may result in errors for some of the models.
Cs%Cs21
m'31[
C,t
(kg m-3)
(kg
20'
20
16
12
12
/..,
4
_
k-
10
15
Time (h)
20
s2
10
15
Time (h)
-.
2O
Fig. 10. Simulations of diauxic growth according to the model for the enzyme concentration
regulation. Plots of biomass concentration Cx, and the concentrations of the substrates, Csl and C~z,
against time
97
..~
.~
r~
9 .-O a.a
0 . ,~
0 t,.] 0
..~<
O ..g~ O
~Z
m
~ ~ .~
t~
8
<~
t~
O
czT.
~oo~
.o
o'
..~
[.,,
[-
>,
~<_~
98
A. Harder, J. A. Ro
Z
g
0
~,g
~
Zz~
o.~
<~
!Z
g
0
0
0
"0
[.I
Z~
6"~<
99
One final remark needs to be made. In the treatment of the various controls no
account was taken of the dilution caused by growth of the various intracellular
constituents active in these control processes. This is a direct consequence of the
assumption that the levels of these constituents are regulated by processes with very
small relaxation times. Hence, their levels are always equal to the steady-state values
corresponding to a certain set of external conditions.
8 Conclusion
The large-scale use of the activities of organisms in an industrial environment
calls for a quantitative understanding of their behavior. As in chemical engineering
where a well developed theory of reaction kinetics provides an adequate framework for
such endeavours, biotechnology needs adequate models for the description of the
behavior of organisms. These models can be applied to process development,
process optimization, process control and can be useful guidelines for the formulation
of desirable properties of organisms as far as industrial application is concerned.
In modelling the behavior of organisms, the problems to be overcome are
concerned with the complexity of biological systems. A great reduction of this
complexity is needed to obtain models which are suitable for engineering applications.
The danger inherent to such a procedure is that it may lead to a loss of realism, i.e.
the model may no longer provide a sufficiently accurate description of the organisms
behavior. In the modelling of microbial metabolism unstructured models have become
quite widely accepted in literature as well as in industry. These models do not allow
for the changes in the compositional variables of the organisms.
There are a variety of situations in which the composition of the organism
is subject to changes which do affect their behavior to a significant extent. In such
cases, structured models have to be used, i.e. models which allow for changes in the
compositional variables of the organisms.
Accounts of the development of structured models of varying complexity have
appeared since 1965. However, in our opinion the success of these endeavours remains
uncertain and they certainly have not become widely accepted in industrial
practice.
Two important factors play a role in this respect. Firstly, the theory is rather
intricate and subject to errors. Secondly, it is still difficult to find the necessary
balance between the avoidance of unnecessary complexity and assurance of sufficient
realism. These problems are not specific for the application of models in biotechnology
but also exist in the other engineering sciences.
In this review attention is first focussed on the intricacies of the theory. It is shown
that the theory seems to be sufficiently advanced to avoid problems of the first
kind mentioned above. The problems of the second type may be tackled by
combining adequate knowledge about the biochemistry of cellular regulation processes
with notions derived from a comparison of the relaxation times of environmental
changes with those of the regulatory processes. These concepts are evaluated.
The field seems now to be open for the development of specific applications.
Two examples of such applications are described in this review and used as a framework for the evaluation of the relevant literature. It has to be expected that the use of
100
A. Harder, J. A. Roels
structured models will become increasingly important in the near future. However,
these applications will call for two specific kinds of developments:
- - A certain degree of 'containment' on the investigators' side. The use of too
complex models serves little purpose if they lack careful experimental evaluation.
Complex simulation studies are of little use if the realism of the conclusion is not
carefully checked experimentally. In our opinion structured models in which more
than three compartments are specified will almost never be needed in view
of the applications encountered in biotechnology.
-- Mathematical models should be carefully evaluated by parameter sensitivity
studies and not in the last place through verification by carefully planned and
discriminating experiments. This is the main aspect which seems to be lacking if
the available literature is critically evaluated.
9 Symbols
C
Ci
Csi
D
e
K
Keat
Ki
Ka
KM
Kj
k
ki
mATP
ms
mi
N
Nt
P/O
Q
Q*
(kg m-3)
(kg m-3)
(kg m -3)
(h -1)
(kg)
(h -l)
(kg m -3)
(kg m -3)
(--)
(kg m - 3)
(--)
h- 1
(mol (kg DS)- 1 h- i)
(kg (kg DS)-1 h - 1 )
(h -l)
(m -3)
(m -3)
(--)
(--)
(--)
Q~, Q2
Q~
ql
qi, max
q*
qij
R
Re
r
rE
rA
ri
(ri),
rij
rATP, i
t
W
W
WD
Wi
w*,
W~o
W~o
W*
(mRNA)0
X
101
(-)
(-)
(kg (kg DS)- 1 h - l)
(kg (kg DS) -1 h -1)
(kg (kg DS) -~ h -~)
(kg (kg DS) -~ h -~)
(kg h -1)
(kg h -1)
(kg m -a h -1)
(kg m -a h -1)
(kg m -3 h -1)
(kg m -s h -1)
(kg m -a h -1)
(kg m-3 h - l )
(mol m -3 h -i)
(h)
(kg)
(-)
(-)
(-)
(-).
(-)
(-)
(--)
(kg m -3)
(kg m -3)
A. Harder, J. A.
102
Yij
(-)
YATP,i
kg (tool ATP)-1
(YATP)max
~x
~y
~c
~mRNA
kt
~max
~Ji
(D x
~y
O) i
CO
Subscripts
ATP
CAP
adenosine-5'-triphosphate
catabolite activator protein
kg (mol ATP) -1
(-)
(-)
(-)
(-)
(mol kg -1)
(h)
(--)
h
h-i
h-1
(-)
(-)
(kg m -3 h -1)
(kg m -3 h -1)
(kg m -3 h -l)
(kg m -3 h -l)
(kg m -3 h -1)
(kg)
(kg)
(kg)
Roels
Other Symbols
p*
(P>
103
complex o f C A P and c A M P
cyclic A M P
enzyme or effector
intermediate complex o f enzyme and substrate
G-compartment
K-compartment
operator
operator-repressor complex
promoter
product
activated p r o m o t e r
product-formation activity c o m p o u n d
messenger R N A
cytoplasmic repressor o r R - c o m p a r t m e n t
repressor-effector complex
substrate o f a constitutive enzyme
substrate o f an inducible enzyme
substrate
biomass
steady-state value o f variable p
average value o f variable p
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