Environmental Control of Cell Synthesis and Function. The 5th International symposium on the Continuous Culture of Micro-Organisms, Held at St. Catherine's College, University of Oxford, July 1971

311 Pages·1972·5.862 MB·English

by  A. Dean (Eds.)

Environmental Control of
Cell Synthesis and Function
(The 5th International Symposium on the Continuous Culture of Micro-organisms, held at
St. Catherine's College, University of Oxford, July 1971)
Edited by
A. C. R. DEAN
University of Oxford
S. J. PIRT
Queen Elizabeth College, University of London
D. W. TEMPEST
Microbiological Research Establishment
For ton Down
The papers in this volume were originally published in the Journal of Applied
Chemistry and Biotechnology, Volume 22, Issues 1 to 4, between January and April
1972. They are reprinted as they appeared in the original publication—hence the fact
that the pages are numbered in four sequential sets with gaps between the sets.
Published for the
SOCIETY OF CHEMICAL INDUSTRY
by
ACADEMIC PRESS
1972
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Copyright © 1972 Society of Chemical Industry
Second Printing 1973
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Library of Congress Catalog Card Number: 72-79140
ISBN: 0-12-208050-5
Printed in Great Britain by The Whitefriars Press Ltd., London and Tonbridge
Preface
The 5th International Symposium on Continuous Culture of Micro-organisms was
held at St. Catherine's College, University of Oxford, from the 19th to 24th July
1971. The scientific programme was divided into four main areas of interest (namely,
Kinetics of Growth, Recent Advances in Equipment Design and Operation, Influence
of Environment on the Control of Cell Synthesis, and Physico-chemical Effects on
Cell Structure and Functioning) and some 20 individual topics were discussed. Each
topic was introduced by a full-length review-type lecture. It is hoped that this collec-
tion of papers provides an up to date and comprehensive survey of the application
of Continuous Culture to research in Microbiology; particularly to problems of
microbial physiology. The programme was designed to illustrate the great extent to
which the structure and functioning of microbial cells is influenced by the chemical
and physical nature of the growth environment. Since continuous culture techniques
provide not only controlled environments, but a wide range of unique environments,
the central role which they can play in microbiological research (and in their applica-
tion to microbiological processes) is clearly evident, and amply illustrated.
D
/. appl. Chem. Biotechnol. 1972, 22, 55-64
Introductory Lecture
Prospects and Problems in Continuous Flow Culture
of Micro-organisms
S. J. Pirt
Microbiology Department, Queen Elizabeth College {University of London),
Camp den Hill, London W.8
1. Introduction
12
This symposium marks the 21st anniversary ' of the publication of the theory of
chemostat continuous-flow culture. The theory marks a turning point in studies on
the physiology of cell growth and, as a result, the chemostat has become a major
method for studies on the dynamics of function in growing populations of microbes
and cells. This paper considers: (i) the basic concepts of continuous-flow culture,
(ii) the need for extension of the theory of chemostat cultures, (iii) some conditions
under which the theory breaks down, in particular, at slow growth rates, and
(iv) future problems.
1.1. Terminology
There is a need to be more precise about the meaning of the term "continuous cul-
ture". There are many different types of continuous culture, all derived from two
basic types: (i) the chemostat and (ii) plug-flow culture. In the chemostat, ideally
the culture is completely mixed, whereas in the ideal plug-flow culture the culture
flows along a tubular vessel without mixing. "Continuous-flow culture" is a preferable
generic term for all the methods, especially since it has been extended to tissue cell
culture where the term "continuous culture" has a quite different meaning.
1.2. Open and closed systems
3
Herbert introduced the concepts of open and closed culture systems. An open
culture system is defined as one which has both input of material (substrates) and
output of material (biomass and products). A closed system is one which has no
input and output of materials. The open systems have, in theory, the possibility that
biomass growth and output will balance and the system reaches a steady state in
which constant conditions can be maintained indefinitely. In a closed system only
transient states are possible in which conditions continually change and approach
a static final state. The term "batch culture" is used as a synonym for a closed system
of culture but the latter term would be more exact. The chemostat and plug-flow
56 S. J. Pirt
cultures are open systems which differ fundamentally in the cultural conditions and
in the nature of the steady states realised in them. Plug-flow culture simulates a batch
culture, the only difference being that the sequence of conditions temporally separated
in a closed system are spatially separated in a plug-flow culture. Thus the biomass
in a plug-flow culture is subjected to changing conditions as it passes along the
vessel. Plug-flow culture with feedback may be convenient as a means for the auto-
matic renewal of the cycle which occurs in a closed system, or for maintenance of
particular phases of the cycle in the culture vessel. It suffers from the disadvantage
that it requires complex apparatus difficult to realise in practice.
The chemostat greatly extends the range of conditions possible in a culture. Many
of the advantages of the chemostat stem from the fact that it is a simple means for
obtaining substrate-limited growth, that is, growth rate limited by the supply of an
essential nutrient whilst maintaining a constant environment. In a closed system
such as the simple batch culture with common media, most of the growth occurs
with an excess of substrate, growth proceeding at the maximum rate until the substrate
is virtually exhausted. Substrate-limited growth may be maintained for a period
in a closed system by the use of a substrate feed, but the environment will not be
constant, which may make it difficult or impossible to discover the effects of a given
environmental condition. It is misleading to regard the chemostat as a means for
extending the period of exponential growth. The latter, in most people's minds,
refers to the period of growth at a constant maximum rate which occurs in a batch
culture while there is excess of nutrients; this is only one extreme case of the constant
conditions possible in a chemostat.
The basic advantages of the chemostat over other means of culture are five in
number.
(i) It provides a means of controlling growth rate. This is achieved not by
changing either the nature of the substrate or the physical conditions of
the culture but by changing the concentration of growth-limiting substrate
4
in the medium. This principle was applied by Herbert to determine the
effect of growth rate on the synthesis of RNA, DNA and cell size in a
5
bacterial culture; similarly, Tempest and Herbert determined the effect
of growth rate on the activity of respiratory enzymes.
(ii) The growth rate can be held constant whilst physical and nutritional con-
ditions are changed; this is the converse of (i). Thus the effect of temperature
6
on RNA synthesis was elucidated.
(iii) It provides a means of achieving "substrate-limited growth" with constant
concentration of the limiting substrate. The great importance of substrate-
limited growth in metabolic control is now emerging largely as a result of
chemostat studies. The wide-reaching effects of substrate limitation is
illustrated by the effect of phosphate limitation on growth of Gram-positive
bacteria. Species of Bacillus** and Staphylococcus (Tempest, private communi-
cation) change their cell wall composition, having teichuronic acid present
with phosphate limitation and teichoic acid with excess phosphate. The
difference between substrate-limited growth and the state of an exponentially
Prospects and problems in continuous flow culture 57
growing batch culture was unexpected and consequently has proved difficult
to appreciate.
(iv) The chemostat permits the biomass in a culture to adjust itself to a steady
state in any given environment. This is a unique feature of the chemostat
made possible because a given environment can be maintained indefinitely.
In a plug-flow culture, in contrast, although the system as a whole can
reach a steady state, the biomass does not because it is moving through an
environment which is changing faster than the organism can adapt its
structure and metabolism. An instance of this is the change in catalase
7
content of cells throughout a batch culture. In contrast, in a chemostat
the enzyme activities settle down to constant values, e.g. Tempest and Her-
5
bert. Only by achieving the steady state in the biomass can one separate the
effects of a given environment from the effects of the history of the organism.
(v) The final advantage of the chemostat is that it permits the most rapid
conversion of substrate into biomass plus growth-limited products such
8
as carbon dioxide. For this reason the chemostat is required for large-scale
biomass production and for the bio-degradation of wastes such as effluents.
2. VALIDITY OF CHEMOSTAT THEORY
1
The Monod relation of growth and substrate utilisation holds reasonably well for
chemostat culture of a single type of organism with a single growth-limiting substrate.
910 4
The deviations due to maintenance energy ' and to storage products are generally
agreed upon. Deviations which seem to depend on biomass concentration are less
11
well understood. With carbon-limited growth, Contois found that the experimental
value of the biomass became progressively less than the predicted value as the biomass
concentration increased. To account for this he proposed that in the Monod relation
between growth rate (μ) and the concentration of growth-limiting substrate )s)
9
that is, μ = /w/C? + ^s), the K value depends on the biomass concentration (x).
H
So he postulated that K = Bx, where Β is a constant. The tests of this model, how-
s
ever, depended on the assumption that the substrate concentration s could be cal-
culated from the equation s = s — x\ Y, where the growth yield Y was assumed
T
constant. In fact, Y for carbon and energy sources is known to decrease at high
growth rates through incomplete oxidation, e.g. see reference 12, and this could
13
account for the deviations which Contois observed. Jannasch observed that the
growth of a spirillum at very low biomass concentration (<15 mg dry wt/1) was less
than that predicted. It appeared that at low biomass concentrations the growth yield
Ffrom lactate decreased and K increased. This effect could be attributed to inhibition
s
14
by oxygen which decreased the value of // . Meers and Tempest observed an increase
m
2 8
in the limiting Mg + concentration at concentrations of Bacillus sp. below 10 bacilli/
2+
ml. The decrease in the limiting Mg concentration with increase in biomass con-
centration was attributed to the secretion of an activator. This activatory substance,
it was postulated, increased the maximum growth rate according to the relation
58 S. J. Pirt
where ρ is the concentration of the activatory product and λ a constant. A similar
expression has been derived from Michaelis-Menten enzyme kinetics to account
for activation of an enzyme (see reference 15, p. 323). The chemical nature of the
activator for uptake of magnesium ions remains unknown.
Much effort in studies with the chemostat has been wasted because it was directed
towards ad hoc studies on substrate utilisation and product formation rather than
systematic tests of theory. As a result, systematic tests of theory have been limited
to studies on a few of the more common energy sources such as glucose and by
ammonia, potassium, magnesium and phosphate limitation, and nearly all the tests
have been done with bacteria. These tests need to be extended to the fungi,
protozoa, algae and tissue cells of animals and plants. Deviations from the model
behaviour bring to light unexpected properties of the organism. For instance, in the
author's laboratory it has been observed that in filamentous mould cultures the
16
critical dilution rate instead of being equal to the maximum growth rate is much
less (about 50%). The cause of this deviation remains to be discovered.
1
3. EXTENSIONS TO CHEMOSTAT THEORY: EFFECTS OF INHIBITORS
3.1. ASSOCIATIONS OF ORGANISMS
Theoretical models for the dynamics of associations of protists and cells in chemostat
culture need to be developed and tested experimentally. Of the different systems
which can be conceived, only a few can be mentioned here.
(i) Interdependence of two organisms A and Β with different growth-limiting
substrates and organism A requiring a growth factor produced by B. Systems
such as this may exist among the lactobacilli and streptococci which often
occur together.
(ii) Interdependence in which one organism (A) produces a growth factor for
organism B whilst Β removes a substance which is toxic for A. Such systems
9
may occur with methane-oxidising bacteria which are known to grow poorly
alone but seem to flourish in mixed cultures with bacteria which cannot
utilise methane.
(iii) Predator-prey relations such as protozoa ingesting bacteria: it is important
to note that the relation between the growth rate of protozoa and the
bacterial substrate conforms to a Monod type of relation in which K is of
s
17
the same order as that for the carbon substrates of bacteria. The protozoa-
bacteria predator-prey system is highly relevant to studies on effluent
purification. Although the protozoa may perform a useful function in
disposing of the bacteria at the end of the process, presumably they are
detrimental if they prey on the bacteria in the early stage before the bacteria
have finished their task. Assuming the protozoa have a lower D nt than the
c
bacteria, the protozoa could be eliminated from the first stage by making
the dilution rate above the D vit for the protozoa.
C
Prospects and problems in continuous flow culture 59
3.2. Inhibitor effects
The dynamics of inhibitor effects on chemostat cultures need theoretical study. It
may be anticipated from the important role inhibitors have played in enzyme studies
and in chemotherapy that they will be important in control of metabolism and product
formation in chemostat cultures.
The basis of the theory of inhibitor effects is either Michaelis-Menten enzyme
18
kinetics as used, for instance, by Van Uden in a study of competitive inhibition
of glucose uptake for yeast growth, or pure mathematical modelling of the type used
19
by Aiba, Shoda and Nagatani in a study of alcohol inhibition of yeast growth.
However, the results of Aiba et al. show that the alcohol effect corresponds to the
case of non-competitive inhibition in enzyme kinetics. Cases of product inhibition
of growth must frequently be met and we need to know how this will modify the
relation between biomass and dilution rate in a chemostat. Also growth inhibition
by substrates is becoming increasingly important in studies on the microbial degrada-
tion of toxic compounds such as phenols and hydrocarbons. A theoretical study of
20
substrate inhibition has been given by Edwards.
3.3. Inhibitor added to medium
The addition to the medium of a substance which competitively inhibits uptake of
18
the growth-limiting substrate is expected from Michaelis-Menten kinetics to
affect the biomass-dilution rate relation as shown in Figure 1. Van Uden used L-
sorbose as the inhibitor of glucose uptake and in general one would expect non-
JT, ι = 0
Ο 0.2 0.4 0.6
1
Dilution rate (h~ )
Figure 1. Effect of a competitive inhibitor of uptake of growth-limiting substrate (s). The effect
was modelled by substituting / f , rr / 1 + * \ \
in the Monod theory, where / = inhibitor concentration; K= 0.01 g/1; Ki = 0.01 g/1; χ = biomass
s
concentration; s = limiting substrate concentration.
metabolisable analogues of the substrate to be competitive inhibitors and to act
specifically at the first step in the metabolic pathway of the substrate. It seems possible,
however, that some analogues might undergo the first one or two steps of metabolism
60 S. J. Pirt
and act as competitors at each of these steps. For example, a glucose analogue might
be phosphorylated but not subject to further metabolism.
Non-competitive inhibition of growth seems more likely than competitive inhibition
because there are obviously more sites for non-competitive effects. The expected
effect of a non-competitive inhibitor of growth in the chemostat is shown in Figure 2.
The predominant effect is a decrease in the maximum growth rate. The latter effect
might be exploited in studies on mixed cultures to eliminate one organism from the
population.
1 1 1 1 1 1 1 1 1 1
—
1
1-0
-
\
1 I
_ 1 1
/ 1
1 1
J 1
- / /
/
5, , Ao J
/=*; /
Ο I .I 0.2 1 L0.'4 0.16 1 .01. 8 1 ^ 1.0
Dilution rate ( h- I)
Figure 2. Effect of a non-competitive inhibitor of growth. The effect was modelled by substituting
μ = BumsKs + K), where Β = 1/(1 + i/Ki) in the Monod theory: / = inhibitor concentration;
s
K = 0.01 g/1; Ki = 0.01 g/1; χ = biomass concentration; s = limiting substrate concentration.
B
The addition of an inhibitor would be expected to bring about both genetic and
phenotypic changes in the population. In this way, the organism might be constrained
to change its enzyme content or excrete intermediary metabolites. Some of the
possibilities might be elucidated by studies with the chemostat of some of the classical
inhibitors of glycolysis or the respiratory pathway.
3.4. TWO OR MORE GROWTH-LIMITING SUBSTRATES
If substrate uptake follows Michaelis-Menten kinetics, then, when two substrates
are growth-limiting, the rate of growth, by analogy with enzyme kinetics (see reference
15, p. 72), would be expected to conform to the relation
1
where μ = specific growth rate (HR ), μ = maximum specific growth rate, a and b
ΊΆ
are the concentrations of the two growth-limiting substrates and AT and Kt> are the
A
saturation constants. It is clear from the above equation that if a and b are of the
same order as and A^, respectively, μ may be much less than it would be if either
a or b were large compared with its respective saturation constant. For example,
if a = Κ and b = Κχ>, then μ = 0.25 /z , whereas it would be 0.5 // if either a or b
Ά m m
Prospects and problems in continuous flow culture 61
were present in excess. Fortunately, for economy of substrate, because growth-
limiting concentrations are generally very low (for example, only a few parts/million
for carbon sources and much less for sources of nitrogen and other substrates), it
should usually be possible to work with all substrates but one in excess. There seems
to be no reason why this consideration should not apply even in effluent purification
where one aim is to free the effluent from substrates as far as possible.
4. MINIMUM GROWTH RATE
There are a number of reports which suggest that the growth rate of micro-organisms
can only be decreased to a finite limit and that if the nutrient feed rate is insufficient
for this then all or a part of the cell population ceases to grow. Another possibility
if there is a minimum growth rate and the dilution rate is below this, is that there
could be bursts of growth followed by periods of no growth. The evidence for a
finite minimum growth rate (//MIN) is as follows.
4.1. IN FUNGI
In a glucose-limited Pénicillium chrysogenum culture a marked change in the properties
1
of the mould occurs when the growth rate is <0.014 h" : the penicillin production
21
rate decays to zero; conidia formation begins and extensive macromolecular change
22 22
occurs. More recently, Bainbridge et al. > have shown that Aspergillus nidulans
does not grow if supplied glucose at 1.5 χ maintenance ration which would corres-
_1
pond to a specific growth rate of 0.007 h . Again, extensive hyphal and macro-
molecular changes were observed although no conidia were formed. These results
indicate that there is a minimum growth rate of moulds which is about 5% of the
maximum rate (//MAX). Below this critical growth rate differentiation into a resting
state occurs.
4.2. IN BACTERIA
The evidence for a "minimum growth rate" in bacteria was reviewed and added to
24
by Tempest, Herbert and Phipps. They showed that the growth rate of Klebsiella
_1
aerogenes tended to a minimum of 0.009 h at 37 °C irrespective of whether glycerol
or ammonia were the growth-limiting substrates. Their estimate of the minimum
growth rate took into account the non-viability (that is, inability to grow) of a part
of the population. However, the "viability" they determined may not have been a
valid estimate of the number of growing bacteria in the chemostat since it was deter-
mined by plating out the bacteria on rich (complete) media instead of the minimal
_1
media used in the culture. Hence the value of 0.009 h could be a serious under-
estimate of the minimum growth rate. A reconsideration of the data of these experi-
ments shows that a sharp discontinuity in the properties of the bacteria occurred at
_1 _1
a growth rate of 0.06 h . Figure 3 shows that below a growth rate of 0.06 h the RNA
and DNA in the cells decreased abruptly whilst the graphs of the QO2 against growth
rate (Figure 4) and of the reciprocal growth yield for glycerol against reciprocal
growth rate (Figure 5) departs from linearity. The linear part of the graph in Figure 5