4.2.1.1 Building Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.2.1.2 Transport Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.2.1.3 Synthesis of the Cell Wall: Chitin . . . . . . . . . . . . . . . . . . . 12
4.2.1.4 Synthesis of the Cell Wall: Glucan . . . . . . . . . . . . . . . . . . . 13
4.2.1.5 Synthesis of the Cell Wall: the Structure . . . . . . . . . . . . . . . 13
4.2.2 Morphology Modelling in General . . . . . . . . . . . . . . . . . . 14
4.2.3 Models for Morphogenesis . . . . . . . . . . . . . . . . . . . . . . 15
4.2.4 Models for the Relation Between Morphology and Production . . 20
4.2.5 Some General Remarks About Models . . . . . . . . . . . . . . . . 21
4.3 Special Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.3.1 Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.3.2 Whole Broth Properties . . . . . . . . . . . . . . . . . . . . . . . . 26
5 Implementation of the Results . . . . . . . . . . . . . . . . . . . . 28
6 Conclusions and Prospects . . . . . . . . . . . . . . . . . . . . . . 29
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
List of Symbols and Abbreviations
C Concentration, kg m
–3
C
X
Concentration of biomass, kg m
–3
DCR Diffusion with chemical reaction
ID Diffusion coefficient, m
2
s
–1
DOT Dissolved oxygen tension, N m
–2
D
r
Stirrer diameter, m
d
h
Diameter of hypha, m
ER Endoplasmatic reticulum (an internal structure element of a cell)
f (x, t) Population density function: number per m
3
with property x at
time t
k
1
,k
2
Lumped parameters
k
l
a Mass transfer parameter, s
–1
L Length of hypha, m
L
e
Length of main hypha in hyphal element, m
L
emax
Maximum length of main hypha capable of withstanding fragmenta-
tion, m
L
equil
Equilibrium length, m
L
t
Length of all hyphae in hyphal element, m
L
hgu
Length of hyphal growth unit (L
t
/n), m
m mass, kg
m
hgu
Mass of a hyphal growth unit, kg per tip
N Rotational speed of stirrer, s
–1
n Number of tips in hyphal element, -
2
N.W.F. Kossen
NADP Nicotinamide adenine dinucleotide phosphate: oxydation/reduction
coenzyme in which NADPH is the reducing substance
P/V Power per unit volume of fermenter, W m
–3
r Distance to stirrer, m
r (C) Reaction rate as function of C, kg m
–3
s
–1
r
l
Rate of vesicle production per unit length of hypha, number m
–1
s
–1)
Rho 1p A GTP-binding enzyme involved in the cell awl synthesis
t
c
Circulation time, s
V Volume, m
3
v Velocity, m s
–1
V
disp
Volume with maximum dispersion potential, m
3
z Vector representing the environmental conditions, varying dimen-
sions
e Power per unit mass, W kg
–1
f
p
Pumping capacity of stirrer, m
3
s
–1
g Shear rate, s
–1
m Specific growth rate, s
–1
t Shear stress, N m
–2
1
General Introduction
Filamentous fungi are fascinating organisms, not only because of the inherent
beauty of their fruiting bodies but also because of their complicated and
scientifically very interesting behaviour. They are also able to produce a large
variety of useful , commercially interesting products.
The use of filamentous fungi as production organisms in industry, originally
as surface cultures, is widespread,. Many scientists once believed that these
fungi could only grow as surface cultures but it became clear in the 1940s
that submerged cultures are also possible and have an enormous production
potential. However, there appeared to be one problem: their form. In their
natural environment filamentous fungi grow in long, branched threads called
hyphae. This form, which is ideal for survival in nature, presents no problem in
surface cultures, but it is often a nuisance in submerged cultures because of the
strong interaction between submerged hyphae. This results in high apparent
viscosities (“applesauce” behaviour) and – as a consequence – in major
problems in the transport of O
2
,CO
2
, and nutrients, as well as in low pro-
ductivities compared with theoretical values and with productivities obtained
with other microorganisms. It was obvious that the control of the form of these
fungi was a real issue that needed further attention in order to make optimal
use of their potential production capacities.
Many scientists have been studying this problem from an engineering point
of view for a number of decades. Simultaneously, many other scientists, working
on morphology mainly because of pure scientific interest or sheer curiosity,
have been very active.
The outcome of the efforts mentioned above is an impressive landscape of
results about what is now called “the morphology of fungi”. This paper is about
The Morphology of Filamentous Fungi
3
this landscape: what it looks like, how it emerged and developed, which tools
were developed, and what are its strengths and weaknesses.
2
The Framework of This Study
As will be clear from the introduction this is not another review on the
morphology of fungi. There are excellent, up-to-date and extensive reviews
available [1]. This is a survey of the main lines of development of a very
interesting area of biotechnology research. based on a limited number of
characteristic publications. These have been selected on the basis of their con-
tributions – either good or debatable ones – to new developments in two areas:
– Improved scientific insight.
– Bioprocess practice – is it useful and usable?
The improvement of scientific insight usually goes hand in hand with a number
of developments in the models used (see Fig. 1). These developments provide
the main yardsticks for the present evaluation.
The trend in the development from unstructured to structured models needs
an introduction. In unstructured models one assumes that the object of study
has no structure: for example, a hyphal element is considered to be a more-
or-less black box without internal detail. If one distinguishes septa, nuclei etc.,
the model then becomes structured. This structuring can go on a long way and
become very detailed, but a limited number of internal “compartments” is
usually sufficient to describe an observed phenomenon properly.
In the literature, models of another useful kind are sometimes mentioned:
segregated – or corpuscular – models. In that case, a population is not con-
sidered to be a unit with average properties, but a collection of different in-
dividuals, each with its own properties: form, size, respiration rate, etc.
The methods used for the parameter optimization and the validation of the
models will also be part of the evaluation.
Three classes of subjects will be discussed:
1. Methods: image analysis, microelectrodes, single hyphal elements, staining.
2. Models: models for morphogenesis and for the relation between morphology
and production.
3. Special aspects: genetics, transport phenomena.
Now that the subjects and the yardsticks have been presented, just one word
about the the author’s viewpoint. This point of view is that of a former uni-
4
N.W.F. Kossen
Fig. 1.
Development of models
versity professor, who started research after the morphology of moulds in 1971
and – inspired by problems he met as a consultant of Gist-brocades – worked in
this particular area of biotechnology for about 10 years. After 17 years at the
university, he went to Gist-brocades and worked there for 10 years. Most of the
time as a director of R&D, in which position he became heavily involved with
technology transfer among all of the disciplines necessary for the development
of new products/processes and the improvement of existing ones.
3
Introduction to Morphology
3.1
What Is Morphology?
Morphology is the science of the form of things. It is a wide spread field of
attention in a large number of sciences: biology as a whole, geology, crystal-
lography, meteorology, chemistry – biochemistry in particular, etc. It usually
starts as a way of classifying objects on the basis of their form. When the
scientist becomes curious about the “why” of the development of a form he/she
gets involved in the relationship between form and function.In the end, this can
result in the prediction of properties given a particular form, or in the control
of form/function.
First, we need several definitions. A hypha (plural: hyphae) is a single thread
of a hyphal element. A hyphal element consists of a main hypha, usually with a
number of branches, branches of branches etc., that originates from one spore.
A flock is a loosely packed, temporary agglomerate of hyphal elements. A pellet
or layer is a dense and –- under normal process conditions – almost permanent
configuration of hyphae or hyphal elements (see Fig. 2).
The Morphology of Filamentous Fungi
5
Fig. 2.
Several definitions and forms
Furthermore, the “form of things” is a rather vague concept that needs
further specification. The morphology of fungi is usually characterized by a
limited number of variables, all related to one hyphal element: the length of the
main hypha (L
e
), the total length of all the hyphae (L
t
), the number of tips (n)
and the length of a hyphal growth unit (L
hgu
). The L
hgu
is defined as L
t
/n.
3.2
The Morphology of Filamentous Fungi
The various forms of filamentous fungi have advantages and disadvantages
in production processes as regards mass transport properties and the
related overall (macro) kinetics, in particular at concentrations above 10–20 kg
m
–3
dry mass (see Table 1). As has already been mentioned, the poor trans-
port properties are the result of the strong interaction between the single
hyphal elements at high biomass concentrations, often resulting in fluids
with a pronounced structure and a corresponding yield stress. This results
in poor mixing in areas with low shear and in bad transport properties
in general.
Morphology is strongly influenced by a number of environmental condi-
tions, i.e. local conditions in the reactor:
1. Chemical conditions like: C
O
2
,C
substrate
,pH.
2. Physical conditions like: shear, temperature, pressure.
We will use the same notation as Nielsen and Villadsen [2] to represent all these
conditions by one vector (z).Thus morphology(z) means that the morphology
is a function of a collection of environmental conditions represented by the
vector z. If necessary z will be specified.
Also, genetics must have a strong influence on the morphology, because the
“genetic blueprint” determines how environmental conditions will influence
morphology. We will return to this important issue later on. For the time being,
it suffices to say that at present, despite impressive amounts of research in this
area, very little is known that gives a clue to the solution of production problems
due to viscosity in mould processes. This situation shows strong similarity with
the following issue.
6
N.W.F. Kossen
Table 1.
Transport properties of various forms of moulds
Form of element Transport to element Transport within Mechanical strength
within broth element of element
Single hyphal –/+
a
+±
elements
Flocs –/++
b
±–
Pellet/layer + – +
a
Depending on the shape, size and flexibility of the hyphal element.
b
Depending on kinetics of floc formation and rupture.
A very important practical aspect of the morphology of filamentous fungi is
the intimate mutual relationship between morphology and a number of other
aspects of the bioprocess. This has already been mentioned by Metz et al. [3], in
the publication on which Fig. 3 is based. The essential difference is the inclusion
of the influence of genetics. In this figure, viscosity is positioned as the central
intermediate between morphology and transport phenomena. Arguments in
support of a different approach are presented in Sect. 4.3.2.
This close relationship, which – apart from genetics to some extent – is
without any “hierarchy”, makes it very difficult to master the process as a whole
on the basis of quantitative mechanistic models. The experience of the
scientists and the operators involved is still invaluable; in other words: empiri-
cism is still flourishing.
Morphology influences product formation, not only via transport properties
– as suggested by Fig. 3 – but can also exert its influence directly. Formation of
products by fungi can be localized – or may be optimal.– in hyphae with a
specific morphology, as has been observed by Megee et al. [4], Paul and Thomas
[5], Bellgardt [6] and many others.
4
Overview of the Research
This chapter comprises three topics: methods, models, aspects.
The Morphology of Filamentous Fungi
7
Fig. 3.
Mutual influences between morphology and other properties
4.1
Methods
Methods are interesting because they provide an additional yardstick for
measuring the development of a science. Improved methods result in better
quality and/or quantity of information, e.g. more structural details, more in-
formation per unit time. This usually results in the development of new models,
control systems etc. The different aspects that will be mentioned are: image
analysis (Sect. 4.1.1), growth of single hyphal elements (Sect. 4.1.2), micro-
electrodes (Sect. 4.1.3) and staining (Sect. 4.1.4).
4.1.1
Image Analysis
Much of the early work on morphology was of a qualitative nature. Early papers
with a quantitative description of the morphology of a number of fungi under
submerged, stirred, conditions have been published by Dion et al. [7] and Dion
and Kaushal [8] (see Table 1 of van Suijdam and Metz [9]). A later example is
the early work of Fiddy and Trinci [10], related to surface cultures and that of
Prosser and Trinci [11]. Measurements were performed under a microscope, by
either direct observation or photography. The work can be characterized as
extremely laborious.
In their work, Metz [12] and Metz et al. [13] made use of photographs of
fungi, a digitizing table and a computer for the quantitative analysis of the
above-mentioned morphological properties of filamentous fungi (L
e
,L
t
,n and
L
hgu
) plus a few more.Although the image analysis was digitized, it was far from
fully automated. Therefore, the work was still laborious, but to a lesser extend
than the work of the other authors mentioned above.
The real breakthrough came when automated digital image analysis (ADIA)
was developed and introduced by Adams and Thomas [14]. They showed that
the speed of measurement – including all necessary actions – was greater than
the digitizing table method by about a factor 5. A technician can now routinely
measure 200 particles per hour. Most of the time is needed for the selection of
free particles.
Since then, ADIA has been improved considerably by Paul and Thomas [15].
These improvements allow the measurement of internal structure elements,e.g.
vacuoles [16], and the staining of parts of the hyphae, in order to differentiate
various physiological states of the hyphae by Pons and Vivier [17].
Although the speed and accuracy of the measurements, as well as the amount
of detail obtained, show an impressive increase, there are areas , e.g. models,
where improvement of ADIA is essential for further exploration and im-
plementation. An important area is the experimental verification of population
balance, in which case the distribution in a population of more than 10,000
elements has to be measured routinely [18]. This is not yet possible, hampering
the verification of these models. For average-property models, where only
average properties have to be measured, 100 elements per sample are sufficient,
and this can be done well with state-of-the-art ADIA.
8
N.W.F. Kossen
Closely related to ADIA is automated sampling, which allows on-line sam-
pling and measurement of many interesting properties, including morphology.
This method is feasible but is not yet fast and accurate enough [17].
Needless to say, in all methods great care must be taken in the preparation of
proper samples for the ADIA. Let this section end with a quotation from the
thesis of Metz [12] (p. 37) without further comment. It reads: “The method for
quantitative representation of the morphology proved to be very useful. About
60 particles per hour could be quantified. A great advantage of the method was
that the dimensions of the particles were punched on paper tape, so automatic
data analysis was possible”.
4.1.2
Growth of Single Hyphal Elements
Measurement of the growth of single hyphal elements is important for under-
standing what is going on during the morphological development of mycelia. It
allows careful observation , not only of the hyphae such as hyphal growth rate,
rate of branching etc., but also – to some extent – of the development of micro-
structures inside the hyphae, such as nuclei and septa. This has contributed con-
siderably to the development of structured models. There are early examples of
this method [10], in which a number of hyphal elements fixed in a surface cul-
ture were observed.An example of present work in this area has been presented
by Spohr [19]. A hyphal element was fixed with poly-L-lysine in a flow-through
chamber. This allows for the measurement of the influence of substrate condi-
tions on the kinetics of morphological change in a steady-state continuous cul-
ture with one hyphal element. This work will be mentioned again in Sect. 4.2.3.
4.1.3
Staining
Another technique that has contributed to the structuring of models is the use
of staining. This has a very long history in microbiology, e.g. the Gram stain, in
which cationic dyes such as safranin, methylene blue, and crystal violet were
mainly used. Nowadays, new fluorescent dyes and/or immuno-labelled com-
pounds are also being used [17, 20], allowing observation of the internal
structure of the hyphae. A few examples are listed in Table 2:
The Morphology of Filamentous Fungi
9
Table 2.
Staining
Dye What does it show?
Neutral red Apical segments
Methylene blue/Ziehl fuchsin Physiological states in P. c hr y so g enum
Acridine orange (AO) fluoresc. RNA/DNA (single or double stranded)
Bromodeoxyuridine (brdu) fluoresc. Replicating DNA
Neutral red Empty zones of the hyphae
Methylene blue/Ziehl fuchsin
Applications in morphology have been mentioned [17, 20]. Several examples
are:
– Distinction between dormant and germinating spores; location of regions
within hyphae – as well as in pellets – with or without protein synthesis (AO).
– Propagation in hyphal elements (BrdU) in combination with fluorescent
antibodies).
– These techniques contribute to the setup and validation of structured
models.
– Measurement of NAD(P)H-dependent culture fluorescence, e.g. for state
estimation or process pattern recognition, is also possible [21].
4.1.4
Micro-Electrodes
As has already been mentioned in Sect. 3a (Table 1), filamentous fungi, among
others, can occur as pellets or as a layer on a support. This has both advantages
and disadvantages. An example of the latter is limitation of mass transfer
and, therefore, a decrease in conversion rate within the pellet or layer compared
with the free mycelium. The traditional chemical engineering literature had
developed mathematical models for this situation long before biotechnology
came into existence [22] and these models have been successfully applied by a
whole generation of biotechnologists. The development of microelectrodes for
oxygen [23], allowing detailed measurements of oxygen concentrations at every
position within pellets or layers, opened the way to check these models.
Hooijmans [24] used this technique to measure the O
2
profiles in agarose pellets
containing an immobilized enzyme or bacteria. Microelectrodes have also been
used to measure concentration profiles of O
2
and glucose (Cronenberg et al.
[25]) as well as pH and O
2
profiles [26] in pellets of Penicillium chrysogenum.
These measurements were combined with staining techniques (AO staining
and BrdU immunoassay). This resulted in interesting conclusions regarding a
number of physiological processes in the pellet.
Much of what has been mentioned above about methods , such as staining
and microelectrodes, has been combined in Schügerl’s review [20]. This
publication also discusses a number of phenomenological aspects of the in-
fluence of environmental conditions (z), including process variables, on
morphology and enzyme production in filamentous fungi, mainly Aspergillus
awamori.
4.2
Models
4.2.1
Introduction
A majority of the models describing the morphogenesis of filamentous fungi
deal with growth and fragmentation of the hyphal elements. Structured models
have been used from early on. A number of them will be shown in this
10
N.W.F. Kossen
paragraph, but some physiological mechanisms of cell wall formation are pre-
sented first
The basis for mechanistic, structured, mathematical models describing the
influence of growth on the morphogenesis of fungi is physiology. At least, the
basic assumptions of the model should not contradict the physiological facts.
Therefore, a brief overview of the physiology of growth, based mainly on a
publication of Gooday et al. [27], is presented here. Emphasis is on growth of
Ascomycetes and Basidiomycetes, comprising Penicillium and Aspergillus, inter
alia. In other fungi, the situation may be different.
Growth of fungi manifests itself as elongation – including branching – of the
hyphae, comprising extension of both wall and cytoplasm with all of its
structural elements: nuclei, ER, mitochondria and other organelles. The
morphology of fungi is determined largely by the rigid cell wall [28]; therefore,
this introduction is limited to cell-wall synthesis.
Cell-wall synthesis in hyphae is highly polarized, because it occurs almost
exclusively at the very tip, the apex.
4.2.1.1
Building Blocks
The major components of the cell wall are chitin and glucan. Chitin forms
microfibrils and glucan the matrix material in between them. The resulting
structure is very similar to glass-fiber reinforced plastic.
Vesicles, containing precursors for cell wall components and enzymes for
synthesis and transformation of wall materials, are formed at the endo-
plasmatic reticulum (ER), along the length of the active part of the hyphae. The
concentration of vesicles in the hyphal compartment increases gradually from
base to tip by about 5% by volume at the base, to 10% at the tip, with the
exception of the very tip, where a rapid increase in the vesicle concentration is
observed. At that point, up to 80% by volume of the cytoplasm may consist of
vesicles.
4.2.1.2
Transport Mechanisms
This subject deserves some attention, because it is a common mechanism in all
polarized growth models. Vesicles are transported to the tip by mechanisms
that are still obscure. A number of suggestions for this transport mechanism
have been summarized [27]
1. Electrophoresis due to electropotential gradients.
2. A decline in concentration of K
+
pumps towards the tip, resulting in a
stationary gradient of osmotic bulk flow of liquids and vesicles to the tip.
3. A flow of water towards the tip, due to a hydrostatic pressure difference
within the mycelium.
4. Cytoplasmic microtubules guiding the vesicles to the tip.
5. Microfilaments involved in intracellular movement.
The Morphology of Filamentous Fungi
11
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