Art 1

Biotechnology Advances 22 (2004) 189 259
www.elsevier.com/locate/biotechadv
Review
Fungal morphology and metabolite production in

submerged mycelial processes
Maria Papagianni *
Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine,
Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece
Accepted 29 September 2003
Abstract
The use of fungi for the production of commercial products is ancient, but it has increased rapidly
over the last 50 years. Fungi are morphologically complex organisms, differing in structure at
different times in their life cycle, differing in form between surface and submerged growth, differing
also with the nature of the growth medium and physical environment. Many genes and physiological
mechanisms are involved in the process of morphogenesis. In submerged culture, a large number of
factors contribute to the development of any particular morphological form. Factors affecting
morphology include the type and concentration of carbon substrate, levels of nitrogen and phosphate,
trace minerals, dissolved oxygen and carbon dioxide, pH and temperature. Physical factors affecting
morphology include fermenter geometry, agitation systems, rheology and the culture modes, whether
batch, fed-batch or continuous. In many cases, particular morphological forms achieve maximum
performance. It is a very difficult task to deduce unequivocal general relationships between process
variables, product formation and fungal morphology since too many parameters influence these
interrelationships and the role of many of them is still not fully understood.
The use of automatic image analysis systems during the last decade proved an invaluable tool for
characterizing complex mycelial morphologies, physiological states and relationships between
morphology and productivity. Quantified morphological information can be used to build
morphologically structured models of predictive value. The mathematical modeling of the growth
and process performance has led to improved design and operation of mycelial fermentations and has
improved the ability of scientists to translate laboratory observations into commercial practice.
However, it is still necessary to develop improved and new experimental techniques for
understanding phenomena such as the mechanisms of mycelial fragmentation and non-destructive
measurement of concentration profiles in mycelial aggregates. This would allow the establishment of
* Fax: +30-2310-999829.
E-mail address: mp2000@vet.auth.gr (M. Papagianni).
0734-9750/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2003.09.005
190 M. Papagianni / Biotechnology Advances 22 (2004) 189259
a process control on a physiological basis. This review is focused on the factors influencing the
fungal morphology and metabolite production in submerged culture.
D 2003 Elsevier Inc. All rights reserved.
Keywords: Filamentous fungi; Morphology; Shear effects
1. Introduction
The use of fungi for the production of commercially important products has increased
rapidly over the past half century. The exploitation of fungi by man is not a recent
phenomenon. Numerous examples which indicate that man has been aware of the value of
fungi since the dawn of civilization are known. The fermentation of alcoholic beverages,
practiced in the days of the Pharaohs, is one of the earliest known examples of the
exploitation of the biochemical activities of a fungus by humans. The use of yeast to
leaven bread also dates back to biblical times. The higher fungi have long been used by
man. Fruit bodies of basidiomyces and ascomyces have been collected and eaten by
civilizations throughout the world (Hayes and Nair, 1978). The production of alcoholic
beverages, biomass and the manufacture of therapeutic compounds, together with the
production of simple organic compounds, still remain the major fields in which fungi are
used.
Apart from the rather unsophisticated techniques used for the production and mainte-
nance of yeast in the brewing and baking industries, the deliberate growth of fungi for
commercial purposes did not commence until well into the twentieth century. The
development of the sulfite process for the production of glycerol by a yeast fermentation,
which was widely used during World War I, probably marks the beginning of industrial
mycology. However, it is since the advent of the submerged culture techniques used in the
penicillin fermentation that the greatest expansion in the use of fungi in the industry has
taken place. At present, increasing numbers of commercially important products are being
produced from fungi. Tables 1 3 present the major classes of filamentous fungal enzymes,
antibiotics and organic acids of commercial importance and some of their sources. For
extensive lists of industrial products and the preferred names of the filamentous fungi that
produce them, the reader is referred to the ATCC names of industrial fungi (Jong et al.,
1994).
Filamentous fungi are morphologically complex microorganisms, exhibiting different
structural forms throughout their life cycles. The basic vegetative structure of growth
consists of a tubular filament known as hypha that originates from the germination of a
single reproductive spore. As the hypha continues to grow, it frequently branches
repeatedly to form a mass of hyphal filaments referred to as mycelium. When grown in
submerged culture, these fungi exhibit different morphological forms, ranging from
dispersed mycelial filaments to densely interwoven mycelial masses referred to as pellets.
The particular form exhibited is determined not only by the genetic material of the fungal
species but also the nature of the inoculum as well as the chemical (medium constituents)
and physical (temperature, pH, mechanical forces) culturing conditions (Atkinson and
Daoud, 1976; Kossen, 2000). With some fungal fermentations a particular morphological
M. Papagianni / Biotechnology Advances 22 (2004) 189259 191
Table 1
Major classes of filamentous fungal enzymes of commercial importance and some of their sources
Enzyme Sources
a-Amylase As. niger, As. oryzae, As. awamori, Au. pullulans, Rhizopus oryzae,
Trichoderma viride
h-Amylase R. niveus
Amyloglucosidase As. niger, As. oryzae, As. awamori, A. phoenicis, Au. pullulans, R. niveus,
Rhizop. oryzae
Catalase As. niger, Eupenicillium javanicum, Penicillium vitale
Cellulase As. niger, A. soyae, As. terreus, P. citrinum, P. funiculosum,
T. longibrachiatum, T. reesei, Tr. viride
Dextranase A. carneus, Chaetomium gracile, P. funiculosum, P. lilacium, P. pinophilum
a-Galactosidase As. awamori, As. niger, As. oryzae, Mortierella vinaceae, P. dupontii
h-Galactosidase As. awamori, As. nidulans, As. niger, As. oryzae, Fusarium oxysporum,
N. crassa, P. funiculosum
h-Glucanase Acremonium persicinum, As. niger, As. oryzae, E. javanicum, G. candidum,
Tr. viride, T. reesei
Glucoamylase As. niger, As. awamori, As. oryzae, Au. pullulans, Rhizop. oryzae, R. niveus
Glucose aerohydrogenase As. niger
Glucose oxidase As. niger, E. javanicum, P. amagasakiense, P. simplicissimum, P. vermiculatum
a-Glucosidase As. awamori, Aspergillus flavus, A. fumigatus, As. niger, Mu. circinelloides
a-D-Glucosidase As. niger
h-Glucosidase As. niger, As. oryzae, T. reesei
Hemicellulase As. niger, As. oryzae, A. phoenicis, T. longibrachiatum, Tr. viride
Hesperidinase As. niger
Invertase As. awamori, As. niger, As. oryzae, N. crassa
Lipase As. niger, As. oryzae, G. candidum, Humicola sp., Rhizomucor miechei,
Rhizomucor sp., R. arrhizus, R. niveus, P. roqueforti
Pectinase A. alliaceus, As. niger, Aspergillus sp., Rhizop. oryzae, Rhizopus sp.,
Sclerotinia libertiana
Phytase As. niger, A. ficuum
Protease As. niger, A. melleus, As. oryzae, A. saitoi, P. dupontii, Penicillium sp.,
Rhizopus sp.
Rennets Cryphonectria parasitica, R. meihei, R. pusillus
Tanase As. niger, A. tamarii
Xylanase T. reesei
form may be preferred to achieve maximal performance. Filamentous growth of Asper-

gillus niger is preferred for pectic enzyme production, whereas the pelleted form is
preferred for citric acid production (Steel et al., 1954; Kristiansen and Bullock, 1988).
Table 2
Major classes of filamentous fungal antibiotics of commercial importance and some of their sources
Antibiotics Sources
Cephalosporins Acremonium chrysogenum, A. kiliense, Ac. persicinum, Fusarium solani, Nectria lucida
Cyclosporins F. solani, Tolypocladium geodes, T. inflatum, Trichoderma polysporum
Echinocandin B As. nidulans, A. rugulosus
Fusidic acid Calcarisporium arbuscula, F. coccophilum, Mortiella ramannianua
Griseofulvin Penicillium aurantiogriseum, P. griseofulvum, P. italicum
Penicillins Ac. chrysogenum, Ac. persicinum, As. flavus, A. giganteus, As. nidulans, As. niger,
As. oryzae, A. parasiticus, Aspergillus sp., P. baculatum, Pe. chrysogenum, P. turbatum
Table 3
Major classes of filamentous fungal organic acids of commercial importance and some of their sources
Organic acid Sources
Citric acid Aspergillus citricus, A. clavatus, As. niger, A. phoenicis, Penicillum decumbens,
P. isariiforme, Tr. viride
Fumaric acid Rhizop. oryzae, Rhizop. stolonifer
Gluconic acid A. carbonarius, As. niger, As. oryzae, A. wentii, E. javanicum, Pe. chrysogenum,
P. luteum, P. simplicissimum
Itaconic acid A. itaconicus, As. terreus
Kojic acid A. candidus, As. flavus, As. oryzae, A. parasiticus, A. tamarii, P. jensenii,
R. microsporus, Rhizop. oryzae, Rhizop. stolonifer
D-Lactic acid Rhizop. oryzae
L-Malic acid A. atroviolaceus, As. citricus, As. flavus, As. niger, A. ochraceus, As. oryzae,
A. wentii, Rhizop. oryzae
D-Araboascorbic acid Pe. chrysogenum
Erythorbic acid P. griseoroseum
Also, Konig et al. (1982) showed that the pelleted form of Penicillium chrysogenum was
desired for production of penicillin in a tower bioreactor.
The change in morphology during growth affects nutrient consumption and oxygen
uptake rate in submerged culture (Schugerl et al., 1983). Also, the morphological growth
forms can have a significant effect on the rheology of the fermentation broth and thus the
performance of the bioreactor. Filamentous growth results in highly viscous broths with
non-Newtonian, pseudoplastic flow behavior (Kristiansen and Bullock, 1988). The high
viscosity has a negative impact on the mass transfer properties of the broth, specially the
gas liquid mass transfer rate. Pelleted growth exhibits low viscosities and approach
Newtonian flow behavior (Chain et al., 1966). As expected, higher power inputs are
required for filamentous versus pelleted growth in achieving adequate agitation and
oxygen transfer. However, frequently, the central region of larger pellets undergoes
autolysis as a result of nutrient limitation. This autolysis can have a significant effect
on both cellular metabolism and product synthesis (Philips, 1966; Elmayergi et al., 1973).
Thus, small pellets as opposed to large ones would generally be considered desirable in
developing filamentous fungal fermentations.
The successful production of a fungal metabolite requires a detailed knowledge of the
growth characteristics and the physiology of the fungus in question. Not only does the
production of different metabolites require different physiological conditions but also each
fungus is unique in its anatomical, morphological and physiological development. Thus,
for each fermentation, the precise physiological conditions and the correct stage of
development must be established for maximal product formation. In other words, the
control of the form of these microorganisms is a real issue that needs great 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 more than five decades,
while the contribution from physiologists is growing steadily as more and more details of
the transport processes and the kinetics involved in the morphogenesis become known.
The outcome is an impressive landscape of results about fungal morphology and
metabolite overproduction and this extensive review is about this landscape. It is a survey
of all the main lines of development of a very interesting area of biotechnology research:
growth mechanisms, dynamics of mycelial aggregation, transport phenomena, fragmen-
tation, growth in submerged culture and process parameters, quantification of morphology
and modeling of the growth and product formation.
2. Growth mechanisms in filamentous fungi
Under appropriate conditions, the vegetative mycelium gives rise to a reproductive

mycelium that supports the production of reproductive spores. The type of sporulation and
the morphology of the spores and spore-bearing structures are key characteristics in fungus
identification. The fungal spore, therefore, can be considered as the beginning and the end
of the differentiation process. In mycelial fungi, hyphae extend by a highly polarized
process of cell extension known as tip extension. As the tip extends, periodic branches are
formed at or near the apex of the tip. These branches also extend in a polarized manner as
new tips. The two processes of tip extension and branching permit the organism to
colonize and efficiently utilize the substrate, and they are rarely found in organisms other
than fungi, leading to their being termed hallmarks of the fungal kingdom (Heath, 1995).
2.1. Hyphal tip extension
Spore germination results in the formation of a germ tube, whose early growth is
supported by mobilization and utilization of storage compounds in the spore. As the germ
tube develops, it contributes to biosynthesis and extension by uptake and metabolism of
nutrients from the medium. Hyphal extension is an extreme example of polarized cell
growth since cell extension is restricted to a narrow zone defined by the tapering hyphal
apex. The rate of wall synthesis in the apical 1 Am of a hypha may be 50 times that 50 Am
behind it (Gooday, 1971; Gooday and Trinci, 1980). Although this apical growth pattern
has been known for more than a century (Reinhardt, 1892), the underlying mechanisms
that account for polarized hyphal growth are not yet understood. Extension rate accelerates
as germ tube length increases and growth becomes autocatalytic.
Tip growth is a process that has many similarities in diverse walled cells such as
hyphae, pollen tubes and hairs. Much research has focused on the mechanism of tip
extension. Analysis of the literature on fungi, with selected comparison with other tip-
growing plant cells, shows that the growth rate and morphology of hyphae are sensitive to
factors, which influence intracellular Ca2 +. These factors include variations in extracel-
lular Ca2 + concentrations, Ca2 + ionophores, inhibitors of Ca2 + transport and buffers
introduced into the cytoplasm (Jackson and Heath, 1993; Parton et al., 1997). The effects
of these agents appear to be mediated by a tip-high gradient of cytoplasmic free Ca 2 +,
which is obligatorily present and involved in active growth. Most recent observations
agree that the gradient is very steep, declining rapidly within 10 20 Am of the tip (Jackson
and Heath, 1993). This gradient seems to be generated by the combined effects of an
influx of Ca2 +, via plasma membrane, possibly stretch-activated channels localized in the
hyphal tip, and subapical expulsion or sequestration of these ions. It is suggested that the
regulation of the Ca2 + gradient, in turn, modulates the properties of the actin-based
component of the cytoskeleton, which then controls the extensibility, and, possibly the
synthesis of the hyphal apex (Heath and Geitmann, 2000).
Mitosis, septation and branching have been studied in undifferentiated mycelia and
main hypha of Aspergillus nidulans, which forms incomplete septa (Fiddy and Trinci,
1976). Following spore germination, nuclei divide synchronously until germ tube hyphae
contain either 8 or 16 nuclei. Mitosis occurs when the volume of cytoplasm per nucleus is
about 60 Am3. Intercompartment development will not synchronize and mitosis in the
mycelium as a whole eventually becomes asynchronous. During the stage of asynchronous
compartment development, the nuclei, septa, branches and total length of undifferentiated
mycelia are increased exponentially at approximately the same specific rate (Fiddy and
Trinci, 1976). The mean time required for the formation of a group of septa is reported to
be about 9 min. Each hypha forms up to nine septa in a group at a time. The mean interval
between successive cycles of septation in a hypha is approximately the same as the
doubling time of the organism. A very high correlation coefficient was reported between
septation, branch initiation and most intercalary compartments initially formed a single
branch (Fiddy and Trinci, 1976).
Synchronous mitosis has also been observed during the early stages of growth of As.
nidulans from spores (Rosenberger and Kessel, 1967) and in apical compartments of main
hypha of Alternaria solanis (King and Alexander, 1969) and As. nidulans (Clutterbuck,
1970). In both species, mitosis in main hyphae is followed after a small interval by
septation. Such observations suggest the existence of a duplication cycle in apical
compartments which is analogous to the cell cycle of uninucleate cells. The main events
of this duplication cycle are the following: (1) reduction of the apical compartment to
almost half of its length by septation; (2) the newly formed apical compartment continues
to increase in length at a linear rate; (3) the volume of cytoplasm per nucleus increases up
to a critical ratio when the nuclei are induced to divide more or less synchronously; and (4)
mitosis followed by septation, which is completed when the apical compartment is about
twice its original length (Fiddy and Trinci, 1976).
Consequently, hyphal length increases exponentially at a constant specific rate which
may be significantly greater than the maximum specific growth rate in the equivalent
liquid medium because of contribution from endogenous spore reserves. Exponential
growth cannot proceed indefinitely and extension rate eventually reaches a constant value,
i.e., extension is linear. This occurs when the tip can no longer incorporate the increasing
amount of material being supplied or, more likely, when transport of material from regions
distant from the tip is limited. The latter may result from a breakdown in apical polarity,
such that transport occurs at a rate less than the hyphal extension rate (Prosser, 1995). A
more common explanation is the formation of septa which prevent transport of material to
the tip. Extension rate will then be dependent on biosynthesis within the apical
compartment only (Prosser, 1995).
Both hyphal extension and the length of hypha supporting tip growth vary considerably
within fungi. For example, linear growth occurs in Rhizopus stolonifer when the germ tube
is only 40 Am in length, whereas exponential growth of sporangiophores of Phycomyces
blakesleeanus continues until they are 4 mm in length (Trinci, 1969). Hyphal extension
rate will depend on the amount of material supplied to the tip and on the surface area of the
extension zone, which will increase with hyphal diameter and with increased tapering of
the tip. Little work has been done on the relationship between tip shape and extension rate,
but the extension rate is generally found to increase with hyphal diameter within a single
organism. The relationship is not always well defined. Zhu and Gooday (1992) found a
direct relationship between hyphal extension rate and the square of the diameter for hypha
of Botrytis cinerea, suggesting extension rate to be dependent on the rate of supply of
material to the tip. In Mucor rouxii, however, although extension rate increased with
diameter, no precise quantitative relationship could be identified (Zhu and Gooday, 1992).
2.2. Branching
2.2.1. Exponential growth

Microbial growth is normally associated with exponential increases in biomass when
conditions are favorable for growth and when nutrients are in excess. Exponential growth
requires that all, or a constant percentage of the mass of the microorganisms present,
contribute to new growth. If all growth takes place in the apical segment of the hyphae and
the individual hypha extend at a constant linear rate, then exponential growth will require
that new branches are produced at a rate proportional to the rate of increase in cell mass.
The first branch is usually formed from the germ tube towards the end of the period of
exponential extension (Prosser, 1995). This rarely affects the extension rate of the parent
hypha, even though early growth of the branch is supported by the material provided by
the parent hypha. Branch length increases at an accelerating rate before reaching a constant
value, which, in young mycelia, is equal to that of the parent hypha. In terms of growth
kinetics, branch formation may be considered equivalent to cell division in unicellular
organisms.
It has been reported that the frequency of branching in As. nidulans was proportional to
the specific growth rate when growth on different media was compared (Katz et al., 1972).
Individual hyphae may also grow exponentially rather than linearly in certain circum-
stances. Exponential growth has been reported during germ tube outgrowth in As. nidulans
and also in the critical period of growth after branch formation. Fungal growth continues at
a rate proportional to the length of the hypha until it reaches the maximum characteristic of
the organism (Katz et al., 1972). The rate of apical growth can be considered to depend
upon the biosynthetic capacity of the hypha. When it exceeds the capacity of the apical
region to utilize the products of biosynthesis a new branch is initiated. Exponential growth
therefore occurs through an exponential increase in the number of branches, each of which
extends at the same constant rate. This was first demonstrated experimentally for Geo-
trichum candidum, Neurospora crassa, Pe. chrysogenum and As. nidulans by Trinci
(1974). This work also demonstrated that the specific rates of increase in total mycelial
length and the total number of branches were equal to the specific growth rate of these
organisms growing in equivalent media but in liquid culture, with biomass concentrations
determined by the dry weight measurements.
A linear rate of extension imposes a restriction to overall growth, which in unicellular
organisms proceeds at an exponential rate, and this is solved by branch formation: Smith
(1924) observed an exponential increase in the combined lengths of parent and branch
hyphae, though individual hyphae were extending linearly, and Plomley (1959) observed
an exponential increase in total mycelial length in Chaetomium globosum.
Trinci (1974) studied early colony growth kinetics of As. nidulans, N. crassa, Mucor
hiemalis and G. candidum on agar surface and found that all four species showed similar
kinetics. Four features are exhibited: (1) total mycelial length increases exponentially for at
least 11 h; (2) the first branch is formed before the germ tube hypha is grown linearly,
though possibly not before the deceleration phase; (3) branch production is initially
discontinuous, but after 10 branches have been formed the number increases exponentially
with a specific rate equal to that for the total mycelial length; and (4) this specific rate is
found to be equal to the specific growth rate during exponential growth in liquid medium,
measured in terms of dry-weight increase. The results in that study (Trinci, 1974)
supported the hypothesis that mycelial growth involves the duplication of a growth
unit containing a tip and a certain mean length of hyphae. Plomley (1959) first suggested
that filamentous fungi have a growth unit, which is duplicated at a constant rate (linear
growth) while the whole mycelium grows exponentially.
Caldwell and Trinci (1973) studied the hyphal growth unit (HGU) of G. candidum
grown in batch culture. The mycelium grew in the filamentous form while hyphal
fragmentation occurred during growth. During the early part of the stationary phase, the
hyphal fragments had a mean length of 300 400 Am. The dry weight, total hyphal length,
number of tips and the turbidity of the culture increased exponentially in a similar trend to
the specific growth rate. The results suggested a functional unit of growth consisting of a
hyphal tip associated with a constant mean length of hyphae. The hyphal growth unit, G,
was defined as the ratio of total hyphal length to the total number of branches and is
therefore the average length of hypha associated with a growing tip. For G. candidum, this
unit was about 100 Am and remained constant, while the specific growth rate varied by
changes in temperature and the source of carbon (Steele and Trinci, 1975). This unit
therefore represents the mean length of the hypha required to support tip growth, while the
peripheral growth zone represents the maximum length. Whereas the peripheral growth
zone and the specific growth rate are related by the maximum rate of extension, the hyphal
growth unit ( G) is related to the specific growth rate by the mean extension rate (Steele
and Trinci, 1975). This is calculated as:
2Ht H0
E 1
B0 Bt
where E is the mean extension rate, H0 and Ht represent the total hyphal lengths 0 and 1
h later, and B0 and Bt represent the respective numbers of hyphal tips. This is related to the
specific growth rate by the equation:
E Gl 2
Values of G increase during mycelial development and then oscillate until branches are
formed continuously, when G reaches a constant value. Thus, whereas the specific growth
rate provides an indication of the kinetics of branch formation, the hyphal growth unit
provides information on the branching density, increasing as branching becomes sparser
(Bull and Trinci, 1977). The hyphal growth unit is a property of the mycelium and its
mathematical properties and its relationship to other hyphal and mycelial growth
parameters have been extensively analyzed by Kotov and Reshetnikov (1990). The
relative constancy of the hyphal growth unit length indicates the existence of a regulatory
mechanism, in that a branch is formed somewhere in the mycelium when the value of G,
characteristic of the organism and growth conditions, is exceeded.
The above discussion refers to hyphae in young developing mycelia. As a colony
forms, the kinetics of hyphal growth and branching at the center of the colony will be
influenced by reduction in concentrations of nutrients and oxygen, accumulation of
inhibitory end products, production of secondary metabolites and changes in environ-
mental factors such as pH (Prosser, 1995). The relative importance of these factors on
biomass formation in developing mycelia is unknown, but they will obviously lead to a
decrease in growth rate, with associated effects on branch formation and the morphology
of freely dispersed hyphal elements.
Trinci (1971) has related the rate at which fungal colonies grew on agar to their growth
rate in submerged culture by using the concept of a peripheral annulus in which the
mycelium grows exponentially. By measuring the width of the peripheral growth zone (w)
and the linear colony radial growth rate (Kr), Trinci used the equation:
dr
lw Kr 3
dt
to calculate the specific growth rate (l) for nine species of fungi. He found that the
computed value was equal to the specific growth rate measured in submerged culture. The
colony radial growth rate would also be affected by the hyphal density or branching
frequency, which may be a determinant of the width of growth zone. Bainbridge and Trinci
(1971) noted that a mutant of As. nidulans was more branched than its parent organism
under the same conditions. It had an almost identical specific growth rate in submerged
culture but a substantially lower colony radial growth rate and a smaller peripheral growth
zone when grown on agar.
Eq. (3), used by Trinci (1971) and Pirt (1967) to express the linear colony radial growth
can be re-written as:
dr
lbk 4
dt
where b is the hyphal density and k a constant. Hyphal density can be directly related to
branching frequency if the internode length is used or the total hyphal length divided by
the number of growing tips. Morisson and Righelato (1974) have used Eq. (4) to relate the
measurements of the specific growth rate and hyphal branching in submerged culture to
colony radial growth rate on agar. Their results suggest that the width of the peripheral
growth zone of colonies growing on agar could change as the specific growth rate changes.
Recently, the use of image analysis in the study of Christiansen et al. (1999) permitted
an on-line determination of the growth kinetics of the single hyphae of Aspergillus oryzae
in a flow-through cell at different glucose concentrations. The tip extension rate of the
individual hyphae were described with saturation type kinetics with respect to the length of
the hyphae. It was observed that the maximum tip extension rate was constant for all
hyphae measured at the same glucose concentration, whereas the saturation constant for
the hyphae varied significantly between the hyphae even within the same hyphal element.
The tip extension rate decreased temporarily when apical branching occurred. The number
of branches formed on a hypha was proportional to the length of the hypha that exceeds a
certain minimum length required to support the growth of a new branch.
Attempts to understand tip growth and branching have employed various approaches.
Cytological analysis has identified several key substances involved in the process, most
notably actin and calcium (Heath, 1995; Grinberg and Heath, 1997; Hyde and Heath,
1997; Jackson and Heath, 1993; Kaminsky and Heath, 1996). Ultrastructural studies have
demonstrated the importance of tip-growth vesicles (Bartnicki-Garcia, 1990; Bartnicki-
Garcia et al., 1989; Prosser and Trinci, 1979; Trinci, 1969). Genetic analysis of induced
and naturally occurring mutants has identified over 100 loci that encode products that can
affect tip growth and branching in N. crassa (Perkins et al., 1982; Scott, 1976). Tip
extension proceeds via the polarized exocytosis of tip-growth vesicles (Bartnicki-Garcia,
1990; Bartnicki-Garcia et al., 1989; Prosser and Trinci, 1979; Scott, 1976). Vesicle
deposition appears to be orchestrated by the Spitzenkorper, a loose collection of vesicles
near the hyphal apex (Grove and Bracker, 1970; Howard, 1981).
2.2.2. Modeling tip growth and branching

Any comprehensive model for tip growth and branching must incorporate the main
phenomenology associated with these processes. Tip extension occurs via apical exocy-
tosis of tip-growth vesicles manufactured subapically and transported to the tip. Thus, the
tip concentration of vesicles and any other tip extension factors depends on the balance
between the rates of supply (synthesis and transport) and consumption (either deposition
or destruction). Branching, which is triggered by the rate of accumulation at the tip, is
proportional to the excess of vesicle production over tip deposition. Branching has been
shown to be at least partially controlled by factors at or proximal to the previous branch
point (Watters et al., 2000a).
The vesicular basis of hyphal growth and branching was incorporated into a model by
Trinci (1974). A key element of this model was the hyphal growth unit. The initiation of a
new branch has been proposed to be controlled by changes in the cytoplasmic volume, so
that branching occurs when a critical value of the mean hyphal growth unit is attained. In
this way, the protoplasm considerably distant from the growing tip could have a
contributing role in branch initiation. In a further elaboration of this model, Prosser and
Trinci (1979) proposed that the concentrations of vesicles and nuclei regulate the increase
in hyphal length and the occurrence of branches and septa. Prosser (1979) has also
developed a model for hyphal growth and branching, which relates cytological events to
growth kinetics. The model quantifies qualitative theories of hyphal growth that vesicles
containing wall precursors and/or enzymes required for wall synthesis are generated at a
constant rate throughout the mycelium and travel to the tips where they fuse with the
plasma membrane. This is followed by the liberation of their contents into the wall and
increasing of the surface area of the hyphae to give elongation. The hypothesis states that
there is a duplication cycle in the hyphae, which is equivalent to the cell cycle observed in
unicellular organisms.
Watters et al. (2000b) showed that in N. crassa the distribution of branch intervals is
independent of tip extension rate, as controlled by temperature. Although rapid cooling
disturbs this distribution, the normal default distribution of branch intervals was soon
restored at the new temperature. Thus, the statistical distribution of branch-to-branch

intervals along a hypha seems to constitute a homeostatic set point. The lack of
dependence of branch distribution on temperature (or growth rate) was explained in the
work of Watters and Griffiths (2001) who developed and tested a model in which the
formation of a lateral branch in N. crassa was determined by the accumulation of tip-
growth vesicles caused by the excess of the rate of supply over the rate deposition at the
apex. The model explains how branching can be independent of tip extension rate under
steady-state conditions while responding dramatically to changing conditions.
Apart from the above described vesicular models, many other different approaches have
been adopted in modeling the early growth of filamentous fungi. Hyphal population
models, in which the average properties of all hyphae within a mycelium are considered,
have been described by Edelstein (1982), and Edelstein and Segal (1983). Stochastic
models, which take account of natural variability within a system and the consequences of
such variability, have been described by Hutchison et al. (1980). Yang et al. (1992)
proposed a model for mycelial growth which combines the model of Prosser and Trinci
(1979) with the stochastic approach adopted by Hutchison et al. (1980) to describe the
direction of tip growth and branching, and the site of branch formation.
A link between mycelial population models and macroscopic growth was recently
provided by the model of Viniegra-Gonzales et al. (1993), which is based on the
mathematics of symmetric trees. The model considers the mycelium as a population of
interbranch segments of average length Lav, defined as:
Lt
Lav 5
Ns
where Lt is the total hyphal length and Ns the number of segments and equals 2(Nt 1),
where Nt is the total number of tips. This model predicts that the specific growth unit G
will be greater at higher levels of branching. This has been suggested by Caldwell and
Trinci (1973), but model expressions provide quantitative relationships which show good
agreement with the experimental data on growth of young mycelia of G. candidum. This
model is developed further to allow prediction of the specific growth rate, determined in
terms of biomass, incorporating a frequency distribution for the proportion of biomass
which is inactive. This macroscopic model predicts the observed difference in specific
growth rate between germ tubes and exponentially growing mycelia. It is also used to
predict growth during batch culture of As. niger and provides a new approach to
descriptions of mycelial growth and it is valuable in linking morphological properties to
kinetics. Testing of the model will be facilitated with image analysis techniques now used
for quantification of mycelial morphology.
Quantified morphological information obtained with on-line image analysis was
employed in the model reported by Christiansen et al. (1999) for the early growth of
As. oryzae. The kinetics observed in that study were used to simulate the outgrowth of a
hyphal element from a single spore using a Monte Carlo simulation technique. The
simulation showed that the observed kinetics for the individual hyphae resulted in an
experimentally verified growth pattern with exponential growth in both total hyphal length
and number of tips.
3. Dynamics of mycelial aggregation
In submerged cultures, many filamentous microorganisms tend to aggregate and grow

as pellets the compactness of which varies considerably. Pellets are spherical or ellipsoidal
masses of hyphae with variable internal structure, ranging from loosely packed hyphae,
forming fluffy pellets, to tightly packed, compact, dense pellets (Yanagita and Kogane,
1963). Wittler et al. (1986) proposed the existence of four regions. The outer region
consists of viable hyphae and surrounds a layer of hyphae showing signs of autolysis. In
hollow pellets, a third layer is found containing hyphae with irregular wall structure, while
the center of the pellet contains no recognizable mycelia. The density of hyphae within
pellets is of significance for diffusion of nutrients and oxygen to the mycelial biomass,
with consequent effects on growth, particularly at the center of compact pellets.
Control of mycelial morphology in fermentation is often a prerequisite for industrial
application. In some processes, free mycelia are required, as in the production of penicillin
from Pe. chrysogenum. Whereas in other processes, pellets or immobilized cells are
preferred for increased yields, as in the production of citric acid from As. niger (Solomons,
1980; Metz, 1976). The disadvantages of dispersed mycelial growth have been discussed
and include a reduction in efficiency of mixing and oxygen supply as well as increased
wall growth. These problems may be solved to some extent by growth in the form of
pellets, which also improve harvesting through improved filtration characteristics of the
broth. The methodology of pellet formation for several microorganisms has been reviewed
(Steel et al., 1954; Whitaker and Long, 1973; Brown and Zainudeen, 1977; van Suijdam et
al., 1980).
Application of mycelial aggregates to metabolite production depends upon obtaining
uniform pellets of a desired size. This is not easily accomplished, since many factors
influence pellet formation. Among the factors influencing cellular aggregation are
inoculum size, type and age (Steel et al., 1954; Calam, 1987; Gerlach et al., 1998;
Papagianni et al., 1999d, 2001; Papagianni and Moo-Young, 2002), genetic factors and
ability to produce bioflocculants (Prosser and Tough, 1991; Braun and Vecht-Lifshitz,
1991), medium composition (Charley, 1981; Gerlach et al., 1998; Papagianni et al.,
1999d; Papagianni, 1999), biosynthesis or addition of polymers, surfactants and
chelators (Pirt and Callow, 1959; Jones et al., 1989; Papagianni, 1999), shear forces
(van Suijdam and Metz, 1981; Braun and Vecht-Lifshitz, 1991), temperature and
pressure (Smith and Anderson, 1973; Bull and Bushell, 1976) and medium viscosity
(Gerlach et al., 1998; Papagianni et al., 2001). The morphology of a filamentous fungus
developing in any fermentation system could be considered as a final result of
competing influences, an equilibrium between forces of cohesion and disintegration.
Shear forces may be unambiguously assigned the role as disintegrating factors. At pH
values above 5.5, cell walls of most microorganisms are negatively charged, tending to
cause separation of aggregating cells by electrostatic repulsion. This may be suppressed
by an increase in ionic strength, or bridging cells with Ca2 + ions (Braun and Vecht-
Lifshitz, 1991). Addition of polycations usually induces aggregation, whereas poly-
anions suppress it (Elmayergi et al., 1973; Elmayergi, 1975; Domingues et al., 2000).
The chemico-thermodynamical basis for the effect of growth conditions on the pellet
formation of As. niger was investigated by Ryoo and Choi (1999). The surface
thermodynamic balance between fungal cell and liquid media was found to be
responsible for pellet formation, since the Gibbs free energy of pellet formation of
the initial culture media ( 73 to 81 ergs/cm2) were increased to 13 to 46 ergs/
cm2 at 48 h. FTIR analysis showed that factors inducing pellet formation simultaneously
increased the cell wall hydrophobicity of As. niger.
Genetic factors influence the cell wall composition and surface properties and
determine the formation and composition of a slime layer. Genetic and environmental
factors are responsible for the production of surface-active agents and of lectins. Both of
these affect forces of cohesion and/or repulsion between cells. According to the
classification of Takahashi et al. (1958) and Takahashi and Yamada (1959), two types
of pellet formation may be distinguished. Pellet formation in As. niger belongs to the
coagulative type (Yanagita and Kogane, 1963), where spores coagulate while germinating
and give rise to a net of intertwining hyphae. Pellets of Pe. chrysogenum belong to the
non-coagulative type (Metz, 1976), where one pellet is produced from one spore. Spore
coagulation may play a role in pellet formation, provided that trap-nets develop, although
pellets frequently form in the absence of any spore agglutination. Even in As. niger, the
number of pellets equals the number of initial spore clumps only at low power input; with
increased power input, the spore to pellet ratio tends towards unity in As. niger (Vecht-
Lifshitz et al., 1989), while in other pellet-forming organisms, this ratio may reach several
orders of magnitude below unity (Elmayergi et al., 1973).
Assessing the factors that influence pellet formation in filamentous fungi, it is often
difficult to define a mechanism for pellet formation from reported results, as often more
than one parameter is adjusted by changing only one variable. Even for the most studied,
industrially important Aspergillus and Penicillium species reports are contradictory
(Solomons, 1980; Whitaker and Long, 1973; Metz et al., 1979; Gomez et al., 1988).
Attempts to treat pellet formation as a general phenomenon are frequently met by
industrial microbiologists with mistrust. Remarkably, the effect of various fermentation
parameters on pellet formation seems to be quite similar in filamentous systems as
genetically remote as fungi and actinomycetes. For example, in Pe. chrysogenum (Metz,
1976), As. niger (van Suijdam et al., 1980), Streptomyces tendae (Vecht-Lifshitz et al.,
1989) and S. griseous (Braun and Vecht-Lifshitz, 1991), pellets are formed at inoculum
levels below 1011 spores m 3, while at higher inocula, filamentous growth predominates.
Similarly, factors favoring increased growth rates, such as media rich in easily assimilable
nutrients, reduce pellet formation in fungi (Hemmersdorfer et al., 1987) and actinomycetes
(Vecht-Lifshitz et al., 1989). Such observations led to a limitation hypothesis which
suggested that the lack of any nutrient, including oxygen, induces pellet formation
(Hemmersdorfer et al., 1987). Indeed, increased mycelial aggregation was noted as a
consequence of nitrogen limitation in many cases (Vecht-Lifshitz et al., 1989; Hemmers-
dorfer et al., 1987). There are, however, a few reports contradicting the limitation
hypothesis with respect to oxygen. Hockenhull (1980) stressed that pelleted morphologies
predominate in the early life of a culture when oxygen supply is sufficient, while older
cultures tend to be filamentous.
Formation of mycelial pellets is considered, in some instances, a prerequisite for
successful production of certain metabolites, such as the itaconic and citric acids (Metz et
al., 1979; Gomez et al., 1988), and some fungal enzymes such as glucose oxidase (Zetelaki
and Vas, 1968), polygalacturonidase (Hemmersdorfer et al., 1987), phytase (Papagianni et

al., 1999d), and glucoamylase (Papagianni and Moo-Young, 2002). Differentiation of
mycelia during pellet formation results in striking effects on enzyme production. Poly-
galacturonidase synthesis is well associated with the fungal morphology of As. niger. The
more compact the pellet, the greater the polygalactorunidase synthesis. Regardless of the
medium used, an increase of two orders of magnitude in enzyme concentration and rate of
production between the free filamentous mycelium and the pelleted type was observed
(Hemmersdorfer et al., 1987). Similar increases were observed in glucoamylase production
rates by pellets of As. niger (Papagianni and Moo-Young, 2002). Such phenomena may be
related to diffusional limitations in pellets, which either reduce the extent of catabolic
repression in pellets or limit the oxygen supply, preventing this way an oxidative
inactivation of a specific set of enzymes.
The magnitude of the difference between enzyme production in pellets and in free
filamentous mycelia at different concentrations of catabolites (Hemmersdorfer et al., 1987)
seems to indicate the existence of additional factors, such as gradients of metabolic
products in pellets which serve as biological signals (modulators). In fact, the high level of
cell-to-cell interaction and signaling resulting from short diffusional distances in mycelial
aggregates leads to a state of differentiation qualitatively different from that of free
filamentous mycelia. In a review on the factors affecting mycelial aggregation, Braun and
Vecht-Lifshitz (1991) have stated that mycelial aggregates may be viewed not merely as
mechanical conglomerates, but rather as complex differentiated tissues phenotypically
characterized by specific metabolic activities. According to them, the closest analogy to
this concept would be the obvious distinction between the unicellular and multicellular
forms of a slime mould.
4. Growth of fungal pellets
Pelleted cultures are traditionally assumed to follow cube-root kinetics, following the
early observations of Emerson (1950), on growth of N. crassa, and Marshall and
Alexander (1960) who investigated a number of fungi. These kinetics are described by
the equation:
1=3
M 1=3 M0 kt 6
where M represents the biomass concentration and k is a constant. Pirt (1966)

explained these kinetics by considering the heterogeneity within pellets in similar
manner to that within colonies growing on solid substrate: A pellet is considered as a
spherical mass of non-growing mycelia surrounded by an outer shell of active hyphae.
Thus, a pellet is assumed to increase in radius at a constant rate through exponential
growth of mycelia within the outer shell of active hyphae. The width of the outer shell,
w, is determined by the diffusion properties of material through the mycelium and
depends on the pellet structure. Therefore, it is not directly equivalent to the peripheral
growth zone. Thus, steady-state concentrations of biomass and substrate and the critical
dilution rate depend not only on diffusivity but also on the pellet size and fragmen-
tation and the growth conditions reflecting on them. Changes in pellet radius are
described by the equation:
r r0 wlt 7
where, r and r0 represent pellet radii at times t and 0. The outer active shell has a w width
and l specific growth rate. If the pellet is assumed to be spherical and to have a constant
biomass density q, the above equation can be written in terms of biomass, as:
1=3
4
M M0 pqn wlt 8
3
where M and M0 are the biomass of pellets at times t and 0, respectively, and n is the
number of pellets. This is equivalent to Eq. (6), with
1=3
4
k pqn lt 9
3
The model predicts exponential growth in batch culture until restrictions to diffusion of
nutrients through the pellet mass reduce growth rates in the center of the pellets.
Subsequent growth will then follow cube-root kinetics. Cube-root kinetics have been
observed experimentally (Trinci, 1970), but practical difficulties in accurately measuring
biomass concentrations make distinction of different types of growth kinetics difficult.
Although cube-root kinetics are predicted for pellet growth, it is difficult to distinguish
them from exponential kinetics. Koch (1975) constructed a model that provides a unified
approach for growth in liquid and solid substrate and predicts the majority of growth
kinetics observed experimentally, such as linear colony expansion, exponential, square and
cube-root kinetics for biomass and explains these in terms of the capacity of mycelia to
colonize unoccupied regions of substrate.
Koch (1975) fitted Trincis data to the logistic equation, modified to account for
mycelial growth. The logistic equation was originally constructed to describe growth of
individuals within a population and it is based on the assumption that the specific growth
rate decreases as a negative linear function of population size, having the following form:
dN rN 2
rN 10
dt k
where N is the number of individuals, r the intrinsic rate of increase and k the yield or the
carrying capacity of the environment. The above equation gives:
kN0 exprt
N 11
k N0 N0 exprt
where N0 is the initial cell number. The equation predicts a sigmoidal growth curve. To
describe mycelial growth, Koch replaced the number of individuals by the mycelial
biomass W, occupying unit volume of space, dV. N0 and k were replaced by the initial (S)
and maximum (K) mycelial biomass per unit volume and t was replaced by (T t). T is the
time since growth of the colony started, while t is the time at which growth first occurred
within the volume element considered. The first equation can then be rewritten for
(T t)>0, as
dW SKexprT t
dV 12
dt K S SexprT t
Restriction of growth to two dimensions to form a colony of height h, which expands at

a constant radial growth rate a, and considering the lag and exponential growth periods as
negligible, leads to the expression:
Z T
SKexprTt
W 2ph2 tdt 13
0 K S SexprT t
while an equivalent expression was derived for three dimensional growth, describing
growth in liquid media in the form of pellets. The model was solved using numerical
approximation techniques. The growth kinetics were fount to depend on the ratio of
maximum to initial biomass density, K/S. For K/S values in the range 102 104, growth is
predicted to be exponential and the curve linear. Experimental data for As. nidulans pellets
were best characterized by a K/S value of about 1 and predicted exponential growth
followed by cube-root kinetics for total biomass and linear radial expansion. Pellet growth
is characterized by rapid and simultaneous colonization of the medium surrounding the
pellet by densely packed hyphae covering the pellet surface. This effectively leaves no
unoccupied space for subsequent growth, giving the low observed K/S ratio.
5. Effects of diffusional limitations inside pellets
Although the above approach provides information on changes in pellet density, there is
a little indication of the heterogeneity characteristic of pellets and a missing link between
the microscopic description of mycelial and pellet growth, and the overall process. The
major cause of heterogeneity within pellets is diffusional limitation of nutrients and
oxygen which arises from dense hyphal packing. The extent of this limitation depends on
the density of the structure, thus in compact pellets, biomass production in the center of the
pellet will cease, and, eventually, cell autolysis will occur. This pellet morphology has
been observed frequently, e.g., in submerged citric acid fermentation (Clark, 1961). Dense
As. niger pellets at the end of fermentation (140 h) consisted of a shell of mycelium
occupying less than 50% of the pellet volume (Clark, 1961). The proportion of the
metabolically active biomass in this case is restricted to the outer zone of less dense
mycelium. In less dense pellets, the actively growing outer zone is wider, substrates diffuse
freely inside the pellet and mass transfer may occur via turbulent diffusion and convective
flow according to Wittler et al. (1986) and Gerlach et al. (1998).
The importance of nutrient and oxygen limitation in pellet growth and pelleted biomass
physiological heterogeneity has been taken into account in modeling the pellet growth.
Pirts (1966) model of pellet growth was based on growth of an active peripheral zone
surrounding a spherical pellet and was used to derive an expression for the pellet radius,
Rc, at which diffusion of nutrients to the core is limited by increased biomass density. The
nutrient which is likely to limit growth first is oxygen and use of the diffusion coefficient
for oxygen and growth kinetic parameters for Pe. chrysogenum predicted a critical radius
which responded well with the experimentally determined radius. The critical radius, Rc,
was given by the expression
1=2
6DVYsm
Rc m 14
ql
where DV is the diffusion coefficient, Ysm is the yield of biomass, q its density and l the
specific growth rate.
More detailed models for oxygen diffusion and utilization inside pellets were presented
and elucidated the importance of oxygen limitation in pellet growth. In the model
described by Aiba and Kobayashi (1971), both respiration and inward diffusion were
considered in calculating the oxygen balance within a pellet. Diffusion equations were
applied and the relative rates of respiration within the pellet and in the surrounding
medium qVO2 =qO2 were determined using the expression:
2
d C 2dC C
D 2qq O2 15
dr2 rdr Km C
where r is the pellet radius and q the biomass density inside the pellet.
In another model, Kobayashi et al. (1973) introduced an effectiveness factor to describe
the reduction in respiration with increasing distance from the pellet surface. They
investigated several situations, with constant respiration within the pellet, respiration
varying with age and also respiration dependent on adaptation of mycelium to oxygen
limitation. Experimental data derived from pelleted cultures of As. niger indicated the last
of these situations to be the most likely.
Michel et al. (1992) compared experimental data from pelleted cultures Phanerochaete
chrysosporium with model predictions for Vmax and Km for oxygen inside the pellets.
These values were then used to predict oxygen limitation as a function of pellet size and
dissolved oxygen concentration. Predictions of CO2 evolution from populations of pellets
in batch culture were also obtained and agreed well with experimental data.
6. Cell aging and autolysis
An important aspect of pellet growth is fragmentation, or breakup of pellets. It has been

observed that the initial increase in pellet concentration in fungal cultures is followed by a
rapid decrease which coincides with a decrease in the specific growth rate (Nielsen et al.,
1995). This breakup is caused by cell lysis within pellets, whereby the stability of the
pellet is lost, and it becomes more susceptible to damage by mechanical forces. Besides
pellet breakup, hyphal elements are torn off at the pellet surface, weakened by the natural
aging process of vacuolation. Due to the fragmentation of pellets and the loss of hyphal
elements from the pellet surface, the macroscopic morphology of a fungal culture grown in
liquid substrate changes drastically, e.g., from the pelleted to the dispersed form (Justen et
al., 1996; Paul et al., 1999), thus changing the rheological properties of the broth and its
mass transfer capabilities.
In spite of the differences brought about by the cultivation conditions, hyphae retain
some characteristics, which can be indicated as symptoms of aging. A growing septate
hypha can be separated into at least three zones: (1) the apical zone (see Section 1); (2) the
subapical zone, rich in plasma components; and (3) the vacuolation zone in which the size
of vacuoles increases with distance from the apex, i.e., with the age of the compartments.
The cell wall also undergoes an aging process. Structural differences between the wall of
the apex and that of the lower part of the hypha have been indicated by Marchant and
Smith (1968) and Strunk (1963). Autoradiography studies of wall synthesis showed a
progressive thickening of the walls in parts more distant from the apex. Similarly, the
autolysis of the walls is relative to their age. Observations on the compositional and
structural inequality of the hyphal walls caused by aging have been made by many
researchers (Katz and Rosenberg, 1971; Gooday, 1971; Gull and Trinci, 1974; Chang and
Trevithich, 1974), and the relationship between the morphological and physiological
functions of hyphal parts of different age is apparent. However, it is often difficult to
distinguish whether the observed biochemical changes in hyphae during the course of
aging are evoked by aging or are only due to cultivation conditions. Autoradiography
studies on As. niger during different phases of culture development showed that during the
stationary phase some parts of the hypha irreversibly lose their ability to synthesize RNA
and protein and they begin to autolyse. In other parts, the rate of synthesis was decreased
by 15 20% as compared to hyphae from the exponential phase (Fencl, 1978). This was
explained by the assumption of protoplasmic streaming and transport of nutrients from the
older to the younger parts of the hyphae, resulting in an exposure to starvation of the older
parts. The apical parts are not exposed to starvation and they retain a higher regeneration
ability. However, this hypothesis is not generally valid since in some older parts of hyphae,
there is the potential possibility of passing to a physiologically younger state if a
compartment forms a new branch. Then this part will increase its rates of RNA and
protein synthesis to the levels of the apical part (Machek and Fencl, 1973).
In contrast to a culture growing in the free filamentous form, in pellets, there is a more
substantial differentiation in the filaments caused by the transport of nutrients from the
outer zone to the inside, and by the excretion of the metabolic products to the outside of
the pellets. This differentiation may be influenced by self-toxicity induced by excreted
metabolites, as observed with a N. crassa mutant which secretes a mucopolysaccharide
that inhibits growth (Reining and Glasgow, 1971). Only a thin layer in the peripheral zone
of pellets is biosynthetically active, as shown in studies with Aspergillus and Penicillium.
Autolysis is considered as the last stage of culture development even when it is
observed in parts of hyphae during the stationary phase (Fencl, 1978). The main cause of
lysis is the material imbalance in hyphae caused either by internal or external factors. Of
the internal factors, it could be a disturbance of the organelles or an accumulation of toxic
metabolites. The external could be physical and chemical factors, the lack of nutrients, as
well as enzyme influences which disturb the cell wall structures (Fencl, 1978; Nombela et
al., 1993). Exhaustion of nutrients from the medium represents one of the most common
reasons for a culture gradually reaching autolysis. The transition to autolysis is gradual and
lack of nutrients is reflected in the hyphae during the pre-autolytic phase by increased
differentiation. Righelato et al. (1968) observed that glucose supply at the maintenance
rate in continuous cultures of Pe. chrysogenum caused increased vacuolation and
predisposed the culture to mechanical damage and increased fragmentation. Righelato et
al. (1968) concluded that the age of the mycelium does not appear to control the aging
process but rather that aging and lysis are determined by the amount of available energy.
Transition to the pre-autolytic phase in Pe. chrysogenum occurs at the moment when the
amount of glucose supplied to the culture approaches the maintenance energy which was
calculated to be 0.022 g glucose/g mycelium dry weight/h. Below this level, autolysis sets
in. The rate of degeneration of the individual biomass components depends on the
cultivation conditions under which the culture has been grown.
Nutrient limitation resulted in heavily vacuolated hyphae and subsequent fragmentation
in fed-batch cultures of Pe. chrysogenum in the work of Paul et al. (1994a). During the
rapid growth phase there was little vacuolation and the mean main and total hyphal lengths
and the mean number of tips per mycelium rose or remained steady. Following glucose
limitation, the values of the parameters declined sharply, indicating a fragmentation
process which was more severe when significant hyphal vacuolation was established,
i.e., during the production phase. The authors based on quantitative information on
vacuolation and morphology obtained by image analysis, concluded that, in addition to
shear, physiological effects can enhance fragmentation, and this supports the idea that
shear might only be effective when the hyphae have been significantly weakened by
internal decay processes. The process of autolysis in batch cultures of Pe. chrysogenum
under a range of stirrer speeds was also investigated by Harvey et al. (1998) who reported
degradation of penicillin V as a result of culture autolysis. The relationship between
vacuolation, fragmentation and citric acid production by As. niger was investigated in
batch and fed-batch culture by Papagianni et al. (1999a,b,c,d). Quantitative information on
morphology and vacuolation obtained by image analysis, together with specific growth
and production rates, were used to establish a link between these under various agitation
conditions and glucose levels. Increased vacuolation and low specific production rates
were observed at low glucose levels, while vacuolation weakened the hyphae and made
the mycelium more susceptible to shear forces at increased agitation levels.
When the fungus is grown in excess nitrogen and growth is limited by the carbon
source, the protein component of the cell is degraded most rapidly (Lahoz et al., 1970;
Lahoz and Miralles, 1970). Conversely, if excess carbon is present in the media, autolysis
raises the amount of reducing substances. Furthermore, while polysaccharides are
normally lysed slowly, lipids are preferentially degraded (Lahoz et al., 1967). Mono-
saccharides, thus, can always be detected in the lysing mycelium (Lahoz et al., 1970).
Growth of Pe. chrysogenum in excess of the penicillin precursor phenylacetic acid, has
been associated with increased cellular autolysis, reduced biomass and penicillin produc-
tion levels, while precursor concentration controlled within the optimal range for penicillin
production has little impact on differentiation or degradation within an industrial culture of
Penicillium (White et al., 1999).
Autolysis does not proceed synchronously in the entire filament but only in its
individual compartments (Trinci and Righelato, 1970). In a lysed compartment, the
resistance of individual organelles is not the same. Mitochondria are much more stable
than ribosomes, and the decomposition of organelles of the same type is synchronous, i.e.,
it is catalyzed by cell-free enzymes (Trinci and Righelato, 1970). In contrast with other
organisms, lysosomes and autophagy do not play a substantial role in the autolysis of
cytoplasm in fungi (Fencl, 1978). Hyphal protoplasm undergoes lysis because of either a
decreased amount or defects in composition of organelles, or to a lack of provided
maintenance energy. Vacuolation and disruption of organelles, including an increased
hydrolase activity may be observed in the older parts of hyphae, however, the physio-
logical age and the age of the compartment need not coincide (Fencl, 1970). For this
reason, there is no regularity in the lysis of hyphae from the base toward the tip and the
lysed compartments are localized irregularly in different parts of the hyphae. Excretion of
lytic enzymes, such as h-N-acetyl-glucosamidase, h-1-3 glucanase, chitinase, invertase
and acid phosphatase was found to be consistent with the degree of autolysis (Lahoz et al.,
1967), however, hyphae contain their own protective substances through which they
withstand lysis by their own enzymes and it has been demonstrated that only after removal
of their protective barrier will intracellular glucanases and chitinases attack the walls
(Wessels and Koltin, 1972). Although the autolytic enzymes are localized directly in
hyphal walls, because of its protection the cell wall is only slowly autolysed (Trinci, 1974).
Both Lahoz et al. (1986) and Trinci and Righelato (1970) noted the persistence of intact
fungal cell walls after many days of autolysis, which involved extensive proteolysis. In
contrast to the inside of hyphae, where autolysis affects compartments irregularly, the lysis
of walls proceeds regularly from the tip to the older sections, thus reflecting the chemical
composition of the walls.
Despite its importance, fungal autolysis has received much less attention compared to
the lysis of bacteria and commercially important yeasts. The methods used to assess the
extent of autolysis in fungal cultures involved the mean decline of biomass, cellular
breakdown products, e.g., NH4+ release enzyme activity assays, and direct measurement of
the autolysing regions by image analysis techniques. The latter, relatively recent improve-
ments in our ability to follow the processes of growth and differentiation in submerged
fungal cultures, allowed extraction of detailed quantitative information on the micro-
morphology of filamentous fungi grown in dispersed form in submerged fermentations.
The studies of Packer and Thomas (1990), Makagiansar et al. (1993), Nielsen et al. (1995),
Paul et al. (1994a,b), Papagianni et al. (1999b) and McIntyre et al. (2001) describe image
analysis studies which measure growth and differentiation in submerged culture and
investigate the relationship between hyphal degeneration and vacuolation, and productiv-
ity. Image analysis is, therefore, a more direct method than the filtration probe of
Nestaas and Wang (1983) for implementing control strategies for antibiotic fermentations
based on simple differentiation.
7. Fragmentation of hyphal elements and pellets
Fragmentation of hyphal elements and pellets during submerged fermentations often

results in growth renewal since fragments may act as centers for new growth, enabling
reseeding of the pellet population. Unfortunately, despite their importance, there have been
a few studies on the physical properties of pellets and dispersed mycelia, because of
experimental and technical difficulties. In recent years, use of image analysis techniques
allowed quantification of fragmentation behavior of fungal cultures, and investigation of
the relationship between viscosity and hyphal tensile strength and morphology. Since
shear forces represent the main cause of hyphal damage and fragmentation, the process of
fragmentation and regrowth with respect to hyphal activity will be discussed under the
agitation section.
In modeling the growth of filamentous fungi, mycelial fragmentation has been
considered in only a few cases. Fragmentation of the mycelium will result in a population
of individual hyphal elements which may have a varying morphology, e.g., different length
and number of tips and pellets of varying size distributions. Empirical models that consider
pellet fragmentation, relatively simple in form and easily simulated, have been presented
by Taguchi (1971) and van Suijdam et al. (1982). The first, described the effect of impeller
speed and diameter on pellet diameter and number of non-disrupted pellets, considering
both removal of material from the pellet surface and complete rupture of pellets. The
second, based on the assumption of oxygen limitation, used an effectiveness factor to
correct specific growth rate and biomass production within pellets for the effects of
diffusional limitation. A major assumption of these models is that cultures are homoge-
neous with respect to pellet size and density and that dispersed growth is negligible. This
assumption is not valid, and variation in pellet size will result from differences in number
of spores aggregating to give original pellets and from fragmentation, leading to
production of smaller fragments of pellets and to dispersed mycelia. The distribution of
pellet size and pellet number is critical as it determines the proportion of actively growing
mycelium in contact with substrate, and the proportion of biomass within pellet which may
be active in secondary metabolite production, but not growth. Also, dispersed mycelia
produced through fragmentation will have different growth kinetics and substrate
utilization characteristics to pellets.
Edelstein and Hadar (1983) were the first to consider pellet size distributions in
modeling growth and biomass production of Sclerotium rolfsii. They were also the first to
consider reseeding of pellet populations by mycelial fragments produced through
fragmentation. The model describes pellet growth as passage of spherical particles through
a series of size classes, with the increase in pellet size modeled by partial differential
equations describing changes in pellet radius (r). The model is presented by the following
equation:
dp dpK br ed2 p

drp 2 16
dt dr dr
where p is equivalent to p(r, t), the number of pellets of radius r at time t. K is the rate of
increase in r due to growth, and b is the rate of increase due to shear. d(r) is a death term,
representing the loss of pellets from each size class through washout or shear effects. e, is a
dispersion coefficient which accounts for the distribution in growth rates among pellets. At
any time, reseeding is determined by the proportion of viable fragments, /, resulting from
pellet fragmentation.
The complexity of this model prevents analytical solution and its basic form is
relatively inflexible. An initial size distribution was defined, based on experimental data,
and size distributions were then derived for a series of time intervals during growth in
batch culture. High values of /, e.g., 50% reseeding, resulted in the development of a
broad range of pellet sizes and the size distribution was characterized by a shoulder. At
lower / values, 10 30% reseeding, the size distribution showed a peak and the rate of
biomass accumulation was reduced.
In the model presented by Nielsen (1992), fragmentation was considered in the
estimation of the morphology of the individual hyphal elements for a number of
filamentous microorganisms. Combining a morphologically structured model and a
population model, growth of filamentous microorganisms both on a solid medium and
in submerged culture, and the properties of the hyphal elements in submerged culture were
described successfully by the complete model which may be valuable for interpretation of
the rapidly increasing number of experimental data obtained using image analysis. The
conclusion was drawn that for some species, fragmentation may be neglected, but for Pe.
chrysogenum, it is important to consider it when morphological data are to be evaluated. A
linear correlation between the specific rate of fragmentation and the energy input to the
bioreactor clearly indicated that fragmentation was mainly caused by physical factors. A
proper evaluation of the hydrodynamic and other physical forces in the submerged culture
environment of a bioreactor is therefore important in understanding and correlating the
fragmentation behavior of at least some fungi. Characterization of the various hydrody-
namic and other forces in bioreactors has been discussed in depth by Chisti (1999a).
A structured kinetic model describing growth, differentiation and penicillin production
in submerged fed-batch Pe. chrysogenum fermentations was reported by Paul and Thomas
(1996a,b). The model incorporated the physiological structure of fungal biomass and the
most novel part of the study was the mechanistic description of vacuolation and
degeneration. The description of differentiation and penicillin production was in the light
of greater understanding of the underlying processes, based on quantitative information
obtained by image analysis. The autolysis and subsequent fragmentation process were
expressed by first-order kinetics with respect to the amount of degenerated regions. In this
model, the parameter agitation speed was excluded since all fermentations were performed
at the same speed, the observed fragmentation was therefore the result of enhanced
vacuolation caused by glucose limitation (1994a). When the glucose feed rate to the
production culture is switched between a high and low value, the model can successfully
predict the dynamic changes of differentiation and the resulting penicillin production
caused by variations in the nutrient conditions.
8. Growth in submerged culture
It is often stated that growth kinetics of filamentous fungi in submerged culture are
quite similar to those of unicellular organisms that reproduce by binary fission. In fact, due
to practical difficulties that hinder studies of filamentous organisms in submerged culture,
growth kinetics are based mainly on studies with unicellular organisms. Attachment and
growth on bioreactors walls, agitators, probes and baffles lead to a degree of heteroge-
neity within the biomass which is more pronounced in the case of pelleted growth. Areas
of growing and non-growing biomass inside the bioreactor influence the overall growth
kinetics. In addition to this, the mechanism of hyphal growth itself contributes to
heterogeneity with extension, but little de novo biosynthesis at the tip, active biosynthesis
behind the tip and reduced activity in more distant regions as hyphae age and vacuolate
(Prosser, 1995).
8.1. Batch culture
Batch growth is typically divided into a number of phases. The lag phase represents
a period during which the fungal cells or spores adapt to a new environment.
Adaptation includes formation of enzymes and intermediates to support resumption of
growth. The length of this phase is dependent not only on the physiological state of the
fungus, but also on the morphology and level of inoculum. Spore inocula require a
germination period (Smith and Calam, 1980), while pelleted inocula may require a
certain degree of mechanical disruption prior to inoculation (Greasham, 1991). Phys-
iological adaptation of the organism includes synthesis of enzyme systems required for
substrate utilization, or removal of inhibitory compounds carried over with the inoculum
(Prosser, 1995).
The exponential phase is characterized by a significant increase in cell mass. The rate of
hyphal growth depends not only on the strain of the fungus, but also on the physico-
chemical environmental conditions. As on solid media, exponential growth results from
autocatalysis through exponential production of branches, each of which extends at a
linear rate. A reduction in the specific growth rate occurs when the fungus begins to
experience an unfavorable growth environment such as the limitation of a required
nutrient, the development of an adverse pH value or the accumulation of end products
of metabolism that are inhibitory. Provided that the latter two are not reducing growth, the
effect of limiting substrate concentration on the specific growth rate (l) may be expressed
by the Monod (1942) model for microbial growth:
S
l lmax 17
Ks S
where lmax is the maximum specific growth rate, S is the substrate concentration and Ks is
the saturation constant equal to the substrate concentration at l = 0.5lmax. Pirt (1975)
reported the saturation constant, Ks, for growth of Aspergillus on glucose to be 5.0 mg/l.
Thus, when the glucose concentration drops below 10 mg/l, a reduction in the specific
growth rate is observed. Although eucaryotes tend to grow more slowly than bacteria,
filamentous fungi such as Aspergillus and Penicillium have maximum specific growth
rates in the order 0.1 0.3 h 1, equivalent to doubling times of 2 7 h.
The many factors governing the entry into the following deceleration phase and the
growth during it have been given little attention. Growth kinetics during this phase is
largely uncharacterized, despite the importance of this phase for biotechnological
processes, as the period when secondary metabolite production begins. Deceleration in
growth rate due to oxygen limitation is of particular importance for cultures of
filamentous fungi because of the influence of their morphology on rheological properties.

Dispersed growth leads to non-Newtonian rheological behavior. The apparent viscosity
increases with growth, reducing the transport of nutrients, oxygen and heat. The increased
energy required for efficient mixing and oxygen transfer significantly increases the cost of
large-scale fungal fermentations. Nutrient limitations can accelerate entry into the
deceleration phase, in comparison with unicellular cultures of equivalent biomass and
activity.
The stationary phase may be defined simplistically as the balance between hyphal mass
increase and decrease. However, if the hyphal mass accumulates intracellular storage
material during the reduced growth phase, a slight increase in hyphal mass may be
observed during endogenous metabolism of these storage materials. Also, if the hyphae
begin to autolyse, new growth could be expected from the products of autolysis, e.g.,
release of a limited nutrient.
8.2. Continuous culture
Continuous culture is usually preceded by growth of the fungus in batch culture to

stationary phase. When supply of fresh medium is initiated growth proceeds and material
from the vessel is washed out, until the concentration of the medium is reduced to a level
at which it limits specific growth rate. In physiological studies, the medium is designed to
determine which substrate limits growth by providing all other components in excess. The
relationship between specific growth rate and the concentration of a liming substrate, S,
can be described by the Monod (1942) model (Eq. (17)). Alternatives have been proposed
in many cases to describe experimental data which do not obey the Monod model.
Deviations from the Monod equation may provide useful information regarding the
physiology of organisms growing under substrate limitation. Alternative descriptions of
growth limitation are usually more complex than Eq. (17) and are therefore of less
descriptive and practical use, although some (Dabes et al., 1973) may have a more sound
mechanistic basis.
The changes in biomass and substrate concentrations during growth in continuous
culture can be described by the following equations:
dX
lX DX 18
dt
and
dS lX
DSr DS 19
dt Y
where l is described by the Monod equation, Sr is the concentration of the limiting

substrate in the inflowing medium, X is the biomass concentration and D is the dilution
rate, defined as the flow rate divided by the volume of culture fluid. If D is less than lmax a
steady state is established. By setting the above equations equal to zero, expressions for
steady-state biomass and substrate concentrations as functions of dilution rate can be
obtained. Also, in steady state, the specific growth rate will equal the dilution rate. Thus,
specific growth rate may be controlled by altering the dilution rate, most conveniently by
adjusting the inflow rate. The composition of the inflowing medium may be manipulated
to obtain limitation of growth by different nutrients, allowing investigation of their role in
growth and product formation.
The problems of filamentous growth in submerged batch culture are exacerbated in
continuous culture. Wall growth increases continuously and maintenance of steady state is
difficult. Breakage of mycelia to provide new centers for growth may be a solution.
Another problem of great importance is the maintenance of stability of filamentous fungi
in prolonged culture. The importance of stability maintenance has increased recently
because of the use of such organisms as hosts for the expression and secretion of
heterologous proteins (Peberdy, 1994). Continuous culture studies have been used to
determine values for growth parameters for filamentous fungi, such as Y and Ks and also
the ability to control the specific growth rate. These studies have provided invaluable
information on the kinetics of branch formation. Robinson and Smith (1979) in studies
with G. candidum grown under glucose limitation showed that an increase in specific
growth rate results in increased hyphal diameter and decreased hyphal growth unit length
but have no significant effect on hyphal growth unit volume. Also, lateral branches are
formed at specific growth rates less than 0.4 h 1, whereas apical branches and extension
rate increase at higher dilution rates. Smith and Robinson (1980) also demonstrated
similarities between cell dimensions and hyphal extension rates in glucose-limited
continuous cultures and in cultures grown on solid media with low initial glucose
concentrations.
Withers et al. (1994) studied the development of morphological heterogeneity in
glucose-limited chemostat cultures of As. niger. Four morphological mutants were isolated
from chemostats in that study and among them a more sparsely branched one than the
parental strain which appeared in high concentrations at the end of the chemostats
suggesting that it was well adapted for growth in stirred-tank reactors. The selection of
appropriate morphological mutants may be a major determinant of the biological
performance of cultures of filamentous microorganisms. Withers et al. (1994) suggest
that fermenter adapted mutants may lead to more stable inocula for industrial
fermentations and because of the complex effects of mycelial morphology on culture
rheology and (perhaps) protein secretion, variants selected in continuous flow fermenta-
tions may give enhanced biological performance in industrial fermentations and be an
important method of strain selection.
At low dilution rates, steady-state biomass concentration decreases, whereas the Monod
equation predicts effectively constant biomass concentrations as D approaches zero. This
is due to consumption of substrate for cell maintenance and requires modification of Eq.
(19) to give (Pirt, 1965):
dS lX
DSr DS mX 20
dt Yg
where Yg is the true growth yield and m is the maintenance coefficient. Eq. (20) provides
the simplest and most widely applicable description of changes in substrate concentration
in continuous culture and enables calculation of the proportion of substrate contributing to
maintenance. Righelato (1979) showed that during growth of Pe. chrysogenum in glucose-
limited chemostats, 10% of substrate is utilized for maintenance at the maximum specific
growth rate of 0.8 h 1 and 70% at a specific growth rate of 0.05 h 1. Studies on growth
and product formation with As. niger and a glucoamylase transformant in chemostat and
recycling cultures performed by Schickx et al. (1993) showed that maintenance require-
ments were dependent on the specific growth rate over the whole range of measured
growth rates. Two regions of specific growth rates were observed in that study, one at
specific growth rates lower (domain I) and one at specific growth rates higher than 0.12
h 1 (domain II). In domain I, changes in morphology and conidia formation were
observed. The authors suggested that deviations in linearity in the linear equation of
substrate utilization should be considered when continuous cultures with filamentous fungi
are performed.
The factors that affect fungal morphology in continuous culture could be numerous and
interrelated, e.g., mechanical parameters of fermentation, the nature of the limited
substrate, the dilution rate. The number of studies on the effect of dilution rate on
mycelial morphology is limited. Wiebe and Trinci (1990) studied two strains of Fusarium
graminearum in continuous culture, a relatively sparsely branched parental strain (A 3/5)
and a relatively highly branched colonial variant (C106). At any given dilution rate, the
concentration of mycelial fragments present at steady state of both strains remained
approximately constant with time, suggesting that mycelial fragmentation occurred in a
regular manner. However, for both strains, fragments decreased with increasing dilution
rate. The length of hyphal growth unit of A 3/5 increased with increasing dilution rate,
while at all dilution rates, C106 produced at least 10 times more macro-conidia than A 3/5.
8.3. Fed-batch culture
The majority of large-scale industrial fungal fermentations involve fed-batch culture in

which biomass is grown initially in batch culture until a chosen component of the substrate
is fully utilized. Fresh nutrient is then added and the culture volume is continually
increasing. It is more common a concentrated form of a component of the original medium
to be supplied rather that complete medium. The aim is to promote product formation
instead of biomass. Substrate is converted immediately on entry into the bioreactor by high
levels of active biomass. Thus, metabolism is directed towards product formation rather
than growth, and substrate utilization and catabolite repression are minimized. One major
difference between fed-batch and continuous culture is that specific growth rate is not
constant in the former. It rather decreases as the culture volume increase. The fed-batch
mode of culture represents a useful tool in studies of substrate limitations and various
inhibition phenomena. However, physiology studies under transient conditions have
received little attention and reliable quantitative growth kinetics have not been character-
ized. Information on morphological development under fed-batch culture conditions is
also limited. Papagianni (1995) and Papagianni et al. (1999c) studied the morphological
development of citric acid producing As. niger in fed-batch culture to find that the main
morphological observations were related to the specific growth rate, which was restricted
by the concentration of glucose and, thus, indirectly related to the concentration of glucose
in the fermentation media.
9. Influence of process parameters on the fungal morphology and productivity
9.1. Inoculum
Among the factors that determine the morphology and the general course of fungal
fermentations, the amount, type (spore or vegetative) and age of the inoculum are of prime
importance. Attempts were made to standardize inocula for citric acid production in
submerged culture by Martin and Waters (1952), Steel et al. (1954) and Clark (1961). van
Suijdam et al. (1980) reported that As. niger pellets would only form at inoculum sizes
below 108 spores/ml, while according to Calam (1987), Pe. chrysogenum forms pellets at
inoculum sizes below 104 spores/ml. In the last case, penicillin production in production
flasks raised sharply from 500 to 5000 U/ml as inoculum size increased from 102 to 104
spores/ml. van Suijdam et al. (1980) working with strains of Pe. chrysogenum, Sporo-
trichum pulverulentum and As. niger, developed an inoculum preparation technique for the
production of fungal pellets in a bubble column. The technique made use of filamentous
mycelium from a preculture as inoculum, yielding many small pellets with a fairly
homogeneous size distribution. At an early stage of growth, the presence of the polymer
Carbopol-934 proved to be very important for the way spores germinated and lowered the
agglomeration tendency, while at a later stage of growth, the influence of shear forces
became more predominant.
During the last decade, the application of image analysis techniques for quantification
of morphology in fungal fermentations resulted in more systematic studies on the
inoculum effects. In earlier studies, the effect of the inoculum was assessed mainly by
the presence or absence of pellets and their characteristics (Vecht-Lifshitz et al., 1989;
Smith and Calam, 1980) due to a lack of an adequate method to monitor mycelial
morphology during fermentations. Morphology was quantified by an image analysis
method (Tucker et al., 1992) in the work of Tucker and Thomas (1992), where a sharp
transition from pelleted to dispersed forms of growth for Pe. chrysogenum grown in batch
culture was reported, as inoculum levels rose towards 5 105 spores/ml, but above that
level, there was little additional effect.
The critical role of the inoculum in the development of fungal morphology during
fermentation and its relation to metabolite production was shown in the work of Nielsen et
al. (1995) with Pe. chrysogenum. The influence of initial spore concentration and agitation
rate on agglomeration leading to pellet formation was studied. For Pe. chrysogenum, pellet
formation occurs by the agglomeration of dispersed mycelia, and the number of hyphal
elements in a pellet is on the order of 2 4, depending on spore concentration. At low
concentrations, agglomeration of hyphal elements was limited and small pellets were
formed. At higher spore concentrations, agglomeration increased and large pellets were
formed. This is different from what is observed for microorganisms where pellets are
formed by the agglomeration of spores (Yanagita and Kogane, 1963; Vecht-Lifshitz et al.,
1989). In the work of Tucker and Thomas (1992), it was found that very few pellets were
formed at high spore concentrations (above 5 105 spores/ml), but some agglomeration
was observed at low agitation rates. The conclusion can be made that agglomeration
leading to pellet formation is not simply determined by the probability of physical contact
between hyphal elements. The same effect of inoculum size was reported for the steroid
transforming filamentous fungus Rhizopus nigricans by Znidarsic et al. (2000). The size of
pellets was found to depend mainly on the spore inoculum size, while the structure of
pellets depended mainly on cultivation temperature. A certain morphological type, that of
smooth pellets, induced by high agitation rates, lower temperatures and high nitrogen
concentrations, was suggested as a prerequisite for further application in the process of
steroid biotransformation.
Inoculum spore concentration appears to be a critical factor for the process outcome
also in immobilized cultures. The effects of spore loading on the growth of Pe.
chrysogenum immobilized in kappa-carrageenan beads were studied by Mussenden et
al. (1991). At high spore loading (103 104 viable spores/bead) the biomass concentration
was low and the majority of the actively respiring biomass was located at the bead
periphery. Reducing the spore loading to 50 viable spores per bead, resulted in a fourfold
increase in immobilized biomass concentration. The spore loading also affected the
morphology of the growing hyphae and the extent of free cell growth.
The type of the inoculum, spore or vegetative, and its effects on the development of
morphology in fermenter culture and metabolite production, was investigated in the
processes of phytase and glucoamylase production by As. niger (Papagianni et al., 1999d,
2001; Papagianni and Moo-Young, 2002). In the presence of wheat bran, a slow-releasing
phosphate source, in the inoculum, the fungus grew in the form of fine pellets and clumps,
which gave a more suitable inoculum in terms of biomass and phytase production in both
submerged and solid-state fermentations. In the absence of bran, large pellets were formed
and productivities were lowered in both types of fermentations. In the process of
glucoamylase production by a wild-type As. niger strain, fungal morphology was
manipulated by means of inoculum level and quality in an attempt to minimize harmful
protease secretion. Different levels of spore inocula and vegetative inocula were used for
the development of distinctive morphological forms in main culture, and it was found that
large pellets were associated with increased specific glucoamylase activities and lower
specific protease activities compared with filamentous morphologies.
Fragmented mycelia of the fungus Cunninghamella echinulata were used as inocula in
g-linolenic acid (GLA) production by Chen and Liu (1997). Mycelial pellets were easily
formed from fragmented hyphae. The floc massiveness of the inoculum and the inoculum
to broth ratio determined the number, size and density of pellets in the final culture.
Smaller pellets were formed from higher floc massiveness and higher inoculum ratios.
Growth of the mycelium in the form of small and compact pellets led to enhanced biomass
and lipid production because of lower oxygen and nutrient transfer limitation in which
case a synergistic increase in GLA was observed. The conclusion was drawn that from the
economic and practical application viewpoints, the medium for GLA production must be
inoculated with a sufficient bulk of mycelial flocs to prevent formation of larger pellets or
mycelial mats which are low in both biomass density and the lipid content.
9.2. Medium composition
The media used in submerged industrial fermentations favor both growth and product
formation at high yields: conidial fungi require water, molecular oxygen, an organic source
of carbon and energy, a source of nitrogen other than molecular nitrogen and several other
elements. At least 13 elements are essential for growth, namely oxygen, carbon, hydrogen,
nitrogen, phosphorus, potassium, sulfur, magnesium, manganese, iron, zinc, copper and
molybdenum. The first eight are needed in relatively large quantities (macronutrients). The
latter five are required in small amounts (micronutrients). Fermentation media can be
chemically defined (synthetic) or complex. When raw materials are used, they may require
prior purification as some trace metals affect certain processes, such as the extensively
studied citric acid fermentation by As. niger. In antibiotic fermentations, the media contain,
in addition to the usual sources of carbon, nitrogen, minerals and buffers, precursors to
increase the yield of the antibiotic, as in the case of penicillin (Atkinson and Mavituna,
1991).
For the industrial production of enzymes and antibiotics, complex media with a solid
substrate are often used (Chisti, 1999b); however, the information on such fermentations
with respect to growth, kinetics and morphology is scarce in literature. The use of complex
media in submerged fermentations of filamentous fungi has been shown to influence the
morphology and growth kinetics. Comparing with work on synthetic medium, Cui et al.
(1998b) found major differences in fermentations with Aspergillus awamori using wheat
bran as the carbon source. The wheat bran was not consumed completely as glucose or
sucrose and fungal growth and adhesion varied depending on the inocula used. Using
spore concentrations higher than 1.8 104 spores/ml, adhesion growth dominated, while
at lower levels, wheat bran free pellets were formed. Solid substrate suppressed the growth
in the free filamentous form. The kinetics with complex medium appeared to be essentially
the same as with synthetic medium. Aspects of the use of complex media for submerged
fermentation of xylanase producing As. awamori has been the subject of investigations of
Schugerl et al. (1998). In cultivations in stirred tank reactors, the predominant part of the
fungus was attached to the wheat bran particles, which protected it from the shear stress.
By changing the stirrer speed, the morphology did not change. Pellets were always
formed, which were surrounded by the wheat bran. However, at the highest stirrer speed
(750 rpm), the fungus was partly separated from the wheat bran and with increasing
cultivation time, the bran was gradually decomposed and disintegrated and the fungal
mycelia were partly lysed, which reduced their intimate contact and protection of the
hyphae from the shear effect. Enzyme activities were lower when milled wheat bran was
used. Under the same cultivation conditions, the use of milled wheat bran led to formation
of both pellets and clumps, which increased the viscosity of the broth and caused mass
transfer problems.
Solids in the liquid substrate have been shown in many cases to induce pellet formation
in filamentous fungi (Braun and Vecht-Lifshitz, 1991; Schugerl et al., 1998). However,
this was not the case in the study of Papagianni et al. (1999d) on phytase fermentation by
As. niger, where addition of wheat bran induced filamentous morphologies and enhanced
both growth and phytase production. This was regarded as an effect of an increased
availability of phosphorus in the medium. During the decomposition of wheat bran,
phosphorous is liberated by the phytase already produced by the mycelium and its slow
release ensures a continuous presence of phosphorous in the medium and prevention of
phosphorus limitation. Studies on other fermentations, e.g., the citric acid fermentation by
As. niger (Charley, 1981; Papagianni, 1995, 1999; Papagianni et al., 1999a) and the
xylanase fermentation by As. awamori (Schugerl et al., 1998), have shown that increased
phosphate concentrations cause a shift from metabolite overproduction to overgrowth of

the organism. Characteristics of this condition include increased specific growth rates,
increased side reactions and unwanted morphologies. However, no shift from product to
biomass formation was observed since phytase production was markedly increased. This
was either due to the slow release of phosphates from the wheat bran or to a phytase
induction by the presence of phytic acid in the medium.
The use of complex media may affect not only the formation but also the degradation
rate of the product. Christensen et al. (1994) showed that the rate of penicillin-V
degradation was significantly higher when media containing corn-steep liquor (CSL), a
crude source of nitrogen which contains phosphate, were used instead of chemically
defined media. This may be a severe problem when carrying out repeated fed-batch
fermentations where corn-steep liquor, and consequently phosphate also, is added at times
in which the penicillin concentration is relatively high.
9.3. Type and concentration of the carbon source
Like all fungi, filamentous fungi are heterotrophic. This means that they require organic
compounds as a source of carbon and energy. A few exceptional reports indicate that
filamentous fungi can fix carbon dioxide. Mirocha and De Vay (1971) reported that
Fusarium sp. and Cephalosporium sp. not only fix carbon dioxide but also grow on an
inorganic salts medium without added carbon. Organic compounds supporting most
growth are usually sugars (e.g., D-glucose, D-fructose, sucrose) which are rapidly taken
up. Polysaccharides, amino acids, lipids, organic acids, proteins, alcohols and hydro-
carbons are also used. A small amount of exogenous carbon may be required to maintain
the fungus even when it is not growing. Carter et al. (1971) estimated that at zero growth
rate As. nidulans consumed 0.029 g of glucose per gram of fungal biomass per hour. The
affinity of As. nidulans for glucose (Ks glucose) determined in chemostat by Carter and
Bull (1969) was 80 120 mg/l.
Carter and Bull (1971) have published results of the first systematic analysis of
environmental factors (dilution rate, dissolved oxygen tension (DOC)) on carbon catabolic
pathways in fungi. They found functional Embden Meyerhof Parnas (EMP) and pentose
phosphate (PP) pathways in chemostat cultures of As. nidulans and that the flux through
the PP pathway was enhanced at dilution rates approaching lmax and at low dissolved
oxygen tension values. Increased activity of the PP pathway was also characteristic of
exponentially growing batch cultures and periods preceding sporulation. These changes in
glucose catabolism were interpreted in terms of the biosynthetic demands of the fungus in
response to changing circumstances of growth. These studies were extended to glucose-
starved and glucose-maintained cultures of As. nidulans (Bainbridge et al., 1971) to find
that all the glucose was catabolized via the EMP pathway within 10 h of terminating the
glucose supply or reducing it to near the maintenance rate, a change which paralleled the
complete decay of PP activity. Findings of this sort have been confirmed and extended by
Ng et al. (1972) with As. niger. In another study, Ng et al. (1974) described the growth rate
dependent changes in enzymes of the EMP and the PP pathways, the glyoxylate shunt and
the tricarboxylic acid cycle under conditions of glucose and citrate limitation. Under
glucose limitation, the TCA cycle enzymes increased in activity as the dilution rate was
raised, a result corroborating the less detailed observations in As. nidulans (Carter and
Bull, 1971; Bull and Bushell, 1976). For both species, evidence for glucose induction of
certain EMP and PP pathway enzymes was obtained.
The focus of much study has been the carbon source for citric acid fermentation,
frequently with a view to the utilization of polysaccharide sources (Gupta et al., 1976;
Hossain et al., 1984; Xu et al., 1989a). The nature of the source has been shown in many
cases to affect citric acid production since it exerts a strong effect on levels of enzymes
within the TCA cycle. The final molar yield of citric acid increases when the initial sugar
concentration is increased and only certain carbohydrates are good carbon sources for
citric acid accumulation (Xu et al., 1989b). A general interpretation of this observation is
that only carbon sources allowing fast growth of As. niger lead to rates of carbon
catabolism necessary for citrate overflow (Wolschek and Kubicek, 1999). Hossain et al.
(1984) suggested that high concentrations of appropriate carbon sources lead to repression
of a-ketoglutarate dehydrogenase, hence explaining the effect of the sugar concentration
and source in terms of enzyme repression. In their benchmark paper, Shu and Johnson
(1984) reported that the final concentration of citric acid increases with increased initial
sugar concentration in the range of 14 22%. Results presented by Honecker et al. (1989)
are in agreement with this. The superiority of sucrose over glucose and fructose has been
documented by Gupta et al. (1976), Hossain et al. (1984), and Xu et al. (1989a). The latter
tested maltose, sucrose, glucose, mannose, and fructose to observe the highest yields at
sugar concentration of 10% w/v, with the exception of glucose, where 7.5% gave the best
results. No citric acid was produced on media containing less than 2.5% sugar. It was also
noted that the increase in sugar concentration from 1% to 14% increased the lag time for
growth from 12 to 18 h and decreased the growth rate by around 20%.
The effects of the type of the carbon source on fungal morphology and ultrastructure
are illustrated in the work of Cundell et al. (1976) with Pe. chrysogenum. The fungus grew
as hollow mycelial pellets surrounding individual hydrocarbon droplets on n-hexadecane
and as solid pellets on peptone. A dense layer of fungal mycelium that showed irregular
forms, fusion, and increase in hyphal size formed at the hydrocarbon water interface.
Inclusion were present in the hexadecane-grown fungus that were absent when the fungus
was grown on peptone.
Studies in conventional batch culture confirmed that initial glucose concentration in the
fermentation medium affected both the rate of citric acid fermentation by As. niger and the
morphology of the producer organism (Papagianni et al., 1999c). To eliminate the effects
of decreasing sugar concentration during batch experiments, a series of fed-batch
(glucostat) experiments were carried out in which the concentration of glucose was kept
constant whereas all other conditions were allowed to change. The level of glucose had a
marked effect on production rates; the specific growth rate of citric acid formation
increased with increasing initial glucose concentration (batch culture) or glucose levels
(glucostat culture), and in both culture methods, the specific growth rate increased with
decreasing glucose concentration for the first 48 h of fermentation. The reduction observed
in the mean length of filaments at low glucose levels was explained by increased
branching frequency as a result of the increasing specific growth rate at the early stages
of fermentation. The main morphological observations in both batch and glucostat cultures
seemed to be related to the growth rate, which is restricted by the glucose concentration,
and thus, indirectly related to the concentration of glucose in the fermentation media. Also,
the glucose levels in the medium were shown to lead a non-catalyzed entry of glucose into
the mycelium, which accounted for the citric acid productivity and growth rate changes.
Muller et al. (2000) investigated the role of the carbon source on mitosis and hyphal
extension in As. niger and As. oryzae grown in submerged culture. In the two Aspergillus
species, the length of the apical compartment, the number of nuclei in the apical
compartment, and the hyphal diameter were regulated in response to the glucose
concentration in the medium. A long apical compartment with many nuclei was the result
of high glucose concentration, whereas a short apical compartment with few nuclei was the
result of a low glucose concentration.
As discussed in Section 6, carbon limitation contributes greatly to the process of cell
aging and autolysis in fungal cultures, resulting in heavily vacuolated hyphae and
fragmentation.
9.4. Nitrogen and phosphate
Nitrogen may be supplied as ammonia, as nitrate or in organic compounds, such as

amino acids or proteins. Beet or cane molasses, corn-steep liquor, Pharmamedia, whey
powder, soy flour, yeast extract and others are used as industrial raw materials, rich in
nitrogen. Phosphate is a convenient and readily used source of phosphorus, while organic
forms are also used in industrial processes. Both play an important role in metabolite
overproduction and affect fungal morphology. In the citric acid process by As. niger, in
order to accumulate citric acid, growth has to be restricted, but it is still not clear whether
phosphate or nitrogen is the necessary limiting factor. According to Shu and Johnson
(1984), phosphate does not have to be limiting, but when trace metals are not limiting,
additional phosphate results in side reactions and increased growth. Kubicek and Rohr
(1977) showed that citric acid accumulated whenever phosphate was limited even when
nitrogen was not. In contrast Kristiansen and Sinclair (1979), using continuous culture
concluded that nitrogen limitation was essential for citric acid production.
Omission of nitrogen in the medium greatly affects fungal growth and metabolite
production. Schrickx et al. (1995) studied the growth behavior and glucoamylase
production by a wild-type As. niger strain and a glucoamylase oveproducer transformant
in recycling culture without a nitrogen source. In both strains, hyphal tip extension and
glucoamylase production still occurred, but overproduction of glucoamylase by the
transformant strain stopped. The mycelium retained low metabolic activity and the hyphae
appeared partially empty and broken at the tip. As fermentation proceeded, fragmentation
became the dominant characteristic in the culture.
Among the factors considered to induce pellet formation in filamentous fungi is the
limitation of particular nutrients, including nitrogen (Braun and Vecht-Lifshitz, 1991). On
the other hand, factors favoring increased growth rates, including excess phosphate
concentrations, have been shown to reduce pellet formation (Katz et al., 1972). Contra-
dictions exist however, as in the work of Znidarsic et al. (2000), where increased nitrogen
concentrations led to larger and denser pellet structures of Rhizop. nigricans, while at the
low nitrogen levels, pellets appeared small, very light and fluffy, and the fungus showed an
increased tendency to form clumps. The effect of the nitrogen source on morphology and
antibiotic production was studied with another Rhizopus strain, Rhizopus chinesis, by Du
et al. (2002). In media containing different nitrogen compounds, the morphology, in terms
of hyphal length and degree of branching, varied significantly. The activity of the
antibiotic also varied with the nitrogen source. The highest antibiotic production,
accompanied by pellet growth, was achieved in a medium containing corn-steep liquor.
The pellets were fluffy with a compact core and a loose outer zone and an average
diameter of 3 mm. When ammonium sulfate was used as nitrogen source, the pellets were
larger (4 mm), compact, with a smooth surface and antibiotic productivity was lower. The
same was the effect with Pe. chrysogenum in the study of Pirt and Callow (1959);
ammonium sulfate in the medium caused compact pellet growth, while CSL produced
looser structures.
Quantitative relationships between phosphate concentrations in the media and As. niger
morphology and citric acid production were investigated by Papagianni (1995) and
Papagianni et al. (1999a) in a tubular loop bioreactor. A small increase in phosphate
concentration in the medium (from 0.1 to 0.5 g/l KH2PO4) led to a sharp drop in citric acid
productivity (from 70% at the circulation time of 18 s, to 39%) while the biomass
concentration at the end of the 7-day process was doubled. Drastic were the changes in
morphology, which were monitored by image analysis. The perimeter of clumps became
almost three times larger, while their cores remained small at increased phosphate
concentrations, giving a loose appearance to mycelial aggregates which accounted for
increased broth viscosities, lower dissolved oxygen levels and, consequently, lower
productivities. Branching frequency and the length of filaments increased with increasing
phosphate concentrations. In another study (Papagianni et al., 1999d), a slow release of an
organic phosphate form during the As. niger phytase fermentation led to a change of
morphology from pelleted to filamentous and enhanced biomass production, while in
contrast to the citric acid case, enzyme production was also increased.
9.5. Metal and other ions
Certain essential metals are required for fungal growth. Extensive information exists on
the effects of metal and other ions on fungal growth and metabolite production. Most of
this information was obtained in batch experiments. The effects of mono-and divalent
cations on the growth of the marine fungus Dendryphiella salina have been examined by
Allaway and Jennings (1970). Growth was reduced by 40% and 25% in the presence of
200 mM NaCl and KCl, respectively. Sodium ions inhibited the uptake of glucose by
inhibiting its catabolism which, in turn, appeared to result from a leakage of K+ from the
mycelium. The inhibition was reversed almost completely by 10 mM CaCl2, which
reduced the loss of K+, Na+ and sugar alcohols from the fungus. An inhibition of glucose
transport was found to be the basis of the potassium effect.
Alberghina et al. (1971) studied the Mg2 + concentration dependency of growth rate in
N. crassa and reported Ks for Mg2 + of 7 Amol/l. A significant increase in polyamine
concentration occurred in mycelia of Mg-limited batch cultures as soon as intracellular
levels of Mg2 + fell, but the RNA content and growth were not affected (Viotti et al., 1971).
Fluctuations in polyamine and Mg2 + concentration in response to dilution rate have been
observed in carbon-limited chemostat cultures of As. nidulans (Bushell and Bull, 1974a).
Magnesium concentration in the mycelium fell from about 100 to about 20 Amol/g
biomass, as the dilution rate was raised from 0.05 to 0.18 h 1. In contrast, spermidine
concentrations increased over this dilution rate such that the molar ratio of polyamine plus
Mg2 + to RNA remained constant at approximately 2. Maintenance of this ratio may be
critical for the stability of ribosomes or for RNA synthesis.
Much work has been done on trace metals with As. niger concerning the growth and
citric acid production. Zinc, manganese, copper, iron, heavy metals and alkaline metals
have been shown to affect both fungal morphology and citric acid production. Shu and
Johnson (1984) reported optimal levels of Zn and Fe at 0.3 and 1.3 ppm, respectively. The
levels of Fe and Zn are probably related to the diversion of carbon between biomass and
citric acid (Mattey, 1992). Addition of zinc to cultures at accumulation phase in the work
of Wold and Suzuki (1976) resulted in their reversion to growth phase. Haq et al. (2002)
reported on the effect of copper ions on As. niger morphology and citric acid production.
Addition of 2 10 5 M CuSO4 to the medium reduced the Fe2 + concentration, counter-
acting its deleterious effects on fungal growth. The copper ions also induced a loose-
pelleted form of growth, reduced biomass levels and increased the volumetric productivity
of citric acid.
Any addition of Mn at a concentration as low as 3 Ag/l drastically reduces the yield of
citric acid under otherwise optimal conditions (Clark et al., 1966). Bowes and Mattey
(1979) reported that addition of 10 mg/l Mn2 + halved the citric acid accumulation.
Investigations by Clark (1961) and Kisser et al. (1980) confirmed the key regulatory
nature of Mn ions. The influence of Mn ions on protein synthesis was considered to be of
major importance since cycloheximide, an inhibitor of de novo protein synthesis, was
found to antagonize the effect of manganese addition. Cellular anabolism of As. niger is
impaired under manganese deficiency and/or nitrogen and phosphate limitation. The
protein breakdown under Mn deficiency results in a high intracellular NH4 + concentration
which causes inhibition of the enzyme phosphofructokinase (an essential enzyme in the
conversion of glucose and fructose to pyruvate), leading to a flux through glycolysis and
the formation of citric acid (Kubicek and Rohr, 1977; Habison et al., 1979).
Manganese ions are known to be specifically involved in many cellular processes, such
as cell wall synthesis and sporulation. Kisser et al. (1980) studied the development of
morphology and cell wall composition of As. niger under conditions of manganese
deficient and sufficient cultivation in an otherwise citric acid producing medium. Omission
of manganese ions (less than 10 7 M) from the nutrient medium resulted in abnormal
morphological development, characterized by increased spore swelling and squat, bulbous
hyphae. The inhibition of glucoprotein turnover caused by the absence of manganese ions
in the medium led to the loss of hyphal polarity and increased branching and chitin
synthesis. Fig. 1 shows the characteristic morphology of citric acid producing mycelium of
As. niger in a manganese-deficient medium. The abnormal morphological development
with bulbous hyphae is obvious in the photograph, which was taken at 120 h of
fermentation in a stirred tank bioreactor at 500 rpm and pH 1.6 (Papagianni, 1995). Clark
et al. (1966) also discussed changes in As. niger morphology following the addition of Mn.
The authors noticed an undesirable change in morphology from the pelleted form to
filamentous with the addition of 2 ppb Mn to ferrocyanide-treated molasses. Morphological
changes, which included prevention of clumping, absence of swollen cells and reduced
Fig. 1. The characteristic morphology of citric acid producing mycelium of As. niger in a manganese deficient
medium. The photograph (400 ) was taken at 120 h of fermentation in a stirred tank bioreactor, at 500 rpm and
pH 1.6.
hyphal diameters, accompanied by a 20% reduction in citric acid yield, following the
addition of 30 Ag/l Mn to Mn-free medium, have also been reported (Papagianni, 1999a).
9.6. Dissolved oxygen tension
Tabak and Bridge Cooke (1968) reviewed the earlier literature on effects of gaseous
environments on fungi. On the basis of present information it appears that most fungi
require molecular oxygen to grow. Fungi can grow over very wide ranges of oxygen
tensions and many of the early investigations of oxygen requirements were limited by the
inability to measure very low oxygen concentrations and to establish rigorous anaerobic
conditions (Bull and Bushell, 1976).
Industrial production of various metabolites by filamentous fungi is susceptible to
regulation by the dissolved oxygen tension of the medium (Kubicek et al., 1980). As these
products in most cases are produced by differentiated cells, it is evident that the critical
DOT for growth and the critical DOT for product formation are distinct parameters and in
general the latter is significantly higher. This is a common feature of fungal metabolism
and points to a general role of oxygen in metabolic regulation. In the case of citric acid
production by As. niger, the influence of oxygen has been well established (Shu and
Johnson, 1984; Clark and Lentz, 1961). Interruptions of aeration up to 20 min do not
reduce the viability of As. niger, but result in complete loss of ability to produce citric acid
(Kubicek et al., 1980). Oxygen acts as a direct regulator of citric acid accumulation.
Kubicek et al. (1980), investigating the mechanism of the control of citric acid
accumulation by oxygen, by means of pilot plant As. niger fermentations, reported the
critical DOT for oxygen uptake at 18 21 and 23 26 mbar for trophophase and idiophase,
respectively. Minimal DOT for citric acid production was about 25 mbar, while production
increased steadily between 40 and 150 mbar. However, reports on the effect of DOT on the
morphology of As. niger suggest that no direct relationship exists between the two. Gomez
et al. (1988) found that no difference in morphology for pellets and filaments could be
ascribed to DOT levels, although production of citric acid was enhanced, particularly from
pellets, by increasing the dissolved oxygen at different fermentation stages. Similarly, in
the work of Carter and Bull (1971) with As. nidulans, morphology was unaffected by
DOT. Cultures in which the specific growth rate was held constant at 0.05 h 1 and the
DOT varied from 1 to 156 mm Hg developed filamentous morphology and both the mean
hyphal segment length and the degree of branching appeared to be independent of the
DOT.
Penicillin production by Pe. chrysogenum is also very sensitive to DOT. Vardar and
Lilly (1982) estimated the specific penicillin production rates ( qpen) at different constant
DOT levels and found that below 30% air saturation qpen decreased sharply, while no
production was observed below 10% DOT. Oxygen uptake of the culture was affected
significantly below 7% DOT, demonstrating that the critical DOT values for penicillin
production and oxygen uptake are different. Again, no direct relationship between DOT
and the morphology of Penicillium has been reported so far. van Suijdam and Metz (1981)
showed that oxygen tension in the range of 12 300 mg Hg had no influence on the
morphology of Pe. chrysogenum. These reports contradict the limitation hypothesis of
Hemmersdorfer et al. (1987), which suggests that a lack of any particular nutrient,
including oxygen, induces pellet formation in filamentous fungi.
Fungal morphology appears to be more sensitive to DOT in the process of arachidonic
acid production by the filamentous fungus Mortiella alpina. Higashiyama et al. (1999), in
an attempt to maintain a DO concentration above 7 ppm, used two methods, the oxygen
enrichment (OE) method (25 90% oxygen gas supplied) and the pressurization (PR)
method (180 380 kPa headspace pressure). As a result, the optimum DO concentration
range was found to be 10 15 ppm. In this range, the arachidonic acid yield was enhanced
about 1.6-fold compared to that obtained at 7 ppm DO, and there was no difference in
production between the two methods. When the DO concentration was maintained at 20
50 ppm using the OE method, the morphology changed from filamentous to pelleted, and
the product yield decreased drastically because of the limited mass transfer rates through
the pellet walls. When the DO concentration was maintained at 15 20 ppm using the PR
method, the morphology did not change and the yield decreased gradually.
Oxygen was found to be the overriding effector of morphological development of the
dimorphic fungus Mucor circinelloides in the study of McIntyre et al. (2002). Growing in
batch cultures, it was possible to induce the dimorphic shift by controlling the influent gas
atmosphere. Pure cultures of the multi-polar budding yeast form were obtained under
anaerobic conditions (with 70% N2/30% CO2 or 100% N2 as the sparged gas and without
aeration), while purely filamentous cultures resulted from aerobic cultivations.
9.7. Dissolved carbon dioxide tension
The processes of carbon dioxide evolution and uptake are fundamental to the activity of
many microorganisms (Dixon and Kell, 1989). Since the assertion by Rockwell and
Highberger (1927) that CO2 is an essential prerequisite to the growth of bacteria, yeasts
and fungi, a number of reports have described its stimulatory effects upon fungal growth,
e.g., Rhizopus (Barinova, 1954), Fusarium (Stover and Freiberg, 1958) and soil fungi
(Macauley and Griffen, 1969). Other authors have considered CO2 as an inhibitor of
fungal growth. Tabak and Bridge Cooke (1968) concluded that gas mixtures containing in
excess of 95% CO2 are, in general, inhibitory. Carbon dioxide fixation has a major role in
the production of commercially important organic acids by filamentous fungi. The
formation of citric acid from hexoses by As. niger proceeds via a split into two C3
compounds, one of which is decarboxylated to C2 and the other is carboxylated to C4.
Condensation of the C2 and C4 products then occurs to form citric acid (Bentley and
Thiessen, 1957; Kubicek and Rohr, 1989). Itaconic acid produced by Aspergillus terreus,
is formed in a similar manner (Bentley and Thiessen, 1957; Wilke and Vorlop, 2001), cis-
aconitate is formed from citrate which is then decarboxylated to form itaconate. Overman
and Romano (1969) were the first to demonstrate the importance of pyruvate carboxylase
in fumaric acid production by Rhizop. nigricans. The continued activity of the enzyme
after the cessation of growth appeared to provide a carbon source for fumaric acid
synthesis in the form of fixed CO2.
In practice, ventilation parameters in organic acid fermentations are arrived at
empirically. The term ventilation was coined by Nyiri and Lengyel (1968) in describing
CO2 transfer processes to distinguish them from aeration effects which are concerned with
oxygen transfer and availability. The optimal conditions should provide a sufficient
concentration of gaseous CO2 in the culture to prevent the solubility equilibrium between
dissolved bicarbonate and gaseous CO2 becoming a limiting factor in CO2 fixation. In
studies of the penicillin fermentation Nyiri and Lengyel (1965) found a CO2 concentration
of 1% in oxygen saturated cultures to be optimal for growth and antibiotic production,
above this critical level penicillin production was impaired. Bushell and Bull (1974b) in
studies with As. nidulans concluded that a critical CO2 partial pressure was required for the
successful establishment of continuous cultures from spore inocula. CO2 fixation by
pyruvate carboxylase and phosphoenol pyruvate carboxylase was responsible for biomass
yield increases of 23% when the bicarbonate concentration in the culture broth was raised
from 2.5 to 7.0 mM. Enzyme activity and resultant rates of CO2 fixation were found to be
strongly growth rate dependent. Pyruvate carboxylase decreased in activity as the dilution
rate was increased while the phosphoenol pyruvate carboxylase activity reached a
maximum at a dilution rate equivalent to approximately 1/2lmax.
Considerable debate still surrounds the mechanisms by which CO2 inhibition of growth
and metabolism is mediated (Dixon and Kell, 1989; Jones and Greenfield, 1982). In
submerged culture, it is also unclear which form of CO2 is responsible for the effects, with
CO2 (aq.) [dissolved CO2], HCO3 (bicarbonate ion), and even a short-lived dimeric
H2C2O being implicated (Covington, 1985). For filamentous fungi, pronounced effects
on both morphology and product formation have been associated with elevated levels of
CO2. Studies with Pe. chrysogenum (Pirt and Mancini, 1975; Smith and Ho, 1985; Ho and
Smith, 1986) showed that levels lower than 5% CO2 in the inlet gas stream had no marked
effects on morphology and production; as CO2 concentration was increased, penicillin
production fell to 10% of the control at 20% CO2 while development of an aberrant
morphology was noted. Light microscopy led to the observation that dissolved carbon
dioxide affected the morphology of Pe. chrysogenum with the normal, healthy hyphae
becoming long, thin and diffuse when exposed to increased concentrations (Smith and Ho,
1985). It was proposed that the effects observed on Pe. chrysogenum were due to the fact
that high levels of CO2 could affect membrane transport properties of the cell or that the
cells were subject to osmotic swelling (Smith and Ho, 1985; Ho and Smith, 1986). The
study of Edwards and Ho (1983) focused on the effect of CO2 on Pe. chrysogenum chitin
synthesis. This process and the resulting chitin fibrils largely determine the shape and
direction of growth of individual hyphae (Steward and Rogers, 1983). Increased CO2
concentrations have been shown to lead to a reduction in chitin synthesis sites causing
spherical or yeast-like growth to occur. Apart from the process of chitin synthesis, other
proposed sites of CO2 action include biological membranes (Sears and Eisenberg, 1961)
and cytoplasmic enzymes. A likely factor in the efficacy of CO2 is the ability to penetrate
bacterial membranes causing intracellular pH changes. It is possible that the resultant pH
changes might be sufficient to disrupt internal enzymatic equilibria.
McIntyre and McNeil (1997a) studied the effects of CO2 on morphology, growth and
citric acid production by As. niger in batch cultures and reported quantitative information
on morphological changes by using image analysis. In processes where the inlet gas
stream contained more that 3% CO2, raised CO2 was associated with decreased biomass
and citrate concentrations, decreased substrate consumption and morphological changes.
An increase in the value of the hyphal growth unit, mean hyphal length and mean branch
length was observed as influent CO2 was increased above 5%. Extending their study in
chemostat cultures (McIntyre and McNeil, 1997b), the same authors reported that although
elevated CO2 did inhibit growth and acid formation and generally led to an increase in the
value of the morphological parameters, these effects were more modest than might have
been expected based on results from their previous study. The differences observed
between the batch and chemostat culture studies were explained in terms of the nature of
the experimental systems themselves.
9.8. Culture pH
The pH of the medium is a very important but often neglected environmental factor.
It can profoundly affect any activity being studied. The pH of a solution is a measure of
the concentration of H+ ions present. Different points on a pH growth plot may be
interpreted in terms of effects on transport of nutrients, nutrient solubilities, enzyme
reactions or surface phenomena. Conidial fungi can grow over a wide range of pH. Most
tolerate a pH range from 4 to 9 but grow and sporulate maximally near neutral pH
(Cochrane, 1958).
The composition of the medium can affect the initial pH and the extent and direction of
pH drifts during growth of the fungus. Poorly buffered media containing ammonium salts
are likely to become more acidic during growth, while media containing nitrate are likely
to become alkaline. Minimizing pH drifts during growth is a desirable objective that is
often difficult to achieve. The high concentrations of ions such as phosphate that are
required to achieve some measure of pH stability often appreciably influence the
biological activity being measured (e.g., growth and enzyme activity). As mentioned in
the previous section, the culture pH influences the concentration of dissolved bicarbonate
formed from gaseous CO2. In this way, the prevailing pH and buffer capacity of the culture
medium can influence fungal growth and product formation.
The effect of varying pH in steady-state chemostat cultures of As. nidulans on biomass
yield has been studied by Bull and Bushell (1976), while keeping the dissolved
bicarbonate concentration constant. The optimum pH for growth under conditions of
glucose limitation and at 30 jC was 6.9; the yield fell sharply at pH values on either side
of the optimum. In a study of melanin production by As. nidulans, Rowley and Pirt (1972)
found that pH 3.0 was the lower limit for growth and that washout from the chemostat
occurred at pH values between 3.0 and 2.7.
Culture pH also affects fungal morphology. Pirt and Callow (1959) reported that
increasing the pH of steady-state cultures of Pe. chrysogenum above pH 6.0 caused a
corresponding decrease in hyphal length which reached a minimum value at pH 7.0 7.4.
At higher pH values, extensive formation of swollen cells was observed. One interpre-
tation of these results is that a pH-dependent change in hyphal wall structure takes place,
resulting in a decrease in the resistance of hyphae to impeller shear. As the optimum pH
for penicillin production is about 7.4, this led Pirt and Callow (1959) to conclude that an
ideal continuous-flow culture system for antibiotic production would comprise two stages,
the first for growth with a pH value not exceeding 7.0 and a second stage with a higher pH
for penicillin production. Similar two-staged strategies have been proposed for the glucan
synthesizing fungus Aureobasidium pullulans (Lacroix et al., 1985), and the exopolysac-
charide producing fungus Sclerotium glucanicum (Wang and McNeil, 1995). The effect of
pH on Pe. chrysogenum morphology in batch and chemostat cultures was studied by Miles
and Trinci (1983). Wall thickness and the length of the hyphal growth unit were found to
be very sensitive to pH changes. Mycelia grown in chemostat culture attained their
maximum hyphal growth unit length at pH 6.0.
The phenomenon of pellet formation is strongly influenced by the pH (Whitaker and
Long, 1973; Gerlach et al., 1998; Braun and Vecht-Lifshitz, 1991). In general, the
tendency of the mycelium to form pellets increases as the pH value of the culture raises.
Galbraith and Smith (1969) have found for As. niger that pH of the growth medium plays
an important role in the process of pellet formation, specially in the coagulation of spores.
At pH 5.0, coagulation prevailed, while at pH 2.0, no coagulation was observed. The
authors concluded that the surface properties of spores were influenced by pH. Similar
were the observations of Carlsen et al. (1995), who carried out batch cultivations of As.
oryzae in a stirred tank reactor with an inoculum consisting of 4 108 spores/ml. At pH
values below 2.5, the mycelium grew heavily vacuolated and the cells appeared swollen,
resulting in poor growth. At pH 3.0 3.5 freely dispersed hyphal elements resulted, while
at pH 4.0 5.0, the broth consisted of both pellets and freely dispersed hyphae. At pH
values higher than 6.0, only pellets were observed and the pellet size increased with
increasing pH value. Pellet formation was caused by agglomeration of the spores at pH
values above 4.0. Steel et al. (1954) also observed that filamentous growth for As. niger
occurred at pH values below 5.0.
In addition to the influence on growth and morphology, Carlsen et al. (1995)
investigated the pH influence on a-amylase formation. a-amylase production had a sharp
maximum at about pH 6.0, in contrast to the growth rate, which had a broad maximum
between pH 3.0 and 7.0. The enzyme production of As. awamori according to Smith and
Wood (1991) is a strong function of the pH value. Organic acid production by Aspergillus
strains is also a strong function of the pH, since certain enzymes within the TCA cycle are
pH sensitive (Mattey, 1992; Smith and Wood, 1991). The maintenance of low pH during
the citric acid fermentation is vital for a good yield, and it is generally considered
necessary for the pH to fall around 2.0 within a few hours of the initiation of the process,
otherwise the yields are reduced (Mattey, 1992). The effect of the pH upon the
morphology of citric acid producing As. niger has been investigated by Papagianni et
al. (1994, 1999a). Quantitative information was provided on the time courses of certain
morphological parameters using a semi-automatic image analysis system (Papagianni et
al., 1994, 1999a). In fermentations performed in a stirred-tank reactor at 500 rpm, the
highest values of specific production rates (0.35 h 1) were obtained at pH controlled at
2.1. Lower (pH 1.6) and higher (pH 3.0) culture pHs reduced specific production rates to
0.18 h 1 and gave rise to morphological forms associated with poor productivities. At
very low pHs ( < 2.0), an unusually high number of swollen cells and tips were obtained;
the mycelium formed small clumps and the filaments were short. Increasing the culture pH
to 3.0, the size of the mycelial aggregates doubled, as did the length of filaments.
9.9. Temperature
Studies of temperature effects on growth and metabolite production are few with
respect to filamentous fungi. Closed culture systems are highly unsuitable for the
elucidation of temperature effects on fungal growth. Although temperature is an environ-
mental parameter that is easy to control, changes in temperature produce simultaneous
changes in other culture variables. An increase in incubation temperature within physi-
ological ranges enhances the growth rate and Q10 values of 2 and 30 are common in the
literature (Bull and Bushell, 1976). [ Q10 is the factor by which the death rate increases for
a 10 jC increase in temperature.] Dissolved oxygen tension is also temperature dependent
and varies inversely with increasing temperature. Similarly, nutritional and pH require-
ments for growth may be influenced by the temperature.
The rate of medium evaporation is also greatly affected by temperature, specially in
continuous culture. If evaporation is not accounted for or compensated, errors can be made
in measurement of growth constants such as Y and m. According to King et al. (1972),
these errors could be particularly serious at low dilution rates which are commonly
established with fungal cultures, and at high temperatures. The factors determining
minimum growth temperature appear to be the inactivation of solute transport systems
and the proton gradient formation which results from freezing of the protoplasmic
membrane. Temperature sensitivity of the protoplasmic membrane in the presence of
metabolizable carbon substrates may be an important determinant of the maximum growth
temperature (Madigan et al., 2000).
Rowley and Pirt (1972) observed that as the temperature of steady-state cultures of As.
nidulans was raised from 23 jC to 37 jC the qO2 increased from 1.54 to 3.24. The authors
interpreted these results in terms of an increased maintenance energy requirement due to
higher turnover rates of protein and nucleic acids at elevated temperatures. Over a similar
range of temperatures the specific growth rate of As. nidulans increased from 1.54 to 3.24
h 1, and a comparable increase in the colony growth rate, Kr, was observed by Trinci
(1969). Trinci concluded that Kr was a reliable measure of the optimum temperature of
fungal growth, a conclusion which has been substantiated by analysis of N. crassa (Trinci,
1973). The influence of cultivation temperature on growth kinetics of As. oryzae has been
investigated by Carlsen et al. (1995) in a series of batch experiments. The specific growth
rate of the fungus, grown in the form of pellets with approximately the same size
distribution, increased as the temperature raised from 27 to 35 jC to reach its maximum
value at 35 jC. Cohen et al. (1969) have described a mutant of As. nidulans which
produces walls with reduced chitin content when grown at high temperatures. Anderson
and Smith (1971) have shown that elevated temperatures produce marked morphological
changes in the conidia and newly formed hyphae of As. niger. Miles and Trinci (1983)
studied the effect of temperature on the morphology of Pe. chrysogenum in batch and
chemostat cultures. The hyphal growth unit length and wall thickness increased with
increasing temperatures in the range of 15 30 jC in both batch and chemostat cultures.
This effect was more pronounced at increased dilution rates.
Pellet formation is affected by temperature. Braun and Vecht-Lifshitz (1991) suggest
temperature reducing, among other possible approaches, for inducing mycelial pellet
formation in fermentation cultures. This is in agreement with the study of Schugerl et al.
(1998) on the production of xylanase by As. awamori. Shake flask studies at constant
rotation speed revealed that cell volume and the amounts of various morphological
forms were determined by temperature in the range of 25 35 jC. The highest cell
volume was obtained at 25 jC, where only pellets were observed. At 30 jC, the pellets
initially formed decomposed after 50 h to filamentous mycelium and at 35 jC, mainly
filamentous mycelium and only a low amount of pellets and clumps were formed.
However, the mycelium turned into clumps after 20 h, the size of which decreased with
time. According to the authors, these investigations indicated that at higher temperature,
the oxygen supply of the cells was inadequate. Therefore, the pellets were transformed
to filamentous mycelium at 30 jC, but at 35 jC, because of the higher shear stress,
clumps were formed. Temperature also affected the production of xylanase, with the
maximum productivity obtained at 35 jC. In the investigated temperature range of 27
40 jC by Carlsen et al. (1995), at pH above 4.0 and with inoculum consisting of spores,
pellets were formed and the pellet size distribution was found to be temperature-
independent.
9.10. Mechanical forces
In submerged fermentations, agitation is important for good mixing and mass and heat
transfer. In aerobic processes, mixing is required to ensure sufficient oxygen transfer
throughout the vessel. In the case of a conventional stirred tank bioreactor, the vessel can
be divided into two regions. The region around the impeller represents a zone of high
energy dissipation and, hence, good mass and heat transfer. The remainder of the
fermentation volume may be subject to an inadequate supply of oxygen because of poor
mixing. In large fermenters, mass transfer gradients across the vessel become significant
and this can affect mycelial growth and product formation. Agitation applied during
fermentation creates shear forces which can affect microorganisms in several ways, e.g.,
damage to cell structure, morphological changes, as well as variations in growth rate and
product formation. The magnitude and variation of shear and other forces in submerged
bioreactors have been discussed extensively in the literature (Chisti 1999a).
Markl and Bronnenmeier (1985) suggested that damage to microorganisms in a
turbine fermenter could be the result of rapid pressure fluctuations around the blade
and of shear stress created by the blade tips of a Rushton turbine. They considered
several possibilities for the way microorganisms respond to these stresses. Microorgan-
isms exposed to the high energy dissipation zone around the impeller may be damaged
but may manage to recover. Exposure to a high shear zone may not cause instantaneous
damage, but damage may occur gradually due to hydrodynamic stresses. This implies that
the microorganism has the ability to adapt to a certain level of mechanical stress. Finally,
these effects may depend on the age of the cell. Potential damage to microorganisms can
limit the impeller speed or power input and consequently the oxygen and nutrient transfer
capability of a fermenter, and, ultimately, the volumetric productivity. Furthermore,
changes in morphology can alter the viscosity of filamentous fermentation broths, with
additional effects on mixing and mass transfer. For each culture, optimum conditions of
agitation will exist that will partly depend on the resistance of the hyphae to mechanical
forces and also on their physiological state (Moo-Young et al., 1992; Paul et al., 1994a;
Papagianni et al., 1999b).
Several studies have demonstrated the effects of mechanical forces on the morphology
of filamentous microorganisms and the overall process productivities. However, for the
dispersed form of filamentous growth, the superimposed effects of agitation are difficult to
quantify. For example, in many reports, improved mycelial growth due to increased
oxygen transfer rates is claimed as an agitation effect, possibly counteracting shear
damage. For Pe. chysogenum in a 24-l fermenter, agitation speeds of 1250 and 1500
rpm resulted in lower penicillin production and higher cell growth in comparison to those
at 900 and 100 rpm (Konig et al., 1981). At 700 rpm, oxygen limitation was observed and
penicillin production was as low as that found at 1500 rpm. However, the results were
insufficient to conclude unequivocally that intense agitation caused low productivities.
High impeller speeds were found to promote growth and possibly to stimulate the
metabolic pathways that resulted in low productivities of penicillin.
Dion et al. (1954) investigated changes in morphology and penicillin production of Pe.
chrysogenum over range of fermentation scales (0.01, 3, and 12 m3). It was found that
biomass concentration did not vary with agitation speed. Penicillin was best produced by
short hyphae with sufficient aeration and optimum agitation. Under intensive agitation
conditions, the cultures consisted of predominantly short, thick and highly branched
mycelia which were assumed to be relatively young hyphae. Metz et al. (1981) in studies
with Pe. chrysogenum showed that the length of the mycelial particles decreased with
increasing power input per unit mass in the reactor, as the increased agitation caused the
hyphae to become shorter, thicker and highly branched. High agitation rates reduced the
agglomeration of Pe. chrysogenum hyphal elements and caused a reduction in both pellet
diameters and the concentration of pellets in the work of Nielsen et al. (1995). However,
no relationship between macroscopic morphology and penicillin production by Pe.
chrysogenum was identified. Smith et al. (1990) also observed a drop in productivity of
Pe. chrysogenum and reduced hyphal lengths as agitation increased. Their data for
penicillin production rate (units/ml h) from 10- and 100-l working volume fermenters
showed good qualitative agreement with their proposed model parameter P/D3tc, based on
a combination of the average power input per unit volume per impeller ( P/D3) and
circulation frequency (1/tc).
Reduction of the mean hyphal length of Pe. chrysogenum and specific penicillin
production rate at high agitation speeds was also reported by Makagiansar et al. (1993),
who investigated the influence of mechanical forces resulting from the rotation of turbine
impellers on fungal morphology and penicillin production at three scales of fermentation
(5, 100 and 1000 l) with the impeller tip speed ranging from 2.5 to 6.3 m/s. The dissolved
oxygen concentration never fell below the critical level for maximum penicillin production
throughout all fermentations. Morphological measurements using image analysis showed
that the mean main hyphal length and the mean hyphal growth unit increased during the
period of rapid growth and then decreased to a relatively constant value dependent on the
agitation intensity. The specific rate of penicillin production and the average main hyphal
length during the linear penicillin production phase were lower at high agitation speed,
which promoted more rapid mycelial fragmentation and a higher branching frequency.
Comparison of the results of the three scales showed that impeller tip speed is a poor scale
up parameter, whereas a term based on mycelial circulation through the zone of high
energy dissipation fitted the data well.
The effect of stirrer speed on the growth and productivity of three As. niger strains was
reported by Ujcova et al. (1980). Higher speeds resulted in thicker and more densely
branched filaments. Citric acid production was optimum within a narrow range of speeds.
There was a drop in productivity at higher speeds although growth remained rapid. Gomez
et al. (1988) presented results of As. niger morphology and citric acid yield as a function of
the stirring conditions. At higher stirrer speeds (1000 rpm) the form of small, compact
pellets predominated, while at lower speeds (450 rpm) a mixture of free filaments and
loose pellets existed. The cultures of small and compact pellets yielded higher levels of
citric acid than did the cultures composed mainly of free filaments and loose pellets.
Highly branched mycelium and small aggregates of compact structure were observed
depending on the shear rate in fermentations of As. niger (Mitard and Riba, 1988).
However, under very strong shear rates the specific growth rates were low and at the same
time a release of nucleotides was observed. A relationship between the specific growth
rates of the organism and the rupture of mycelial aggregates was also observed. As the
aggregates were broken, the specific growth rate reduced; it increased again as the
liberated filaments went on growing.
Reduced productivities of recombinant enzyme at increased impeller power in a
production-scale As. oryzae fed-batch fermentation were reported by Li et al. (2002c).
This was attributed to altered fungal morphology, increased fragmentation, reduced
biomass, as well as undesirable mixing or mass transfer effects. The effects of agitation
and fragmentation of a recombinant strain of As. oryzae and its consequential effects
on protein production were investigated by Amanullah et al. (1999) in chemostat cultures
(5.3 l) at a dilution rate of 0.05 h 1. Protein production was found to be independent of
agitation speed in the range 550 1000 rpm, despite significant changes in morphology.
Mycelial morphology did not directly affect protein production at a constant dilution rate
and therefore specific growth rate. In another set of operating conditions (Amanullah et al.,
2002) in fed-batch cultures, at biomass concentrations up to 34 g dry cell weight per liter
and three agitation speeds (525, 675 and 825 rpm), similar results were obtained. Even
though mycelial morphology was significantly affected by changes in agitation intensity,
enzyme titers under conditions of substrate limited growth and controlled dissolved
oxygen of >50% did not change.
The effect of agitation on As. niger morphology and citric acid production has been
investigated by Papagianni (1995) and Papagianni et al. (1994, 1998) in a stirred tank and
a tubular loop bioreactor, through a series of batch and fed-batch experiments. Morpho-
logical measurements using image analysis showed that by increasing the intensity of
agitation, the size of clumps decreased, as did the length of the filaments that arose from
the cores of the clumps, while the diameters of the filaments increased. In both fermenters,
specific rates of citric acid formation increased with agitation; the amount of citric acid
produced at the end of the runs (168 h) was dependent on the stirrer speed and the
circulation time. In the stirred tank reactor, citric acid production increased with stirrer
speed up to a point (500 rpm) beyond which production remained constant. Varying the
intensity of agitation, different patterns of morphological development were observed in
both fermenters. Fig. 2, 3 and 4 show the characteristic development of As. niger
morphology under different agitation levels. Fig. 2 shows a photograph of the mycelium
at inoculation time (vegetative inoculum grown for 30 h in shake flasks). In Fig. 3, the
photograph was taken at the end of a run (168 h) at 200 rpm in the stirred tank bioreactor,
while in Fig. 4, at the end of a run at 500 rpm in the same bioreactor. Under intensive
agitation conditions a cycle of fragmentation and regrowth was observed, while at low
agitation intensities, a gradual aging process predominated and the filaments grew long,
with few branches and gave higher mean values of clump perimeters. The mean diameters
of filaments also changed during fermentation; filaments became thinner with time in all
experiments performed in both fermenters. The reduction of hyphal diameter was found to
Fig. 2. As. niger mycelium at inoculation: vegetative inoculum grown for 30 h in shake flasks (100 ).
Fig. 3. Characteristic morphology of As. niger grown in a stirred tank bioreactor for 168 h at 200 rpm and pH 2.0
(100 ).
be dependent on agitation: The faster the broth circulation and the higher the stirrer speed,
the more rapid was the reduction.
In another study with As. niger (Papagianni et al., 1999b), time profiles of morpho-
logical parameters (i.e., mean perimeter of clumps, length of filaments, vacuoles) obtained
by image analysis, along with specific growth and production rates, were used to establish
Fig. 4. Characteristic morphology of As. niger grown in a stirred tank bioreactor for 168 h at 500 rpm and pH 2.0
(100 ).
a relationship between vacuolation, fragmentation and product formation under various

agitation conditions and glucose levels. Under intensive agitation conditions (500 rpm)
and during the early fermentation stages, the characteristics observed were the increased
specific growth rates and hyphal branching, along with low vacuolation levels. These were
followed by fragmentation of the highly vacuolated parts of filaments and regrowth at later
stages. Contrary to this, low agitation levels created conditions that limited the renewal of
the mycelium, which appeared in a decaying condition with a large proportion of its
volume degenerated. At 200 rpm, a high degree of agglomeration was observed and
dissolved oxygen concentrations fell to 40% at 72 h of fermentation and remained low
thereafter. At 400 and 600 rpm, the dissolved oxygen concentration was above 80% of air
saturation throughout the fermentations. The morphology obtained at 200 rpm created
conditions of reduced mass transfer, not compatible with increased citric acid production.
Increased vacuolation and low specific production rates were observed at low glucose
levels in fed-batch culture. The results showed that vacuolation weakened the hyphae and
the low glucose levels created the conditions that favored fragmentation and made the
mycelium more susceptible to it when exposed to increased agitation.
Ayazi Shamlou et al. (1994), investigating the impact of bioreactor hydrodynamics on
hyphal breakage of Pe. chrysogenum, suggested that in a mechanically agitated bioreactor,
hyphal breakage is likely to occur in the impeller zone as a result of the direct fluctuating
stresses acting on the opposite sides of the hypha. These stresses can be related to the
mean turbulent characteristics of the fluid in the reactor by assuming a constant turbulence
factor. Their experimental data suggested that hyphal breakage is approximately a first-
order kinetic process with a rate constant which appears to be moderately dependent on
the product of the mean energy dissipation rate and the reciprocal of the impeller
circulation time. A similar first-order hyphal fragmentation has been reported by many
others (Chisti, 1999a).
Li et al. (2000) studied the relationship between mycelial fragmentation and agitation at
production scale (80 m3) with As. oryzae using image analysis. In fed-batch fermentations,
performed at two different impeller power levels (one 50% greater than the other), they
found that, contrary to literature reports (Makagiansar et al., 1993; Markl and Bronnen-
meier, 1985; Smith et al., 1990; Johansen et al., 1998), increased impeller power had little
effect on biomass concentration, fungal morphology or fragmentation behavior. In
addition, clumps were not as abundant as has been previously reported (Tucker et al.,
1992; Olsvik and Kristiansen, 1992a,b), and fragmentation was found to dominate fungal
growth and branching. At the end of each of the fermentations studied, most of the fungal
biomass ( f 80%, as measured by projected area) existed as small, sparsely branched, free
hyphal elements. Using a population balance model to analyze the morphological data,
they concluded that fragmentation was the dominant process in those fed-batch fermenta-
tions. The same investigators in another work (Li et al., 2002b) with the same organism
and under the same culture conditions, used the turbulent hydrodynamic theory to develop
a correlation that allowed experimental data of morphology and hydrodynamics to be used
to estimate relative (pseudo) tensile strength of filamentous fungi. They found that the
kinematic viscosity, v, increased over 100-fold during fermentations, and hence, Kolmo-
goroff microscale (k) also changed significantly with time. In the impeller discharge zone,
where hyphal fragmentation is thought to actually take place, k value was calculated to be
700 3500 Am, which is large compared to the size of typical fungal hyphae (100 300
Am). According to them, this implied that eddies in the viscous subrange are responsible
for fragmentation. Also, the hyphal tensile strength was found to change significantly over
the course of fungal fermentations (Li et al., 2002a). The authors suggested that existing
fragmentation and morphology models may be improved by accounting for variations in
hyphal tensile strength with time (Li et al., 2002a).
It is obvious from the above that the pellet size is influenced to a large extent by the
level of agitation applied to the mycelial suspension. Strong agitation results in smaller and
more compact pellets. It is generally reported in the literature (Joshi et al., 1996) that
pellets will be broken if they are larger than a critical size and the critical size is a function
of the agitation intensity. Two mechanisms of changing the size of a pellet were reported
by Taguchi et al. (1968) and Nielsen et al. (1995); one of these is the decrease in diameter
by chipping off pellicles from the surface of the pellets, and the other is the direct break up
of the pellet structure at higher hydrodynamic loads. However, the study of Cui et al.
(1998a) with As. awamori does not support the above mechanism. Fermentations
performed in stirred tank reactors of 2-, 15- and 100-l working volumes showed that
the pellet size was influenced by the amount of available substrates per pellet, the density
of the pellets, agitation intensities and the time period of pellet growth. For the same
density and the same agitation intensities, a higher sugar concentration in the bulk, a lower
number of pellets and a longer period of growth led to the formation of a larger pellet than
when the sugar concentration was lower, a larger number of pellets were present and the
growth period was shorter. The size of the pellets was controlled more by growth than by
breakage.
Changes in fungal morphology associated with mechanical forces produced by Rushton
turbines have been observed in many cases (Dion et al., 1954; Metz et al., 1981;
Makagiansar et al., 1993; Shamlou et al., 1994; Cui et al., 1998a) and have sometimes
been modeled (van Suijdam and Metz, 1981; Nielsen, 1993; Shamlou et al., 1994). A
particularly interesting study for penicillin fermentations was conducted by Smith et al.
(1990), as mentioned earlier in this section. Their data for penicillin production rate (units/
ml h) from 10- and 100-l working volume fermenters showed good qualitative agreement
with their proposed model parameter P/D3tc at the different scales, although this could not
be tested in production scales. Makagiansar et al. (1993) showed that the same parameter
might also be used as a correlating parameter for morphology changes as well as for
productivity at scales up to 1000 l. In addition to the power input, particular impeller
geometrical parameters, such as impeller diameter to vessel diameter ratio (D/T), blade
height to impeller diameter ratio (W/D), number of blades (n) and blade angle (a) have also
to be considered in order to correlate mechanical damage (Justen et al., 1996). Justen et al.
(1996) examined the influence of the agitation conditions on the morphology of Pe.
chrysogenum using radial flow impeller (Rushton turbines, paddles), axial flow impellers
(pitched blades, propeller, Prochem Maxflow T) and counterflow impellers (Intermig). To
characterize the intensity of the damage caused by different impellers, the mean total
hyphal length for the freely dispersed form and the mean projected area for all dispersed
forms including the aggregates were measured using image analysis. At 1.4 l scale and a
given P/VL (power input per unit volume of liquid in the tank), changes in the morphology
depended significantly on the impeller geometry. The morphological data obtained with
different geometries and various P/VL levels was correlated on the basis of equal tip speed
and two other less simple mixing parameters. One was based on the specific energy
dissipation rate in the impeller region, while the other was based on a combination of the
specific energy dissipation rate in the impeller swept volume and the frequency of mycelial
circulation through that volume. The function arising from the latter concept was called the
energy dissipation/circulation function (EDC). To test the broader validity of these
correlations, scale-up experiments were carried out in mixing tanks of 1.4, 20 and 180
l using a Rushton turbine and broth form a fed-batch fermentation. The EDC function was
found to be a reasonable correlating parameter for hyphal damage over this range of scales,
whereas tip speed, P/VL and specific energy dissipation rate in the impeller region were
poor. Further work by the same group (Amanullah et al., 2000) demonstrated that the EDC
function can be successfully applied to correlate mycelial fragmentation of species taken
from chemostat culture other than Pe. chrysogenum, namely As. oryzae, again using
different agitation intensities and different impellers.
Apart from hyphal fragmentation, release of intracellular material into the fermentation
broth, due to changes in membrane structure and permeability, is also ascribed to
mechanical forces. Tanaka (1976) reported the results of extensive experiments with
mycelial suspensions of Mucor javanicus and Rhizopus javanicus. Agitation resulted in
leakage of intracellular substances consisting of RNA-related nucleotides, mostly mono-
nucleotides, with a maximum absorption at 260 nm. This phenomenon was not accom-
panied by any fracture of mycelia. The composition of the nucleotides was the same
regardless of culture apparatus and culture age. The rate of observed leakage was
dependent on the agitation speed and at any constant agitator speed, the nucleotides
leaked from mycelia in direct proportion to agitation time. Tanaka et al. (1975) studied the
effect of agitation on mycelial broths of eighteen strains of filamentous microorganisms
which included species of Mucor, Rhizopus, Aspergillus, Penicillium, Neurospora,
Pircularia, Cephalosporium and Streptomyces. It was found that the leakage of low
molecular nucleotides in stirred mycelial suspensions is a common phenomenon in
filamentous microorganisms. The rate of leakage correlated well with the culture
conditions, age of mycelia, viscosity of the broth, Reynolds number, power input and
impeller tip speed. The leakage of nucleotides affected the growth. Mitard and Riba (1988)
also concluded that the loss of nucleotides was responsible for low specific growth rate
under strong shear stresses.
Adaptation phenomena are observed in growing cultures. During cultivation of As.
niger S59, Ujcova et al. (1980) found a lower leakage of intracellular material at higher
stirring speeds. The possible reason for this observation is suggested by Musilcova et al.
(1981). High agitation leads to a morphologically compact and strong mycelium, and in
addition, the cell wall provides a greater resistance to the action of hydrolases. This
effect was shown in experiments with two As. niger mutants by measuring the quantity
of protoplasts released after a 2-h action of snail gastric juices. The quantity of
protoplasts released from hyphae of As. niger was smaller after cultivation at higher
impeller speed. Hyphae cultivated at the lowest impeller speed were the most sensitive
to lytic enzymes.
Most of the published literature on the effects of agitation on fungal morphology and
process productivities is based on experiments performed in stirred tank bioreactors.
Comparison of data obtained from different reactor types (e.g., pneumatically agitated
versus mechanically agitated or reactors with different geometries) is limited. Since a
direct comparison cannot be made, it is often a difficult task to identify a parameter that
can be used successfully for the characterization of the hydrodynamic situation in
geometrically dissimilar bioreactors (Viesturs and Vanags, 1996). Trager et al. (1989)
compared the morphologies of As. niger growing in a stirred tank and an airlift
fermenter. The formation of fine pellets predominated in the airlift, where shear stress
was milder compared to that in the stirred tank, while in the stirred tank, the
microorganism grew as loose mycelia. This contradicts the study of Gomez et al.
(1988) who obtained good pellet formation of As. niger at high turbulence achieved by
exposing the culture in a stirred tank reactor to a higher stirrer speed. Schugerl et al.
(1998) and Gerlach et al. (1998) compared results obtained from different reactor types
and types of shear stress (shake flasks, stirred tank, airlift bioreactors) in studies with the
xylanase fermentation by As. awamori. Large, loose, hairy pellets were formed in
pneumatically mixed reactors (airlift tower loop), while small and compact pellets
formed in shake flasks and stirred tank reactors. In the first case of pellets, internal mass
transfer was enhanced due to the high flexibility of the pellets. On account of this high
mass transfer rate, the supply of the nutrients to cells in the pellet center was
satisfactory, as long as the substrate and dissolved oxygen concentrations in the liquid
medium were high.
The influence of two mixing geometries (at the same scale) with different flow energy
distributions on the performance of the gibberellic acid fermentation and on the
morphology of the producing fungus Fusarium monilliforme was investigated by Priede
et al. (1995). Fermentations were performed using a turbine mixing system (TMS) and a
counterflow mixing system (CMS), which were high and low power number mixing
systems, respectively. Morphology of both freely dispersed mycelia and clumps was
characterized by image analysis. A higher proportion of clumped mycelia with clumps of
larger area, perimeter, and roughness was observed in the TMS. A correlation between the
morphology and productivity was found, and TMS favored the development of more
productive mycelia with longer and thinner hyphae.
Papagianni et al. (1998) investigated the relationship between the morphology of As.
niger and citric acid production in two reactor systems with different configurations, a
tubular loop and a conventional stirred tank bioreactor, with operating volumes of 6 and
8 l, respectively. Morphology was characterized by image analysis. In each system,
morphology, characterized by the parameter P (mean convex perimeter of clumps), and
citric acid production were agitation dependent and closely linked. Increased agitation
caused a reduction of clump sizes and results from both reactors demonstrated that P
should not exceed a threshold value in order to achieve increased productivities. The
results obtained from the two reactors were in agreement, both qualitatively and
quantitatively. Reducing the fundamentally different mixing conditions of the two systems
to the order of the dimensionless relative mixing time (tm), results showed that the loop
simulated the stirred tank. Also, relationships valid for one system accurately described
the results obtained from the other system, demonstrating the validity of the relationship
between morphology and productivity for the particular fermentation, regardless of the
reactor type.
10. Fungal morphology and rheology of fermentation broths
The flow behavior of fermentation broths containing filamentous microorganisms

differs considerably from that of most bacterial cultures because of the complex
morphology of these microorganisms. Several studies have shown that fermentation
broths containing increased concentrations of filamentous microorganisms are highly
viscous and are characterized by shear rate-dependent viscosities (Charles, 1978; Ruess et
al., 1982; Ju et al., 1991; Olsvik and Kristiansen, 1992a) and by a yield stress (Olsvik and
Kristiansen, 1992b; Berovic et al., 1993). Fermentation broth rheology greatly affects the
transport phenomena in the bioreactor which, in turn, have a strong influence on the
efficiency and productivity of the entire process. Hence, an understanding of the broth
flow behavior is necessary in order to develop design strategies that will help to overcome
possible limitations in mass, momentum and heat transfer in fermenters.
Although pelleted fermentation broths may be Newtonian and of low viscosity,
problems might arise with the transport of nutrients inside the pellets, thus reducing
productivity. The dispersed forms therefore predominate in most industrial fermentations
(Riley et al., 2000). Small increases in biomass concentration in such fermentations can
cause large increases in broth viscosity. Even in early stages of many fungal fermentations
the broth can exhibit several non-Newtonian characteristics, possibly including a yield-
stress. The rheological properties of a filamentous fermentation are normally considered to
be determined by the biomass concentration and the morphological form of the mycelia
(Metz et al., 1979; Charles, 1978), which is partially affected by the operating conditions
in the fermenter (Metz et al., 1979; van Suijdam and Metz, 1981). To control fermenter
performance, it is necessary to know how the operating conditions are influencing the
rheological properties of the filamentous fermentation broth. As these depend on the
morphology of the fungus, the relationship between rheology and morphology is vitally
important.
Correlations between parameters describing rheological parameters of the broth and its
biomass concentration have been suggested by many investigators (Deindoerfer and
Gaden, 1955; Carilli et al., 1961; Solomons and Weston, 1961; Metz et al., 1979). For
the dispersed growth forms, it has been suggested that (in the case the broth has power
law-like behavior) the consistency index varies with biomass concentration, typically to
the power of 0.3 3.3 (Fatile, 1985; Allen and Robinson, 1990; Kim and Yoo, 1992;
Olsvik et al., 1993; Tucker and Thomas, 1993; Olsvik and Kristiansen, 1994; Tucker,
1994). Comparatively fewer are the reports on correlations between morphological
parameters and broth rheological properties, and this is due to the large variability that
exists in the morphology of mycelia grown in submerged cultures (Metz et al., 1981),
which makes its characterization difficult, and also due to the lack, until recently, of the
appropriate instrumentation for making morphological measurements not only on the
individual hyphae but also on mycelial aggregates. Using image analysis, Paul and
Thomas (1998) showed that for many Pe. chrysogenum strains, the mycelial clumps
can account for >90% (in terms of percentage projected area, which is used as an estimate
of clump size) of the mycelial biomass within the fermentation broth and this is also true
for other fungi (Papagianni, 1995). It was suggested, therefore, that the rheology of fungal
fermentation broths should be related to clump properties rather than to the morphology of
small amounts of freely dispersed mycelia (Tucker and Thomas, 1993). Quantitative
studies on morphology and rheology include the works of Roels et al. (1974), Metz et al.
(1979), Fatile (1985), Kim and Yoo (1992), Liu and Yu (1993), Olsvik et al. (1993),
Tucker and Thomas (1993), Olsvik and Kristiansen (1994), Tucker (1994), Mohseni and
Allen (1995), Johansen et al. (1998), Bocking et al. (1999), Riley et al. (2000), Sinha et al.
(2001a,b) and Pollard et al. (2002).
Tucker and Thomas (1993) investigated the separate influences of biomass concentra-
tion and mycelial morphology on broth rheology using an industrial strain of Pe.
chrysogenum and suggested the correlation
RP constant Cma roughnessb compactnessv
where RP is the rheological parameter under examination, Cm is the biomass concentration

and a, b and c are the exponents for each rheological parameter. Compactness and
roughness are clump properties that can be quantitatively characterized by image analysis.
The above correlations were fairly successful at predicting broth rheology for batch
fermentations. Tucker (1994) later established that a was essentially constant throughout a
batch fermentation. Olsvik et al. (1993) used the image analysis method of Tucker et al.
(1992) on As. niger broths from 7-l chemostats with varying dilution rates and dissolved
oxygen tensions. Changes in the rheological properties of the broths (measured on-line),
represented by the power law consistency index, depended on biomass concentration and
clump roughness. As the latter increased, so did the consistency index. Later, Kristiansen
and Olsvik (1993) proposed a similar correlation for batch and fed-batch fermentations of
As. niger.
Mohseni and Allen (1995) used image analysis to examine the influence of biomass
concentration and particle morphology on the yielding properties of filamentous broths of
As. niger. Correlations were found with the freely dispersed form of the biomass
concentration, the mean dimensionless length, and the mean hyphal growth unit. The
studies by Mohseni and Allen (1995), Olsvik et al. (1993), Kristiansen and Olsvik (1993),
Tucker et al. (1992) and Tucker and Thomas (1993) appear to agree that clump
morphology and in particular clump roughness is important in determining fungal broth
rheology. However, in all cases, the data were limited, and no indication was made
concerning the statistical significance of the predictions made using the rheological
parameter correlations. A much broader range of data was presented by Riley et al.
(2000) from which another correlation for the consistency index was proposed, with error
analysis:
K Cm2 5 103 D 103 21
where K is the consistency index, Cm is the biomass concentration and D is the mean
maximum dimension.
Johansen et al. (1998) studied the relationship between As. awamori morphology and
heterologous protein production during a series of fermentations with a batch phase
followed by a fed-batch phase. Agitation rate and inoculum concentration were used as
controlled variables to generate different fungal morphologies in 20-l stirred tank reactors.
A threefold increase in hyphal length increased the apparent viscosity of the broth by a
factor of 7. The observed morphological differences had only a limited effect on product
formation, suggesting that the structural features such as hyphal length and number of tips
were of less importance for product formation. The primary effect of morphology on
product formation was due to viscosity.
11. Characterization of morphology in fungal fermentations
Many attempts have been made to relate morphology of filamentous fungi and
metabolite production. Although the morphology of these organisms reflects their
physiology during fermentation and is closely linked with process performance, little
quantitative work to evaluate its importance was undertaken until the 1970s. Initial
investigations on mycelial morphology relied upon manual measurements performed
under a microscope, by either direct observation or photography. Subsequently, simple
digitizing tables were used by Metz et al. (1981) and van Suijdam and Metz (1981).
Photographs of mycelial particles were made through a microscope and projected on an
electronic digitizing table attached to a computer. The digitizing table determined the x and
y coordinates of any point touched by a cursor. Morphological parameters such as main
hyphal length, total hyphal length and number of tips were calculated. The method was
labor-intensive, time-consuming and not particularly precise or accurate. It was also
difficult to automate.
To quantify the morphology throughout a fermentation, an automated method of
analyzing a significant number of mycelia from each sample is desirable. In 1988, Adams
and Thomas presented a method for morphological measurements based on image analysis
(Adams and Thomas, 1988). The method was faster and more accurate than the digitizing
tables although considerable manual intervention was still required. It was clear that the
full potential of image analysis could only be achieved by automation. The power of image
analysis lies in extracting quantitative information from a picture in a reproducible,
automated manner. Many applications of image analysis in studies of morphology and
differentiation have appeared since the first work of Adams and Thomas (1988). The
principle stages of image analysis are image capture and enhancement, segmentation,
object detection, measurement and analysis (Paul and Thomas, 1998). In image capture, a
television camera is mounted on a microscope and the image from the camera is digitized,
through a digitizer, both in space and tone producing an array of picture elements or pixels.
In segmentation, the region of interest is separated from the background, i.e., all tones
below a selected level might be treated as black and all above as white. Object detection is
a data reduction step which produces a description of the object of interest for measure-
ments and analysis. Repetitive measurements can be automated and thus made more
accurately and reproducibly.
In 1990, Packer and Thomas presented a software for a fully automated system using a
general purpose image analyzer (Packer and Thomas, 1990). The method was tested on
Streptomyces clavuligerus and Pe. chrysogenum samples. The automation and speed of
the process made it practical to measure distributions of hyphal parameters within a
sample. It also revealed that filamentous fermentation broths contain significant amounts
of aggregated material (clumps)an observation not apparent using earlier methods.

Because of the prevalence of clumping in filamentous fermentations, and the fact that
mycelia in clumps cannot be isolated for independent measurements, Tucker et al. (1992)
developed a method that could give important measurements on clumps. Detailed
information such as projected area (clump area), perimeter, compactness and roughness,
circularity, as well as degree of branching was presented quantitatively for St. clavuli-
gerus and Pe. chrysogenum. The method of Tucker et al. (1992) was used by Amanullah
et al. (2001) to describe the dynamics of mycelial aggregation in batch and chemostat
cultures of As. oryzae.
Paul et al. (1993) developed an automatic image analysis method to test the viability
and characterize the germination of fungal spores in submerged processes. Swelling spores
and germination processes constitute a major part of the lag phase and the subsequent
culture morphology and productivity can be greatly influenced by the initial concentration
and condition of spores. The method of Paul et al. (1993) offers a number of advantages
over photomicroscopy or colony counting. It is rapid, more accurate and can discriminate
between non-germinated and just-germinated spores. In particular, this can be used on
spores germinating in actual submerged fermentation medium. Structural variations during
germination, e.g., swelling, germ tube formation and elongation were measured in terms of
their distribution of spore volume and germ tube length and volume.
The structural complexity of the mycelium grown in dispersed form in submerged
processes was quantified for the first time by Paul et al. (1992) using a fully automatic
image analysis method. The method is capable of differentiating between cells completely
filled with cytoplasmic materials, vacuolated cells, and degenerated or empty cells.
Proportions of these three regions were quantified. Vacuolation was also expressed in
terms of the distributions in volume and shape (circularity) of vacuoles and empty cells.
The method was tested by monitoring these parameters throughout a laboratory scale fed-
batch penicillin fermentation. Later, a method for the simple differentiation of Pe.
chrysogenum was developed by the same group (Paul et al., 1994b). The resulting
quantitative information about the biomass structure permitted a powerful structured
model for the penicillin fermentation to be devised (Paul and Thomas, 1996a,b). These
image analysis techniques permit the rapid and accurate measurement of simple cellular
differentiation of fungi and represent a valuable tool in studies of the links between
differentiation and metabolite production, and might be used in process control of fungal
fermentations.
There are a number of image analysis methods available to characterize pellets from
submerged fermentations. Reichl et al. (1992) using an image analysis system described
pellet morphology during growth of St. tendae by numerous features, such as frequency
distributions, mean sizes, content of pellets and shape of pellets. The shape factor was the
first proposed for qualitative discrimination between pellets. Durant et al. (1994b)
discriminated between pellet zones by rinsing out a stain which made image analysis
much easier. The method was further improved using color-image processing (Durant et al.
1994a). Pichon et al. (1993) and Cox and Thomas (1992) proposed image analysis
methods to characterize pellets based on the presence of a central core. Cox and Thomas
(1992) further classified the pellets into smooth and hairy types using automatic image
analysis.
Image analysis applications in fungal fermentations have been most recently reviewed
by Paul and Thomas (1998) and Thomas and Paul (1996). Image analysis methods have
found applications in studies of early growth of filamentous fungi, morphological
development during submerged fermentations, e.g., dynamics of aggregation, vacuolation,
fragmentation, autolysis, physiology and rheology studies. Most of these applications have
been reviewed in this work. Other applications include the estimation of mycelial cell
volume and biomass, as well as the connection between surface and submerged
morphologies, the screening for favorable morphologies, mutant selection and culture
maintenance.
Nestaas and Wang (1983) developed a filtration probe and through a theoretical
analysis of the filtration behavior of the mycelia were able to estimate mycelial cell
volume. Also, from the filter cake volume, the total cell mass could be estimated.
However, the filtration probe did not provide a direct measurement of cell volume and
morphology. The methodology devised by Packer et al. (1992) using image analysis for
direct quantification of mycelial differentiation and measurement of cell volume results
additionally in an estimate of biomass in fermentation.
The connection between surface and submerged morphologies has been investigated by
Larralde-Corona et al. (1994). These authors developed and tested a model to correlate
morphometric data from Gibberella fujikuroi grown on solid media with observed specific
growth rates in stirred fermenters. However, the connection between surface and
submerged morphologies is still poorly understood, although it might be exploited in
screening for favorable morphologies, mutant selection and culture maintenance. A major
problem in the selection of improved mutants of fungi that over-produce enzymes is to
have in situ and non-destructive techniques to evaluate their phenotypes that may be
related to their ability to produce and excrete enzymes. Carlsen et al. (1994, 1995) have
used image analysis of submerged cultures of As. oryzae to relate their morphology to a-
amylase production. Minjares-Carranco et al. (1997) showed that image analysis of
colonies of some As. niger strains helped to relate their morphological features (sporu-
lation and apical extension rates) to the pectinase production features. Loera and Viniegra-
Gonzalez (1998) used image analysis to classify (on the basis of the specific growth rate)
pectinase over-producing mutants of As. niger in selective culture media. Such pheno-
typical classification can be related to enzyme production levels, helping to correlate the
effect of certain mutations on different kinds of physiological processes. The authors
suggested that their approach may help to improve the selection procedures for enzyme
over-producing strains specially adapted to different kinds of culture media with different
water activity values, for instance, to solid-state or submerged fermentations.
Image analysis can be combined with various staining techniques in physiology and
differentiation studies of filamentous fungi in order to visualize the organelles or the cell
wall. Fluorescent stains coupled with image analysis have been used by Vanhoutte et al.
(1995), Agger et al. (1998) and Hamanaka et al. (2001). Vanhoutte et al. (1995) presented
a quantitative image analysis method for characterizing the physiology of Pe. chrysoge-
num. The method which was based on a differential staining procedure showed and
quantified six physiological states: growing material (zone 1), three differentiated states
characterized by an increasing granulation (zones 2, 3, 4), a highly vacuolized state (zone
5) and dead segments having lost their cytoplasm (zone 6). The image analysis software,
with versions written for monochrome and color images, consisted of a semiautomatic
binary mask computation step and a fully automatic segmentation step based on a fuzzy
classification. The use of a new staining procedure and the fine discrimination of
physiological states by color image analysis shed a new light on the growth and
differentiation of Pe. chrysogenum.
Agger et al. (1998) used fluorescence microscopy and automated image analysis in
studies of the growth and product formation of As. oryzae. The organelles inside the
hyphae as well as the cell wall were stained. The ratio between the projected areas of the
organelles and the entire hyphal element was taken to be proportional to the fraction of
active cells in a morphologically structured model describing the growth and product
formation in fed-batch and chemostat cultures. When applied to fed-batch and chemostat
experiments, the double-staining method confirmed the basic morphological structure of
the model. Fluorescence microscopy and three-dimensional image analysis were used by
Hamanaka et al. (2001) to study the intracellular product distribution inside the pellets of
the arachidonic acid producer Mo. alpina. The combination of these methods allowed the
visualization of the time course of pellet morphology and showed that the lipid was
produced on the edge of pellets, an area where the mycelial density was higher compared
to the other regions.
Morphological characterization of filamentous microorganisms is most commonly
undertaken using image analysis. Other methods of morphological quantification have
also been employed. Liu and Yu (1993) used rheology and filtration methods to
characterize the morphology of Pe. chrysogenum, while flow cytometry can also be used.
Gerlach et al. (1998) used neural networks and cluster analysis in studies of As. awamori
morphology. Image analysis is generally limited by relatively long processing times, the
extent of operator intervention and the need for off-line sample preparation and analysis.
However, recent developments allow the use of image analysis on-line. Christiansen et al.
(1999) studied the growth kinetics of single As. oryzae hyphae by on-line image analysis
using a flow-through cell technique. Treskatis et al. (1997) worked on the morphological
characterization of filamentous microorganisms in submerged cultures by on-line digital
image analysis and pattern recognition. Bittner et al. (1998) used in situ microscopy for
on-line determination of biomass.
12. Modeling the growth and product formation in fungal fermentations
There have been many different types of kinetic models for filamentous microorgan-
isms in the literature (Nielsen, 1992; Krabben and Nielsen, 1998; Bellgardt, 1998;
Wayman et al., 1999). Unstructured models use a single biomass component to describe
the total biomass concentration in steady-state conditions. They are deterministic in their
approach and perform poorly when applied to significantly different operating conditions.
Structured models are also deterministic, but are an improvement on unstructured models
as they may use more than one set of equations to represent different phases of growth and
production. It is also possible to represent biomass as more than one component to
describe the physiological state of the microorganisms during fermentation. Both types of
model involve the specific growth rate (l) as a function of the concentration of substrate
(S), product ( P) and biomass. Mechanistic models describe the conversion from substrates
into products by applying the known concentrations of metabolites and properties of all
the enzymes in the reaction sequence. Such models are closely related to the models used
for metabolic control analysis, where the activity of individual enzymes is related to their
effect upon the overall reaction rate. This process has been carried out for the production
of citric acid through the glycolytic pathway by As. niger by Torres (1994a,b) and was
successful in showing that the activities of glycolytic enzymes have little influence on the
rate of product formation in that system.
Several structured models for fungal fermentations are available that consider hyphal
growth, differentiation and metabolite production. Structured models for Aspergillus
growth and citric acid production have been presented by Kristiansen and Sinclair
(1979), Rohr et al. (1981), Ho et al. (1994) and Torres et al. (1996). In the work of Rohr
et al. (1981), cell growth and product formation were subdivided into several phases, each
described by a simple deterministic model. Product formation was related to the growth
rate by a modified Luedeking Piret equation. Although the same descriptions were
applicable for both growth and acid formation, the acid formation kinetics usually
differentiated from the growth kinetics by a term that represented the lag time, which is
also known as the maturation time, when the culture has taken up all the ammonium ions
but does not yet produce citric acid. The model closely resembled the data from citric acid
production. Ho et al. (1994) developed their model in a similar way to that of Rohr et al.
(1981) by plotting the results of fermentations and identifying phases where simple
expressions could be used to describe ammonium and glucose uptake, the drop of pH and
the accumulation of biomass and citric acid. The model differed from that of Rohr et al.
(1981) because simple linear relationships were used to describe all the features with
respect to one limiting factor per phase. All the phases linked together to achieve a degree
of overlap during the batch. Four different equations were used for the growth rate with
different values of maximum specific growth rate. It was possible for more than one
expression to be valid at any one time, but it was unlikely that a situation would arise when
all expressions were valid. The model was quite complex, but it was still a deterministic
model, and it could not accurately simulate the results of alternative batch conditions.
Growth and metabolite production from filamentous fungi are associated with complex
and not well-understood processes. The multicellular structure of the mycelium, the
morphological heterogeneity and the heterogeneity in physiology and differentiation along
the length of the hyphae and during fermentation makes it difficult to construct
mathematical models of fungal fermentations to use for predictive simulations or process
control. To take into account these variations in the state of biomass, differentiation
including structural features should be incorporated into models of growth and metabolite
production. In such models, the structural features would ideally be related to the
underlying physiological states of the hyphae and, consequently, to metabolite production
(Paul and Thomas, 1996a,b).
The work of Megee et al. (1970) was a fundamental approach in which the hyphae of
As. awamori were divided into five differentiation states, assuming different activities and
metabolite synthesis of the hyphal compartments. In submerged fermentation, it was
assumed that the active growth of hyphae occurred at the tips. This apical region was
assumed to give rise to a second region characterized by the production of a growth-
associated product. The other three regions were classified on the basis of three different
types of non-growth-associated products. The model involved a large number of
parameters that were difficult to experimentally validate. Later, a simplified but similar
structured model was developed by Nestaas and Wang (1983) for the penicillin
fermentation. The hyphae were divided into three regions: actively growing tips, non-
growing regions and degenerated regions. This model was also difficult to apply to real
processes and required one set of equations for the growth phase and another set for
production. A unified set of equations for the whole fermentation was used by Cagney et
al. (1984). However, it was not possible to verify the model because of the lack of
experimental data.
Nielsen (1992) described a structured model for the growth of filamentous micro-
organisms. The differentiation mechanism considered in the model was similar to that
described by Megee et al. (1970). The hyphae were divided into three regions: apical,
subapical and hyphal. The model described the growth mechanisms of filamentous
microorganisms, i.e., tip extension and branching, and it also considered fragmentation
in the estimation of the morphology of the individual hyphal elements in a submerged
culture. The model was a combination of a simple morphologically structured model
describing the growth kinetics of the individual hyphal elements and a population
model, which described the properties of the hyphal elements in the culture. The two
parts were described separately before they were combined into the complete model.
Comparison of the complete model with experimental data for several different species
of filamentous microorganisms (G. candidum, S. hygroscopicus and Pe. chysogenum)
illustrated the predictive power of the complete model. A certain drawback of the model
was that no mechanism was considered for the formation of product as a consequence of
differentiation.
Paul and Thomas (1996a,b) reconsidered the structured modeling approach originally
proposed by Megee et al. (1970) and later simplified by Cagney et al. (1984) for the
penicillin fermentation. The differentiation of the hyphae was incorporated in a model on
the basis of quantitative information obtained by image analysis measurements. In this
model, the vacuole formation was considered as an important physiological process during
growth and aging of hyphae. The model divided the biomass into three distinct regions
according to the activities and structure of hyphal compartments: the actively growing tips,
the non-growing penicillin-producing regions and the degenerated or metabolically
inactive regions that were subject to fragmentation and autolysis. A mechanistic approach
was taken to give quantitative descriptions of differentiation and degeneration as a
consequence of vacuolation. The model assumed that newly generated vacuoles appeared
by differentiation of healthy regions, grew in size with limitation of available substrate and
eventually gave rise to empty hyphal compartments. Penicillin production was related to
the amounts of the non-growing regions of the hyphae. The model was used for successful
predictions of the amounts of the four hyphal regions and the penicillin production rate in
fed-batch fermentations of an industrial Pe. chrysogenum strain under different glucose
feeding regimes. When the glucose feed rate was switched between a high and a low value
the model could successfully predict the dynamic changes of differentiation and the
resulting penicillin production caused by the variations in the nutrient conditions. The use
of image analysis to characterize differentiation as a basis for structured modeling of the
penicillin fermentation appears to be a powerful method that has a great potential for use in
process simulation and control of antibiotic fermentations.
13. Commercial use of morphological data
The amount of research carried out on the morphology of filamentous fungi has been
impressive. However, the implementation of these results in the fermentation industry is
limited. Although highly structured models exist for morphogenesis and for the relation
between morphology and productivity, the application of these models in the industry
seems to be rather limited for practical reasons. The pressure of economics and time in the
fermentation industry does not allow for time-consuming practices. This means that very
often morphology problems are solved by empirical methods based on extensive
experience. For extensively structured models, parameter estimation remains a big
problem since it is often more time-consuming than the setup of the model itself.
Concerning industrial applications, the effect of scale-up on the values of parameters is
often neglected. Fungal morphology is very dependent on environmental conditions,
which are themselves very often dependent on scale. The majority of parameters have been
obtained from small-scale experiments, because data from production scale operations are
not usually available for publication. Other factors related to scale-up and able to influence
the behavior of a fungal culture are the types of substrates (substrate can be quite different
on production scale compared to those used on laboratory scale) and the number of
inoculation steps. The latter increase from one on the laboratory scale to about four on
production scale.
Present models and practices are based almost entirely on the influence of environ-
mental factors on morphology. Increasingly, the contribution of pure physiologists to
knowledge of morphology is growing steadily as more details of the transport processes
and the kinetics involved in the development of morphology become known. What is
unfortunate is that structured mechanistic models, based on intracellular reactions at a
molecular level, are absent and information on the genetics that govern the development of
morphology is limited. Manipulation of morphology through inoculation protocols,
process parameters, and mass screening of cultures need to be relied on until basic
genetics knowledge of morphology has developed sufficiently.
14. Concluding remarks
Mycelial fungi are widely used to produce antibiotics, enzymes and many other
useful metabolites. Increasingly, fungi are being used to produce recombinant proteins
and other compounds. In many cases, the fungal morphology directly or indirectly
influences the productivity of fungal fermentations. A large number of factors contribute
to the development of any particular morphological form in submerged fermentation and
it is difficult to deduce unequivocal general relationships between process variables,
product formation and fungal morphology. Too many parameters influence these
interrelationships and the role of many of them is still not fully understood. However,
the relatively recent use of image analysis has proved a valuable tool for characterizing
complex mycelial morphologies, physiological states, and their interactions with fer-
mentation conditions. Quantitative information of mycelial differentiation can be used to
build structured models of predictive value and this understanding should lead to
improved design and operation of mycelial fermentations. Although the models can
help make best use of the available experimental data, it is absolutely necessary to
develop improved and new experimental techniques and measuring methods to under-
stand phenomena such as the breakage of hyphae and its relationship to mechanical
stress. This would allow further progress towards a better understanding of production
processes involving filamentous fungi and help establish process control schemes on a
true physiological basis.
Although any particular morphological type is the result of the genotype of the
organism and the environment, present models and practices are based almost entirely
on the influence of the environmental factors on morphology. Little knowledge is available
about the effect of genes on morphology. This makes morphology rather difficult to
control, because environmental factors vary considerably during production and with the
scale of operation. The genetics of morphogenesis can be very complicated and, as Kossen
(2000) pointed out, there is little hope of its elucidation in the near future.
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Biotechnology Advances 22 (2004) 189 259

Fungal morphology and metabolite production in

Accepted 29 September 2003

Keywords: Filamentous fungi; Morphology; Shear effects

form may be preferred to achieve maximal performance. Filamentous growth of Asper-

2. Growth mechanisms in filamentous fungi

Under appropriate conditions, the vegetative mycelium gives rise to a reproductive

2.1. Hyphal tip extension

2.2.1. Exponential growth

2.2.2. Modeling tip growth and branching

restored at the new temperature. Thus, the statistical distribution of branch-to-branch

3. Dynamics of mycelial aggregation

In submerged cultures, many filamentous microorganisms tend to aggregate and grow

and Vas, 1968), polygalacturonidase (Hemmersdorfer et al., 1987), phytase (Papagianni et

4. Growth of fungal pellets

where M represents the biomass concentration and k is a constant. Pirt (1966)

Restriction of growth to two dimensions to form a colony of height h, which expands at

5. Effects of diffusional limitations inside pellets

6. Cell aging and autolysis

An important aspect of pellet growth is fragmentation, or breakup of pellets. It has been

7. Fragmentation of hyphal elements and pellets

Fragmentation of hyphal elements and pellets during submerged fermentations often

dp dpK br ed2 p

8. Growth in submerged culture

8.1. Batch culture

filamentous fungi because of the influence of their morphology on rheological properties.

8.2. Continuous culture

Continuous culture is usually preceded by growth of the fungus in batch culture to

where l is described by the Monod equation, Sr is the concentration of the limiting

8.3. Fed-batch culture

The majority of large-scale industrial fungal fermentations involve fed-batch culture in

9. Influence of process parameters on the fungal morphology and productivity

9.2. Medium composition

phosphate concentrations cause a shift from metabolite overproduction to overgrowth of

9.3. Type and concentration of the carbon source

9.4. Nitrogen and phosphate

Nitrogen may be supplied as ammonia, as nitrate or in organic compounds, such as

9.5. Metal and other ions

9.6. Dissolved oxygen tension

9.7. Dissolved carbon dioxide tension

9.10. Mechanical forces

a relationship between vacuolation, fragmentation and product formation under various

10. Fungal morphology and rheology of fermentation broths

The flow behavior of fermentation broths containing filamentous microorganisms

RP constant Cma roughnessb compactnessv

where RP is the rheological parameter under examination, Cm is the biomass concentration

K Cm2 5  103 D 103 21

11. Characterization of morphology in fungal fermentations

of aggregated material (clumps)an observation not apparent using earlier methods.

12. Modeling the growth and product formation in fungal fermentations

13. Commercial use of morphological data

14. Concluding remarks

Hemmersdorfer H, Leuchtenberger A, Wardsack C, Ruttloff H. Influence of culture conditions on mycelial

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dp dpK br ed2 p

K Cm2 5 103 D 103 21