Strathclyde Fermentation Centre, University of Strathclyde, Glasgow, U.K

Bio~cK Aav. Vol. 12, pp. 1--39,1994 0734-9750/94 $24.
00
Printed in Great Britain.All Rigb.tlReserved, 1993 Perpmoaa Prme Lid
RHEOLOGY OF FILAMENTOUS FERMENTATIONS
E. OLSVIK* and B. KRISTIANSEN
Strathclyde Fermentation Centre, University of Strathclyde, Glasgow, U.K.
ABSTRACT
The performance of a bioreactor containing a filamentous fermentation broth is greatly

influenced by the theological properties of the broth. These properties are determined
mainly by the concentration of biomass, its growth rate and morphology. Included in the
morphology are such factors as the geometry of hyphae (length, diameter, branching
frequency), hyphal flexibility and hyphal-hyphal interactions, which can all be affected by
the operational design of the reactor. Thus, correlations describing viscosity as a function
of biomass only are of limited value. A better understanding of the relations between
morphology and rheology may be achieved by a combination of theological and
morphological studies.
Rheological properties are normally determined using off-line measurements in-spite
of associated problems with sample treatment influencing the results. Equipment for
dynamic, on-line, measurement of morphology and rheology is available, but little used in
filamentous fermentations. Controlling the rheological properties of mycelial fermentations
may be difficult because of the great number of factors influencing mycelial development
and/or hyphal-hyphal interactions.
Polymer solutions are often used to simulate flow behaviour of filamentous
fermentations and scale-up and mass transfer considerations are based on these studies.
Although much information has been gained this way, the predictions developed do not
include the effect of an active biomass on the mass transfer and flow properties of the
culture. It is important to carry out studies on the non-homogeneous fermentation fluids,
and develop correlations based on these studies.
Keywords: Rheology, fermentation broths, dynamic viscosity measurements, fungal

morphology, filamentous fungi.
* Permanent address: SINTEF-SI, P. O. Box 124 Blindern, 0314 Oslo 3, Norway.

2 E. OLSVIK and B. KRISTIANSEN
INTRODUCTION
The morphological growth form of fungi may give fungal broth suspensions characteristics
quite different from those of bacterial and yeast cultures. The mycelial growth forms may
be divided into two main categories: the filamentous and the pellet forms. Between these
two extremes lies a wide range of mycelial morphologies. The pellet growth form consists
of branched hyphae intertwined to a stable aggregate, and this growth form causes less
viscous fluids than the filamentous form. Attention is being given to change the morphology
of filamentous strains to pellet growing strains because this favourably alters the overall
rheology of the fermentation broth. The pellet growth form may, however, impose other
problems such as nutrient limitation in the pellets.
The filamentous growth form is dominant in most processes and long, thin, usually
branched, hyphae create a three-dimensional network which may give very viscous
fermentation broths. The theological properties of a fermentation broth will affect the
turbulence characteristics in the fluid and, hence, heat and mass transfer processes and
mixing [22,52]. This in turn will influence the operating conditions and thus affect growth,
morphology and product formation. The system is complex and difficult to predict because
a number of feed-back relations exist [52].
The influence of fluid flow properties on reactor performance is widely recognised;
however, only a few systematic studies of the theological properties of fermentation fluids
have been reported and the use of rheometry in fermentation processes is limited [22]. The
reasons for this lack of attention appear to be the problems associated with measuring
rheological properties of the non-homogenous fermentation broths and difficulties in
unambiguous interpretation of the results. Not only is there a question of use of the
appropriate instrument, but also the problem of representative sampling [89] of the broth.
Rheological properties are intrinsic properties of the fluids and should ideally not be
influenced by the measuring technique or instrument.
The earlier reviews [22, 52] presented fundamental information on rheology of
filamentous fermentation broths and could be used as the basis for any work in this area.
Metz et al. [52] discussed methods for measuring rheology in mycelial fermentations and
presented correlations for relating the biomass concentration and the morphology of the
fungi to the theological properties of the broth. Still earlier work of Bongenaar et al. [14]
and Roels et al. [79] had already pointed out the difficulties of using conventional
viscometers in measuring the rheological properties of fungal fermentation broths, and new
RHEOLOGYOF FILAMENTOUSFERMENTATIONS 3
impeller viscometers were presented. These impeller viscometers were meant to prevent
settling of the hyphae in the measuring cylinder (which was thought to be a problem with
conventional rheometers) and destruction of the floes in the annulus of the apparatus.
Settling or restructuring of the mycelium in the impeller viscometer was observed [52] but
it was much reduced compared to other instruments. On-line instruments for dynamic
determination of the theological properties of fermentation broths were developed based
on the impeller system [44,47]. Dynamic measurements of filamentous fermentations have
also been carried out using pipeline rheometers [3,11,32]. In-situ instruments have been
developed [31,73], although not used in fungal fermentations.
Since the work of Metz et al. [52], no significant contributions to the understanding
of the effect of the operating conditions on broth theology have been reported. Papers
showing the effect of operating conditions on the morphology of the fungi have been
published [53,56,102,105,108,109], but no direct information on broth rheology can be
derived from these morphology studies because the rheological properties of the broth are
not only a result of the morphology of the fungi, but also of hyphal-hyphal interactions in
the broth [22,52,101]. The broth rheology may be a factor limiting the productivity in many
fungal fermentations [22,52]. Despite this, it appears from the available literature that very
little work on control of broth rheology, on the effects of operating conditions on broth
theology or on the relations between bulk rheological properties and fungal morphology
has been carried out.
RHEOLOGICAL MODELS
Fluid mechanics can be defined by solving a set of constitutive equations, including the
constitutive rheological equation or a rheological model [68]. Newtons law is the simplest
of rheological models. Here, viscosity, rl, is defined as the relationship between the applied
shear stress and the resulting movement of the fluid (shear rate).
rl- x y-l, (1)
where rI is the viscosity (N m "2 s), x is the shear stress (N m -2) and y is the shear rate (s-l).
The viscosity of a Newtonian fluid is independent of the shear rate. For
non-Newtonian fluids the relationship is more complex. Mycelial fermentation broths
generally have pronounced non-Newtonian rheological characteristics and show shear
thinning or pseudoplastic behaviour [25,26,90]. Pseudoplastic fluids exhibit a decrease in

viscosity with increasing shear rate. The viscosity in such fluids is shear dependent (or
apparent) viscosity.
Three mathematical models commonly used for describing the rheology of mould
suspensions are given in Table 1. These models describe fermentation broths in terms of
time independent, non-viscoelastic fluids. No rheological models proposed so far describe
the time-dependent behaviour of microbial broths, although experimental results have
shown that such behaviour exists [14,26,68,79]. Since it is impossible to have complete
knowledge of shear stress/shear rate relations and the elastic properties in the different
flow fields for non-Newtonian fluids, the rheological models may be regarded as an
empirical fit to the experimental data [68].
Table 1 Rheologicai models
Bingham x = x0 + n Y (2)
Casson x0.5 = ~00.5 + K y 0.5 (3)
Power Law x =Kyn (4)
In Table 1 ~o is the yield stress (N m-2), K is the consistency index (N m "2 sn), n is the flow
behaviour index and Kc is "Casson" viscosity (N m -2 s)'5.
The power Law model is commonly used to describe rheological behaviour of
fermentation fluids [5,26,40,76,92,99]. This model does not include a yield stress, but an
adaptation of the equation to include yield stress has been proposed by Deindorfer and
West [26].
The Bingham model includes the often observed yield stress, which has to be
exceeded before the fluid will flow. The existence of yield stress is important, because it
will determine start-up power requirement in pumping through pipelines and mixers and
the existence of dead regions in fermenters [7,23]. A number of authors have described the
rheological properties of mould suspensions by the Bingham model [25,26,87,88]. A review
of the different aspects of the yield stress phenomenon was given by Cheng [23], who
pointed out that the existence of yield stresses was debatable. The paper showed that a
fluid can exhibit a range of yield stresses and how these stresses can be measured. The
RHEOLOGY OF FILAMENTOUS I~r..RMENTATIONS 5
yield stress phenomenon in filamentous fermentation fluidswas discussed by Moo-Young
et al. [58].They found that polymer solutions could be used for predicting the bchaviour
of fermentation fluids if no yield stress existed in the fermentation fluids, but failed to
describe fermentations where yield stresswas measured. However, no clear answers have
emerged as to whether yield stress exists in filamentous fermentation fluids.
The Casson model is also used to describe filamentous fermentation fluids. It is
claimed that the fit to experimental data at low shear rates is better using the Casson
model than the power law model [14,35,52,68,79]. The shear stress-shear rate behaviour
of different theological models is shown in Figure 1.
Scott Blair [84] demonstrated by statistical testing, that if the data ranges over only
one or one-and-a-half decades, alternative models can be used which will describe the data
equally well. Reuss et al [75] compared the Casson, Herschel and Bulkley and the power
law plots for the apparent viscosity of a suspension of Penicillium ctuysogemtrn. They
concluded that on the basis of measurements in the small range of shear rates which could
be measured with the impeller rheometer, it was not possible to distinguish between these
models. Other work also showed that the two-parameter model was sufficient for describing
data measured in the shear rate range of 2 to 650 s-1 (pipeline measurements) [6]. Addition
of a third parameter did not significantly improve the model fit to the data.
(N rn -=)
~ tic
> 9" (s -~)
Figure 1. Rheological models.

6 E. OLSVIKand B. KRISTIANSEN
The consistency index, K, have also been used as the sole indicator for the viscosity
of filamentous fermentations [63,64,83]. This is often a valid assumption, because the value
of the flow index (n) is affected by the operational design to a smaller extent than the
value of K [5,66]. However, because n is an exponent, changes in n will have a larger effect
on shear stress than a similar change in K. Thus, K-values can only be used as an indicator
for fluid viscosity if n is constant or the change in n is constant [66]. Correlations between
the consistency index and the biomass concentration for strains of Penicillium, Aspergillus
and Streptomyces have been reported, but no correlation was found between the flow index
and the biomass level [5].
EFFECTS OF VISCOSITY ON TRANSPORT PHENOMENA

The viscosity of a fluid influences the turbulence as expressed by the Reynolds number for
mixing (Re):
Re = Di 2 N p ( qa )-1, (5)
where D i is the impeller diameter (m), N is the impeller speed (s-l), p is the broth density
(g L -t) and qa is the shear dependent viscosity (N m "2 s).
High turbulence in the system is needed to promote mass and heat transfer [89]. As
the viscosity in fungal fermentations may increase a hundredfold over the course of a batch
process [25], the mass and heat transfer rates and the degree of mixing are adversely
affected. High viscosities lead to high mixing time, especially in non-Newtonian fluids [57],
chemical gradients in the reactor broth, stagnant zones and loss of productivity due to loss
in active volume [22]. Mixing is also important for control purposes and affects the stability
of control processes. Temperature control in viscous fermentation broth may be
problematic because the viscosity is highest in regions of low shear near the cooling
coils [52].
Normally, the most critical transfer operation in aerobic fermentations is supply of
oxygen to the organisms. The solubility of oxygen in water is low, and the fungi may
therefore experience severe oxygen starvation in viscous broths where the mixing times may
be in the order of minutes. The transfer of oxygen from the gas to the liquid phase is
usually the limiting step in supplying oxygen to the cells [52], but the transport limitation
may also be in the bulk liquid (poor bulk mixing) or between the bulk liquid and the
cell [90].
RHEOLOGYOFFILAMENTOUSFERMENTATIONS 7
The shear rates and power dissipation rates are very high near the impeller, but
decrease rapidly with distance from the impeller in viscous fermentation broths. This means
that oxygen transfer takes place mainly in the impeller region. Depletion of oxygen,
especially in stagnant zones, leads to loss of productivity [55].
Gas-liquid mass transfer coefficient (kLa) correlations for filamentous fermentation

broths. Deindorfer and Gaden [25] noted an 85% reduction in kLa as the mycelial cell
concentration increased, and claimed this was because the broth viscosity increased by
100% during the fermentation run. Decrease in oxygen transfer rate with increasing
mycelial cell concentration has also been shown elsewhere [17,21,34,87]. The adverse
affects on kLa are diminished if the cells assume the pellet morphology [25]. Since the
oxygen transfer rates determines the production capacity of most aerated fermenters, there
is great interest in methods for measurement and evaluation of the oxygen transfer
coefficient in the fermenters. Empirical correlations are normally used for estimating kLa
in non-Newtonian fluids [80]:
kLa --- k (PN) a (V s )b (Vla)C' (6)
where kLa is the overall oxygen transfer coefficient (s-l), P is the power consumption (W),
V is the volume (L), V s is the superficial gas velocity ( m s "l) and a, b, c and k are
constants.
Available mass transfer coefficients are often scale dependent, and one reason for
this is that they do not include explicit measures of mixing qualities such as mixing time
[22]. Metz et al. [52] gave the following connection between mass transfer and flow for
orders of magnitude estimation of kLa:
kLa = 1/tc, (7)
where t c is the circulation time (s).
Because of a shortage of experimental data in real fermentation broths, the
significance of rheology in bioreactor design and performance has often been quantified
using homogeneous polymer solutions to simulate flow behaviour of heterogeneous
fermentation broths. None of these model media can simulate the course of kLa in fungal
fermentations well [4,24,58]. The difficulties involved in quantifying the rheological
properties of fermentation broths may result in widely different values obtained from the
kLa-correlations [22,52,58]. Moo-Young et al. [58] has reported, however, that the
correlations obtained from model media may agree with data for fungal fermentation
8 E. OLSVIKand B.KRISTIANSEN
broths if no yield stress exists in the culture broth.
Oxygen transfer rates increase with the superficial gas velocity, Vs, and with
increasing impeller speed until maximum oxygen transfer rates for the system are reached
[9,17,39]. Increasing the gas flow rate has been reported to be less effective in increasing
kLa with increasing mould concentrations and viscosity [33,34]. Gbewonyo et aL [34]
reported that the effectiveness of agitation and aeration to promote oxygen transfer is
diminished in filamentous fermentations.
Increased kLa in fermentation broths as compared to aqueous fluids having the
same rheology and surface tension have also been observed, and the following explanations
for this effect have been presented: Chemical enhancement by the microorganisms [97];
direct gas uptake by the organisms [86]; hydrodynamical effects (equally important with
dead cells or inert particles) [8,28]; and increasing kLa with increasing ionic strength
[40,43,77].
Tsao [97] showed that active biomass can enhance kLa. Linek et aL [51] and Yagi
et aL [110], however, found that neither dead nor resting or growing cells enhanced the
overall oxygen transfer coefficient, and argued that the enhancement effect observed by
Tsao was mostly due to errors in the measuring technique used to obtain kLa 0. Other
authors have also described a higher kLa in fermentation broths than anticipated from
polymer studies [58]. Voigt and Schugerl [106] described an increase in kLa in the early
stages of the fermentation runs, followed by a decline in the kLa value. This enhancement
effect was explained by the secretion of surface active compounds from the cells.
Attempts to separate the main effects contributing to the rheological properties of
a fermentation fluid, the biomass and the morphology, have been made [52,79,101], but
have not been successful. The effect of the density of the broth on kLa has been included
in some kLa- correlations [36,42,62,77,110], but not the effect of biomass concentration.
In our own work [65] it was also noted that the general trend was a decrease in kLa
with increasing consistency index, as shown in an A. niger batch fermentation given in
Figure 2. This trend was reversed as K continued to increase from 2 to 2.5 N m -2 s n. The
observed increase in kLa could have been caused by the changing biomass concentration
or growth rate or by changes in the physical properties of the broth resulting from the
biomass activity. To eliminate the effect of the biomass concentration, a factor called kLa/x
was defined, and Figure 3 shows a plot of kLa and kLa/x vs. K for continuous culture
experiments run at constant dilution rate (D = 0.03 h-l). The biomass concentration varied
between 5.5 and 9.0 g L "1 in these experiments. The relationship between kLa/x and K can
be described by an exponential expression. These results suggest that the presence of cells
(particles) can affect kLa positively.
Continuous experiments also showed that the consistency index, K, increased with
increasing dilution rate if DO was in excess, but decreased with the dilution rate in oxygen-
limiting experiments (Figure 4) [63]. However, kLa/x was found to increase with the dilution
rate, irrespective of whether K was increasing or decreasing at the same time (Figure 4),
and the results thus suggest an enhancement effect arising from the presence of growing
cells. This effect might have been caused by surface active compounds secreted by the cells
or the cells themselves acting as surfactants. However, the observed influence of the cells
or their activity both in batch and continuous culture does suggest the need for further
experimentation on oxygen transfer rates in filamentous fermentation broths.
kca
100
~0
0 0
0
0
@0
0 O~
0
0 0 0 0000 0(~
0
40
O0
o I I I
0 1 2 3
K(N IT1-2 Sn
Figure 2. kLa vs. the consistency index, K, for a filamentous A. niger batch
fermentation.
10 E. OLSV1Kand B.KRISTIANSEN
kLa/x
100
+
80
15
+
+
60
10
++ +
40
sl
20
0 I , I I 0
0 1 2 3 4
K (N m -2 s")
Figure 3. kt,a (+) and kLa/x ([3) vs. K for continuous A. niger fermentations.
k.a/x K ( N m -2 s"
25 4
20
3
15
10
I1
0 I I I I 0
ODO 0.20 0.40 (X60 080 1.00
~-1)
Dilution rate (h-')
Figure 4. kLa/x at DO=40% (O) and DO=limiting (I-3) and K at DO=40% ( t )

and DO=limiting (11) vs. dilution rate for continuous culture experiments.
VISCOSITY AS A FUNCTION OF SUSPENSION CHARACTERISTICS
It is normally considered that the rheological properties of mycelial cultures are determined
by the concentration and the morphological state of the mycelium [22,52], which is affected
by the operating conditions in the fermenter [52,53]. High biomass concentration will in
general lead to high viscosity.
Influence of biomass concentration. A number of correlations between the biomass
concentration and viscosity (or shear stress) have been proposed. Typical correlations are
shown in Table 2.
Table 2 Relations between biomass and viscosity parameters
Takahashi and Yamada [93] 11 ~ x 1.1 (8)

Deindorfer and Gaden [25] Xo ~ x2'3"2'5 (9)
Solomons and Weston [88] x ~X 2.65 (10)
Roels et al. [79] Xo ~ ~ x 2.5 (11)
Blakebrough et al. [12] plotted K and n from the power law model against the dry
weight (x) for an A. niger culture grown in an airlift fermenter. They found an increase in
K and a decrease in n as the biomass increased, but the rheological properties were said
to be influenced by growth rate as well as biomass concentration; therefore, no simple
relationship could be given between rheological properties and solids content. Plots of both
n and K vs. the biomass concentration for three different filamentous fermentations were
reported by other workers [5]. A summary of model parameter variations, using either the
power law, Bingham or Casson model parameters, with the biomass concentration was also
given. The correlations of K vs. x which were shown to be dependent on the type of strain,
and thus on mycelial morphology were as follows [5]:
A. niger K = 4.3 10-4x 3.3 (12)
P. chrysogenum K = 3.6 10-3 x 2.5 (13)
S. levoris K = 0,27 x 0.7 (14)
Recently, we reported that process variables, such as the specific growth rate, the
DO-tension in the broth and the mixing time (operating volume, V s and tin) influenced the
relationship between the biomass concentration and the consistency index [63,64]. Figure 5
shows the relationship between the biomass level and the consistency index in four different
experiments. The experiments shown are one batch experiment and three fed-batch
experiments with nitrogen-limited growth. The variables in the fed-batch experiments were
the medium feed rate and the DO-tension in the broth. (The growth curves were very
similar for the three fed-batch experiments.) It appears from the figure that describing the
viscosity as a function of the biomass only, according to the correlations shown in Table 2,
will be of limited value.
Ju et al. [40] also reported that plotting apparent viscosity vs. biomass concentration
in a penicillin fermentation could not be used to obtain a general relationship between
biomass concentration and viscosity, since different plots were obtained as the reactor
conditions changed. However, their plot of viscosity vs. biomass concentration showed that
exposing the cells to different partial pressures of CO 2 influenced broth viscosity at a given
biomass concentrations. It was also shown that the higher the n, the shorter the mean
hyphal length. In a previous publication [63] we showed that the "morphology-index", K/x,
increased with the biomass, which implies that the influence of the biomass concentration
on broth viscosity increases as the biomass level rises. It was also shown that n was
approximately constant when increasing the biomass concentration from 7 to 9 g L q [66].
Morphological studies [68] showed that the mean hyphal length increased with the biomass
concentration, although n was constant, indicating no relationship between n and hyphal
length. It should be mentioned that 90% of the hyphae existed in small clumps and any
relationships between hyphal geometry of freely dispersed hyphae and rheological
parameters may thus not be reliable.
K ( N r n -2 S~ )
21
,,..-"
I
3 6 9 12 15
x(cJl-')
Figure 5. Consistency index, K, vs. biomass concentration for batch ( .......), fed-
batch I ( ), fed-batch II ( - ' - ' - ' - ) and fed-batch II1 ( . . . . ).
RHEOI.DGY OF FILAMENTOUS FERMENTATIONS 13
Influence of morphology. Morphology is a term which describes the external form and
structure of an organism, but itis also used to describe the nature of the microbiological
system, consistingof the mycelial network and the homogeneous phase. These two terms
were distinguished as microscopic and macroscopic morphology by van Suijdam [101].
Microscopic morphology may be determined by microscopic examination, whilst
macroscopic morphology involvesthe determination of viscosity.The mycelial morphology
is influenced by a number of process parameters, such as the growth rate, the medium
composition, the DO-tension and the shear stress. The microscopic and macroscopic
morphology may respond differently to changes in some of these factors.
The microscopic morphology of filamentous micro-organisms may be quantitatively
determined by a description of the geometry of the hyphae. The morphology parameters
may be the length, the diameter or the branching frequency of the hyphae or combinations
of the individual dimensions [102]. These parameters can be determined automatically by
image analysis techniques [1,70,95], which can also be used to determine the size and shape
of mycelial aggregates and to differentiate between freely suspended mycelia and
aggregates present in the fermentation broths [98]. For several filamentous fermentations
it has been shown that 80-90% of the mycelium exists in such aggregates, despite appearing
fully filamentous by vi.'sual examination [70,98]. In such cases, attempts to relate broth
rheological properties to the morphology of only freely dispersed hyphae have been found
to be of little value [98].
Image analysis techniques have also been used to estimate biomass concentrations
in P. chrysogenum fermentations [71]. The estimation was based on measurements of the
volume of the cytoplasmic and the degenerated regions including vacuoles. The biomass
concentration was calculated by multiplying the density of various cell components by their
fractional volume. Paul et al. [72] used image analysis for a complete characterisation of
vacuolation in terms of the percentage by volume of vacuoles and vacuole size and shape,
whereas Reichl et aL [74] used image analysis for studying septation in filamentous
fermentations. The increasing use of fluorescent markers and immunofiuorescent labelling
in microbiology will most likely increase the use of, and need for, image analysis techniques
[59,74,78]. Based on a number of batch fermentations, Metz et al. [52] suggested the
following correlation between the Casson parameters Kc and x0 and the length of the
hyphae, Le, and the length of the hyphal growth unit, Lhg,,:
K c = 5.454 x 1"0 Lhgu0"6 (15)
Xo = 1.67 10 -4 x2'5 (Le) '8 (16)
The correlations gave a close fit in batch experiments for a number of strains in different
reactors at different impeller speeds, media and pH values for different media, but were
found inadequate for describing the relationship between morphology and rheology in
continuous culture [52]. This was explained in terms of changes in hyphal flexibility caused
by differences in culture conditions in batch and continuous culture. Differences in culture
conditions can lead to differences in hyphal-hyphal interactions, therefore, it is difficult to
draw conclusions about rheology from morphological measurements alone even when no
aggregates are found in the culture broth. Changes in the flexibility of the hyphae may be
due to [52] changes in cell wall composition, changes in branching pattern or diameter of
the hyphae, or variations in osmotic pressure.
Correlations between morphological parameters, biomass concentration and
rheological parameters have been presented by many authors [32,52,79,101]. The
correlation given by Metz et al. [52] was based on the determination of morphological
parameters, whereas van Suijdam [101] defined an apparent morphology factor, MF, as
follows:
M F = lim x~O (Vl/Vl -1)/x, (17)
The M F factor could not be measured directly, but was determined from viscosity data of
mycelial broth diluted to biomass concentrations at which hyphal-hyphal interactions could
be excluded. In Roels' correlations (see Table 2), the morphology parameter (6) was
determined from viscosity measurements of undiluted broth combined with a mycelial dry
weight determinations [79]. As shown in Figure 4, this morphology parameter depends
upon the operating conditions.
Fatile [32] correlated the diameter of mycelial aggregates, dp, and the biomass
concentration, x, with the rheological properties of the broth using the power law. The
average diameter of these aggregates was calculated as described by Roels et al. [79]. The
shape of the aggregates was not measured. The following correlations were suggested:
K = 0.3 x 0.3 dp 0.2 (18)

RHEOLOGY OF FILAMENTOUS FERMENTATIONS 15
n = 0.5 x -O.O6dp~.O8 (19)
Using image analysis techniques, Packer and Thomas [70] showed that more than 90 % of
the mycelia in P. chtysogenum cultures existed as clumps, although the fermentation broth
appeared to be fully filamentous by visual examination. These results were later confirmed
for Aspergillus and Streptomyces broths [98]. Since the clumps could not easily be
disaggregated by dilution, it was assumed that they existed in a similar form within the
fermenter. The image analysis technique has recently been extended to determine
parameters describing the size and shape of these aggregates, such as their projected area,
perimeter, circularity, compactness and roughness [98]. Packer and Thomas [70] and
Tucker et aL [98] suggested that the hyphal dimensions of free mycelia may influence broth
viscosity to a lesser extent in such fermentations. Thus, the theological properties should
be correlated with morphological parameters describing the size and shape of these clumps.
Olsvik et aL [68] correlated the shape of the mycelial aggregates with the rbeological
properties of the broth and the biomass concentration for continuous fermentations of A.
niger. The changes in the hairiness or roughness, R, of the mycelial aggregates were found
to correspond to changes in the measured consistency index, K. Correlations of consistency
index with other morphological parameters were sought, but could not be developed for
all the fermentations. The reasons for this discrepancy are discussed in a later section
together with a discussion of the effects of operating conditions on morphology and/or
rheology. The following correlation was suggested for the continuous culture experiments:
K -- -0.56 + 1.8 10-3 R x 1.7 (20)
Results from batch and fed-batch fermentations (unpublished data) produced the following
correlation:
K = 0.38 + 4.8 10-5 R x 2.9 (21)
Values for the roughness were in the range of 15 to 40. The correlations show that the
consistency index depends on the biomass concentration. Although the exponent on x varies
according to the cultivation mode, the values are in the range 0.7 - 4 as suggested by other
authors [5,52]. The work was based on a limited number of experiments and it is possible
that other forms of equations may correlate K, x and R just as well. Further
experimentation will be necessary to establish the definitive form of the equations, and to
find the cause of the differences in the correlations obtained in different reactors.
Differences in hyphal flexibility or hyphal cell wall composition may be a possible
explanation.
MEASUREMENTS OF RHEOLOGICAL PROPERTIES
It is difficult to compare literature results on measurements of rheological properties of
fungal fermentations, and the reasons for this were listed by Charles [22]: In most cases
morphology was not described, the rheological fundamentals were overlooked in several
instances, different types of viscometers and measurement techniques were used by
different investigators studying the same type of broth, the inappropriate instrument was
used, or the instrument operating problems were overlooked.
Static (off-line) vs dynamic (on-line) measurements. Conventional viscometers, usually the

concentric cylinder or the cone and plate type, are most often used to determine the
rheological properties of mycelial fermentation broths. The main problems with these
viscometers are [14,79]: The size of particles in suspension (pellets) may be of the same
order of magnitude as that of the annulus of the apparatus, resulting in the destruction of
pellets and flocks during the experiment; formation of less dense layers next to the surface
of the measuring bodies; a tendency of the suspension to become inhomogeneous because
of settling of particles; and particle-particle interaction.
The uncertainties inherent in the measurement techniques suggest caution in
interpreting the results obtained with concentric cylinders or cone and plate viscometers
on filamentous broths [52]. It has been shown that tube viscometers with large pipe
diameters cope with the first of the listed problems, but the others may still be significant
[14,52,79]. To solve the problem of settling and water layers next to the surfaces,
Bongenaar et al. [14] and Reels et al. [79] used a Rushton turbine instead of the concentric
cylinder system. The treatment of the sample before measurement, and the time-lag
between sampling and taking the measurements have been shown to be important [52,65].
Problems with obtaining meaningful static measurements of filamentous fermentation
broths have been well documented both in concentric cylinder as well as in impeller devices
[22,52,65]. It was found that torque measurements in the impeller cell decreased even after
applying a constant shear rate for 5 minutes [52,65] (Figure 6).
RI-I~OLOGYOF FILAMENTOUSFERMENTATIONS 17
.T ( N rn-= )
'I
7
i I
100 ~O0
Time ($)
Figure 6. Time dependence of torque in impeller viscometer

with filamentous suspension at constant shear rate
( ,30 sa; -.-.-., 50 s'l; --'.-'.- 100 s'l).
Metz et aL [52] explained this effect in terms of sedimentation or a change in the structure
of the mycelium in the measuring cylinder. Flow-through cells for rheological measurements
have been developed using a Rushton turbine [2,44,45] or a helical ribbon impeller [46,47]
to get an uniform treatment of the samples before measurements and to minimise aligning
and settling of hyphae in the measuring cell. Pipeline systems have also been used for
on-line measurements of the rheological properties of fungal fermentation broths [3,11,32].
Changes in the mycelial properties (sedimentation or a change in the mycelial
structure) may occur to a different degree in all types of cells with time or degree of
mixing, and a comparison between different off-line cells or on- and off-line instruments
is thus very difficult and of limited value. A comparison between different on:line cells in
different publications is also difficult, even when measuring the same culture broth. This
is because even though on-line impeller cells give internally consistent and reproducible
results, and this suggests that an intrinsic property is being measured, different values may
be obtained in the different instruments giving only relative measurements. Differences in
the physical and chemical environment in the reactor will also affect viscosity
measurements, as discussed later, and unless experiments are carried out under equal
growth conditions, different viscosity curves will be obtained.
Impeller viseometers. Rheological measurements are carried out under laminar flow
conditions. In this context, the helical ribbon impeller system is said to be an improvement
on the Rushton turbine version, because the laminar shear rate range is increased and thus
allows for more accurate determination of the rheological properties [46,75]. The
disadvantage of using impeller systems, such as the helical ribbon or the Rushton turbine,
is that they establish complex flow patterns with no straightforward calculation of shear.
All results will thus be empirical. The shear rate in the viscometer is assumed to be
proportional to the impeller speed according to the Metzner and Otto equation [54]:
~, = k N (22)
The calibration constant, k, in Equation (22) has been shown to be dependent on the type
and concentration of the calibration fluid [15,61]. Brauer [15], referred to the work of
Schilo [82] who found the average shear constant to be a function of the power law index:
k = 4.4 n q.6~ (23)
Thus, the largest error when assuming a constant value for k will be found when n is
changing markedly.
Taguchi and Myamato [91] presented a figure showing the course of K and n,
measured off-line, throughout a filamentous Endomyces fermentation (Figure 7). It appears
that control of broth theology based on such a limited number of measurements will be
difficult. The first continuous and automatic measurements of filamentous fermentation
broths using an helical screw impeller system were presented by Kemblowski et al. in
1990 [47]. On-line, continuous measurements of broth theology give uniform treatment of
the samples and statistically more "correct" measurements. On-line, continuous
measurements of K and n for an A. niger fermentation are given in Figure 8. The shear
rate in the fermenter varies from very high values in the region close to the impeller to low
values some distance away, and it is therefore difficult to define an average shear rate in
the broth. However, an average shear rate is often estimated using the Metzner and Otto
equation (22), assuming a constant k-value of 10. The shear stress measured at a shear rate
of 50 s"1 (N = 300 rpm for this fermentation) is given in Figure 8 to show the relationship
between shear stress and consistency index for a filamentous fermentation. It appears from
the figure that K and are proportional when the variations in n are small or when n is
constant [66].
RHEOI.,OGYOF FILAMENTOUSI~RMENTATIONS 19
t.oo,I 40
30 A
c 0,50
,
10
! !
0.00 0
0 50 IO0 150
Time (h)
Figure 7, K and n measured off-line using a coaxial cylinder viscometer. The data are
from an Endomyces fermentation [91].
x (gl-') DO 10 -2 (%)
1"( N m -z } n K ( I,#n"~" )
!o
16 q
0,60
I
I
12-
1q
8 -
1
I
t
0
I
A
0 2O 40 60 aO 100 120 140 leo ~aO
F e r m e n t a t i o n t i m e (h)
Figure 8. K (. ), n ( ...........) and (-.---.-) measured dynamically for a filamentous A.

niger fermentation. The figure also shows the biomass concentration ([2]) and the DO-
tension (O) in the broth.
20 E.OLSVIK and B.KRISTIANSEN
Pipeline instruments. Pipeline viscometers have been used for on-line determination of
rheological properties of filamentous organisms by a number of authors [3,5,6,11,12,75].
Slip in pipeline viscometers does not affect the measurements once the pipe diameter
exceeds a certain value (11.6 ram i.d) [5]. The broth is pumped around in pipeline
viscometers, but no shear damage to the mycelium by the pumping has been visually
observed [5].This agrees with results from on-line impeller systems in which pumping the
broth through the system caused no observed change to the mycelium [65].
In comparison of viscometers, measurements with Rushton tyl~ impellers were
found to give higher readings than helical ribbon impeller systems, concentric cylinder
systems and pipeline viscometers [3,5,6].This has also been reported by others [12,75].The
effect was most pronounced at high biomass concentrations. It was reported that no change
in the structure of the broth was observed in the coaxial or pipeline viscometers, and
rotating cylinder systems or pipeline viscometers was recommended for filamentous
fermentations [3].
The explanation often given for the higher readings in the turbine instruments is that
the proportionality constant for conversion of impeller speed to shear rate is not invariant
with rheological properties as assumed in the calibration method. This was discussed above.
The inaccuracy of assuming an average shear rate has been found to be most pronounced
for strongly pseudoplastic fluids (low n values) or fluids which possesses a yield stress [5].
However, the assumption of an average shear rate is also made for helical screw impeller
systems, so this cannot be used as the explanation for the difference between the two
systems. It has been found that the dependence of the proportionality constant k on the
rheological properties of the fluid is more pronounced in turbine impeller viscometers than
in helical ribbon instruments [5], and this has been attributed to a more uniform shear
stress distribution in the helical impeller system [6]. Pace [69] suggested that the higher
readings in the turbine impeller system was caused by energy dissipation due to distortion
of streamlines. The flow pattern in helical ribbon instruments allows measurements at
higher shear rates than in the Rushton turbine system before the flow becomes turbulent.
Blakebrough et al. [12] also found a lack of consistency between pipeline
measurements and Rushton impeller measurements. The explanation given was that
pipe-flow instruments measure fluid properties at the wall of the measuring device, whereas
the turbine systems measured properties in the bulk of the fluid. No evidence for this
hypothesis was given.
RHEOLfX;YOFFILAMENTOUSFERMENTATIONS 21
In-situ measurements. In-situ measurements of rheologieal properties may be
advantageous, especially in terms of process control [22]. Wang and Fewkes [107] used the
fermenter itself as a viscometer for measuring the rheologieal properties of S. niveus
culture broths. The impeller speed was reduced to ensure laminar flow, and the power
input was then measured as a function of impeller speed. The results were not consistent,
however, and the execution of the measurements invariably influenced the mixing pattern
in the fermenter and hence reactor performance [22].
The in-situ probe developed by Pique et aL [73] involved the excitation of a vibrating
rod placed inside the fermenter. The probe could be steam sterilised and the
reproduc~ility and stability was said to be satisfactory. The heterogeneity of filamentous
fermentation fluids, and the high risk of emerging surface growth, may cause calibration
problems and disturbances in the measurements. This device may thus be difficult to use
in fungal fermentations. Endo et al. [31] developed an in-situ sensor, utilising a quartz
crystal to monitor the viscosity of fermentation broth. The system consisted of a quartz
crystal and an oscillating circuit supplying an signal to one of the electrodes on the crystal.
The output signal was monitored by a peak level meter. The viscometer was tested in a
polysaeeharide bacterial fermentation process. The cell could be steam sterilised and only
a minor increase in the sensor signal was observed when NaCI (up to 2.0 M) was added
to increase the conductivity of the solution. A good correlation between the sensor signal
and measurements of the viscosity with off-line impeller viscometers was found. No
absolute values could be obtained using these in-situ probes.
CONTROL OF THE RHEOLOGICAL PROPERTIES OF FUNGAL FERMENTATIONS

Influence of process parameters on the rheology of the broth. Fermenter type and mixing
device will influence the mixing and theological properties of a fermentation fluid. This has
been extensively studied in the literature and will not be dealt with here. For industrial
production, increasing the mixing and thereby the oxygen transfer rate by lowering the
broth viscosity may be more profitable than trying mechanical improvements [49]. Attempts
have been made to influence the viscosity of a fermentation broth by diluting it. Taguchi
and Myamato [91] found that a 10-15% dilution of an Endomyces fermentation broth
halved the consistency index, and showed thereby a non-linearity between biomass
concentration and broth rheology. For a shear thinning broth of Nocardia, Buckland et al.
[18] claimed a large improvement in kLa by the use of water additions to reduce broth
viscosity. Sato [81] reported a 20% increase in kanamycin yield by diluting the broth with
water to 5% (v/v). Blanch and Bhavaraju [13] referred to observations of large reductions
of the viscosity of the broth by water dilutions, and suggested that nutrients should be fed
semi-continuously to viscous fermentation broths. The shot-wise additions should be
followed by withdrawal of the same volume from the vessel. Shot-wise additions were said
to reduce the viscosity in the broth to a far greater extent than continuous medium feed.
In the reports on process control based on theological measurements and broth dilutions,
no mention has been made to whether the rheological improvements were permanent or
temporary.
In a previous publication [63], we investigated the effect of diluting a filamentous
fermentation broth with water or a sugar solution when the viscosity of the broth exceeded
a predetermined value. It was found that diluting the broth gave only temporary
improvements in broth viscosity, and that the duration of improvements became lower the
higher the broth viscosity. Diluting the broth reduced viscosity and increased oxygen
transfer rates which in turn led to increase in cell growth and viscosity. Temporary
improvements of oxygen transfer rate (OTR) was also found by K0nig et al. [48] in a
viscous fermentation process when attempting to increase OTR by increasing the total
pressure or aerating the fermenter with pure oxygen. The OTR increase which followed
led to further cell growth, producing a marked increase in the viscosity of the broth.
Figure 9 shows two A. niger fed-batch runs with different starting volumes (6 and
8 L) and different feed volumes (4 and 2 L, respectively) [64]. All other operating
parameters were the same in the two experiments. The figure shows that whereas the
growth curves were similar, the K-curves differed. The lowest K-curve was observed in the
experiment with the lowest starting volume, and this suggests that a higher degree of mixing
per unit volume (until final volumes are reached) will influence the consistency index
positively. Figure 10 shows that a similar effect on K was found by reducing the operating
volume in the fermenter from 10 to 6 L, everything else being kept constant.
X (g I-') K ( N m ~ s"
15
12
0
J I
2O
/
40
/
I
60
I
80
l
100
L
120
I
140
I
100
0
100
Fermentation t i m e (h)
Figure 9. x and K vs fermentation time for fed-batch experiments with

different start and feed volumes ( - - - , K: 6 + 4 L, , K: 8 + 2 L;
- -A- - , x: 6 + 4 L; - + - - , x: 8 + 2 L).
K/x ( N m -2 s" g-' I )
O4O
O.3O
O.2O
0.10
GO0 I |
0 180
Fermentation time (h)
Figure 10. Effect on K/x of reducing the operating volume in the fermenter
from 10 ( ) to 6 L ( . . . . ).
Addition of salts is known to lower the viscosity of polyelectrolytes [30]. Sodium salt
additions have also been shown to reduce the viscosity of P. chrysogenum cultures
considerably [52]. The ionic strength of the medium will influence cell interactions in many
fermentations, because most cell surfaces are negatively charged above pH 5.5. Polycation
addition will usually induce aggregation, whereas polyanions suppresses aggregation. The
effect of adding Ca 2+ ions to a filamentous fermentation broth, which may cause cell
aggregation, will depend on the number of negatively charged groups, but also the
accessibility of these groups will also influence the degree of aggregation. The
hydrophobicity of cell walls may vary among different strains and it has been suggested that
it also will depend strongly on the DO-tension in the broth [16]. Ju et al. [40] controlled
broth morphology by CO 2 addition. Increasing CO 2 pressure has been reported to increase
chitin synthesis, increase the branching frequency and induce pelleted growth [29].
Influence of process parameters on the morphology of the fungus. Morphology is

genetically determined, but is also influenced by a number of microbiological and
physiochemical factors, such as pH, growth rate, medium composition, DO-tension, shear
stress and osmotic pressure [16,102]. These factors may determine the microscopic
morphology, but also the macroscopic morphology by influencing the degree of mycelial
aggregation. Factors affecting mycelial aggregation may be separated into a microbiological
and physiochemical as shown in Table 3. Braun and Vecht-Lifshitz [16] recently presented
a review on effects on mycelial aggregation. Macroscopic and microscopic morphology may
be affected by some of the factors listed in Table 3 differently. Factors which may be more
important for cellular aggregation than for microscopic morphology development include:
cell-wall composition, surface-active agents, ionic strength, suspended solids and divalent
ions such as Ca 2+. The following discussion will be limited to how process engineering
parameters such as growth rate, oxygen tension and shear stress influence microscopic
morphology and/or broth rheology. An earlier review of the effects of these parameters on
microscopic morphology was done by Metz et al [53].
RI-IEOLOGYOF FILAMENTOUSFERMENTATIONS 25
Table 3 Factors affecting aggregation [16]
Microbiological Physioehemical
Genetic Shear forces
Cell-wall composition Surface-active agents
Inoculum size pH, Temperature
Growth rate Ca 2+ ions
Nutrition Ionic strength
Carbon-nitrogen ratio Suspended solids
Carbon-dioxide tension
Growth rate and DO-tension. Van Suijdam and Metz [102] showed that the hyphal length
increased with increasing growth rate in a carbon-limited continuous culture of P.
chlysogenum. The branching frequency remained the same. In batch culture, both Metz et
a/= [53] and Belmar-Beiny and Thomas [10] found the morphology parameters to be
constant in the logarithmic growth phase, whereas fragmentation was found as the growth
rate decreased. Katz et al. [41] reported a reduction in the hyphal growth unit when the
growth rate for a A. nidulans culture was increased. The growth rate was varied by varying
the medium composition in batch culture. Morrison and Righelato [60] and Miles and
Trinci [56] found a similar effect when growing P. chrysogenum in batch and continuous
culture. Others have concluded that the growth rate does not influence the branching
frequency [19,96]. In the latter reports the growth rate was varied by varying the
temperature, by incorporating inhibitors in the medium and by changing the carbon source.
It cannot be discounted that the method used for changing the growth rate may have
influenced the morphology and/or viscosity changes [18,102].
Wiebe and Trinci [108] showed the influence of genetic factors on responses to
changes in the growth rate. The hyphal growth unit length decreased with increasing growth
rate for one strain of the genus Fusarium, whereas the opposite was the case for another
strain. The fungi were grown in a glucose-limited continuous culture at growth rates from
0.07 to 0.19 h -1. The morphology data was related to the rheology of the culture using
Metz's equation (Eq. 15), even though Metz et al. [52] had found this equation inadequate
for describing continuous culture broths. The effect of growth rate on rbeology was not
considered in any of the other mentioned papers. None of the cited papers makes any
mention of the DO tension in the broth, but this may not be a serious omission since the
DO tension may affect hyphal dimensions to a very slight extent, whereas the effect on
viscosity may be much more pronounced [111].
The effects of the growth rate on the morphology and viscosity of an A . niger culture
have been investigated [63,65,67]. The specific growth rate influenced the viscosity of the
culture, but the effect on the consistency index, K, was found to be very dependent on the
DO tension in the broth. At DO concentrations above 10% of air saturation, K increased
with the growth rate, whereas the opposite was the case for DO concentrations below 10%.
The roughness of the aggregates were found to vary with the consistency index, and
correlations between the consistency index, the biomass concentration and the roughness
of the aggregates could be found for batch and continuous experiments [67]. For two
fed-batch experiments with equal feed-rates and growth rates, but with DO controlled at
different levels, it was found that in both experiments the length of hyphae and hyphal
growth unit decreased with time after the initial rapid growth period was over, as observed
elsewhere [10]. However, the consistency index and the roughness of the clumps increased
with fermentation time in one experiment, but decreased in the other. This suggests a close
relationship between clump roughness and viscosity, and that prediction of viscosity based
on the length and branching frequency of the hyphae will be process specific and not as
universal as often suggested. Both the growth rate and the DO tension were found to
influence the roughness of the clumps and hence the consistency index of the broth [67].
The growth rate influenced the geometry of the hyphae and possibly the amount of clump
formation and breakup. This was seen as changes in the roughness and the area of the
clumps with growth rate, but with no changes in clump density. When changing the
DO-tension in the broth, the compactness of the aggregates appeared to change, whereas
no changes in the geometry of the hyphae was observed.
Vecht-Lifshitz et al. [104] suggested that the DO tension in the broth affected the
hydrophobicity of the cell wall in a S. tendae fermentation. Oxygen-limiting conditions
tended to reduce the pellet size and induced pulpy growth, whereas oxygen sufficiency
induced pellet formation. Factors increasing cell wall hydrophobicity simultaneously induced
pellet formation. It was thus suggested that the main forces inducing cellular aggregation
were hydrophobic interactions of cell walls, and that these interactions could be controlled
by the availability of oxygen. We have also reported an observed increase in viscosity with
decreasing DO-tension, and considered that the main reason for this was increasing clump
roughness and less compact aggregates [67].
Van Suijdam and Metz [102] varied the oxygen tension in continuous culture
experiments with P. chrysogenum and observed no effect on the measured morphological
parameters. The specific growth rate was 0.109 h "1 and the oxygen tension was varied from
12 to 300 ram Hg. Carter and Bull [20] found the same forA. nidulans grown in continuous
culture at 0.05 h "1. They said, however, that small changes in oxygen tension could result
in changes in the physiological state of the microorganisms. The oxygen tension was varied
between 1 and 156 mm Hg. Zetelaki and Vas [111] found a reduction in the apparent
viscosity in an A. nidulans culture when increasing the oxygen tension. However, no
differences in morphology parameters were observed. The reduction in the viscosity was
said to be due to cell wall changes (thinner walls) and thus changes in the hyphal flexibility
in the oxygenated culture, that is, a change in the macroscopic morphology. On the other
hand, Dion et al. [27] reported shorter, thicker and highly branched hyphae by aerating the
reactor with oxygen instead of air.
Most papers seem to agree that the DO-tension does not alter microscopic
morphology, and the Vecht-Lifshitz theory of changes in the hyphal-hyphal interactions
appears to have some merit. However, the response of cells to the oxygen partial pressure
is a complex phenomenon. Several fermentation pathways are either induced or repressed
by the presence of dissolved oxygen at various levels. Molecular oxygen may act as a toxin,
and it is generally accepted that oxygen toxicity is caused by univalent reduction of the
O2-molecule which finally leads to damage of enzymes, nucleic acids or lipids [37,38]. These
are factors which may affect the cell wall composition or hyphal-hyphal interactions,
thereby causing changes in broth viscosity.
Metz et al [52] suggested that another effect of poor oxygen transfer could be that
the low oxygen tension in the vessel would result in a change in the mycelial properties and
an increase in the viscosity of the broth. This could lead to a further drop in the oxygen
tension and a negative feed-back loop may thus be initiated. A similar effect was also
observed by Olsvik and Kristiansen [63]. In a continuous A. niger fermentation it was
observed that an increase in the consistency index, K resulted in a reduction in DO which
led to a further increase in K even if the biomass concentration was kept constant. A shift
from low to high DO-tension in a continuous culture resulted in a decrease in the value of
K, whereas a shift the other way resulted in an increase in K again [67]. The large
28 E. OLSVIKand B. KRISTIANSEN
influence of the DO tension on K led to the need for a strict DO-control in the continuous
reactor. At low growth rates and DO concentrations, a 2% change in the DO-tension could
give a 25% change in the consistency index. Buckland et al. [18] also found the physical
response of oxygen through cycle of rpm changes to be reproducible. The cells recovered
from short periods of exposure to low oxygen tension.
Effect of shear stress. Agitation of the culture broth may cause rupture of the cell wall,
changes in broth theology, and influence cell morphology. It is generally considered that
an increase in the power input per unit volume will lead to a reduction in the apparent
viscosity. Both van Suijdam and Metz [102] and Smith et al. [85] reported a decrease in the
effective hyphal length with increased power input per unit mass for a P. chtysogenum
culture. However, van Suijdam and Metz reported on the effect of increasing the shear
stress on the effective hyphal length to be very small [102]. They therefore considered
increases in the power input as a means of reducing viscosity to be of limited value. A
decrease in the mean length of the hyphae with increasing impeller speed was also
reported by Dion [27] for Penicillium and Aspergillus cultures. Ujcova et al. [100] observed
thicker, more twisted, densely branched and septated hyphae with increasing impeller
speed. Belmar-Beiny and Thomas [10] found the fragmentation rate in batch cultures to
accelerate with increasing impeller speed for a strain of Streptomyces. This was observed
as a reduction in the main hyphal length, total hyphal length and number of tips with
increasing shear. Rapid breakdown of the mycelium with increasing impeller speed is
normally not found unless a reduction of the physical strength of the mycelium has
occurred due to substrate exhaustion [85]. Smith et al. [85] suggested that neither the
concepts of impeller tip speed nor the dissipation rate of turbulence has general validity
as a measure of hyphal damage. They suggested a dispersion zone around the impeller in
which mycelia may be damaged, and that the frequency of circulation is proportional to
hyphal damage. Unfortunately, in the presented papers the theological properties of the
fluid were not measured.
We observed a small reduction in broth viscosity with increasing impeller speed in
batch culture [64]. A reduction in the fermenter operating volume from 10 to 6 L gave
significant reductions in K/x, as shown in Figure 6. The morphology of the fungi was not
studied in these experiments, but since the hyphal length appears not to be very sensitive
to changes in the impeller speed [102], the difference in viscosity must have been due to
R H E O L C ~ Y OF FILAMENTOUS FERMENTATIONS 29
changes in the hyphal network or hyphal-hyphal interactions. It was observed that the effect
on broth rheology of changing the impeller speed was influenced by the medium
composition [64]. In a oxygen limited culture, increasing the impeller speed resulted in an
increased oxygen supply to the organisms, an increase in the biomass concentration and an
increase in K. In a nitrogen-limited culture, increasing the impeller speed led to a small
reduction in broth viscosity.
Increasing the impeller speed may also result indamage to the mycelium [92], and
the agitation speed will thus have an upper limit for growth and production. The lower
limit is often set by mass transfer considerations. Fungal damage may, however, be difficult
to assess, due to the fact that vacuolisation and finally autolysis is a natural process. A. niger
cultures are normally very resistant to shear damage [92] but observation of mechanical
damage to A. niger in stirred vessels has been observed [51]. The strength of the applied
shear and its frequency are important in determining whether disruption occurs. Tanaka
and Ueda [94] and Ujcova et al. [100] observed an increase in the nucleotide concentration
in the medium with increasing impeller speed. This effect of the impeller speed may,
however, be strain dependent. Vardar and Lilly [103] observed a shift from the production
of the desired product to cell constituents with intensification of agitation. The explanation
given was that the response of the cells to the increase in the degree of agitation was
strengthening of the cell wall or replacement of leaking cell constituents.
SUMMARY
The morphological growth of fungi may give fungal broth suspensions a pronounced non-
Newtonian character. The filamentous growth form is dominant in most processes, and
long, thin, usually branched hyphae create a three-dimensional network that may give very
viscous fermentation broths. The influence of fluid flow properties on reactor performance
is widely recognized, but few systematic studies of the rheological properties of
fermentation fluids have been reported and the use of rheometry in fermentation processes
is limited. The reasons for this appear to be the problems encountered in measuring
rheological properties of the non-homogeneous fermentation fluids and in unambiguous
interpretation of results.
Much attention has been given to rheological classification of filamentous
fermentation broths. With the techniques normally employed, the measurements are taken
over a very narrow shear rate range and it can be very difficult to separate different types
of non-Newtonian broths. However, this may not be too important from a processing point
of view.
High biomass level will normally lead to very viscous broths, but there appears to
be no simple relationship between biomass concentration and the rheological properties
of the broth as this will be influenced by other operational parameters.
The morphology of the microorganism is the major determinant of the flow
properties of the broth. It appears that more than 80% of the biomass in a visually
filamentous fungal broth is in the form of small aggregates (nominal diameter < 100 I~m)
and the surface dimensions and properties of these decide the rheological properties.
Comparison of rheological data from different publications can be problematic. The
main reasons for this are the differences in the instruments used and unstated differences
in the morphology of the culture. In addition, measurements are normally taken off-line,
introducing the added problems of sample treatment influencing the measurements. The
latter can be combated with dynamic, or on-line measurements but these have not found
wide-spread application in fermentation fluid rheology.
Control of broth rheology is normally attempted by diluting the broth. Normally this
will only lead to very short-term reduction of the apparent viscosity and will be of little
value for process control purposes. There are very few published attempts on control of
broth rheology beyond this technique. A promising approach appears to be using
physiological manipulation of cell growth as a way of controlling the rheology, possibly
through manipulations of the morphology of the mycelial aggregates or the surface
properties of the mycelium. Several parameters, such as nutrient concentrations and the
nature of the limiting nutrient, specific growth rate and mixing quality influence the
morphology and the cell wall composition and hence rheological properties of the
fermentation fluid.
NOMENCLATURE
a,b and c constants in Eq. (6)

Di impeller diameter (m)
dp diameter of mycelial aggregates (mm)
K consistency index (Nm'Zsn)
"Casson" viscosity (Nm'Zs)0"5
k coefficient in Equations (6), (22) and (23)
kLa overall oxygen transfer coefficient (h "1)
kLa0 physical oxygen transfer coefficient (h-1)

length of hyphae (~m)
Lh n length of hyphal growth unit (l~m)
MF morphology factor
N impeller speed (s)
n flow behaviour index
P power consumption (W)
R roughness of mycelial aggregates
tc circulation time (s)
tm mixing time (s)
V volume (L)
v~ superficial gas velocity (m s"l)
X dry weight (g L -I)
Y shear rate (s-1)
8 morphology factor
n viscosity (Nm-2s)
~o viscosity of the suspending fluid (Nm'2s)
rla shear dependent viscosity (Nm'2s)
P broth density (g L"l)
shear stress (Nm -z)
zO yield stress (Nm-2)
REFERENCES
1. Adams, H. I_. and Thomas, C. R. (1988), The use of image analysis for morphological
measurements on filamentous microorganisms. BiotechnoL Bioeng., 32, 707-712.
2. Ajayi, O., Kemblowski, Z., Kristiansen, B. and Larsen, V. F. (1991). On-line

rheometer for the monitoring of polymer fermentations: Operating conditions.
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Bio~cK Aav. Vol. 12, pp. 1--39,1994 0734-9750/94 $24.

RHEOLOGY OF FILAMENTOUS FERMENTATIONS

E. OLSVIK* and B. KRISTIANSEN

Strathclyde Fermentation Centre, University of Strathclyde, Glasgow, U.K.

The performance of a bioreactor containing a filamentous fermentation broth is greatly

Keywords: Rheology, fermentation broths, dynamic viscosity measurements, fungal

* Permanent address: SINTEF-SI, P. O. Box 124 Blindern, 0314 Oslo 3, Norway.

rl- x y-l, (1)

thinning or pseudoplastic behaviour [25,26,90]. Pseudoplastic fluids exhibit a decrease in

Table 1 Rheologicai models

> 9" (s -~)

Figure 1. Rheological models.

EFFECTS OF VISCOSITY ON TRANSPORT PHENOMENA

Gas-liquid mass transfer coefficient (kLa) correlations for filamentous fermentation

Dilution rate (h-')

Figure 4. kLa/x at DO=40% (O) and DO=limiting (I-3) and K at DO=40% ( t )

Table 2 Relations between biomass and viscosity parameters

Takahashi and Yamada [93] 11 ~ x 1.1 (8)

M F = lim x~O (Vl/Vl -1)/x, (17)

K = 0.3 x 0.3 dp 0.2 (18)

K -- -0.56 + 1.8 10-3 R x 1.7 (20)

K = 0.38 + 4.8 10-5 R x 2.9 (21)

Static (off-line) vs dynamic (on-line) measurements. Conventional viscometers, usually the

Figure 6. Time dependence of torque in impeller viscometer

Figure 8. K (. ), n ( ...........) and (-.---.-) measured dynamically for a filamentous A.

CONTROL OF THE RHEOLOGICAL PROPERTIES OF FUNGAL FERMENTATIONS

Figure 9. x and K vs fermentation time for fed-batch experiments with

K/x ( N m -2 s" g-' I )

Fermentation time (h)

Influence of process parameters on the morphology of the fungus. Morphology is

a,b and c constants in Eq. (6)

kLa0 physical oxygen transfer coefficient (h-1)

2. Ajayi, O., Kemblowski, Z., Kristiansen, B. and Larsen, V. F. (1991). On-line

3. Allen, D. G. and Robinson, C. W. (1987). The influence of "slip" on rheological

4. Allen, D. G. and Robinson, C. W. (1989). Hydrodynamics and mass transfer in

5. Allen, D. G. and Robinson, C. W. (1990). Measurement of rheological properties of

6. Allen, D. G. and Robinson, C. W. (1991). The prediction of transport parameters in

Anderson, C., LeGrys, G. A. and Solomons, G. L (1982). Concepts in the design of

10. Belmar-Beiny, M. T. and Thomas, C. R. (1991). Morphology and clavulanic acid

12. Blakebrough, N., McManamey, W. J. and Tart, K. R. (1978). Rheological

14. Bongenaar, J. J. T. M., Kossen, N. W. F., Metz, B. and Meijboom, F. W. (1973). A

16. Braun, S. and Vecht-Lifshitz, S. E. (1991). Mycelial morphology and metabolite

17. Brierly, M. E. and Steel, R. (1959). Agitation-aeration in submerged fermentation. II.

22. Charles, M. (1978). Technical aspects of the rheological properties of microbial

24. Chisti, M. Y. and Moo-Young, M. (1988). Hydrodynamics and oxygen transfer in

25. Deindorfer, F. H. and West, J. H. (1955). Effects of liquid physical properties on

26. Deindorfer, F. H. and West, J. H. (1960). Rheological examination of some

28. Donaldsen, T. L. and Palmer, H. J. (1978). Enhancement of gas absorption in

29. Edwards, A. G. and Ho, C. S. (1988). Effects of carbon dioxide on Penicillium

32. Fatile, I. A. (1985). Rheological characteristics of suspensions of Aspergillus niger.

37. Gotlieb, S. F. (1966). Bacterial nutritional approach to mechanisms of oxygen toxicity.

45. Kemblowski, Z. and Kristiansen, B. (1986). Rheometry of fermentation fluids.

56. Miles, E. A. and Trinci, A. P. J. (1983). Effect of pH and temperature on morphology

60. Morrison, K. B. and Righelato, R. C. (1974). The relationship between hyphal

62. Nakanoah, M. and Yoshida, F. (1980). Gas absorption by Newtonian and

68. Oolman, T. and Blanch, H. W. (1986). Non-Newtonian fermentation systems. Oit.

70. Packer, H. L and Thomas, C. R. (1990). Morphological measurements on filamentous

71. Packer, H. L., Keshavarz-Moore, E., Lilly, M. D. and Thomas, C. R. (1992).

72. Paul, G. C., Kent, C. A. and Thomas, C. R. (1992). Qualitative characterisation of

80. Ruy, D. Y. and Humphrey, A. E. (1972). A reassessment of oxygen-transfer rates in

82. Schilo, D. (1968). Leistungsbedarf beim Ruhren nicht-Newtonscher Flussigkeiten.

87. Solomons, G. L and Perkin, M. P. (1958). The measurement and mechanism of

9. Solomons, G. L (1980). Fermentor design and fungal growth. In FungalBiotechnology,