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i
(C
i
h
i
) e
j
(c
j
h
j
)
2 3
= r
///
o
DH
o
F
//
a
o
oz
h
a
(1)
o
ot
(1 e) (x
ws
C
s
x
wx
C
x
) e C
wg
= r
///
w
F
//
a
o
oz
y
w
(2)
C
i
h
i
and C
j
h
j
indicate enthalpies of all components
in the moist solid matrix and the gas phase, respectively.
The term for convective enthalpy transport is based on
the mass ow rate of dry air (F
///
a
kg/m
2
per s), and takes
the enthalpy of dry air 4 and water vapor into account:
h
a
(T) = c
pa
(T T
ref
) y
w
(T) c
pwv
(T T
ref
) DH
w
(3)
The water vapor fraction in saturated air can be calcu-
lated from:
y
w
=
M
w
(water) p
w
M
w
(air) (P
tot
p
w
)
= 0:622
p
w
P
tot
p
w
(4)
and the water vapor pressure can be calculated from the
following empirical expression, which was obtained by
tting Antoine's law to water vapor pressure data at
temperatures between 273 K and 333 K (Kaye and
Laby, 1995):
p
w
= exp 23:59
4045
T 37:70
(5)
The enthalpy and mass balances can be simplied
considerably (Weber et al., 1999). First, in the accu-
mulation term of the balances, the contributions of
gases and all mass accumulation terms are negligible.
Second, a pseudo-steady state with respect to tempera-
ture and oxygen consumption rate can be assumed, be-
cause the characteristic times for changes in these
variables are much larger than those for convective axial
enthalpy transport. Third, we assume that the air is at
equilibrium with the solid matrix at any point in the bed.
These simplications reduce the enthalpy balance to:
0 = r
///
o
DH
o
F
//
a
d
dz
(h
a
) (6)
For the simplication of the water mass balance, it is
also assumed that the water production rate is propor-
tional to the oxygen consumption rate:
r
///
w
= r
///
o
Y
wo
(7)
This simplies the mass balance to:
o
ot
(1 e) (x
ws
C
s
x
wx
C
x
) [ [ = r
///
o
Y
wo
F
//
a
o
oz
y
w
(8)
Note that this model discriminates between water in
biomass and water in the substrate. However, it is hard
to measure these variables independently. The change in
the total amount of water can be calculated from:
o
ot
x
w
= r
///
o
Y
wo
F
//
a
o
oz
y
w
(9)
Table I shows the physical constants used in our
model. An important parameter, the yield coecient
Y
wo
, is unknown and dicult to measure accurately. We
have estimated Y
wo
from the following stoichiometric
equations for, respectively, glucose and starch:
C
6
H
12
O
6
2:59 O
2
2:85 CH
1:8
O
0:5
3:44 H
2
O 3:15 CO
2
C
6
H
10
O
5
2:59 O
2
2:85 CH
1:8
O
0:5
2:44 H
2
O 3:15 CO
2
These equations were set up using an average biomass
composition (CH
1.8
O
0.5
) (Roels, 1983) and the reported
yield of 0.75 kg biomass per kg of oxygen for C. minitans
(Ooijkaas et al., 2000a). The obtained Y
wo
for growth on
glucose (0.75 kg H
2
O per kg O
2
) is higher than for
growth on 5 starch (0.53 kg H
2
O per kg O
2
) as the hy-
drolysis of starch requires water.
Oxygen Consumption
The growth of microorganisms on a solid substrate can
be described by the logistic law (Okazaki et al., 1980):
r
///
X
= l
max
X 1
X
X
max
(10)
The oxygen consumption rate is described with the
linear-growth model (Pirt, 1965):
r
///
o
q
s
=
1
Y
xo
r
///
x
m
o
X
(11)
Table I. Physical properties used in the 9 models.
Parameter Value Unit Reference
c
pa
1,005 J/kg per K Hamblin (1971)
c
pwv
1,857 J/kg per K Hamblin (1971)
DH
o
1.4 10
7
J/kg O
2
Cooney et al. (1968)
DH
w
2.5 10
6
(at T
ref
) J/kg H
2
O Perry et al. (1984)
P
tot
1.01 10
5
Pa
T
ref
273 K
Y
wo
0.75 (glucose),
0.53 (starch)
kg H
2
O/kg O
2
Assumed
q
s
64 kg dry hemp/m
3
reactor
This study
384 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 77, NO. 4, FEBRUARY 15, 2002
It is assumed that Y
xo
and X
max
are temperature inde-
pendent. The parameters l
max
and m
o
are assumed to be
temperature dependent, and the Ratkowsky equation is
used to describe the temperature dependency (Ratkow-
sky et al., 1983):
l
max
(T) = a
1
T T
min
[ [ 1 e
a
2
(TT
max
)
h i
2
& '
(12)
m
o
(T) = b
1
T T
min
[ [ 1 e
b
2
(TT
max
)
h i
2
& '
(13)
The unknown parameters were obtained by tting
Eqs. (10)(13) on O
2
consumption rates measured at
various temperatures using a three-dimensional regres-
sion (SigmaPlot 4.01, SPSS, USA).
RESULTS AND DISCUSSION
To estimate the heat production rate in a solid-state
bioreactor, the growth rate of the microorganisms
should be known. However, reliable methods to quantify
biomass formation on solid substrates are not available
(Ooijkaas et al., 1998). Oxygen consumption, however, is
easily determined and directly correlated with heat pro-
duction (Cooney et al., 1968). We have therefore mea-
sured the O
2
consumption to estimate growth.
Effect of Temperature on Growth
For C. minitans growing on hemp impregnated with a
nutrients solution, the O
2
consumption was determined
at various temperatures between 12 and 30C. In pre-
liminary experiments, it was observed that temperature
had a remarkable eect on the germination rate of the
conidia. In a solid-state reactor, initially no temperature
gradient will exist and all conidia will germinate at the
same temperature. Only when biomass starts to grow
can an axial temperature gradient develop. Therefore,
we used an initial temperature of 20C for the rst 2 d to
allow germination. Subsequently, the cultures were in-
cubated at temperatures between 12 and 30C, and ox-
ygen consumption was measured (Fig. 1).
The logistic and Pirt equations [Eqs. (10) and (11)]
were used to t the O
2
consumption measurements. A
temperature-independent yield coecient of 0.75 kg
biomass per kg O
2
was used (Ooijkaas et al., 2000a). All
other model parameters were obtained by tting; it was
assumed that the maximum amount of biomass (X
max
) is
the same at all temperatures, and that l
max
and m
o
are
temperature dependent [Eqs. (12) and (13)]. A good
correlation (R
2
= 0.977) between measurements and
model description was obtained (Fig. 1B); which was
conrmed by the parity plot of measured values against
predicted values (Fig. 1C). The tted minimum tem-
perature for growth of C. minitans (261 K) is clearly not
a meaningful value (Table II). This is caused by the low
number of measurements at temperatures near T
min
.
Furthermore, the Ratkowsky equation is an arbitrary
function, and the validity of this function to accurately
describe growth and maintenance of C. minitans over
the entire temperature has not been shown yet. Despite
the nonrealistic value for T
min
, a good description
of the temperature-dependent O
2
consumption in the
Figure 1. Measured and tted O
2
consumption of C. minitans growing on hemp (A) at (
o
w
(
k
g
/
m
3
p
e
r
s
)
D
u
r
a
t
i
o
n
(
d
)
O
2
c
o
n
s
u
m
e
d
(
k
g
O
2
/
m
3
)
M
a
x
i
m
u
m
r
O
2
k
g
O
2
/
m
3
p
e
r
s
F
i
n
a
l
a
a w
C
o
n
i
d
i
a
1
/
m
3
D
e
c
r
e
a
s
e
d
w
a
t
e
r
c
o
n
t
e
n
t
(
k
g
)
M
e
a
s
u
r
e
d
P
r
e
d
i
c
t
e
d
b
1
1
C
.
m
i
n
i
t
a
n
s
H
e
m
p
A
c
1
.
0
1
0
.
0
2
7
1
4
.
1
2
5
.
2
8
.
2
1
0
)
5
1
.
0
0
6
.
2
1
0
1
4
1
.
7
0
1
.
1
6
1
.
1
2
1
8
C
.
m
i
n
i
t
a
n
s
H
e
m
p
A
1
.
0
8
0
.
0
2
7
1
1
.
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2
2
.
9
8
.
3
1
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)
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.
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7
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2
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6
1
9
C
.
m
i
n
i
t
a
n
s
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e
m
p
A
1
.
0
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0
.
0
2
7
1
4
.
0
2
4
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9
6
.
9
1
0
)
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1
.
0
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.
9
1
0
1
4
1
.
9
2
1
.
1
6
1
.
1
7
2
5
C
.
m
i
n
i
t
a
n
s
H
e
m
p
A
1
.
1
8
0
.
0
2
7
1
4
.
0
2
6
.
4
8
.
3
1
0
)
5
1
.
0
0
5
.
3
1
0
1
4
2
.
2
1
1
.
3
7
1
.
3
6
1
2
C
.
m
i
n
i
t
a
n
s
H
e
m
p
A
1
.
0
1
0
.
0
5
5
1
4
.
2
2
4
.
1
7
.
1
1
0
)
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1
.
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4
1
0
1
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6
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.
m
i
n
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t
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n
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e
m
p
B
c
1
.
0
1
0
.
0
2
7
1
7
.
0
5
7
.
3
1
4
1
0
)
5
0
.
9
7
1
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7
1
0
1
4
2
.
9
6
2
.
9
3
2
3
C
.
m
i
n
i
t
a
n
s
H
e
m
p
A
1
.
1
8
V
a
r
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a
b
l
e
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1
.
6
2
8
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6
9
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6
1
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.
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8
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4
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t
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d
O
2
c
o
n
s
u
m
p
t
i
o
n
i
n
t
h
e
r
e
a
c
t
o
r
(
r
s
t
c
o
l
u
m
n
)
o
r
t
h
e
m
e
a
s
u
r
e
d
O
2
c
o
n
s
u
m
p
t
i
o
n
(
s
e
c
o
n
d
c
o
l
u
m
n
)
.
c
H
e
m
p
i
m
p
r
e
g
n
a
t
e
d
w
i
t
h
e
i
t
h
e
r
s
o
l
u
t
i
o
n
A
:
1
0
0
g
/
L
g
l
u
c
o
s
e
a
n
d
2
0
g
/
L
y
e
a
s
t
e
x
t
r
a
c
t
:
o
r
s
o
l
u
t
i
o
n
B
:
3
0
0
g
/
L
g
l
u
c
o
s
e
a
n
d
6
0
g
/
L
y
e
a
s
t
e
x
t
r
a
c
t
.
388 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 77, NO. 4, FEBRUARY 15, 2002
will inevitably lose water during the cultivation due to
evaporation. Ideally, the support should be able to re-
lease this large amount of water without aecting the
water activity. Hemp provided good control of water
activity due to its high water uptake capacity and fa-
vorable sorption isotherm (Weber et al., 1999). A spore
yield of 9 10
14
conidia per m
3
packed-bed was expected
to be feasible (Weber et al., 1999). Our experiments in
the PBR show that the water activity indeed remained
1.0 and that the expected spore numbers were obtained
(Table III). The nal moisture content of impregnated
hemp was suciently high to prevent a drop in the a
w
. It
was anticipated that a higher initial nutrient concen-
tration could be used to impregnate the hemp, which
might lead to an even higher spore yield. However, a
threefold increase in the nutrient concentration resulted
in a drop of the initial water activity. Due to this re-
duction in water activity, the obtained conidia yield was
low (Table III).
The water activity of oats was expected to become
limiting for conidia formation after 24 d of cultivation
(Weber et al., 1999). The water activity at the end of the
cultivation was indeed slightly reduced (a
w
= 0.98), as
was previously predicted (Weber et al., 1999). Despite
the inhibitory a
w
levels at the end of the cultivation, the
conidia production on oats was still about 1.1 10
15
conidia/m
3
(Table III), which is only slightly lower than
that obtained on lab-scale (Weber et al., 1999).
Channeling
Evaporation can negatively aect the cultivation in
various ways. In literature, the attention is primarily
focused on the reduction in water activity. In previous
work, we have emphasized the possibility of shrinkage
and subsequent channeling (Oostra et al., 2000; Weber
et al., 1999). In the current paper, we show that
shrinkage of the solid substrate and channeling can in-
deed have a more signicant eect on the cultivation
than the reduction in water activity.
A disadvantage of grain is that evaporation of water
will cause shrinkage, which we expected to result in
channeling and inhomogeneous aeration (Oostra et al.,
2000; Weber et al., 1999). Indeed, channel formation
was observed when A. oryzae was grown on wheat in the
packed-bed reactor. Evaporation caused shrinkage, and
large air channels appeared between the tightly bound
substrate and the reactor wall (Fig. 6). As a consequence
of the inhomogeneous aeration, the temperature in the
center of the bed could not be controlled and quickly
reached 45C. At the end of the cultivation, almost no
mycelium was visible in the center of the bed. Only near
the channels, where the temperature could be controlled,
was abundant growth observed (Fig. 6). Surprisingly, no
channeling was observed when C. minitans was culti-
vated on oats in the packed-bed reactor. Contrary to
A. oryzae, C. minitans does not form hyphal bridges
between particles. As a consequence, the grains re-
mained free-owing and no channels were observed.
Instead, we observed a reduction of the total bed height.
Our mathematical model cannot be used to predict
temperatures in situations where channeling occurs.
However, it might be extended to predict when shrink-
age will occur. The current prediction of the water
content of the substrate bed can be combined with in-
dependent measurements of substrate volume versus
moisture content (Oostra et al., 2000), to generate a
prediction of bed volume. When combined with obser-
vations from small-scale cultivations on the extent of
hyphal bridge formation, this might be translated into a
rough prediction of channel formation.
Limitations of Evaporative Cooling
Although evaporative cooling is a very ecient way to
remove heat, it has several disadvantages. It may aect
the cultivation by causing a reduction in a
w
or shrinkage
of the substrate, which may cause channeling. Another
disadvantage might be that the evaporation rate of
water cannot be controlled, and might become rate
limiting.
In our model, we assumed that the evaporation rate is
not limiting, and, therefore, the air is expected to be in
equilibrium with the solid matrix at any point in the bed.
The o-gas of the cultivations of C. minitans on hemp
was water-saturated during the whole cultivation, indi-
cating that this assumption is valid. However, when oats
were used as solid substrate, the relative humidity of the
o-gas was only 87% around day 12 (Fig. 7). For this
cultivation our assumption is therefore not valid, and as
a consequence, the measured temperature was higher
than the predicted temperature. Only when the mea-
sured relative humidity of the o-gas was included in the
Figure 6. Channeling in an aerated (F
///
a
= 0.069 kg/m
3
per s) packed-
bed reactor with A. oryzae on wheat (experiment 17). Left: Top view
showing the air channel, (C) between the reactor wall, (W) and the bed
(B) Right: Cross section showing poor mycelium distribution in the
bed resulting from the inhomogeneous aeration.
WEBER ET AL.: LARGE-SCALE PACKED-BED SOLID-STATE FERMENTATION 389
calculations was good agreement between predicted and
measured temperature observed (Fig. 7). The observed
dierence between hemp and oats might be attributed to
the smaller characteristic particle size of the hemp,
which causes (1) a higher specic surface area for mass
transfer, and (2) a smaller mass transfer resistance inside
the particles.
Aspergillus oryzae has a higher growth rate than C.
minitans: consequently, a higher aeration and evapora-
tion rate is required to remove sucient metabolic heat
when A. oryzae is cultivated on hemp. During cultivation
of A. oryzae on hemp in the PBR, it was observed that
evaporation of water also became rate limiting (Fig. 8).
The o-gas was not water-saturated during the whole
cultivation: after about 35 h, the relative humidity was
only 92%. This observation that the air at the outlet is not
always water-saturated implies that the transfer of water
from particle to air is rate limiting. As evaporation of
water is the main mechanism to remove heat, the
eectiveness of evaporative cooling can be seriously
Figure 7. Temperatures (left) and relative humidity of outlet gas (right) in an aerated (F
///
a
= 0.027 kg/m
3
per s) packed-bed reactor with C.
minitans growing on oats (experiment 15). Symbols: () measurements, bottom to top, of temperature of inlet air at 5, 15, 25, and 35 cm bed height
and of outlet air; (
)
predicted outlet temperature using measured O
2
consumption and RH of outlet air.
390 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 77, NO. 4, FEBRUARY 15, 2002
limited by the transfer rate of water from the particle
to air. It is therefore expected that the evaporation rate
required to minimize axial temperature gradients cannot
always be reached. Especially for fast growing fungi,
such as A. oryzae, this means that axial temperature
gradients cannot always be prevented in large-scale
packed-bed reactors. As temperature can have a tre-
mendous eect on the production of, for instance, ex-
tracellular enzymes (Suh et al., 1988) or secondary
metabolites (Cooney et al., 1997), the overall produc-
tivity in a full-scale packed-bed reactor might be lower
than would be expected on the basis of small-scale ex-
periments.
This unexpected limitation demonstrates very clearly
the necessity to experimentally validate a scale-up
strategy. Recently, Mitchell and colleagues (1999) also
proposed a scale-up strategy for packed-bed reactors,
and assumed that air is always water-saturated at su-
percial aeration rates up to 0.10 m/s. We 7 already ob-
served limitations in the evaporation rate at 0.011 m/s
with oats and at 0.021 m/s with hemp. Clearly, the
packed-bed models need to be extended with solids-to-
gas mass transfer kinetics for water.
Predicting Moisture Content
Desiccation of the substrate during cultivation cannot
be prevented and might result in unfavorable conditions.
Our model predicts the moisture content of the substrate
during cultivation. This prediction can be used to eval-
uate whether unfavorably low water activities or sub-
strate shrinkage are expected to occur when a process is
scaled up. The model discriminates between water in
biomass and extracellular water. However, as only the
overall water content could be measured, we have only
compared the predicted and measured overall water
content. Although the model predicts moisture content
during cultivation (Weber et al., 1999), moisture content
measurements can only be done by taking samples from
the reactor bed. As the moisture loss will be highest at
the end of the cultivation, we compared the predicted
and measured decrease in overall water content at the
end of the various cultivations (Table III).
For all cultivations of C. minitans on hemp impreg-
nated with 100 g glucose/L, the measured decrease in
water content was higher than predicted. For all other
cultivations, the measured and predicted values corre-
spond well (Table III). The fact that the deviations be-
tween predicted and measured water content are only
observed for the cultivations of C. minitans on hemp is
remarkable. Both evaporation and metabolic water
production aect the water content of the substrate. As
the model accurately described temperatures in the
PBR, it is expected that the predicted evaporation rates
are correct. The metabolic production of water is small
compared to evaporation. It is therefore not expected
that a fault in this prediction can account for the ob-
served dierence in predicted and measured water con-
tent. The observed dierences could also be caused by
an error in the water-content measurements. Although
the error in the water-content measurements is relatively
large, it is uncertain whether this accounts for the dif-
ferences. The deviation between measurement and pre-
diction is the same for all cultivations of C. minitans
with hemp, and for the other cultivations the predictions
are similar to the measurements. Therefore, it is unlikely
that the error in the moisture analysis causes the ob-
served deviation.
Another striking dierence between the cultivations
on hemp is the higher amount of O
2
consumed by C.
minitans compared to A. oryzae (Table III). The glucose
concentration is expected to be similar for all the ex-
periments, as the same impregnation procedure was
used. C. minitans is known to produce various cell-wall
degrading enzymes (Jones et al., 1974). It is therefore
expected that C. minitans also uses the hemp as sub-
strate, resulting in the higher O
2
consumption. The mi-
crobial degradation of hemp will require water, and this
might cause the observed deviation between measured
and predicted water content. The fact that the nal
water content in the cultivation with C. minitans on
hemp with a higher glucose concentration was correctly
predicted supports this hypothesis. Due to the higher
glucose concentration, it is expected that less hemp is
degraded. However, to explain the large deviation be-
tween measured and predicted water content, the
amount of water used for the degradation of hemp
should have been extremely large. The cause for the
dierence therefore remains uncertain.
CONCLUSIONS
Several models predicting the performance of packed-
bed bioreactors for solid-state fermentation have been
published in recent decades. Most of these models cannot
be used for scale-up studies, as they do not take into
account the most important heat transfer mechanism:
evaporation (Gutie rrez-Rojas et al., 1995; Sangsurasak
and Mitchell, 1995; Saucedo-Castan eda et al., 1990).
Another shortcoming in all previous models is the lack of
a water balance to predict moisture losses (Sangsurasak
and Mitchell, 1998). Also, none of the proposed models
have been properly validated. Several authors tried to
verify their model by tting the model predictions to the
measured temperatures (Sangsurasak and Mitchell,
1998; Saucedo-Castan eda et al., 1990). A t is, of course,
no proof that the models are valid. The results of the
same authors already indicate that their ``validation''
was not properly performed, because when they used
independently determined parameters in their models,
unsatisfactory model predictions were obtained (Sang-
surasak and Mitchell, 1998; Saucedo-Castan eda et al.,
WEBER ET AL.: LARGE-SCALE PACKED-BED SOLID-STATE FERMENTATION 391
1990). The urgent need for proper validation experiments
was also recognized by Mitchell and colleagues (2000) in
their review on design and operation of SSF reactors.
Our model consists of two parts: a biological part and
a physical part. The biological part predicts the activity
of the fungus in response to changing temperatures at
various positions in the bed. The physical part of the
model uses these activities to predict temperatures at
various positions in the bed. Both parts of the model
have been veried.
Reasonable agreement between predicted and mea-
sured O
2
consumption rates was observed. The devia-
tions that were observed may be attributable to the
dynamic response of fungi to changes in temperature,
which was neglected in the model and in the underlying
isothermal kinetic experiments. An eect of the tem-
perature history on fungal growth rates has been re-
ported previously, but remains to be explained (Ikasari
et al., 1999). More research into this area would be re-
quired to further improve the kinetic model.
Very good predictions were obtained when the phys-
ical part of the model was validated using respiration
rates measured in the packed bed. Only at certain con-
ditions was a limitation of the current model identied.
Incorporation of gas-solids water and heat transfer ki-
netics to account for deviations from equilibrium ob-
served with fast-growing fungi would improve the
current model.
Our results clearly show that a proper choice of the
solid medium is essential for a successful process. As-
pergillus oryzae could be successfully cultivated on
hemp, but the cultivation failed when wheat was used as
solid substrate. The high water uptake capacity of hemp
provided good control of water activity, and shrinkage
and channeling were not observed. Also, hemp allowed
higher evaporation rates and thus better control of
temperature. Another important advantage of hemp is
that it can be impregnated with an optimized medium
supporting high product yields (Ooijkaas et al., 2000b).
We believe that our model and the previously pre-
sented design approach using a simplied version of the
current model (Weber et al., 1999) are valuable tools to
evaluate
:
scale-up of SSF processes in packed-bed re-
actors. We hope that continued development of vali-
dated models of this type will contribute to the
development of SSF technology to a feasible alternative
for submerged fermentation.
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NOMENCLATURE
8
a
1
, a
2
b
1
, b
2
Fit parameters see Table II
C
s
concentration of support
in bed
kg dry support/m
3
support
C
wg
concentration of water in
gas phase
kg water/m
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air
C
x
concentration of biomass in
bed
kg dry biomass/m
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support
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specic heat of dry air J/kg dry air per K
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pwv
specic heat of water vapor J/kg per K
F
a
supercial aeration rate kg dry air/m
2
per s
h
a
enthalpy of (moist) air J/kg dry air
DH
o
reaction enthalpy J/kg O
2
DH
w
evaporation enthalpy water J/kg H
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O
M
w
molecular weight kg/mole
m
o
maintenance requirements
biomass
kg O
2
/kg dry biomass per s
P
tot
atmospheric pressure Pa
p
w
water vapor pressure Pa
r
///
o
oxygen production biomass kg O
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/m
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reactor per s
r
///
w
water production biomass kg H
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O/m
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r
///
x
biomass formation rate kg dry biomass/ kg dry
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t time s
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T
min
minimum temperature
for growth
K
T
max
maximum temperature
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K
T
ref
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X
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maximum content biomass kg biomass/kg dry support
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x
wx
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xo
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O/kg O
2
y
w
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e void fraction m
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air/m
3
reactor
l
max
maximum specic growth rate 1/s
q
s
density support kg dry support/m
3
reactor
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