Heat and Mass Transfer Effects in Static

Solid-Substrate Fermentations: Design of

Fermentation Chambers

B. L. RATHBUN” and M. L. SHULER,t School of Chemical Engineering,

Cornell University, Ithaca, New York 14853

summary
An experimental device was constructed to allow nearly simultaneous measurements to be
made on temperature and gas composition at different depths in a solid-substrate fermentation
bed. The time-dependent values of temperature, mol 700,and mol 70C 0 2 were measured at
five positions in beds 6.35 cm (2.5 in.) deep. With a tempeh fermentation (Rhiopus oligosporus
growing on soybeans) the temperature gradient could be as steep as 3’C/cm during active mold
growth and concentration of CO, could reach 21 vol. 70in the bottom layer.

INTRODUCTION

In nature most molds grow on solid surfaces. Such surfaces provide a

satisfactory nutritional base and a competitive advantage over most bacteria
which require a higher moisture environment.
However, most mold products made on a large scale employ submerged
culture techniques which provide a high level of spatial uniformity in culture
conditions. Such uniformity is important in designing a large-scale process
allowing the operator the opportunity to exert timely control over the en-
vironment and consequently to control cellular physiology. Since the
submerged system is sterilized, problems due to competition from bacteria
are avoided. The Japanese “Koji” fermentation is the only exception to the
use of submerged cultures for large-scale production of mold products. “Koji”
fermentations can operate on a continuous basis with reactor volumes of
200 ft3. The “koji” fermentations serve as a starting basis for making sakC,
soya sauce, miso, and certain enzymes. Other potential uses for solid-
substrate fermentations (SSF) include upgrading agricultural residues as
feeds, 1-5 conversion of agricultural products or residues for ethanol produc-
tion,6 and production of medicinals, enzymes, and food grade pigments.’-’*
Hesseltine9 has made an extensive listing of the potential advantages and
disadvantages of solid-substrate fermentations. They normally take the form
*Current address: IBM Corp. Owego, New York 13126.
tTo whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. XXV, Pp. 929-938 (1983)

0 1983 John Wiley & Sons, Inc. CCC 0006-3592/83/040929-10$02.00
930 RATHBUN AND SHULER

of molds growing on the surfaces of grains. The most exciting advantages are
associated with improved product recovery and with reduced energy re-
quirements and wastewater outputs. For certain molds only the low moisture
environment of solid-substrate fermentation can be tolerated. Consequently,
certain mold products can be made only with SSF or made in higher
y i e l d ~ . ~ .The
~,’~
disadvantages of large-scale SSF center on problems of heat
buildup and process control and scaleup.
The use of rotating solid-substrate fermenters can reduce system
heterogeneity in comparison to static bed f e r m e n t a t i ~ n . ~ JHowever,
~J~ such
“improvements” are not always desirable. Certain SSF products (e.g.
tempeh) are used as foods, and the texture developed in a static bed is an im-
portant attribute of the product. In other cases the abrasion encountered in
rotating systems can disrupt mold development and the formation of secon-
dary products.1sJ6 Although the macrogradients of a static bed are
eliminated in the rotary reactor, both types of SSF still have microgradients
(within the grain and the mold on the surface and the gas boundary layer
about the particle).
A detailed knowledge of the inter-relationship of heat and mass transfer
with mold growth kinetics is essential for the rational design and control of
SSFs.14 The techniques used to gain such knowledge would also be ap-
plicable to related problems such as composting rates for solid wastes” and
such as degradation rates and nutrient release in natural ecosystems.
The purpose of this article is to report on a fermentation device which
should be suitable to develop fundamental information on the interaction of
growth kinetics with macrogradients in heat and mass transfer. For the
preliminary study the tempeh fermentation was chosen as a model system.
Tempeh is a fermented soybean product which is used as a primiative meat
analog in Southeast Asia.18 The normal fermentation depends primarily on
the mold Rhizopus oligosporus and is usually conducted in static beds
2.5-6.5 cm deep. Tempeh is currently enjoying an increased popularity in
Western countries such as the United States. The microbiological features
and description of the enzymatic degradation of soybean in this fermentation
has been reviewed elsewhere.l9

MATERIALS AND METHODS

Preparation of Soybeans
One kilogram of dry beans were soaked overnight in 3 L of 1% lactic acid
solution. The liquid was decanted and saved for the cooking process. The
beans were then rubbed between the fingers to remove their hulls. Hulls were
separated from the cotyledons using gravity filtration.
Dehulled beans were put into a metal sieve, mesh number four, to separate
out the smaller pieces resulting from the handling of the beans. The beans re-
maining in the sieve were all half or whole beans, about one centimeter long by
 STATIC SOLID-SUBSTRATE FERMENTATIONS 93 1

0.5 cm in width. These were returned to the soak water, covered with foil, and
autoclaved for ten minutes at 120°C. This cooking both sterilized the beans
and destroyed the trypsin inhibitor inherent in the beans which could effect
mold growth. The final pH of the beans ranged from 4 to 5. Upon cooling to
ambient temperature, the liquid was decanted and the beans inoculated.

Organism
A pure culture of Rhizopus oligosporus (obtained from Professor K. H.
Steinkraus, Cornell University, Ithaca, NY) was employed in all experiments.
The inoculum had been prepared by lyophilizing and grinding a single batch
of tempeh fermented until well sporulated. The inoculum contained both
spores and fragments of mycelia as well as fragments of undigested bean par-
ticles. For each kilogram of dry beans, approximately 0.5 g of powdered in-
oculum was used.

Chamber Design
The purpose of the study was to develop a chamber which would allow the
fundamental evaluation of mass and heat transfer effects on mold growth.
The device ultimately developed is shown in Figure 1. Among its virtures are

TOP VIEW
THERMOCOUPLE PORT
PORT
,sI w
5.1 cm

r o p SECTION
(PLASTIC)

‘ I I

AIR SPACE
(ALUMINUM)

1 I 1 I
I

SIDE RINGS
(PLASTIC)

BOTTOM PLATE
(ALUMINUM)

Fig. 1. Design of the fermentation chamber. Measurements are approximate metric

equivalents of English units [e.g. height of side ring was 0.50 in. (1.27 cm)].
932 RATHBUN AND SHULER

1) the ability to vary the depth of the fermentation bed without altering the
fluid dynamics of gas flow; 2) the ability to measure temperatures and gas
composition at various depths with minimum disturbance of the fermenta-
tion; and 3) the ability to establish concentration and temperature gradients
primarily in one direction (axial to gas flow).
The spacers (or sides of the chamber) and top plate were made of Lexan (a
clear plastic). The air space unit and the bottom plate were aluminum. Three
inlet and outlet ports for gas flow were provided in an attempt to achieve
more uniform conditions in the gas phase. The clear plastic top and sides
allow for direct observation of the fermentation’s progress. Since the
aluminum bottom is a much better thermal conductor than the plastic, its
presence encourages the formation of thermal gradients as a function of
depth rather than radial distance from the center of the bed.
The height of the fermentation bed was set by the number of spacers used.
The gas volume above the bed could be maintained at a constant value irre-
spective of the bed’s depth. Air-tight connections were made between
spacers, the air space plate, and the bottom plate by sealing with Dow Corn-
ing high-vacuum grease. All pieces of the chamber were clamped in place by
four nuts and bolts in the corners. In some experiments it was desirable to
observe the progress of the experiment by recovering representative samples.
The nuts and bolts were removed and horizontal layers of the tempeh cor-
responding to the height of the spacer unit could be sequentially removed by
slicing with a knife. Fully assembled chambers were autoclaved for 15 min at
121OC prior to inoculation.
The top plate contained one central thermocouple port. Baily Type MT-23/8
microprobes were used (Bailey Instruments, Inc., Saddle Brook, NJ). The
device was said to be accurate to k 0.25”C, and each thermocouple will be
within 0.1OC of each other. The central thermocouple was arranged to detect
temperatures at various depths in the tempeh by slowly raising the probe to
predetermined positions.
The top plate contained eight sampling ports. These ports were designed
to allow the extraction of samples from the tempeh bed by a cork borer (size
No. 3). They were also used for thermocouple placement to allow the simulta-
neous measurement of temperatures at the same depth but at various posi-
tions in the bed. Such information was important in determining whether
“radial” gradients were significant.
Gas samples could be obtained at various depths in the reactor through spec-
ially designed gas sampling ports (Fig. 2). A 2.5-in., size 17 syringe needle was
attached to a modified filter holder (Millipore Swinnex-13, Millipore Corp.,
Bedford, MA). The interior ring of the filter was removed and replaced with a
gas partitioner septum to prevent gas from passing through the needle. The
entire assembly was secured in a small cork stopper and placed in a hole
drilled in the side of a spacer ring. The needle portion extended inside the
bean bed place prior to filling the chamber with inoculated beans. The ar-
rangement of the ports was all on one side of the chamber on a diagonal run-
ning from top to bottom.
 STATIC SOLID-SUBSTRATE FERMENTATIONS 933

Swinnex 13
? Beans

! Cork Side Rtnq

i
Septum i 1
1 XI7 Needle

Fig. 2. Side view of gas sampling assembly. Gas sample is taken by inserting small bore needle
of a gas tight syringe through septum into No. 17 outside needle.

To take a sample of gas at any of the layers, a Pressure-lok gas-tight sy-

ringe (Precision Sampling Corp., Baton Rouge, LA) and No. 27 needle was
used. The needle would pierce the septum and enter the larger needle chan-
nel. The syringe chamber was slowly flushed twice, and a volume of approx-
imately 0.30 cm3 was slowly withdrawn. Before injecting into the gas parti-
tioner, the sample volume was reduced to 0.25 cm3. Such a gas sampling
system allowed a gas sample to be obtained with minimal disturbance of the
fermentation bed and of the internal gas phase.

System Design
Ideally, an investigator would want to incorporate such a fermentation into
a constant temperature environment with control of gas composition and
humidity. In our experiments the fermentation chambers were placed in a
dry type bacteriological incubator (Blue M Electric Co., Blue Island, IL)
which was modified by placing a small fan inside (Dayton Model 2C782,
Dayton Electric Mfg. Co., Chicago, IL). The fan maintained more uniform
temperature throughout the incubator.
A mixture of air and prepurified nitrogen (99.8% nitrogen, Airco, New
York, NY) could be supplied to the chambers. The gas composition and
overall flow rate was determined by using rotameters. Model 10A3565A
(Fischer and Porter Co., Westminster, PA) was used for low flow rates, while
model 7262 by Markson (Del Mar, CA) was used for higher rates. Since C 0 2
evolution could be indicative of growth, the inlet gas was passed through a
6M NaOH solution to remove C02. To prevent drying of the beans the inlet
gas was passed through a humidifier. A saturated solution of (NHJ2-PO4
was used in the humidifier; the exit gas would contain 95% relative humidity
if equilibrium was achieved. The humidified and C02-strippedgas was passed
through a glass-wool autoclaved air filter prior to entrance to the fermenta-
tion chamber.
Typical gas flow rates were 0.4-10 standard L/min. Higher gas flow rates
would make the measurement of C02 evolution difficult and tend to dry the
beans too quickly. These problems might be eliminated at high gas flow rates
if a gas recycle system could be perfected; attempts at such recycle proved
fruitless since gas leakage was significant. At low gas flow rates, as used here,
the gas composition and temperature could vary perceptibly from the en-
934 RATHBUN AND SHULER

trance to the exit of fermentation chamber (e.g., up to a decrease in O2 from

20.5% at the inlet to 15.5% at the midpoint of the chamber).
It was necessary to have a water trap and condenser on the effluent gas line
to remove excess moisture from the gas. The fermentation itself can release
water vapors. This excess water can condense in the gas outlet lines. To pre-
vent this condensate from flowing back into the chamber, a water trap was
placed immediately following the exit point. A condenser on the effluent line
further removed water vapor prior to gas analysis.

Gas Analysis
A Fisher model 1200 gas partitioner (Fisher Scientific Co., Pittsburgh,
PA) was used. Helium (Airco, New York, NY, research grade) was the car-
rier. Two columns were employed; one contained molecular sieve 13X and
the other 30% of di-2-ethyl hexylsebacate on 60/80 mesh Chromosorb P. Ex-
cellent resolution of N2, 02,and C 0 2 could be obtained within three
minutes. The sample size was 0.25 cm3. Samples from within the fermenter
were injected using a syringe as described previously. A sampling line from
the effluent gas could be activated with a pushbutton valve to send a 0.25 cm3
sample to the partitioner.

Growth Rate
Direct, unambiguous measurement of the cell mass in SSFs is extremely
difficult. The glucosamine determination has been used2”; but since the
amount of glucosamine in fungal cell walls varies with the age of mycelia,I4it
can be regarded only as a rough approximation. Several authors have sug-
gested using C 0 2 evolution as an indirect rnea~urement.~l-~~
The method used
by Carrizalez et a1.22relied on the continuous collection of C 0 2 from the ef-
fluent gas using absorption in a NaOH solution. The C 0 2 measurements are
essentially an integration of rate data and would not be very sensitive to
changes in growth rate toward the end of the growth cycle. Direct measure-
ment of rate (gas flow times mole fraction C02) is more sensitive to changes
in growth rate and was used in these experiments. Because C 0 2 can evolve
from endogenous metabolism and also possibly from anaerobic metabolism
of sugars,16 the relationship of C 0 2 evolution to growth is rather tenuous.
Nonetheless, the method is rapid and no worse than alternate methods of
growth estimation.

RESULTS AND DISCUSSION

The characteristics of the chamber were examined using a variety of

thicknesses of the fermentation layer. In each case measurements of
temperature and gas concentration were made vertically through the bed.
The chamber design was to encourage gradients along the “z-axis” (Fig. 1)
rather than longitudinally (x-axis in Fig. 1). How well this worked is
 STATIC SOLID-SUBSTRATE FERMENTATIONS 935

demonstrated by the data in Table I for a 5.1-cm (2.0-in.) deep bed. Clearly,
the longitudinal gradients (less than 0.15"C/cm) are much smaller than the
vertical ones (ca. 3.2"C/cm).
A number of experiments were done on thin beds (0.5 cm thick) in an at-
tempt to discern what the "intrinsic" kinetics of growth were. Growth-rate
measurements based on C 0 2 evolution were not precise enough to justify
mathematical correlation. Endogenous C 0 2 evolution, dispersion of ger-
mination times due to using a mixture of spores and mycelial fragments, and
fluctuations in inlet gas composition probably contributed to this variability.
From these experiments it was concluded that growth would take place at
47°C but not at 50°C. Relatively rapid growth was obtained using oxygen
concentrations of 1.0 to 6.5 vol. 9'0 in the inlet gas with perhaps some slight
increase in rate at higher oxygen values. At oxygen concentrations of 0.23 to
0.32% no growth would occur. The optimum temperature appeared to be
near 40°C. The exponential growth phase was short in duration in most
cases. At 40°C and oxygen concentrations of about 3-3.570, the specific
growth rates was 0.25 & 0.05 h-l.
The effects of temperature and gas composition must manifest themselves
in deeper bed fermentations. Such effects are displayed in Figure 3 for a bed
6.35 cm in depth. Mycelial growth was clearly visible at 17 h after inocula-
tion, and this observation corresponds to a period of rapid O2 consumption.
Coupled with 0 2 consumption is a rapid rise in the in situ C 0 2 values-up to
21 Yo. It is conceivable that such high C 0 2 values would be growth inhibitory.
In this experiment, the maximum temperature observed was 47°C in the
middle layer. In another experiment with a 5.1-cm-deep bed incubator

TABLE I
Longitudinal Temperature Gradients 1.9 cm from the Bottom
of a 5.1 Centimeter Deep Tempeh Fermentation

Hours of incubation time

Positiona 17 34.5 42 65

A 34.5"C 47.9OC 38.6"C 37.4"C

B 34.5 48.3 38.6 37.5
C 34.5 48.2 38.5 37.4
D 34.4 48.2 38.5 37.5
E 34.2 48.1 38.4 37.2
F 34.4 47.9 38.6 37.4
G 34.3 47.8 38.5 37.2
H 34.4 48.1 38.6 37.4
Incubator 34.0 38.0 36.0 36.0
~~ ~ ~~

aPosition refers to horizontal location with respect to the air inlet to

the chamber. Letters start with A located at center of inlet air side and
proceed clockwise around a circle. See Figure 1 for exact placement.
936 RATHBUN AND SHULER

temperature of 36°C and a gas flow rate of 0.47 Standard L/min, the mid-
point temperature reached 49.6"C-sufficiently high to inhibit growth.
After 25 h postinoculation, the system entered a period of relatively slow
change which lasted about 15 h. From 40 to 47 h, postinoculation growth
slowed somewhat with slight increases in O 2 in most of the bed. At the latter
stages of fermentation the top layers had shrunk, leaving an empty space be-

w
LL 40-
3
c
a
a 38.
. -8

W
n

l l l l l l
20 25 30 35 40 45
T l M t (hr)

22 -
oo.o-o- 0 0
LO -
~

15 20 25 30 35 40 45
TIME (hr)

Fig. 3. Mass and heat transfer effects on a tempeh fermentation bed 6.35 cm (2.5 in.) deep.
The incubator temperature was set at 3SoC, and CO,-stripped air was used at a flow rate of 0.71
standard L/min. Figure (A) displays temperature profiles in various layers, while 0, and CO,
concentrations are given in (B) and (C). In all three graphs, the following symbols are used to
denote the position of the sample: ( 0 )middle of bottom ring 1 (0.6 cm from bottom); (0)
middle of
ring 2 (1.9 cm from bottom); (a) middle of ring 3 (3.2 cm from bottom); ( v ) middle of ring 4
(4.4 cm from bottom); (L) middle of top ring 5 (5.7 cm from bottom); (a)middle of air space
above tempeh; ( 0 )inlet air.
 STATIC SOLID-SUBSTRATE FERMENTATIONS 937

I
6

<Y5 210 215 o; is 40 415

TIME lhr)

Fig. 3 Continued.

tween the knitted bean cake and the walls of the chambers. Thus, gas could
diffuse into the bed from the sides as well as the top. The temperature and
COz profiles are consistent with the possibility that the bottom layers ex-
perienced a renewal in growth with the increased gas exchange at this point.
The carbohydrate in the beans in the top layer had been probably been ex-
hausted-visual appearance and odor were indicative of protein break-
down-and further mold growth was limited by substrate availability.
The depth of 5 to 6 cm used for the tempeh fermentation is also typical of
the shoyu fermentation. Recent innovations in the koji fermentation typically
use depths of 30 to 40 cm and sometimes values up to 80 cm are used. The
deep beds in the koji fermentation are typically not totally static but are slowly
turned over. Nontheless, the large gradients observed in these experiments are
likely to exist in commercial units for other solid-substrate fermentations.
Fermentation devices of the type described in this article may prove useful
in developing methodology for more rational design of large-scale SSF units.
In principle the design of a deep bed unit could be accomplished with a
knowledge of the intrinsic kinetics of mold growth, the diffusivity of O2 and
COz in the gas phase, the thermal conductivity of the mold and bean, the
structure of the bed, and the fluid dynamics of the gas phase. Of particular
importance would be the development of a general and accurate kinetic
model for mold growth on solid surfaces.

This work was supported in part by a grant from Sun Oil Corporation to the School of Chemical
Engineering. The technical assistance of A. Tannahill and S. Rollins is greatly appreciated. K. H.
Steinkraus’svaluable advice on the tempeh fermentation is particularly appreciated.
938 RATHBUN AND SHULER

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Accepted for Publication October 19, 1982