Agricultural and Biological Chemistry

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Growth and Respiratory Activity of Aspergillus

oryzae Grown on Solid State Medium

Yonekichi Sakurai, Shaw Misawa & Hideo Shiota

To cite this article: Yonekichi Sakurai, Shaw Misawa & Hideo Shiota (1985) Growth and
Respiratory Activity of Aspergillus oryzae Grown on Solid State Medium, Agricultural and Biological
Chemistry, 49:3, 745-750, DOI: 10.1080/00021369.1985.10866800

To link to this article: https://doi.org/10.1080/00021369.1985.10866800

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Agric. BioI. Chern., 49 (3), 745 -750, 1985 745

Growth and Respiratory Activity of Aspergillus oryzae

Grown on Solid State Medium

Yonekichi SAKURAI, Shaw MISAWA and Hideo SHIOTA

Department of Agricultural Chemistry. Faculty of Agriculture.
Iwate University. Morioka 020. Japan
Received August 14, 1984

Growth and respiratory activity of Aspergillus oryzae KB were examined on bread medium as
a model solid state medium with different combinations of water activity (Aw) of the medium and
incubation temperature conditions. The growth, evaluated as to the glucosamine content of the
culture, of A. oryzae KB increased logarithmically at an early incubation stage, and in spite of
different Aw conditions the maximum specific growth rate was observed at 30 0 e and the actual
maximum growth was seen at 2ye on prolonged incubation. The O2 uptake rate increased within
the logarithmic growth phase and showed a peak at an RQ value of around 1.0, followed by a
parallel decrease with the RQ value. The closing stage of the logarithmic growth of A. oryzae KB
coincided with the incubation period at which the O2 uptake rate at a peak and the RQ value started
to decrease. The O2 uptake rate per unit of glucosamine was nearly constant with the same
incubation temperature regardless of the Aw condition during early logarithmic growth. The O2
uptake rates increased with increasing incubation temperature, and the maximum value for the
rates was expected to be between incubation temperatures of 35 and 40 0 e which was higher than the
optimum temperature for growth.

Aspergillus fungi are able to grow on cereals
and their crushed forms, and on the surface of
a nutrient solution in stationary conditions or
solidified conditions with agar, and also in the
submerged state with shaking or bubbling with
air conditions. But the development of
Aspergillus fungi on a solid state or solidified
material was recognized to be more natural
from their bionomical status. In eastern and
southeastern regions of Asia, the processing of
many traditional fermented foods applying
fungal activities is based commonly on the
technical advantages of mildewed products of
grains and beans, and cakes of their crushed
form kneaded with water in the unheated or
heated state. In Japan the utilization of
Aspergillus fungi grown on a solid state sub-
strate especially as koji has been very impor-
tant in the fermented food industry, and only a
few reports are available concerning the
various physiological activities in connected
quantitatively with the growth of Aspergillus
fungi on a solid state substrate because the
mycelium was difficult to separate quanti-
tatively from the solid state substrate.
Research reports concerning the quanti-
tative analysis of growth and enzyme activities
of Aspergillus fungi with simulated solid state
culture models have been published; with an
asbestos koji method by Sakaguchi et al./) a
nylon-paste method by Yamamoto,2) and a
synthetic poly-urethane sponge method by
Fujishima et al. 3 ) The mycelial weight of
practical rice-koji for sake brewing was de-
termined with the residue after amylase diges-
tion of the rice-koji by Terui and Morimoto. 4 )
Arima and Uozumi 5 ) proposed the estimation
of glucosamine as a useful parameter for
measuring the growth of Aspergillus fungi on a
solid state substance, and its application and
improvement were reported for practical prod-
ucts of rice-koji for sake brewing by Ohuchi
et al.,6) for shoyu-koji for shoyu brewing by
Kozaki and Kitahara,7) Katou et al.,8) and
Nakadai,9) and for several kinds of koji with a
modified convenient assay method by Sakurai
746 Y. SAKURAI, S. MISAWA and H. SHIOTA

et al. 10 ) distilled water in a measuring vessel were set in the bath,

Recently a mechanical incubation system and the volumetric change monitored for an appropriate
for Aspergillus fungi on a solid state substance time. Then and after, the moistened filter paper in the
vessel was replaced by a new filter paper moistened with
in a growth cabinet has become available with
20% KOH solution, and the volumetric change followed
closed forced-air conditions under automatic repeatedly in the same manner as above. During these
control. Using this apparatus, evaluation of determinations, another vessel without the sample and
the growth of the fungus was attempted on the filter paper was used as a thermo barometer. The res-
basis of the total amount of CO 2 evolved or O2 piratory quotient (RQ) and O2 uptake rate were calcu-
absorbed during incubation, and several re- lated from the volumetric difference in each case after an
appropriate period. O2 uptake rate was expressed as O2
sults for rice-koji were reported by Sugama mljhr per 5.0 g of original bread weight (equal to per flask)
and Okazaki/1) Koyama et al./ 2 ) and with the corrected volume at 20°C.
Narahara et alY)
In present experiment, market bread was Estimation of growth. The sample in the measuring
vessel of the O2 UP-TESTER was used for estimation of
chosen as a model solid state substance be- .the glucosamine content after determination of the re-
cause of its nutritional abundance accom- spiratory activity. The glucosamine content as a growth
panied with homogenous and porous structure parameter of the sample was determined by the method
which did not obstruct physically to apical previously reported,9) and expressed as glucosamine mg
elongation of the Aspergillus mycelium unlike per 5.0 g of original bread weight (equal to per flask).
on cereal grains. We describe the changes in
the growth determined from the glucosamine
RESULTS
content and the respiratory activity of A.
oryzae grown on the bread medium under A. oryzae KB was cultured Oll the opt.
controlled water activity (Aw) and incubation and low Aw media at 20, 25, 30, 35 and 40°C
temperature conditions. for prolonged periods, and RQ, O2 uptake
rate and glucosamine content were deter-
MATERIALS AND METHODS mined, respectively. The changes in these de-
terminants at 20 and 30°C are shown in Figs.
Organism and culture conditions. Bread from a market 1 and 2, respectively. These changes at other
was cut into about 5 mm cubics and dried in a drafting incubation temperatures were highly analo-
chamber. Five g of air-dried bread was put in a 200ml
gous like those at 30°C.
Erlenmeyer flask, and moistened with 3 ml of tap water
(low Aw; Aw was about 0~97 after sterilization) or 5 ml
(optimum Aw, opt. Aw; Aw was nearly 0.99 after sterili- Growth
zation, the best Aw condition for hyphal development of As shown in Figs. la and 2a, the lag periods
Aspergillus oryzae KB 14»), and sterilized at I kgjcm2 for of growth curves on opt. Aw media at· the
15min to obtain the bread medium. A slant culture of respective incubation temperatures were short-
Aspergillus oryzae KB maintained in our laboratory was
used as the test strain. The low Aw bread medium was
er than on the low Aw media, and the growth
inoculated with 2 or 3 loopfuls of conidia from the test was retarded on the low Aw media comparing
strain slant, and incubated at 35°C for 4 or 5 days until full to the opt. Aw in all cases. Glucosamine con-
conidiation for the inoculation seed. The opt. and low Aw tents of the cultures on opt. Aw media in-
media were inoculated with a piece of the inoculation seed, creased logarithmically within about 50 to
and incubated at the temperature noted in the text for an
75 mg per flask, and also on the 10wAw media
appropriate period.
within 35 to 50 mg per flask in an early period
Determination of respiratory activity. For a portion of incubation, and a maximum value was
(about 500mg wet weight) of the culture after the in- attained on prolonged incubation, respec-
cubaton, respiratory activity was determined with a con- tively. Table I shows the specific growth rate
venient horizontal manometer, TAIYO O2 UP-TESTER
calculated from the doubling time of the glu-
(Taiyo Scientific Co., Tokyo), immersed in a thermostatic
water bath at the same temperature as for the incubation. cosamine content in the logarithmic growth
At first, the sample' and.a filter paper moistened with phase. and. the presumable maximum growth
 Growth and Respiratory Activity of Aspergillus oryzae 747

~ 0,8

'1
0,6

20 b
~ 20
""0
~

~ ~
"- "-
~ ~

"- "-
~ ~
'"
[i 1O '"
[i

""'"0 fO
Q

N
ON 0

""
.§
~
"-
50 •
~
N
~
z
u
, N
~
~ ,
Z
U
~

50 100 10
Incubation Period in oours
50 100
FIG. I. Changes in Growth and Respiratory Activities
Incubation Period in hours
of A. oryzae KB Incubated at 20°e.
The solid line with open circles (-0-) shows the culture FIG. 2. Changes in Growth and Respiratory Activities
of A. oryzae KB grown on the opt. Aw media, and the of A. oryzae KB Incubated at 30°e.
solid line with solid circles (-e-) shows the culture on Symbols and representation in the figure are the same as in
the low Aw media, and a, shows growth (glucosamine Fig. 1.
content); b, O2 uptake rate; c, RQ (respiratory quotient).

comparison of the curve for glucosamine con-

value deduced from the Figs., respectively. The
specific growth rate increased with increasing
incubation temperature for both Aw con-
ditions except at 40°C, but some low values
were observed on the low Aw media compared
to the opt. Aw condition. Although the highest
value for the specific growth ratewas obtained
on 30°C incubation, the highestglucosamine
content realized was observed at 25°C in both
Aw conditions on prolonged incubation.
O2 uptake rate
As shown in Figs. 1band 2b, the O2 uptake
rate in each culture increased nearly linearly
with the incubation period in the early stage,
and reached a peak followed by a decrease. On
tent with the curve for O 2 uptake rate in the
figures (compare Ia to lb, and so on), the
incubation period at which the peak of O 2
uptake rate was seen in the curve was found to
be nearly the same period as the closing stage
of the logarithmic increase phase of the glu-
cos amine content. Table II shows the highest
O2 uptake rate and the incubation period
when the O2 uptake rate was at its peak in
every culture. As can be seen in Table II, the
highest O2 uptake rate in every culture increas-
ed with increasing incubation temperature un-
der both Aw conditions, but the rate was
some degree low under the low Aw condition,
and was also decreased at 40°C compared to
at 35°C. These results indicate that the maxi-
748 Y. SAKURAI, S. MISAWA and H. SHIOTA

TABLE I. GROWTH PARAMETERS OF Aspergillus oryzae KB GROWN ON BREAD MEDIA

Doubling time Specific growth Maximum growth

Incubation Aw a
of glucosamine rate presumed
temperature condition
(hr) (hr- 1 ) (Glucosamine mg/flask)

opt. 19.0 0.036 100

low 33.0 0.021 45
opt. 9.5 0.073 160
25°e
low 11.0 0.063 90
opt. 9.0 0.077 140
low 11.0 0.063 70
opt. 9.6 0.072 100
low 11.0 0.063 60
opt. 14.0 0.049 80
low 16.0 0.043 40

a Aw values of the bread media were noted in the text.

TABLE II. THE HIGHEST O2 UPTAKE RATE AND for the cultures at 20°C, and the RQ values at
INCUBATION PERIOD AT WHICH THE O2 30°C were between 0.95 and 1.0 independent
UPTAKE RATE WAS AT A PEAK
of the Aw condition in the early stage of the
Incubation The highest culture, and a gradual decline followed, and
Incubation Aw
temperature condition a
period b O2 uptake rate' also analogous shift of RQ were observed in
(hr) (02 ml/hr/flask) cultures at 25, 35 and 40°C, respectively. In
opt. 90~96 17
these cultures, the start of the decrease in RQ
20 0 e value coincided with closing stage of the loga-
low 96 11
opt. 40 18 rithmic increase in the glucosamine content
25°e
low 40 17 and/or the incubation period when the peak of
30 e 0
opt. 36 25 the O 2 uptake rate was seen. During incu-
low 36 24 bation at 20°C, RQ values fluctuated but
opt. 36 42 remained around 1.0.
35°e
low 36 35
opt. 40 34
40 0 e Relationship of O2 uptake rate to growth
low 40 25
Figure 3 shows the relationships of O 2 up-
a Aw values of the bread media were noted in the text. take rate to the growth evaluated on the basis
b The period was determined from the figures. of glucosamine content. As shown in Fig. 3,
, The highest O2 uptake rate are expressed as the
the O2 uptake rate per unit of glucosamine
corrected rate at 20 0 e for comparison.
formed in the early growth phase increased
mum O2 uptake rate of the test strain occurs with increasing incubation temperature to
between 35 and 40°e. The incubation peri- nearly the same degree for both Aw condi-
ods showing the highest O2 uptake rate un- tions at the respective temperature, and reach-
der both Aw conditions were very similar ed a peak value followed by a rapid decrease.
regardless of the Aw condition at the respec- But on the low Aw medium a lower value
tive incubation temperature, but these peri- of the O 2 uptake rate for the peak was seen
ods were longer at 25 and 40°C, and more with less growth that was equivalent to the
delayed at 20°e. value on the opt. Aw medium. At 25°C, the
changes were analogous to in the case at 20
Changes in respiratory quotient (RQ) or 30°C and were in-between at 20 and 30°C,
Changes in RQ values are shown in Figs. lc and also the results at 40°C were actually sim-
and 2c. Though the shown values fluctuated ilar to those at 35°C.
 Growth and Respiratory Activity of Aspergillus oryzae 749

Sugama 17) reported that the total amount of

40 O 2 consumed could be used to evaluate the
growth of A. oryzae grown on rice because the
total amount of O2 consumed increased loga-
rithmically with the RQ value of 0.9 to l.09
(average 0.98) in an early stage of incubation
30
(within about 22 hr).
~
The present report describes that an obvious
"-
0
logarithmic increase in the glucosamine con-
"-
~
"- tent is seen in every culture of A. oryzae KB
§
20 grown with the various combinations of Aw
0'" and incubation temperature conditions on the
~
0
'" bread medium which has a porous and nearly
,§
homogenous structure that allows free apical
6'
elongation and respiration of mycelium and
10 which contains abundant nutrients required
for growth. In spite of the different Aw con-
ditions, the maximum specific growth rates
were observed at 30°C, but the maximum
growth presumed was seen at 25°C, respec-
Glc-NH 2
50
(mg/flaskl
100 tively. Therefore the optimal growth tempera-
ture for the test strain was between 25 and
FIG. 3. Relationships of O 2 Uptake Rate to Growth. 30°C, presumably near 30°C. It was confirmed
O 2 Uptake rate is plotted against the glucosamine content that the mycelial growth of A. oryzae on a
of the cultures with different combinations of Aw and
solid state medium was independently influ-
incubation temperature conditions. The solid line with
open symbols shows O 2 uptake rates of the cultures
enced with the A w condition notwithstanding
incubated on the opt. Aw medium, and the dotted line changes in the incubation temperature as pre-
with closed symbols shows those on the low Aw medium. viously noted. 14 ) These results lead to the
The symbols indicate the incubation temperature as fol- supposition that an apically elongating hypha
lows: oe, at 20°C; 1:,£', at 30°C; and <>+, at 35°C. on the solid state medium produces a new
apical branch per a definite hyphal length to
DISCUSSION
result logarithmic increase in cell mass (e.g.
glucosamine) under circumstances where there
As to the growth of A. oryzae on a wheat are no mechanical or nutritional limitations to
bran medium, a logarithmic increase in hypha I the hyphal growth.
weight inferred from the glucosamine content In every culture, the O2 uptake rate in-
was recognized in an early incubation stage by creased under an RQ value of around 1.0, and
Kurimoto et aUS) Sugama et al. 161 reported a the highest O2 uptake rate was seen at a time
logarithmic increase in the total amount of that coincided with the closing stage of loga-
CO 2 evolved during incubation of A. oryzae on rithmic growth, and there followed a decrease
a highly polished and steamed rice medium, parallel with the decline in RQ value. These
and proposed that the total amount of CO 2 results suggest that the logarithmic growth of
evolved could be used as a substitute param- A. oryzae on a solid state medium is sustained
eter for the mycelial weight because the ratio with a sufficient supply of sugar, and re-
of the total amount of CO 2 evolved to the tardation of growth occurs with a sugar de-
mycelial weight calculated from the glucos- ficiency reflected as a decline in RQ value in
amine content was practically constant during parallel with a decrease in O2 uptake rate.
the incubation. Furthermore, Okazaki and The RQ values during koji processing of A.
750 Y. SAKURAI, S. MISAWA and H. SHIOTA
oryzae on highly polished and steamed rice
were reported to be relatively constant at
about 1.0 by Terui and Morimoto,41 Oka-
zaki and Sugama,17l Koyama et al.,121 and
Narahara et alYI Moreover, Narahara etal. 13 )
pointed out that the growth of A. oryzae on
rice medium was suppressed and stopped by
accumulation of glucose produced on hy-
drolysis of rice starch resulting in an Aw drop
of rice medium to 0.9 during incubation. This
evidence suggests' a special character of rice-
koji in that the growth of A. oryzae is self-
controlled on highly polished rice through Aw
mediation, and growth ceases within the loga-
rithmic growth phase with anRQ value of
around 1.0 without a sugar deficiency.
As the nearly linear increase in O2 uptake
rate reflected the glucosamine content in the
early growth phase as shown in Fig. 3, it is
obvious that the integral amount of O2 ab-
sorbed would show a logarithmic manner as
in the case of the results reported by Okazaki
and Sugama. 17 ) In the same situation with a
sufficient supply of sugar with an RQ value
of around 1.0, the O2 uptake rate per unit of
glucosamine increased with increasing, incu-
bation temperature, and the highest rate was
expected to be between 35 and 40°C. The result
was in good agreement with the result that the
maximum specific growth rate determined
from the O 2 absorption was recognized at
37SC for A. oryzae grown on highly polished
rice medium as reported by Okazaki et al./ 81
but the growth estimated from the glucos-
amine content should be distinguished from
the growth on the basis of the respiratory
activity.
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