Appl Microbiol Biotechnol (1989) 31:346-350
Microbiology
Continuous fermentation of soy sauce by immobilized cells
of Zygosaccharomyces rouxii in an airlift reactor
Takashi Hamada, Takashi Ishiyama, and Hiroshi Motai
Research Division, Kikkoman Corporation, Noda-shi, Chiba, 278, Japan
Summary. The optimum conditions for contin-
uous alcohol fermentation of soy sauce with im-
mobilized Zygosaccharomyces rouxff cells were in-
vestigated using an airlift reactor. The optimum
pH and temperature of the fermentation were 4.5-
5.5 and 250-27.5 ° C, respectively. Ethanol content
in the fermented liquid was increased with in-
creasing height to diameter ratio of the reactor
and the ratio of air to nitrogen in the supplied gas
(total supplied gas: 0.08 vvm). A notable decrease
in ethanol content was observed when only ni-
trogen gas was supplied. The products fermented
by supplying air (0.02 vvm) had a higher conent of
aroma components than that by supplying only
nitrogen gas, and the aroma of the former prod-
ucts was similar to that of conventional soy sauce.
This alcohol fermentation using an airlift reactor
was continued for about 50 days without prob-
lems even if conditions such as residence time
and aeration were altered.
Introduction
In the conventional method of brewing soy sauce,
cooked soybean and roasted wheat a r e mixed
with seed spores of Aspergillus sojae and/or A.
oryzae to make koji i n 2 days of solid Culture.
Then, koji is mixed with brine to make moromi,
which is hydrolysed by enzymes from the koji,
such as proteinases, peptidases and amylases.
In general, the salt content of moromi mash
juice is kept around 17%. The high salt concentra-
tion effectively limits the growing microorganisms
to a few desirable salt-tolerant ones. At the first
stage of moromi mash, Pediocoecus halophilus
Offprint requests to: T. Hamada
Applied
Biotechnology
© Springer-Verlag 1989
grows and produces lactic acid which causes a
drop in the pH. Accompanying the decrease in
the moromi pH, vigorous alcohol fermentation by
Zygosaccharomyces rouxii occurs, and 2%-3%
ethanol and many aroma components are pro-
duced. At the same time phenolic compounds,
which add some aroma to soy sauce, are also pro-
duced by other yeasts such as Candida versatilis
and C. etchelsii under the anaerobic conditions
without aeration. It takes over 6 months for the
entire fermentation and ageing of the mororni
mash.
Recently, immobilized microorganisms have
been used for various processes in food produc-
tion such as beer (Onaka et al. 1985; White and
Portno 1978), wine (Gestrelius 1982; Totsuka and
Hara 1981), vinegar (Mori 1985) and soy sauce
(Osaki et al. 1985) because of the rapid fermenta-
tion, the high production efficiency and ease of
both continuous operation and system control.
Osaki e t al. (1985) have reported a method for
shortening the period for brewing soy sauce by
continuous fermentation using a fluidized bed
reactor with immobilized whole cells of three
kinds of microorganisms entrapped in calcium al-
ginate gel. However, the optimum conditions for
fermentation with immobilized cells have not
been well clarified yet.
In this paper, we have used an airlift type
bioreactor to investigate the effects of pH, tem-
perature, NaC1 and aeration on the continuous
fermentation of soy sauce with immobilized cells
of Z. rouxii, the most important yeast for develop-
ment of the aroma of soy sauce.
Materials and methods
Materials. Sodium alginate was obtained form Kimitsu Chem-
ical Industries, Tokyo, Japan.
T. Hamada et al.: Soy sauce fermentation by immobilized Z. rouxii cells 347

pH of the solution obtained from the hydrolysates were ad-

justed as described in the text (total nitrogen, 2%; glucose,

a,e
v
4.0

3.0
I I Feren
s liquid
"3
7%).

Analytical methods, Ethanol was measured by a gas chromato-

graphic method (Hamano et al. 1971) and glucose was deter-
mined enzymatically by the glucose oxidase-peroxidase
e e o
method (Trinder 1969). Aroma components were analysed by
l.- "1- a gas chromatographic method as described by Yokotsuka et
~J2.0
al. (1980). The number of viable cells in the gel was counted as
follows. The gels were homogenized in 0.1 M citrate buffer
a 3.57 (pH 6.0) at 5°C for 4 min. The cell suspension obtained was
1.0 I I
I I I I serially diluted and grown on agar plates.
20 30 40 50 uU
RESIDENCE TIME ( S ) Raw liquidGa s
Measurement of oxygen transfer rate. To 0.4 M Na2SO3 solu-
I*- D -M tion 1 mM CuSO4 was added as a catalyst. The oxidation rate
Fig. 1. Diagram of the airlift reactor and effect of its height to of sulphite was determined by the usual iodometric procedure
diameter ratio ( H / D ) on alcohol fermentation by immobilized of back-titration with 0.05 M Standard NazS203 solution.
Zygosaccharomyces rouxii cells. Conditions were as follows:
pH, 5.0; NaCI, 12.5%; temperature, 27°C; gas flow rate, air
0,02 w m and nitrogen gas 0.06 vvm Results and discussion

Reactors. Three airlift reactors with a draft tube (total volume: Effect of H / D of the reactor and residence time
1 1; height to diameter ratio ( H / D ) = 1.47, 2.50, 3.57; Kawasaki on alcohol fermentation
Heavy Industries, Tokyo, Japan) were used to obtain good
three-phase contact (gas-liquid-gel). The packed gel volume We employed three types of airlift reactor of dif-
was 300 ml and the gas (80 ml/min) was supplied through the
perforated plate to produce fluidizing conditions (Fig. 1).
ferent H / D values. Figure 1 shows the amount of
ethanol produced by immobilized Z. rouxii cells
Microorganism. Zygosaccharomyees rouxii no. 13 from our at different residence times in the three reactors.
stock culture was grown aerobically at 30 ° C for 48 h in a soy The ethanol content in the fermented liquid; often
sauce medium containing 15% raw soy sauce, 7% glucose, and used as an index of alcohol fermentation in soy
8.5% NaC1 (pH 5.2).
sauce brewing, increased with increasing H / D
Immobilization. A 3% (w/v) alginate-cell mixture was dropped values and residence times. The standard ethanol
into a 5% CaC12 solution, and the gel beads (final diameter, content in conventionally brewed soy sauce (2%-
2.5 mm) entrapping the cells were propagated statically in soy 3%) was produced at a residence time of 28 h,
sauce medium at 30°C for 48 h.
which is considerably shorter than the conven-
Scanning electron microscopy. The fixation and dehydration of tional fermentation period (3-4 months).
the gel beads were performed as described by Fukushima et al. It has been reported that the oxygen transfer
(1988). rate increases in proportion to the superficial vel-
Preparation of raw liquid. A mixture ofkoji, NaC1 and tap wa-
ocity of the supplied gas (Sharma and Mashelkar
ter was enzymatically hydrolysed at concentrations of NaCI 1968), so the increased ethanol production in the
below 10% and a temperature below 55 ° C in a stirred tank as higher H / D reactor might be closely related to the
described by Osaki et al. (1985). Concentrations of NaCI and oxygen transfer rate. From these results the reac-

A B
5.0
4.0
3.0 ~ O ~ o . . . ~
0 ~ 0 ~ 0 ~ 0 ~ 0 ~
~2.0 :/ O__o ~ O

u 1.0
O
0 I I I I I I I I I I
~9.0
~d
~
~m
8.0 Fig. 2. Effects of pH (A) and NaC1 (B) on alcohol
~ 7.0 "/ ~ fermentation by immobilized Z. rouxii cells in the air-
lift reactor. Conditions were as follows: (A) NaC1,
owq~ 6 . 0
~5.0
2/ ' j ' .I0 i i , , ~ ,
12.5%; temperature, 27 ° C; residence time, 21 h; gas
flow rate, air 0.08 vvm. (B) pH, 5.0; temperature,
3.0 4.0 50 6.0 7 8 10 12 14 16 18 27°C; residence time, 28 h; gas flow rate, air 0.08
pH NaCI (~) vvm. Cell number: rq, in gel; I , in fermented liquid
348 T. Hamada et al.: Soy sauce fermentation by immobilized Z. rouxii cells

B
4.0

0 ~ 0 ---------_.0..~"
3.0 /0--0--0-~_ 0 Fig. 3. Effects of temperature (A) and aeration (B)
on alcohol fermentation by immobilized Z. rouxii ceils
~ 2.o °/° ~"'~o in the airlift reactor. Conditions were as follows: (A)
pH, 5.0; NaCI, 12.5%; residence time, 28 h; gas flow
1.0 I I I I I I I I I
rate, air 0.08 vvm. (B) pH, 5.0; NaC1, 12.5%; temper-
~9.0 ature, 27°C; residence time, 28 h. Cell number: H, in
~u
w~ 0 o--O--o~.o..~.~.o_.. ~
gel; I , in fermented liquid
z~8.0 o o
~J
zj
-Jo • - I--I~II~I~l~i~ I
. ~ 7.0
oS
~6.0 0 I I I I I
2 25 30 35 40 0'02 004 oh6 o'.o8,2
0.08 0.06 0.04 0.02 0 AIR
TEMP. (*C)
AERATION (VVM)

tor with an H / D of 3.57 was used in the following Figure 3B shows the effect of the ratio of air to
studies. nitrogen in the supplied gas on fermentation by im-
mobilized Z. rouxii cells and the viable cell num-
ber in the gel and the fermented liquid. The etha-
Effects of pH, NaCI, temperature and aeration on nol content decreased gradually with increase in
alcohol fermentation the proportion of nitrogen gas. When only ni-
trogen gas was used, the ethanol content and the
In order to determine the optimum conditions for number of viable cells both in the gel and the fer-
continuous alcohol fermentation by immobilized mented liquid decreased drastically. The oxygen
Z. rouxii cells in the airlift reactor, the effects of transfer rate at different ratios of air to nitrogen in
pH, NaC1, temperature and aeration on the fer- the supplied gas were measured as shown in Ta-
mentation were investigated. High ethanol pro- ble 1. The oxygen transfer rate decreased in pro-
duction was observed over a wide p H range (3.5- portion to the partial pressure of oxygen in the
5.5) while the number of viable cells in the gel and supplied gas. These results suggest that a supply
the fermented liquid was maximal at p H 4.5-6.0 of air is necessary for vigorous alcohol fermenta-
(Fig. 2A). From these results it appears that the tion by immobilized Z. rouxii cells and that the
optimum p H was between 4.5 and 5.5. degree of fermentation is affected by the oxygen
With respect to NaCI concentration, the etha- transfer rate. It is considered that an oxygen
nol content and the number of viable cells both in transfer rate above 0.15 mmol/l per hour is
the gel and the fermented liquid decreased with
increasing concentrations of NaC1 above 10%, as
shown in Fig. 2B. However, 10%-13% NaC1 was
employed to diminish the risk of bacterial con- AERATION(VVM)
tamination at levels of NaC1 below 10%. AIR: 0.02 0.08 0,04 0.02
N2: 0.06 ,, 0 ~ 0.04~, 0.06 .
With respect to temperature, the ethanol con-
tent was maximal at 25°-30°C while the number 5.0 ' 50 ~; 28 " 21:; 28 '
RESIDENCE TIME(H)
of viable cells in the gel was maximal at 18 °- ~4.0 o%~, 9 \
27.5 ° C (Fig. 3A). From these results the optimum
temperature was considered to be 250-27.5 ° C. g3.0 i? ooO \
1 o \ I',.. r~_

~~2.o "O; ~ -~L.~qF ~^ _~ ..

/6 ~ -1,- ,-.,~ooocx:x:~oo.~
Table 1. Oxygen transfer rate in the airlift reactor 1.0 " i s

Gas flow rate (vvm) Oxygen 0 i I I I

transfer rate 10 20 30 40 50
Air Nitrogen (mmol/I/h) TIME (DAY)

0.08 0 0.78 Fig. 4. Continuous alcohol fermentation by immobilized Z.

0.04 0.04 0.38 rouxii cells in the airlift reactor. Conditions were as follows:
0.02 0.06 0.15 pH, 5.0; NaCI, 12.5%; temperautre, 27°C. 0 , ethanol; ©, glu-
cose
T. Hamada et al.: Soy sauce fermentation by immobilized Z. rouxii cells 349

1 1 1 1
1oO.~ 2 9 10 2 9 100.~ 2 1~i~ / ~2

8 3

6 5 6 5 6 5 6 5
A B C D
Fig. 5 A-D. Analysis of aroma components extracted from the fermented products. Conditions were as same as in Fig. 3B. The
concentrations of aroma components are expressed as mg/1. A. Raw liquid. B. Air 0.02 vvm and nitrogen gas 0.06 vvm. C.
Nitrogen gas 0.08 vvm. D. Conventional soy sauce. Peaks 1, isobutyl alcohol; 2, n-butyl alcohol; 3, isoamyl alcohol; 4, furfuryl
alcohol; 5, methionol; 6, 2-phenyl-ethanol; 7, 4-hydroxy-2,5-dimethyl-3(2H)-furanone; 8, 4-hydroxy-2-(or5)-ethyl-5(or2)-rnethyl-
3(2H)-furanone; 9, 4-hydroxy-5-methyl-3(2H)-furanone

needed to obtain about 3% ethanol by 28 h resi-
dence time.
The number of viable cells reached 1-4 x 108/
ml gel and 2-4 x 107/ml fermented liquid, respec-
tively, under the optimum conditions and these
numers correspond to 10-100-fold of that present
in moromi mash. It appears that the possibility of
a short period of fermentation using our method,
which would be of great value in soy sauce pro-
duction, is due to the high density of viable cells
in the gel and fermented liquid.
Figure 4 shows a profile of continuous alcohol
fermentation by immobilized Z. rouxii cells in the
airlift reactor. This alcohol fermentation contin-
ued for about 50 days without problems even if
the residence time and aeration were altered. Glu-
cose was consumed in proportion to the ethanol
produced.
Aroma component of fermented products
Figure 5 shows the amounts of aroma compo-
nents in the fermented products under varying
conditions of aeration. With supplied air (0.02
vvm), isobutyl alcohol, isoamyl alcohol and 2-
phenylethanol were present in higher concentra-
tions than when supplying only nitrogen gas, and
the aroma profiles also differed significantly from
each other. Furthermore, the content of aroma
components and aroma profiles between the
products fermented under these conditions and
the conventional soy sauce were different. In par-
ticular, 4-hydroxy-2-(or5)-ethyl-5(or2)-methyl-
3(2H)-furanone was less in the former two prod-
ucts than in the latter. However, the aroma of the
products with supplied air was similar to that of
conventional soy sauce as compared with that ob-
tained by supplying only nitrogen gas, although
some differences were observed. The aroma of the
products obtained by supplying only nitrogen gas
was not much altered from that of the raw liquid.
However, more detailed studies may be needed to
evaluate aroma production with respect to the
conditions of fermentation.
Because of the constant production of ethanol,
the airlift reactor is more suitable for the fermen-
tation of soy sauce and soy sauce-like seasoning

Fig. 6. Scanning electron micrographs of the surface of a gel bead entrapping Z. rouxii cells after 40 h (A) and 7 days (13) of
fermentation. Bar= 10 Ixm
350 T. Hamada et al. : Soy sauce fermentation by immobilized Z. rouxii cells
by immobilized Z. rouxii cells than either the plug
flow reactor or the fluidized bed reactor (data not
shown).
Scanning electron microscopy of immobilized cells
To examine the state of Z. rouxii cells in the gel
during fermentation, the gel beads were observed
by electron microscopy. Forty hours after entrap-
ment a small number of the cells were present on
the surface of the gel, but after 7 days the number
of the cells on the gel surface increased markedly,
as shown in Fig. 6. This feature of the immobil-
ized growing cells of Z. rouxii was similar to that
of Saccharomyces cerevisiae (Osumi 1986; Fuku-
shima et al. 1988).
Acknowledgements. We thank Dr. E. Ichishima for his kind ad-
vice and also Dr. M. Nagasawa, Mr. H. Kashima, Dr. M. Ki-
kuchi and Mr. K. Nagayasu for their valuable suggestions.
Thanks are also due to Dr. M. Sasaki for gas chromatographic
analysis of aroma components, Mr. S. Ushijima for the elec-
tron microscopic observations and Mrs. Y. Kuramochi for
technical assistance.
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Received 25 November, 1988/Accepted 3 April, 1989