JOURNAL OF BIOSCIENCE

Vol. 87, No. 6, 825-827.

AND BIOENGINEERING
1999

Effect of Soybean Oil on Oxygen Transfer in the Production of

Tetracycline with an Airlift Bioreactor
SHIRU JIA, GUIBIN CHEN, PRIHARDI

KAHAR,2 DU BOK CHOL3

AND

MITSUYASU

OKABEZ*

Department of Biochemical Engineering, Tianjin University of Light Industry, 300222 Tianjin, China, Laboratory
of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka
University, 836 Ohya, Shizuoka 422-8529, Japan,2 and Institute of Life Science, Chosun
University, 375 Seo-Suk Dong, Dong-Gu, Kwang-Ju, South Korea3
Received 14 January 1999/Accepted 15 March 1999

Corn starch and soybean oil are suitable carbon sources for the production of tetracycline by Streptomyces
aureofacience CG-1. However, it could not produce more than 6 g/l of tetracycline even if initial corn starch
concentration was increased to more than 100 g/Z. It was confirmed by shaking flask experiments that the kLa
in a mixture of 296 soybean oil in water was four folds compared with that without soybean oil. With the
addition of soybean oil to the starch medium in a shaking flask, tetracycline production was significantly improved. By scaling-up to a 55Zairiift bioreactor from SOO-mlErlenmeyer flask, more than 10 gNof tetracycline
was produced with the addition of 60 g/Z of soybean oil to the medium contsining 100 g/Z of corn starch. The
dissolved oxygen level in the airlift bioreactor containing soybean oil was higher than that without soybean oil.
This suggeststhat soybean oil is not only a suitable carbon source but is also a surface-active agent which may
accelerate the oxygen transfer. This may lead to the possibility of the enhanced production of tetracycline at a
low cost in airlift bioreactor.
[Key words: tetracycline, soybean oil, Streptomycesaureofaciens, airlift bioreactor]

Tetracycline (TC), which is a broad-spectrum antibiotic, can inhibit the growth of gram-positive and gramnegative bacteria, some kinds of Rickettsia, viruses and
protozoa. Moreover, TC can be used as an intermediate
and synthesised into some excellent semi-synthetic derivatives by modifying the chemical structure. Therefore, TC
has a vast scope of use. We are attempting to confirm
the possibility of enhancing the production of TC using
an airlift bioreactor, because airlift bioreactors have
many economical implications in regard to reactor construction, maintenance and scaling-up as previously
reported (1, 2). However, the ability of airlift bioreactors to supply oxygen is generally lower than that of conventional type of bioreactors, i.e., aeration and stirred
tank bioreactors. Therefore, it is necessary to determine
the optimal culture conditions suitable for the airlift bioreactor as previously reported.
In this study, the medium and culture conditions were
designed so that TC may be produced efficiently at low
oxygen supply conditions in a 500-ml Erlenmeyer flask,
followed by scaling-up to 5.5-l airlift bioreactor (3).
The TC-producing strain used in this study was Streptomyces aureofaciens CC-l. For production of TC, the
following media were used. (a) Slant medium (g/l distilled water): bran, 36; MgS04.7H20, 0.1; KH2P04, 0.3;
agar, 20; (b) seed medium (g/l tap water): soybean meal,
20; corn starch, 40; peptone, 5; yeast extract, 5; CaC03,
4; (NH4)2S04, 3; NaBr, 2; MgS04. 7H20, 0.25; KH2P04,
0.2; and (c) production medium (g/l tap water): soybean
meal, 30; corn starch, 100; yeast extract, 2.5; peptone,
15; CaC03, 7; (NH4)2S04, 2.5; NaBr, 1.5; MgS04.7H20,
0.25. These media were sterilized at 121C for 30min.
In the case of using corn starch as a carbon source, the
corn starch was partially hydrolyzed by the addition of
0.2% cY-amylase (HS, NAGASE Biochem. Ind. Ltd.,
* Corresponding

Kyoto) at 95C for 40 min.

One loopful of Streptomyces aureofaciens CC-1 was
transferred to the slant medium and cultured at 31C
for 5 d. Then one loopful of the slant culture of Streptomyces aureofaciens was inoculated into a 500-ml Erlenmeyer flask containing 40ml of the seed medium and
cultivated at 31C for 17 h on a reciprocating shaker
at 150rpm. For the production of TC, 10% of the seed
culture was inoculated into a 500~ml Erlenmeyer flask containing 40ml of the production medium, and to a 5.5-l
airlift bioreactor containing 4 I of the production medium. The schematic structure of the 5.5-l airlift bioreactor was previously reported (3). Cultivation in the Erlenmeyer flask was carried out under the above-mentioned
conditions for 144 h. The airlift bioreactor was operated
at 31C for 144 h. The aeration rate during the cultivation in the airlift bioreactor was fixed at 2 vvm. Since
this aeration rate was such that the working liquid
volume could be decreased due to evaporation, the culture volume was maintained at 4 I by the addition of
sterilised water to the reactor using a level sensor. The
dissolved oxygen concentration in the airlift bioreactor
was measured with a DO meter (Tianjin University of
Light Industry, Tianjin, China). The TC concentration
was determined by a spectrophotometric method (4).
The concentrations of cells and the carbon sources were
measured as described previously (5). The volumetric oxygen transfer coefficient, kLa was measured by the method
of Linek and Vacek (6).
In generally, corn starch is often used as the sole
carbon source for the production of various kinds of
antibiotics. Table 1 shows the effect of initial concentration
of corn starch on TC production in a shaking 500-ml
Erlenmeyer flask. TC production was the highest at an
initial corn starch concentration of 100 g/l. At concentrations higher than lOOg/l of corn starch, the TC production was rapidly decreased, possibly due to limitation of

author.

825

826

JIA ET AL.

TABLE

J.

BIOSCI.

BIOENG.,

1. Effect of initial concentration of starch as the sole carbon

source on the production of TC

Starch (g/f)
Tetracycline (g/f)
Yield from starch (%)

0
0.8
-

40
3.0
7.6

60
5.5
9.2

80
5.8
7.3

100
6.1
6.1

120
5.0
4.2

TABLE 2. Effect of the addition of soybean oil to the production

medium containing 100 g/l of corn starch on the production of TC
0 20 30 40 50 60 80 100
Soybean oil (g/l)
Tetracycline (g/[)
6.1 7.2 8.5 9.0 10.0 10.8 9.2 6.4
Yield from soybean oil (%) - 36.2 28.2 22.5 19.9 20.0 11.5 6.4

10

20

30

40

Soybean oil (g/O

FIG. 1. Effect of addition of soybean oil on the oxygen transfer
coefficient, k~a in a 500-ml Erlenmeyer flask with water.

the oxygen supply as viscosity of the culture broth increased. Therefore, it is very difficult to increase TC
production only by increasing the initial concentration
of corn starch. We have previously reported that addition of perfluorocarbon or n-dodecane efficiently improved the oxygen transfer rate coefficient, thereby eventually increasing the yield of yeast (3). However, it is
very difficult to use them for the commercial production
of TC, since they are expensive. Therefore, we also attempted to confirm whether soybean oil, which is often
used as a carbon source for the production of various
kinds of antibiotics (7-9), can improve the oxygen transfer coefficient, kLa, as well as the aforementioned in a
water-air system in 500-ml shaking flask experiments.
As clearly shown in Fig. 1, soybean oil can significantly enhance kLa. In this model experiment, the kLa value
was the highest when 2Og/l of soybean oil was added
to a 500-ml Erlenmeyer flask with water. However, it
decreased rapidly when more that 20 g/Z of soybean oil
121
10.

OJ

1.01
=
3
m

1.0-

-70
-60

loo-

0.8.

0.0'

oJ L
0

120-

24

48

72

%
120
Time (h)

0.d

144

96 120 144
Time (h)

of TC in a shaking flask without (a) and with the addition of 60 g/l of soybean oil (b).

IO-

(b)

(a)

(b)

1201

FIG. 2. Time courses of production

12-

added. Although the reason why the kLa was decreased

at concentrations higher than 20 g/l of soybean oil in
this model experiment is not clear yet; however, it was
confirmed that soybean oil can increase the kLa if added
at an appropriate concentration.
In order to increase the TC production to higher than
6 g/l, soybean oil was added to the production medium
containing lOOg/l of corn starch in the shaking 500-ml
Erlenmeyer flask. As clearly shown in Table 2, TC production was significantly increased to more than 10 g/l
with the addition of 6Og/l of soybean oil. However,
the addition of more than 60 g/l of soybean oil rather led
to a decrease in the TC production. Figure 2 shows the
time-course of TC production when 60 g/l of soybean oil
was added to the production medium containing 100 g/l
of corn starch (b) and without addition of soybean oil (a).

0
Time (h)

Time (h)

FIG. 3. Time-courses of production of TC in a 5.5 I-airlift bioreactor without (a) and with the addition of 60 g/i of soybean oil (b).

VOL. 87, 1999

NOTES

As clearly from the figure, 45 g/l of soybean oil was efficiently consumed, suggesting that soybean oil might be a
suitable carbon source for the growth of Streptomyces
aureofaciens CG-1 and production of TC; TC production was higher in medium with soybean oil than in that
without it (a). Since we can increase TC production to
more than log/l by adding soybean oil in a shaking
culture in a WO-ml Erlenmeyer flask, we attempted then
to reproduce the data in a 5.5 f-airlift bioreactor.
As shown in Fig. 3, although the DO concentration
decreased rapidly with increasing cell concentration, it
was maintained at higher than 10% from the first 48 h to
96 h (b), resulting in a higher TC production more than
log/l at 144 h. The final soybean oil concentration was
less than 2Og/l, suggesting that the soybean oil was consumed as rapidly as the corn starch. On the other hand,
when soybean oil was not added, the DO rapidly
decreased to nearly zero at 24 h (a), resulting in a lower
TC production (6.2g/l). Thus, a higher TC production
of more than log/l obtained from the Erlenmeyer flask
cultivation was successfully reproduced in the 5.5-I airlift
bioreactor by adding soybean oil as an enhancer of oxygen transfer and a carbon source.
REFERENCES
1. Yahiro, K., Takahama, T, Jia, S. R., Park, Y. S., and Okabe,
M.: Comparison of airlift and stirred tank reactors for itaconic

827

acid production by Aspergillus terreus. Biotechnol. Lett., 19,

619-621 (1997).
Choi, D. B., Tamnra, S., Park, Y. S., and Okabe, M.: Improvement of tylosin production from Streptomyces fradiae
culture by decreasing the apparent viscosity in an airlift bioreactor. J. Ferment. Bioeng., 86, 413-417 (1998).
Jia, S. R., Li, B., Park, Y. S., and Okabe, M.: Enhanced oxygen transfer in tower bioreactor on addition of liquid hydrocarbons. J. Ferment. Bioeng., 82, 191-193 (19%).
Sahav, A. J.: J. Assoc. Anal. Chem., 70, 686 (1973).
Choi, D. B., Tamura, S., Park, Y. S., Okabe, M., Seriue, Y.,
and Takeda, S.: Efficient tylosin production from Streptomyces
fradia using rapeseed oil. J. Ferment. Bioeng., 82, 183-186
(1996).
Linek, V. and Vacek, V.: Chemical engineering use of catalyzed sulfite oxidation kinetics for the determination of mass
transfer coefficient by dynamic model moment analysis.
Biotechnol. Bioeng., 60, 835-965 (1977).
Eiki, H., Gushima, H., Saito, T., Ishida, H., Oka, Y., and
Osoao, T.: Production inhibition and its removal on josamycin
fermentation by Streptomyces narnbonensis var. josamyceticus.
J. Ferment. Technol., 66, 559-565 (1988).
Ohta, N., Park, Y. S., Yahiro, K., and Okabe, M.: Comparison of neomycin production from Streptomyca fradiae cultivation using soybean oil as the sole carbon source in an airlift
bioreactor and a stirred-tank reactor. J. Ferment. Bioeng., 79,
443-448 (1995).
Park, Y. S., Momose, I., Tsunoda, K., and Okabe, M.: Enhancement of cephamycin C production using soybean oil as
the sole carbon source. Appl. Microbial. Biotechnol., 40, 773779 (1994).