Appl Microbiol Biotechnol (1988) 27:470--473
Microbiology
Production of vitamin B12 by a fermentor
with a hollow-fiber module
Hidetaka Hatanaka*, Enhao Wang, Masayuki Taniguchi, Shinji Iijima, and Takeshi Kobayashi
Department of Chemical Engineering, Faculty of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464, Japan
Summary. To improve the microbial production
of vitamin BI2, we applied a hollow-fiber module
to cultivation of the vitamin producers. By the re-
moval of growth inhibitors, very high concentra-
tions of cells and vitamin BI2 were obtained com-
paring to the batch culture. We obtained 227 g dry
cells/1 and 52 mg vitamin Bl2/l with Propionibac-
terium shermanii and 33.4 g dry cell/l and 92.5 mg
vitamin B12/1 with Butyribacterium methylotrophi-
cure by this cultivation.
Introduction
Vitamin B~2 is an important cofactor for metabol-
ism of carbohydrates, lipids, amino acids and nu-
cleic acids. The vitamin is thus an important addi-
tive in animal feeds. Vitamin BI2 is also used in
chemotherapy, especially against pernicious
anaemia. Up to now vitamin B12 has been pro-
duced by fermentation on an industrial scale,
since chemical synthesis of the vitamin is very dif-
ficult (Florent 1986). For the microbial produc-
tion of the vitamin, Rhodopseudomonas (Florent
1986), Propionibacterium (Yongsmith and Api-
raktivongse 1983), Butyribacterium (Zeikus et al.
1980), Pseudomonas (Florent 1986) and Methano-
sarcina (Mazumder et al. 1986) were used and
cultivated in batch or fed-batch system. By these
fermentations, relatively low concentration of the
vitamin was produced.
High concentration cultivation seemed likely
to be the most effective means of achieving higher
* On leave from Morita Co. Kosugaya, Tokoname-shi, Ai-
chi-ken, 479, Japan
Offprint requests to: T. Kobayashi
Applied
Biotechnology
© Springer-Verlag 1988
productivity because vitamin B12 usually accumu-
lates in the cells. We tried high concentration cul-
tivation (Yamauchi et al. 1983; Mori et al. 1981)
and have also recently established a cultivation
system with a filtration unit. The latter was very
effective for the high concentration cultivation of
the microorganisms that themselves produce a
growth inhibitor (Taniguchi et al. 1987a, b). In
the case of the vitamin producers we used, or-
ganic acids such as propionate and butylate were
produced, which inhibited bacterial growth
(Nanba et al. 1983). Therefore, we applied a culti-
vation system with a hollow-fiber module. The
apparatus enabled the metabolites which inhi-
bited the cell growth to be removed and high con-
centrations of cell mass and vitamin B12 to be ob-
tained.
Materials and methods
Microorganisms. Propionibacterium shermanii PZ-3 and Escher-
ichia coli 215 were kindly provided by Drs. A. Nanba and K.
Sato of Hiroshima University, respectively. Butyribacteriurn
methylotrophicum (ATCC33266) (Zeikus et al. 1980) was ob-
tained from American Type Culture Collection.
Batch cultivation. P. shermanii was cultivated in a tube con-
taining 10 ml of a medium at 30 o C for 24 h. The medium con-
tained 1% peptone, 0.5% yeast extract, 0.05% KzHPO4,
0.0015% COSO4 • 7 H 2 0 , 2% glucose, pH 6.5. The seed culture
was then transferred to a jar-fermentor (working volume
600 ml) containing the same medium (except that it had 3%
glucose) under N2-gas atmosphere. The fermentor was agi-
tated slowly (100 rpm) and pH was controlled at 6.5 by adding
14% w / v of ammonia solution.
B. methylotrophicum was cultivated in the medium containing
2% peptone, 0.6% yeast extract, 0.09% N H n P O 4 , 0.15%
K2HPO4, 0.003% FeSO4- 0.0006% COC12 • 6H20, 0.0002% re-
sazurin, 0.45% Na2CO3, 9 ml/l of the trace mineral solution
(Zeikus et al. 1979), 5 ml/1 of the vitamin solution (Wolin et al.
1963), pH 7.5. After autoclaving the medium, methanol, cys-
H. Hatanaka et al.: Vitamin BI2 production with hollow-fiber bioreactor
teine. HCI and Na2S-9H20 were added to final concentra-
tions of 1%, 0.025% and 0.025%, respectively. The bacterium
was cultivated in a tube with a stopper containing 7 ml of the
medium at 37 ° C for 24-48 h, then the bacterium was inocu-
lated to the 200 ml of the medium in a glass bottle with a tight
cap and cultivated at 37°C for 24-28 h. Then, a 100 ml of this
seed culture was transferred to the fermentor containing
600 ml of the medium and the bacterium was cultivated at
37°C and pH 7.5 under N2-gas atmosphere. Since B. methylo-
trophicum is obligate anaerobe, the bacterium was cultivated
under a strict anaerobic condition.
High concentration cultivation with filtration. Figure 1 shows
the schematic diagram of the cultivation system with a hollow-
fiber module (2) (Microza PW103 made of polyolefin, Asahi
Kasei Co., fiber inner diameter: 0.7 mm, 400 fibers, effective
filtration area: 0.2 m 2, pore size: 0.1 Ixm). Before use, the fiber
was treated with 3% formalin, then with 0.3-1% sodium hy-
pochlorite solution and washed with sufficient autoclaved hot
water at about 80 ° C. The broth was circulated with a roller
pump (7) (Tokyo Rika Kikai Co., RP-60). The filtration flux
was controlled with the valve (8). The fresh medium was reple-
nished with the pump (6) by the aid of a level controller (4).
For the cultivation of B. methylotrophicum, NazS - 9H20 was
omitted from the replenishment medium and only 0.075% cys-
teine was added as a reducing agent.
Analyses. Methanol, acetate, propionate and butyrate concen-
trations were measured by a gas-chromatograph with Chro-
mosorb 101 after removal of the cell from the broth. Glucose
was measured by the method of Somogyi and Nelson (Nelson
1944). Vitamin B12 was determined by a bio-assay with E. coil
215 after the extraction of the broth with K C N (Sato 1983).
The optical density of the broth at 570 n m (OD57o) was mea-
sured by a spectrophotometer and the values were multiplied
by a conversion factor to obtain dry cell weight per liter of
broth. The conversion factor was 0.289 for P. shermanii and
0.275 for B. methylotrophicum. For the measurement of the op-
tical density, B. methylotrophicurn cells were washed with 0.9%
NaCI solution and suspended in the same solution to remove
resazurin which hampered the spectrophotometric measure-
ment.
Results and discussion
Cultivation of Propionibacterium shermanii
Figure 2 shows the results of the batch cultivation
of P. shermanii. The bacterium grew logarithmi-
cally with a specific growth rate of 0.138 h -~ for
initial 25 h, then the growth rate decreased grad-
ually, possibly due to growth inhibition by meta-
bolites and 6.5 g dry cells/1 was finally obtained.
The final vitamin B12 concentration was 2.14 rag/
1, which corresponded to 0.33 m g / g dry cells. The
concentrations of propionate and acetate in-
creased with cell growth and reached 8.4 and
3.4 g/1 at 50 h, respectively (Fig. 2). The ratio of
these acids (acetate/propionate) was 0.41, which
was the similar ratio reported previously (Nanba
et al. 1983).
471
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I-
Fig. 1. Schematic diagram of the fermentor equipped with hol-
low-fiber module. 1: fermentor (1 1), 2: hollow-fiber module,
3: fresh medium, 4: level controller, 5: pH-controller, 6: feed
pump, 7: circulation pump, 8: valve
0.4- 10 100
4O
? o.~-
~0 o°
_
1 +- c
o 0
°.,
5"P" o - o O.Ol o1
0 20 40
CuLtivation time [hi
Fig. 2. Batch cultivation of P. shermanii. (O) dry cell weight,
( 0 ) CN-B12 concentration, (/x) accumulated CN-B~2 per cell,
( • ) propionate concentration, ([]) acetate concentration and
( • ) glucose concentration
In the batch cultivation, we attained some pro-
duction of vitamin B12, however, the growth stop-
ped at a low cell concentration. Since vitamin B12
accumulated in the cells, high concentration culti-
vation is expected to improve the productivity as
described previously (Iijima et al. 1987; Tanigu-
chi et al. 1987a, b). We applied the cultivation sys-
tem with a hollow-fiber module to remove acetate
and propionate that may be major growth inhibi-
tors (Nanba et al. 1983) and to attain high cell
concentration. Figure 3 shows the cultivation re-
sults with the hollow-fiber module. The filtration
was started at 27 h before the growth inhibition
became evident. By the removal of the medium
containing metabolites, the cells grew logarithmi-
cally up to 59 h with the specific growth rate of
0.136h -~ which was substantially the same as
that obtained with the initial part of the batch cul-
ture (Fig. 2). The filtration flux was increased