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
®
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
472 H. Hatanaka et al.: Vitamin B~z production with hollow-fiber bioreactor

tration was 0.91 g/1 (OD57o=3.3). The concentra-

tion of vitamin B12 was 3.65 mg/l which corre-
sponded to 4.0 m g / g of dry cells. This vitamin
~.~ I ~, I I = content was ten times higher than that of P. sher-
o~ ~oo i 1ooo
manii (0.33 m g / g of dry cells) in Fig. 2.
20 ~ Since the cell concentration was very low in
~ 1o 1oo
the batch cultivation of B. methylotrophicum, the
bacterium was cultivated in the fermentor with
0,2- ~ 1 10 ~ o the filtration unit so as to remove butylate which
.T= +.o may inhibit growth. The filtration was started at
°o.1 1 ~ ]1 27 h in late-logarithmic growth phase. As shown
in Fig. 5, the bacterium grew logarithmically and
0.1- o o ~ I the final dry cell concentration at 131 h was
33.4g/1 (OD57o = 121.5). This cell concentration
z 0 - 0.0011 20 40 60 0.01 0.1 ~-
CuLtivation time [h] 5- 1
o

Fig. 3. Cultivation of P. shermanii with filtration. Symbols, see

Fig. 2 ~ 4- ~ 20
2 0.5 ~= 10
/
gradually with cell growth by controlling the
valve in Fig. 1 and it was adjusted at the maxi-
"-: 2-- 1
mum (1.4 l/h) in the late stage of the cultivation.
However, the flux decreased gradually due to 0.2 a 6 ~
o
.7 0.5
high cell concentration. Therefore, acetate and
propionate concentrations increased in the latter m ~0.2 '~
stage of the cultivation. Forty litres of fresh me- zo 0 - . I I I 0.I ~ 3.1
0 10 20 30
dium was fed during the cultivation using hollow-
fiber module. A dry cell concentration of 227 g/1 CuLtivation time [hi
(OD57o=787) and a vitamin B~2 concentration of Fig. 4. Batch cultivation of B. methylotrophicum. (O) dry cell
52 mg/1 were obtained at the end of the cultiva- weight, ( 0 ) CN-B~2 concentration, (A) accumulated CN-B12
tion. The cell and vitamin concentrations were 35- per cell, (A) butyrate concentration, (D) acetate concentra-
tion, and ( I ) methanol concentration
fold and 24-fold respectively of those in the batch
cultivation. Final vitamin B12 content was 0.23
m g / g of dry cells. The vitamin content per cell in-
creased with the growth in fed-batch culture o~" I
(Fig. 3). On the other hand, it decreased slightly p~0.5
after starting the filtration. This may be due to the ~ 0 / , 1 1 I I I I
physical stress by circulation of the broth or re- 4 100 . ~
moval of activators and precursors of the vitamin
due to the filtration.
~ lO- <
Cultivation orB. methylotrophicum
=
o

Since B. methylotrophicum is known as a good

producer of vitamin B12 (Zeikus et al. 1980), we
also cultivated the bacterium in the fermentor.
Figure 4 shows the results of the batch cultivation.
The bacterium grew logarithmically up to 10 h 0.1
and then the growth rate decreased gradually. z 0 • II I I I I I 1oo5 0.1 ~
Since B. methylotrophicum produces butylate (Zei- 0 40 80 120
kus et al. 1980), the decrease of growth rate may cultivation time [hl
be due to accumulation of the acid as shown in Fig. 5. Cultivation of B. methylotrophicum with filtration. Sym-
Fig. 4 (1.76 g/l at 30 h). The final dry cell concen- bols are the same as shown in Fig. 4
H. Hatanaka et al.: Vitamin Bt2 production with hollow-fiber bioreactor
was 37-fold that in the batch cultivation shown in
Fig. 4. Vitamin B12 concentration also increased
logarithmically with the cell growth and reached
92.5 mg/1 at 131 h. This was 1.8-times as much as
the amount obtained by the high-concentration
cultivation of P. shermanii shown in Fig. 3. The
vitamin content in the cells was 2-3 mg/g-dry cell
through the cultivation. However, the content de-
creased just after starting the filtration, which
may also partly be due to the same reason as with
P. shermanii. To replenish the filtrate, 60 1 of the
fresh medium was fed through the cultivation.
In the cultivation accompanied by the filtra-
tion (Figs. 3 and 5), a large amount of fresh me-
dium was supplied. This lowers the efficiency of
the carbon source. In the practical application,
this problem must be overcome. We are now try-
ing to reduce the replenishing medium by using
an on-line glucose analyser (Mizutani et al. 1987)
and controlling acid concentration in the fermen-
tor (Wang et al. 1988).
By applying the hollow-fiber module, we
could obtain a high concentration of vitamin B12
and cell mass. This is due to the effective removal
of inhibitory metabolites produced by cell growth.
We succeeded in the high concentration cultiva-
tion of lactic acid bacteria (Taniguchi et al. 1987a)
and Bifidobacterium longum (Taniguchi et al.
1987b) using a similar filtration system made of
ceramic filter. We also succeeded in the on-off
regulation of the gene expression from trp pro-
moter with E. coli strain carrying a recombinant
plasmid (Iijima et al. 1987). In this case, the ex-
tent of the gene expression was profoundly af-
fected by the concentration of tryptophan in the
medium. The tryptophan level in the medium was
therefore controlled by the cross-flow filtration.
To obtain cells continuously, chemostat cul-
ture is widely used. However, cell concentration is
usually low in chemostat culture, which results in
lower productivity. To improve this, we have ap-
plied the filtration system, in which the cell con-
centration could be kept at a constant value with
the aid of a turbidimeter and a pump coupled
with a microcomputer. Streptococcus cremoris was
cultivated continuously by using this apparatus at
constant cell and lactate concentrations (Wang et
al. 1988). As shown in Fig. 3, P. shermanii grew
logarithmically with the specific growth rate of
0.136h -1 for over 59 h by the removal of the
growth inhibitor. At 59 h, cell concentration was
126 g/l. From these, dry cell production rate at
59 h was calculated to be 17.1 g/1 per hour. There-
fore, if cells could be withdrawn continuously at
this rate from the fermentor using the filtration
473
system described above, we would obtain a high
production rate of the vitamin (3.9 mg/1 per
hour), which would continue for a certain period.
From the similar consideration and the data at
122 h shown in Fig. 5, 3.3 mg/1 per hour of the
vitamin would be produced with B. methyltrophi-
cum. We are trying to establish this type of bio-
reactor which can produce the vitamin contin-
uously.
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Received April 9, 1987/Accepted August 4, 1987