JOURNAL Or FERMENTATION AND BIOENGINEERING

VOI. 73, No. l, 76-78. 1992

Vitamin B12 Production by Acetobacterium sp. and Its

Tetrachloromethane-Resistant Mutants
KOICHI INOUE, t SADAO KAGEYAMA, LKATSUYA MIKI, I TSUYOSHI MORINAGA, 2 YOICHI KAMAGATA,3.
KAZUNORI NAKAMURA, 3 AnD EIICHI MIKAMI 3
Tsukuba Research Center, Daicel Chemical Industries, Ltd.. Tsukuba, lbaraki 305, ~ Research Center, Daicel
Chemical Industries, Ltd., Aboshi-ku, Himeji, Hyogo 671-12, 2 and Fermentation Research Institute,
Agency of Industrial Science and Technology, Tsukuba, lbaraki 305, 3 Japan
Received 12 September 1991/Accepted 12 October 1991

A strictly anaerobic, homoacetogenic bacterium strain 69, which produces high amounts of intraeellular
vitamin B~2, was isolated from sea sediment. Taxonomical and physiological studies revealed that the isolate
should be classified in the genus A c e t o b a c t e r i u m .
The isolate grown on methanol produced c a . 11 mg of
cyanocobalamin per gram dry cells after 7-d cultivation. Tetrachloromethane (TCM) resistant mutants, strain
69-7 and 69-23, were obtained from the isolate by ethylmethanesulfonate treatment, and produced c a . 23 mg
of cyanocobalamin per gram dry cells for strain 69-7 and 20 mg for strain 69-23 in the presence of 10 pM of

TCM.

nm by UV detector) and/or gas chromatography (column,

PEG-6000, 1.5 mm 3 m; detection, FI-detector). The
tubes showing growth were transferred to fresh medium.
After several transfers, isolation was conducted using
the roll agar tube method (10).
Of the 800 isolates, a strictly anaerobic bacterium, tentatively designated strain 69 isolated from sea sediment, was
selected and used for further experiments. Strain 69 was a
rod-shaped, Gram-positive, non-spore forming bacterium.
The mean dimensions of single cells were 0.8 by 2.5/~m.
Cells were motile by peritrichous flagella (Fig. 1). Colonies
on roll agar tubes were smooth, colorless or sometimes
slightly yellowish, and convex with a size up to 3 mm in diameter. The optimum temperature for growth was 30C.
The DNA base composition determined by HPLC (1 I) was
48 molJo guanine plus cytosine. The isolate utilized fructose, glucose, mannose, galactose, arabinose, xylose,
cellobiose, methanol, lactate, pyruvate, glycerol, formate,
and H2/CO2, but did not utilize rhamnose, lactose,
raffinose, melibiose or ethanol. The only fermentation
product from the substrates described above was acetate.
Formation of acetate from methanol coincided with the

A number of microorganisms have been considered to

be potential sources for vitamin B~2 production (1, 2). Of
the microorganisms studied, Propionibacterium freudenreichii, P. shermanii, and Pseudomonas denitrificans are
known to be high vitamin Bt2-producers (2), and the actual
production of vitamin B~2 by these organisms has been
extensively investigated (1-3). Recently, Mazumder et al.
found that Methanosarcina barkeri Fusaro, a methyltrophic methanogen, produces high amounts of corrinoids,
and the effect of culture conditions on the improvement of
productivity has been investigated (4-7). Stupperich et al.
showed that acetogenic anaerobic bacteria, such as Clostridium spp., Acetobacterium woodii, and Sporomusa
ovata form corrinoids with various types of derivatives
including unusual cobamides (8).
We also found one of the acetogens isolated from sea
sediment produced significant amounts of intracellular
vitamin Bt2 with a complete form. In the present paper, we
describe some characteristics of the isolate, and the productivity of vitamin B~2by the isolate and its alkylhalide-resistant mutants.
Approx. 0.1 g of mud samples was inoculated into test
tubes containing 5 ml of methanol medium with N2/CO2
(80/20, vol/vol) as a gas phase. The methanol medium
based on a previous report (9) contained the following
(g/0: KH2PO4, 0.685; NH4CI, 1.0; MgSO4-7H20 , 0.1;
COC12.6H20, 0.01; 5,6-dimethylbenzimidazole, 0.01;
NaHCO3, 10.0; Na2S.9H20, 0.25; cysteine-HC1, 0.5,
resazurin, 0.001; yeast extract, 0.4; methanol, 5.0 (ml);
and mineral solution, 40 (ml). The mineral solution contained (g/0: MgSO4.7H20, 3.1; MnSO4. H20, 0.28; NaC1,
0.5; FeSO4.7H20, 0.05; CaC12.2H20, 0.07; ZnSO4.7H20,
0.09; CuSO4, 0.03; A1K(SO4)2.12H20, 0.009; H3BOa, 0.005;
Na2MoO4.2H20, 0.006; NiCI2.6H20, 0.006; NaEWO42H20, 0.083; and nitrilotriacetic acid, 0.25. Enrichment
was performed at 30C for two weeks by monitoring absorbance at 600 nm spectrophotometricaily and determining fermentation products by HPLC (column, Shimadzu
SCR-101H, 7.9x300mm; detection, absorbance at 210

FIG. 1. Electron micrograph ofAcetobacterium sp. strain 69 negatively stained with uranyl acetate. Bar indicates 1 pm.

* Corresponding a u t h o r .

76

VOL. 73, 1992

NOTES

TABLE I. Production of corrinoid by Acetobacterium sp. strain

69 and its mutants 69-7 and 69-23
Strain
69
69
69-7
69-7
69-23
69-23

CC14
(/~M)
-10
-10
-10

Dry cell yield

(g//)
0.31
no growth
0.33
0.33
0.33
0.31

Cyanocobalamin
(mg/g dry cells)
11
-13
23
15
20

Organisms were cultivated with 500 ml bottles containing 300 ml of

methanol medium prepared as described in the text. Values are the
average of triplicates after 7-d cultivation.
following equation:
4CH3OH + 2CO 2 -->- 3CH3COOH + 2H20
These results indicated that the isolate should be included
in the genus A c e t o b a c t e r i u m (12).
Corrinoids in the cells were extracted by extraction
buffer (0.25% KCN in 0.5 M acetate buffer, pH 4.5: metha n o l = 4 : 6, vol/vol) based on the methods of Stupperich
et al. (13), and determined by H P L C according to the
method of Binder et al. (14) (column, Waters/2Bondapak
Cjs, 3.9 x 300 ram; carrier, water: acetic acid: 2-propanol
= 9 0 : 1 : 9 , by vol.; detection, UV-detector; absorbance
at 365 nm). Strain 69 grown on 0.5% methanol (vol/vol)
was found to produce ca. 11 mg corrinoid, detected as
cyanocobalamin, per gram dry cells intracellularly (Table
1). Other derivatives such as Factor B and Factor III were
not detected (data not shown).
Alkylhalides are known to inhibit the corrinoid enzymes
involved in methyl transfer reactions by alkylation of Co
atoms (15). We thus attempted to obtain an alkylhalide-resistant m u t a n t giving a higher corrinoid productivity than
the parent strain. We selected tetrachloromethane (TCM)
as the alkylhalide, and mutation was performed with
ethylmethanesulfonate (EMS) as the mutagen. All operations for mutation were performed in an anaerobic box
(Forma Scientific, model 1024) under an atmosphere of N2/
H2/CO 2 (90/5/5, vol/vol). Cells harvested at the late log
phase (7-d culture) were suspended in the medium, treated
with 5 m g / m l of EMS at 30C for 3 min with gentle
shaking, and spread over on an agar plate containing 0.5%
methanol and 10/~M TCM. After cultivation at 30C for
7 d under an atmosphere of N2/CO2 (80/20), colonies appeared on the agar plates with a frequency of 1.2 10 -6.
Thirty-five of the colonies were selected and inoculated
into the methanol medium containing TCM to verify T C M
resistance and to determine their corrinoid productivity.
Of the mutants, numbered from 69-1 to 69-35, strains
69-7 and 69-23 were found to produce higher amounts of
corrinoid as cyanocobalamin than the parent strain. The
productivities of corrinoid were 23 mg per gram dry cells
for strain 69-7 and 20 mg for strain 69-23 (Table 1). However, both strains produced higher amounts of corrinoid
only in the presence of 10 #M TCM (Table 1). The effect of
the T C M concentration on corrinoid production was investigated. Table 2 shows that the productivity by both
mutants was affected by the T C M concentration, with 10
/zM T C M being optimum for production.
The reason why TCM is required for the enhancement
of production is not known. Alkylhalides are generally considered to alkylate corrinoids, resulting in inhibition of corrinoid enzymes which catalyze methyl transfer reactions

77

TABLE 2. Effect of the CC14 concentration on corrinoid production in Acetobacterium sp. mutants 69-7 and 69-23
Strain
69-7
69-7
69-7
69-7
69-7
69-23
69-23
69-23
69-23
69-23

CC14
(pM)
-0.1
1
10
I00
-0.1
1
10
100

Dry cell yield

(g//)
0.28
0.30
0.28
0.28
no growth
0.25
0.30
0.30
0.30
no growth

Cyanocobalamin
(mg/g dry ceils)
6.5
13
I1
24
-9.6
12
14
23
--

Cultivation was carried out as described in Table 1. Values are the

average of triplicates.
for the metabolism of methyl compounds such as methanol (15). The mutants which we obtained in the present
work might thus overproduce corrinoid by increasing the
level of total activity of corrinoid synthesis to compensate
for the inhibitory effect of TCM. Further study will be required to obtain hyperproducing mutants in the absence of
aikylhalides and to elucidate the effect of alkylhalides on
methyl transfer reactions and corrinoid synthesis.
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INOUE ET AL.

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