Production of extracellular vitamin B-12 compounds from methanol by Methanosarcina barkeri

Appl Microbiol Biotechnol (1987) 26: 511 -- 516 Applied Microbiology

Biotechnology © Springer-Verlag 1987

Production of extracellular vitamin B-12 compounds from methanol by Methanosarcina barkeri

Tapan Kumar Mazumder, Naomichi Nishio, Satoshi Fukuzaki, and Shiro Nagai

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Saijo, Higashi-Hiroshima 724, Japan

Summary. Production of vitamin B-12 compounds from methanol was carried out by Metha- nosarcina barkeri Fusaro, an anaerobic methanogen. The methanogen released about 40% to 70% of corrinoids irrespective of the culture medium used. The use of cysteine instead of NaES as the sole sulphur source for cell growth led to an increase in the cobalt chloride concentration in the culture medium up to 16 times the normal (0.6 mg-I -~) without medium precipitation. This in turn resulted in an intracellular vitamin B-12 content of 5.6 rag- g dry cel l- 1, the rest being discharged into the culture supernatant; this was 87 mg-l-~, 73% of the total corrinoids after 20 repeated intermittently fed cultures and the final cell concentration was 5.8 g dry cell.l-~. Taking advantage of this, continuous production of extracellular vitamin B-12 compounds was attempted with a fixed-bed bioreactor (carrier: diatomaceous clay). At a steady state operation at space velocity of 9 to 11 d a y - ~, the concentration of the discharged corrinoid was 6.8 to 7.9 mg-1-1, hav- ing a vitamin B-12 activity of about 4 m g . l - k To- tal cell mass retained in the reactor was 39.6 g dry cell 1-reactor-k Identification of the corrinoids revealed that 19% of the total corrinoids was com- prised of the vitamin B-12 Factor III (5-hydroxybenzimidazolyl cobamide) and the remainder were mainly the base-free vitamin B-12 Factor B (cobinamide and its derivatives).

Introduction

For the industrial production of vitamin B-12 Pro- pionibacterium spp. and Pseudomonas denitrifi-

Offprint requests to: Shiro Nagai

cans have previously been cultivated on sugars. Although the methanogens, particularly the methanol fermenter Methanosarcina barkeri Fusaro are known to be potent producers of vitamin B-12 (Krzycki and Zeikus 1980; Zeikus 1983), few at- tempts have so far been made with methanogens because of the difficulties of cultivation and of attaining a high cell mass under strictly anaerobic conditions. During the course of cultivation of M. barkeri, it was found that the organism could uti- lize cysteine as the sole sulphur source instead of the commonly used inorganic sulphide, since cysteine was intracellularly hydrolysed to HzS, NH3 and pyruvate (Mazumder et al. 1986a). This suggested the possibility of mass production of the methanogen and hence of vitamin B-12 production.

Vitamin B-12 production by the methanogen may have the following advantages over those of usual B-12 producers: (1) it is easy to maintain a pure culture since methanol is the sole carbon and energy source under strictly anaerobic conditions; (2) more than 90% of methanol consumed is con- verted to methane, where vitamin B-12 plays a significant role as a coenzyme; (3) the main product, methane, does not inhibit growth, and (4) methanol is inexpensive, water miscible, neutral, re- latively stable and renewable.

With these considerations in mind, we attempted the production of vitamin B-12 compounds by M. barkeri in a methanol minimum medium containing cysteine as the sole sulphur source for growth (Mazumder et al. 1986a). This study shows that M. barkeri excretes corrinoids into the extracellular culture fluid during growth, suggesting that the continuous production of extracellular corrinoids by means of an immobilized cell system is possible. Hence, in this investigation, the production of extracellular vitamin B-12

512 T .K. Mazumder et al.: Extracellular corrinoid production by M. barkeri

compounds from methanol was carried out using M. barkeri in a fixed-bed bioreactor.

Materials and methods

Organism and culture medium. Methanosarcina barkeri, strain Fusaro (DSM 804), obtained from the Deutsche Sammlung von Mikroorganismen (G6ttingen, FRG), was used throughout this study.

All manipulations of medium and cultures were carried out under the O2-free atmosphere of N2 gas. Medium prepara- tions, culture techniques and culture conditions were the same as reported previously (Mazumder et al. 1986a). The culture medium (pH 6.4 to 6.8) contained the following materials (per liter of deionized water): imidazole, 2.72 g; K2HPO4, 0.7 g; KH2PO4, 0.45g; MgSO4.7H20, 1.0g; NH4CI, 1.0g; NaCI, 2.25 g; CaCI~.2 H20, 0.25 g; L-cysteine. HCI, 0.3 g; FeSO4.7H20, 2rag; ZnSOn.7H20, 0.6rag; H3BO3, 1.8 rag; CUC12-2 HeO, 0.06 mg; MnCI2.4 H20, 0.25 mg; Na2MoO4.2 H20, 0.18 mg; NazSeO3, 0.04 mg; CoC12.6 HzO, 0.6 rag; NiCI2.6 H20, 0.06 mg; vitamin solution (Wolin et al. 1963) without vitamin B-12, 20ml; Ti(III)-citrate (Zehnder and Wuhrmann 1976), 0.075 mM and methanol, 5 to 10 ml. This medium was a modification of the previous one (Ma- zumder et al. 1986a). Modifications were based on the growth and vitamin B-12 contents of the organism.

Batch culture. Batch culture experiments for production of vitamin B-12 were carried out in 125- or 1000-ml serum vials containing 50 ml or 100 ml of medium. Experimental vials were inoculated with 5% to 10% (vol/vol) of a late log phase culture grown in the same medium. Cultures were incubated at 37 ° C without shaking.

Fed-batch culture. Fed-batch culture of M. barkeri for accumulation of high concentration of vitamin B-12 compounds was carried out in a glass fermentor (approximately 1.2 1) equipped with pH and temperature control, a magnetic stirrer (150 rpm) and a gas outlet. Culture pH was controlled at 6.5 to 6.8. A batch culture was started with 500 ml of medium and after about 60 h of cultivation, when the gas (CH4+CO2) production ceased (that is, methanol exhaustion), a sterile, concen- trated, whole medium was added to give the same concentra- tions as in the beginning of the batch culture. In this way, manual feeding of nutrients was repeated twice a day and the culture was continued for 11 days, that is, with 20 intermittent additions (Mazumder et al. 1986b).

e )

f

Fig. 1. Schematic diagram of the fixed-bed bioreactor, a, sterile fresh medium; b, Tedlar bag filled with N2 gas; c, peristaltic pump; d, anaerobic reactor; e, gas-liquid separator; f, liquid collector; g, gas collector

Fixed-bed cell culture. A cylindrical glass column reactor (2.7 cm diameter x 17 cm length) (Fig. 1) with a working volume of 85 ml was packed with Nagao Porcell (diatomaceous clay; particle size, 4 to 14ram [diameter]; apparent density, 0.38 g/ml; true density, 2.17 g/ml; porosity, 81%; average pore diameter, 128 lim) as a support material by leaving about 57% to 60% of void space. Medium was supplied from bottom by a peristaltic pump (Decarf N-10, Taiyo, Japan) and the effluent (gas plus liquid) was discharged from top of the reactor. Tem- perature was maintained at 37°C by a hot water stream through the reactor jacket. During the continuous operation pH was not controlled.

Initially, the immobilized cell culture, supporter-packed into the reactor, was soaked in the basal medium (without methanol and cysteine) for 1 h. After removal of excess medium, the packed reactor was autoclaved at 121 °C for 20 min, made O2-free by flashing N2 and then appropriate amounts of methanol and cysteine were added to it prior to inoculation. A batch culture was carried out after inoculating with a 48-h culture of M. barkeri (50% inoculum culture, vol/vol). When gas (CHaq-CO2) production was going to cease due to methanol exhaustion, continuous culture was started by feeding fresh medium at an initial space velocity (SV=volumetric flow rate/ working volume of reactor) of 1.13 d a y - ' . By checking the gas production and effluent methanol concentration, space velocity was increased stepwise to establish the desired steady state.

Analytical procedures. Cell growth activity was monitored by gas (CH4+CO2) production and quantitatively measured by cellular protein content. Reactor performance was monitored by gas production and effluent methanol concentration. Evolved gas and methanol were determined by previous methods (Nishio et al. 1984). Cell mass was calculated from cellular protein content (60% on the basis of cell dry wt). To determine the total cell mass fixed on the support materials, the support- ers were taken out of the reactor, suspended in 1 M NaOH solution (aqueous) and heated in a boiling water bath for 15 rain. After removing the solid particles and cell debris by centrifugation (10,000g for 20 rain), protein concentration of the supernatant was determined by a dye-binding method as reported (Mazumder et al. 1986a). To measure the extent of cell lysis and/or cell leakage during culture, protein was also investigated in culture supernatant and reactor effluent.

Isolation and quantification of corrinoid. Corrinoids in the culture broth or culture supernatant and cells were extracted in the cyano form, as reported previously (Mazumder et al. 1986b). Vitamin B-12 activity of corrinoid solution was determined by using Escherichia coli 215, a vitamin B-12-requiring auxotroph, as a test organism (Kamikubo 1956) and 5-hydroxybenzimidazolyl cobamide (vitamin B-12 Factor III) cyanide as the standard.

To isolate the extracellular corrinoids in the reactor effluent, the liquid (containing few or no bacterial cells) was directly passed through an Amberlite XAD-2 (Fenton and Ro- senberg 1978; Kamikubo and Narahara 1968a) column (2.5 cm diameter x 40 cm length). After washing the column with water, the adsorbed corrinoids were eluted with methanol containing 0.01% (wt/vol) KCN. Corrinoid fractions were flash- evaporated to dryness and dissolved in a small volume of water. Corrinoids were then extracted with l-butanol and further purified and separated by paper electrophoresis on Toyo filter paper No. 527 (Toyo Roshi Ltd., Japan) with 1 N acetate buffer (pH 2.4 to 2.6) and borate buffer (0,2 M, pH 10.5 to 11) (Dellweg et al. 1956) and by ascending paper chromatography on Toyo filter paper No. 50 with solvents, water saturated 2-

T. K. Mazumder et al.: Extracellular corrinoid production by M. barkeri 513

butanol:acetic acid:5% aqueous KCN solution (100:1:0.2) and/or water saturated 2-butanol:5% aqueous KCN solution (100:0.2) (Dellweg et al. 1957).

Total corrinoid fractions were quantitatively determined as the dicyano form based on the molar extinction coeffi- cients, E367=30.4X103 M - ' . c m - ' (Giannotti 1982)and/or 6580 = 10.2x 103 M-].cm -] (Pol et al. 1982).

Chemicals. Vitamin B-12 Factor III (5-hydroxybenzimidazolyl cobamide) and vitamin B-12 Factor B (cobinamide) were purified from digestion sludges (Kamikubo and Narahara 1968b). 5,6-Dimethylbenzimidazole (DBI) was the product of Aldrich Chemical Co., USA. 5-Hydroxybenzimidazole (HBI) and Nagao Porcell were the gifts from Daicel Chemical Indus- tries Ltd., Japan and Nagao Soda Ltd., Japan, respectively. All gases (Chugoku Teisan, Japan) had a purity greater than 99.999% (vol/vol). All other chemicals were of reagent grade and obtained from commercial sources.

Results and discussion

Effect of cobalt ion concentration on vitamin B-12 production by Methanosarcina barkeri

Since Methanosarcina barkeri can grow with cysteine as sole su lphur source in place of Na2S (Ma- z u m d e r et al. 1986a), the concen t ra t ion in the me- d ium of some essential t race e lements (Co, Ni, Fe etc.) could be increased several fold by avo id ing prec ip i ta t ion with the inorgan ic sulphide. The effect o f cobal t concen t ra t ion on growth and vitamin B-12 conten t o f the m e t h a n o g e n was thus s tudied in ba t ch cultures. Vi tamin B-12 content increased with increas ing concen t ra t ions o f cobal t chlor ide in the cul ture m ed i um , reaching a maxi- m u m value o f 5.8 m g . g dry cell -1 ( 4 m g - 1 - 1 o f cul ture bro th) at 9.6 m g . 1 - 1 o f cobal t chlor ide (Fig. 2). At this cobal t concen t ra t ion cell g rowth

2 0 0

0 U +

~" 1 0 0 u

0

o/YO~'O ~ O

O ~ 3 3

,O<A/A ~- ~4~

I i f I i i i i i

2 4 6 8 i0

Cobalt chloride (mg/l)

Fig. 2. Effect of cobalt chloride on gas formation and vitamin B-12 contents of Methanosarcina barkeri Fusaro after 76-h batch culture. Culture medium (50ml) contained: L-cysteine- HCI, 0.3 g/l; methanol, 8 g/l; COC12.6 H20, between 0 and 10 mg/1. (©): gas (CH4+CO2); (0): vitamin B-12 content (mg/g dry cell); (A): vitamin B-12 concentration (mg/1 culture broth)

was only slightly inhibi ted ( < 10%) as shown by the gas (CH4-t-CO2) p roduc t i on curve. Any further increase in cobal t level drast ical ly inhibi ted cell g rowth (not shown in Fig. 2). A cobal t chloride concen t ra t ion o f 9.6 rag .1-1 m e d i u m was therefore chosen for subsequen t exper iments . Al- though suff icient gas p roduc t i on occur red at added cobal t level o f zero, B-12 content was very low (0.26 m g . g dry ce l l - l ) . This gas p roduc t i on might be due to the small a m o u n t ( < 0.03 mg- 1-1) of ca r ryover cobal t with the inocu lum culture.

M. barkeri contains two other t e t rapyr ro le c o m p o u n d s , namely , Ni -con ta in ing Fac tor F430 and Fe-conta in ing cy tochromes . For o p t i m u m vi- t amin B-J2 content it is essential to ma in ta in nickel and iron levels in the cul ture m e d i u m at the m i n i m u m (see the cul ture med ium) required for cell growth (unpub l i shed results). Dur ing active methanogenes i s , the cul ture bro th gradual ly tu rned light yel low to greenish b rown which, u p o n exposure to air, t u rned reddish. This obser- va t ion indicates that M. barkeri p r o d u c e d the reduced fo rm of corr inoids ([Co-II]-B-12) dur ing me thanogenes i s and these were conver ted to red corr inoids ([Co-III]-B-12) u p o n ox ida t ion with air.

Extracellular corrinoid production by M. barkeri

In the above exper iments it was obse rved that the cul ture supe rna tan t f luid con ta ined cons iderab le quant i t ies o f corr inoids , whereas no p ro te in was de tec ted in it, suggest ing that M. barkeri re leased corr inoids into the culture. To de te rmine whe the r

Table 1. Extracellular corrinoid production by Methanosarcina barkeri Fusaro in batch culture

Medium Dry Corrinoid in Total used cell corrinoid

(g/l) Cell Super- (rag/1 (mg/g natant culture dry cell) (mg/l) broth)

Original 0.66 1.14 0,59 1.35 medium" Modified medium b 0.77 2.16 1.23 2.90

Culture was carried out in 1000-ml serum vial containing 100 ml medium. Culture time: 55 h. No protein was detected in the culture supernatant

Medium described previously (Mazumder et al. 1976a) without resazurin

b Medium described in Materials and methods with cobalt chloride = 9.6 mg/l

514 T.K. Mazumder et al.: Extracellular corrinoid production by M. barkeri

this interesting phenomenon was due to the modification of the culture medium, batch cultures were carried out in both the original medium (Mazumder et al. 1986a) and the present modified medium (see "Materials and methods"). To avoid the possibility of cell lysis, cultures were continued up to mid-log phase of growth (55 h) when methanol still remained. Table 1 shows that the methanogen released more than 40% of corrinoids during growth, irrespective of the medium used. In neither case was protein detected in the culture supernatant, confirming that the extracellular corrinoids were due to excretion by cells rather than cell lysis. This phenomenon might be of interest from practical viewpoints.

Fed-batch culture of M. barkeri for vitamin B-12 production

tion, and gas production was resumed as soon as methanol was supplied to the culture (results not shown), indicating that the methanogen was active through the cultivation.

Corrinoid contents of cells were almost constant (5.6 to 5.8 mg- g dry cell- 1) both in the batch culture (Fig. 2) and in the fed-batch culture (Table 2), suggesting that the higher level of extracellular corrinoids in the fed-batch culture might be due to over production by the methanogen. Whether this phenomenon was due to the use of cysteine as the sulphur source cannot be ascertained from the present results, although there are reports of induction of metabolite production by S-containing compounds including cysteine (Watanabe et al. 1985). Nevertheless, such a high level of corrinoid (40% to 70%, extracellular) production from methanol or by a methanogen has not been reported previously.

To avoid the inhibitory effect of methanol on cell growth a fed-batch culture was carried out with the im[groved medium (CoCI:. 6 H20, 9.6 mg. 1-~) for attaining a high cell mass with vitamin B-12 accumulation. After 20 intermittent additions during 280 h cultivation, the total concentration of corrinoids reached as much as 120mg-1-1 (42.7 mg. 1-1 of vitamin B-12 activity by E. coli) of which more than 70% was released into the culture supernatant and the remaining 27% (5.6 mg. g dry cell-~) was found in the cell (Table 2). In this experiment, total cell mass obtained was only 5.8 g dry cell-1-1 after consumption of about 145 g. 1-1 of methanol. Culture supernatant fluid contained about 20% of total culture protein, suggesting that the extracellular corrinoids were mainly due to excretion by the cells, although a very minor fraction might be due to cell lysis during this long cultivation period (11.6 days). At each addition of medium, added methanol was completely utilized even in the final 20th addi-

Table 2. Distribution of corrinoids in cell and culture supernatant in fed-batch culture of M. barkeri Fusaro

Total corrinoid In supernatant In cell (mg/l culture broth) (rag/l) (mg/g dry cell)

120 87.6 5.6 (100%) (73%) (27%)

Culture time, 280 h (20-time intermittent fed culture); Total methanol consumed, 145 g/l; Total cell mass obtained, 5.8 g (dry cell wt)/1. Culture supernatant contained less than 20% of total culture protein. Total corrinoid had a vitamin B-12 activity of 42.7 mg/l (by E. coli 215)

Production of extracellular corrinoid in fixed-bed bioreactor

Although a high corrinoid concentration was obtained in fed-batch culture, the methanogen cell density was still very low (5.8 g dry cell.1-1 in Ta- ble 2). In order to attain a higher cell density for continuous production of extracellular vitamin B- 12 compounds and its simultaneous isolation, a fixed-bed bioreactor was considered. In fact, various solid support materials are known effectively to retain microbial cells, including methanogens (Nishio et al. 1985).

Figure 3 shows the performance of a fixed-bed bioreactor (solid supporter: diatomaceous clay) in terms of extracellular corrinoid productivity and methanol consumption rate. Continuous cultures were conducted at various space velocities (1.1 to 11.6 day-~) as shown in the figure. At the highest SV (11.6 day-l) , the reactor was operated for about 10 days, but still methanol appeared in the effluent which was undesirable from the view- point of simultaneous isolation of extracellular corrinoid, since it would pass through the Amber- lite column in the presence of methanol. The SV was therefore manually reduced, until the effluent methanol concentration was reduced to zero. Un- der these conditions, the reactor was stably operated for at least 20 days at space velocities of 11 to 9 day-~, by maintaining the effluent methanol level at zero and with almost constant levels of extracellular corrinoids (6.8 mg. 1-~) and vitamin B- 12 activity (4 mg.l-~). This range of SV (9 to 11 day-1) might be considered suitable for the pro-

T. K. Mazumder et al.: Extracellular corrinoid production by M. barkeri 515

12o 12i:i i 1 2 12 120 100 f l0 l0 tl0 tl00

~ q x 11

O[ OL 0 ," ~ 0 JO dO

0 10 20 30 40 50

Fermentation time (day)

Fig. 3. Corrinoid production by M. barkeri Fusaro in a fixed- bed bioreactor with Nagao Porcell as the support material. Methanol concentration in the fresh medium: 8g / l . ( - - ) : space velocity; (A) : methanol consumption rate; (©): corrinoid concentrat ion in the effluent; (A) : methanol concentration in the effluent; (O) : vitamin B-12 activity; (13): corrinoid productivity

duction of extracellular corrinoids. At SV=9 day - l , the methanol consumption rate, corrinoid productivity and vitamin B-12 productivity ac- counted for 72 g. I - 1. d a y - 1, 61 mg. 1-1. d a y - 1 and 36 mg- 1 - ~. d a y - 1, respectively.

Such a high corrinoid productivity with other B-12 producers has not yet been reported (Busaba et al. 1982). Very low protein level (10 to 70 mg. l -1) in the effluent (not shown in Fig. 3) and high methanol consumption rate indicated that very little cell lysis or cell leakage was taking place during the continuous operation at space time (1/SV) of 2.7 to 2.2 h.

Total cell mass retained in the reactor during 55 days of continuous operation was about 39.6 g dry cell . l-reactor-1. This cell density which was at least 150 times higher than that of chemostat cultures of methanogens (Nishio et al. 1984) was responsible for higher methanol consumption rate and vitamin B-12 productivity.

As to the types of corrinoids produced by M. barkeri in the continuous system (see Fig. 3), paper electrophoretic and paper chromatographic separation and identification revealed that vitamin B-12 Factor III consisted about 19% of the corrinoids (Table 3, experiment 1) and it was the only complete form of B-12 compound produced by the methanogen (Pol et al. 1982; Mazumder et al. 1986b). Residual corrinoids (80%) were base- free vitamin B-12 Factor B, such as cobinamide, cobyrinic acid and /o r cobinic acid, containing

Table 3. Effect of supplementat ion of exogenous base moieties to fresh medium on corrinoid formation by M. barkeri Fu- saro in continuous system

Expt. Base added (50 ~tM) Corrinoid Amount of No. with 1-amino-2- isolated corrinoid

propanol (25 I-tM) (mg/1 effluent)

1 None F I I l 1.47 B-12 ND F B 5.81 Others" 0.17 Total 7.45

2 5-hydroxybenz- F I l i 3.16 imidazole (HBI) B-12 ND

F B 4.58 Others" 0.16 Total 7.90

3 5,6-dimethylbenz- F III 0.83 imidazole (DBI) B-12 0.42

F B 1.85 Others ~' 1.48 Total 4.58

Experiments were carried out in fixed-bed bioreactor with Na- gao Porcell as the support material. Medium was supplied to the reactor at a space velocity of SV=10.8 d a y - L F I I I : 5- hydroxybenzimidazolyl cobamide; B- 12: 5,6-dimethylbenzimi- dazolyl cobamide; F B: base-free corrinoids such as cobinamide and its derivatives containing one or more carboxylic groups on the corrin nucleus; a), one or more unidentified corrinoids; ND: not detectable

one or more carboxylic groups on the corrin nucleus. Such compounds are assumed to be precur- sors of cobamide coenzyme (Bernhauer et al. 1963). There was an unidentified corrinoid (2% to 4%) which could also support the growth of E. coli 215, and was not further investigated.

Effect o f exooenous bases on corrinoid formation by M. barkeri

In order to determine whether exogenous base moieties can influence the types and amount of corrinoid formed in the continuous system, HBI or DBI (50 lxM, each) together with 1-amino-2- propanol (25 p.M) were supplemented to the fresh medium to be fed into the bioreactor at a steady state condition (SV=10.8 day- l ) . When a new steady state was established with the changed medium, corrinoids in the effluent liquid were ana- lysed. In response to HBI supplementation, vitamin B-I2 Factor III was double the amount usually obtained without any added base and Factor B fraction was reduced by about 20%, suggesting that the exogenous base could be transformed into corresponding corrinoid (Table 3, ex-

516 T.K. Mazumder et al.: Extracellular corrinoid production by M. barkeri

periment 2). Total corrinoid level remained almost unchanged which was in contrast to the previous finding that the exogenous base stimulates corrin biosynthesis (Scherer et al. 1984). Besides the naturally occurring base moiety HBI, the other base such as DBI was also transformed into the corresponding corrinoid (5,6-dimethylbenzim- idazolyl cobamide) by M. barkeri Fusaro, although the total corrinoid biosynthesis was sup- pressed by about 40% (Table 3, experiment 3), as the previous study suggested (Scherer et al. 1984). In this case, vitamin B-12 Factor III level was also reduced by about 50% when compared with that of the control experiment (Table 3, experiment 1). In both cases, the base-free corrinoids were still more than 40%. With a view to transforming these base-free corrinoids into complete forms (F III or B-12), studies with other potential microorgan- isms along with the methanogen are in progress.

Although the extracellular corrinoid concen- trations in the effluent (at SV=9 to 10.8 day -1) were low (6.8 to 7.9 mg-l-1), these corrinoids could still be conveniently and economically isolated by passing directly through an Amberlite XAD-2 column and then eluting with methanol or acetone (Fenton and Rosenberg 1978; Kamikubo and Narahara 1968a). The Amberlite column could also be used repeatedly.

The results presented in this paper show that although the fixed-bed reactor has its inherent problem of pressure drop or plugging after a long term operation, Methanosarcina barkeri Fusaro could be applied for continuous production of extracellular vitamin B-12 compounds from cheap and renewable methanol by using more suitable immobilization systems. Studies in this regard are also in progress and will be reported elsewhere.

Acknowledgements. We gratefully acknowledge the gifts of HBI and Nagao Porcell, respectively, by Daicel Chemical In- dustries Ltd., and Nagao Soda Ltd., Japan.

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Production of extracellular vitamin B-12 compounds from methanol by Methanosarcina barkeri