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

Summary Production of vitamin B-12 compounds from methanol was carried out by Methanosarcina 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 Na₂S 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·l^-1) without medium precipitation. This in turn resulted in an intracellular vitamin B-12 content of 5.6 mg·g dry cell^-1, the rest being discharged into the culture supernatant; this was 87 mg·l^-1, 73% of the total corrinoids after 20 repeated intermittently fed cultures and the final cell concentration was 5.8 g dry cell·l^-1. 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 day^-1, the concentration of the discharged corrinoid was 6.8 to 7.9 mg·l^-1, having a vitamin B-12 activity of about 4 mg·l^-1. Total cell mass retained in the reactor was 39.6 g dry cell l-reactor^-1. Identification of the corrinoids revealed that 19% of the total corrinoids was comprised 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).