Improvement of vitamin B12 production by anaerobic ecosystem

虽然从丙酸杆菌的细胞中经糖发酵纯化出维生素B12，但由于产生的丙酸盐和醋酸盐对其生长有很强的抑制作用，维生素B12难以获得较高的细胞浓度。以甲醇/CO2为底物生成乙酸，在细胞内具有较高的B12含量。但是醋酸也会抑制细菌的生长。因此，我们试图通过在厌氧条件下利用这些酸的微生物生态系统来改进这些细菌的培养，以获得高浓度的B12。实验结果如下：在反硝化条件下与反硝化副球菌或球形红杆菌共培养弗氏丙酸杆菌的培养。（1） .在与反硝化假单胞菌混合培养中，即使在葡萄糖存在的情况下，由于假单胞菌对丙酸盐和乙酸盐的选择性利用，B12的浓度也能增加约2倍。（2） .在与球孢霉的混合培养中，丙酸盐和乙酸盐也得到了充分利用，产生了N2。因此，B12的产量增加了4倍。但由于球孢霉对这些酸的利用不完全，其细胞浓度仍为10 g/l。乙杆菌与产甲烷菌Methanothrix sp共培养的研究(1)。虽然所使用的醋酸杆菌细胞中B12含量很高（5 mg/g细胞），但乙酸表现出较强的生长抑制作用（Kp=270 mM）。(2)由于产甲烷菌的生长速度比Acetobacterium低10倍，因此与Methanothrix sp.在单独的生物反应器系统中共培养。首先，建立了固定床的甲烷菌反应器，在反应器中填充聚丙烯/陶瓷混合物以粘附甲烷菌细胞。然后，将该反应器与醋酸杆菌的恒化培养液循环连接。采用该组合反应器系统，甲烷菌细胞消耗乙酸，可使恒化培养中乙酸水平基本为零，B12产量可达20 mg/l/d。少

Although vitamin B12 has been purified from the cells of Propionibacterim spp.after fermentation of sugars, it is difficult to get a high cell concentration due to strong growth inhibition by propionate and acetate prouced. Acetogens produce acetate from methanol/CO2 as substrates and have a high B12 content in the cells. But acetate also inhibits the bacterial growth.Therefore, we attempted to improve these bacterial cultivations to get high concentration of B12 by using microbial ecosystem which can utilize these acids under anaerobic condition. The results were as follows.1.Cultivation of Propionibacterium freudenreichii in coculture with Paracoccus denitrificans or Rhodobacter sphaeroides under denitrification.(1) .In mixed culture with P.denitrificans, B12 concentration could increase ca.2-fold due to the selective utilization of propionate and acetate by P.denitrificans even in the presence of glucose.(2) .In mixed culture with R.sphaeroides, propionate and acetate were also util … More ized and N2 was produced. Therefore, B12 production increased 4-fold. But the cell concentration of P.freudenreichii was still 10 g/l due to imcomplete utilization of these acids by R.sphaeroides.2.Cultivation of Acetobacterium sp.in coculture with a methanogen, Methanothrix sp.(1) .Although B12 content in the cells of Acetobacterium sp.used was very high (5 mg/g cell) , acetate exhibited strong growth inhibition (Kp=270 mM) .(2) Coculture with Methanothrix sp.was carried out in separate bioreactor system, since growth rate of the methanogen was 10 times lower than Acetobacterium. First, a fixed bed reactor of the methanogen was set up, where polypropyrene/ceramic mixture was packed to adhere the methanogen cells. Then, this reactor was conected to the chemostat culture of Acetobacterium with liquid recycling. By using this combined reactor system where Methanothrix cells consumed acetate, acetate level in the chemostat culture could be kept almost zero and B12 productivity reached to 20 mg/l/day. Less