Blautia coccoides GA-1的传代培养及其同化H_2/CO_2产乙酸特征

Sub-culturing of novel Blautia coccoides GA-1 and acetate synthesis from H_2/CO_2 in subcultures

为了解同型产乙酸菌异养代谢与自养代谢的相互作用与机制,并为快速获得具有较强自养代谢能力的菌体细胞提供培养方法,以H_2/CO_2和(或)葡萄糖为碳源,考察了Blautia coccoides GA-1在连续传代培养中的代谢特征。结果表明,以H_2/CO_2作为唯一碳源进行连续传代培养时,菌株GA-1长势较弱,其子代的自养代谢能力也逐渐下降;在葡萄糖培养基中,菌株GA-1增殖旺盛,但高浓度的葡萄糖对其自养代谢能力有显著抑制作用,这种抑制作用可能是自养代谢和异养代谢对辅酶A和ATP的竞争、酸性环境造成的代谢抑制以及辅酶I的氧化还原平衡调节等综合作用的结果。以体积比为4:1的H_2/CO_2混合气为气相条件,用200 mg·L-1葡萄糖培养基对菌株GA-1进行传代培养,不仅可获得稳定的子代培养物,而且可以将其利用H_2/CO_2产乙酸的能力维持在2.16g乙酸·(g干细胞)-1的水平。

Though homoacetogens have been defined as autotrophic anaerobes using acetyl-Co A Wood-Ljungdahl carbon fixation pathway（WLP） for energy conservation, acetate production and biomass formation from CO_2, these anaerobes can also utilize organic compounds heterotrophically to produce acetate and form biomass by Embden-Meyerhof-Parnas（EMP） glycolysis. A better understanding of the interrelationship between autotrophy and heterotrophy in homoacetogens will be helpful for acetate biosynthesis from exhaust rich in CO_2. However, interaction of the two metabolisms has not been well understood though both of which have been extensively investigated respectively. Blautia coccoides GA-1, a novel homoacetogen strain obtained in a previous study, was introduced to the present research and the interrelationship between autotrophy and heterotrophy in the strain was investigated by sub-culturing with glucose and（or） H_2/CO_2（vol, 4：1） as carbon source（s）. The results showed that the cell production of strain GA-1 in H_2/CO_2 cultures was too low to be sub-cultured after the 5th generation due to the energy produced was very limited in WLP. On the contrary, the strain could grow very well in glucose cultures and could be sub-cultured steadily. However, the autotrophic capability of the subcultures decreased by generations, even lost since the 7th generation in 1000 mg·L^（-1） glucose cultures. This may be resulted in：（1） the competition for Coenzyme A（Co A） between the autotrophic WLP and the heterotrophic EMP glycolysis,（2） ATP produced in the strain used up for biosynthesis rather for acetate synthesis from H_2/CO_2,（3） inhibition of lower p H less than 5.5 to the activity of hydrogenase,（4） negative effect of the lower p H on intracellular balance of NADH/NAD＋. In mixotrophic cultures with H_2/CO_2 and 200 mg·L-1 glucose as co-carbon sources, strain GA-1 could not only be sub-cultured steadily but also keep autotrophic activity well in the generations with a specific acetate production of about 2.16 g·（g dry cell）-1 from H_2/CO_2.