Physiological and cultural characteristics of a succinate-producing Anaerobiospirillum succiniciproducens and a novel succinate-producing Mannheimia sp., isolated from bovine rumen, were studied to develop the biological systems for succinic acid production. Among several environmental and physiological factors affecting cell growth and end-product formation, the external supply of $CO_2$ and $H_2$ enhanced the growth of A. succiniciproducens and its succinic acid formation. Steady-state parameters obtained by the continuous cultures of A. succiniciproducens indicated that both succinic acid production and acetic acid production were found to be growth-associated and that the formation of unwanted acetic acid reduced the succinic acid yield and cell growth. Acetic acid formation could be avoided by using glycerol as a carbon source during the batch and continuous cultures. Accordingly, a high yield and productivity of succinic acid could be obtained. Inexpensive materials such as whey, wood hydrolysate, and corn steep liquor could be effectively utilized by A. succiniciproducens. A novel succinate-producing Mannheimia sp. 55E isolated from bovine rumen is a metabolically versatile bacterium capable of growing either anaerobically and aerobically. It fermented glucose to succinic, acetic, formic, and lactic acids in response to environmental conditions. Under 100% $CO_2$ condition, more succinic acid and less lactic acid were formed, and cells grew well. The culture pH did not affect the ratio of end-products formed under 100% $CO_2$condition. Whereas, under 100% $N_2$ condition, more lactic acid and less succinic acid were formed, and cells grew poor. Aerobically, Mannheimia sp. 55E produces lactic and acetic acids without exhausted $CO_2$ gas and irrespective of initial glucose concentration, which indicated that this bacterium may not have tricarboxylic acid cycle (TCA). The pckA gene encoding phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme in the central metabolic pathway of the metabolically versatile Mannheimia sp. 55E, was cloned, sequenced, and characterized. The gene encoded a 538-residue polypeptide with a calculated molecular mass of 58.8 kDa and a calculated pI of 5.03. The deduced amino acid sequence of the Mannheimia sp. 55E PEPCK was similar to those of all known ATP-dependent PEPCKs. Mannheimia sp. 55E effectively utilized whey and corn steep liquor. Consequently, the data obtained in this study provide physiological and genetic information on succinate-producing bacteria in general, and also form the basis for the economical and feasible production of succinic acid from renewable sources.
