Breeding of ammonium-tolerant mutants of Actinobacillus succinogenes for succinic acid production and effect of ammonium

An ammonium-tolerant mutant of Actinobacillus succinogenes, YZ25, was obtained in the medium containing 61-242 mmol/L NH₄⁺ after DES mutagenesis. Succinic acid produced by the mutant YZ25 reached 32.68 g/L when the medium contains 50 g/L glucose and 121 mmol/L ammonium, which was increased by 180.5% compared with that of the parent strain. The effects of different ammonium salts on the growth of the mutant and its metabolic response to high ammonium concentrations were investigated. The results showed that low ammonium concentration could improve the specific growth rates of the mutants, while high ammonium concentration inhibited cell growth. The ammonia-nitrogen half-inhibition constants (K_i) for different ammonium salts were as follows: 215 mmol/L for (NH₄)₂SO₄, 265 mmol/L for NH₄HCO₃, 235 mmol/L for NH₄Cl, and 210 mmol/L for NH₄NO₃. The process of ammonium inhibition on the mutant YZ25 was investigated in 3.0 L stirred fermentor. When NH₄OH was used to buffer the pH, cell growth was not inhibited. However, production of succinic acid and consumption of glucose gradually decreased when cells entered the stationary phase, and the glucose could not be utilized completely at the end of fermentation. The possible ammonium inhibition mechanism was discussed based on the metabolic pathway of A. succinogenes.