耐铵型产琥珀酸放线杆菌的选育及铵离子对其生长代谢的影响

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

摘要:

经硫酸二乙酯 (DES) 诱变，在含61~242 mmol/L NH4+梯度平板中，筛选到一株耐铵型突变株YZ25，该菌株在含121 mmol/L NH4+发酵培养基中，琥珀酸产量达32.68 g/L，转化率为65.4％，比出发菌提高了180.5％。进一步考察了不同形态铵盐对YZ25生长的影响，结果表明添加少量铵盐能够提高突变菌的生长速率，但当超过一定量后菌株生长受到抑制，不同铵盐对菌株的抑制程度不同，硫酸铵、碳酸氢铵、氯化铵和硝酸铵对突变株YZ25的半抑制浓度分别为：215 mmol/L、265 mmol/L、235 mmol/L、210 mmol/L。为了考察铵离子对YZ25发酵产琥珀酸的影响，在3.0 L发酵罐以氨水作为pH的调控剂发酵，结果表明在稳定期前菌株生长基本不受铵离子抑制，生物量能够达到正常水平，但是进入稳定期后铵离子抑制作用越来越明显，导致菌株生长提前结束，耗糖不完全，产酸受阻。最后结合产琥珀酸放线杆菌Actinobacillus succinogenes代谢途径分析了铵离子对菌株抑制作用的机理。

Abstract:

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 (Ki) 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.