Isolation of NH₄+-tolerant mutants of Actinobacillus succinogenes for succinic acid production by continuous selection.

Actinobacillus succinogenes, a typical succinic acid producing microorganism, was inhibited seriously by ammonium ion, which hampered industrialization of A. succinogenes with ammonium ion based material as the pH controller. In this study, we have isolated an ammonium ion-tolerant mutant of A. succinogenes by continuous-culture technique in which all environmental factors beside the stress (ammonium ion) were maintained constant. In this technique, the mutant-generating system was not operated as a nutrient-limited chemostat, but as a nutrient-unlimited system where cells were continuous cultured at the maximum specific growth rate. Mutants were isolated on agar plates containing a acid-base indicator bromothymol blue and high level of ammonium ion which gives 100% killing of the parent strain. When cultured in anaerobic bottles at ammonium ion concentration of 354 mmol/L, the mutant YZ0819 could produce succinic acid 40.21 g/L with yield 80.4%, while the parent strain NJ113 did not grow. With NH4OH being used to buffer the culture pH in 3.0 liter stirred bioreactor, YZ0819 produced 35.15 g/L succinic acid with yield 70.3%, 155% higher than that obtained by NJ113. In addition, the morphology of YZ0819 in fermentation broths was changed. Cells of YZ0819 were aggregated from the beginning to the end of fermentation. These results indicate that YZ0819 would be used to be the potential strain produced efficiently succinic acid with NH4OH as the pH controller and the formation of aggregates may be useful as an aid in the transferring of cells from a cultivation medium for various industrial applications.