High-throughput screening coupled with laboratory adaptive evolution to enhance acid-tolerance and succinic acid production of Actinobacillus succinogenes.

The development of efficient bio-based succinic acid production technology holds significant importance for reducing carbon emissions and advancing the industrial production of biodegradable plastics. Actinobacillus succinogenes is a natural high-yield succinic acid producer. However, its fermentation is limited by low acid tolerance and suboptimal productivity. To address these challenges, this study employed laboratory adaptive evolution to isolate acid-tolerant A. succinogenes strains capable of synthesizing elevated levels of succinic acid. A pH indicator-based high-throughput screening method was developed and successfully applied to facilitate the evolution of A. succinogenes. After 561 days of low-pH adaptation and high-throughput screening, strains with varying degrees of acid tolerance and enhanced succinic acid production were obtained. Among them, the optimal strain, A. succinogenes S5-79, exhibited robust growth and superior succinic acid production performance at pH 5.8. Furthermore, optimization of the fermentation medium and neutralizing reagent significantly improved the succinic acid yield. In fed-batch fermentation using a 5  L bioreactor with the optimized medium, S5-79 produced 108  g/L of succinic acid with a yield of 0.82  g/g at pH 5.8, representing increases of 87.8 % and 74.5 % over the wild-type strain at pH 6.8, respectively. The evolved strain also demonstrated a markedly accelerated production rate, achieving 100  g/L of succinic acid within 41  h. By enabling efficient succinic acid production at low pH, the evolved A. succinogenes strains developed herein provide a feasible approach to lowering process costs and carbon emissions, accelerating the industrial adoption of bio-based succinic acid for biodegradable plastic production.