Adaptive Laboratory Evolution of <i>Bacillus subtilis</i> 168 for Efficient Production of Surfactin Using NH₄Cl as a Nitrogen Source

<i>Bacillus subtilis</i> strain 168 is commonly used as a host to produce recombinant proteins and as a chassis for bio-based chemicals production. However, its preferred nitrogen source is organic nitrogen, which greatly increases production costs. In this study, adaptive laboratory evolution (ALE) was used to improve <i>B. subtilis</i> 168 growth using NH₄Cl as the sole nitrogen source. The cell density (OD₆₀₀) of a mutant strain LJ-3 was 208.7% higher than that of the original strain. We also optimized the metal ions in the medium and this resulted in a further increase in growth rate by 151.3%. Reintroduction of the <i>sfp+</i> gene into strain LJ-3 led to the LJ-31 clone, which restored LJ-3’s ability to synthesize surfactin. The fermentation system was optimized (C/N, aeration, pH) in a 5 L bioreactor. Dry cell weight of 7.4 g/L and surfactin concentration of 4.1 g/L were achieved using the optimized mineral salt medium after 22 h of batch fermentation with a Y_P/S value of 0.082 g/g and a Y_P/X of 0.55 g/g. HPLC analysis identified the surfactin isoforms produced by strain LJ-31 in the synthetic medium as C₁₃-surfactin 13.3%, C₁₄-surfactin 44.02%, and C₁₅-surfactin 32.79%. Hence, the variant LJ-3 isolated by ALE is a promising engineering chassis for efficient and cost-effective production of a variety of metabolites.