Exploiting the ability of Bacillus subtilis to synthetize surfactin under oxygen limitation

Surfactin production by Bacillus subtilis is typically performed under aerobic conditions, requiring high aeration and agitation, which leads to mechanical stress for the cells and therefore promotes excessive foaming. As a process-oriented alternative to full aerobic operation, an aerobic to micro-aerobic switching strategy was developed that aims to decouple biomass formation from surfactin synthesis by controlling oxygen availability. Promoter activities relevant to nitrate respiration and surfactin biosynthesis were first characterised in shake flasks using transcriptional reporter strains and online monitoring of dissolved oxygen. The nitrite reductase promoter showed the strongest induction under oxygen-limited conditions and was used to construct an oxygen-responsive production strain in which the native P_srfA promoter was replaced to suppress surfactin formation during aerobic growth and shift production to the micro-aerobic phase. The switching concept was subsequently evaluated in 30-L stirred-tank bioreactor cultivations using stepwise reductions of dissolved oxygen setpoints combined with exponential glucose feeding and nitrate supplementation. The engineered strain B. subtilis MG19 enabled stable transitions into micro-aerobic operation without apparent loss of biomass, while a reference strain B. subtilis MG17 with the native regulation showed surfactin formation already during the aerobic phase and pronounced process disturbance after switching, characterised by glucose accumulation and a strong decrease in surfactin concentration. Overall, the study demonstrates oxygen switching as a scalable process engineering tool for controlling surfactin production phases and highlights key constraints for robust micro-aerobic bioreactor operation. KEY POINTS: • Oxygen availability decoupled growth and surfactin formation. • Micro-aerobic process control was evaluated in 30-L bioreactor cultivations. • The engineered strain shifted surfactin production to oxygen-limited conditions.