Addressing the critical challenge for rhamnolipid production: Discontinued synthesis in extended stationary phase

Rhamnolipids are commonly produced using N-limited Pseudomonas aeruginosa fermentations, with active production in the stationary phase. The production, however, stops after certain period. This causes lower productivity and yield because of downtime between batches and the substrate consumed for cell growth. This discontinued production is the significant problem addressed in this study. Rhamnolipid synthesis involves complex regulatory mechanisms, including quorum-sensing systems and alternative sigma factors. Current knowledge on these mechanisms, however, cannot adequately explain the discontinued rhamnolipid production at extended stationary phase. Four hypotheses to causes of this discontinued production were examined here with carefully planned fermentation designs using different initial N-source concentrations and different ways of adding oil and nutrients. Results rejected three of the hypothetical causes: accumulation of rhamnolipids or other inhibitory metabolites, presence of high amounts of oil phase, and exhaustion of non-N nutrients, and supported the hypothesis that the stopped production was caused solely by the extended N-starvation experienced by the cells. The discontinued production was found to be fully reactivated by partial broth replacements with fresh media containing N-source. The finding is important to production economics and promotes new designs to maximize rhamnolipid productivity and yield using extended stationary-phase production instead of short repeated batches.