(23d) Enhancing Lipid Production in Yarrowia Lipolytica through CO2-Derived Substrate Co-Feeding and Continuous Fermentation

Microbial biomanufacturing offers a sustainable way for producing biofuels and high-value chemicals, yet carbon loss as CO₂ during fermentation undermines yield and environmental benefits. This research introduces an innovative approach to enhance lipid production in the oleaginous yeast Yarrowia lipolytica by integrating CO₂-derived C₁/C₂ chemicals (formate and acetate) with glucose co-feeding and optimizing continuous fermentation processes. Addressing the challenge of low lipid yields from traditional glucose-based batch fermentation, we demonstrate that co-feeding acetate with glucose reduces carbon loss by 40-50%, achieving a lipid titer of 35.6 g/L and a lipid-to-cell dry weight (CDW) ratio of 71% in fed-batch trials with the wild type and engineered Y. lipolytica. To overcome labor-intensive optimization, predictive kinetic modeling was developed, forecasting optimal single-stage continuous fermentation conditions, yielding a simulated lipid productivity of 1.832 g/L/h and biomass exceeding 113 g/L. Experimental validation in the single-stage continuous fermentation confirmed enhanced biomass (up to 120 g/L) and lipid titers, with adaptive laboratory evolution (ALE) further improving lipid/CDW to 76% in screened mutants. These advancements signify a shift toward zero-carbon-release biomanufacturing, leveraging CO₂ recycling and continuous processing to boost efficiency. The methodology, grounded in metabolic engineering and process simulation, advances the state of knowledge in chemical engineering by offering a scalable, eco-friendly framework for lipid production. Ongoing work will refine two-stage continuous systems and downstream recovery, positioning this platform as a transformative solution for sustainable biofuel and chemical synthesis.