Optimization of Trichosporon oleaginosus cultivation paving the way toward a continuous two-stage operation for advanced biofuel feedstock production

This research focuses on optimizing the cultivation of the oleaginous yeast Trichosporon oleaginosus as part of an integrated heterotrophic (yeast)–autotrophic (microalgae) cultivation system for the sustainable generation of lipid-rich biomass to serve as feedstock for aviation and marine biofuel manufacture. The aim is to refine process parameters governing both the growth and lipogenic phases to facilitate the transition toward a continuous two-stage operation and enhance overall process efficiency and scalability. Major issue of the study is the investigation of the nitrogen source and supplementation strategy during the growth phase, along with the fine-tuning of dissolved oxygen (DO) levels to achieve optimal performance in each phase. The design of experiments (DoE) initially included diagnostic shaking-incubator trials examining different nitrogen sources (ammonium sulfate, yeast extract, and mixtures thereof) during the growth phase of the microorganism. An isonitrogenous ammonium sulfate–yeast extract mixture was identified as the most effective and economically tolerable option. Subsequently, lab-scale bioreactor experiments were conducted to evaluate the effect of DO levels during both the growth and lipogenic phases. The DoE included three 72-h trials in which DO was maintained at 10 %, 20 %, and 30 % of its saturation value throughout both phases. The growth phase in all experiments lasted 24 hours, during which nitrogen (the ammonium sulfate–yeast extract mixture) and carbon (glucose) were frequently supplemented to ensure nutrient sufficiency, while maintaining a low C/N ratio. The transition to the lipogenic phase was then triggered by effectuating complete nitrogen depletion around 24 h after the beginning and applying a concentrated glucose pulse (60 g L⁻¹), followed by fed-batch carbon feeding. The 20 % and 30 % DO yielded the highest and nearly identical biomass concentrations during the growth phase (21.2 g L⁻¹), markedly surpassing the 10 % DO trial (16.3 g L⁻¹), while the contained lipids remained below 20 % wt. in all cases, consisting mainly of structural lipids. At the end of the lipogenic phase, all trials exceeded 54 g L⁻¹ of biomass, with lipid contents averaging close to 60 %, representing highly promising preliminary outcomes. Based on these observations, a final set of experiments is currently being conducted by maintaining the less energy-demanding DO level (20 %) during the growth phase, followed by the application of either 10 % or 30 % DO during the lipogenic phase, to further refine the effect of oxygen availability on lipid productivity and overall process efficiency. All these trials were conducted to support the design and execution of the forthcoming continuous two-stage operation. Thus, a meticulous monitoring strategy was applied throughout cultivation. In particular: carbon (glucose) and nitrogen concentrations (in the form of ammonium, free amino nitrogen-FAN, and total nitrogen) were measured every two hours during growth, and at 4–8-hour intervals in the lipogenic phase. Continuous exhaust gas analysis was also performed, as this is essential for scaling up the integrated system, where the emitted CO₂ will support microalgal growth, while also providing valuable information on the metabolic activity of the yeast cells. In parallel, frequent macromolecular analyses of lipids and carbohydrates captured the metabolic profile(s) toward lipogenesis. Importantly, all collected data will also feed a hybrid mechanistic-data driven model that will further support the design and control of the continuous cultivation strategy.