Optimizing the Enzymatic Hydrolysis of Microchloropsis salina Biomass for Single-Cell Oil Production

There is an increasing industrial demand for sustainable resources for lipid-based biofuels and platform chemical production. A promising, CO₂-efficient resource is autotrophically cultivated microalgae, either for direct single-cell oil (SCO) production or as a biomass substrate for fermentative SCO production via organisms like yeasts. Regarding the latter, chemical biomass hydrolysis typically results in high sugar yield and high salt concentrations due to the required neutralization prior to fermentation. In contrast, enzymatic hydrolysis is often lacking in mass efficiency. In this study, the enzymatic hydrolysis of both nutrient-replete and lipid-rich autotrophic Microchloropsis salina biomass was optimized, testing different pre-treatments and enzyme activities. Hereby, the protease treatment to weaken the cell wall integrity and the dosing of the Cellic CTec3 was identified to have the highest effect on hydrolysis efficiency. Sugar yields of 63% (nutrient-replete) and almost 100% (lipid-rich) could be achieved. The process was successfully scaled-up in mini bioreactors at a 250 mL scale. The resulting hydrolysate of the lipid-rich biomass was tested as a substrate of the oleaginous yeast Cutaneotrichosporon oleaginosus in a consumption-based acetic acid fed-batch setup. It outperformed both the model substrate and the glucose control, demonstrating the high potential of the hydrolysate as feedstock for yeast oil production. The presented sequential and circular SCO-producing value chain highlights the potential for mass- and space–time-efficient biofuel production, combining the autotrophic cultivation of oleaginous algae with decoupled yeast oil fermentation for the first time.