Microalgae Path to Premium Vegetable Oil: Fermentation strategy to produce Auxenochlorella protothecoides single cell oil analogue to olive oil

Microalgae are increasingly recognized as a sustainable oil source due to unique biological and ecological advantages. This study aims to develop a scalable heterotrophic high cell density fermentation strategy for microalgae Auxenochlorella protothecoides ( AP ) to produce single-cell oil analogue to olive oil. A batch fermentation yielded a dry cell weight (DCW) concentration of 20.0 g L -1 with 18.7% (w/w) oil content at a high glucose to yeast extract (YE) ratio of 3:1 (g/g). However, the resulting oil composition differed from that of olive oil. Additionally, the specific growth rate decreased at glucose concentration above 30.0 g L -1 . To address this, a dissolved-oxygen (DO)-pH state fed-batch fermentation with controlled glucose feeding was developed, extending exponential growth and enabling tuning fatty acid composition. This approach increased DCW concentration to 80 g L -1 and oil content to 66.2%, predominantly composed of potentially high-value unsaturated fatty acids (87.3%). The fatty acid profile of AP oil varied with fermentation time. After 119 h of cultivation, AP oil consisted of oleic acid (72.85±1.17%), ω-6 linoleic acid (12.28±0.20%), ω-3 linolenic (1.46±0.02%), stearic acid (3.32±0.05%) and palmitic acid (9.45±0.14%), similar to that of virgin olive oil. The process was scaled-up using 10 L bioreactor, where AP achieved average cell biomass productivity of 0.36 g L -1 h -1 and DCW of 76.3 g L -1 , comparable to the 2 L scale. The developed process demonstrates strong potential for producing virgin olive oil-like oil by customizing microalgae fatty acid profiles, offering a promising alternative to traditional plant-derived premium oils.