Integrated downstream processing of Auxenochlorella protothecoides biomass: Pilot scale implementation and biomass fractionation for protein and functional ingredients development

The growing demand for sustainable protein sources has driven interest in microalgae, particularly Auxenochlorella protothecoides , due to its high protein content and ability to utilize agro-industrial byproducts. This study presents a circular biorefinery approach integrating heterotrophic cultivation of A. protothecoides on white grape pomace (WGP) sugars with optimized high-pressure homogenization (HPH) and advanced fractionation techniques. Biomass was produced at pilot scale, achieving a yield of 0.47 g dry weight per g of reducing sugars. HPH parameters were optimized (210 MPa and six passes) to maximize cell disruption and protein solubilisation. Downstream processing enabled the separation of three main fractions: a protein-rich extract, a pellet, and a bioactive effluent. The protein extract exhibited high protein content (46%), balanced amino acid profile, improved protein digestibility (81.5%), and superior foaming and emulsifying properties compared to pea protein. The pellet contained lower protein levels (25.5%) and was enriched in minerals and pigments, while the effluent exhibited antioxidant capacity (up to 162 mg TE L -1 ) and broad-spectrum of antimicrobial activity. This integrated strategy valorises food industry byproducts, enhances protein recovery, and generates multifunctional ingredients for food applications, supporting the development of sustainable alternative proteins within a circular bioeconomy framework. • Pilot scale cultivation of A. protothecoides achieved using grape pomace sugars • Optimized HPH treatment at 210 MPa and 6 passes enhanced protein solubilization • Fractionation of the biomass led to a protein extract, pellet, and bioactive effluent • Protein extract showed high digestibility and functional properties • Effluent exhibited antioxidant and antimicrobial activities