Mild One-Step Protein Recovery from Microalgae Cultivated in Swine Wastewater Using Natural Deep Eutectic Solvent-Based Aqueous Biphasic Systems

Photobioreactor-based microalgae cultivation offers an integrated approach for nutrient-rich wastewater treatment while producing valuable biomass. One of the main microalgae components is proteins, making them a biotechnological target. In this work, to develop efficient and greener extraction methodologies, aqueous two-phase systems (ATPSs) based on natural deep eutectic solvents (NADESs) were evaluated for one-step protein extraction from microalgae cultivated in swine wastewater. Six ATPSs combining two NADES-betaine:levulinic acid (Bet:2LA) and choline chloride:urea (ChCl:2Urea)-and their individual components (Bet or ChCl) with phosphate salts were compared. Systems {NADES + K₃PO₄ + water} were characterized and reported for the first time. Protein recovery yield (PRY) and selectivity (protein-to-carbohydrate mass ratio, R) were assessed for three extraction times and at room temperature. The ATPS {Bet:2LA + K₃PO₄ + H₂O} achieved a PRY of 16.4% and remarkable selectivity after 30 min (R = 2.17 g·g^-1), with proteins concentrated in the NADES-rich phase, and negligible recovery in the salt-rich phase. Although the maximum PRY (18.2% at 120 min) was achieved with the precursor betaine, the ATPS with Bet:2LA at 30 min offered an optimal balance between efficiency and process time. With a water content of up to 50%, these systems underscore the potential of NADES-based ATPSs as sustainable platforms for protein recovery.