Cascade Biorefinery of Chlorella vulgaris Optimized Extraction Sequencing for Sustainable Whole-Biomass Valorization

An integrated biorefinery model was developed and experimentally validated for the multiproduct valorization of Chlorella vulgaris biomass through the sequential and polarity-driven extraction of high-value bioactive fractions. The extraction of C. vulgaris biomass was based on integration of mechanical disruption with selective aqueous, ethanol-based, and alkaline extraction steps. Six different extraction sequences were applied to high-quality biomass cultivated in an indoor photobioreactor system to obtain polysaccharides (Po), pigments (Pi), and proteins (Pr). The resulting fractions were compared in terms of extraction yields, biochemical marker content, and postextraction residual biomass also characterized in term of morphology and solid state. The novelty of the work lies in a systematic comparison of all six possible extraction permutations within the polarity-driven cascade. Our results demonstrate that, among all configurations, the Po → Pi → Pr sequence provided the most balanced outcome, yielding 20.7% polysaccharides, 16.2% pigments, and 27.6% proteins. At the same time, 36% (w/w) of residual biomass was preserved, a significantly higher retention compared to the most existing strategies relying on lipid-first or fixed-sequence extraction schemes. The considerable residual biomass (36% w/w), retained after processing remains available for potential further valorization, including lipid extraction or agronomic reuse as a biostimulant. Biochemical integrity was maintained across all fractions, as confirmed by reference markers: 411.07 mg g–1 total carbohydrates (Dubois method), 392.53 mg g–1 proteins (Bradford assay), and 0.88 mg g–1 lutein (UV–Vis). Overall, the findings indicate that a rationally designed cascade biorefinery can maximize product recovery, minimize material losses, and preserve extract quality.