Exploiting indigenous self-flocculating alkaliphilic Desmodesmus subspicatus for robust photosynthetic biogas upgrading under temperate climatic regimes

• Desmodesmus subspicatus was bioprospected for photosynthetic biogas upgrading. • Maximum biomass productivity of 83.4 mg/L/d was achieved at pH 9 and 1.5 gIC/L. • A self-flocculation of 85% was observed at high pH and alkalinity. • Self-flocculation was driven by pH, EPS biosynthesis and precipitating divalent ions. • A carbon balance resulted in the 86-89% carbon recovery at the end of the process. Photosynthetic biogas upgrading using algae presents a sustainable approach by co-generating biomethane via anaerobic digestion and valuable algal biomass. To enable application of this technology in temperate climatic conditions (with typical temperature of 5-20°C), suitable algal species should additionally grow optimally at alkaline pH (9-10) and sustain growth at high alkalinity of 1.5-2.5 grams inorganic carbon per litre (gIC/L). It should also facilitate low-cost harvesting. This study addressed these constraints by bioprospecting indigenous temperate algae and identified Desmodesmus subspicatus as a robust candidate for photosynthetic biogas upgrading. D. subspicatus tolerated pH 9-10 and alkalinity 1.5-2.5 gIC/L at 20°C in Bold's Basal Medium (BBM), thereby meeting key physiochemical requirements for this technology. The maximum biomass productivity achieved was 83.4 mg/L/d at an initial pH 9.0 and alkalinity 1.5 gIC/L, as compared to 68.4 mg/L/d at an initial pH 9.5 and alkalinity 2.5 gIC/L. However, precipitation of calcium (Ca 2+ ) and magnesium (Mg 2+ ) ions was observed at high pH and high alkalinity, limiting nutrient availability to the algae. The strain exhibited self-flocculation of 85% of cells within 3 hours due to pronounced secretion of extracellular polymeric substances (EPS), peaking at 182 mg EPS/gram biomass. Metabolite profiling showed a shift in carbon flux towards biosynthesis of carbohydrates (29-35%) at the expense of proteins under high alkalinity. Future strategies to improve the strain’s performance and process economics should address precipitation of divalent ions, employ mixotrophic cultivation, and use adaptive laboratory evolution techniques.