Enhanced Biomass, Paramylon, and Lipids Production by Non-Axenic Cultivation of Euglena gracilis in Anaerobically Digested Livestock Wastewater

Wastewater-based microalgal cultivation enables coupling environmental remediation with the production of sustainable, value-added biomass. In this study, <i>Euglena gracilis</i> was cultivated under non-axenic conditions in a 2% anaerobically digested livestock wastewater (LSWW)-based medium to enhance biomass accumulation, paramylon storage, and biodiesel precursor production, while simultaneously removing residual nitrogen and phosphorus. The LSWW medium was strongly phosphate-limited relative to ammoniacal nitrogen (N:P mass ratio ~39:1), which constrained growth. Adjustment of the N:P ratio to ~10:1 by NaH₂PO₄ supplementation, together with MgSO₄·7H₂O addition, significantly enhanced biomass production, whereas trace metals and CaCl₂ provided minimal benefit. Cultivation at an initial pH of 3 resulted in substantially higher biomass accumulation than at pH 7 under xenic conditions. Under these optimized conditions, total phosphate and ammonia were efficiently removed, decreasing from 5.27 to 0.009 mg/L (99.8%) and from 57.40 to 2.11 mg/L (96.3%), respectively. Although paramylon accumulation was low in LSWW alone (~4% dry weight), short-term ethanol supplementation (0.095%, <i>v</i>/<i>v</i>, 24 h) enhanced paramylon content to ~20% dry weight. Subsequent anaerobic treatment further enhanced lipid conversion, increasing fatty acid methyl ester (FAME) content to ~45% dry weight. Collectively, low-pH non-axenic cultivation of <i>E. gracilis</i> in LSWW, combined with minimal nutrient supplementation, provides an integrated platform for enhanced biomass, paramylon, and biodiesel precursor production with efficient nutrient removal.