Journal of Biology and Nature

The rising demand for sustainable bioresources has prompted interest in wastewater-based microalgal cultivation for bioactive compound production. In this study, municipal wastewater (MWW) was evaluated as a sole culture medium for biomass production by the double mutant Chlorococcum humicola MCH4 following ultrasonic pretreatment. Response Surface Methodology (RSM) based on a Central Composite Design (CCD) was employed to optimize ultrasonic intensity (0.2-0.5 W/mL) and exposure time (10–30 min). The model was statistically significant (p = 0.05), with high determination coefficients (R² = 99.1%, R²adj = 98.4%). Optimal pretreatment conditions were identified at 0.35 W/mL for 20 min, resulting in a maximal biomass yield of 3.98 g/L, compared to 1.76 g/ in untreated 50% MWW and 1.32 g L⁻¹ in BBM control. Ultrasonic pretreatment increased dissolved organic matter from 12.41 ± 0.11 to 35.12 ± 0.14 mg/L, while eliminating bacterial colonies (reduced from 272 CFU to 0 CFU), demonstrating its dual role in nutrient solubilization and disinfection. Biomass production using varying UPMWW strengths showed the highest yield at 75% UPMWW (4.29 g/L), significantly higher (p = 0.05) than other concentrations. FTIR analysis confirmed the presence of proteins, polysaccharides, lipids, pigments, and astaxanthin in the biomass, suggesting suitability for value-added bioproducts. To the best of our knowledge, this is the first report characterizing UPMWW-grown double mutant C. humicola MCH4 for sustainable bioactive feedstock production. Overall, UPMWW presents a promising low-cost substrate for circular bioeconomy-driven microalgal bioprocessing.