Zinc Oxide Nanoparticles Assisted Extraction of Antioxidant Rich Phenolic Compounds from Double Mutant Chlorococcum humicola Biomass by Submerged Fermentation

This study presents an integrated approach for solvent free recovery of total phenolic compounds (TPC) from the biomass of double mutant microalga Chlorococcum humicola MCH4 using microwave-assisted pretreatment (MAPT) followed by submerged fermentation (SmF) with Bacillus subtilis. This microalga strain was developed through UV-C and EMS-induced mutagenesis and cultivated in ultrasonically pretreated municipal wastewater to obtain biomass, which was then subjected to MAPT to improve the release of TPC. Key fermentation conditions such as temperature, pH, fermentation time, agitation speed and zinc oxide nanoparticles (ZnO-NPs) concentration were optimized to maximize TPC yield. Under optimal conditions-microwave power of 600 W, pH 7.0, temperature 35 °C, agitation at 150 rpm, fermentation time of 48 h, and ZnO-NPs supplementation at 150 mgL-1, the process achieved a maximum TPC yield of 60.72 ± 0.11 mg GAE g-1 DW. The IC50 values of 38.7 µgmL-1 and 41.2 µgmL-1 were obtained for antioxidant activity of the recovered TPC by DPPH and ABTS assay, respectively, indication strong antioxidant potential. This work highlights the synergistic effect of MAPT and bacterial fermentation for effective solvent free recovery of bioactive phenolics from microalgal biomass. The process is both scalable and environmentally sustainable, indicating robust possible for use in the nutraceutical and pharmaceutical industries.