Influence of zinc oxide nanoparticles on bioethanol production from hydrolysate of defatted <i>Neodesmus pupukensis</i> DMA5 biomass under optimized fermentation conditions

Bioethanol, a sustainable fossil fuel alternative, is produced by microbial fermentation of carbohydrate-rich biomass, requiring cost-effective production strategies. This study investigates a comprehensive approach to sustainable bioethanol production by utilizing the defatted biomass (DB) of the double mutant green alga Neodesmus pupukensis DMA5 (Accession number-PQ084675.1), isolated from the ancient temple pond, and cultivated in ultrasonically pretreated municipal wastewater, as a renewable feedstock. Microwave-assisted alkaline pretreatment (MAAPT) enhanced carbohydrate recovery from the DB, with 500 W identified as the optimal power level, yielding 37.47 ± 0.21% (w/w) total carbohydrates-over twice that of the untreated control. Sugar profiling of the hydrolysate showed glucose as the major component (76.11%), followed by xylose, rhamnose, and fucose, confirming efficient disruption of cellulose and hemicellulose. The resulting hydrolysate served as a substrate for bioethanol production using immobilized Saccharomyces cerevisiae NITTS1(Accession Number-MG255132.1). Process parameters including pH, temperature, agitation speed, and fermentation time were optimized via Response Surface Methodology (RSM) using Central Composite Design (CCD). The highest ethanol yield (193.09 mgg-1DB) was achieved at pH 5.0, 35 °C, 150 rpm, and 48 h. Supplementation with zinc oxide (ZnO) nanoparticles (NPs) further enhanced ethanol production, with 150 mg L-1 ZnO NPs yielding 198.34 ± 0.11 mg g-1 DB, attributed to zinc’s role as a metabolic cofactor in S. cerevisiae. Higher ZnO NPs concentrations, however, were inhibitory due to potential oxidative stress. This study demonstrates that the synergistic integration of MAAPT, optimized fermentation, and nanoparticle supplementation provides an effective strategy for maximizing bioethanol production from DB, thereby advancing economically viable and eco-friendly biofuel technologies.