Deciphering Melatonin-Driven Bioenergy Reprogramming Through Integrated Physiological and Metabolomic Analyses in Dunaliella salina.

Despite their immense promise as renewable bioenergy resources, microalgae continue to face major barriers to industrial deployment due to low biomass output and constrained lipid accumulation. In this regard, the regulatory role of melatonin (MT) in modulating carbon flux allocation and redox homeostasis was investigated in the oleaginous, D. salina using a two-stage cultivation strategy. During Stage I, supplementation with 20 µM MT enhanced biomass accumulation and chlorophyll a content relative to the control. However, during Stage II, MT supplementation triggered pronounced metabolic reprogramming, leading to substantial enrichment of lipids (41.6%) and carbohydrates (17.52%), accompanied by a concomitant decline in protein content under nitrogen starvation. This metabolic shift was coupled with strong activation of the antioxidant defense system, as evidenced by elevated SOD, CAT, and APX activities, while DCFDA-based flow cytometric analysis revealed a tightly regulated ROS profile, indicative of maintained redox homeostasis. In addition, FTIR spectroscopy showed increased absorbance at 2929 cm⁻¹, and 1726 cm⁻¹, corroborating neutral lipid enrichment. Interestingly, untargeted HR-MS-based metabolomics further revealed a pronounced upregulation of fatty acid biosynthesis pathways, accompanied by enrichment of saturated fatty acids and a concomitant reduction in polyunsaturated fatty acids. These findings indicate that MT coordinates a finely tuned redox-metabolic network in which controlled ROS dynamics, together with reinforced antioxidant defense, synergistically redirect carbon flux toward neutral lipid biosynthesis. This study provides mechanistic insights into MT-mediated biochemical engineering and highlights its potential to enhance the commercial feasibility of vehicular grade biofuel production.