Phytohormone augmented biomass and lipid production in Dunaliella salina under two-stage cultivation strategy: A comprehensive characterization of biomass and lipid using DLS, FTIR, CHNS and NMR for bioenergy prospects

Increasing population, climatic shifts, and decreasing fossil-fuels worldwide led scientists to explore microalgae as a renewable energy resource. The present study unravels the impact of gibberellic acid (GA3) on biomass production, enzymatic activities, macromolecular synthesis, and metabolome analysis of Dunaliella salina using two-stage cultivation strategy. The results indicated a marked increase in biomass (1.44-fold), photosynthetic yield (Fv/Fm 0.68), along with RuBisCO activity (1.66-fold), with an increased in absolute value of zeta potential (zeta) in cultures treated with 10 mu M GA3 compared to controls (Stage I). Interestingly, 15 mu M GA3-treated cells significantly enhanced lipid content (42.36 %), and carbohydrates (16.56 %) with elevated antioxidative enzymes (SOD, CAT, and APX) activities (Stage II). Enhanced lipid content was further confirmed through FTIR spectra (2928 cm-1, and 1740 cm-1) and 1H NMR signals (1.0- 2, 3.5-4.5 ppm), indicating an increase in both saturated (SFA) and polyunsaturated fatty acids (PUFAs). Elemental analysis demonstrated higher carbon and hydrogen percentages, reflected in higher heating value (HHV) and H/C ratio at 15 mu M GA3. The increased lipid content was found to be significantly correlated with ACCase and GPAT activities, suggesting the allocation of carbon flux toward lipid biosynthesis pathways. To substantiate the carbon flux, HR-MS-based metabolomic analysis was performed, indicating a reduction in TCA and Calvin cycle intermediates with a significant rise in carotenoid biosynthesis, as well as metabolic shift towards fatty acid biosynthesis reflecting in the enhanced SFA and PUFAs. Thus, these findings highlighted GA3 significant role in the carbon flux allocation towards the neutral lipid biosynthesis for bioenergy prospects.