Unveiling the bioenergy potential of oleaginous Monoraphidium contortum: Biomass and lipid hyperaccumulation and its valorization for biodiesel production under sequential nitrogen cultivation

• In stage I, nitrate boosted CO 2 fixation, pigments, photosynthesis, and biomass. • N 2 deprivation increased carbohydrates, lipids, and antioxidants in stage I. • In stage II, 4dN − maximized lipid (52.6%), neutral lipid (46.6%), and hydrocarbons. • CHNS showed higher HHV, CV, and TMP in 4dN − cultures. • FAME profiling showed dominant C16–C18 SFAs (32.65%), suiting biodiesel. Despite the promise of microalgal bioenergy, the trade-off between biomass production, lipid accumulation, and biodiesel yield under nutrient stress remains a key limitation. This study used M. contortum to assess the effects of nitrate supplementation on biomass yield (stage I) and of nitrate deprivation on lipid biosynthesis (stage II) using growth kinetics and metabolite profiling to harness bioenergy potential. During stage I, nitrate supplementation at 0.25 g L −1 enhanced growth, CO 2 fixation, biomass, pigments, photosynthetic efficiency (Fv/Fm, Y(II), α, Ik, and ETRmax), and protein content, whereas nitrate deprivation (0 g L −1 ) increased carbohydrate (3.6-fold) and lipid content (4.3-fold), along with elevated carotenoids, Y(NO), MDA, and antioxidant activities (CAT, APX, TFC, and TPC). In stage II, the 4-day nitrogen-deprived culture (4dN − ) achieved the highest lipid content (52.6%) and neutral lipid fraction (46.6%), along with the lowest glycolipid (9.5%) and phospholipid (7.1%) contents. Elevated lipid content was supported by FTIR peaks at 2920, 2852, and 1739 cm −1 , indicating metabolic reprogramming of M. contortum toward TAG accumulation and hydrocarbon synthesis. Additionally, CHNS analysis showed higher carbon and hydrogen, with lower nitrogen, sulfur, and oxygen, resulting in increased C/N and H/C ratios, HHV, CV, TMP, COD, and TEF in 4dN − cultures, indicating enhanced bioenergy potential. P-XRD analysis revealed an enhancement in the amorphous characteristics of the biomass. Furthermore, GC–MS analysis showed that SFAs (32.65%), mainly C16–C18, were predominant over MUFAs (18.08%) and PUFAs (23.11%) in 4dN − cultures. The resulting biodiesel met the EN 14214 and ASTM D6751 standards, highlighting M. contortum potential to achieve SDG 7.