A High-Resolution Mass Spectrometry Approach to Promote Sorghum bicolor Biomass Valorisation

Sorghum (L. Moench ) subsp. bicolor (hereafter S. bicolor ) is an edible grain species native to Africa widely cultivated worldwide due to its adaptability to difficult environment and diverse secondary metabolism, that make it a valuable resource for food, feed and biomass re-use (Stefoska-Needham, 2024). Despite its potential, a great part of S. bicolor biomass still remains largely undervalued and is often treated as waste. Recent growing interest in its nutritional value and possible health benefits has further highlighted its high potential but the understanding of how environmental conditions influence its metabolic composition is still not complete. Therefore, this study aims to develop a robust mass-spectrometry method for the analysis of the impact of three among the most relevant environmental factors triggering S . bicolor develop and growth, the species secondary metabolism, through a comprehensive metabolomic analysis.Twelve pots were prepared according to a fully cross-cutting experimental design to assess the effects of three considered environmental factors which were water availability, soil enrichment with phosphorous (Ca(H 2 PO 4 ) 2 ) and growth-promotion, obtained using an ad-hoc consortium of fungi and bacteria (Micosat F 002). Endogenous metabolites in the leaves of the plants were identified using liquid chromatography (Agilent Technologies, 1200 Series) coupled to high-resolution mass spectrometry (Orbitrap Q-Exactive, Thermo Scientific), operated in both positive and negative heated electrospray ionization (HESI) mode. Analyses were run at four time points to monitor the metabolite production during plant growth phases. Our work starts from a workflow previously applied to study Lepidium sativum (cress) metabolism that was adapted and further optimized for the untargeted identification of Sorghum bicolor metabolites. A linear mixed model was fitted to the longitudinal data to account for repeated measurements and to assess statistical significance across different conditions and time points. Preliminary analysis revealed an extended panel of metabolites belonging from ammino acids, lipids, carbohydrates, phenols and carotenoids classes. These preliminary findings also demonstrated the enhanced efficiency and reliability of this workflow in plant metabolomics. The analysis should highlight key metabolic shifts, providing valuable insights into strategies for enhancing metabolite synthesis. Overall, these findings contribute to the valorisation of underexploited plant resources (Van Nguyen et al. , 2022). Within the framework of the circular economy, valorising plant biomass is essential to minimize waste production, promote environmental sustainability, and obtain high-valuable bioactive compounds. Overall, this study establishes a basis for the metabolic characterization of Sorghum bicolor under different environmental conditions and supports its potential for future valorisation.