Sequential Inoculation of Indigenous Starmerella bacillaris and Saccharomyces cerevisiae to Modulate the Volatile Profiles and Lower Ethanol Yields in Romanian Aromatic Wines.

The industrial use of non-Saccharomyces yeasts offers a robust bioprocess strategy for modulating volatile biochemical profiles and reducing climate-driven ethanol production in fermented beverages. This study explores the metabolic diversity of indigenous non-Saccharomyces, with a particular focus on Starmerella bacillaris MI151, selected for sequential inoculation with Saccharomyces cerevisiae MI118. Pilot-scale bioreactor fermentations (25 L) of two Romanian matrix-specific aromatic grape cultivars, Busuioacă de Bohotin and Tămâioasă Românească, exhibited clear strain-dependent carbon flux changes. The sequential culture actively triggered the glycerol-pyruvic pathway, which consistently reduced the final ethanol concentration to up to 0.94% (v/v) and maintained a balanced profile of volatile compounds. GC-MS-based metabolomic profiling revealed substantial changes in the esterification kinetics and glycosidic precursor cleavage. The sequential fermentation resulted in a synergistic increase in n-hexyl acetate (up to 2310.01 µg/L) and bypassing of the standard enzymatic repression, freeing highly volatile monoterpenes (β-citronellol and β-geraniol) in the analyzed matrices. This targeted microbial system successfully modulated the organic acid-phenolic balance, neutralizing harsh structural finishes, as confirmed by quantitative sensory mapping. Finally, these specific local yeast strains exhibit strong bioprocess scalability, offering a predictable, non-engineered strategy to lower the ethanol yield while driving targeted flavor enhancement in climate-vulnerable viticultural regions.