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.