Single-cell phenotyping and sequencing uncover metabolically active low-abundance yeasts in thermophilic fermentation.

Microbiota-driven fermentation is a global biomanufacturing process that often operates under extreme and fluctuating temperatures. To understand how such systems maintain productivity, this study investigated the Chinese fermentation starter high-temperature Daqu (HTD) as a model system. By combining metagenomics and Raman microspectroscopy, the analysis revealed a drastic decoupling between phylogenetic composition and metabolic activity, with only 10-32 % of yeast species detected by sequencing remaining metabolically active under heat stress. Raman-activated cell sorting and culture (RACS-Culture) recovered three yeasts that consistently maintained viability throughout HTD production: Pichia kudriavzevii, Wickerhamomyces anomalus, and Saccharomycopsis fibuligera. Mono-species and synthetic-community fermentation further revealed a sophisticated mechanism of temporal niche partitioning: in the moderate-temperature early and late stages, S. fibuligera and W. anomalus dominated substrate degradation and flavor precursor biosynthesis, respectively. However, as temperatures rose above 45 °C, both species exhibited low metabolic activity and survival rates. In contrast, only P. kudriavzevii sustained robust growth at this elevated temperature. Genomic analysis revealed a remarkable expansion of heat-resistance and cell-clustering-related genes of wos2 and FLO8 in P. kudriavzevii. These genetic characteristics underpin its enhanced viability, which enables the initially low-abundance species to thrive as a primary ethanol producer and ultimately establish numerical dominance. Thus, temporally overlaying single-cell metabolic vitality profiles onto the corresponding metagenomes can unravel novel functional species and reveal their ecological roles in a complex ecosystem.