Petruzziello, Ernesto (2025) Wild Yeasts as a Source for Designing Specific Consortia for Wine Fermentation. [Tesi di dottorato]

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Abstract

Yeasts and microbial diversity are essential components of terroir and serve as a resource for more sustainable, place-based oenology. This doctoral thesis aims to characterize yeast communities associated with Campanian grape varieties, particularly the Falanghina grape, and with natural habitats, and to develop autochthonous microbial consortia for use in wine fermentation. The approach views microbial biodiversity through a terroir perspective, accounting for both spatial differences (among vineyards and soils) and temporal changes (successions during spontaneous fermentation), to promote the use of natural microorganisms in creating distinctive and sustainable wines. Characterization was conducted by integrating metataxonomic DNA sequencing (amplicons of ribosomal regions, ITS1, ITS2 and 26S) with traditional culture-based yeast isolation. Grapes and musts were sampled from several autochthonous varieties (Falanghina, Fiano di Avellino, Aglianico Lasco, Camaiola) across multiple Campanian terroirs and at different time points, and environmental matrices, such as soils, oak bark and olive drupes were also included as potential ecological reservoirs of yeasts. Numerous autochthonous isolates of Saccharomyces cerevisiae and non-Saccharomyces yeasts underwent phenotypic and technological characterization (tolerance to fermentative stresses; production of metabolites such as ethanol, glycerol, volatile acidity, H₂S; and enzymatic activities of oenological interest) to assess their applicative potential. An innovative strategy was also developed to study yeast interactions during fermentation: an autochthonous S. cerevisiae strain (isolated from Fiano grapes) was modified via CRISPR-Cas9 to insert a fluorescent reporter gene (GFP) by replacing the HO locus with a constitutively expressed cassette. Thanks to this tagged strain and flow cytometry (FACS), it was possible to monitor in real time the dynamics of co-cultures of S. cerevisiae with non-Saccharomyces yeasts, distinguishing live and dead cells and detecting the relative composition of populations during controlled micro-fermentations. Finally, pilot fermentations of Falanghina must be co-inoculated with autochthonous mixed consortia (S. cerevisiae together with selected Metschnikowia pulcherrima) to verify feasibility and effectiveness at a semi-industrial scale. Spontaneous fermentation revealed clear microbial successions: fermentation time emerged as the main factor shaping yeast community composition, driving the shift from initial populations dominated by non-Saccharomyces (oxidative/environmental) yeasts to consortia dominated by Saccharomyces cerevisiae by the second week. In parallel, terroir-related differences were observed: each vineyard displayed a distinctive microbiological “signature” (e.g., in initial abundances and presence of minor species). However, the effect of the site proved secondary to temporal fermentative dynamics. The observed taxonomic diversity was broad, with numerous non-Saccharomyces genera identified (Hanseniaspora, Metschnikowia, Starmerella, etc.), often present in the early phases of spontaneous fermentation. Some species (Lachancea, Torulaspora) were also detected in environmental matrices, suggesting ecological links between the vineyard and its surroundings and the presence of rare strains potentially useful for winemaking. These results open the way to fermentation protocols based on terroir-driven, autochthonous yeast consortia that can enhance local oenological features while improving process sustainability. From an applicative standpoint, a framework is proposed that combines: (i) targeted selection of autochthonous species/strains according to enological objectives (e.g., acidity, roundness, aromatic profile, attenuation), (ii) definition of appropriate inoculation timing and modes (simultaneous co-inoculation of multiple species or sequential inoculation, with S. cerevisiae introduced in the second phase), and (iii) quantitative, non-invasive monitoring of fermentation progress (weight/CO₂, HPLC for sugars, acids, ethanol, FACS for viability and composition) with clear intervention thresholds. Using such locally defined consortia makes it possible to valorize local biodiversity, obtain wines more closely aligned with their place of origin, and, at the same time, reduce corrective interventions and unnecessary chemical additives. Looking ahead, multi-vintage and multi-site campaigns will be valuable for testing the stability of these microbiological terroir “signatures” and the reproducibility of co-fermentation protocols across different contexts, alongside scale-up trials under real operating conditions to validate effectiveness at industrial scale. Integrating more extensive sensory and chemical analyses (e.g., volatilome/volatilomics studies and larger tasting panels) will enable tighter correlations between the fermentative microbiota and wine sensory quality, consolidating a sustainable oenological approach that leverages local microbial biodiversity.

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