Bacterial and Fungal Community Profiles During Spontaneous Agave cupreata Must Fermentation for Ancestral Mezcal Production

Spontaneous fermentation is central to ancestral mezcal production, yet the bacterial and fungal communities associated with Agave cupreata must remain insufficiently characterized. This exploratory study described community composition and predicted functional profiles at three stages of a traditional fermentation defined by sugar depletion: 10 °Brix (0 h), 5 °Brix (72 h), and 2 °Brix (144 h). Ambient temperature ranged from 21.7 to 23.7 °C, must temperature from 28.5 to 29.9 °C, pH from 4.6 to 5.2, and relative humidity from 51.3% to 58.7%. At each stage, material collected from two wooden vats and three vertical positions was pooled into one composite sample. Bacterial 16S rRNA gene and fungal ITS1 amplicons were sequenced and analyzed using QIIME 2. At 10 °Brix, five dominant bacterial genera accounted for more than 93% of relative abundance. Lactic and acetic acid bacteria represented more than 91% at 5 °Brix, while Pediococcus, Lactiplantibacillus, and Loigolactobacillus were prominent at 2 °Brix. Seven fungal genera represented more than 98% of the initial profile, whereas Saccharomyces, Debaryomyces, and Maudiozyma accounted for more than 98% at 2 °Brix. PICRUSt2 predicted pathways related to carbohydrate degradation and fermentation, whereas FUNGuild assignments were predominantly saprotrophic at the later stages. Because one pooled sample was analyzed per stage, these patterns are descriptive and require validation in replicated fermentations. Despite these limitations, this study provides baseline information on the microbial communities associated with ancestral A. cupreata fermentation and identifies taxa and predicted functions that merit future isolation and experimental validation.