Agave Species Influence the Taxonomic and Predicted Functional Structure of Traditional Pulque Inoculum Microbiomes

Abstract

Pulque is a traditional Mexican beverage produced by the spontaneous fermentation of “aguamiel”, the sap of several Agave species. Fermentation is driven by adding a previously fermented inoculum, locally known as “semilla”, whose microbial community contributes to the sensory and physicochemical properties of the beverage. The objective of this study was to provide a preliminary characterization of the microbial taxonomic composition and functional potential of pulque inoculant prepared from Agave mapisaga and Agave salmiana using shotgun metagenomic sequencing. Six inoculum samples were sequenced on the DNBSEQ using 150 bp paired-end reads. Metagenomic DNA was extracted using a CTAB-based protocol and analyzed in the Galaxy platform. The workflow included quality filtering, host-sequence removal, taxonomic classification with Kraken2, assembly with MEGAHIT, and functional annotation with eggNOG Mapper. Bacterial communities in inoculum from A. mapisaga and A. salmiana sap were dominated by Acetobacter (68.1% and 58.3%) and Leuconostoc (19.5% and 21.9%). Komagataeibacter was more abundant in A. mapisaga inoculum (3.8%), whereas Zymomonas was more abundant in A. salmiana inoculum (11.7%). The greatest species-level difference was observed for Zymomonas mobilis, whose mean relative abundance was 6.4-fold higher in A. salmiana. Fungal communities were dominated by Saccharomyces (91.4% and 72.7%) and Kluyveromyces (6.6% and 25.5%) in A. mapisaga and A. salmiana, respectively. Across all replicates, the most abundant species were Acetobacter sp. AC2005 (23.9%), Saccharomyces paradoxus (17.0%), and Zymomonas mobilis (12.9%), together accounting for approximately 54% of the total relative abundance. Kluyveromyces marxianus was 3.9-fold more abundant in A. salmiana inoculum, whereas Saccharomyces paradoxus was 1.26-fold more abundant in A. mapisaga inoculum. Alpha-diversity analysis indicated higher bacterial diversity in A. mapisaga inoculum, with a Shannon index of 2.74 and 35 exclusive species, whereas A. salmiana inoculum showed greater fungal diversity, with a Shannon index of 0.68. These differences were not statistically significant (p > 0.05). However, beta-diversity analysis suggested substantial separation between the microbial communities associated with the two Agave species (R2 ≈ 0.92). Functional annotation identified genes potentially associated with carbohydrate metabolism, sucrose degradation, and secondary metabolite biosynthesis. These findings suggest that the agave species used as the sap source may influence the taxonomic composition and functional potential of microbial communities involved in pulque fermentation.