The aim of this pioneering study was to determine the biodiversity of non-Saccharomyces yeasts in ancient distilleries located in the La Mancha region, which is the principal area for the production of bioethanol and grape-based distillates in Spain. In this study, the yeast populations that were present during the process of extraction of alcohol and residual sugars from the byproducts of vinification, such as piquettes, pomace and grape skins, were studied. Non-Saccharomyces yeasts were identified by PCR-RFLP analysis of the 5.8S rRNA genes and, when necessary, by sequencing the D1/D2 domain of the 26S and/or 5.8S rRNA genes. Further, fermentation and the assimilation of carbon compounds were studied, to identify potential industrial applications. Phylogenetic trees and heat-maps were constructed for the genetic and phenotypic traits, respectively. Twenty yeast species belonging to eight genera were identified (Torulaspora, Candida, Zygosaccharomyces, Pichia, Hanseniaspora, Kluyveromyces, Ogataea and Saccharomycodes). Pichia galeiformis, Candida lactis-condensi, Hanseniaspora osmophila and Torulaspora delbrueckii were the most abundant species and were found principally in sweet and fermented piquettes.
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