AbstractIn Colombia, coffee growers use a traditional method of fermentation to remove the cherry pulp surrounding the beans. This process has a great influence on sensory quality and prestige of Colombian coffee in international markets, but has never been studied. Here we use an Illumina-based amplicon sequencing to investigate bacterial and fungal communities associated with spontaneous coffee-bean fermentation in Colombia. Microbial-derived metabolites were further analysed by high–performance liquid chromatography and gas chromatography–mass spectrometry. Highly diverse bacterial groups, comprising 160 genera belonging to 10 phyla, were found. Lactic acid bacteria (LAB), mainly represented by the genera Leuconostoc and Lactobacillus, showed relative prevalence over 60% at all sampling times. The structure of the fungal community was more homogeneous, with Pichia nakasei dominating throughout the fermentation process. Lactic acid and acetaldehyde were the major end-metabolites produced by LAB and Pichia, respectively. In addition, 20 volatile compounds were produced, comprising alcohols, organic acids, aldehydes, esters, terpenes, phenols, and hydrocarbons. Interestingly, 56 microbial genera, associated with native soil, seawater, plants, insects, and human contact, were detected for the first time in coffee fermentation. These microbial groups harbour a remarkable phenotypic diversity and may impart flavours that yield clues to the terroir of Colombian coffees.