High-Throughput rRNA Gene Sequencing Reveals High and Complex Bacterial Diversity Associated with Brazilian Coffee Bean Fermentation

Fermentacija kave je spontani proces koji se odvija već na plantaži, a u kojeg su uključene različite skupine mikroorganizama, poput bakterija i plijesni. U ovom je radu ispitana raznolikost i dinamika odnosa bakterija unutar populacije uključenih u fermentaciju brazilske kave, i to postupkom sekvenciranja visoke propusnosti. Iz uzoraka fermentirane kave izolirana je DNA u različitim vremenskim razmacima, te je pomoću visokopropusne metode Illumina sekvenciran gen 16S rRNA sa segmentima oko V4 varijabilne regije. Pronađeno je više od 80 vrsta bakterija, od kojih su mnoge po prvi put otkrivene tijekom fermentacije kave, poput vrsta Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas i Hymenobacter. Prisutnost bakterija vrste Fructobacillus potvrđuje njihov utjecaj na razgradnju fruktoze tijekom fermentacije. Temporalnom je analizom otkrivena dominacija bakterija mliječno-kiselog vrenja, i to pretežno vrsta Leuconostoc i Lactococcus, u više od 97 % sekvencija pri završetku fermentacije. HPLC analiza pokazala je da je zbog metaboličke aktivnosti bakterija mliječno-kiselog vrenja nastala veća količina mliječne kiseline tijekom fermentacije. Rezultati potvrđuju da je broj bakterijskih vrsta uključenih u fermentaciju kave bitno veći od očekivanog. Nove skupine bakterijskih vrsta pronađene u ovom istraživanju mogu poslužiti kao funkcionalne starter kulture za preradu kave na plantaži.Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97 % of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing