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Temporal shotgun metagenomics of multiple samples of an Arabica wet coffee fermentation process, which was examined microbiologically and metabolomically before, was performed to complement these microbiological and biochemical data with an in-depth in silico analysis of the structure and functions of the coffee microbiome. The taxonomic analysis of a massive sequence dataset of ca. 16 Gbp identified >150 microbial species and distinguished three successive, microbial phases: (i) enterobacteria, acetic acid bacteria, and some yeasts prevailing at the start; (ii) lactic acid bacteria (LAB) in the second phase; and (iii) acid-tolerant LAB at the end of the fermentation process. The functional analysis of prokaryotic genes, based on ca. 138,000 coding sequences, facilitated the gene repertoire overview of this ecosystem, showcasing differences in each fermentation stage. Initial prototrophic characters, plant cell wall-degrading activities, and pectinolytic activities were followed by a more auxotrophic and saccharolytic microbial profile, due to an increasing relative abundance of LAB. The almost full genome of 22 bacterial species was reconstructed and additional coffee-specific features were identified. This temporal metagenomic analysis of a case of reproducible wet coffee fermentation processes carried out in a coffee plantation near Nanegal, Ecuador, constitutes a breakthrough for the study of wet coffee processing and the contribution of the growth and activities of different microorganisms, in particular LAB, to this process.

Post-harvest wet coffee processing is a commonly applied method to transform coffee cherries into green coffee beans through depulping or demucilaging, fermentation, washing, soaking, drying, and dehulling. Multiple processing parameters can be modified and thus influence the coffee quality (green coffee beans and cup quality). The present study aimed to explore the impacts of these parameters, including processing type (depulping or demucilaging), fermentation duration, and application of soaking, on the microbial community dynamics, metabolite compositions of processing waters (fermentation and soaking) and coffee beans, and resulting cup quality through a multiphasic approach. A large-scale wet coffee processing experiment was conducted with Coffea arabica var. Catimor in Yunnan (China) in duplicate. The fermentation steps presented a dynamic interaction between constant nutrient release (mainly from the cherry mucilage) into the surrounding water and active microbial activities led by lactic acid bacteria, especially Leuconostoc and Lactococcus. The microbial communities were affected by both the processing type and fermentation duration. At the same time, the endogenous coffee bean metabolism remained active at different stages along the processing, as could be seen through changes in the concentrations of carbohydrates, organic acids, and free amino acids. Among all the processing variants tested, the fermentation duration had the greatest impact on the green coffee bean compositions and the cup quality. A long fermentation duration resulted in a fruitier and more acidic cup. As an ecological alternative for the depulped processing, the demucilaged processing produced a beverage quality comparable to the depulped one. The application of soaking, however, tempered the positive fermentation effects and standardized the green coffee bean quality, regardless of the preceding processing practices applied. Lastly, the impact strength of each processing parameter would also depend on the coffee variety used and the local geographical conditions. All these findings provide a considerable margin of opportunities for future coffee research.

A cup of coffee is the final product of a complex chain of operations. Wet postharvest processing of coffee is one of these operations, which involves a fermentation that inevitably has to be performed on-farm. During wet coffee processing, the interplay between microbial activities and endogenous bean metabolism results in a specific flavor precursor profile of the green coffee beans. Yet, how specific microbial communities and the changing chemical compositions of the beans determine the flavor of a cup of coffee remains underappreciated. Through a multiphasic approach, the establishment of the microbial communities, as well as their prevalence during wet processing of Coffea arabica, was followed at an experimental farm in Ecuador. Also, the metabolites produced by the microorganisms and those of the coffee bean metabolism were monitored to determine their influence on the green coffee bean metabolite profile over time. The results indicated that lactic acid bacteria were prevalent well before the onset of fermentation and that the fermentation duration entailed shifts in their communities. The fermentation duration also affected the compositions of the beans, so that longer-fermented coffee had more notes that are preferred by consumers. As a consequence, researchers and coffee growers should be aware that the flavor of a cup of coffee is determined before as well as during on-farm processing and that under the right conditions, longer fermentation times can be favorable, although the opposite is often believed. IMPORTANCE Coffee needs to undergo a long chain of events to transform from coffee cherries to a beverage. The coffee postharvest processing is one of the key phases that convert the freshly harvested cherries into green coffee beans before roasting and brewing. Among multiple existing processing methods, the wet processing has been usually applied for Arabica coffee and produces decent quality of both green coffee beans and the cup of coffee. In the present case study, wet processing was followed by a multiphasic approach through both microbiological and metabolomic analyses. The impacts of each processing step, especially the fermentation duration, were studied in detail. Distinct changes in microbial ecosystems, processing waters, coffee beans, and sensory quality of the brews were found. Thus, through fine-tuning of the parameters in each step, the microbial diversity and endogenous bean metabolism can be altered during coffee postharvest processing and hence provide potential to improve coffee quality.

The postharvest treatment and processing of fresh coffee cherries can impact the quality of the unroasted green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through two different wet and dry methods to monitor differences in the microbial community structure and in substrate and metabolite profiles. The changes were followed throughout the postharvest processing chain, from harvest to drying, by implementing up-to-date techniques, encompassing multiple-step metagenomic DNA extraction, high-throughput sequencing, and multiphasic metabolite target analysis. During wet processing, a cohort of lactic acid bacteria (i.e., Leuconostoc , Lactococcus , and Lactobacillus ) was the most commonly identified microbial group, along with enterobacteria and yeasts ( Pichia and Starmerella ). Several of the metabolites associated with lactic acid bacterial metabolism (e.g., lactic acid, acetic acid, and mannitol) produced in the mucilage were also found in the endosperm. During dry processing, acetic acid bacteria (i.e., Acetobacter and Gluconobacter ) were most abundant, along with Pichia and non- Pichia ( Candida , Starmerella , and Saccharomycopsis ) yeasts. Accumulation of associated metabolites (e.g., gluconic acid and sugar alcohols) took place in the drying outer layers of the coffee cherries. Consequently, both wet and dry processing methods significantly influenced the microbial community structures and hence the composition of the final green coffee beans. This systematic approach to dissecting the coffee ecosystem contributes to a deeper understanding of coffee processing and might constitute a state-of-the-art framework for the further analysis and subsequent control of this complex biotechnological process. IMPORTANCE Coffee production is a long process, starting with the harvest of coffee cherries and the on-farm drying of their beans. In a later stage, the dried green coffee beans are roasted and ground in order to brew a cup of coffee. The on-farm, postharvest processing method applied can impact the quality of the green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through wet and dry processing in four distinct variations. The microorganisms present and the chemical profiles of the coffee beans were analyzed throughout the postharvest processing chain. The up-to-date techniques implemented facilitated the investigation of differences related to the method applied. For instance, different microbial groups were associated with wet and dry processing methods. Additionally, metabolites associated with the respective microorganisms accumulated on the final green coffee beans.