## 3 Results and Discussion  
### Farm Microbiome  
To elucidate the influences of the environmental microbiota on the fermentation process, coffee fruits and leaves (collected from the coffee farm and on the ground), over-ripe fruits, cherries before de-pulping, depulped beans and water used for de-pulping beans, were collected to analyze the bacterial and fungal communities. A total of 282.288 sequences from the hypervariable region V3-V4 of the 16S rRNA gene and 158.316 from the Internal Transcribed Spacer (ITS) rDNA gene were obtained from all samples. Sequences that presented identity above 97% were considered the same Operational Taxonomic Unit (OTUs). Thus, 299 prokaryotes and 189 eukaryotes were identified (Table S1). The rarefaction curves were satisfactory, suggesting that most bacterial and fungal communities were covered (Figure 1). The presence and abundance of the major bacteria and fungi, defined as taxa with proportional abundance \(\geq\)2%, are reported in Figure 2. In general, Enterobacteriaceaeae and Saccharomyces were the most abundant microbial groups. To facilitate visualization of the results, PCA analysis was constructed to group the coffee farm samples according to microbial abundance and diversity (Figure 3). Among the eukaryotes, the samples were divided into three clusters; (i) cherries before de-pulping, depulped beans and water, characterized by the marked presence of the Saccharomyces family; (ii) soil, leaves and fruits collected on the ground, and fruits collected from the coffee tree, characterized by a low frequency of Saccharomyces and dominance of specific groups such as Pleosporales, Mycosphaerellaceae and _Colletotrichum_; and (iii) fruits collected on the ground, leaves collected from the coffee tree and over-ripe fruits, characterized by the dominance of _Candida_, Lecanoromycetes, and _Fusarium_, respectively. These results suggest that the first grouping (mainly cherries before de-pulping and depulped beans) may favor the fermentative process due to the high incidence of Saccharomyces. This order comprises about 1000 known species, including the yeasts _Pichia_, _Saccharomyces_, _Meyerozyma_, _Candida_ and _Hansenispora_ that have been widely selected as starter cultures for coffee fermentation [8; 18; 23]. Moreover, it is likely that most of the yeasts found in these samples are endophytic, since the coffee cherries collected from the coffee trees had a low incidence of Saccharomyces (1.33%), while the depulped fruits contained a high frequency (69.15%) (Figure 2B). On the other hand, the marked presence of filamentous fungi, such as _Cladosporium_, _Colletotrichum_, _Fusarium_ and Mycosphaerellaceae are undesirable in the fermentation process and control measures should be adopted to reduce the contact of soil, leaves, fruits collected from the ground and over-ripe fruits with the fermentation tank. Interestingly, the genera _Cladosporium_ and _Fusarium_ were also observed at high frequency in soil from a Honduran coffee farm [10]. The bacterial group was also divided into three clusters; (i) leaves and fruits collected from the ground, cherries collected from the coffee tree and over-ripe fruits, by the high incidence of Enterobacteriaceaeae and _Erwinia_; (ii) cherries before de-pulping, depulped beans and water, with high frequencies of Enterobacteriaceaeae and _Leuconstoc_; and (iii) soil and leaves collected from the coffee tree, with high dominated of _Annyclatoposis_ and _Methylobacterium_, respectively, and low frequency of Enterobacteriaceaeae. _Lactococcus_ and _Lactobacillus_ were also identified in high populations in the coffee leaves (Figure 2A). Figure 1: Alpha rarefaction curves of observed OTUs (operational taxonomic units) from the environmental samples. (**A**) Bacterial analysis, (**B**) Fungal analysis.