## 4 Discussion  
In order to characterize the microbial composition and contribution to the volatile dynamics of Nacional x Trinitario fine-flavor cacao bean fermentation, a multiphasic approach was employed. First, the microbial communities were analyzed by isolating LAB, AAB, yeasts and _Bacillus_ spp. in specific culture media. Then, pure isolates were identified by Sanger sequencing and inoculated into cacao beans that had been spontaneously ferrented for 0, 24, 28, 72, or 96 h to assess the role of each individual microorganism in the production of volatile compounds at each fermentation stage. Additionally, high throughput sequencing based on the ITS (internal transcribed spacer), LSU (Large subunit) and 16S rRNA regions was used to characterize the fungal and bacterial communities in spontaneously fermented cacao beans. The results showed a high but variable microbial diversity during fermentation. The cacao bean samples fermented for 72 h showed the highest diversity index with 3.065, while the samples from 96 h of fine-flavor cacao fermentation exhibited the lowest diversity values with 1.45. The differences in the Shannon diversity values suggest that the proportion of predominant communities fluctuate as fermentation progresses. In general, the decrease in the diversity at the end of the fermentation may be explained by the accumulation of various microbial inhibitors, such as organic acids. A culture-dependent analysis, followed by Sanger sequencing, showed that the first 24 h of the fermentation were dominated by the yeast _Saccharomyces cerevisiae_. On the other hand, Illumina-based sequencing revealed that the most abundant fungal species within the initial stage of fermentation was _Candida glabrata_, which was reported in previous studies [13]. In our work, a high number of fungal taxa were detected using NGS, as this technique allows the detection of species with relatively low abundance [18]. During the lab-scale fermentation, the inoculation of _S. cerevisiae_ showed a significant production of alcohols, such as benzene ethanol, 2-propylecan-1-ol and 2,3-butanediol, at 0 h and 24 h of fermentation. Different yeast species have been identified as alcohol producers in previous reports [16; 37] and have been associated with flowery and sweet aromas [3; 38]. During the start of the spontaneous fermentation process (0-24 h), the presence of different LAB species was detected by NGS analyses, including _Limosilactobacillus fermentum_ and _Lacticaseobacillus zeae_. In contrast, the most abundant species observed by culture-based methods was _Liquorilactobacillus nagelli_. The NGS data revealed a low abundance of AAB, which included species such as _Acetobacterium_ sp., whereas culture-dependent strategies only identified _Acetobacter pasteurianus_ during this fermentation period, similar to what was observed in previous reports [39]. The production of compounds linked to floral, almond and fruity notes, such as acetophenone, benzaldehyde and acetaldehyde [4; 40], was observed after the inoculation of _A. pasteurianus_ and _Liquorilactobacillus nagelli_ in cacao beans that were previously spontaneously fermented for 0-24 h. These species have been reported in cacao bean box fermentation from Mexico and Brazil [41; 42], but the production of volatile compounds during fermentation was not reported. However, the formation of acetic acid with an unpleasant sour aroma was detected in cacao beans spontaneously fermented for 24 h and inoculated with _A. pasteurianus_. The Acetobacter genus was represented by the abundant presence of _A. syzygii_ detected through culture-dependent techniques after 48 h of spontaneous fermentation, similar to what was observed in other reports [11; 43]. The production of benzaldehyde was observed when fermented cacao beans (48 h of spontaneous fermentation) were inoculated with this species.