## 3 Results
### Evolution of Target Bacteria during the 72-th Fermentation  
In order to monitor the evolution of fifteen target bacterial taxa during the three days of colonic fermentation, qPCR analyses were conducted. Thus, Figure 2 presents, indistinctly, the results of the microbial community composition analysis for Bacteroidetes, Firmicutes, and Actinobacteria phylum, as well as for the _Gammaproteobacteria_ class, the communities _Bacteroides-Prerotella_, _Clostridium coccoides_ (Cluster IV), _Clostridium leptin_ (Cluster XIVa), and butyrate-producing bacteria based on the detection of butyryl-coenzyme A transferase genes (ButCoA). The _Christensenellaceae_ family, some selected genera, and the _Akkermans muciniphila_ species are also represented. Changes over time were analyzed independently using the daily harvested fermentation samples.  
Figure 1: Microbial community analysis in stool samples from donors 1, 2, and 3, as assessed using 16S rRNA Illumina Sequencing. (**a**) Genera abundance histogram (%, left panel) and genus classification (right panel) based on quality-controlled OTU reads. Only bacterial genera with relative abundance >1% in at least one sample are shown, and the rest are included as others * (see details in Supplementary File S2, Table S2). Diversity analysis of the sequencing data is presented as (**b**) [PERSON] \(\alpha\)-diversity index, (**c**) [PERSON] estimator of genus richness, (**d**) [PERSON] reciprocal \(\alpha\)-diversity index, and (**e**) [PERSON] abundance-derived evenness.  
Figure 2: Relative quantification of fifteen target taxa assessed by qPCR analysis. Data are presented as mean \(\pm\) SD fold-changes calculated using \(2^{-\Delta\text{MCq}}\) method [42]. Comparisons were performed by means of one-way ANOVA, followed by Bonferroni post-test, and statistical significance is indicated as \(p<0.05\) (*), \(p<0.01\) (**), \(p<0.001\) (***), and \(p>0.05\) (ns).  
The Firmicutes phylum (Figure 2b) and _Akkermansia muciniphila_ species from the Verrucomicrobia phylum (Figure 2o) both decreased during the fermentation, and such diminution was significantly higher by the third day of the experiment. In parallel, levels of the Gammaproteobacteria class and the _Enterococcus_ genus significantly increased during the three days of colonic fermentation, as shown in Figure 2d,l, respectively.  
The _Streptococcus_ genus increased significantly during the first and second day (Figure 2m). However, by the third day, _Streptococcus_ levels returned to values not significantly higher than those present initially (Day 0). The analyzed ButCoA genes did not reveal any important changes in butyrate-producing bacteria (Figure 2g).  
The _Roseburia_ genus (Figure 2h), as well as the Cluster IV group (Figure 2f) that includes important genera (e.g., _Ruminococcus_, _Faecalibacterium_, _Oscillibacter_, etc.), were significantly reduced during the experiment. The Cluster XIVa group (Figure 2e) decreased, but only at significant levels on the last day. A significant decrease in the _Christensenellaceae_ family was identified in fermentation samples after the first and second days of the simulation (Figure 2k).  
The _Lactobacillus_ genus increased during the first day of incubation, but after that, it significantly decreased (Figure 2i). In contrast, _Bacteroides-Prerotella_ (Figure 2n) and its phylum Bacteroidetes (Figure 2a) were identified only by the end of the fermentation (third day). However, these last two taxa were not quantified at statistically important levels when compared with the fecal inoculum.  
Finally, the levels of the Actinobacteria phylum did not have any significant variation (Figure 2c) during the experiment. Nevertheless, the _Bifidobacterium_ genus increased on the third day with respect to the day of inoculation and the first day after inoculation (Figure 2j).