## 3 Results and Discussion
### Anaerobic Digestion of the BWW and PE Mixture
#### 3.2.2 Molecular Characterization of Microbial Communities in the Hybrid Anaerobic Reactor  
To compare distribution of microbial populations during the experiment, a microbial analysis of effluents and samples, collected at the beginning of HRT 6.7 d IN and at the end of HRT 3 d OUT, was conducted. The sample collected at the end of the first phase (HRT 6.7 d OUT) of anaerobic digestion failed library preparation and was excluded from the sequencing run. After NGS, the samples generated were between the minimum of 609,126 and maximum of 682,804 raw sequence reads, which corresponded to samples HRT 6.7 d (IN) and HRT 3 d (OUT), respectively. A total of 777,827 sequences (775,651 bacterial, 2123 archaeal, and 53 unassigned) were retrieved and analyzed (Table 5). Libraries were composed of a total 2634 observed OTUs, that were detected in each sample. The highest Shannon-Wiener indices (4.95 and 5.28 for bacteria and archaea, respectively, Table 5), suggested that microbial communities were the most diverse at the beginning of experiment (HRT 6.7 d IN). The further decrease observed in Shannon-Wiener index values, until the end of the anaerobic digestion, confirms the occurrence of the microbial acclimation (bacteria and archaea) to the operating hydraulic condition, that is, shortening HRT to 3 d with concomitant increase in OLR up to 10 g COD/L-d. The decrease in microbial diversity means that the microbiota suffers a specialization shift in the microbial structure, consistent with the good performance of the hybrid anaerobic reactor. In the case of microbial diversity analysis, it is necessary to know whether the number of reads of the sequencing reaches a reasonable amount so that more sequencing does not significantly increase species diversity. Figure 4 indicates that, with 97% similarity  
Figure 3: Hybrid anaerobic reactor profile: (**a**) COD, TS, and VS; (**b**) total VFA and pH; (**c**) VFA composition. Sampling ports: P3 (14 cm), P2 (37 cm), P1 (48 cm). COD—Chemical oxygen demand; TS—total solids; VS—volatile solids; VFA—volatile fatty acids. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Samples** & **No. of Sequences** & **OTU** &
\begin{tabular}{c} **Shannon–Wiener Index** \\ **Bacteria** & **Archaea** \\ \end{tabular} \\ \hline BWW & 194,323 & 334 & 3.25 & - \\ PE & 193,742 & 1623 & 3.52 & 2.95 \\ HRT 6.7 d (IN) & 168,868 & 1589 & 4.95 & 5.28 \\ HRT 3.0 d (OUT) & 220,894 & 891 & 4.03 & 1.23 \\ \hline \hline \end{tabular} BWW—brewry wastewater; PE—piggery effluent; HRT—hydraulic retention time; OUT—operational taxonomic unit.  
\end{table} 
Table 5: Sequencing summary and microbial community (bacteria and archaea) diversity index of samples. levels, the rarefaction curve was asymptotic; that is, a flat portion of observed OTUs was attained, representing that the sequencing was sufficient for all samples, and the dataset of sequences had thoroughly sampled diversity in this analysis and sufficient sequence depth was achieved. Relative abundance of bacterial and archaeal groups was determined in terms of the percentage of total number of sequences in each sample. As predictable, bacteria predominated the microbial populations in all samples, accounting for more than 99.7% of relative abundance at domain level (Table 6). The discrepancy found between Bacteria and Archaea is normal, real and has already been reported by other authors in anaerobic digestion processes [59; 60; 61]. Most of the microorganisms presented in the substrate (HRT 6.7 d IN) belonged to the Bacteria domain, with 0.7% represented by the Archaea domain (Table 6). During the second phase (HRT 3 d) of the anaerobic digestion process in the hybrid anaerobic reactor, archaea populations drastically decreased yet, at a later phase, HRT 1 d, they recovered to the initial values of 0.7% (data not shown), confirming the maintenance quality of the biogas, at methane levels of around 80%.