# Structure of Microbial Communities When Complementary
## 3 Results and Discussion
### Molecular Characterization of Microbial Communities  
To compare the differences in the distribution of microorganisms, a microbial analysis of samples collected at the beginning (IN) and end (OUT) of the anaerobic digestion was conducted. Samples for microbial characterization were selected according to the best biogas/methane production, PE and 70P + 30O (IN and OUT). After NGS analysis of microbial communities, the samples generated between 615,732 and 679,254 raw sequence reads, which corresponded to samples 70P + 30O (IN) and PE (OUT), respectively. A total of 723,289 sequences (721,887 bacterial and 1402 archaeal) were retrieved and analyzed. Libraries containing all samples were composed of a total 2522 OTUs (Table 9). Samples collected after anaerobic digestion (OUT) exhibit lower diversity indices than samples at the beginning of the assay (IN), which is confirmed by Shannon-Wiener index in Table 9. The decrease observed in index values for both samples, during anaerobic digestion period, confirms the occurrence of the microbial acclimation to these samples, to a good performance reactors achievement. The greater microbial diversity, both bacterial and archaeal, observed in the 70P + 30O mixture samples, justifies the higher methane yield and COD removals obtained for these samples. 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 2 indicates that, with 97% similarity levels, the rarefaction curve was asymptotic, that is, a flat portion of observed OTUs was \begin{table}
\begin{tabular}{c c c} \hline \hline
**Mixture** & \
\begin{tabular}{c} **Cumulative CH4 Yield** \\ **(L CH4/kg SVin)** \\ \end{tabular} &
\begin{tabular}{c} **Energy Content** \\ **(KWh/kg SVin)** \\ \end{tabular} \\ \hline PE & \(434\pm 3\) & 4.32 \(\pm\) 0.03 \\
70P + 30O & \(489\pm 8\) & 4.87 \(\pm\) 0.08 \\
50P + 50O & \(178\pm 29\) & 1.77 \(\pm\) 0.29 \\
20P + 80O & 6.3 \(\pm\) 0.3 & 0.06 \(\pm\) 0.00 \\ \hline \hline \end{tabular}
\end{table} 
Table 8: Energy potential of PE + OMW mixtures used as feedstock in anaerobic digestion. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Sample** & \begin{tabular}{c} **No. of** \\ **Sequences** \\ \end{tabular} & \
\begin{tabular}{c} **OTU** \\ **Bacteria** \\ \end{tabular} &
\begin{tabular}{c} **Shannon–Wiener Index** \\ **Archaea** \\ \end{tabular} \\ \hline PE (IN) & 193,742 & 1623 & 3.52 & 2.95 \\ PE (OUT) & 206,714 & 779 & 1.38 & 1.45 \\
70P + 30O (IN) & 181,834 & 1819 & 4.79 & 4.45 \\
70P + 30O (OUT) & 140,999 & 610 & 1.64 & 2.16 \\ \hline Total & 723,289 & 4831 & - & - \\ \hline \hline \end{tabular}
\end{table} 
Table 9: Sequencing summary and microbial community (bacteria and archaea) diversity index of samples. 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. As expected, bacteria predominated the microbial populations in all samples, accounting for more than 99.7% of relative abundance at domain level (Table 10). The discrepancy found between bacteria and archaea is usual, real, and has already been reported by other authors in anaerobic digestion processes [49; 50; 51]. After anaerobic digestion, relative abundance of archaeal domain decreased, being the most pronounced reduction observed in the mixture containing OMW. Concerning bacterial sequences analyzed at phylum level, a majority (about 84%) were classified within three main phyla, Firmicutes, Bacteroidetes, and Proteobacteria (Figure 3). Most of the classes within the Proteobacteria were represented, Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Tenericutes, Synergistetes, Spirochaeta, and Actinobacteria sequences were classified within minor phyla.