## 3 Results
### Molecular Characterization of Microbial Communities
The inoculum shows the predominance of _Bacteroidales_ (11%, Figure 3a), _Anaerolinales_ (12%, Figure 3b), and _Clostridiales_ (4%, Figure 3c), and the presence of members of all classes of the phylum Proteobacteria (Figure 3d), with dominance attributed to \(\alpha\)-Proteobacteria (9%), \(\beta\)-Proteobacteria (11%), and \(\delta\)-Proteobacteria (9%). The predominant populations in H (IN) units were _Pseudomonas_ (77%, Figure 3d). After AD, a change was observed in the microbiota composition. Although the predominance of Proteobacteria remained, this phylum decreased to 59% (Figure 2) and became mainly constituted by _Rhizobiales_ (39%, Figure 3d). Simultaneously, _Bacteroidales_ (11%, Figure 3a) increased together with _Clostridiales_ (10%, Figure 3c) in the bacterial populations, which is in line with what has been detected by other authors as the main bacterial populations present in anaerobic digesters [38]. The role of most members of these microbial populations is crucial in acid and acetogenesis since they can convert amino acids, sugars, and alcohols into volatile fatty acids. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Units**} & \multicolumn{3}{c}{**Relative Abundance (\%)**} \\ \cline{2-3}  & **Bacteria** & **Archaea** \\ \hline I & 98.31 & 1.69 \\ H (IN) & 100.0 & 0.00 \\ H (OUT) & 99.70 & 0.30 \\ HPU (IN) & 99.90 & 0.09 \\ HPU (OUT) & 99.13 & 0.87 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Relative abundance of bacteria and archaea domains. A quite different behavior was seen in the HPU units, after AD, in which Proteobacteria showed a slight increase from 44% to 47%, and since these microorganisms are described as secondary degraders of polysaccharides [39], it suggests the availability of more complex substrates in this unit. _Bacteroidales_ decreased from 37% to 4.9% (Figure 3a), and _Clostriidales_ increased from 7% to 9% (Figure 3c), whose order consisted mainly of members of the family _Ruminococcaceae_. These populations can hydrolyze compounds such as cellulose, starch, and proteins into acetate, improving the methane yield [40; 41]. Moreover, it is important to highlight that _Bacilluses_, which became part of the composition of this microbiota (7.5%, Figure 3c), are known as good carbohydrate-utilizing species [40]. The other populations, more specifically Bacteroidetes, known as proteolytic bacteria, became almost non-existent, suggesting that members of these two phyla, Bacteroidetes and Firmicutes, might compete for the same resources and energy. Methylotrophic, hydrogenotrophic, and acetoclastic are the three major pathways of methanogenesis [42]. In an anaerobic digester, the methane production depends on the methanogens and the substrates available for the process. Figure 4 shows the changes that occurred in the archaeal structure and composition during AD in the H and HPU units. Figure 2: Relative abundance of bacterial communities and taxonomy at phylum level. “Other” are phyla that were not considered due to their low presence (0.01–2%) in the composition: Acidobacteria; Actinobacteria; Armatimonadetes; Atribacteria; BRC1; Candidatus Berkelbacteria; Chlorobi; Cloactinetes; Deferribacteres; Deinococcus-Thermus; Elusmicrobia; Fusobacter; Gemmatimonadetes; Gracilibacteria; Hydrogenedentes; Latescibacteria; Lentisphaerae; Microgenonates; Nitrospirae; Omnitrophica; Parcubacteria; Planctomycetes; SR1 (Absconditobacteria); Sacchanibacteria; TA06; TM6 (Dependentiae); Tenericutes; Thermotogae; WS6; WWE3; and unidentified bacteria. Figure 3: Cont. Figure 3: Relative abundance of bacterial communities at the four main order levels: (**a**) Bacteroidetes; (**b**) Chloroflexi; (**c**) Firmicutes; and (**d**) \(\alpha\)-Proteobacteria, \(\beta\)-Proteobacteria, \(\delta\)-Proteobacteria, \(\varepsilon\)-Proteobacteria, and \(\gamma\)-Proteobacteria. The relative abundance is a percentage of the total number of sequences in each unit. All archaeal sequences were assigned to the phylum Euryarchaeota.