## 3 Results
### Molecular Characterization of Microbial Communities  
A molecular characterization was conducted for inoculum and samples collected at the beginning (IN) and at the end (OUT) from H and HPU units to observe changes in the microbial populations during the experiment. After NGS, the samples created between 600,188 and 837,438 raw sequence reads, corresponding to sample H (OUT) and to the inoculum (I), respectively. Table 5 shows a total of 1,074,985 sequences (1,068,111 bacterial, 6850 archaeal, and 24 unassigned) which were recovered and analyzed. A total of 3224 operational taxonomic units (OTUs) that comprised libraries were detected in each sample. The sludge used for inoculation had the highest Shannon-Wiener diversity index value (8.52) for bacteria, and a suitable archaeal diversity index value (2.50), suggesting that it is a reliable source of diverse bacterial and archaea populations for the AD process (Table 5). In both experiments, H and HPU, samples OUT exhibited higher diversity indices than samples IN, which is confirmed by the Shannon-Wiener index. The higher increase \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Units**} & **Number of** & \multirow{2}{*}{**OTUs**} & \multicolumn{2}{c}{**Shannon–Wiener Index**} \\ \cline{3-5}  & **Sequences** & & **Bacteria** & **Archaea** \\ \hline I & 258,364 & 1248 & 8.52 & 2.50 \\ H (IN) & 236,493 & 65 & 1.64 & 0.00 \\ H (OUT) & 199,089 & 558 & 4.03 & 4.44 \\ HPU (IN) & 192,070 & 727 & 3.89 & 2.68 \\ HPU (OUT) & 188,969 & 626 & 4.44 & 3.81 \\ Total & 1,074,985 & 3224 & - & - \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Sequencing data results and microbiota diversity index. observed either in the OTUs or in the diversity index values for H units, during the anaerobic digestion period, indicates that there was a lower microbial acclimation to digest the heterotrophic algae than in the HPU units, which is consistent with the higher methane yields obtained in these samples (Table 3). Moreover, there was not any type of inhibition of the microbial growth in these units, and this is consistent with the reactor's satisfactory performance. During the analysis of microbial diversity, it is appropriate to guarantee that the number of readings reached a satisfactory value, so that more sequencing does not significantly increase species diversity, since the rarefaction curves are an estimate of species richness. Figure S4 indicates that, at similarity levels of 97%, the rarefaction curve grows rapidly at the beginning, when the most common OTUs were found, and then stabilizes, becoming asymptotic, since only the rarest species remain to be sequenced. Sequencing was sufficient for all samples, and the sequence dataset had complete sample diversity in this analysis, and sufficient sequence depth was achieved. The relative abundance of groups of bacteria and archaea was determined in terms of the percentage of the total number of sequences in each sample. Table 6 shows that, as in other anaerobic digestion processes [33; 34; 35], the bacteria domain predominates over the archaea domain in all studied units, accounting for more than 98.3% of the relative abundance. The relative abundance of the archaeal domain increased during AD and was especially pronounced in the digestion of ultrasound pretreated heterotrophic algae (HPU), which confirmed the highest methane yield obtained for this substrate. The compositions of bacterial communities with a relative abundance greater than 2%, in at least one sample, show 11 major phyla (Figure 2). A majority of about 82% were assigned to Bacteroidetes, Firmicutes, Proteobacteria, Spirochaeta, and Synergistetes, which are known to efficiently convert complex compounds into methane-containing biogas [36]. Proteobacteria, Firmicutes, and Bacteroidetes are known to be abundant in anaerobic digestion processes [34; 37].