## 3 Results
### Global Overview of the Microbial Ecology in the Monitored AD Plants  
The monitoring of bacterial and archaeal compositions of the four selected AD plants was obtained using 16S rRNA gene sequencing. The main results are reported in Table 2. About 48% of the sequenced reads from the digesters D2 and D3 have been assigned to OTUs, while for D1 and D4 the percentage is slightly lower, 41 and 40%, respectively. Alpha diversity indexes were similar for D2-D4 and slightly higher in the case of D1. Rarefaction curves (Figure S2) show that adequate sequencing depth was achieved to thoroughly describe the microbial community. Given the negligible impact of the less abundant OTUs on the production of intermediate compounds and biogas, only the 73 OTUs with a relative abundance higher than 0.5% in at least one of the three replicates have been considered in the following analyses. Particularly, the dominant OTUs account, on average, for 85% of the diversity of the microbial community in the biogas plants. The beta diversity was represented in Figure 2a as a principal coordinate analysis (PCoA) plot. PCo1, PCo2, and PCo3 explained 39.3, 17.2, and 13.6% of the total diversity, respectively. As expected, samples collected from agricultural plants were clearly clustered in restricted areas and well-separated from samples of reactor D4. This finding can be \begin{table}
\begin{tabular}{c c c c c} \hline  & **D1** & **D2** & **D3** & **D4** \\ \hline Raw reads & 241,360 & 227,000 & 235,940 & 225,250 \\ Assigned reads & 100,600 & 109,500 & 113,700 & 90,510 \\ OTUs & 387 & 389 & 395 & 393 \\ Alpha diversity & 86.6 & 83.5 & 83.2 & 84.3 \\ \hline \end{tabular}
\end{table} 
Table 2: Summary of sequencing results and alpha diversity indexes. ascribed to the major contribution of the feedstocks (agricultural _vs._ municipal waste) in shaping the microbiome (Table 1). The marked difference in feedstock composition and microbial structure resulted also in a different digestate composition (Figure 1a). Moreover, the three agricultural AD plants were characterized by a stable microbial composition, whereas the D4 showed clear microbial changes throughout the monitored  
Figure 2: (a) PCoA plots representing the similarity in the microbial communities of the four biogas plants obtained from the beta-diversity values (each spot is the average of the three biological replicates as described in Section 2.1); (b) Venn diagram representing the main OTUs shared among biogas plants and those specifically identified in one of the four microbiomes. OTUs reported in bold started populating D4 reactor only after COVID-19 related issues. period (Figure 2a). This agrees with recent surveys on the monitoring of microbiomes of agricultural and food waste-treating AD plants [28; 29]. The progressive shift in the microbiome composition experienced by D4 is evident in Figure 2a where the dots form a long tail diverging from the central cluster. At a higher taxonomic level, Firmicutes, followed by Bacteroidetes, and Synergistetes were found to be the predominant phyla in all the AD plants (Figure S3), in agreement with previous studies [10; 28], while Thermotogae specifically contributed to the hydrolysis and fermentation of nutrient sources in D1 and D4. Among the methanogenic archaea, the identified microbes belong to the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales. The microbial structure of D2 and D3 was nearly identical, dominated by Firmicutes (approximately 50.6%), Bacteroidetes and Synergistetes (about 21.2 and 8.6%, respectively), while Euryarchaeota was less than 1.6% on average. D1 had a slightly different microbial distribution with a lower frequency of Firmicutes and Bacteroidetes but an increased abundance of Thermotogae. Due to its specific feedstock (OFMSW), and in line with the data displayed in the PCoA (Figure 2a), plant D4 exhibited a different microbial population.