## 3 Results and Discussion
### Microbial Compositions in Anaerobic Digester  
To achieve efficient and stable AD, a diverse microbiome is required. The design, the chemical and physical composition of the feedstock, and the operational parameters of the reactors such as OLR, HRT, pH, and temperature have the greatest impact on the biogas-producing microorganisms [62]. The specific environmental conditions enhance the formation of different groups of microorganisms, and the stability of AD plays a key role in maintaining a delicate balance in microbial populations, which consequently ensures high biogas production [63]. Metagenomic analyses provide insight into the taxonomic diversity and the physiological potential of microbiomes, among which the identification of the biogas-producing microbiomes is the most important [64]. Links between bacterial and archaeal populations and AD process parameters have been studied by several authors [64; 65; 66; 67]. However, these correlations did not directly and clearly identify factors that promote AD or act as a barrier [63]. Nowadays, the most important issue is to identify the diversity and composition of individual microorganisms forming under different environmental conditions in order to better understand their behavior in response to AD process disturbances, and the main goal of these studies is to effectively improve the efficiency and stability of AD [63; 67]. Thus, there is now a high potential for microbial-based management in AD to determine a set of microbial indicators. A total of 23 taxonomic phyla were observed in anaerobic granular sludge samples collected from methanogenesis tanks operated at OLRs of 4 g COD/L-d, 8 g COD/L-d, and 12 g COD/L-d, represented by V1, V3, and V5, respectively (Figure 6). The abundance of Archaea in the anaerobic granular sludge was 4.85%, 2.95%, and 3.92% in V1, V3, and V5, respectively. Unclassified reads constituted only less than 0.5% at this level in all samples. Bacteria dominated, even if the anaerobic granular sludge samples were collected from the methanogenesis tank. The abundance of Archaea in the two-stage system operated in this study was similar to that observed in solid-state anaerobic digestion [68] and full-scale anaerobic digesters operated in municipal wastewater treatment plants [69]. Most of the Archaea were Euryarcheota, and among them, the domination of Methanomicobia (1.5% in V1, 1.6% in V2, 2.6% in V5) and Methanobacteria (3.3% in V1, 1.3% in V3, 1.2% in V3) was observed. The abundance of the former increased with the increase in OLR, and the abundance of the latter decreased with an increase in OLR (Figure 7). The abundance of Archaea was similar in all operating variants; therefore, bacteria providing the substrate for Archaea were probably more responsible for observed differences in biogas production. These different supplies of substrates could affect the activity of Archaea and thus the efficiency of biogas production. Among methanogens in all tanks, the most abundant species was acetoclastic Methanosaeta concilii. Methanosaeta grow in stable habitats characterized by low concentrations of acetate, due to their affinity for acetate [70; 71]. [PERSON] and co-workers [72] investigated six different samples of anaerobic sludges, treating wastewater containing simple and complex compounds with low- and high-strength treatments at psychrophilic (10-14 \({}^{\circ}\)C), mesophilic (37 \({}^{\circ}\)C), and thermophilic (55 \({}^{\circ}\)C) temperatures. The authors observed domination of Methanosaeta spp. irrespective  
Figure 6: The abundance of phyla in anaerobic granular sludge in V1, V3, and V5 in stage 1.  
of wastewater type or operating conditions, showing underlying potential for effective growth of Methanosaeta sp. in different environments and ensuring efficient operation of an anaerobic bioreactor [72]. At the phylum level, Bacteroides were the most abundant in V1 (30.7%) and V3 (38.7%) (Figure 6).