## 3 Results and Discussion
### Microbial Compositions in Anaerobic Digester
In V5, the abundance of Firmicutes increased so that they constituted the majority (30.7%); however, the abundance of Bacteroides was still high (28.8%). The abundance of Firmicutes was 16.1% in V1, and it was the second most abundant phylum. In V3, the abundance of Firmicutes increased to 30.7% and remained at that level also in V5 (which was mentioned above). Firmicutes are characterized by the ability to degradation of different kinds of organic compounds under various conditions [73]. The domination of Firmicutes was observed in a reactor with high acidification conditions (at OLRs of 11.0, 12.9, and 14.0 g COD/L-d) during the anaerobic digestion process of two-phase olive mill residue [74]. However, some other studies reported by the same authors showed that Firmicutes were the predominant bacteria at a low OLR [75]. This is in contradiction with the observations in the present study, where the abundance of Firmicutes increased at higher OLRs. It turns out that such statements on the phylum level are probably too generalized, due to phyla being represented by numerous microorganisms, which can be replaced one by another. Bacteroidetes are responsible for hydrolytic and fermentative reactions in a range of organic substrates such as municipal waste and manure organic materials with complex carbohydrates [76; 77; 78; 79; 80]. Figure 7: The abundance of classes in anaerobic granular sludge in V1, V3, and V5 in stage 1. In V1, the abundance of phyla was characterized by an even distribution, except for the domination of Bacteroides (Figure 6). The abundance of Proteobacteria and Chloroflexi was about 10%. Next, Euryarcheota, Nitrospirae, Actinobacteria, and Synergistates had an abundance of about 3-5%. In V3, the domination of Bacteriotes and Firmicutes (both at about 30%) above other phyla was significantly marked. The phyla Proteobacteria, Synergistates, Chloroflexi, Actinobacteria, Nitrospirae, and Euryarcheota (the abundance in this order) constituted 3-5%. In V5, the domination of Bacterioidetes and Firmicutes was also clearly marked; however, the abundance of Proteobacteria increased to 13%. In V5, the abundance of Thermotogae was 6.87%, which constituted less than 1% in V1 and V3. Only the abundance of Euryarcheota (3.91%) and Chloroflexi (2.41%) exceed 2%, and the abundance of other phyla was less than 2% in V5. The conditions in V1 (low OLR, neutral pH) were more favorable for a more diverse community than the more restricted conditions in V5 (high OLR, acidic pH), which caused most of the species to be less abundant. Therefore, an increase in OLR reduced microbial richness and the even distribution of populations. This evenest distribution of phyla in V1 ensured high biogas production. The phylum Bacteroidetes was represented by classes Flavobacteria, Sphingobacteria, and Bacteriadia at all applied conditions. The abundance of Flavobacteria was the highest in V1 (14.1%); in V3 and V5, its abundance was 1.7% and 1.3%, respectively (Figure 7). Flavobacteria were recognized as bacteria responsible for the degradation of carbohydrates and proteins [81], which were also observed in an acidogenic reactor [82]. The presence of Sphingobacteria and Bacteriadia, in the present study, was characterized by a similar tendency; however, they differed in numbers. These classes were most abundant in V3 (22.8% and 12.7%, respectively), and then their abundance decreased in V5 (17.5% and 9%, respectively) and was the lowest in V1 (11% and 4.8%, respectively) (Figure 7). Class Bacteria was recognized as important hydrolyzers and fermenters, which can grow in extreme pH conditions [83; 84]. The phylum Firmicutes was represented by classes Clostridia and Bacilli. The abundance of Clostridia was 12.7% in V1 and about 27% in V3 and V5 (Figure 7). Clostridia are mostly recognized as cellulose-hydrolyzing and protein-hydrolyzing bacteria [85]. These bacteria also perform acidogenesis and produce short-chain fatty acids, CO\({}_{2}\), and H\({}_{2}\).