## 3 Results and Discussion
### Two-Stage Anaerobic Digestion of Wheat Straw and Algal Biomass
#### 3.2.4 Microbial Communities' Composition
Another species with a relatively high share in BR-1 was _Californonas adultescens_ (Figure 9). This is a thermophilic species that produces ethanol in addition to acetate and CO\({}_{2}\) during growth on complex substrates, which is also typical for _Caldanaerobius_ and _Thermomaerobacterium_ genera [38]. Differing from BR-1, the methane-generating BR-2 contains a bacterial community dominated by representatives of _Flavobacteriaia_ (30.28%) and _Bacteroidia_ (27.81%), as well as _Opittate_ (13.13%), _Synergistia_ (2.43%), _Desulfurononadia_ (2.22%), and, to a lesser extent, _Negativictates_ (0.97%), _Anaerolineae_ (0.51%), _Verrucomicrobiae_ (0.41%), _Syntrophia_ and _Tissierella_ (0.24% each), followed by _Syntrophobacteria_ (0.08%), _Erysipelotrichia_ (0.04%), and _Ignavibacteria_ (0.02%), and _Actinomycetes_, _Myxococcus_, and _Spirochaeta_ (with one-tenth of a percent each) (Figure 10). _Clostridia_ constituted 6.03% of the community, in contrast to the hydrogen-producing fermenter. An analysis of the archaeal community showed the presence of four species, with one of them completely conquering BR-2. The most abundant archaeal representative possessed a high similarity (99.2% 16S rRNA homology) with the phylum _Bathyarchaeota_ and comprised 99.5% of the archaeal community. Other archaea in BR-2 were _Methanobacterium formicicum_, _Methanoplasma termium_, and an unidentified species of the genus _Thermoproteota_ (Figure 11). _Methanobacterium formicicum_ is a representative of the phylum _Euryarchaeota_ and is often found in anaerobic biogas-producing systems [39]. However, the presence of _Bathyarchaeota_ and _Methanoplasma_ in the anaerobic digester is quite unusual. Both methanogens belong to _Proteoarchaeota_, of the phylum _Thermoproteota_, a lineage of hyperthermophilic _Crenarchaeota_, which is abundant in deep marine subsurface sediments [40]. Although _Bathyarchaeota_ has not yet been cultivated in pure cultures, there are data on their metabolism, thanks to reconstructed genomes. According to [PERSON] et al. (2015) [41], these archaea are methylotrophic methanogens, feeding on a wide variety of methylated compounds. They break down peptides, glucose, fatty acids, and glucose. The identification of key genes related to methanogenesis reveals mechanisms close to those of _Euryarchaeota methanogens_. Figure 10: Bacterial diversity in BR-2, analysed via metagenomics. The species are listed in order of their decreasing share of the bacterial community in percent. _Crenarchaeota_ is one of the most abundant thermophilic archaeal phyla in terrestrial environments. However, nonthermophilic _Crenarchaeota_ can be found in soils and marine freshwater sediments. New discoveries are anticipated, because archaea have become one of the most exciting current topics of microbial aquatic research [42]. The natural abundances of uncultured archaea are substantial; thus, for elucidating metabolic pathways and fully understanding archaea, combinations of new cultivation strategies with high-resolution molecular technologies and bioinformatics tools must be used. The dominant development of _Crenarchaeotes_ in our case can be attributed to the high temperatures during the process. A spontaneous initial inoculation of BR-2 with _Crenarchaeota_, along with the algal part of the substrate, probably occurred during operation of the two-stage system.