Characterization of organisms recovered from metagenome sequence data of microbial biofilms residing in mesophilic and thermophilic biogas reactors

Members of the recently proposed archaeal phylum Bathyarchaeota are currently discussed to be capable of methylotrophic methanogenesis, indicating that methane metabolism also exists outside the phylum Euryarchaeota. To identify members of phylum Bathyarchaeota participating in biotechnological biogas fermentations and to unravel their putative role during anaerobic digestion and biomethanation of crop residues, microbial biofilms residing in mesophilic (37 °C) and thermophilic (55 °C) biogas fermenters were sampled. Metagenome shotgun libraries of corresponding microbiomes were prepared and sequenced on the Illumina MiSeq system. Taxonomic classification of the biofilm microbiomes revealed that between 0.1% and 2% of all classified sequences could be assigned to the phylum Bathyarchaeota. Individual metagenome assembly of the biogas microbiomes followed by genome binning resulted in reconstruction of five Bathyarchaeota genome bins (GBs) estimated to be 65% to 92% complete, featuring contamination rates of less than 10% and ranging in the genome sizes from 1.1 Mb to 2.0 Mb. Phylogenetic classification of these GBs based on core gene sets confirmed their placement within the novel archaeal phylum Bathyarchaeota. Moreover, Bathyarchaeota GBs originating from the biogas reactors cluster together within the tree and thus represent a separate group that diverged from other Bathyarchaeota groups. The genetic repertoire of the analyzed Bathyarchaeota GBs indicated an energy metabolism based on carbohydrate and amino acid fermentation with potential for extracellular hydrolysis of cellulose, cellobiose as well as proteins and corresponding transporter systems. Neither hydrogenotrophic nor aceticlastic or methylotrophic methanogenesis pathways were completely encoded in the Bathyarchaeota GBs. However, all GBs harbor genes encoding enzymes for utilization of carbon monoxide and /or carbon dioxide via the Wood-Ljungdahl pathway. Based on these findings, a hydrolytic lifestyle is proposed for the five Bathyarchaeota members represented by the GBs analyzed. This is the first study indicating that Bathyarchaeota members presumably contribute to fermentation of organic substrates and hydrolysis within the biogas production process.