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The methanogenic strain Mx-05 T was isolated from the human fecal microbiome. A phylogenetic analysis based on the 16S rRNA gene and protein marker genes indicated that the strain is affiliated with the order Methanomassiliicoccales . It shares 86.9% 16S rRNA gene sequence identity with Methanomassiliicoccus luminyensis , the only member of this order previously isolated. The cells of Mx-05 T were non-motile cocci, with a diameter range of 0.4-0.7 μm. They grew anaerobically and reduced methanol, monomethylamine, dimethylamine, and trimethylamine into methane, using H 2 as an electron donor. H 2 /CO 2 , formate, ethanol, and acetate were not used as energy sources. The growth of Mx-05 T required an unknown medium factor(s) provided by Eggerthella lenta and present in rumen fluid. Mx-05 T grew between 30 °C and 40 °C (optimum 37 °C), over a pH range of 6.9-8.3 (optimum pH 7.5), and between 0.02 and 0.34 mol.L -1 NaCl (optimum 0.12 mol.L -1 NaCl). The genome is 1.67 Mbp with a G+C content of 55.5 mol%. Genome sequence annotation confirmed the absence of the methyl branch of the H 4 MPT Wood-Ljungdahl pathway, as described for other Methanomassiliicoccales members. Based on an average nucleotide identity analysis, we propose strain Mx-05 T as being a novel representative of the order Methanomassiliicoccales , within the novel family Methanomethylophilaceae , for which the name Methanomethylophilus alvi gen. nov, sp. nov. is proposed. The type strain is Mx-05 T (JCM 31474T).

Members of the archaeal order Methanomassiliicoccales are methanogens mainly associated with animal digestive tracts. However, environmental members remain poorly characterized as no representatives not associated with a host have been cultivated so far. In this study, metabarcoding screening combined with quantitative PCR analyses on a collection of diverse non-host-associated environmental samples revealed that Methanomassiliicoccales were very scarce in most terrestrial and aquatic ecosystems. Relative abundance of Methanomassiliicoccales and substrates/products of methanogenesis were monitored during incubation of environmental slurries. A sediment slurry enriched in Methanomassiliicoccales was obtained from a freshwater sample. It allowed the reconstruction of a high-quality metagenome-assembled genome (MAG) corresponding to a new candidate species, for which we propose the name of Candidatus 'Methanomassiliicoccus armoricus MXMAG1'. Comparison of the annotated genome of MXMAG1 with the published genomes and MAGs from Methanomassiliicoccales belonging to the 2 known clades ('free-living'/non-host-associated environmental clade and 'host-associated'/digestive clade) allowed us to explore the putative physiological traits of Candidatus 'M. armoricus MXMAG1'. As expected, Ca. 'Methanomassiliicoccus armoricus MXMAG1' had the genetic potential to produce methane by reduction of methyl compounds and dihydrogen oxidation. This MAG encodes for several putative physiological and stress response adaptations, including biosynthesis of trehalose (osmotic and temperature regulations), agmatine production (pH regulation), and arsenic detoxication, by reduction and excretion of arsenite, a mechanism that was only present in the 'free-living' clade. An analysis of co-occurrence networks carried out on environmental samples and slurries also showed that Methanomassiliicoccales detected in terrestrial and aquatic ecosystems were strongly associated with acetate and dihydrogen producing bacteria commonly found in digestive habitats and which have been reported to form syntrophic relationships with methanogens.