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.

A novel bacterial strain, designated HK31-P ^T , was isolated from a deep subsurface geothermal aquifer in southwestern Iceland. Phylogenetic analyses, based on 16S rRNA gene sequences, revealed that the strain was affiliated to the genus Phreatobacter . Cells of the strain were Gram-negative, rod-shaped, motile by means of a polar flagellum and granule-containing. Colonies were small, circular, convex, smooth and white in colour. Growth under chemoorganoheterotrophic and aerobic conditions was observed at the following ranges: 20–35 °C (optimum, 30–35 °C), pH 7–9 (optimum, 8) and 0–0.5% salinity (optimum, 0%). The strain was also capable of growing under chemolithoautotrophic and (micro)aerobic conditions with Fe(II) as an electron donor. The major respiratory quinone identified was ubiquinone Q-10 (97.0%), and the predominant fatty acid was C _18:1  ω 7c. The dominant polar lipids consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid, along with several unidentified glycolipids, phospholipids and lipids. The G+C content of the genomic DNA of strain HK31-P ^T was 67.66 mol%, and its complete genome was 4.34 Mbp in size. The values of the digital DNA–DNA hybridization and average nucleotide identity indices between strain HK31-P ^T and the type species of the genus Phreatobacter ranged, respectively, from 21.30 to 27% and from 78.25 to 86.32%. These values are well below the accepted species delineation thresholds, thereby clearly indicating that strain HK31-P ^T represents a distinct genomic species within the genus Phreatobacter , strongly supporting that the former represents a new species belonging to the genus Phreatobacter . Therefore, based on this polyphasic approach, strain HK31-P ^T represents a new species within the genus Phreatobacter , for which the name Phreatobacter aquiterrae sp. nov. is proposed. The type strain is HK31-P ^T (DSM 116433 ^T =UBOCC-M-3430 ^T ).

Les Methanomassiliicoccales (MX) sont des archées méthanogènes hydrogénotrophes méthyle-dépendantes (MHMD) réparties en deux clusters phylogénétiques reflétant leurs adaptations aux écosystèmes digestifs (host-associated) ou non-digestifs (free-living). Nos connaissances les concernant se limitent surtout à des études centrées sur des milieux digestifs. De nombreuses questions demeurent sur le mode de vie et les adaptations des représentants du cluster free-living dans des habitats non-digestifs. Cette thèse visait à améliorer nos connaissances sur leur biologie en étudiant leur distribution, leur diversité, leur abondance et potentiels métaboliques et physiologiques dans des habitats non-digestifs. Des analyses de diversité et d’abondance sur un panel d’échantillons de natures et d’origines variées ont permis de mettre en évidence la faible abondance et diversité taxonomique des MX dans les échantillons criblés. Des approches culturales ont permis d’obtenir un enrichissement significatif en MX à partir d'un microcosme inoculé avec des sédiments d'eau douce. Une analyse en métagénomique de cet enrichissement a permis la reconstruction d'un metagenome assembled genome (MXMAG1) de grande qualité correspondant à une nouvelle espèce candidate affiliée au cluster free-living. Son analyse a révélé la présence des gènes impliqués dans la réponse et la régulation de stress environnementaux (ex : pH, métaux lourds) et ceux impliqués dans la MHMD. Enfin, des analyses en réseaux de cooccurrence basées sur des données de diversité ont révélé la cooccurrence des MX avec des taxa bactériens inféodés aux tractus digestifs et connus pour entretenir des relations syntrophiques avec des archées méthanogènes.