Phenotypic and genomic characterization of the first alkaliphilic aceticlastic methanogens and proposal of a novel genus Methanocrinis gen.nov. within the family Methanotrichaceae

Abstract

Highly purified cultures of alkaliphilic aceticlastic methanogens were collected for the first time using methanogenic enrichments with acetate from a soda lake and a terrestrial mud volcano. The cells of two strains were non-motile rods forming filaments. The mud volcano strain M04Ac was alkalitolerant, with the pH range for growth from 7.5 to 10.0 (optimum at 9.0), while the soda lake strain Mx was an obligate alkaliphile growing in the pH range 7.7–10.2 (optimum 9.3–9.5) in the presence of optimally 0.2–0.3 M total Na+. Genomes of both strains encoded all enzymes required for aceticlastic methanogenesis and different mechanisms of (halo)alkaline adaptations, including ectoine biosynthesis, which is the first evidence for the formation of this osmoprotectant in archaea. According to 16S rRNA gene phylogeny, the strains possessed 98.3–98.9% sequence identity and belonged to the obligately aceticlastic genus Methanothrix with M. harundinaceae as the most closely related species. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein-coding marker genes clearly indicated a polyphyletic origin of the species included in the genus Methanothrix. We propose to reclassify Methanothrix harrundinacea (type strain 8AcT) into a new genus, Methanocrinis gen. nov., with the type species Methanocrinis harrundinaceus comb. nov. We also propose under SeqCode the complete genome sequences of strain MxTs (GCA_029167045.1) and strain M04AcTs (GCA_029167205.1) as nomenclatural types of Methanocrinis natronophilus sp. nov. and Methanocrinis alkalitolerans sp. nov., respectively, which represent other species of the novel genus. This work demonstrates that the low energy aceticlastic methanogenesis may function at extreme conditions present in (halo)alkaline habitats.

Peloplasma aerotolerans gen. nov., sp. nov., a Novel Anaerobic Free-Living Mollicute Isolated from a Terrestrial Mud Volcano

“…Strain M4Ah was isolated from the enrichment culture containing "Methanocrinis alkalitolerans" as the dominant population [16]. This enrichment culture was obtained from a sample of mud collected from the active gryphon (ca.…”

“…1 year, strain M4Ah was isolated as a satellite bacterium with the relative abundance of 2.5% [16]. A further enrichment and isolation of the strain M4Ah were performed in the anaerobic liquid medium of the following composition (per liter of distilled water): KH 2 PO 4 , NH 4 Cl, KCl, and MgCl 2 •6H 2 O (0.33 g each); CaCl 2 •6H 2 O, 0.033 g; NaCl, 10.00 g; NaHCO 3 , 2.00 g; and 1 mL of a trace element solution [16] and 1 mL of a vitamin solution [17] and resazurin solution (0.001 g L −1 ). The medium was boiled and cooled under N 2 flow before adding NaHCO 3 , vitamins, and Na 2 S•9H 2 0 (0.5 g L −1 ).…”

“…The strain M4Ah was isolated from a culture highly enriched in the methanogenic archaeon "M. alkalitolerans" [16]. After serial 10-fold dilutions in the same liquid medium performed consequently for three times, profiling of 16S rRNA gene amplicons by using whole-genome sequencing and a metagenomic bioinformatics analysis in the highest growth positive dilution (10 −7 ) revealed the presence of sequences related to Methanothrix and the Acholeplasmataceae bacterium with the relative abundance of 97.5 and 2.5%, respectively [16]. During the cultivation of this co-culture on the PYG medium, multiple small round cells were observed in the light microscope.…”