Maria A. Khomyakova

A novel facultatively anaerobic halotolerant strain C05BenzN^T was isolated from a terrestrial mud volcano Gnilaya Gora (Taman Peninsula, Russia). Cells were rod-shaped, motile and Gram-stain-negative. The temperature range for growth was 14–42°C (optimum at 25°C). The pH range for growth was 7.5–11.0 (optimum at pH 8.0). NaCl range for growth constituted up to 20% with the optimum 2.0–3.0%. Strain C05BenzN^T grew aerobically, microaerobically with 1% O₂ or anaerobically with nitrate as an electron acceptor. Carbohydrates, proteinaceous substrates, organic acids and various aromatic compounds were used as electron donors during aerobic growth and acetate under microaerobic and anaerobic conditions. The G+C content of the genomic DNA was 67.4%. The fatty acid profile of strain C05BenzN^T was characterized by the presence of C_18:1 cis11, C_16:1 cis9, C_16:0 as the major components. The strain possessed genes related to variable carbon and nitrogen metabolism, hinting at its substantial metabolic flexibility. The results of phylogenetic analysis based on 120 bacterial single copy conservative marker genes revealed that the closest phylogenetic relatives of strain C05BenzN^T were Halomonas heilongjiangensis, Halomonas flagellata and Halomonas kalidii (with ANI range 88.4–88.6%). Based on the phenotypic, genotypic and phylogenetic characteristics of the isolate, strain C05BenzN^T is considered to represent a novel species of the genus Halomonas, with proposed name Halomonas terrestris sp. nov. The type strain of Halomonas terrestris is C05BenzN^T (=KCTC 8548^T = CGMCC 1.61916^T).

A novel aerotolerant anaerobic bacterium (strain M4Ah^T) was isolated from a terrestrial mud volcano (Taman Peninsula, Russia). Cells were small, cell-wall-less, non-motile cocci, 0.32–0.65 μm in diameter. The isolate was a mesophilic, neutrophilic chemoorganoheterotroph, growing on carbohydrates (D-glucose, D-trehalose, D-ribose, D-mannose, D-xylose, D-maltose, D-lactose, D-cellobiose, D-galactose, D-fructose, and D-sucrose), proteinaceous compounds (yeast extract, tryptone), and pyruvate. Strain M4Ah^T tolerated 2% oxygen in the gas phase, was catalase-positive, and showed sustainable growth under microaerobic conditions. The dominant cellular fatty acids of strain M4Ah^T were C_16:0 and C_18:0. The G+C content of the genomic DNA was 32.42%. The closest phylogenetic relative of strain M4Ah^T was Mariniplasma anaerobium from the family Acholeplasmataceae (order Acholeplasmatales, class Mollicutes). Based on the polyphasic characterization of the isolate, strain M4Ah^T is considered to represent a novel species of a new genus, for which the name Peloplasma aerotolerans gen. nov., sp. nov. is proposed. The type strain of Peloplasma aerotolerans is M4Ah^T (=DSM 112561^T = VKM B-3485^T = UQM 41475^T). This is the first representative of the order Acholeplasmatales, isolated from a mud volcano.

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 8Ac^T) 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 Mx^Ts (GCA_029167045.1) and strain M04Ac^Ts (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.

Bathyarchaeia are widespread in various anoxic ecosystems and are considered one of the most abundant microbial groups on the earth. There are only a few reports of laboratory cultivation of Bathyarchaeia, and none of the representatives of this class has been isolated in pure culture. Here, we report a sustainable cultivation of the Bathyarchaeia archaeon (strain M17C^Ts) enriched from anaerobic sediment of a coastal lake. The cells of strain M17C^Ts were small non-motile cocci, 0.4–0.7 μm in diameter. The cytoplasmic membrane was surrounded by an S-layer and covered with an outermost electron-dense sheath. Strain M17C^Ts is strictly anaerobic mesophile. It grows at 10–45°C (optimum 37°C), at pH 6.0–10.0 (optimum 8.0), and at NaCl concentrations of 0–60 g l⁻¹ (optimum 20 g l⁻¹). Growth occurred in the presence of methoxylated aromatic compounds (3,4-dimethoxybenzoate and vanillate) together with complex proteinaceous substrates. Dimethyl sulfoxide and nitrate stimulated growth. The phylogenomic analysis placed strain M17C^Ts to BIN-L-1 genus-level lineage from the BA1 family-level lineage and B26-1 order-level lineage (former subgroup-8) within the class Bathyarchaeia. The complete genome of strain M17C^Ts had a size of 2.15 Mb with a DNA G + C content of 38.1%. Based on phylogenomic position and phenotypic and genomic properties, we propose to assign strain M17C^Ts to a new species of a novel genus Bathyarchaeum tardum gen. nov., sp. nov. within the class Bathyarchaeia. This is the first sustainably cultivated representative of Bathyarchaeia. We propose under SeqCode the complete genome sequence of strain M17C^Ts (CP122380) as a nomenclatural type of Bathyarchaeum tardum, which should be considered as a type for the genus Bathyarchaeum, which is proposed as a type for the family Bathyarchaeaceae, order Bathyarchaeales, and of the class Bathyarchaeia.

An Erratum to this paper has been published: https://doi.org/10.1134/S0026261723090011

A novel alkaliphilic sulfate-reducing bacterium, strain M08but^T, was isolated from a salsa lake of terrestrial mud volcano (Taman Peninsula, Russia). Cells were rod-shaped, motile and Gram-stain-negative. The temperature range for growth was 15–42 °C (optimum at 30 °C). The pH range for growth was 7.0–11.0, with an optimum at pH 8.5–9.0 Strain M08but^T used sulfate, thiosulfate, sulfite, dimethyl sulfoxide and arsenate as electron acceptors. Acetate, formate, butyrate, fumarate, succinate, glycerol and pyruvate were utilized as electron donors with sulfate. Fermentative growth was observed with fumarate, pyruvate, crotonate. Strain M08but^T grew chemolithoautotrophically with H₂ and CO₂. The G + C content of the genomic DNA was 60.1%. The fatty acid profile of strain M08but^T was characterized by the presence of anteiso-C_15:0 as the major component (68.8%). The closest phylogenetic relative of strain M08but^T was Desulfatitalea tepidiphila (the order Desulfobacterales) with 96.3% 16S rRNA gene sequence similarity. Based on the phenotypic, genotypic and phylogenetic characteristics of the isolate, strain M08but^T is considered to represent a novel species of the genus Desulfatitalea, with proposed name Desulfatitalea alkaliphila sp. nov. The type strain of Desulfatitalea alkaliphila is M08but^T (= KCTC 25382^T = VKM B-3560^T = DSM 113909^T = JCM 39202^T = UQM 41473^T).