Ecology of Methanonatronarchaeia

Abstract

Methanonatronarchaeia represents a deep-branching phylogenetic lineage of extremely halo(alkali)philic and moderately thermophilic methyl-reducing methanogens belonging to the phylum Halobacteriota. It includes two genera, the alkaliphilic Methanonatronarchaeum and the neutrophilic Ca. Methanohalarchaeum. The former is represented by multiple closely related pure culture isolates from hypersaline soda lakes, while the knowledge about the latter is limited to a few mixed cultures with anaerobic haloarchaea. To get more insight into the distribution and ecophysiology of this enigmatic group of extremophilic methanogens, potential activity tests and enrichment cultivation with different substrates and at different conditions were performed with anaerobic sediment slurries from various hypersaline lakes in Russia. Methanonatronarchaeum proliferated exclusively in hypersaline soda lake samples mostly at elevated temperature, while at mesophilic conditions it coexisted with the extremely salt-tolerant methylotroph Methanosalsum natronophilum. Methanonatronarchaeum was also able to serve as a methylotrophic or hydrogenotrophic partner in several thermophilic enrichment cultures with fermentative bacteria. Ca. Methanohalarchaeum did not proliferate at mesophilic conditions and at thermophilic conditions it competed with extremely halophilic and moderately thermophilic methylotroph Methanohalobium, which it outcompeted at a combination of elevated temperature and methyl-reducing conditions. Overall, the results demonstrated that Methanonatronarchaeia are specialized extremophiles specifically proliferating in conditions of elevated temperature coupled with extreme salinity and simultaneous availability of a wide range of C₁ -methylated compounds and H₂ /formate.

FIGURE 1

Potential methanogenic activity in sediment slurry from hypersaline soda lakes in Kulunda Steppe (Altai, Russia) incubated at moderately thermophilic conditions. (A) Tanatar‐1 (2014) at 48°C; (B) Stamp Lake (11KL‐015) at 54°C, soda crystallizer (7KL‐015) and Crooked Lake (12KL‐014) at 48°C. Substrates were added at 5 mM. The endogenous rates (without substrates) were subtracted. Mean values from duplicate experiments. MeOH (or Me), methanol; TMA, trimethylamine; (+f), with formate addition as an external electron donor

FIGURE 2

Potential methanogenic activity of methylotrophic methanogenesis at moderately thermophilic conditions in anaerobic sediment slurries from hypersaline salt lakes with neutral pH (Russia). (A) Mixed sediments from four hypersaline lakes in Kulunda Steppe, Altai (2014); (B) salt concentrator in Eupatoria (Crimea, 2015); (C) lake Baskunchak and (D) lake Elton (south Russia, 2016). Incubation temperatures: (A, B) = 48°C, (C, D) = 50°C. The endogenous rates were subtracted. The methanogenesis with formate alone was not observed. Mean values from duplicate experiments.

FIGURE 3

Differential influence of incubation temperature on the potential rates of classical methylotrophic (V1) and methyl‐reducing (V2, with formate as e‐donor) methanogenesis in sediment slurries from hypersaline lakes. MA, methylamine, DMA, dimethylamine, TMA, trimethylamine. (A) Hypersaline soda lakes in Kulunda Steppe (Altai, Russia); (B, C), hypersaline salt lakes with neutral pH in Kulunda Steppe (mixed sample) and in south Russia. The endogenous rates were subtracted. The methanogenesis with formate alone was not observed. Mean values from duplicate experiments

FIGURE 4

Methyl‐reducing methanogenesis in sediment slurries from the mix Kulunda Steppe hypersaline soda lake sediments at 50°C, 4 M Na⁺/pH 9.5 amended with formate as e‐donor and different methyl acceptors (5 mM each). The numbers at the curve end‐points indicate % of the AMET‐like 16S rRNA amplicon sequences from the total archaeal population and the closest related AMET strains known in pure culture are shown in parentheses. AMET1 and AMET4/5 represent the type species Methanonatronarchaeum thermophilum, while AMET6‐2 belongs to a putative new species of Methanonatronarchaeum. At the start of incubation the abundance of Methanonatronarchaeia determined by qPCR and 16S rRNA gene amplicon sequencing was ~1% from total archaeal population

FIGURE 5

Results of mcrA‐DGGE analysis of methanogenic populations developing in sediment slurry incubations from hypersaline soda (A, B) and salt (C) lakes at classical methylotrophic conditions with methanol (Me) or trimethylamine (TMA) and methyl‐reducing conditions with formate addition (marked as Me⁺ and TMA⁺) and at different temperatures. At conditions used, the mcrA genes from single organisms resolved in bundles of 2–6 separate bands with identical sequences. The mcrA band bundles belonging to two different Methanonatronarchaeum species (A, B) are framed in red for the type species M. thermophilum‐like and in purple for a putative new species Methanonatronarchaeum sp. AMET6‐2. The results were obtained from duplicate sediment incubations pooled together for DNA extraction. Salt lakes abbreviations in (C): Elt—lake Elton; Eupt—Eupatorian solar saltern

FIGURE 6

Phylogenomic placement of Methanonatronarchaeum durum AMET6‐2 sp. nov. based on concatenated partial amino acid sequences of 122 archaeal single‐copy conserved marker genes with taxonomic designations according to the GTDB (Release 07‐RS207). Bootstrap consensus tree is shown with values above 90% placed at the nodes. Bar, 0.1 changes per position