Methanonatronarchaeum thermophilum gen. nov., sp. nov. and 'Candidatus Methanohalarchaeum thermophilum', extremely halo(natrono)philic methyl-reducing methanogens from hypersaline lakes comprising a new euryarchaeal class Methanonatronarchaeia classis nov.

Abstract

Methanogenic enrichments from hypersaline lakes at moderate thermophilic conditions have resulted in the cultivation of an unknown deep lineage of euryarchaeota related to the class Halobacteria . Eleven soda lake isolates and three salt lake enrichment cultures were methyl-reducing methanogens that utilize C₁ methylated compounds as electron acceptors and H₂ or formate as electron donors, but they were unable to grow on either substrates alone or to form methane from acetate. They are extreme halophiles, growing optimally at 4 M total Na⁺ and the first representatives of methanogens employing the ‘salt-in’ osmoprotective mechanism. The salt lake subgroup is neutrophilic, whereas the soda lake isolates are obligate alkaliphiles, with an optimum around pH 9.5. Both grow optimally at 50 °C. The genetic diversity inside the two subgroups is very low, indicating that the soda and salt lake clusters consist of a single genetic species each. The phylogenetic distance between the two subgroups is in the range of distant genera, whereas the distance to other euryarchaea is below 83 % identity of the 16S rRNA gene. These isolates and enriched methanogens, together with closely related environmental clones from hypersaline habitats (the SA1 group), form a novel class-level clade in the phylum Euryarchaeota. On the basis of distinct phenotypic and genetic properties, the soda lake isolates are classified into a new genus and species, Methanonatronarchaeum thermophilum, with the type strain AMET1^T (DSM 28684^T=NBRC 110805^T=UNIQEM U982^T), and the salt lake methanogens into a candidate genus and species ‘Candidatus Methanohalarchaeum thermophilum’. These organisms are proposed to form novel family, order and class Methanonatronarchaeaceae fam. nov., Methanonatronarchaeales ord. nov. and Methanonatronarchaeia classis nov., within the phylum Euryarchaeota .

In hypersaline habitats, methylotrophic methanogenesis is usually considered to be the dominant pathway [1, 2]. The organisms responsible for this process are members of the order Methanosarcinales . In neutral pH conditions, they are represented by the moderately salt-tolerant genus Methanohalophilus and extremely halophilic genus Methanohalobium that can grow at up to 5 M NaCl [2–4], while a single moderately halophilic genus, Methanosalsum , has been identified that can grow in hypersaline soda brines [5–8]. Both Methanohalophilus and Methanohalobium produce organic-compatible solutes, but also accumulate intracellular potassium at high concentrations in their cytoplasm [9–11], thus employing a mixed osmotic strategy, resembling what has been documented in extremely salt-tolerant bacteria and in some haloarchaea [12, 13].

Our recent exploration of methanogenic archaea in sediments of hypersaline inland lakes has shown that, at elevated temperatures, a previously unknown group of extremely halo(natrono)philic methanogens started to outcompete the salt-tolerant Methanosarcinales members when formate was supplied on the top of C₁ methylated compounds as methanogenic substrate. This suggested the methyl-reducing nature of the novel group and has been experimentally confirmed both in growing cultures and in resting cells [14]. In this hybrid methanogenic pathway, the C₁-methylated compounds are used as electron acceptors only, whereas external H₂ is required as the electron donor. This obligate demand for both methyl acceptor and H₂ as the external electron donor, until recently, had been considered rare, having been characterized in only two species of methanogens, Methanosphaera stadtmanae ( Methanobacteriales ) and Methanomicrococcus blatticola ( Methanosarcinales ) [15–17]. However, virtually all recent discoveries of novel deep lineages of methanogens involve methyl-reducers, including the Thermoplasmata methanogens from the order Methanomassiliicoccales [18–22], the candidate class ‘Methanofastidiosa’ [23] and the candidate phyla ‘Bathyarchaeota’ [24] and ‘Verstraetearchaeota’ [25]. These findings indicate that methyl-reduction has so far been overlooked as an important methanogenic pathway that might be able to compete with both classical methylotrophic and lithotrophic pathways. Using combination of C₁-methyl acceptor and formate or H₂ as the electron donor, moderately high temperature and nearly saturated salt concentrations, allowed us to either isolate in pure culture or to highly enrich two groups of obligately methyl-reducing extremely halophilic methanogens that form a deep phylogenetic lineage most closely related to haloarchaea [14]. This novel group of extremely halophilic methanogens was able to proliferate and to form methane only when both a methyl acceptor (methanol or trimethylamine) and either H₂ or formate (shown for the first time as a possible electron donor for methyl-reducers) were present together. This was confirmed by the genomic analysis that showed a lack of two out of the six essential genes encoding enzymes of the ‘upper branch’ of the CO₂-reduction/methyl group oxidation pathway [14].

The main purpose of this paper is to formalize the novel lineage taxonomically using a combination of already determined physiological and phylogenetic data [14] and to propose to place the isolates from soda lakes into a novel genus Methanonatronarchaeum and the enrichments from salt lakes into a candidate genus ‘Candidatus Methanohalarchaeum’. Furthermore, it is proposed that the two genera form a novel family, Methanonatronarchaeaceae, a novel order, Methnanonatronoarchaeales, and a novel class, Methanonatronarchaeia, within the phylum Euryarchaeota .

The source of the isolates was the surface layer (5–15 cm) of anaerobic sediments from hypersaline chloride–sulfate and soda lakes from various geographical locations as shown in Table 1. Overall, 11 pure cultures of haloalkaliphilic and three highly enriched cultures of halophilic methyl-reducing methanogens were obtained at 4 M total Na⁺ and 37–60 °C.

Table 1. Extremely halophilic and moderately thermophilic mixotrophic methanogens isolated from hypersaline lakes at 4 M total Na⁺ .

Strain	Lake	Area	Brine parameters			Enrichment conditions
			pH	Total salt, g l−1	Soluble carbonate alkalinity, M	Substrate	pH	T, °C
AMET1	Mix from five soda lakes*	Kulunda Steppe (Altai, Russia) 2013–2015	9.6–10.1	120–400	0.6–3.0	MeOH+formate	9.6	48
AMET3	Tanatar-1		10.1	350	2.8			48
AMET4	Picturesque Lake		9.8	250				48
AMET5	Mix from six soda lakes		9.6–10.2	50–380	0.5–3.4	TMA+formate		48
AMET6-2	Tanatar-1		10.25	380	3.4	MeOH+formate		60
AMET7	Soda crystallizer		9.6	350	3.8			55
AMET8	Mix from six soda lakes		9.6–10.2	50–380				30
AMET9	Soda crystallizer		10.1	340	3.9			43
AMET10	Stamp Lake		9.1	325	0.2			54
AMET2	Mix from eight lakes†	Wadi al Natrun (Egypt, 2000)	9.1–9.9	200–360	0.1–0.9	MeOH+formate		60
AMET-Sl	Searles Lake	California	9.8	350	0.2	MeOH+formate	9.2	48
HMET1 (mixed culture)	Mix from four salt lakes‡	Kulunda Steppe 2014	7.5–8.1	280–340	–	TMA+H2	7.0	48
HMET-El (mixed culture)	Lake Elton	Southa Russia 2015	6.7	320	–	MeOH+formate		54
HMET-Eu (mixed culture)	Salt crystallizer	Crimea (Russia) 2015	7.2	220	–			55

MeOH, methanol; TMA, trimethylamine; T, temperature.

*The Kulunda Steppe soda lakes in the mix included Tanatar-1,-2,-3,-5; Bitter-1,-3 lakes and a soda crystallizer.

†The Wadi Natrun soda lakes mix included lakes Fazdah, Umm-Risha, Hamra, Beidah, Gaara, Khadra, Rusita, and Zugm.

‡The Kulunda Steppe salt lakes in the mix included Cock Salt Lake, Lake Lomovoe, Lake Hummocky and Crimson lake.

Eleven extremely haloalkaliphilic methyl-reducing isolates (designated AMET) were enriched and further purified by serial dilution using mineral base medium containing 4 M total Na⁺ (2 M Na⁺ as sodium carbonates+2 M NaCl), 5 g l⁻¹ KCl and and 1 g l⁻¹ K₂HPO₄ at pH 9.5 (4 mM NH₄Cl was added after sterilization). Three extremely halophilic, neutrophilic methyl-reducing cultures (designated HMET) were enriched in 4 M NaCl/5 g l⁻¹ KCl, buffered at pH 7 by K₂HPO₄-KH₂PO₄ (total 3 g l⁻¹) and supplemented with 0.5 g l⁻¹ NH₄Cl. Further details of the enrichment and isolation protocols are as described previously [14].

Phase contrast microphotographs were obtained using a Zeiss Axioplan Imaging 2 microscope. For the total-cell electron microscopy, the cells were centrifuged and resuspended in 3 M NaCl, fixed with paraformaldehyde (final concentration 3 %, v/v) for 2 h at room temperature, then washed again with the same NaCl solutions. The fixed cells were positively contrasted with 1 % (w/v) uranyl acetate. For thin sectioning, the cell pellets were fixed in 1 % (w/v) OsO₄ containing 3.0 M NaCl for 1 week at 4 °C, washed and resuspended in 3 M NaCl, stained overnight with 1 % (w/v) uranyl acetate, dehydrated in ethanol series, and embedded in Epon resin. Thin sections were post-stained with 1 % (w/v) lead citrate. The core membrane lipids were obtained by acid hydrolysis (5 % HCl in methanol by reflux for 3 h) of the freeze-dried cells and subsequent analysis by high-performance liquid chromatography–mass spectrometry (HPLC–MS) for glycerol dialkyl glycerol tetraethers (GDGTs) and archaeol derivatives according to Weijers et al. [26]. Intact polar lipids (IPLs) were obtained by Bligh Dyer extraction of freeze-dried cells and subsequent HPLC–MS analysis as described by Sinninghe Damsté et al. [27]. The presence of intracellular organic compatible solutes in the lyophilized cells of strain AMET1^T was analysed by HPLC and ¹H-NMR after extraction with EtOH and the intracellular potassium concentration was measured using inductively coupled plasma–mass spectrometry. The cell protein was analysed by the Lowry method after removal of cell-bound FeS by washing several times with acidic 4 M NaCl solution.

Table 1 summarizes the results on enrichment and isolation of extremely halophilic methyl-reducing methanogens from hypersaline lakes. In addition to the extreme salinity, all but one AMET strain were enriched and isolated at elevated temperatures, between 43 and 60 °C. They were successfully purified from bacterial satellites using a combination of antibiotic treatment and prefiltration of the inoculum through 0.45 µm membrane syringe filters [14]. Although this also worked out for HMET cultures from salt lakes, a small fraction (approx. 5 %) of other, non-methanogenic haloarcheal cells persisted in the serial dilutions (flat rods easily distinguishable from the dominant fraction of small coccoid cells; the dominant among them was identified as a member of the genus Halanaeroarchaeum ; [28]). Furthermore, the growth rate and yield of the HMET cultures were extremely low compared to the AMET isolates, which made their purification problematic. The growth of AMET cultures was markedly stimulated by addition of up to 50 µM CoM, while the HMET cultures did not grow at all without such addition, which can be explained either by the incompleteness (AMET1^T) or the lack (HMET1) of necessary genes responsible for the biosynthesis of this important co-factor [14]. Furthermore, both AMET and HMET cultures exhibited obligate dependence on external FeS. However, only three of the 11 AMET isolates (1, 3 and 4) grew in the minimal medium (mineral base/MeOH+formate/CoM/yeast extract) with these additions, whereas the rest were dependent on the presence of sterilized sediments either from soda (AMET) or salt (HMET) lakes (0.5 ml 1 : 1 sediment–brine/100 ml). What exactly these organisms need from the sediments remains unclear, although a test with pore brines (separated by high speed centrifugation) and the solid phase demonstrated that the latter was far more efficient as a growth factor. To our knowledge, similar observations have been reported in only one other case, for an unidentified methylotrophic methanogenic culture obtained from alkaline saline Mono Lake, but that culture most probably belonged to a classical methylotroph, because it grew in the presence of methanol as the only substrate [29].

In a single case of strain AMET1^T, which can grow in presence of FeS alone, colonial growth was achieved. The colonies were obtained by the soft agar dilution technique on plates after 40 days incubation in an anaerobic jar under argon with 0.2 atm H₂ gas overpressure. They were disc-shaped, up to 1 mm in diameter and yellow-coloured. The typical cell morphologies of strain AMET1^T and enriched culture HMET1 are shown on Figs 1 and 2. The cells are irregular angular cocci of a characteristic small size (mean cell diameter is 0.4 µm). The cells of all AMET strains were motile and in the type strain AMET1^T multiple archaella were detected, whereas the cells in HMET enrichments were not motile. Both groups have a thin, monolayer cell wall covered with a thick EPS layer. In addition, invaginations of cytoplasmic membrane and large electron transparent inclusions were visible in the cells of HMET1 enrichment. The cells lysed immediately upon downshift in salinity below 2 M Na⁺ and in the presence of ≥0.2 % (w/v) SDS. The blue autofluorescence was not detected in the AMET and HMET cells by standard fluorescence microscopy, indicating the absence or low concentrations of ‘F₄₂₀’ (deazoflavine) normally present in classical methylotrophic and hydrogenotrophic methanogens. This is corroborated by the absence of genes coding for the F₄₂₀-dependent hydrogenases in the genomes of AMET1^T and HMET1 [14].

Fig. 1.

Cell morphology of Methanonatronarchaeum thermophilum strain AMET1^T grown with MeOH+formate at pH 9.5, 4 M total Na⁺ and 48 °C. (a) Phase contrast microscopy. (b and c) Electron microscopy of total cells and thin sections, respectively. N, nucleoide; CM, cytoplasmic membrane; CW, cell wall.

Fig. 2.

Cell morphology of ‘Ca. Methanohalarchaeum thermophilum’ strain HMET1 grown with TMA+H₂ at pH 7, 4 M NaCl and 50 °C. (a) Phase contrast microscopy. (b and c) Electron microscopy of total cells and thin section, respectively. N, nucleoide; ICPM, intracytoplasmic membrane; Stg, inclusion granule; cw, cell wall.

The analysis of organic compatible solutes in cells of strain AMET1^T (grown at 4 M Na⁺, pH 9.5) gave negative results. However, intracellular cation analysis demonstrated molar concentrations of K⁺ [14]. These observations indicated that the novel methanogens employ the haloarchaeal type (‘salt-in’) osmoprotection mechanism [30], which has not yet been demonstrated for other well-characterized halophilic methanogens, such as Methanohalobium , Methanohalophilus and Methanosalsum . This was in line with the absence of genes responsible for the biosynthesis of organic osmolytes commonly present in halophilic procaryotes, including halophilic methanogens [14].

The core membrane lipids of strains AMET1^T and HMET1 enriched culture are primarily composed of a mixture of GDGTs (dibiphytanylglycerol tetraethers) and archaeol (diphytanylglycerol diether) (Table S1, available in the online version of this article). The GDGTs were dominated by GDGT-0 (archaeol). AMET1^T also contained small quantities of GDGT-1, which was not detected in the HMET1 enrichment. In addition to archaeol, minor amounts of two monophytanyl glycerol ethers (2-C₂₀ MGE and 1-C₂₀ MGE) in strain AMET1^T and only 1-C₂₀ MGE in HMET1 enrichment culture were detected. The complete absence of extended archaeols (C₂₀-C₂₅ and C₂₅-C₂₀ DGE) in the membrane lipids differentiated the extremely halophilic methanogens from haloarchaea [31]. The IPL composition of the cells in two strains were clearly different. In the alkaliphilic strain AMET1^T, the dominant IPLs were phosphatidylglycerol (PG) and GDGT-0 containing two PG groups. In halophilic enrichment culture HMET1, the dominant IPLs were identified as dihexose derivatives of both archaeol and GDGT-0 (Table S1).

Both AMET strains and HMET enrichments used the methyl-reducing pathway of methanogenesis, whereby the C₁ methylated compounds, such as methanol, methylamines or methylated sulfides are used only as electron acceptors, whereas H₂ served as the external electron donor [14]. For the AMET strains, the best electron acceptor was methanol. Methylamines, including mono-, di- and tri-methylamines and tetramethylammonium, can also be utilized in ammonia-free media but were highly toxic at alkaline conditions and the growth was much less active, although the activity of washed cells pregrown on methanol+formate, was nearly in the same range as with methanol [14]. The growth with dimethylsulfide (DMS) demanded gradual adaptation starting from 2 mM, but after several steps, the best adapted strain, AMET6-2, was able to grow in presence of up to 20 mM DMS (with formate as electron donor). On the other hand, although possible in principle, methanethiol as acceptor (5–10 mM) with formate as donor was irregularly used and no adaptation was observed to this toxic methylated sulfide. The neutrophilic HMET enrichment cultures preferred trimethylamine as the acceptor over methanol, while growth with other C₁ methyl compounds (methylamine, dimethylamine and methylated sulfides) was not observed. The two groups also differed in their preferred electron donor: while the AMET strains clearly preferred formate, the HMET enrichments used H₂ more actively. Utilization of formate as the electron donor, as well as DMS as the acceptor, have not been demonstrated previously for any cultured methyl-reducing methanogens, including Methanosphaera , Methanomicrococcus , ‘Candidatus Methanoplasma’ and Methanomassiliicoccus . Both the soda and the salt lake organisms required a limited amount of organic carbon (in addition to electron donor/electon acceptor pair) in the form of either yeast extract (both) or acetate (AMET strains) to support growth. The biomass increase was observed within the concentrations of yeast extract from 20 to 100 mg l⁻¹, with further increase having no significant effect.

A unique property of the novel methyl-reducing methanogens is their extreme halophily. Both AMET and HMET groups grew at extremely high Na⁺ concentrations, which is a typical characteristic of cultured haloarchaea, i.e. from 3 to 5 M, with an optimum at approximately 4 M. This preferred range of salt concentration is compatible with the evidence indicating that these organisms employ the ‘salt-in’ strategy for osmoprotection, that include the absence of any recognizable organic osmolytes in the cells of AMET1^T, the presence of high concentrations of potassium in the cells of AMET1^T and the absence of genes encoding synthesis of common organic osmolytes in halophilic prokaryotes [9, 10, 14]. The AMET group from soda lakes belongs to obligate alkaliphiles growing within the pH range (at 4 M Na⁺ and 48 °C) from 8.2 to 10.2 (optimum at 9.5–9.8). In contrast to most of the extremely natronophilic bacteria isolated from hypersaline soda lakes, the new archaea depend on molar concentrations of NaCl and grow optimally in a medium containing 2 M NaCl and 2 M (Na) carbonates. The HMET enrichment cultures were typical neutrophiles, with a pH range for growth from 6.5 to 8. Furthermore, both groups preferred elevated temperatures for growth despite being isolated from moderate habitats. They grew optimally at 50 °C and five out of 11 AMET strains tolerated up to 60 °C. The lowest growth temperature among the AMET isolates was 30 °C, while the HMET1 enrichment did not grow below 40 °C.

At optimal growth conditions (MeOH+formate, 4 M total Na⁺, pH 9.5 and 50 °C) the values of maximum specific growth yield (Y_max) and maximum specific growth rate (μ_max) for strain AMET1^T were estimated as 1.5 mg cell protein (mM MeOH)⁻¹ and 0.012–0.015 h⁻¹, respectively [14].

The maximum-likelihood phylogenetic trees based on 16S rRNA gene analysis and on the key methanogenic marker McrA (methyl-coenzyme M reductase) sequences were reconstructed using PhyML 3.0 with the Smart Model Selection [32], the subtree pruning and regrafting) type of tree improvement [33] and the approximate likelihood-ratio test for branch support [34]. Nearly complete sequences of 16S rRNA genes from the silva database [35] and McrA from the genomic databases (GenBank) were included in the calculation. The 16S rRNA gene phylogeny showed that the AMET and HMET groups form two compact clades, with a maximum distance inside the groups of 1.5 %. The distance between the two groups was about 10 %, indicating that they represent two distinct genera of the same family. However, no close relatives of these organisms were identified among the cultivated members of Euryarchaeota , whereas among uncultured archaeal clones the novel methanogens were clearly related to the SA1 group (sequence identity in the range of 85–94 %) detected in various hypersaline habitats [36–38]. Further phylogenetic reconstruction [14] showed that the closest relatives of the AMET-HMET group in Euryarchaeota were haloarchaea of the class Halobacteria (Fig. 3a), that, again, is consistent with the extreme halophily and the likely ‘salt-in’ osmotic strategy of the novel methanogens.

Fig. 3.

Phylogeny of novel halo(alkali)philic methanogens from hypersaline lakes based on the 16S rRNA gene (a) and full amino acid sequences of the α-subunit of methyl coenzyme M reductase (McrA or MrtA) (b). The trees were built with the PhyML program and the approximate likelihood-ratio test for branches [33]. Bootstrap values above 70 % are shown at the nodes. Bar, 0.10 changes per position.

In contrast to the majority of known methanogenic euryarchaea, the phylogeny of the key functional marker McrA of the novel group was significantly different from the 16S- and 23S-rRNA gene-based phylogeny [14]. Two different McrA proteins from strain AMET1^T and a single copy from the HMET enrichments clustered with the methyl-reducing representatives from the order Methanomassiliicoccales with the protein sequence identity ranging from 61 to 67 % (Figs 3b and S1). The same branching pattern has also been validated by using a concatenated sequence analysis of McrABDG obtained from the genomes of strain AMET1^T and HMET1 enrichment, with the remarkable discrepancy between the ribosomal and the key metabolic gene phylogenies of the extremely halophilic methyl-reducing methanogens being explained by the gene loss evolutionary scenario [14].

Overall, on the basis of phylogenetic analysis and unique phenotypic properties, the novel moderately thermophilic and extremely haloalkaliphilic methyl-reducing methanogenic isolates from hypersaline soda lakes are proposed to form a new genus and species Methanonatronarchaeum thermophilum. The highly enriched methyl-reducing methanogenic cultures from salt lake are proposed to form a candidate genus and species ‘Candidatus Methanohalarchaeum thermophilum’. These two taxons together are proposed to be classified in a new family Methanonatronarchaeaceae, a new order Methanonatronarchaeales and a new class a class Methanonatronarchaeia within the phylum Euryarchaeota .

Description of Methanonatronarchaeum gen. nov.

Methanonatronarchaeum (Me.tha.no.na.tron.ar.chae′um. N.L. neut. n. methanum methane; N.L. pref. methano- pertaining to methane; N.L. n. natron, arbitrarily derived from the Arabic n. natrun natron, soda; N.L. neut. n. archaeum [from Gr. adj. archaios, -e, -on] ancient archaeon; N.L. neut. n. Methanonatronarchaeum a soda-loving archaeon-forming methane).

Extremely halo(alkali)philic and moderately thermophilic methanogens that use the methyl-reducing pathway of methanogenesis. Accumulate high intracellular concentrations of potassium. Found in hypersaline alkaline lakes. Member of the family Methanonatronarchaeaceae. The type species is Methanonatronarchaeum thermophilum.

Description of Methanonatronarchaeum thermophilum sp. nov.

Methanonatronarchaeum thermophilum [ther.mo′phi.lum. Gr. adj. thermos hot; N.L. neut. adj. philum (from Gr. adj. philos -ê –on), friend, loving; N.L. neut. adj. thermophilum, thermophilic].

The species description is based on eleven isolates. Cells are small irregular cocci, 0.4–0.5 µm in size, motile by 1–5 archaella. The cell wall is a thin monolayer covered with EPS. The cells lyse at salinity below 2 M Na⁺ and in the presence of >0.2 % SDS. Accumulate potassium as the main compatible solute. The F₄₂₀-dependent cell autofluorescence is not detected by a standard epifluorescence microscopy. The colonies are yellowish, lens-shaped and up to 1 mm. The core lipids are dominated by archaeol (C₂₀-C₂₀ DGE). Strictly anaerobic methanogens utilizing MeOH, methylamines and dimethylsulfide as electron acceptor and formate or H₂ as electron donor. Cannot grow with C1 methyl compounds alone or H₂/formate+CO₂ alone and cannot use acetate as methogenic substrate. Heterotrophic, utilizing yeast extract or acetate as carbon source. Growth depends on external CoM, FeS/or sterilized anaerobic sediments from soda lakes. Obligately alkaliphilic with a pH range for growth from 8.2 to 10.2 (optimum at pH 9.5–9.7) and extremely halophilic, growing between 3 and 4.8 M total Na⁺ (optimum at 4 M). Moderately thermophilic, with the growth temperature range between 30–34 and 55–60 °C (optimum at 50 °C). The G+C content of the genomic DNA in the type strain is 38 mol% (genome). The type strain, AMET1^T (DSM 28684=NBRC 110805=UNIQEM U982), was isolated from sediments of hypersaline soda lakes in Kulunda Steppe (Altai, Russia).

Description of ‘Candidatus Methanohalarchaeum thermophilum’

‘Candidatus Methanohalarchaeum thermophilum’ [Me.tha.no.hal.ar.chae′um. N.L. neut. n. methanum methane; N.L. pref. methano-, pertaining to methane; Gr. n. hals halos salt; N.L. neut. n. archaeum (from Gr. adj. archaios, e, -on ancient archaeon; N.L. neut. n. Methanohalarchaeum a salt-loving archaeon forming methane); ther.mo′phi.lum. Gr. adj. thermos hot; N.L. neut. adj. philum (from Gr. adj. philos -ê –on), friend, loving; N.L. neut. adj. thermophilum, thermophilic].

A member of the phylum Euryarchaeota , class Methanonatronarchaeia, order Methanonatronarchaeales, family Methanonatronarchaeaceae. The description is based on three highly enriched monomethanogenic cultures. Cells are small, irregular, non-motile cocci, 0.4–0.5 µm. The cell wall is a thin monolayer covered with EPS. The cells lyse at salinity below 2 M NaCl and in the presence of >0.2 % SDS. The F₄₂₀-dependent cell autofluorescence is not detected by a standard epifluorescence microscopy. The core lipids are dominated by archaeol (C₂₀-C₂₀ DGE). Colony formation was not observed. Strictly anaerobic methanogens utilizing MeOH and trimethylamine as an electron acceptor and H₂ or formate as an electron donor. Cannot grow with C1 methyl compounds alone or H₂/formate+CO₂ alone and cannot use acetate as methogenic substrate. Heterotrophic, utilizes yeast extract as a carbon source. The growth depends on external CoM and sterilized anaerobic sediments from salt lakes. Extremely halophilic, grow optimally at 4–5 M NaCl. The pH optimum for growth is 7–7.5. Moderately thermophilic with an optimum at 50 °C and the upper limit for growth at 60 °C. The G+C content of the genomic DNA in the type strain is 35.4 mol% (genome). The type strain, HMET1^T, was enriched from sediments of hypersaline lakes in the Kulunda Steppe.

Description of Methanonatronarchaeaceae fam. nov.

Methanonatronarchaeaceae (Me.tha.no.na.tron.ar.chae.a.ce′ae. N.L. neut. n. Methanonatronarchaeum the type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Methanonatronarchaeaceae the Methanonatronarchaeum family).

The properties of the family are same as for the representative genera Methanonatronarchaeum and ‘Candidatus Methanohalarchaeum’. The type genus of the family is Methanonatronarchaeum.

Description of order Methanonatronarchaeales ord. nov.

Methanonatronarchaeales (Me.tha.no.na.tron.ar.chae.a′les. N.L. neut. n. Methanonatronarchaeum the type genus of the order; -ales ending to denote an order; N.L. fem. pl. n. Methanonatronarchaeales the Methanonatronarchaeum order).

The properties of the order Methanonatronarchaeales are same as for the representative genera Methanonatronarchaeum and ‘Candidatus Methanohalarchaeum’. The type genus of the order is Methanonatronarchaeum.

Description of Methanonatronarchaeia classis nov.

Methanonatronarchaeia (Me.tha.no.na.tron.ar.chae′i.a. N.L. fem. pl. n. Methanonatronarchaeales the type order of the class; -ia ending to denote a class; N.L. neut. pl. n. Methanonatronarchaeaia the Methanonatronarchaeales class).

The class Methanonatronarchaeia is defined on the basis of comparative sequence analysis of the 16S rRNA gene of the genus Methanonatronarchaeum, highly enriched cultures of ‘Candidatus Methanohalarchaeum thermophilum’ and the cloned sequences from an uncultured SA1 group found in various hypersaline habitats of terrestrial and marine origin.

Type order: Methanonatronarchaeales ord. nov.