# Methanobacterium alkalithermotolerans sp. nov., a novel alkaliphilic and hydrogen-utilizing methanogen isolated from an alkaline geothermal spring (La Crouen, New Caledonia)

Nan Mei, Anne Postec, Manon Bartoli, Céline Vandecasteele, Laura Wils, Lisa Gil, Christophe Monnin, Bernard Pelletier, Gael Erauso, Marianne Quéméneur

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Nan Mei, Anne Postec, Manon Bartoli, Céline Vandecasteele, Laura Wils, et al. Methanobacterium alkalithermotolerans sp. nov., a novel alkaliphilic and hydrogen-utilizing methanogen isolated from an alkaline geothermal spring (La Crouen, New Caledonia). International Journal of Systematic and Evolutionary Microbiology, 2022, 72 (10), 10.1099/ijsem.0.005554. hal-04114150

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1 *Methanobacterium alkalithermotolerans* sp. nov., a novel alkaliphilic and hydrogen-utilizing methanogen isolated from an alkaline geothermal spring (La Crouen, New Caledonia)

3

4 Nan Mei¹†, Anne Postec¹, Manon Bartoli¹, Céline Vandecasteele², Laura Wils¹, Lisa Gil²,
5 Christophe Monnin³, Bernard Pelletier⁴, Gael Erauso¹, and Marianne Quéméneur¹*

6

7 ¹ Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO, Marseille, France

8 ² INRAE, US 1426, GeT-PlaGe, Genotoul, Castanet-Tolosan, France

9 ³ GET UMR5563 (CNRS/UPS/IRD/CNES), Géosciences Environnement Toulouse, 14 Avenue Edouard Belin, 31400 Toulouse, France

10

11 ⁴ Centre IRD de Nouméa, 101 Promenade Roger Laroque, BP A5 – 98848 Nouméa cedex,
12 Nouvelle-Calédonie.

12

13

14 †Present address: School of Chemistry, Chemical Engineering, and Life Science, Wuhan University of Technology, Wuhan 430070, PR China

15

16 *Corresponding author: Marianne Quéméneur; E-mail: marianne.quemeneur@ird.fr

17

18 Running title: *Methanobacterium alkalithermotolerans*

19

20 **Keywords:** Alkaliphile, hydrogenotrophic, methanogen, alkaline thermal spring,
21 *Methanobacteriaceae, Methanobacterium alkalithermotolerans*

21

22

23 **Section/Category:** New taxa – "Methanobacteriota"

24 ** Repositories: The GenBank accession number for the 16S rRNA gene sequence of strain CAN**<sup>T</sup>
25 is KR349725, and the one for the whole genome sequence is CP058560.

26 The word count of the article is 2689.
27

28

29 An anaerobic, hydrogenotrophic methane-producing archaeon was isolated from an alkaline
30 thermal spring (42°C, pH 9.0) in New Caledonia. This methanogen, designated strain CAN<sup>T</sup>, is
31 alkaliphilic, thermotolerant, with Gram-positive staining non-motile cells. Strain CAN<sup>T</sup> grows
32 autotrophically using hydrogen exclusively as an energy source and carbon dioxide as the sole
33 carbon source (without the requirement of yeast extract or other organic compounds). It grows
34 at 20-45°C (optimum 45°C) and pH 7.3-9.7 (optimum 9.0). NaCl is not required for growth
35 (optimum 0%) but is tolerated up to 1.5%. It resists to novobiocin, streptomycin, and
36 vancomycin but is inhibited by ampicillin and penicillin, among other antibiotics. The genome
37 consists of a circular chromosome (2.2 Mb) containing 2,126 predicted protein-encoding genes
38 with a G+C content of 36.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence
39 indicated that strain CAN<sup>T</sup> is a member of the genus *Methanobacterium*, most closely related
to the alkaliphilic *Methanobacterium alcaliphilum* WeN4<sup>T</sup> with 98.5% 16S rRNA gene
40 sequence identity. The genomes of strain CAN<sup>T</sup> and *M. alcaliphilum* DSM 3459, sequenced in
41 this study, share 71.6% average nucleotide identity (ANI) and 14.0% digital DNA-DNA
42 hybridization (dDDH). Therefore, phylogenetic analysis and physiological features determined
43 strain CAN<sup>T</sup> as a novel species, for which the name *Methanobacterium alkalithermotolerans*
44 sp. nov. is proposed, and strain CAN<sup>T</sup> (=DSM 102889<sup>T</sup> = JCM 31304<sup>T</sup>) is assigned as the type
45 strain.
46

47

48
Methanogens are a functional group of *Archaea* that produce methane as a by-product of their energy metabolism (1, 2). *Methanobacterium* belongs to the *Methanobacteriaceae* family of the "Methanobacteriota" phylum (3, 4). *Methanobacterium* species are mesophilic or thermophilic and occur in various anoxic environments, such as anaerobic digesters (5) and marine sediments (6). More than 41 species of the genus *Methanobacterium* have been described to date. Some of them can use organic compounds, such as formate, to produce methane (5, 7), while other *Methanobacterium* species grow exclusively through the reduction of carbon dioxide (CO2) by hydrogen (H2); examples of such include *Methanobacterium aarhusense* H2-LR<sup>T</sup> (6), *Methanobacterium alcaliphilum* WeN4<sup>T</sup> (8), and *Methanobacterium movens* TS-2<sup>T</sup> (7).

In this study, we describe the isolation and characterization of an autotrophic, hydrogenotrophic, alkaliphilic methanogen, which has phenotypic, phylogenetic, and phylogenomic characteristics that allow its assignment to a novel species of the genus *Methanobacterium*. This novel archaeal species was isolated from ‘La Crouen’ geothermal spring, located north of the southern peridotite nappe of New Caledonia (9). This spring discharges alkaline (pH 9.0), anoxic (O₂(aq)<1 mg/L) and warm (42°C) waters (10, 11), enriched in dinitrogen (N₂) with traces of methane (CH₄ <3%) (12). This archaeon is the first *Methanobacterium* member isolated and characterized from terrestrial thermal sources (or hot springs) and the first example of methanogens isolated and characterized from the alkaline springs of New Caledonia.

For enrichment experiments, a sediment slurry sample was collected in November 2014 using both sterilized spatula and glass bottle from the geothermal spring of La Crouen (165° 53' 20.6" E 21° 32' 06.6" S), located next to the city of Canala (New Caledonia) (13). For preserving anoxic conditions, the bottle was immediately hermetically sealed and stored at 4°C. In the laboratory, anaerobic enrichments were carried out using a culture medium containing
(per liter of distilled water): 0.1 g KH₂PO₄, 0.1 g K₂HPO₄, 0.5 g NH₄Cl, 0.5 g NaCl, 0.002 g FeSO₄·7H₂O, 0.1 g CaCl₂·2H₂O, 0.1 g MgSO₄·7H₂O, 2 g sodium formate, 1 g sodium acetate, 0.1 g cysteine hydrochloride, and 1 mL trace element solution SL-10 (14), supplemented with 25 mM Tris-Base. The initial pH was adjusted to 9.0 with NaOH. This medium was boiled for 5 min and then cooled to room temperature under a flow of O₂-free N₂ gas. The medium (5 mL) was dispensed into Hungate tubes, degassed under a flow of N₂, and subsequently autoclaved (20 min, 120°C). The following sterile solutions were injected in each tube: 0.1 mL of 2% Na₂S·9H₂O, 0.1 mL of 8% Na₂CO₃, 0.1 mL of 10% tryptone and 0.1 mL of 10% yeast extract.
Inoculation at 10% vol. was done with 0.5 mL of the sediment slurry sample. The suspensions were serially diluted in decimal steps using the same medium (up to 10⁻⁴), and each tube was supplemented by H₂/CO₂ gas mixture (80/20 v/v at 2 bars). The suspensions were incubated for one month at 37°C under shaking (100 rpm). The Hungate technique for anaerobic cultivation was used throughout this study (15).

Isolation and growth experiments were performed using the medium described above (without sodium acetate, sodium formate, yeast extract, and tryptone) with the addition of Balch vitamin solution (16), and H₂/CO₂ (80/20 v/v, 2 bars) as the only sources of energy and carbon, and supplemented with 25 mM Tris-Base (except for testing the optimal pH for growth). For the optimum pH tests, the initial pH of the culture media (ranging between 6.9 and 10.1) was adjusted with an anaerobic sterile stock solution of 1M NaOH. The pH was always checked after H₂/CO₂ injection (following inoculation) and at the end of incubation. Water baths were used for incubation at temperatures ranging from 10 to 60°C. For studies of NaCl requirements (tested between 0 and 3%), NaCl was weighted and added directly into the tubes before medium addition. Each test culture was sub-cultured at least once under the same experimental conditions before determining growth rates. The following compounds were tested as potential substrates: (i) acetate, ethanol, formate, iso-butanol, methanol, trimethylamine, or 2-propanol
(at a final concentration of 20 mM) with N₂/CO₂ (80/20, v/v; 1 bar), or (ii) only H₂/CO₂ (80/20, v/v) replacing N₂/CO₂ as the gas phase, or (iii) H₂/CO₂ (80/20, v/v) in the presence of acetate (2 mM) as carbon source. Sensitivity to antibiotics (ampicillin, bacitracin, chloramphenicol, cycloserine, kanamycin, metronidazole, novobiocin, penicillin, rifampicin, streptomycin, tetracycline, vancomycin) was tested by adding each antibiotic separately to the basal medium at a final concentration of 25 µg/mL or 50 µg/mL. The effect of sulfide concentration (0–10 g/L) on the growth rate of the isolate was also tested by adding an increasing concentration of a Na₂S solution. Each growth test and control, carried out in parallel, was run in duplicate. Hungate tubes. Controls were (i) inoculated tubes without tested compounds and (ii) uninoculated tubes with tested compounds. Growth was monitored by measuring the turbidity increase at 600 nm by inserting Hungate tubes into the tube holder of a spectrophotometer (Cary 50, Varian). Gaseous and liquid end-products of metabolism were periodically measured by gas chromatography (Shimadzu GC 8A) and high-performance liquid chromatography (Thermo Separation Products), as described by Mei *et al.* (17).

The presence of putative methanogens from CH₄-producing enrichment cultures was checked by observing the autofluorescence of the cofactor F420 characteristic of methanogens. For this purpose, cell observations were performed under UV light using a Nikon Eclipse E600 equipped for epifluorescence and an F420 emission filter. Cellular morphology and purity check of the strain were examined using a phase-contrast microscope (Optiphot; Nikon). For transmission electron microscopy (using a MET FEI Tecnai G2), exponentially grown cells were fixed with glutaraldehyde and osmium tetraoxide, epon-embedded, and stained with uranyl acetate and lead citrate (to watch the cell wall), as described by Quéméneur *et al.* (18).

The purity of the strain was further checked after DNA extraction (19) by the amplification of partial 16S rRNA gene using the bacteria-specific primers 341F/518R (20) to verify the absence of any bacterial contaminant. Near-complete 16S rRNA genes were amplified using
the primer pairs A109F/1492R for *Archaea* domain (21, 22), then sequenced by GATC-Biotech (Konstanz, Germany). The 16S rRNA gene sequence was deposited in Genbank and aligned with available sequences from the NCBI database using the MUSCLE program (23), implemented in MEGA7 software (24). Evolutionary analyses were conducted from 16 nucleotide sequences (including the isolate, fourteen *Methanobacterium* type species, and *Methanopyrus kandleri*) using three different methods: maximum-likelihood (ML) (25), maximum-parsimony (MP) (26), and neighbor-joining (NJ) (27).

High molecular weight DNA of the isolate and *Methanobacterium alcaliphilum* DSM 3459 was extracted using a phenol-chloroform method (28) modified by adding a step including several cycles of grinding the frozen cell pellet in liquid nitrogen to optimize cell wall lysis. Whole genomes were then sequenced at the GeT-PlaGe platform (INRAe Toulouse, France) by combining long reads (Oxford Nanopore) and short reads (Illumina MiSeq) technologies, as described by Quéméneur *et al.* (13). The genomes of the isolate and *Methanobacterium alcaliphilum* DSM 3459 were assembled with Unicycler v0.4.6 (29) and SPAdes v3.7.1 (30). Their sequences were deposited in the NCBI database and annotated using the MaGe system (Microscope Genome Annotation) (31). The average nucleotide identity value (ANI) was calculated using OrthoANI with Usearch (http://www.ezbiocloud.net/tools/ani). Digital (or *in silico*) DNA-DNA hybridization (dDDH) was calculated using the Genome-to-Genome Distance Calculator (GGDC) 2.1 (http://ggdc.dsmz.de/) (32). Whole-genome- and proteome-based taxonomic analyses were performed using the bioinformatics platform Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de) (33).

Methane production and a simultaneous decrease in both H₂ and CO₂ were detected in all enrichment cultures of the ‘La Crouen’ slurry sample after two weeks of incubation at 37°C. The highest dilution culture (10⁻⁴) was transferred into a minimum liquid medium with H₂/CO₂ as sole energy and carbon sources. The Roll-tube method described originally by Hungate (15)
149 was used for strain isolation in a solid medium. The culture was ten-fold serially diluted in roll tubes containing a molten 1.6% agar medium. Single white and circular colonies (1 mm in diameter after 2-week incubation) were resuspended in basal medium with the addition of Balch vitamins, which serves to inoculate a second round of ten-fold serially diluted in roll tubes.
150
151
152
153 After the third round of roll tube isolation, one single colony was finally grown in liquid culture, resulting in the strain designated CAN<sup>T</sup>, further characterized physiologically and 154
155 metabolically.

156 Cells of strain CAN<sup>T</sup> are straight rods 1.3-4.0 µm long and 0.5-0.7 µm wide, occurring singly, in pairs, or chains (Fig. 1A and 1B). They are non-motile and Gram-positive staining (Fig. 1B).
157
158

159 Strain CAN<sup>T</sup> grows only under strictly anaerobic conditions. It is mesophilic but 160 thermotolerant and grows at temperatures ranging from 20°C to 45°C (Table 1). The optimal 161 temperature for growth is 45°C. The pH range for growth is 7.3-9.7, with an optimum growth 162 at pH 9.0 (checked 15 min after H<sub>2</sub>/CO<sub>2</sub> addition and at the end of the experiments). NaCl is 163 not required for growth, and the NaCl range for growth is 0-1.5% (optimum at 0%), indicating 164 that strain CAN<sup>T</sup> is adapted to the low-salinity, warm, and high-pH fluid emitted by the alkaline 165 spring of La Crouen (New Caledonia).
166

166 H<sub>2</sub> and CO<sub>2</sub> were the only substrates used for growth and methanogenesis (Table 1, Fig. 2).
167 Acetate, ethanol, formate, isobutanol, methanol, trimethylamine, and 2-propanol were not used 168 as substrates for growth or methane production. Yeast extract and tryptone were not required 169 for growth. The maximal growth rate was found to be 0.02 h<sup>-1</sup> using H<sub>2</sub>/CO<sub>2</sub> as sole energy and 170 carbon sources for growth in optimal conditions (45°C, pH 9.0, and 0% NaCl).
171

171 Strain CAN<sup>T</sup> was resistant to antibiotics such as novobiocin, streptomycin, and 172 vancomycin added at 25 µg/mL or 50 µg/mL in the medium. It also grew in the presence of 25 173 µg/mL of tetracycline, but it was sensitive to this antibiotic at 50 µg/mL. Ampicillin, bacitracin,
chloramphenicol, cycloserine, kanamycin, metronidazole, penicillin, and rifampicin at 25 µg/mL inhibited cell growth. Sulfide concentration greater than 1.5 g/L also inhibited cell growth of strain CAN<sup>T</sup>.

Phylogenetic analysis based on the almost full-length 16S rRNA gene sequence (1227 nt) indicated that strain CAN<sup>T</sup> is a member of the genus *Methanobacterium*, in the order *Methanobacteriales*, class *Methanobacteria*, phylum "*Methanobacteriota*" (Fig. 3). The phylogenetic position of strain CAN<sup>T</sup> was confirmed by using three different methods: maximum-likelihood (ML, Fig 3), maximum-parsimony (MP, Fig. S1A), and neighbor-joining (NJ, Fig. S1B). The closest relatives of strain CAN<sup>T</sup> are type strains of the species: *Methanobacterium alcaliphilum* WeN4<sup>T</sup> (98.5% identity) isolated from sediments of a low-salt alkaline lake of the Wadi el Natrun (Egypt) (8), *Methanobacterium movens* TS-2<sup>T</sup> (98.4% identity) isolated from sediments of the alkaline saline Tuosu Lake (Qaidam basin, China) (7), and *Methanobacterium flexile* GH<sup>T</sup> (97.5% identity) isolated from sediments of an alkaline saline lake, the Gahai Lake (Qaidam basin, China) (7). It is more distantly related to the other alkaliphilic *Methanobacterium* type strains of the species: *Methanobacterium subterraneum* A8p<sup>T</sup> (95.1% identity).

The 16S rRNA gene sequence of strain CAN<sup>T</sup> was closely related (>99% identity) to a few environmental sequences retrieved in March 2022 by NCBI web BLAST search (34) and originated from deep subsurface gas-associated water (DQ841242) (35) as well as H<sub>2</sub>-fed bioreactor operated at halo-alkaline conditions (KJ491002) (36). Sequences of the 16S rRNA gene affiliated to the genus *Methanobacterium* were also detected in the alkaline spring of La Crouen (representing 4.5% of prokaryotes in the sample studied) (11) and in the hyperalkaline springs of the Ligurian Alps ophiolite massif and in subsurface fluids of the Sultanate of Oman ophiolite massif (19, 37-39), where the methanogen could potentially consume H<sub>2</sub> produced by serpentinization reactions.
The complete genome sequence of strain CAN<sup>T</sup> is a unique circular chromosome 2,2
Mb long with a 36.4 mol% G+C content (Table 2). Its G+C content is identical to that of *M. flexile* GH<sup>T</sup> (36.4 mol%), higher than that of *M. alcaliphilum* WeN4<sup>T</sup> (33.5 mol%), and lower
than *M. movens* TS-2<sup>T</sup> (39.1 mol%). Its size is much smaller than that of its closest phylogenetic
neighbor *M. alcaliphilum* WeN3 (2.4 Mb). The ANI and dDDH values between CAN<sup>T</sup> and *M. alcaliphilum* WeN3 were 71.55% and 14.0%, i.e., far below the species boundaries of 95~96%
and 70%, respectively, recommended by Chun *et al.* (40). The CAN<sup>T</sup> strain genome comprises
a single chromosome and 2,189 genes, including 2,126 protein-encoding genes and 49 total
RNA encoding genes. A complete pathway for hydrogenotrophic methanogenesis was found
using MaGe with both genes encoding formylmethanofuran dehydrogenase and methyl
coenzyme-M reductase that catalyze the initial and final step of methanogenesis on H<sub>2</sub>/CO<sub>2</sub>,
respectively. The genome of *M. alcaliphilum* WeN3 comprises 2,375 genes, including 2,325
protein-encoding genes and 46 total RNA encoding genes. The distribution of protein-encoding
genes of both strain CAN<sup>T</sup> and *M. alcaliphilum* WeN3 into COGs functional categories is very
similar (Supplementary Table S1). In a whole-proteome-based phylogenetic analysis (Fig. S2),
strain CAN<sup>T</sup> is also closely related to *M. alcaliphilum* (consistently with the 16S rRNA gene-
based phylogenetic analysis) and clustered with other members of the family
*Methanobacteriaceae*, including the genera *Methanobacterium*, *Methanobrevibacter*, and
*Methanothermobacter* (previously placed in the genus *Methanobacterium* (41)).

Table 1 summarizes the physiological and biochemical differences between strain
CAN<sup>T</sup> and several type species of the genus *Methanobacterium* (including the four closest
phylogenetic neighbor type species and other alkaliphilic members). Strain CAN<sup>T</sup> shares
similar properties with several *Methanobacterium* members, such as the exclusive utilization
of H<sub>2</sub>/CO<sub>2</sub> for methanogenesis. However, strain CAN<sup>T</sup> differs from its closest phylogenetic
neighbors, *M. alcaliphilum* WeN4<sup>T</sup>, *M. movens* TS-2<sup>T</sup>, and *M. flexile* GH<sup>T</sup>, by its ability: (i) to
224 grow at 45°C (its optimum temperature), similarly to *M. aarhusense* H2-LR<sup>T</sup> (6), and (ii) to
225 grow autotrophically in mineral medium without any organic additions (e.g., yeast extract),
226 similarly to *Methanobacterium subterraneum* A8p<sup>T</sup> (5). To our knowledge, strain CAN<sup>T</sup> is also
227 singular by its ability to grow optimally at higher pH (i.e., pH 9.0) than other type species of
228 the genus *Methanobacterium*. Indeed, the two other alkaliphilic *Methanobacterium* members,
229 i.e., *M. alcaliphilum* WeN4<sup>T</sup> (8) and *M. subterraneum* A8p<sup>T</sup> (5), display optimal growth at pH
230 8.4 and pH 7.8–8.8, respectively. Based on morphological, phenotypic, phylogenetic, and
231 genomic analyses, strain CAN<sup>T</sup> is proposed to represent a novel species of the genus
232 *Methanobacterium*, *M. alkalithermotolerans* sp. nov.

233

234 Description of *Methanobacterium alkalithermotolerans*

235

236 *Methanobacterium alkalithermotolerans* (al.ka.li.ther.mo.to'le.rans. N.L. n. *alkali*, alkali (from
237 Arabic article *al*, the; Arabic n. *qaliy*, ashes of saltwort); Gr. fem. adj. *therme*, heat; L. pres.
238 part. *tolerans*, tolerating, enduring; N.L. part. adj. *alkalithermotolerans*, thermophilic alkali-
239 tolerating).

240

241 Displays similar traits as described for the genus. Cells are non-motile, straight rods 1.3-4.0
242 µm long and 0.5-0.7 µm wide, occurring singly, in pairs, or in chains. Colonies are white and
243 circular, 1 mm in diameter after two weeks of incubation. Alkaliphilic, mesophilic but
244 thermotolerant. Growth occurs at 20-45°C (optimum 45°C), at pH 7.3-9.7 (optimum pH 9.0)
245 with 0-1.5% NaCl (optimum 0%). Added NaCl and yeast extract were not required for growth.
246 Hydrogen and carbon dioxide were used as substrates for growth and methanogenesis. Acetate,
247 ethanol, formate, isobutanol, methanol, trimethylamine, and 2-propanol were not used for
248 growth. Resistant to novobiocin, streptomycin, and vancomycin (at 50 µg/mL) but sensitive to
249 ampicillin, bacitracin, chloramphenicol, cycloserine, kanamycin, metronidazole, penicillin,
250 rifampicin (at 25 µg/mL), and tetracycline (at 50 µg/mL). Most closely related to
251 *Methanobacterium alcaliphilum* WeN4T (98.5% 16S rRNA gene sequence identity). The G+C
252 content of the genomic DNA is 36.4%. The GenBank accession number for the 16S rRNA gene
253 sequence of strain CANT is KR349725, and that for the whole genome sequence is CP058560.
254 The type strain CANT (=DSM 102889T = JCM 31304T) was isolated from a sediment slurry of
255 La Crouen geothermal spring near Canala, New Caledonia.

256

257 **Conflicts of interest**

259 The authors declare that there are no conflicts of interest.

260

261 **Funding Information**

262 This project was financially supported by the French Research Institute for Development (IRD),
263 the INTERVIE CNRS INSU program, and the H2NAT project funded by the Midi-Pyrénées
264 Observatory (OMP, Toulouse) and ANR MICROPRONY (N°19-CE02-0020-02). We thank
265 Céline Roques at the GeT core facility, Toulouse, France (http://get.genotoul.fr), for the
266 genome sequencing of the CAN<sup>T</sup> strain, supported by France génomique. We thank Mathieu
267 Gonnet at the ISP (INRAE, University of Tours, France) for his assistance in the sequencing of
268 *M. alcaliphilum* DSM 3459. The LABGeM (CEA/Genoscope & CNRS UMR8030) is
269 acknowledged for support within the MicroScope annotation platform.

270

271
272 **Figures legends**

273

**Fig 1:** Phase-contrast micrographs of cells of strain CAN<sup>T</sup> (A; bar 10 µm) and transmission electron micrograph showing cells of strain CAN<sup>T</sup> (B; bar 1 µm), with Gram-positive type cell wall (C; bar 0.5 µm).

277

**Fig. 2:** Kinetics of hydrogen (H₂) consumption, methane (CH₄) production, and growth (optical density OD 600 nm × 10) of strain CAN. This monitoring was determined at pH 9.0 and 37°C in the minimal medium with the addition of H₂/CO₂ (80/20 v/v, 2 bars) as the only source of energy and carbon. Values are means ± standard deviations of triplicated cultures.

282

**Fig. 3:** Maximum-likelihood (ML) phylogenetic tree based on 1227 aligned bp of 16S rRNA gene sequences showing the position of strain CAN<sup>T</sup> among its closest neighbors belonging to the genus *Methanobacterium*. *Methanopyrus kandleri* AV19<sup>T</sup> (NR_074539) was used as an outgroup (not shown). Bootstrap values (based on 1000 replicates) are shown at branch nodes. Accession numbers are indicated in brackets. Bar: 0.02 substitutions per 100 nucleotides.

288

289
<table><caption>**Table 1.** Differential characteristics between strain CAN<sup>T</sup> (obtained in this study) and data obtained from previous studies for several type species of the genus *Methanobacterium*. Strains: 1, CAN<sup>T</sup>; 2, *M. alcaliphilum* WeN4<sup>T</sup> (8); 3, *M. movens* TS-2<sup>T</sup> (7); 4, *M. flexile* GH<sup>T</sup> (7); 5, *M. aarhusense* H2-LR<sup>T</sup> (6); 6, *M. subterraneum* A8p<sup>T</sup> (5).</caption><thead><tr><th>Characteristics</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th></tr></thead><tbody><tr><td>Origin</td><td>Sediment of thermal spring, New Caledonia</td><td>Alkaline lake sediment, Egypt</td><td>Sediment of Tuoso Lake, China</td><td>Sediment of Gahai Lak, China</td><td>Marine sediment, Denmark</td><td>Deep groundwater, Sweden</td></tr><tr><td>Cell size (µm)</td><td>0.5–0.7 x 1.3–4.0</td><td>0.5–0.6 x 2–25</td><td>0.4–0.5 x 2–5</td><td>0.3–0.5 x 2–5</td><td>0.7 x 5–18</td><td>0.1–0.15 x 0.6–1.2</td></tr><tr><td>Gram staining</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Motility</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Growth substrates</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>H<sub>2</sub> + CO<sub>2</sub></td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Formate</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Alcohols</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Acetate requirement</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Yeast extract requirement</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Optimum temperature (°C)</td><td>45</td><td>37</td><td>35–38</td><td>35–38</td><td>45</td><td>30–40</td></tr><tr><td>Optimum pH for growth</td><td>9.0</td><td>8.4</td><td>7.2–7.5</td><td>7.0–5</td><td>7.5–8.0</td><td>7.8–8.8</td></tr><tr><td>Optimum NaCl (%)</td><td>0</td><td>0–0.6</td><td>0–0.3</td><td>0–0.1</td><td>0.05–0.9</td><td>0.1–0.2</td></tr><tr><td>DNA G+C content (mol%)<sup>1</sup></td><td>36.4<sup>1*</sup></td><td>57<sup>2</sup></td><td>39.5<sup>2</sup></td><td>37.3<sup>2</sup></td><td>34.9<sup>2</sup></td><td>39.5<sup>1</sup> (54.5<sup>2</sup>)</td></tr></tbody></table>

+, Positive; -, negative.

293

294

295

<footnote n="1">¹ G+C content determined from genomic data.</footnote>

<footnote n="2">² G+C content determined by using HPLC.</footnote>
<table><caption>**Table 2.** Genome statistics of strain CAN<sup>T</sup>, and *Methanobacterium alcaliphilum* DSM3459 (both genomes obtained in this study).</caption><thead><tr><th>Genome / Sample Name</th><th>Strain CAN<sup>T</sup></th><th><i>Methanobacterium alcaliphilum</i> DSM 3459</th></tr></thead><tbody><tr><td>GenBank accession number</td><td>CP058560</td><td>JALKIF000000000</td></tr><tr><td>MaGe Genome ID</td><td>MAGE_000013558.1</td><td>MAGE_000016954.1</td></tr><tr><td>Genome Size (bp)</td><td>2,187,077</td><td>2,377,238</td></tr><tr><td>Contig count</td><td>NA</td><td>65</td></tr><tr><td>Scaffold count</td><td>1</td><td>65</td></tr><tr><td>GC (%)</td><td>36.4</td><td>33.4</td></tr><tr><td>Gene Count</td><td>2,189</td><td>2,375</td></tr><tr><td>Protein count</td><td>2,126</td><td>2,325</td></tr><tr><td>rRNA Count</td><td>7</td><td>4</td></tr><tr><td>tRNA Count</td><td>40</td><td>40</td></tr><tr><td>Other RNA</td><td>2</td><td>2</td></tr></tbody></table>
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