Six novel species of the genus Methanoculleus isolated from various environments in Taiwan.

Taiwan is situated in the subtropical region and its geographical location and topographical features contribute to a rich ecological diversity and scenic landscapes. We investigated the diversity of methanogens in different environments of Taiwan using a culture-dependent method. This report presents the characterization and taxonomy of six hydrogenotrophic methanogens obtained from cold seep sediments (strain FWC-SCC1T and FWC-SCC3T), marine sediments (strain CWC-02T and YWC-01T), estuarine sediments (strain Afa-1T), and a hot spring well (strain Wushi-C6T) in Taiwan. The proposed names of the six novel species are Methanoculleus frigidifontis (type strain FWC-SCC1T=BCRC AR10056T=NBRC 113993T), Methanoculleus oceani (CWC-02T=BCRC AR10055T=NBRC 113992T), Methanoculleus methanifontis (FWC-SCC3T=BCRC AR10057T=NBRC 113994T), Methanoculleus nereidis (YWC-01T=BCRC AR10060T=NBRC 114597T), Methanoculleus formosensis (Afa-1T=BCRC AR10054T=NBRC 113995T), and Methanoculleus caldifontis (Wushi-06T=BCRC AR10059T= NBRC 114596T).

Methanofollis fontis sp. nov., a methanogen isolated from marine sediment near a cold seep at Four-Way Closure Ridge offshore southwestern Taiwan.

A mesophilic, hydrogenotrophic methanogen, strain FWC-SCC2T, was isolated from deep-sea sediments collected by a real-time video multiple-corer at the C5-6 station near a cold seep at Four-Way Closure Ridge region during R/V Ocean Researcher III ORIII-1900 cruise in 2015. The cells were irregular cocci, non-motile and 0.8-1.2 µm in diameter. The methanogenic substrates utilized by strain FWC-SCC2T were formate or H2+CO2, but not acetate, methanol, ethanol or methylamines. Strain FWC-SCC2T was lysed in SDS (0.01 %, w/v). The M r of surface-layer protein was 116 400. The optimum growth conditions of strain FWC-SCC2T were 37 °C, 0.17 M NaCl and pH 6.7-7.0. The genomic DNA G+C content calculated from the genome sequence of strain FWC-SCC2T was 59.5 mol %. Phylogenetic analysis revealed that strain FWC-SCC2T was a member of the genus Methanofollis, and was most closely related to Methanofollis tationis Chile 9T (97.6 % similarity of 16S rRNA gene sequence) and shared 97.4, 95.9, 95.9 and 95.4 % with Methanofollis liminatans GKZPZT, Methanofollis formosanus ML15T, Methanofollis aquaemaris N2F9704T and Methanofollis ethanolicus HASUT, respectively. The genome relatedness values between strain FWC-SCC2T and M. tationis DSM 2702T were estimated by average nucleotide identity and digital DNA-DNA hybridization analyses and the results were 79.4 and 21.2 %, respectively. Based on the differences in physiological and biochemical properties, 16S rRNA gene phylogeny and genome relatedness presented here, it is suggested that strain FWC-SCC2T represents a novel species of the genus Methanofollis, and the name Methanofollis fontis sp. nov. is proposed. The type strain is FWC-SCC2T (=BCRC AR10052T=DSM 107935T= NBRC 113164T).

The time response of anaerobic digestion microbiome during an organic loading rate shock.

Knowledge of connections between operational conditions, process stability, and microbial community dynamics is essential to enhance anaerobic digestion (AD) process efficiency and management. In this study, the detailed temporal effects of a sudden glycerol-based organic overloading on the AD microbial community and process imbalance were investigated in two replicate anaerobic digesters by a time-intensive sampling scheme. The microbial community time response to the overloading event was shorter than the shifts of reactor performance parameters. An increase in bacterial community dynamics and in the abundances of several microbial taxa, mainly within the Firmicutes, Tenericutes, and Chloroflexi phyla and Methanoculleus genera, could be detected prior to any shift on the reactor operational parameters. Reactor acidification already started within the first 24 h of the shock and headed the AD process to total inhibition in 72 h alongside with the largest shifts on microbiome, mostly the increase of Anaerosinus sp. and hydrogenotrophic methanogenic Archaea. In sum, this work proved that AD microbial community reacts very quickly to an organic overloading and some shifts occur prior to alterations on the performance parameters. The latter is very interesting as it can be used to improve AD process management protocols.

A novel archaeal species belonging to Methanoculleus genus identified via de-novo assembly and metagenomic binning process in biogas reactors.

Recently, a first comprehensive catalogue of microbial genomes populating biogas reactors treating manure and agro-industrial residues was determined by sequencing samples collected from 22 biogas reactors including laboratory and full scale. Among the archaeal community, one of the most abundant methanogens belongs to Methanoculleus genus and for this reason it was provisionally named Methanoculleus sp. DTU006. Its full length 16S rRNA sequence is 97% similar to Methanoculleus marisnigri JR1 and to Methanoculleus palmolei DSM 4273. Despite the high similarity of the 16S gene sequence, Average Nucleotide Identity calculation (ANI) calculated on all protein encoding genes indicated that the two most similar species, Methanoculleus bourgensis MS2T and Methanoculleus sp. MAB1, are divergent enough to define Methanoculleus sp. DTU006 as new archaeal species. Its genome (2.15 Mbp) has an estimated completeness around 93%. Analysis of the metabolic pathways using KEGG confirmed that it is a hydrogenotrophic methanogen and therefore it is proposed the Candidatus status by naming it as "Candidatus Methanoculleus thermohydrogenotrophicum".

Methanomicrobium antiquum sp. nov., a hydrogenotrophic methanogen isolated from deep sedimentary aquifers in a natural gas field.

A mesophilic, hydrogenotrophic methanogen, designated strain MobHT, was isolated from sediments derived from deep sedimentary, natural-gas-bearing aquifers in Japan. Strain MobHT utilized H2/CO2 or formate, but not ethanol, 1-propanol, 2-propanol, 2-butanol or cyclopentanol, for growth and methane production. In addition, acetate and tungsten were required for growth. Yeast extract stimulated the growth, but was not required. The cells were weakly motile with multiple flagella, presented as a curved-rod-shaped (0.8×2.0 µm) and occurred singly or in pairs. Strain MobHT grew at 15-40 °C (optimum 35 °C) and at pH 5.9-7.9 (optimum pH 7.0-7.5). The sodium chloride range for growth was 0-5.8 % (optimum 2 %). The G+C content of the genomic DNA was 37.6 mol%. In the phylogenetic tree based on the 16S rRNA gene sequences, strain MobHT clustered together with Methanomicrobium mobile (95.4 % in sequence similarity), and formed a distinct clade from Methanolacinia petrolearia SEBR 4847T (95.6 %) and Methanolacinia paynteri G-2000T (95.4 %). The two species of the genus Methanolacinia utilized 2-propanol, whereas strain MobHT and Methanomicrobium mobile, the sole species of the genus Methanomicrobium, do not. Based on phenotypic and phylogenetic features, we propose a novel species for the isolate with the name, Methanomicrobiumantiquum sp. nov. The type strain is MobHT (=DSM 21220T=NBRC 104160T).

Methanoculleus taiwanensis sp. nov., a methanogen isolated from deep marine sediment at the deformation front area near Taiwan.

A mesophilic, hydrogenotrophic methanogen, strain CYW4(T), was isolated from deep-sea sediment obtained by the Ocean Researcher I cruiser, ORI-961, in 2011. The sediment was from the deformation front area offshore of south-western Taiwan. Here, seismic reflections indicated that methane hydrates were abundant. The methanogenic substrates utilized by strain CYW4(T) were formate and H2/CO2, but not acetate, secondary alcohols, methylamines, methanol and ethanol. Cells of strain CYW4(T) were non-motile, irregular cocci and 0.6-1.5 µm in diameter. The S-layer protein had an Mr of 112 000. The optimum growth conditions were at 37 °C, pH 8.1 and 0.08 M NaCl. Growth of the strain was stimulated by acetate. The G+C content of the chromosomal DNA of strain CYW4(T) was 61 mol%. Phylogenetic analysis revealed that strain CYW4(T) was most closely related to Methanoculleus marisnigri JR1(T) (96.82 % 16S rRNA gene sequence similarity). Based on the morphological, phenotypic and phylogenetic characteristics presented here, it is evident that strain CYW4(T) represents a novel species of the genus Methanoculleus, and the name Methanoculleus taiwanensis sp. nov. is proposed. The type strain is CYW4(T) ( = BCRC AR10043(T) = NBRC 110782(T)). The optical density of cultures of strain CYW4(T) dropped abruptly upon entering the stationary growth phase. During this time numerous particles of approximately 50 nm in diameter were observed on and around the cells. This suggests that strain CYW4(T) harbours a lytic virus that is induced in the stationary phase, which is of interest because only a few lytic viruses have been reported in methanogens.

Methanoculleus sediminis sp. nov., a methanogen from sediments near a submarine mud volcano.

A mesophilic, hydrogenotrophic methanogen, strain S3Fa(T), was isolated from sediments collected by Ocean Researcher I cruise ORI-934 in 2010 near the submarine mud volcano MV4 located at the upper slope of south-west Taiwan. The methanogenic substrates utilized by strain S3Fa(T) were formate and H2/CO2 but not acetate, secondary alcohols, methylamines, methanol or ethanol. Cells of strain S3Fa(T) were non-motile, irregular cocci, 0.5-1.0 µm in diameter. The surface-layer protein showed an Mr of 128,000.The optimum growth conditions were 37 °C, pH 7.1 and 0.17 M NaCl. The DNA G+C content of the genome of strain S3Fa(T) was 62.3 mol%. Phylogenetic analysis revealed that strain S3Fa(T) was most closely related to Methanoculleus marisnigri JR1(T) (99.3% 16S rRNA gene sequence similarity). Genome relatedness between strain S3Fa(T) and Methanoculleus marisnigri JR1(T) was computed using both genome-to-genome distance analysis (GGDA) and average nucleotide identity (ANI) with values of 46.3-55.5% and 93.08%, respectively. Based on morphological, phenotypic, phylogenetic and genomic relatedness data, it is evident that strain S3Fa(T) represents a novel species of the genus Methanoculleus, for which the name Methanoculleus sediminis sp. nov. is proposed. The type strain is S3Fa(T) ( = BCRC AR10044(T) = DSM 29354(T)).

Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples.

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-(13)C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of (13)C into DNA was detectable at in situ acetate concentrations (~7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the (13)C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

Methanoculleus horonobensis sp. nov., a methanogenic archaeon isolated from a deep diatomaceous shale formation.

A methanogenic organism from the domain Archaea, designated strain T10(T), was isolated from groundwater sampled from a deep diatomaceous shale formation located in Horonobe, Hokkaido, Japan. The strain utilized H2/CO2 and formate as substrates for methanogenesis. Cells were strictly anaerobic, Gram-negative-staining, flagellated, irregular coccoids, 0.7-1.6 µm in diameter, and occurred singly. The strain grew at 25-45 °C (optimum 37-42 °C), at pH 5.8-8.2 (optimum pH 6.7-6.8) and in the presence of 0-1.3 M NaCl (optimum 0.1-0.2 M NaCl). The G+C content of the genomic DNA was 62.9 mol%. 16S rRNA gene sequencing revealed that, although the strain is a member of the genus Methanoculleus, it clearly differed from all described species of this genus (95.5-98.3 % sequence similarity). Values for DNA-DNA hybridization with type strains of closely related Methanoculleus species were less than 50 %. Phenotypic and phylogenetic features of strain T10(T) clearly indicate that it represents a novel species of the genus Methanoculleus, for which the name Methanoculleus horonobensis sp. nov. is proposed. The type strain is T10(T) ( = DSM 21626(T) = JCM 15517(T)).

Complete genome sequence of the hydrogenotrophic, methanogenic archaeon Methanoculleus bourgensis strain MS2(T), Isolated from a sewage sludge digester.

Methanoculleus bourgensis, of the order Methanomicrobiales, is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2(T), isolated from a sewage sludge digester.

Dominance of Methanomicrobium phylotype in methanogen population present in Murrah buffaloes (Bubalus bubalis).

AIMS: To study the diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India by using 16S rRNA gene libraries obtained from the pooled rumen content from four animals and using suitable software analysis. METHODS AND RESULTS: Genomic DNA was isolated and PCR was set up by using specific primers. Amplified product was cloned into a suitable vector and the positive clones were selected on the basis of blue-white screening and sequenced. The resulting nucleotide sequences were arranged in the phylogenetic tree. A total of 108 clones were examined, revealing 17 different 16S rRNA gene sequences or phylotypes. Of the 17 phylotypes, 15 (102 of 108 clones) belonged to the genus Methanomicrobium, indicating that the genus Methanomicrobium is the most dominant component of methanogen populations in Murrah buffaloes (Bubalus bubalis) from North India. The largest group of clones (102 clones) was more than 98% similar to Methanomicrobium mobile. BLAST analysis of the rumen contents from individual animals also revealed 17 different phylotypes with a range of 3-10 phylotypes per animal. CONCLUSION: Methanomicrobium phylotype is the most dominant phylotype of methanogens present in Murrah buffaloes (Bubalus bubalis). SIGNIFICANCE AND IMPACT OF THE STUDY: Effective strategies can be made to inhibit the growth of Methanomicrobium phylotype to reduce the methane emission from rumen contents and thus help in preventing global warming.

Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea.

Although of limited metabolic diversity, methanogenic archaea or methanogens possess great phylogenetic and ecological diversity. Only three types of methanogenic pathways are known: CO(2)-reduction, methyl-group reduction, and the aceticlastic reaction. Cultured methanogens are grouped into five orders based upon their phylogeny and phenotypic properties. In addition, uncultured methanogens that may represent new orders are present in many environments. The ecology of methanogens highlights their complex interactions with other anaerobes and the physical and chemical factors controlling their function.

Isolation and characterization of Methanoculleus receptaculi sp. nov. from Shengli oil field, China.

Three strictly anaerobic, thermophilic methanogens (ZC-2T, ZC-3 and ZC-6) were isolated from Shengli oil field, China. The 16S rRNA gene sequences of the three strains were nearly identical, possessing > 99.8% sequence similarity. They also possessed high sequence similarity, 97.4%, to Methanoculleus palmolei strain INSLUZ(T) (97.4% and 97.5%, respectively), indicating that they represented a novel species within the genus Methanoculleus. Cells of strain ZC-2T were nonmotile cocci, 0.8-1.7 microm in diameter, and always occurred singly or in pairs. The three strains used H2/CO2 or sodium formate as substrates for methanogenesis but not sodium acetate, trimethylamine, monomethylamine, ethanol, dimethyl sulfide, isopropanol, isobutanol, butan-2-ol or H2/CO. Optimum growth of strain ZC-2T occurred in the presence of 0.2 M NaCl, pH 7.5-7.8 and temperature 50-55 degrees C with a specific growth rate of 0.084 h(-1). The mol% G+C content of the genomic DNA was 55.2 mol%. Based on these phenotypic and phylogenetic characteristics, strains ZC-2T, ZC-3 and ZC-6 are proposed to represent a novel species in the genus Methanoculleus and named Methanoculleus receptaculi sp. nov. The type strain is ZC-2T (CGMCC 1.5087T=DSM 18860T).

Links between archaeal community structure, vegetation type and methanogenic pathway in Alaskan peatlands.

Although northern peatlands contribute significantly to natural methane emissions, recent studies of the importance and type of methanogenesis in these systems have provided conflicting results. Mechanisms controlling methanogenesis in northern peatlands remain poorly understood, despite the importance of methane as a greenhouse gas. We used 16S rRNA gene retrieval and denaturing gradient gel electrophoresis (DGGE) to analyse archaeal communities in 15 high-latitude peatland sites in Alaska and three mid-latitude peatland sites in Massachusetts. Archaeal community composition was analysed in the context of environmental, vegetation and biogeochemical factors characterized in a parallel study. Phylogenetic analysis revealed that Alaskan sites were dominated by a cluster of uncultivated crenarchaeotes and members of the families Methanomicrobiaceae and Methanobacteriaceae, which are not acetoclastic. Members of the acetoclastic family Methanosarcinaceae were not detected, whereas those of the family Methanosaetaceae were either not detected or were minor. These results are consistent with biogeochemical evidence that acetoclastic methanogenesis is not a predominant terminal decomposition pathway in most of the sites analysed. Ordination analyses indicated a link between vegetation type and archaeal community composition, suggesting that plants (and/or the environmental conditions that control their distribution) influence both archaeal community activity and dynamics.

Thermophilic methanogenic Archaea in compost material: occurrence, persistence and possible mechanisms for their distribution to other environments.

Since compost is widely used as soil amendment and the fact that during the processing of compost material high amounts of microorganisms are released into the air, we investigated whether compost may act as a carrier for thermophilic methanogens to temperate soils. All eight investigated compost materials showed a clear methane production potential between 0.01 and 0.98 micromol CH(4) g dw(-1)h(-1) at 50 degrees C. Single strand conformation polymorphism (SSCP) and cloning analysis indicated the presence of Methanosarcina thermophila, Methanoculleus thermophilus, and Methanobacterium formicicum. Bioaerosols collected during the turning of a compost pile showed both a highly similar SSCP profile compared to the corresponding compost material and clear methane production during anoxic incubation in selective medium at 50 degrees C. Both observations indicated a considerable release of thermophilic methanogens into the air. To analyse the persistence of compost-borne thermophilic methanogens in temperate oxic soils, we therefore studied their potential activity in compost and compost/soil mixtures, which was brought to a meadow soil, as well as in an agricultural soil fertilised with compost. After 24h anoxic incubation at 50 degrees C, all samples containing compost showed a clear methanogenic activity, even 1 year after application. In combination with the in vitro observed resilience of the compost-borne methanogens against desiccation and UV radiation we assume that compost material acts as an effective carrier for the distribution of thermophilic methanogens by fertilisation and wind.

Abundance and diversity of methanogens: potential role in high arsenic groundwater in Hetao Plain of Inner Mongolia, China.

To investigate the community diversity and abundance of methanogens and their potential role in high arsenic groundwater, 17 groundwater samples from Hetao Plain of Inner Mongolia were investigated with an integrated method including 16S rRNA gene clone library, quantitative polymerase chain reaction and geochemistry analyses. Total arsenic (AsTot) concentrations were 82.7-1088.7 µg/L and arsenite (AsIII) mostly dominated in these samples with percentages of 0.04-0.79. CH4 concentrations ranged from 0.01 to 292 µg/L and distinctly elevated only when AsTot were relatively high and SO4(2-) were distinctly low. Principal component analysis indicated that these samples were divided into three groups according to the variations of AsTot, CH4 and SO4(2-). AsTot concentrations were distinctly high in the group with high CH4 and low SO4(2-) comparing to the other two groups (one with high CH4 and high SO4(2-), the other with low CH4 and SO4(2-)). The mcrA gene (methyl coenzyme-M reductase gene) based phylogenetic analysis of methanogens population showed that methanogenic archaea was diverse but mainly composed of Methanomicrobiales, Methanosarcinales, Methanobacteria and unidentified groups, with Methanomicrobiales being distinctly dominant (50.6%). The mcrA gene abundance in high arsenic groundwater ranged from 3.01 × 10(3) to 3.80 × 10(6)copies/L and accounted for 0-30.2% of total archaeal 16S rRNA genes. The abundance of mcrA genes was positively correlated with the concentrations of AsTot (R=0.59), AsIII (R=0.57) and FeII (R=0.79), while it was negatively correlated with oxidation-reduction potential (R=-0.66) and SO4(2-) concentration (R=-0.64). These results implied that methanogenic archaea might accelerate As release in groundwater aquifers in Hetao Plain.

Ammonia effect on hydrogenotrophic methanogens and syntrophic acetate-oxidizing bacteria.

Ammonia-rich substrates can cause inhibition on anaerobic digestion process. Syntrophic acetate-oxidizing bacteria (SAOB) and hydrogenotrophic methanogens are important for the ammonia inhibitory mechanism on anaerobic digestion. The roles and interactions of SAOB and hydrogenotrophic methanogens to ammonia inhibition effect are still unclear. The aim of the current study was to determine the ammonia toxicity levels of various pure strains of SAOB and hydrogenotrophic methanogens. Moreover, ammonia toxicity on the syntrophic-cultivated strains of SAOB and hydrogenotrophic methanogens was tested. Thus, four hydrogenotrophic methanogens (i.e. Methanoculleus bourgensis, Methanobacterium congolense, Methanoculleu thermophilus and Methanothermobacter thermautotrophicus), two SAOB (i.e. Tepidanaerobacter acetatoxydans and Thermacetogenium phaeum) and their syntrophic cultivation were assessed under 0.26, 3, 5 and 7 g NH4 (+)-N L(-1). The results showed that some hydrogenotrophic methanogens were equally, or in some cases, more tolerant to high ammonia levels compared to SAOB. Furthermore, a mesophilic hydrogenotrophic methanogen was more sensitive to ammonia toxicity compared to thermophilic methanogens tested in the study, which is contradicting to the general belief that thermophilic methanogens are more vulnerable to high ammonia loads compared to mesophilic. This unexpected finding underlines the fact that the complete knowledge of ammonia inhibition effect on hydrogenotrophic methanogens is still absent.

Biogas production from coumarin-rich plants--inhibition by coumarin and recovery by adaptation of the bacterial community.

Plants like sweet clover (Melilotus spp.) are not suitable as fodder for cattle because of harmful effects of the plant secondary metabolite coumarin. As an alternative usage, the applicability of coumarin-rich plants as substrates for biogas production was investigated. When coumarin was added to continuous fermentation processes codigesting grass silage and cow manure, it caused a strong inhibition noticeable as decrease of biogas production by 19% and increase of metabolite concentrations to an organic acids/alkalinity ratio higher than 0.3(gorganic acids) gCaCO3 (-1). Microbial communities of methanogenic archaea were dominated by the genera Methanosarcina (77%) and Methanoculleus (11%). This community composition was not influenced by coumarin addition. The bacterial community analysis unraveled a divergence caused by coumarin addition correlating with the anaerobic degradation of coumarin and the recovery of the biogas process. As a consequence, biogas production resumed similar to the coumarin-free control with a biogas yield of 0.34 LN g(volatile solids) (-1) and at initial metabolite concentrations (∼ 0.2 g(organic acids) gCaCO3 (-1)). Coumarin acts as inhibitor and as substrate during anaerobic digestion. Hence, coumarin-rich plants might be suitable for biogas production, but should only be used after adaptation of the microbial community to coumarin.

[Methanocalculus natronophilus sp. nov., a new alkaliphilic hydrogenotrophic methanogenic archaeon from a soda lake, and proposal of the new family Methanocalculaceae].

A mesophilic hydrogenotrophic methanogenic archaeon, strain Z-7105(T), was isolated from the bottom sediments of a collector in the vicinity of a soda lake Tanatar II (Altai, Russia). The cells were motile, irregular cocci 0.2-1.2 µm in diameter. The organism was an obligate alkaliphile, growing within a pH range from 8.0 to 10.2 with the optimum at pH 9.0-9.5. It was obligately dependent on carbonates, growing at 0.5 to 1.6 M total carbonates with the optimum at 0.7-0.9 M. Sodium ions were also obligately required at concentrations from 0.9 to 3.3 M Na+ (optimum at 1.4-1.9 M). The organism was halotolerant, but Cl- ions were not required. Hydrogen and formate were used as electron donors. Acetate was required for anabolism. The DNA G + C content was 50.2 mol %. According to the results of its 16S rRNA gene sequence analysis, the isolate belonged to the genus Methanocalculus, being the first known alkaliphilic member of this genus. Its similarity to the neutrophilic and halotolerant Methanocalculus species (M. halotolerans, M. taiwanensis, M. pumilus, and M. chunghsingensis) was 98.2-97.1%, which is within the interspecific range for this genus. The level of DNA-DNA hybridization between strain Z-7105(T) and the Methanocalculus type species M. halotolerans DSM 14092(T) was 32%. The genus Methanocalculus, including the new isolate and the previously described species, is distant from other genera of methanogens (< 90% 16S rRNA gene similarity). Based on significant phenotypic differences and the results of phylogenetic analysis, including DNA-DNA hybridization, it is proposed to assign strain Z-7105(T) (= DSM 25006(T), = VKM B-2765(T)) to the new species Methanocalculus natronophilus sp. nov. and to incorporate the genus into the new family Methanocalculaceae fam. nov.