Hanako Mochimaru

Evidence for syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the high-temperature petroleum reservoir of Yabase oil field (Japan).

The methanogenic communities and pathways in a high-temperature petroleum reservoir were investigated through incubations of the production water and crude oil, combined with radiotracer experiments and molecular biological analyses. The incubations were conducted without any substrate amendment and under high-temperature and pressurized conditions that mimicked the in situ environment (55°C, 5 MPa). Changes in methane and acetate concentrations during the incubations indicated stoichiometric production of methane from acetate. Rates of hydrogenotrophic methanogenesis measured using [(14)C]-bicarbonate were 42-68 times those of acetoclastic methanogenesis measured using [2-(14) C]-acetate, implying the dominance of methane production by syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the environment. 16S rRNA gene sequence analyses of the incubated production water showed bacterial communities dominated by the genus Thermacetogenium, known as a thermophilic syntrophic acetate-oxidizing bacterium, and archaeal communities dominated by thermophilic hydrogenotrophic methanogens belonging to the genus Methanothermobacter. Furthermore, group-specific real-time PCR assays revealed that 16S rRNA gene copy numbers of the hydrogenotrophic methanogens affiliated with the order Methanobacteriales were almost identical to those of archaeal 16S rRNA genes. This study demonstrates that syntrophic acetate oxidation is the main methanogenic pathway in a high-temperature petroleum reservoir.

Methanolobus profundi sp. nov., a methylotrophic methanogen isolated from deep subsurface sediments in a natural gas field

A mesophilic, methylotrophic methanogen, strain MobM(T), was isolated from a natural gas field in Japan. Strain MobM(T) grew on methanol and methylamines, but not on H(2)/CO(2), formate, acetate or dimethyl sulfide. The cells were motile, irregular cocci (diameter, 0.9-1.2 microm) and occurred singly, in pairs, as tetracocci or (occasionally) as aggregates. Strain MobM(T) grew at 9-37 degrees C (optimally at 30 degrees C) and at pH 6.1-7.8 (optimally at pH 6.5). Sodium and magnesium were required for growth, at 0.1-1.0 M Na(+) (optimally at 0.35 M) and 10-400 mM Mg(2+) (optimally at 15-25 mM). The G+C content of the genomic DNA was 42.4 mol%. 16S rRNA gene sequencing revealed that the isolate is a member of the genus Methanolobus, but distinct from its closest neighbours, Methanolobus tindarius DSM 2278(T) (sequence similarity, 98.0 %) and Methanolobus vulcani DSM 3029(T) (98.1 %). On the basis of phenotypic and phylogenetic features of MobM(T), it is clear that this strain represents a novel species of the genus Methanolobus, for which the name Methanolobus profundi sp. nov. is proposed. The type strain is MobM(T) (=DSM 21213(T)=NBRC 104158(T)).

Methanogen Diversity in Deep Subsurface Gas-Associated Water at the Minami-Kanto Gas Field in Japan

Methanogen diversity and methanogenic potential in formation water obtained from the Minami-kanto gas field in Japan were investigated by using 16S rRNA gene libraries and culture-based enrichment methods, respectively. This region is the largest gas field that produces natural gases of dissolved-in-water type in Japan. Although the microbial population density was below statistical quantification limits (1 × 104 cells ml−1), autofluorescent coccoid and rod-shaped cells indicative of methanogens were observed. The represented genera in the archaeal 16S rRNA genes libraries were comprised of Methanobacterium, Methanospirillum, Methanocalculus, Methanococcus, Methanolobus and Methanosaeta. The dominant archaeal sequences were related to the hydrogenotrophic methanogens in the genus Methanobacterium. Of the methanogenic substrates tested using the formation water-based medium,H2-CO2 yielded the highest methane production. These results strongly suggest that the formation water of the Pleistocene strata in the gas fields harbor viable hydrogenotrophic methanogens and have possibly been making a contribution to ongoing methanogenesis.

Development of 16S rRNA gene‐targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs)

Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.

Methane production from coal by a single methanogen.

Microbes make methane from coal Methane associated with coal beds is an important global resource of natural gas. Much of the methane in coal comes from microbial methanogenesis. Mayumi et al. characterized a strain of Methermicoccus shengliensis that, unexpectedly, is capable of making methane from the dozens of methoxylated aromatic compounds found in a variety of coal types (see the Perspective by Welte). Isotope tracer experiments showed that this organism could also incorporate carbon dioxide into methane. Science, this issue p. 222; see also p. 184 Microbes can produce methane directly from the complex aromatic compounds found in a variety of coal substrates. Coal-bed methane is one of the largest unconventional natural gas resources. Although microbial activity may greatly contribute to coal-bed methane formation, it is unclear whether the complex aromatic organic compounds present in coal can be used for methanogenesis. We show that deep subsurface–derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs. In contrast to known methanogenesis pathways involving one- and two-carbon compounds, this “methoxydotrophic” mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl–coenzyme A metabolism. Because MACs derived from lignin may occur widely in subsurface sediments, methoxydotrophic methanogenesis would play an important role in the formation of natural gas not limited to coal-bed methane and in the global carbon cycle.

Methanothermobacter tenebrarum sp. nov., a hydrogenotrophic, thermophilic methanogen isolated from gas-associated formation water of a natural gas field.

A thermophilic and hydrogenotrophic methanogen, strain RMAS(T), was isolated from gas-associated formation water of a gas-producing well in a natural gas field in Japan. Strain RMAS(T) grew solely on H(2)/CO(2) but required Casamino acids, tryptone, yeast extract or vitamins for growth. Growth of strain RMAS(T) was stimulated by acetate. Cells were non-motile, straight rods (0.5×3.5-10.5 µm) and occurred singly or in pairs. Bundles of fimbriae occurred at both poles of cells and the cell wall was thick (approximately 21 nm, as revealed by ultrathin section electron microscopy). Strain RMAS(T) grew at 45-80 °C (optimum, 70 °C), at pH 5.8-8.7 (optimum, pH 6.9-7.7) and with 0.001-20 g NaCl l(-1) (optimum, 2.5 g NaCl l(-1)). Phylogenetic analysis revealed that Methanothermobacter thermautotrophicus ΔH(T) was most closely related to the isolate (95.7 % 16S rRNA gene sequence similarity). On the basis of morphological, phenotypic and phylogenetic characteristics, it is clear that strain RMAS(T) represents a novel species of the genus Methanothermobacter, for which we propose the name Methanothermobacter tenebrarum sp. nov. The type strain is RMAS(T) ( = DSM 23052(T) = JCM 16532(T) = NBRC 106236(T)).

Methanohalophilus levihalophilus sp. nov., a slightly halophilic, methylotrophic methanogen isolated from natural gas-bearing deep aquifers, and emended description of the genus Methanohalophilus

A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed.

Petrothermobacter organivorans gen. nov., sp. nov., a thermophilic, strictly anaerobic bacterium of the phylum Deferribacteres isolated from a deep subsurface oil reservoir

A novel thermophilic, anaerobic, chemoheterotrophic, acetate-oxidizing and iron(III)-, manganese(IV)-, nitrate- and sulfate-reducing bacterium, designated strain ANAT, was isolated from a deep subsurface oil field in Japan (Yabase oil field, Akita Pref.). Cells of strain ANAT were Gram-stain-negative, non-motile, non-spore forming and slightly curved or twisted rods (1.5-5.0 µm long and 0.6-0.7 µm wide). The isolate grew at 25-60 °C (optimum 55 °C) and pH 6.0-8.0 (optimum pH 7.0). The isolate was capable of reducing iron(III), manganese(IV), nitrate and sulfate as an electron acceptor. The isolate utilized a limited range of electron donors such as acetate, lactate, pyruvate and yeast extract for iron reduction. Strain ANAT also used pyruvate, fumarate, succinate, malate, yeast extract and peptone for fermentative growth. The major respiratory quinones were menaquinone-7(H8) and menaquinone-8. The strain contained C18 : 0, iso-C18 : 0 and C16 : 0 as the major cellular fatty acids. The G+C content of the genomic DNA was 34.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ANAT was closely related to Calditerrivibrio nitroreducens in the phylum Deferribacteres with low sequence similarities (89.5 %), and formed a distinct clade within the family Deferribacteraceae. In addition, the isolate is the first sulfate-reducing member of the phylum Deferribacteres. Based on phenotypic, chemotaxonomic and phylogenetic properties, a novel genus and species, Petrothermobacter organivorans gen. nov., sp. nov., is proposed for the isolate (type strain=ANAT= NBRC 112621T=DSM 105015T).

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).