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).

Use of microbial indicators combined with environmental factors coupled with metrology tools for discrimination and classification of Luzhou-flavoured pit muds.

AIMS: Pit mud is essential for the quality and yield of Chinese Luzhou-flavoured liquor. A reliable and fast method based on the use of microbial indicators combined with environmental factors coupled with metrology tools is needed to discriminate and classify different maturity levels of Luzhou-flavoured pit muds. METHODS AND RESULTS: Firmicutes, Bacteroidetes, Actinobacteria, Lactobacillus, Bacillus, Methanosarcina, Methanocorpusculum, Methanoculleus and Clostridium kluyveri were microbial indicators in Luzhou-flavoured pit muds. They were detected by real-time quantitative PCR. Environmental factors investigated included moisture content, pH, total acid and ammonia nitrogen. Principal component analysis (PCA) and partial least square-discriminant analysis were employed to explore the structure of the data and construct discrimination and classification models by reduction in the data dimensionality. Pit muds were distinguished and classified as new, trend to-be aged and aged. Moisture content and pH were significantly negatively correlated with new pit mud, while pH, total acid, amino nitrogen, Firmicutes, Bacteroidetes, Actinobacteria, Methanosarcina, Methanoculleus and C. kluyveri were significantly positively correlated with aged pit mud. CONCLUSIONS: Microbial indicators combined with environmental factors coupled to metrology tools can reliably and quickly discriminate and classify different maturity levels of Luzhou-flavoured pit muds. SIGNIFICANCE AND IMPACT OF THE STUDY: Modern techniques and metrology tools verified the correctness of the traditional sensory evaluation used in controlling the quality of pit mud, and will contribute to distinguishing different maturity levels of Chinese Luzhou-flavoured pit muds.

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).

Methanogen communities in stools of humans of different age and health status and co-occurrence with bacteria.

Hydrogenotrophic methanogens live in a synthrophic relationship with the human gut microbiota as the terminal part of the anaerobic food chain. Methanobrevibacter smithii of the Methanobacteriales is the prevailing archaeal species. Recently, methylotrophic archaea of the novel order Methanomassiliicoccales were isolated from human stools. Few data exist on the prevalence, abundance, persistence and ecology of these methanogens in humans. This study investigated methanogen communities in 26 healthy and obese children (8-14 years) and 18 adults (28-78 years) using quantitative PCR. Samples were obtained from nine females before and after giving birth. Bacterial groups linked to the abundance of methanogens in adult females were identified using a 16S rRNA gene amplicon data set. A total of 89% and 65% of adults and children, respectively, carried Methanobacteriales. Methanomassiliicoccales were recovered from 50% of the adults and one child. Mean relative abundance of Methanomassiliicoccales in adults was lower than that of Methanobacteriales (0.10% versus 0.52%). Both Methanobacteriales and Methanomassiliicoccales formed stable communities in females before and after giving birth. Methanobacteriales co-occurred with bacterial taxonomic groups associated with the trophic chain from carbohydrate degradation to hydrogen and formate formation. Relative abundance was inversely correlated to Blautia. Negative correlation with little characterized groups within the Clostridiales indicated possible interactions of Methanomassiliicoccales with the bacterial community.

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)).

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.

¹³C pulse-chase labeling comparative assessment of the active methanogenic archaeal community composition in the transgenic and nontransgenic parental rice rhizospheres.

More and more investigations indicate that genetic modification has no significant or persistent effects on microbial community composition in the rice rhizosphere. Very few studies, however, have focused on its impact on functional microorganisms. This study completed a ¹³C-CO2 pulse-chase labeling experiment comparing the potential effects of cry1Ab gene transformation on ¹³C tissue distribution and rhizosphere methanogenic archaeal community composition with its parental rice variety (Ck) and a distant parental rice variety (Dp). Results showed that ¹³C partitioning in aboveground biomass (mainly in stems) and roots of Dp was significantly lower than that of Ck. However, there were no significant differences in ¹³C partitioning between the Bt transgenic rice line (Bt) and Ck. RNA-stable isotope probing combined with clone library analyses inferred that the group Methanosaetaceae was the predominant methanogenic Archaea in all three rice rhizospheres. The active methanogenic archaeal community in the Bt rhizosphere was dominated by Methanosarcinaceae, Methanosaetaceae, and Methanomicrobiaceae, while there were only two main methanogenic clusters (Methanosaetaceae and Methanomicrobiaceae) in the Ck and Dp rhizospheres. These results indicate that the insertion of cry1Ab gene into the rice genome has the potential to result in the modification of methanogenic community composition in its rhizosphere.

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)).

Biodiversity and composition of methanogenic populations in the rumen of cows fed alfalfa hay or triticale straw.

It is clear that methanogens are responsible for ruminal methane emissions, but quantitative information about the composition of the methanogenic community in the bovine rumen is still limited. The diversity and composition of rumen methanogens in cows fed either alfalfa hay or triticale straw were examined using a full-cycle rRNA approach. Quantitative fluorescence in situ hybridization undertaken applying oligonucleotide probes designed here identified five major methanogenic populations or groups in these animals: the Methanobrevibacter TMS group (consisting of Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii), Methanbrevibacter ruminantium-, Methanosphaera stadtmanae-, Methanomicrobium mobile-, and Methanimicrococcus-related methanogens. The TMS- and M. ruminantium-related methanogens accounted for on average 46% and 41% of the total methanogenic cells in liquid (Liq) and solid (Sol) phases of the rumen contents, respectively. Other prominent methanogens in the Liq and Sol phases included members of M. stadtmanae (15% and 33%), M. mobile (17% and 12%), and Methanimicrococcus (23% and 9%). The relative abundances of these methanogens in the community varied among individual animals and across diets. No clear differences in community composition could be observed with dietary change using cloning techniques. This study extends the known biodiversity levels of the methanogenic communities in the rumen of cows.

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.

Physico-chemical characteristics and methanogen communities in swine and dairy manure storage tanks: spatio-temporal variations and impact on methanogenic activity.

Greenhouse gas emissions represent a major environmental problem associated with the management of manure from the livestock industry. Methane is the primary GHG emitted during manure outdoor storage. In this paper, the variability of two swine and two dairy manure storage tanks was surveyed, in terms of physico-chemical and microbiological parameters. The impact of the inter-tank and spatio-temporal variations of these parameters on the methanogenic activity of manure was ascertained. A Partial Least Square regression was carried out, which demonstrated that physico-chemical as well as microbiological parameters had a major influence on the methanogenic activity. Among the 19 parameters included in the regression, the concentrations of VFAs had the strongest negative influence on the methane emission rate of manure, resulting from their well-known inhibitory effect. The relative abundance of two amplicons in archaeal fingerprints was found to positively influence the methanogenic activity, suggesting that Methanoculleus spp. and possibly Methanosarcina spp. are major contributors to methanogenesis in storage tanks. This work gave insights into the mechanisms, which drive methanogenesis in swine and dairy manure storage tanks.

Pulp mill wastewater sediment reveals novel methanogenic and cellulolytic populations.

Pulp mill wastewater generated from wheat straw is characterized as high alkalinity and very high COD pollution load. A naturally developed microbial community in a pulp mill wastewater storage pool that had been disused were investigated in this study. Owing to natural evaporation and a huge amount of lignocellulose's deposition, the wastewater sediment contains high concentrations of organic matters and sodium ions, but low concentrations of chloride and carbonate. The microbiota inhabiting especially anaerobic community, including methanogenic arhcaea and cellulolytic species, was studied. All archaeal sequences fall into 2 clusters of family Halobacteriaceae and methanogenic archaeon in the phylum Euryarchaeota. In the methanogenic community, phylogenetic analysis of methyl coenzyme M reductase A (mcrA) genes targeted to novel species in genus Methanoculleus or novel genus of order Methanomicrobiales. The predominance of Methanomicrobiales suggests that methanogenesis in this system might be driven by the hydrogenotrophic pathway. As the important primary fermenter for methane production, the cellulolytic community of enzyme GHF48 was found to be dominated by narrower breadth of novel clostridial cellulase genes. Novel anoxic functional members in such extreme sediment provide the possibility of enhancing the efficiency of anoxic treatment of saline and alkaline wastewaters, as well as benefiting to the biomass transformation and biofuel production processes.

[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.

[Isolation and identification of a methanogen from the high temperature oil reservoir water].

To explore new microbial resources in deep subsurface oil reservoirs, strain DL-7 was isolated with Hungate technology from oil reservoir water sampled from Dagang oilfield, China. Physiological and biochemical examinations showed that H2/CO2 is the unique substrate of the strain, which cannot metabolize formate, methanol, trimethylamine, acetate and other secondary alcohols. The optimum growth conditions were further identified to be 60 degrees C, pH 7.0-7.5 and 0.25% NaCl. Moreover, the strain cannot grow without yeast extract. Analysis of its 16S rRNA sequence indicated that a similarity of 99.7% presents between the strain and the model species M. marburgensis DSM2133T (X15364).

Methanoculleus hydrogenitrophicus sp. nov., a methanogenic archaeon isolated from wetland soil.

An obligately anaerobic, methanogenic archaeon, strain HC(T), was isolated from soil of the Zoige wetland on the Tibetan plateau, China. The strain was isolated through construction of an artificial butyrate-degrading consortium in co-culture with a syntrophic bacterium, 'Syntrophomonas erecta subsp. sporosyntropha' JCM 13344. Cells of strain HC(T) were irregular coccoids, 0.8-2 mum in diameter, that occurred singly and utilized only H(2)/CO(2) for growth and methane production. Growth occurred at 18-45 degrees C (optimum around 37 degrees C). The pH for growth was 5.0-8.5 (optimal growth around pH 6.6). The G+C content of the genomic DNA was 60.2 mol%. 16S rRNA gene sequence analysis indicated that strain HC(T) was affiliated to the genus Methanoculleus, with sequence similarities of 94.8-97.2 % to existing members. However, strain HC(T) was distinguished from described Methanoculleus species by not using formate for growth or methane formation and not requiring acetate as a growth factor. On the basis of phylogenetic analysis and phenotypic characteristics, the novel species Methanoculleus hydrogenitrophicus sp. nov. is proposed, with strain HC(T) (=CGMCC 1.5146(T) =JCM 16311(T)) as the type strain.

5'-MGB probes allow rapid identification of methanogens and sulfate reducers in cold marine sediments by real-time PCR and melting curve analysis.

The analysis of microorganism communities in uncultured environmental samples requires laborious and cumbersome techniques such as denaturing gradient gel electrophoresis of amplicons generated with 16S rRNA generic primers with subsequent fragment sequencing. We have developed a simple method for genus identification of methanogen archaea and sulfate-reducing bacteria based on a real-time PCR hybridization probe melting curve analysis. The method takes advantage of a recent explosion of microorganism sequencing data conveniently packaged in the Ribosomal Database Project. Specificity of detection is based on a genus-specific real-time PCR fluorescent 5'-MGB-probe melt. As the probes are designed to have destabilizing mismatches with undesired genera, only samples with a proper melting temperature are called positive.

Design and testing of real-time PCR primers for the quantification of Methanoculleus, Methanosarcina, Methanothermobacter, and a group of uncultured methanogens.

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta, Methanosarcina, and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter, and a group of yet uncultured archaea from anaerobic habitats.

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.

Methanofollis ethanolicus sp. nov., an ethanol-utilizing methanogen isolated from a lotus field.

A novel methane-producing archaeon, designated strain HASU(T), was isolated from a lotus field. Cells were Gram-negative, non-motile, irregular cocci, 2-3 mum in diameter, and occurred singly. Growth was observed at 15-40 degrees C (optimum, 37 degrees C) and pH 6.5-7.5 (optimum, pH 7.0). The G+C content of the genomic DNA was 60.9 mol%. Strain HASU(T) utilized ethanol, 1-propanol, 1-butanol, hydrogen and formate for growth and methane production. It converted ethanol to methane and acetate. Based on comparative 16S rRNA gene sequence analysis, strain HASU(T) was shown to be affiliated with the genus Methanofollis. It was related most closely to the type strain of Methanofollis liminatans (96.1 % 16S rRNA gene sequence similarity). Based on phylogenetic analysis and phenotypic characteristics, strain HASU(T) is considered to represent a novel species of the genus Methanofollis, for which the name Methanofollis ethanolicus sp. nov. is proposed. The type strain is HASU(T) (=NBRC 104120(T)=JCM 15103(T)=DSM 21041(T)).