Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea.

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate-methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.

Methanolobus halotolerans sp. nov., isolated from the saline Lake Tus in Siberia.

A halotolerant, psychrotolerant and methylotrophic methanogen, strain SY-01T, was isolated from the saline Lake Tus in Siberia. Cells of strain SY-01T were non-motile, cocci and 0.8-1.0 µm in diameter. The only methanogenic substrate utilized by strain SY-01T was methanol. The temperature range of growth for strain SY-01T was from 4 to 40 °C and the optimal temperature for growth was 30 °C. The pH range of growth was from pH 7.2 to 9.0, with optimal growth at pH 8.0. The NaCl range of growth was 0-1.55 M with optimal growth at 0.51 M NaCl. The G+C content of the genome of strain SY-01T was 43.6 mol % as determined by genome sequencing. Phylogenetic analysis revealed that strain SY-01T was most closely related to Methanolobus zinderi SD1T (97.3 % 16S rRNA gene sequence similarity), and had 95.5-97.2 % similarities to other Methanolobus species with valid names. Genome relatedness between strain SY-01T and DSM 21339T was computed using average nucleotide identity and digital DNA-DNAhybridization, which yielded values of 79.7 and 21.7 %, respectively. Based on morphological, phenotypic, phylogenetic and genomic relatedness data presented here, it is evident that strain SY-01T represents a novel species of the genus Methanolobus, and the name Methanolobus halotolerans sp. nov. is proposed. The type strain is SY-01T (=BCRC AR10051T=NBRC 113166 T=DSM 107642T).

Methanolobus psychrotolerans sp. nov., a psychrotolerant methanoarchaeon isolated from a saline meromictic lake in Siberia.

A psychrotolerant, methylotrophic methanogen, strain YSF-03T, was isolated from the saline meromictic Lake Shira in Siberia. Cells of strain YSF-03T were non-motile, irregular cocci and 0.8-1.2 µm in diameter. The methanogenic substrates utilized by strain YSF-03T were methanol and trimethylamine. The temperature range of growth for strain YSF-03T was from 0 to 37 °C. The optimum growth conditions were 30-37 °C, pH 7.0-7.4 and 0.17 M NaCl. The G+C content of the genome of strain YSF-03T was 41.3 mol%. Phylogenetic analysis revealed that strain YSF-03T was most closely related to Methanolobus profundi MobMT (98.15 % similarity in 16S rRNA gene sequence). Genome relatedness between strain YSF-03T and MobMT was computed using the Genome-to-Genome Distance Calculator and average nucleotide identity, which gave values of 23.5 and 79.3 %, respectively. Based on the morphological, phenotypic, phylogenetic and genomic relatedness data presented here, it is evident that strain YSF-03T represents a novel species of the genus Methanolobus, for which the name Methanolobus psychrotolerans sp. nov. is proposed. The type strain is YSF-03T (=BCRC AR10049T=DSM 104044T=NBRC 112514T).

The response of archaeal species to seasonal variables in a subtropical aerated soil: insight into the low abundant methanogens.

Archaea are cosmopolitan in aerated soils around the world. While the dominance of Thaumarchaeota has been reported in most soils, the methanogens are recently found to be ubiquitous but with low abundances in the aerated soil globally. However, the seasonal changes of Archaea community in the aerated soils are still in the mist. In this study, we investigated the change of Archaea in the context of environmental variables over a period of 12 months in a subtropical soil on the Chongming Island, China. The results showed that Nitrososphaera spp. were the dominant archaeal population while the methanogens were in low proportions but highly diverse (including five genera: Methanobacterium, Methanocella, Methanosaeta, Methanosarcina, and Methanomassiliicoccus) in the aerated soil samples determined by high throughput sequencing. A total of 126 LSA correlations were found in the dataset including all the 72 archaeal OTUs and 8 environmental factors. A significance index defined as the pagerank score of each OTU divided by its relative abundance was used to evaluate the significance of each OTU. The results showed that five out of 17 methanogen OTUs were significantly positively correlated with temperature, suggesting those methanogens might increase with temperature rather than being dormant in the aerated soils. Given the metabolic response of methanogens to temperature under aerated soil conditions, their contribution to the global methane cycle warrants evaluation.

Biomethane production and microbial community response according to influent concentration of molasses wastewater in a UASB reactor.

This study aimed to investigate the interaction between methane production performance and active microbial community dynamics at different loading rates by increasing influent substrate concentration. The model system was an upflow anaerobic sludge blanket (UASB) reactor using molasses wastewater. The active microbial community was analyzed using a ribosomal RNA-based approach in order to reflect active members in the UASB system. The methane production rate (MPR) increased with an increase in organic loading rate (OLR) from 3.6 to 5.5 g COD·L(-1)·day(-1) and then it decreased with further OLR addition until 9.7 g COD·L(-1)·day(-1). The UASB reactor achieved a maximum methane production rate of 0.48 L·L(-1)·day(-1) with a chemical oxygen demand (COD) removal efficiency of 91.2 % at an influent molasses concentration of 16 g COD·L(-1) (OLR of 5.5 g COD·L(-1)·day(-1)). In the archaeal community, Methanosarcina was predominant irrespective of loading rate, and the relative abundance of Methanosaeta increased with loading rate. In the bacterial community, Firmicutes and Eubacteriaceae were relatively abundant in the loading conditions tested. The network analysis between operation parameters and microbial community indicated that MPR was positively associated with most methanogenic archaea, including the relatively abundant Methanosarcina and Methanosaeta, except Methanofollis. The most abundant Methanosarcina was negatively associated with Bifidobacterium and Methanosaeta, whereas Methanosaeta was positively associated with Bifidobacterium.

Changes in microbial community structures due to varying operational conditions in the anaerobic digestion of oxytetracycline-medicated cow manure.

Management of manure containing veterinary antibiotics is a major concern in anaerobic treatment systems because of their possible adverse effects on microbial communities. Therefore, the aim of study was to investigate how oxytetracycline (OTC) influences bacteria and acetoclastic and hydrogenotrophic methanogens under varying operational conditions in OTC-medicated and non-medicated anaerobic cow manure digesters. Concentrations of OTC and its metabolites throughout the anaerobic digestion were determined using ultraviolet-high-performance liquid chromatography (UV-HPLC) and tandem liquid chromatography-mass spectrometry (LC/MS/MS), respectively. Fluorescent in situ hybridization, denaturing gradient gel electrophoresis, cloning, and sequencing analyses were used to monitor changes in microbial community structures. According to the results of analytical and molecular approaches, operating conditions highly influence active microbial community dynamics and associate with biogas production and elimination of OTC and its metabolites during anaerobic digestion of cow manure in the presence of an average initial concentration of 2.2 mg OTC/L. The impact of operating conditions has a drastic effect on acetoclastic methanogens than hydrogenotrophic methanogens and bacteria.

Temperature impacts differentially on the methanogenic food web of cellulose-supplemented peatland soil.

The impact of temperature on the largely unresolved intermediary ecosystem metabolism and associated unknown microbiota that link cellulose degradation and methane production in soils of a moderately acidic (pH 4.5) fen was investigated. Supplemental [(13) C]cellulose stimulated the accumulation of propionate, acetate and carbon dioxide as well as initial methane production in anoxic peat soil slurries at 15°C and 5°C. Accumulation of organic acids at 15°C was twice as fast as that at 5°C. 16S rRNA [(13) C]cellulose stable isotope probing identified novel unclassified Bacteria (79% identity to the next cultured relative Fibrobacter succinogenes), unclassified Bacteroidetes (89% identity to Prolixibacter bellariivorans), Porphyromonadaceae, Acidobacteriaceae and Ruminococcaceae as main anaerobic degraders of cellulose-derived carbon at both 15°C and 5°C. Holophagaceae and Spirochaetaceae were more abundant at 15°C. Clostridiaceae dominated the degradation of cellulose-derived carbon only at 5°C. Methanosarcina was the dominant methanogenic taxa at both 15°C and 5°C. Relative abundance of Methanocella increased at 15°C whereas that of Methanoregula and Methanosaeta increased at 5°C. Thaumarchaeota closely related to Nitrosotalea (presently not known to grow anaerobically) were abundant at 5°C but absent at 15°C indicating that Nitrosotalea sp. might be capable of anaerobic growth at low temperatures in peat.

Isolation and characterization of a tetramethylammonium-degrading Methanococcoides strain and a novel glycine betaine-utilizing Methanolobus strain.

Two novel strains of methanogens were isolated from an estuarine sediment with the capability to utilize quaternary amines. Based on the 16S rRNA analysis, strain B1d shared 99 % sequence identity with Methanolobus vulcani PL-12/M(T) and strain Q3c shared 99 % identity with Methanococcoides sp. PM1 and PM2, but our current isolates display clearly different capabilities of growth on quaternary amines and were isolated based on these capabilities. Strain Q3c was capable of growth on tetramethylammonium and choline, while strain B1d was capable of growth on glycine betaine. Ml. vulcani PL-12/M(T) was incapable of growth on glycine betaine, indicating an obvious distinction between strains B1d and PL-12/M(T). Strain Q3c now represents the only known tetramethylammonium-utilizing methanogen in isolation. Strain B1d is the first quaternary amine-utilizing methanogen from the genus Methanolobus. This study suggests that quaternary amines may serve as ready precursors of biological methane production in marine environments.

Methanosalsum natronophilum sp. nov., and Methanocalculus alkaliphilus sp. nov., haloalkaliphilic methanogens from hypersaline soda lakes.

Two groups of haloalkaliphilic methanogenic archaea were dominating in enrichments from hypersaline soda lake sediments at pH 10. At moderate salt concentrations with formate or H2 as electron donor, methanogens belonging to the genus Methanocalculus were enriched, while at high salt concentrations with methylated substrates, a group related to Methanosalsum zhilinae was dominating. For both groups, several pure cultures were obtained including the type strains AMF2T for the Methanocalculus group and AME2T for the Methanosalsum group. The Methanocalculus group is characterized by lithoheterotrophic growth with either formate (preferable substrate) or H2 at moderate salinity up to 1.5-2 M total Na+ and obligate alkaliphilic growth with an optimum at pH 9.5. According to phylogenetic analysis, the group also includes closely related strains isolated previously from the low-salt alkaline Lonar Lake. The novel Methanosalsum group is characterized by high salt tolerance (up to 3.5 M total Na+) and obligate alkaliphilic growth with an optimum at pH 9.5. It has a typical methylotrophic substrate profile, utilizing methanol, methylamines and dimethyl sulfide (at low concentrations) as methanogenic substrates. On the basis of physiological and phylogenetic data, it is proposed that the two groups of soda lake methanogenic isolates are assigned into two novel species, Methanocalculus alkaliphilus sp. nov. (type strain AMF2T = DSM 24457T = UNIQEM U859T) and Methanosalsum natronophilum sp. nov. (type strain AME2T = DSM 24634T = NBRC 110091T).

The genus name Methanothrix Huser et al. 1983 and the species combination Methanothrix soehngenii Huser et al. 1983 do not contravene Rule 31a and are not to be considered as rejected names, the genus name Methanosaeta Patel and Sprott 1990 refers to the same taxon as Methanothrix soehngenii Huser et al. 1983 and the species combination Methanothrix thermophila Kamagata et al. 1992 is rejected: Supplementary information to Opinion 75. Judicial Commission of the International Committee on Systematics of Prokaryotes.

The Judicial Commission affirms that the genus name Methanothrix Huser et al. 1983 and the species combination Methanothrix soehngenii Huser et al. 1983 do not contravene Rule 31a and are not to be considered as rejected names. The genus name Methanosaeta Patel and Sprott 1990 applies to the same taxon as Methanothrix Huser et al. 1983 and is therefore a later heterotypic synonym. The combinations Methanothrix thermoacetophila corrig. Nozhevnikova and Chudina 1988 and Methanothrix thermophila Kamagata et al. 1992 are considered to refer to the same taxon, a consequence of which is that Methanothrix thermophila Kamagata et al. 1992 contravenes Rule 51b and is placed on the List of Rejected Names.

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.

Methanococcoides vulcani sp. nov., a marine methylotrophic methanogen that uses betaine, choline and N,N-dimethylethanolamine for methanogenesis, isolated from a mud volcano, and emended description of the genus Methanococcoides.

A novel, strictly anaerobic, methylotrophic marine methanogen, strain SLH33(T), was isolated from deep sediment samples covered by an orange microbial mat collected from the Napoli Mud Volcano. Cells of strain SLH33(T) were Gram-stain-negative, motile, irregular cocci that occurred singly. Cells utilized trimethylamine, dimethylamine, monomethylamine, methanol, betaine, N,N-dimethylethanolamine and choline (N,N,N-trimethylethanolamine) as substrates for growth and methanogenesis. The optimal growth temperature was 30 °C; maximum growth rate was obtained at pH 7.0 in the presence of 0.5 M Na(+). The DNA G+C content of strain SLH33(T) was 43.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLH33(T) within the genus Methanococcoides. The novel isolate was related most closely to Methanococcoides methylutens TMA-10(T) (98.8% 16S rRNA gene sequence similarity) but distantly related to Methanococcoides burtonii DSM 6242(T) (97.6%) and Methanococcoides alaskense AK-5(T) (97.6%). DNA-DNA hybridization studies indicated that strain SLH33(T) represents a novel species, given that it shared less than 16% DNA-DNA relatedness with Methanococcoides methylutens TMA-10(T). The name Methanococcoides vulcani sp. nov. is proposed for this novel species, with strain SLH33(T) ( = DSM 26966(T) = JCM 19278(T)) as the type strain. An emended description of the genus Methanococcoides is also proposed.

Methanosarcinaceae and acetate-oxidizing pathways dominate in high-rate thermophilic anaerobic digestion of waste-activated sludge.

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH4/kg of VS(added). According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the Methanosarcinaceae, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.

Methanomethylovorans uponensis sp. nov., a methylotrophic methanogen isolated from wetland sediment.

A novel mesophilic, methylotrophic, methanogenic archaeon, designated strain EK1(T), was enriched and isolated from wetland sediment. Phylogenetic analysis showed that strain EK1(T) was affiliated with the genus Methanomethylovorans within the family Methanosarcinaceae, and shared the highest 16S rRNA and methyl-coenzyme M reductase alpha-subunit gene sequence similarity with the type strain of Methanomethylovorans hollandica (98.8 and 92.6 %, respectively). The cells of strain EK1(T) were observed to be Gram-negative, non-motile and irregular cocci that did not lyse in 0.1 % (w/v) sodium dodecyl sulfate. Methanol, mono-, di- and trimethylamine, dimethyl sulfide and methanethiol were found to be used as catabolic and methanogenic substrates, whereas H2/CO2, formate, 2-propanol and acetate were not. Growth was observed at 25-40 °C (optimum, 37 °C), at pH 5.5-7.5 (optimum, pH 6.0-6.5) and in the presence of 0-0.1 M NaCl (optimum, 0 M). Growth and methane production rates were stimulated in the presence of H2/CO2 although methane production and growth yields were not significantly affected; acetate, formate, 2-propanol and CO/CO2/N2 did not affect methane production. CoCl2 (0.6-2.0 µM) and FeCl2 (25 mg/l) stimulated growth, while yeast extract and peptone did not. The DNA-DNA hybridization experiment revealed a relatedness of <20 % between EK1(T) and the type strains of the genus Methanomethylovorans. The DNA G+C content of strain EK1(T) was determined to be 39.2 mol%. Based on the polyphasic taxonomic study, strain EK1(T) represents a novel species belonging to the genus Methanomethylovorans, for which the name Methanomethylovorans uponensis sp. nov. is proposed. The type strain is strain EK1(T)(=NBRC 109636(T) = KCTC 4119(T) = JCM 19217(T)).

Choline and N,N-dimethylethanolamine as direct substrates for methanogens.

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.

Phylogenetic diversity of methyl-coenzyme M reductase (mcrA) gene and methanogenesis from trimethylamine in hypersaline environments.

Methanogens have been reported in complex microbial communities from hypersaline environments, but little is known about their phylogenetic diversity. In this work, methane concentrations in environmental gas samples were determined while methane production rates were measured in microcosm experiments with competitive and non-competitive substrates. In addition, the phylogenetic diversity of methanogens in microbial mats from two geographical locations was analyzed: the well studied Guerrero Negro hypersaline ecosystem, and a site not previously investigated, namely Laguna San Ignacio, Baja California Sur, Mexico. Methanogenesis in these microbial mats was suspected based on the detection of methane (in the range of 0.00086 to 3.204 %) in environmental gas samples. Microcosm experiments confirmed methane production by the mats and demonstrated that it was promoted only by non-competitive substrates (trimethylamine and methanol), suggesting that methylotrophy is the main characteristic process by which these hypersaline microbial mats produce methane. Phylogenetic analysis of amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from natural and manipulated samples revealed various methylotrophic methanogens belonging exclusively to the family Methanosarcinaceae. Moderately halophilic microorganisms of the genus Methanohalophilus were predominant (>60 % of mcrA sequences retrieved). Slightly halophilic and marine microorganisms of the genera Methanococcoides and Methanolobus, respectively, were also identified, but in lower abundances.

Complete genome sequence of Methanosaeta concilii, a specialist in aceticlastic methanogenesis.

The genome sequence of the aceticlastic methanoarchaeon Methanosaeta concilii GP6, comprised of a 3,008,626-bp chromosome and an 18,019-bp episome, has been determined and exhibits considerable differences in gene content from that of Methanosaeta thermophila.

Methanogenic archaea isolated from Taiwan's Chelungpu fault.

Terrestrial rocks, petroleum reservoirs, faults, coal seams, and subseafloor gas hydrates contain an abundance of diverse methanoarchaea. However, reports on the isolation, purification, and characterization of methanoarchaea in the subsurface environment are rare. Currently, no studies investigating methanoarchaea within fault environments exist. In this report, we succeeded in obtaining two new methanogen isolates, St545Mb(T) of newly proposed species Methanolobus chelungpuianus and Methanobacterium palustre FG694aF, from the Chelungpu fault, which is the fault that caused a devastating earthquake in central Taiwan in 1999. Strain FG694aF was isolated from a fault gouge sample obtained at 694 m below land surface (mbls) and is an autotrophic, mesophilic, nonmotile, thin, filamentous-rod-shaped organism capable of using H(2)-CO(2) and formate as substrates for methanogenesis. The morphological, biochemical, and physiological characteristics and 16S rRNA gene sequence analysis revealed that this isolate belongs to Methanobacterium palustre. The mesophilic strain St545Mb(T), isolated from a sandstone sample at 545 mbls, is a nonmotile, irregular, coccoid organism that uses methanol and trimethylamine as substrates for methanogenesis. The 16S rRNA gene sequence of strain St545Mb(T) was 99.0% similar to that of Methanolobus psychrophilus strain R15 and was 96 to 97.5% similar to the those of other Methanolobus species. However, the optimal growth temperature and total cell protein profile of strain St545Mb(T) were different from those of M. psychrophilus strain R15, and whole-genome DNA-DNA hybridization revealed less than 20% relatedness between these two strains. On the basis of these observations, we propose that strain St545Mb(T) (DSM 19953(T); BCRC AR10030; JCM 15159) be named Methanolobus chelungpuianus sp. nov. Moreover, the environmental DNA database survey indicates that both Methanolobus chelungpuianus and Methanobacterium palustre are widespread in the subsurface environment.

A new process for efficiently producing methane from waste activated sludge: alkaline pretreatment of sludge followed by treatment of fermentation liquid in an EGSB reactor.

In the literature the production of methane from waste activated sludge (WAS) was usually conducted in a continuous stirred tank reactor (CSTR) after sludge was pretreated. It was reported in our previous publication that compared with other pretreatment methods the methane production in CSTR could be significantly enhanced when sludge was pretreated by NaOH at pH 10 for 8 days. In order to further improve methane production, this study reported a new process for efficiently producing methane from sludge, that is, sludge was fermented at pH 10 for 8 days, which was adjusted by Ca(OH)(2), and then the fermentation liquid was treated in an expanded granular sludge bed (EGSB) for methane generation. First, for comparing the methane production observed in this study with that reported in the literature, the conventional operational model was applied to produce methane from the pH 10 pretreated sludge, that is, directly using the pH 10 pretreated sludge to produce methane in a CSTR. It was observed that the maximal methane production was only 0.61 m(3)CH(4)/m(3)-reactor/day. Then, the use of fermentation liquid of pH 10 pretreated sludge to produce methane in the reactors of up-flow anaerobic sludge bed (UASB), anaerobic sequencing batch reactor (ASBR) and EGSB was compared. The maximal methane production in UASB, ASBR, and EGSB reached 1.41, 3.01, and 12.43 m(3)CH(4)/m(3)-reactor/day, respectively. Finally, the mechanisms for EGSB exhibiting remarkably higher methane production were investigated by enzyme, adenosine-triphosphate (ATP), scanning electron microscope (SEM) and fluorescence in situ hybridization (FISH) analyses. It was found that the granular sludge in EGSB had the highest conversion efficiency of acetic acid to methane, and the greatest activity of hydrolysis and acidification enzymes and general physiology with much more Methanosarcinaceae.

Methanohalophilus profundi sp. nov., a methylotrophic halophilic piezophilic methanogen isolated from a deep hypersaline anoxic basin.

A novel anaerobic methylotrophic halophilic methanogen strain SLHTYROT was isolated from a deep hypersaline anoxic basin called "Tyro" located in the Eastern Mediterranean Sea. Cells of SLHTYROT were motile cocci. The strain SLHTYROT grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L-1 NaCl (optimum 135 g L-1). Strain SLHTYROT was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYROT was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYROT was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYROT should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYROT (=DSM 108854 = JCM 32768 = UBOCC-M-3308).