Mesophilic Methanogen Research Articles

Draft genome sequence of Methanobrevibacter smithii strain B181, isolated from a fecal sample.

A mesophilic methanogen, Methanobrevibacter smithii B181 (DSM 11975) was previously isolated from a human fecal sample, grown on carbon dioxide and hydrogen, and subsequently sequenced. The reconstructed 1.9-Mb genome sequence of Methanobrevibacter smithii B181 contributes to our understanding of hydrogenotrophic, CO2-reducing methanogenesis in the human gut.

Characterization of protein glycosylation in an Asgard archaeon

Archaeal cells are typically enveloped by glycosylated S-layer proteins. Archaeal protein glycosylation provides valuable insights not only into their adaptation to their niches but also into their evolutionary trajectory. Notably, thermophilic Thermoproteota modify proteins with N-glycans that include two GlcNAc units at the reducing end, resembling the "core structure" preserved across eukaryotes. Recently, Asgard archaea, now classified as members of the phylum Promethearchaeota, have offered unprecedented opportunities for understanding the role of archaea in eukaryogenesis. Despite the presence of genes indicative of protein N-glycosylation in this archaeal group, these have not been experimentally investigated. Here we performed a glycoproteome analysis of the firstly isolated Asgard archaeon Promethearchaeum syntrophicum. Over 700 different proteins were identified through high-resolution LC-MS/MS analysis, however, there was no evidence of either the presence or glycosylation of putative S-layer proteins. Instead, N-glycosylation in this archaeon was primarily observed in an extracellular solute-binding protein, possibly related to chemoreception or transmembrane transport of oligopeptides. The glycan modification occurred on an asparagine residue located within the conserved N-X-S/T sequon, consistent with the pattern found in other archaea, bacteria, and eukaryotes. Unexpectedly, three structurally different N-glycans lacking the conventional core structure were identified in this archaeon, presenting unique compositions that included atypical sugars. Notably, one of these sugars was likely HexNAc modified with a threonine residue, similar to modifications previously observed in mesophilic methanogens within the Methanobacteriati. Our findings advance our understanding of Asgard archaea physiology and evolutionary dynamics.

Characterizing the N- and O-linked glycans of the PGF-CTERM sorting domain-containing S-layer protein of Methanoculleus marisnigri.

The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-β-GlcNAc3NGaAN-4-β-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide β-GlcNAc3NAcAN-4-β-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-β-Ribf-3-α-Rha2OMe-3-α-Rha-2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Identification of a novel N-linked glycan on the archaellins and S-layer protein of the thermophilic methanogen, Methanothermococcus thermolithotrophicus

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-β-Man-(1-4)-[β-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-β-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

Hydrogenotrophic methanogen strain of Methanospirillum from anaerobic digester fed with agro-industrial waste

A hydrogenotrophic, mesophilic methanogen designated as strain T53BJ was isolated from anaerobic digester continuously fed with agro-industrial waste. Cells were stained Gram negative, spiral shaped, motile and found resistant to cell lysis by SDS and hypotonic solution. Growth occurred with optimum (i) temperature of 30 °C (20–40 °C) (ii) pH of 7.0 (6.5–8.5), in the presence of 0.0–0.2 M NaCl with a doubling time of 73.43 h and utilized only hydrogen and carbon dioxide (H2:CO2; 4:1) as a sole source of carbon and energy. The phenetic and phylogenetic analysis based on 16S rRNA and mcrA gene sequences revealed close affiliation of T53BJ strain to the genus Methanospirillum spp. This is the first report about isolation of hydrogenotrophic Methanospirillum spp. from anaerobic digester fed with fruit waste, press mud and neem seed cake, suggesting its potential use for bioaugmentation of anaerobic reactors or upgrading biogas/ syngas to methane.

Microbial Diversity and Potential Sulfide Producers in the Karazhanbas Oilfield (Kazakhstan)

Biogenic sulfide production in oilfields results in lower quality of oil and gas and in corrosion of oil-producing equipment. Bactericidal agents are not used at the Karazhanbas oilfield (Kazakhstan), since sulfate concentrations in both injected and formation water are low or zero. However, increasing corrosion of steel equipment and sulfide release into formation water were observed in the course of the oilfield development. The goal of the present work was to reveal the potential agents of microbial corrosion and to investigate the possibility to suppress development of sulfidogens using nitrate. Environmental conditions and the composition of microbial communities in production and injection water were studied for the sites with different temperatures. High-throughput sequencing of the V3–V4 region of the 16S rRNA gene revealed predominance of thermophilic sulfate-reducing bacteria (genera Thermodesulfobacterium, Thermodesulfovibrio, Desulfotomaculum, Thermodesulforhabdus, Desulfovirgula, Defluviitoga, and Desulfonauticus) and archaea (genus Archaeoglobus) at the high-temperature horizon, as well as of thiosulfate-reducing (genera Thermoanaerobacter, Anaerobaculum, and Coprothermobacter) and syntrophic bacteria, while abundance of methanogens (genera Methanolinea and Methanothermobacter) was low. Communities from low-temperature horizons contained mesophilic methanogens (genera Methanococcus, Methanobacterium, and Methanothrix), thermophilic and mesophilic sulfate-reducing bacteria (genera Desulfovibrio, Desulfomicrobium, Desulfosarcina, Desulfoglaeba, Desulfotignum, and Desulfocurvus), syntrophic bacteria (genus Smithella), and members of the genera Marinobacter, Paracoccus, Alcaligenes, Arcobacter, and Halomonas. Enrichment cultures of bacteria producing sulfide were obtained from formation water, as well as the cultures reducing nitrate to nitrite and suppressing growth of sulfate- and thiosulfate-reducing bacteria. These findings may indicate the possibility of using nitrate as a competitive inhibitor of sulfidogenesis in this oilfield. Corrosion-active hydrogen-utilizing methanogens and acetogens were also found, which implies the necessity for monitoring the oilfield microbial communities.

Experimental investigation on the controls of clumped isotopologue and hydrogen isotope ratios in microbial methane

The abundance of methane isotopologues with two rare isotopes (e.g., 13CH3D) has been proposed as a tool to estimate the temperature at which methane is formed or thermally equilibrated. It has been shown, however, that microbial methane from surface environments and from laboratory cultures is characterized by low 13CH3D abundance, corresponding to anomalously high apparent 13CH3D equilibrium temperatures. We carried out a series of batch culture experiments to investigate the origin of the non-equilibrium signals in microbial methane by exploring a range of metabolic pathways, growth temperatures, and hydrogen isotope compositions of the media. We found that thermophilic methanogens (Methanocaldococcusjannaschii, Methanothermococcus thermolithotrophicus, and Methanocaldococcus bathoardescens) grown on H2 + CO2 at temperatures between 60 and 80 °C produced methane with Δ13CH3D values (defined as the deviation from stochastic abundance) of 0.5–2.5‰, corresponding to apparent 13CH3D equilibrium temperatures of 200–600 °C. Mesophilic methanogens (Methanosarcina barkeri and Methanosarcina mazei) grown on H2 + CO2, acetate, or methanol produced methane with consistently low Δ13CH3D values, down to −5.2‰. Closed system effects can explain part of the non-equilibrium signals for methane from thermophilic methanogens. Experiments with M. barkeri using D-spiked water or D-labeled acetate (CD3COO−) indicate that 1.6–1.9 out of four H atoms in methane originate from water, but Δ13CH3D values of product methane only weakly correlate with the D/H ratio of medium water. Our experimental results demonstrate that low Δ13CH3D values are not specific to the metabolic pathways of methanogenesis, suggesting that they could be produced during enzymatic reactions common in the three methanogenic pathways, such as the reduction of methyl-coenzyme M. Nonetheless CH bonds inherited from precursor methyl groups may also carry part of non-equilibrium signals.

Effects of changes in temperature on treatment performance and energy recovery at mainstream anaerobic ceramic membrane bioreactor for food waste recycling wastewater treatment

An anaerobic ceramic membrane bioreactor (AnCMBR) has been attracted as an alternative technology to co-manage various organic substrates. This AnCMBR study investigated process performance and microbial community structure at decreasing temperatures to evaluate the potential of AnCMBR treatment for co-managing domestic wastewater (DWW) and food waste-recycling wastewater (FRW). As a result, the water flux (≥6.9 LMH) and organic removal efficiency (≥98.0%) were maintained above 25 °C. The trend of methane production in the AnCMBR was similar except for at 15 °C. At 15 °C, the archaeal community structure did not shifted, whereas the bacterial community structure was changed. Various major archaeal species were identified as the mesophilic methanogens which unable to grow at 15 °C. Our results suggest that the AnCMBR can be applied to co-manage DWW and FRW above 20 °C. Future improvements including psychrophilic methanogen inoculation and process optimization would make co-manage DWW and FRW at lower temperature climates.

Effect of humic acids on the activity of pure and mixed methanogenic cultures

The impact of humic acid (HA) on methanogenic activity was investigated. Methanogenic crushed granular sludge and pure cultures of mesophilic methanogens were incubated in batch cultures with HA. Initial methane production rates and substrate consumption rates were quantified. In the presence of 1 kg m−3 HA, the methane production rate of all hydrogenotrophic methanogens was inhibited by more than 75%, except Methanospirillum hungatei that was not inhibited up to 5 kg m−3 HA. The acetoclastic Methanosarcina barkeri was completely inhibited by HA ≥1 kg m−3. However, Methanosaeta concilii was only slightly affected by HA up to 3 kg m−3. When methanogenic granular sludge was incubated with HA, the specific methanogenic activity (SMA) tests showed less inhibition, when compared to the pure cultures of methanogens. The SMA test with H2/CO2, formate and acetate showed reduced initial methane production rate of 42%, 23% and 40%, respectively. Differences in HA susceptibility were explained by differences in cell wall structure.

Ammonia inhibition on hydrogen enriched anaerobic digestion of manure under mesophilic and thermophilic conditions

Capturing of carbon dioxide by hydrogen derived from excess renewable energy (e.g., wind mills) to methane in a microbially catalyzed process offers an attractive technology for biogas production and upgrading. This bioconversion process is catalyzed by hydrogenotrophic methanogens, which are known to be sensitive to ammonia. In this study, the tolerance of the biogas process under supply of hydrogen, to ammonia toxicity was studied under mesophilic and thermophilic conditions. When the initial hydrogen partial pressure was 0.5 atm, the methane yield at high ammonia load (7 g NH4+-N L−1) was 41.0% and 22.3% lower than that at low ammonia load (1 g NH4+-N L−1) in mesophilic and thermophilic condition, respectively. Meanwhile no significant effect on the biogas composition was observed. Moreover, we found that hydrogentrophic methanogens were more tolerant to the ammonia toxicity than acetoclastic methanogens in the hydrogen enriched biogas production and upgrading processes. The highest methane production yield was achieved under 0.5 atm hydrogen partial pressure in batch reactors at all the tested ammonia levels. Furthermore, the thermophilic methanogens at 0.5 atm of hydrogen partial pressure were more tolerant to high ammonia levels (≥5 g NH4+-N L−1), compared with mesophilic methanogens. The present study offers insight in developing resistant hydrogen enriched biogas production and upgrading processes treating ammonia-rich waste streams.

Flux measurements and maintenance energy for carbon dioxide utilization by Methanococcus maripaludis.

BackgroundThe rapidly growing mesophilic methanogen Methanococcus maripaludis S2 has a unique ability to consume both CO2 and N2, the main components of a flue gas, and produce methane with H2 as the electron donor. The existing literature lacks experimental measurements of CO2 and H2 uptake rates and CH4 production rates on M. maripaludis. Furthermore, it lacks estimates of maintenance energies for use with genome-scale models. In this paper, we performed batch culture experiments on M. maripaludis S2 using CO2 as the sole carbon substrate to quantify three key extracellular fluxes (CO2, H2, and CH4) along with specific growth rates. For precise computation of these fluxes from experimental measurements, we developed a systematic process simulation approach. Then, using an existing genome-scale model, we proposed an optimization procedure to estimate maintenance energy parameters: growth associated maintenance (GAM) and non-growth associated maintenance (NGAM).ResultsThe measured extracellular fluxes for M. maripaludis showed excellent agreement with in silico predictions from a validated genome-scale model (iMM518) for NGAM = 7.836 mmol/gDCW/h and GAM = 27.14 mmol/gDCW. M. maripaludis achieved a CO2 to CH4 conversion yield of 70–95 % and a growth yield of 3.549 ± 0.149 g DCW/mol CH4 during the exponential phase. The ATP gain of 0.35 molATP/molCH4 for M. maripaludis, computed using NGAM, is in the acceptable range of 0.3–0.7 mol ATP/molCH4 reported for methanogens. Interestingly, the uptake distribution of amino acids, quantified using iMM518, confirmed alanine to be the most preferred amino acids for growth and methanogenesis.ConclusionsThis is the first study to report experimental gas consumption and production rates for the growth of M. maripaludis on CO2 and H2 in minimal media. A systematic process simulation and optimization procedure was successfully developed to precisely quantify extracellular fluxes along with cell growth and maintenance energy parameters. Our growth yields, ATP gain, and energy parameters fall within acceptable ranges known in the literature for hydrogenotrophic methanogens.

Intensification of microbial decomposition of organic fraction of municipal waste: Laboratory and field experiments

Methods of intensifying the anaerobic microbial decomposition of the organic fraction of municipal solid waste (MSW) on an MSW landfill and in anaerobic reactors were studied. It was discovered that it is preferable for the initiation and stabilization of the process of anaerobic digestion of organic waste in laboratory bioreactors at 20 and 50 degrees C to use a mixture of activated suspension of soil from the anaerobic zone of the landfill and digested sewage sludge. Stimulation of methanogenesis was shown in field conditions when digested sewage sludge was added directly into the upper layer of anaerobic zone of the landfill. The investigation of methane production during fermentation of concentrated food waste with a mixture of excessive activated sludge in the laboratory under thermophilic conditions (50 degrees C) has shown that the main problem at the first stage of the process was the acidification of the digested mixture due to the accumulation of volatile fatty acids. It was shown that for stable operation of the bioreactor under thermophilic conditions the amount of inoculum added during the start up should be no less than 30%-50%--based on volatile suspended solids. A sharp decrease in the digestion temperature from 50 to 20 degrees C did not cause methanogenesis termination, since the thermophilically fermented biomass contained both thermophilic and mesophilic methanogens.

Two-stage thermophilic fermentation and mesophilic methanogen process for biohythane production from palm oil mill effluent

A two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent (POME) was investigated. The hydrogen and methane potential from POME were 170–200 L H2 kgCOD−1 and 210–292 L CH4 kgCOD−1, respectively. A continuous two-stage process with hydraulic retention time (HRT) of 2 d for hydrogen reactor and 15 d for methane reactor, obtained 34% higher energy yield than single stage methane production. The hydrogen and methane yields from two-stage were 210 L H2 kgCOD−1 and 315 L CH4 kgCOD−1, respectively with total energy yield of 15.34 MJ kgCOD−1. Mixed hydrogen and methane (biohythane) production rate was 4.4 L biogas L−1d−1 with containing of 51% CH4, 14% H2 and 35% CO2. Hydrogen reactor was dominated with hydrogen producing bacteria of Thermoanaerobacterium thermosaccharolyticum, while acetoclastic Methanoculleus sp. was the dominant methanogen in methane reactor. Two-stage process for biohythane production could efficiently for energy recovery from POME.

Genome sequence of the mud-dwelling archaeon Methanoplanus limicola type strain (DSM 2279(T)), reclassification of Methanoplanus petrolearius as Methanolacinia petrolearia and emended descriptions of the genera Methanoplanus and Methanolacinia.

Methanoplanus limicola Wildgruber et al. 1984 is a mesophilic methanogen that was isolated from a swamp composed of drilling waste near Naples, Italy, shortly after the Archaea were recognized as a separate domain of life. Methanoplanus is the type genus in the family Methanoplanaceae, a taxon that felt into disuse since modern 16S rRNA gene sequences-based taxonomy was established. Methanoplanus is now placed within the Methanomicrobiaceae, a family that is so far poorly characterized at the genome level. The only other type strain of the genus with a sequenced genome, Methanoplanus petrolearius SEBR 4847T, turned out to be misclassified and required reclassification to Methanolacinia. Both, Methanoplanus and Methanolacinia, needed taxonomic emendations due to a significant deviation of the G+C content of their genomes from previously published (pre-genome-sequence era) values. Until now genome sequences were published for only four of the 33 species with validly published names in the Methanomicrobiaceae. Here we describe the features of M. limicola, together with the improved-high-quality draft genome sequence and annotation of the type strain, M3T. The 3,200,946 bp long chromosome (permanent draft sequence) with its 3,064 protein-coding and 65 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

The genome and transcriptome of a newly described psychrophilic archaeon, Methanolobus psychrophilusR15, reveal its cold adaptive characteristics

We analysed the cold-responsive gene repertoire for a psychrophilic methanogen, Methanolobus psychrophilus R15 through genomic and RNA-seq assayed transcriptomic comparisons for cultures at 18°C (optimal temperature) versus 4°C. The differences found by RNA-seq analysis were verified using quantitative real time-PCR assay. The results showed that as in the Antarctic methanogen, Methanococcoides burtonii, genes for methanogenesis, biosynthesis and protein synthesis were all downregulated by the cold in R15. However, the RNA polymerase complex was upregulated at cold, as well as a gene cluster for a putative exosome complex, suggesting that exosome-mediated RNA decay may be cold-accelerated. Unexpectedly, the chaperonin genes for both thermosome and GroES/EL were all upregulated at 4°C. Strain R15 possessed eight protein families for oxygen detoxification, including both anaerobe-specific superoxide reductase (SOR) and the aerobe-typical superoxide dismutase (SOD)-catalase oxidant-removing system, implying the higher oxidative tolerance. Compared with a mesophilic methanogen, R15 survived in higher paraquat, a redox-cycling drug. Moreover, 71 one-component systems and 50 two-component systems for signal transduction ranked strain R15, together with M. burtonii, as being highly adaptive among archaea. Most of them exhibited cold-enhanced expression, indicating their involvement in cold adaptation. This study has added new perspectives on the cold adaptation of methanogenic archaea.

Archaeal Phospholipid Biosynthetic Pathway Reconstructed inEscherichia coli

A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli.

Microbial reduction of Fe(III) in illite–smectite minerals by methanogen Methanosarcina mazei

Clay minerals are ubiquitous in soils, sediments and sedimentary rocks, and could coexist with methanogens in anoxic environments, but whether or not methanogens can reduce structural Fe(III) in clay minerals other than smectite is not known. The objective of this study was to understand the capability of a mesophilic methanogen, Methanosarcina mazei in reducing structural Fe(III) in illite–smectite clay minerals and its potential role in inducing mineralogical changes. Bioreduction experiments were performed in batch systems, where four different clay minerals (nontronite NAu-2, mixed-layer illite–smectite RAr-1 and ISCz-1, and illite IMt-1) were exposed to M. mazei in growth medium with and without anthraquinone-2,6-disulfonate (AQDS) as electron shuttle. The extent and rate of bioreduction were quantified via wet chemistry and mineralogical transformed by X-ray diffraction and scanning and transmission electron microscopy. Our results demonstrated that M. mazei was able to reduce structural Fe(III) in clay minerals with Fe(III) in smectite being the most reducible and illite the least. AQDS enhanced the reduction rate and extent. The bioavailability of structural Fe(III) to M. mazei was correlated to the smectite proportion in each clay mineral . Methanogenesis was inhibited by Fe(III) bioreduction, possibly due to diversion of electrons from the methanogenesis pathway to structural Fe(III) in clay minerals. Bioreduction of Fe(III) in clay minerals induced formation of biogenic mixed-layer illite–smectite, silica, and vivianite. These data collectively showed that mesophilic methanogen is capable of reducing structural Fe(III) in illite–smectite minerals and of inducing a number of mineralogical changes.

Inhibitory Effects of the Divalent Metal Ions on Biomethanation by Isolated Mesophilic Methanogen in AC21 Medium in Presence or Absence of Juices from Water Hyacinth

The effect of divalent metal ions on biomethanation was studied using pre-reduced methanogenic AC21 medium in absence or in presence of leaf or root extracts of Eichornia grassipes (water hyacinth) in vitro. The metal ions like Cu2+, Fe2+, Ni2+, and Co2+ were found to be effective in increasing methane production in vitro at low concentrations, but toxic inhibitory effects were observed at relatively high concentrations. Zn2+ ion was inhibitory in character with exceptions. The bioavailability of metal ions in AC21 medium alone or with juices from leaf or root extracts of plant (water hyacinth in the present case) was limited due to complex formation of metal ions with different constituents in the medium, precipitation, etc. However, the results of the microscopic experiments (in vials) with metal ions for methane production by isolated methanogens in the methanogenic medium should not be compared with the conversion of biomass to methane in biodigesters. In such cases, not only a large number of methanogenic bacteria of different strains but also a consortium of bacteria of different specificities is usually involved. Though the biomass may contain sufficient concentrations of metal ions, it is advisable to add metal ions like Cu2+, Fe2+ , Zn 2+ ,Co2+, etc. in the biomass, but the concentrations of the metal ions should be limited to 10–20 µM of the biomass to make methane generation successful.

Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., mesophilic methanogens isolated from salty environments

Two methane-producing archaea, designated Mic5c12(T) and Mic6c05(T), were isolated from sludge deposited in a crude oil storage tank and a tubercle on the interior of a pipe transporting natural gas-containing brine, respectively. The isolates were Gram-staining-variable, non-motile rods and grew only on H(2)/CO(2). Strain Mic6c05(T) produced methane from some alcohols without showing any growth; strain Mic5c12(T) did not utilize alcohols. The optimum growth conditions for strain Mic5c12(T) were 35 °C, pH 6.5 and 0-0.68 M NaCl and for strain Mic6c05(T) were 40 °C, pH 6.0-7.5 and 0.34 M NaCl. Strain Mic5c12(T) was halotolerant and strain Mic6c05(T) was halophilic. Comparative 16S rRNA gene sequence analysis revealed that strains Mic5c12(T) and Mic6c05(T) belonged to the genus Methanobacterium and their closest relative was Methanobacterium subterraneum A8p(T) (97.3 and 97.9 % 16S rRNA gene sequence similarity, respectively). The findings from the 16S rRNA gene sequence analyses were supported by analysis of McrA, the alpha subunit of methyl-coenzyme M reductase. On the basis of phylogenetic analyses and phenotypic characteristics, two novel species are proposed, Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., with type strains Mic5c12(T) (=NBRC 105198(T) =DSM 22353(T)) and Mic6c05(T) (=NBRC 105197(T) =DSM 21974(T)), respectively.

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.