A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. The new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h-1) using carbon dioxide (CO2) as unique carbon source and hydrogen (H2) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH4+) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO2 conversion efficiency of 97% and a final methane (CH4) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.IMPORTANCEAs a methanogenic archaeon, Methanothermobacter wolfeii uses CO2 as electron acceptor, producing CH4 as final product. The metabolism of M. wolfeii can be harnessed to capture CO2 from industrial emissions, besides producing a drop-in renewable biofuel to substitute fossil natural gas. If used as biocatalyst in new-generation CO2 sequestration processes, M. wolfeii has the potential to accelerate the decarbonization of the energy generation sector, which is the biggest contributor of CO2 emissions worldwide. Nonetheless, the development of CO2 sequestration archaeal-based biotechnology is still limited by an uncertainty in the requirements to cultivate methanogenic archaea and the unknown longevity of archaeal cultures. In this study, we report the adaptation, isolation, and phenotypic characterization of a novel variant of M. wolfeii, which is capable of maximum growth with minimal nutrients input. Our findings demonstrate the potential of this variant for the production of renewable natural gas, paving the way for the development of more efficient and sustainable CO2 sequestration processes.

Methanogenic archaea are a part of the commensal gut microbiota responsible for hydrogen sink and the efficient production of short-chain fatty acids. Dysbiosis of methanogens is suspected to play a role in pathogenesis of variety of diseases, including inflammatory bowel disease (IBD). Unlike bacteria, the diversity of archaea seems to be higher in IBD patients compared to healthy subjects, whereas the prevalence and abundance of gut methanogens declines in IBD, especially in ulcerative colitis. To date, studies focusing on methanogens in pediatric IBD are very limited; nevertheless, the preliminary results provide some evidence that methanogens may be influenced by the chronic inflammatory process in IBD. In this review, we demonstrated the development and diversity of the methanogenic community in IBD, both in adults and children.

UNASSIGNED: Methanogenic archaea represent a less investigated and likely underestimated part of the intestinal tract microbiome in swine.
UNASSIGNED: This study aims to elucidate the archaeome structure and function in the porcine intestinal tract of healthy and H1N1 infected swine. We performed multi-omics analysis consisting of 16S rRNA gene profiling, metatranscriptomics and metaproteomics.
UNASSIGNED: We observed a significant increase from 0.48 to 4.50% of archaea in the intestinal tract microbiome along the ileum and colon, dominated by genera Methanobrevibacter and Methanosphaera. Furthermore, in feces of naïve and H1N1 infected swine, we observed significant but minor differences in the occurrence of archaeal phylotypes over the course of an infection experiment. Metatranscriptomic analysis of archaeal mRNAs revealed the major methanogenesis pathways of Methanobrevibacter and Methanosphaera to be hydrogenotrophic and methyl-reducing, respectively. Metaproteomics of archaeal peptides indicated some effects of the H1N1 infection on central metabolism of the gut archaea.
UNASSIGNED: Finally, this study provides the first multi-omics analysis and high-resolution insights into the structure and function of the porcine intestinal tract archaeome during a non-lethal Influenza A virus infection of the respiratory tract, demonstrating significant alterations in archaeal community composition and central metabolic functions.

Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in Methanococcus maripaludis, a representative archaeon lacking known 3\'-5\' exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors. After splicing, the circular processing intermediates were formed and this was confirmed by quantitative RT-PCR and Northern blot. Ribonuclease assay revealed a specific cleavage at a 10-nt A/U-rich motif at the mature 5\' end of pre-16S rRNA, which linearized circular pre-16S rRNA intermediate. Further 3\'-RACE and ribonuclease assays determined that the endonuclease Nob1 cleaved the 3\' extension of pre-16S rRNA, and so generated the mature 3\' end. Circularized RT-PCR (cRT-PCR) and 5\'-RACE identified two cleavage sites near helix 1 at the 5\' end of 23S rRNA, indicating that an RNA structure-based endonucleolytic processing linearized the circular pre-23S rRNA intermediate. In the maturation of pre-5S rRNA, multiple endonucleolytic processing sites were determined at the 10-nt A/U-rich motif in the leader and trailer sequence. This study demonstrates that endonucleolytic processing, particularly at the 10-nt A/U-rich motifs play an essential role in the pre-rRNA maturation of M. maripaludis, indicating diverse pathways of rRNA maturation in archaeal species.

Methanogenic archaea (methanogens) represent a diverse group of microorganisms that inhabit various environmental and host-associated microbiomes. These organisms play an essential role in global carbon cycling given their ability to produce methane, a potent greenhouse gas, as a by-product of their energy production. Recent advances in culture-independent and -dependent studies have highlighted an increased prevalence of methanogens in the host-associated microbiome of diverse animal species. Moreover, there is increasing evidence that methanogens, and/or the methane they produce, may play a substantial role in human health and disease. This review addresses the expanding host-range and the emerging view of host-specific adaptations in methanogen biology and ecology, and the implications for host health and disease.

Ammonia (NH3) inhibition represents a major limitation to methane production during anaerobic digestion of organic material in biogas reactors. This process relies on co-operative metabolic interactions between diverse taxa at the community-scale. Despite this, most investigations have focused singularly on how methanogenic Archaea respond to NH3 stress. With a high-NH3 pre-adapted and un-adapted community, this study investigated responses to NH3 inhibition both at the community-scale and down to individual taxa. The pre-adapted community performed methanogenesis under inhibitory NH3 concentrations better than the un-adapted. While many functionally important phyla were shared between the two communities, only taxa from the pre-adapted community were robust to NH3. Functionally important phyla were mostly comprised of sensitive taxa (≥ 50%), yet all groups, including methanogens, also possessed tolerant individuals (10-50%) suggesting that potential mechanisms for tolerance are non-specific and widespread. Hidden Markov Model-based phylogenetic analysis of methanogens confirmed that NH3 tolerance was not restricted to specific taxonomic groups, even at the genus level. By reconstructing covarying growth patterns via network analyses, methanogenesis by the pre-adapted community was best explained by continued metabolic interactions (edges) between tolerant methanogens and other tolerant taxa (nodes). However, under non-inhibitory conditions, sensitive taxa re-emerged to dominate the pre-adapted community, suggesting that mechanisms of NH3 tolerance can be disadvantageous to fitness without selection pressure. This study demonstrates that methanogenesis under NH3 inhibition depends on broad-scale tolerance throughout the prokaryotic community. Mechanisms for tolerance seem widespread and non-specific, which has practical significance for the development of robust methanogenic biogas communities. KEY POINTS: • Ammonia pre-adaptation allows for better methanogenesis under inhibitory conditions. • All functionally important prokaryote phyla have some ammonia tolerant individuals. • Methanogenesis was likely dependent on interactions between tolerant individuals.

To date, no research has been done on energy requirements for yaks in Tibetan cold weather. The findings of the current study provide proper energy requirements for yaks would facilitate scientific feeding of fattening yaks in cold weather. The metabolomics and 16s rRNA sequencing technologies were used to explore the underlying mechanism that affects the growth performance of yaks fed with different energy levels of diet in cold weather. Three groups of yaks (141.7 ± 3.34 kg) were fed with diets containing metabolizable energy 7.20, 7.89, and 8.58 MJ/kg DM (dry matter) and named the low-, medium-, and high-energy groups, respectively. The results showed that the average daily feed intake of the high-energy group was higher than that of the low-energy group (p  = 0.006). Plasma aspartate aminotransferase (p  = 0.004), alanine aminotransferase (p  < 0.001), and interferon-γ (p  < 0.001) in the high-energy group were lower than in the low-energy group. In contrast, superoxide dismutase (p  < 0.001), immunoglobulin G (p  < 0.001), and interleukin 2 (p  = 0.002) were higher than the low-energy group. The rumen microbial protein (p  = 0.025), total volatile fatty acids (p  = 0.029), and neutral detergent fiber digestibility (p  = 0.050) in the high-energy group were higher than in the low-energy group, whereas the acetate: propionate ratio (p  = 0.001) and ammonium nitrogen (p  = 0.001) were lower than in the low-energy group. The plasma metabolomics results displayed that yaks fed with a high-energy diet augmented the metabolism of arginine, proline, purine, taste transduction, pyrimidine, and glutathione pathways. The relative abundance of Methanobrevibacter in the high-energy group was lower (p  < 0.001), whereas the relative abundance of Methanosphaera (p  < 0.001) was higher than in the low-energy group. The results of the current study suggest that a high-energy diet in growing yaks during the cold season can improve growth performance, rumen microbial protein synthesis, antioxidants, and immunity.

Free-living anaerobic ciliates are of considerable interest from an ecological and an evolutionary standpoint. Extraordinary tentacle-bearing predatory lineages have evolved independently several times within the phylum Ciliophora, including two rarely encountered anaerobic litostomatean genera, Legendrea and Dactylochlamys. In this study, we significantly extend the morphological and phylogenetic characterization of these two poorly known groups of predatory ciliates. We provide the first phylogenetic analysis of the monotypic genus Dactylochlamys and the three valid species of Legendrea based on the 18S rRNA gene and ITS-28S rRNA gene sequences. Prior to this study, neither group had been studied using silver impregnation methods. We provide the first protargol-stained material and also a unique video material including documentation, for the first time, of the hunting and feeding behavior of a Legendrea species. We briefly discuss the identity of methanogenic archaeal and bacterial endosymbionts of both genera based on 16S rRNA gene sequences, and the importance of citizen science for ciliatology from a historical and contemporary perspective.

Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans have been biochemically characterized and can be genetically engineered by using a variety of existing molecular tools. The anaerobic lifestyle and autofluorescence of methanogens, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives), which require oxygen for chromophore maturation. Recently, the use of a novel oxygen-independent fluorescent activation and absorption-shifting tag (FAST) was demonstrated with M. maripaludis. Similarly, we now describe the use of the tandem activation and absorption-shifting tag protein 2 (tdFAST2), which fluoresces when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5DOM) is present. Expression of tdFAST2 in M. acetivorans and M. maripaludis is noncytotoxic and tdFAST2:HBR-3,5DOM fluorescence is clearly distinguishable from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be distinguished, thereby allowing for the possibility of high-throughput investigations of the characteristic dynamics within single and mixed cultures. IMPORTANCE Methane-producing archaea play an essential role in the global carbon cycle and demonstrate great potential for various biotechnological applications, e.g., biofuel production, carbon dioxide capture, and electrochemical systems. Oxygen sensitivity and high autofluorescence hinder the use of common fluorescent proteins for studying methanogens. By using tdFAST2:HBR-3,5DOM fluorescence, which functions under anaerobic conditions and is distinguishable from the autofluorescence, real-time reporter studies and high-throughput investigation of the mixed culture dynamics of methanogens via flow cytometry were made possible. This will further help accelerate the sustainable exploitation of methanogens.

Apart from their archetypic use in anaerobic digestion (AD) methanogenic archaea are targeted for a wide range of applications. Using different methanogenic archaea for one specific application requires the optimization of culture media to enable the growth of different strains under identical environmental conditions, e.g., in microbial electrochemical technologies (MET) for (bio)electromethanation. Here we present a new culture medium (BFS01) adapted from the DSM-120 medium by omitting resazurin, yeast extract, casitone, and using a low salt concentration, that was optimized for Methanosarcina barkeri, Methanobacterium formicicum, and Methanothrix soehngenii. The aim was to provide a medium for follow-up co-culture studies using specific methanogens and Geobacter spp. dominated biofilm anodes. All three methanogens showed growth and activity in the BFS01 medium. This was demonstrated by estimating the specific growth rates ( μ ) and doubling times ( t d ) of each methanogen. The μ and t d based on methane accumulation in the headspace showed values consistent with literature values for M. barkeri and M. soehngenii. However, μ and t d based on methane accumulation in the headspace differed from literature data for M. formicicum but still allowed sufficient growth. The lowered salt concentration and the omission of chemically complex organic components in the medium may have led to the observed deviation from μ and t d for M. formicicum as well as the changed morphology. 16S rRNA gene-based amplicon sequencing and whole genome nanopore sequencing further confirmed purity and species identity.