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Lignin, a major component of plant biomass, remains underutilized for renewable biofuels due to its complex and heterogeneous structure. Although investigations into depolymerizing lignin using fungi are well-established, studies of microbial pathways that enable anaerobic lignin breakdown linked with methanogenesis are limited. Through an enrichment cultivation approach with inoculation of freshwater sediment, we enriched a microbial community capable of producing methane during anaerobic lignin degradation. We reconstructed the near-complete population genomes of key lignin degraders and methanogens using metagenome-assembled genomes finally selected in this study (MAGs; 92 bacterial and 4 archaeal MAGs affiliated into 45 and 2 taxonomic groups, respectively). This study provides genetic evidence of microbial interdependence in conversion of lignin to methane in a syntrophic community. Metagenomic analysis revealed metabolic linkages, with lignin-hydrolyzing and/or fermentative bacteria such as the genera Alkalibaculum and Propionispora transforming lignin breakdown products into compounds such as acetate to feed methanogens (two archaeal MAGs classified into the genus Methanosarcina or UBA6 of the family Methanomassiliicoccaceae). Understanding the synergistic relationships between microbes that convert lignin could inform strategies for producing renewable bioenergy and treating aromatic-contaminated environments through anaerobic biodegradation processes. Overall, this study offers fundamental insights into complex community-level anaerobic lignin metabolism, highlighting hitherto unknown players, interactions, and pathways in this biotechnologically valuable process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The majority of research in the field of human microbiota has predominantly focused on bacterial and fungal communities. Conversely, the human archaeome has received scant attention and remains poorly studied, despite its potential role in human diseases. Archaea have the capability to colonize various human body sites, including the gastrointestinal tract, skin, vagina, breast milk, colostrum, urinary tract, lungs, nasal and oral cavities. This colonization can occur through vertical transmission, facilitated by the transfer of breast milk or colostrum from mother to child, as well as through the consumption of dairy products, organic produce, salty foods, and fermented items. The involvement of these microorganisms in diseases, such as periodontitis, might be attributed to their production of toxic compounds and the detoxification of growth inhibitors for pathogens. However, the precise mechanisms through which these contributions occur remain incompletely understood, necessitating further studies to assess their impact on human health., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Ruminants are responsible for enteric methane production contributing significantly to the anthropogenic greenhouse gases in the atmosphere. Moreover, dietary energy is lost as methane gas without being available for animal use. Therefore, many mitigation strategies aiming at interventions at animals, diet, and microbiota have been explored by researchers. Specific chemical analogues targeting the enzymes of the methanogenic pathway appear to be more effective in specifically inhibiting the growth of methane-producing archaea without hampering another microbiome, particularly, cellulolytic microbiota. The targets of methanogenesis reactions that have been mainly investigated in ruminal fluid include methyl coenzyme M reductase (halogenated sulfonate and nitrooxy compounds), corrinoid enzymes (halogenated aliphatic compounds), formate dehydrogenase (nitro compounds, e.g., nitroethane and 2-nitroethanol), and deazaflavin (F 420 ) (pterin and statin compounds). Many other potential metabolic reaction targets in methanogenic archaea have not been evaluated properly. The analogues are specifically effective inhibitors of methanogens, but their efficacy to lower methanogenesis over time reduces due to the metabolism of the compounds by other microbiota or the development of resistance mechanisms by methanogens. In this short review, methanogen populations inhabited in the rumen, methanogenesis pathways and methane analogues, and other chemical compounds specifically targeting the metabolic reactions in the pathways and methane production in ruminants have been discussed. Although many methane inhibitors have been evaluated in lowering methane emission in ruminants, advancement in unravelling the molecular mechanisms of specific methane inhibitors targeting the metabolic pathways in methanogens is very limited., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Despite the large number of studies conducted on archaea associated with extreme environments, the archaeal community composition in food products is still poorly known. Here, we investigated a new insight into exploring the archaeal community in several food matrices, with a particular focus on determining whether living archaea were present. A total of 71 samples of milk, cheese and its derived brine, honey, hamburger, clam, and trout were analyzed by high-throughput 16S rRNA sequencing. Archaea were detected in all the samples, ranging from 0.62 % of microbial communities in trout to 37.71 % in brine. Methanogens dominated 47.28 % of the archaeal communities, except for brine, which was dominated by halophilic taxa affiliated with the genus Haloquadratum (52.45 %). Clams were found to be a food with high richness and diversity of archaea and were targeted for culturing living archaea under different incubation time and temperature conditions. A subset of 16 communities derived from culture-dependent and culture-independent communities were assessed. Among the homogenates and living archaeal communities, the predominant taxa were distributed in the genera Nitrosopumilus (47.61 %) and Halorussus (78.78 %), respectively. A comparison of the 28 total taxa obtained by culture-dependent and culture-independent methods enabled their categorization into different groups, including detectable (8 out of 28), cultivable (8 out of 28), and detectable-cultivable (12 out of 28) taxa. Furthermore, using the culture method, the majority (14 out of 20) of living taxa grew at lower temperatures of 22 and 4 °C during long-term incubation, and few taxa (2 out of 20) were found at 37 °C during the initial days of incubation. Our results demonstrated the distribution of archaea in all analyzed food matrices, which opens new perspectives to expand our knowledge on archaea in foods and their beneficial and detrimental effects., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Tetramethylammonium hydroxide (TMAH) is a known toxic chemical used in the photolithography process of semiconductor photoelectronic processes. Significant amounts of wastewater containing TMAH are discharged from electronic industries. It is therefore attractive to apply anaerobic treatment to industrial wastewater containing TMAH. In this study, a novel TMAH-degrading methanogenic archaeon was isolated from the granular sludge of a psychrophilic upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater containing TMAH. Although the isolate (strain NY-STAYD) was phylogenetically related to Methanomethylovorans uponensis, it was the only isolated Methanomethylovorans strain capable of TMAH degradation. Strain NY-STAYD was capable of degrading methylamine compounds, similar to the previously isolated Methanomethylovorans spp. While the strain was able to grow at temperatures ranging from 15 to 37°C, the cell yield was higher at lower temperatures. The distribution of archaeal cells affiliated with the genus Methanomethylovorans in the original granular sludge was investigated by fluorescence in situ hybridization (FISH) using specific oligonucleotide probe targeting 16S rRNA. The results demonstrated that the TMAH-degrading cells associated with the genus Methanomethylovorans were not intermingled with other microorganisms but rather isolated on the granule's surface as a lone dominant archaeon. KEY POINTS: • A TMAH-degrading methanogenic Methanomethylovorans strain was isolated • This strain was the only known Methanomethylovorans isolate that can degrade TMAH • The highest cell yield of the isolate was obtained at psychrophilic conditions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA-DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

Analyses of protein structures have shown the existence of molecular channels in enzymes from Prokaryotes. Those molecular channels suggest a critical role of spatial voids in proteins, above all, in those enzymes functioning under high temperature. It is expected that these spaces within the protein structure are required to access the active site and to maximize availability and thermal stability of their substrates and cofactors. Interestingly, numerous substrates and cofactors have been reported to be highly temperature-sensitive biomolecules. Methanogens represent a singular phylogenetic group of Archaea that performs anaerobic respiration producing methane during growth. Methanogens inhabit a variety of environments including the full range of temperatures for the known living forms. Herein, we carry out a dimensional analysis of molecular tunnels in key enzymes of the methanogenic pathway from methanogenic Archaea growing optimally over a broad temperature range. We aim to determine whether the dimensions of the molecular tunnels are critical for those enzymes from thermophiles. Results showed that at increasing growth temperature the dimensions of molecular tunnels in the enzymes methyl-coenzyme M reductase and heterodisulfide reductase become increasingly restrictive and present strict limits at the highest growth temperatures, i.e., for hyperthermophilic methanogens. However, growth at lower temperature allows a wide dimensional range for the molecular spaces in these enzymes. This is in agreement with previous suggestions on a potential major role of molecular tunnels to maintain biomolecule stability and activity of some enzymes in microorganisms growing at high temperatures. These results contribute to better understand archaeal growth at high temperatures. Furthermore, an optimization of the dimensions of molecular tunnels would represent an important adaptation required to maintain the activity of key enzymes of the methanogenic pathway for those methanogens growing optimally at high temperatures.

Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F 420 -dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr ( Mj Fsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. Mj FsrI reduces sulfite to sulfide with reduced F 420 (F 420 H 2 ), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F 420 H 2 . Instead, it acted as an F 420 H 2 -dependent nitrite reductase (FNiR) with physiologically relevant K m values (nitrite, 5 μM; F 420 H 2, 14 μM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F 420 H 2 -derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E 0 ' [standard potential], +440 mV) but not sulfite (E 0 ', -116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies. IMPORTANCE Coenzyme F 420 -dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F 420 -dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need.

Background: As a recently discovered member of the DPANN superphylum, Woesearchaeota account for a wide diversity of 16S rRNA gene sequences, but their ecology, evolution, and metabolism remain largely unknown., Results: Here, we assembled 133 global clone libraries/studies and 19 publicly available genomes to profile these patterns for Woesearchaeota. Phylogenetic analysis shows a high diversity with 26 proposed subgroups for this recently discovered archaeal phylum, which are widely distributed in different biotopes but primarily in inland anoxic environments. Ecological patterns analysis and ancestor state reconstruction for specific subgroups reveal that oxic status of the environments is the key factor driving the distribution and evolutionary diversity of Woesearchaeota. A selective distribution to different biotopes and an adaptive colonization from anoxic to oxic environments can be proposed and supported by evidence of the presence of ferredoxin-dependent pathways in the genomes only from anoxic biotopes but not from oxic biotopes. Metabolic reconstructions support an anaerobic heterotrophic lifestyle with conspicuous metabolic deficiencies, suggesting the requirement for metabolic complementarity with other microbes. Both lineage abundance distribution and co-occurrence network analyses across diverse biotopes confirmed metabolic complementation and revealed a potential syntrophic relationship between Woesearchaeota and methanogens, which is supported by metabolic modeling. If correct, Woesearchaeota may impact methanogenesis in inland ecosystems., Conclusions: The findings provide an ecological and evolutionary framework for Woesearchaeota at a global scale and indicate their potential ecological roles, especially in methanogenesis.

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O -phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications., Competing Interests: The authors declare no conflict of interest.

Microbial communities in residual slurry left after removal of stored liquid dairy manure have been presumed to increase methane emission during new storage, but these microbes have not been studied. While actual manure storage tanks are filled gradually, pilot- and farm-scale studies on methane emissions from such systems often use a batch approach. In this study, six pilot-scale outdoor storage tanks with (10% and 20%) and without residual slurry were filled (gradually or in batch) with fresh dairy manure, and methane and methanogenic and bacterial communities were studied during 120 days of storage. Regardless of filling type, increased residual slurry levels resulted in higher abundance of methanogens and bacteria after 65 days of storage. However, stronger correlation between methanogen abundance and methane flux was observed in gradually filled tanks. Despite some variations in the diversity of methanogens or bacteria with the presence of residual slurry, core phylotypes were not impacted. In all samples, the phylum Firmicutes predominated (∼57 to 70%) bacteria: >90% were members of Clostridia Methanocorpusculum dominated (∼57 to 88%) archaeal phylotypes, while Methanosarcina gradually increased with storage time. During peak flux of methane, Methanosarcina was the major player in methane production. The results suggest that increased levels of residual slurry have little impact on the dominant methanogenic or bacterial phylotypes, but large population sizes of these organisms may result in increased methane flux during the initial phases of storage. IMPORTANCE Methane is the major greenhouse gas emitted from stored liquid dairy manure. Residual slurry left after removal of stored manure from tanks has been implicated in increasing methane emissions in new storages, and well-adapted microbial communities in it are the drivers of the increase. Linking methane flux to the abundance, diversity, and activity of microbial communities in stored slurries with different levels of residual slurry can help to improve the mitigation strategy. Mesoscale and lab-scale studies conducted so far on methane flux from manure storage systems used batch-filled tanks, while the actual condition in many farms involves gradual filling. Hence, this study provides important information toward determining levels of residual slurry that result in significant reduction of well-adapted microbial communities prior to storage, thereby reducing methane emissions from manure storage tanks filled under farm conditions., (Copyright © 2018 American Society for Microbiology.)

In this study, four different mesophilic and thermophilic full-scale anaerobic digesters treating food wastewater (FWW) were monitored for 1-2years in order to investigate: 1) microbial communities underpinning anaerobic digestion of FWW, 2) significant factors shaping microbial community structures, and 3) potential microbial indicators of process instability. Twenty-seven bacterial genera were identified as abundant bacteria underpinning the anaerobic digestion of FWW. Methanosaeta harundinacea, M. concilii, Methanoculleus bourgensis, M. thermophilus, and Methanobacterium beijingense were revealed as dominant methanogens. Bacterial community structures were clearly differentiated by digesters; archaeal community structures of each digester were dominated by one or two methanogen species. Temperature, ammonia, propionate, Na + , and acetate in the digester were significant factors shaping microbial community structures. The total microbial populations, microbial diversity, and specific bacteria genera showed potential as indicators of process instability in the anaerobic digestion of FWW., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Studies on methanogenesis from freshwater sediments have so far primarily focused on lake sediments. To expand our knowledge on the community composition of methanogenic archaea in river sediments, we studied the abundance and diversity of methanogenic archaea at two localities along a vertical profile (top 50 cm) obtained from sediment samples from Sitka stream (the Czech Republic). In this study, we compare two sites which previously have been shown to have a 10-fold different methane emission. Archaeal and methanogen abundance were analyzed by real-time PCR and T-RFLP. Our results show that the absolute numbers for the methanogenic community (qPCR) are relatively stable along a vertical profile as well as for both study sites. This was also true for the archaeal community and for the three major methanogenic orders in our samples (Methanosarcinales, Methanomicrobiales, and Methanobacteriales). However, the underlying community structure (T-RFLP) reveals different community compositions of the methanogens for both locations as well as for different depth layers and over different sampling times. In general, our data confirm that Methanosarcinales together with Methanomicrobiales are the two dominant methanogenic orders in river sediments, while members of Methanobacteriales contribute a smaller community and Methanocellales are only rarely present in this sediment. Our results show that the previously observed 10-fold difference in methane emission of the two sites could not be explained by molecular methods alone., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or acetate as carbon and energy sources. This method provides an avenue for the first step in identifying new gene functions: associating a genetic mutation with a reproducible phenotype. Mutations in bona fide methanogenesis genes such as corrinoid methyltransferases and proton-translocating F 420 H 2 :methanophenazine oxidoreductase (Fpo) were also generated, opening the door to in vivo functional complementation experiments. Irradiation-based mutagenesis such as from ultraviolet (UV) light, combined with modern genome sequencing, is a useful procedure to discern systems-level gene function in prokaryote taxa that can be axenically cultured but which may be resistant to chemical mutagens., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Methanogens define the archaeal communities involved in anaerobic digestion. Recently, non-methanogen archaeal populations have been unexpectedly identified in anaerobic digestion processes. To gain insight into the ecophysiology of these uncharacterized archaeal populations, for the first time, a phylogenetic analysis was performed on a collection of non-methanogen archaeal 16S rRNA gene sequences from anaerobic digesters of broad geographic distribution, revealing a distinct clade formed by these sequences in subgroup 6 of the Miscellaneous Crenarchaeotal Group in the newly proposed archaeal phylum Bathyarchaeota. This exclusive phylogenetic assemblage enabled the development of a real-time quantitative PCR (qPCR) assay specifically targeting these non-methanogen archaeal populations in anaerobic digestion. Application of the qPCR assay in continuous anaerobic digesters indicated that these archaeal populations were minor constituents of the archaeal communities, and the abundance of these populations remained relatively constant irrespective of process perturbations. Analysis of the archaeal populations in methanogenic communities further revealed the co-occurrence of these non-methanogen archaea with acetoclastic methanogens. Nevertheless, the low abundance of non-methanogen archaea as compared with acetoclastic methanogens suggests that the non-methanogen archaeal populations were not major players in animal waste-fed methanogenic processes investigated in this study and the functions of these archaeal populations remain to be identified.

Methane, which is produced by methanogenic archaea, is the second most abundant carbon compound in the atmosphere. Due to its strong radiative forcing, many studies have been conducted to determine its sources, budget, and dynamics. However, a mechanistic model of methane flux has not been developed thus far. In this study, we attempt to examine the relevance of the abundance of methanogen as a biological indicator of methane flux in three different types of soil ecosystems: permafrost, rice paddy, and mountainous wetland. We measured the annual average methane flux and abundance of methanogen in the soil ecosystems in situ. The correlation between methane flux and the abundance of methanogen exists only under a specific biogeochemical conditions such as SOM of higher than 60%, pH of 5.6-6.4, and water-saturated. Except for these conditions, significant correlations were absent. Therefore, microbial abundance information can be applied to a methane flux model selectively depending on the biogeochemical properties of the soil ecosystem.

The molecular diversity of rumen methanogens was investigated using 16S rDNA gene library prepared from the rumen contents of Nili-Ravi buffaloes. Microbial genomic DNA was isolated from four adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the inserts of positive clones were sequenced. A total of 142 clones were examined, and the analysis revealed 46 species level (0.01 distance) operational taxonomic units (OTUs). Twenty six OTUs comprising 89 clones (63% of the total clones) were taxonomically assigned to Methanobacterium genus and the majority of them had highest percent identity with Methanobacterium flexile among cultured methanogens. Five OTUs comprising 27 clones (19% of total clones) were taxonomically assigned to Methanomicrobium genus and these clones showed highest sequence identity with Methanomicrobium mobile. Only two OTUs comprising 6 clones (4% of total clones) were assigned to Methanobrevibacter genus. A total of 17 clones belonging to 10 species level OTUs showed highest percent identity (ranging from 85 to 95%) with Methanomassilicoccus luminyensis and were taxonomically classified as Methanomassiliicocaceae. Out of the 142 rDNA clones, 112 clones, which constitute 79% of the total clones representing 42 OTUs, had less than 98.5% sequence identity with any of the cultured strains of methanogens and represent novel species of methanogens. This study has revealed the largest assortment of hydrogenotrophic methanogen phylotypes ever identified from the rumen of Nili-Ravi buffaloes. The study indicates that Methanobacterium is the most dominant methanogen in the rumen of Nili-Ravi buffalo. This is also the first report on the presence of methanogens phylogenetically close to M. luminyensis, an H2 dependent methylotrophic methanogen, in the rumen of buffaloes at such a high level of abundance., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

2-Phase anaerobic digestion (AD), where the acidogenic phase was operated at 2day hydraulic retention time (HRT) and the methanogenic phase at 10days HRT, had been evaluated to determine if it could provide higher organic reduction and methane production than the conventional single-stage AD (also operated at 12days HRT). 454 pyrosequencing was performed to determine and compare the microbial communities. The acidogenic reactor of the 2-phase system yielded a unique bacterial community of the lowest richness and diversity, while bacterial profiles of the methanogenic reactor closely followed the single-stage reactor. All reactors were predominated by hydrogenotrophic methanogens, mainly Methanolinea. Unusually, the acidogenic reactor contributed up to 24% of total methane production in the 2-phase system. This could be explained by the presence of Methanosarcina and Methanobrevibacter, and their activities could also help regulate reactor alkalinity during high loading conditions through carbon dioxide production. The enrichment of hydrolytic and acidogenic Porphyromonadaceae, Prevotellaceae, Ruminococcaceae and unclassified Bacteroidetes in the acidogenic reactor would have contributed to the improved sludge volatile solids degradation, and ultimately the overall 2-phase system's performance. Syntrophic acetogenic microorganisms were absent in the acidogenic reactor but present in the downstream methanogenic reactor, indicating the retention of various metabolic pathways also found in a single-stage system. The determination of key microorganisms further expands our understanding of the complex biological functions in AD process., (Copyright © 2015. Published by Elsevier B.V.)

Abundance and diversity of methanogenic archaea were studied at five localities along a longitudinal profile of a Sitka stream (Czech Republic). Samples of hyporheic sediments were collected from two sediment depths (0-25 cm and 25-50 cm) by freeze-core method. Methanogen community was analyzed by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequencing method. The proportion of methanogens to the DAPI-stained cells varied among all localities and depths with an average value 2.08 × 10(5) per g of dry sediment with the range from 0.37 to 4.96 × 10(5) cells per g of dry sediment. A total of 73 bands were detected at 19 different positions on the DGGE gel and the highest methanogen diversity was found at the downstream located sites. There was no relationship between methanogen diversity and sediment depth. Cluster analysis of DGGE image showed three main clusters consisting of localities that differed in the number and similarity of the DGGE bands. Sequencing analysis of representative DGGE bands revealed phylotypes affiliated with members belonging to the orders Methanosarcinales, Methanomicrobiales and Methanocellales. The knowledge about occurrence and diversity of methanogenic archaea in freshwater ecosystems are essential for methane dynamics in river sediments and can contribute to the understanding of global warming process., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Oxygen injection is a commonly used mitigation strategy for sulfide control in sewers. Methane, a potent greenhouse gas, is also produced in sewers. Oxygen injection may reduce methane generation/emission, but could potentially lead to N2O production due to the development of a nitrifying microbial community. The impact of oxygen dosing for sulfide control in sewers on CH4 and N2O production was assessed in this study in laboratory sewer reactors. Results showed that oxygen injection is able to reduce CH4 formation in sewers, although full control of CH4 was not achieved, likely due to partial oxygen penetration into sewer biofilm. The experimental results also revealed a nitrogen loss of around 5 mN/L. However, no significant N2O accumulation was detected., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aims: Crusts forming at the surface of liquid manure (slurry) during storage have been shown to harbour a potential for mitigating CH4 emissions. This study investigated the microbial community in surface crusts, with a focus on micro-organisms related to CH4 metabolism., Methods and Results: Microbial communities in four crusts from cattle and swine slurries were investigated using denaturing gradient gel electrophoresis and tag-encoded amplicon pyrosequencing. All crusts had distinct compositions of bacteria and archaea. The genera Methylobacter, Methylomicrobium, Methylomonas, and Methylosarcina of Type I, and Methylocystis of Type II, dominated the methane-oxidizing bacteria (MOB) community, whereas Methanocorpusculum was the predominant methanogen. Higher numbers of operational taxonomic units (OTUs) representing Type I than Type II MOB were found in all crusts. Potential CH4 oxidation rates were determined by incubating crusts with CH4 , and CH4 oxidization was observed in cattle, but not in swine slurry crusts., Conclusions: Slurry surface crusts harbour a diverse microbial community. Type I MOB are more diverse and abundant than Type II MOB in this environment. The distinct CH4 oxidation rates could be related to microbial compositions., Significance and Impact of the Study: This study is the first to present the overall microbial community structure in slurry surface crusts. A better understanding of microbial community in surface crusts could support strategies for mitigation of CH4 emissions from livestock manure management., (© 2014 The Society for Applied Microbiology.)

Methanogenic archaea lack cysteinyl-tRNA synthetase; they synthesize Cys-tRNA and cysteine in a tRNA-dependent manner. Two enzymes are required: Phosphoseryl-tRNA synthetase (SepRS) forms phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)), which is converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS). This represents the ancestral pathway of Cys biosynthesis and coding in archaea. Here we report a translation factor, SepCysE, essential for methanococcal Cys biosynthesis; its deletion in Methanococcus maripaludis causes Cys auxotrophy. SepCysE acts as a scaffold for SepRS and SepCysS to form a stable high-affinity complex for tRNA(Cys) causing a 14-fold increase in the initial rate of Cys-tRNA(Cys) formation. Based on our crystal structure (2.8-Å resolution) of a SepCysS⋅SepCysE complex, a SepRS⋅SepCysE⋅SepCysS structure model suggests that this ternary complex enables substrate channeling of Sep-tRNA(Cys). A phylogenetic analysis suggests coevolution of SepCysE with SepRS and SepCysS in the last universal common ancestral state. Our findings suggest that the tRNA-dependent Cys biosynthesis proceeds in a multienzyme complex without release of the intermediate and this mechanism may have facilitated the addition of Cys to the genetic code.

The methanogenic community throughout the gastrointestinal tract (GIT) of pre-weaned calves has not been well studied. The current study firstly investigated the distribution and composition of the methanogenic community in the rumen, ileum, and colon of 3-4 week-old milk-fed dairy calves (n = 4) using 16S rRNA gene clone library analysis. The occurrence of methanogens in the GIT of pre-weaned calves was further validated by using PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and quantitative real-time PCR (qPCR) was applied to quantify the methanogenic community in the rumen, jejunum, ileum, cecum, colon and rectum of 8 3-4 week old animals. Both cloning libraries and PCR-DGGE revealed that phylotypes close to Methanobrevibacter were the main taxon along the GIT in pre-weaned sucking calves. The composition and abundance of methanogens varied significantly among individual animals, suggesting that host conditions may influence the composition of the symbiotic microbiota. Segregation of methanogenic communities throughout the GIT was also observed within individual animals, suggesting possible functional differences among methanogens residing in different GIT regions. This is the first study to analyze methanogenic communities throughout the GIT of milk-fed newborn dairy calves and reveal both their diversity and abundance. The identification of methanogens in the lower GIT of pre-weaned dairy calves warrants further investigation to better define methanogen roles in GIT function and their impact on host metabolism and health.

Aims: To gain new insights into the metabolic contribution of bacterial group in the rumen., Methods and Results: Both DNA- and RNA-derived bacterial 16S ribosomal materials from bovine rumen contents were used as the template for bacterial community and analyse microbiota by three methods namely custom phylogenetic microarray, quantitative real-time PCR and denaturing gradient gel electrophoresis techniques. Bacterial analysis showed that genera affiliating with the Proteobacteria apparently made a greater metabolic contribution to rumen function than their population sizes indicated. Analysis of another rumen microbial group, the methanogens, using clone libraries for the expressed methyl coenzyme reductase subunit A (mcrA) revealed that an uncultivated methanogen clade contributes one-third of RNA-derived mcrA sequences based on a limited number of clones analysed. These uncultivated methanogen species were not observed in the mcrA gene library based on the DNA-derived sequences., Conclusions: The comparison of results obtained from DNA- and RNA-derived materials suggests that some of the Proteobacteria and novel methanogen species appeared to be low in abundance in the rumen maintained on grain-based diets might play a greater role in rumen metabolism., Significance and Impact of the Study: These studies provide the first report to compare high-throughput analysis of bacterial 16S rRNA genes from DNA- and RNA-derived materials to indicate differences that species make to community structure and metabolic activity., (© 2013 The Society for Applied Microbiology.)

Methanogenic archaea are a group of microorganisms found in the gastrointestinal tract of various herbivores and humans; however, the quantity (intensity) of methane emissions during feed digestion varies. Macropodids, such as the Eastern Gray Kangaroo (Macropus giganteus), are considered to be low methane-emitting animals, but their gut methanogenic archaea remain poorly characterized. Characterizing methanogens from animals with low methane emissions offers the potential to develop strategies and interventions that reduce methane emissions from livestock. In this study, we describe a novel strain of Methanobrevibacter gottschalkii (EGK), the first Methanobrevibacter isolate from a marsupial host. Comparative analyses with other M. gottschalkii genomes revealed a high degree of gene conservation, along with strain-specific differences in genes related to membrane transport, xenobiotic metabolism, nucleotide metabolism, and the metabolism of cofactors and vitamins. Notably, the M. gottschalkii EGK genome contains multiple copies of large proviral elements, likely acquired through integration events in this strain. M. gottschalkii EGK is the first isolated representative of Methanobrevibacter from a low methane-emitting animal, providing a valuable reference genome to identify metabolic targets for methane mitigation. [ABSTRACT FROM AUTHOR]

Abstract Background The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2−based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory. Results This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5′end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes. Conclusions Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

Background: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2−based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory. Results: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5′end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes. Conclusions: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea. [ABSTRACT FROM AUTHOR]

Methanogenic archaea are a group of microorganisms found in the gastrointestinal tract of various herbivores and humans; however, the quantity (intensity) of methane emissions during feed digestion varies. Macropodids, such as the Eastern Gray Kangaroo (Macropus giganteus), are considered to be low methane-emitting animals, but their gut methanogenic archaea remain poorly characterized. Characterizing methanogens from animals with low methane emissions offers the potential to develop strategies and interventions that reduce methane emissions from livestock. In this study, we describe a novel strain of Methanobrevibacter gottschalkii (EGK), the first Methanobrevibacter isolate from a marsupial host. Comparative analyses with other M. gottschalkii genomes revealed a high degree of gene conservation, along with strain-specific differences in genes related to membrane transport, xenobiotic metabolism, nucleotide metabolism, and the metabolism of cofactors and vitamins. Notably, the M. gottschalkii EGK genome contains multiple copies of large proviral elements, likely acquired through integration events in this strain. M. gottschalkii EGK is the first isolated representative of Methanobrevibacter from a low methane-emitting animal, providing a valuable reference genome to identify metabolic targets for methane mitigation.

This article addresses the misconception that archaea lack peptidoglycan cell walls, specifically in the Methanobacteria and Methanopyri classes. The misunderstanding arose from a misinterpretation of the cell wall structure of methanogens. The recognition of peptidoglycan in archaea has important implications for various fields, such as environmental detection, antibiotic development, and genetics and cell biology research. The article emphasizes the need to correct this misconception and highlights the potential applications of studying archaeal peptidoglycan. The document is a list of references cited in an article about archaea, covering their cell envelopes, diversity, ecology, evolution, and adaptations to different environments. [Extracted from the article]

Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1–2-µm-long and 0.6–0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies. [ABSTRACT FROM AUTHOR]

Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector. [ABSTRACT FROM AUTHOR]

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 byproduct 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. [ABSTRACT FROM AUTHOR]

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼104-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications. • This study fills a gap by specifically addressing the scarcity of genetic element libraries in archaea. • 81 promoters having a ∼104-fold dynamic range and 42 ribosome binding sites (RBSs) with a ∼100-fold range. • The base preference role of strong archaeal promoters and RBSs can be applied to enhance strengths of weaker elements. • The obtained genetic elements greatly improved protein expression and modulated essential genes for their study. • The genetic toolbox facilitates M. maripaludis as an ideal workhorse for biology and biotechnology studies of archaea. [ABSTRACT FROM AUTHOR]

Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector.

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.

Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1–2-µm-long and 0.6–0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.

Analyses by secondary ion mass spectroscopy (SIMS) of 11 specimens of five taxa of prokaryotic filamentous kerogenous cellular microfossils permineralized in a petrographic thin section of the ∼3,465 Ma Apex chert of northwestern Western Australia, prepared from the same rock sample from which this earliest known assemblage of cellular fossils was described more than two decades ago, show their δ13C compositions to vary systematically taxon to taxon from -31‰ to -39‰. These morphospecies-correlated carbon isotope compositions confirm the biogenicity of the Apex fossils and validate their morphology-based taxonomic assignments. Perhaps most significantly, the δ13C values of each of the five taxa are lower than those of bulk samples of Apex kerogen (-27‰), those of SIMS-measured fossil-associated dispersed particulate kerogen (-27.6‰), and those typical of modern prokaryotic phototrophs (-25 ± 10‰). The SIMS data for the two highest δ13C Apex taxa are consistent with those of extant phototrophic bacteria; those for a somewhat lower δ13C taxon, with nonbacterial methane-producing Archaea; and those for the two lowest δ13C taxa, with methane-metabolizing γ-proteobacteria. Although the existence of both methanogens and methanotrophs has been inferred from bulk analyses of the carbon isotopic compositions of pre-2,500 Ma kerogens, these in situ SIMS analyses of individual microfossils present data interpretable as evidencing the cellular preservation of such microorganisms and are consistent with the near-basal position of the Archaea in rRNA phylogenies.

Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.

The glycerol dialkyl glycerol tetraether (GDGT) lipids of archaea and bacteria have been extensively studied in both modern and ancient wetlands, but their specific biological sources and biogeochemical significance in such settings remain unclear. The stable carbon isotopic composition of GDGTs, and associated tetraether lipids such as butanetriol - DGTs (BDGTs), can help reveal aspects of the carbon metabolism of their source organism. Here we present an in-depth compound-specific analyses of archaeal (isoGDGTs and BDGT-0) and bacterial (brGDGTs) tetraether lipid δ13C values from the Florida Everglades wetland, supplemented by 16S rRNA gene profiling of the archaeal community. We interpret this alongside GDGT δ13C analyses from three additional freshwater wetland sediments, and two ancient wetland deposits (lignites) from the Miocene. Our data suggests that isoGDGT-0 δ13C values, which range from –22.5‰ to −36.5‰ across our wetland sites, could to some degree reflect the relative importance of the acetotrophic and CO 2 -reducing hydrogenotrophic methanogenic pathways, though definitively delineating between the two is challenging due to (a) potential incorporation of dissolved inorganic carbon (DIC) by CO 2 reducers, and (b) the diluting effect of GDGTs derived from seemingly heterotrophic/fermentative Bathyarchaeia, the most abundant archaeal class in the Everglades. In the Everglades, which harbours abundant Methanomassiliicoccales, BDGT-0 is significantly more 13C-depleted than all other lipids, with a δ13C signature at certain depths (∼−40.0 to −45.0‰) that is consistent with an important source from these (likely) methylotrophic methanogens. This adds to a growing body of evidence on the underappreciated role of methylotrophic methanogenesis, largely driven by Methanomassiliicoccales , in the freshwater methane cycle. However, slightly more 13C-enriched values (∼−35.0‰) at certain depths in the same site provide evidence for additional, possibly heterotrophic sources under certain conditions, and BDGT-0 δ13C values in the Miocene lignite (≈ δ13C of TOC) are consistent with a solely heterotrophic source. Where available for measurement, δ13C values of isoGDGTs-1 to -3, averaging –32.7 ± 2.3‰, were significantly more 13C-depleted than isoGDGT-0, suggesting a different mixture of sources, likely including an autotrophic/mixotrophic component derived from archaea belonging to the Crenarchaeota, Nitrososphaeria (formerly Thaumarchaeota) and/or MBG-D/DHVEG-1. Finally, brGDGT δ13C values are generally consistent with their proposed bacterial heterotrophic origin and biosynthetic effects, averaging out at around 4‰ depleted relative to total organic carbon (TOC). Importantly, we find no significant variation between the δ13C values of different brGDGTs across our wetland types. This highlights that spatial and temporal changes in commonly applied brGDGT-based proxies likely do not co-occur with changes in the heterotrophic lifestyle of their producer(s), discounting a possible confounding factor in brGDGT palaeothermometry. Taken together, our results yield insights into wetland archaeal communities and the lipids they produce, and underline the applicability of tetraether δ13C analyses in probing aspects of microbial carbon cycling in modern and ancient wetland sediments. [ABSTRACT FROM AUTHOR]

It is known that the physiology of Methanosarcina species can differ significantly, but the ecological impact of these differences is unclear. We recovered two strains of Methanosarcina from two different ecosystems with a similar enrichment and isolation method. Both strains had the same ability to metabolize organic substrates and participate in direct interspecies electron transfer but also had major physiological differences. Strain DH-1, which was isolated from an anaerobic digester, used H2 as an electron donor. Genome analysis indicated that it lacks an Rnf complex and conserves energy from acetate metabolism via intracellular H2 cycling. In contrast, strain DH-2, a subsurface isolate, lacks hydrogenases required for H2 uptake and cycling and has an Rnf complex for energy conservation when growing on acetate. Further analysis of the genomes of previously described isolates, as well as phylogenetic and metagenomic data on uncultured Methanosarcina in anaerobic digesters and diverse soils and sediments, revealed a physiological dichotomy that corresponded with environment of origin. The physiology of type I Methanosarcina revolves around H2 production and consumption. In contrast, type II Methanosarcina species eschew H2 and have genes for an Rnf complex and the multiheme, membrane-bound c-type cytochrome MmcA, shown to be essential for extracellular electron transfer. The distribution of Methanosarcina species in diverse environments suggests that the type I H2-based physiology is well suited for high-energy environments, like anaerobic digesters, whereas type II Rnf/cytochrome-based physiology is an adaptation to the slower, steady-state carbon and electron fluxes common in organic-poor anaerobic soils and sediments. [ABSTRACT FROM AUTHOR]

Hydrologic shifts due to climate change will affect the cycling of carbon (C) stored in boreal peatlands. Carbon cycling in these systems is carried out by microorganisms and plants in close association. This study investigated the effects of experimentally manipulated water tables (lowered and raised) and plant functional groups on the peat and root microbiomes in a boreal rich fen. All samples were sequenced and processed for bacterial, archaeal (16S DNA genes; V4), and fungal (internal transcribed spacer 2 [ITS2]) DNA. Depth had a strong effect on microbial and fungal communities across all water table treatments. Bacterial and archaeal communities were most sensitive to the water table treatments, particularly at the 10- to 20-cm depth; this area coincides with the rhizosphere or rooting zone. Iron cyclers, particularly members of the family Geobacteraceae, were enriched around the roots of sedges, horsetails, and grasses. The fungal community was affected largely by plant functional group, especially cinquefoils. Fungal endophytes (particularly Acephala spp.) were enriched in sedge and grass roots, which may have underappreciated implications for organic matter breakdown and cycling. Fungal lignocellulose degraders were enriched in the lowered water table treatment. Our results were indicative of two main methanogen communities, a rooting zone community dominated by the archaeal family Methanobacteriaceae and a deep peat community dominated by the family Methanomicrobiaceae. [ABSTRACT FROM AUTHOR]

Featured Application: Engineered Methanosarcina acetivorans can be introduced to municipal wastewater to produce renewable bioisoprene. Wastewater biosolids are a promising feedstock for production of value-added renewable chemicals. Methane-producing archaea (methanogens) are already used to produce renewable biogas via the anaerobic treatment of wastewater. The ability of methanogens to efficiently convert dissolved organic carbon into methane makes them an appealing potential platform for biorefining using metabolic engineering. We have engineered a strain of the methanogen Methanosarcina acetivorans to produce the volatile hemiterpene isoprene in addition to methane. The engineered strain was adapted to grow in municipal wastewater through cultivation in a synthetic wastewater medium. When introduced to municipal wastewater the engineered methanogens were able to compete with the indigenous microorganisms and produce 0.97 mM of isoprene (65.9 ± 21.3 g per m3 of effluent). The production of isoprene in wastewater appears to be dependent on the quantity of available methanogenic substrate produced during upstream digestion by heterotrophic fermenters. This shows that with minimal adaptation it is possible to drop-in engineered methanogens to existing wastewater environments and attain value-added products in addition to the processing of wastewater. This shows the potential for utilizing methanogens as a platform for low-cost production of renewable materials without expensive feedstocks or the need to build or adapt existing facilities. [ABSTRACT FROM AUTHOR]

Isoprene is a valuable petrochemical used for a wide variety of consumer goods, such as adhesives and synthetic rubber. We were able to achieve a high yield of renewable isoprene by taking advantage of the naturally high-flux mevalonate lipid synthesis pathway in anaerobic methane-producing archaea (methanogens). Our study illustrates that by genetically manipulating Methanosarcina species methanogens, it is possible to create organisms that grow by producing the hemiterpene isoprene. Mass balance measurements show that engineered methanogens direct up to 4% of total carbon flux to isoprene, demonstrating that methanogens produce higher isoprene yields than engineered yeast, bacteria, or cyanobacteria, and from inexpensive feedstocks. Expression of isoprene synthase resulted in increased biomass and changes in gene expression that indicate that isoprene synthesis depletes membrane precursors and redirects electron flux, enabling isoprene to be a major metabolic product. Our results demonstrate that methanogens are a promising engineering chassis for renewable isoprene synthesis. IMPORTANCE A significant barrier to implementing renewable chemical technologies is high production costs relative to those for petroleum-derived products. Existing technologies using engineered organisms have difficulty competing with petroleum-derived chemicals due to the cost of feedstocks (such as glucose), product extraction, and purification. The hemiterpene monomer isoprene is one such chemical that cannot currently be produced using cost-competitive renewable biotechnologies. To reduce the cost of renewable isoprene, we have engineered methanogens to synthesize it from inexpensive feedstocks such as methane, methanol, acetate, and carbon dioxide. The "isoprenogen" strains we developed have potential to be used for industrial production of inexpensive renewable isoprene. [ABSTRACT FROM AUTHOR]

Oil reservoirs contain microbial populations that are both autochthonously and allochthonously introduced by industrial development. These microbial populations are greatly influenced by external factors including, but not limited to, salinity and temperature. In this study, we used metagenomics to examine the microbial populations within five wells of the same hydrocarbon reservoir system in the Gulf of Mexico. These elevated salinity (149–181 ppt salinity, 4–5× salinity of seawater) reservoirs have limited taxonomic and functional microbial diversity dominated by methanogens, Halanaerobium and other Firmicutes lineages, and contained less abundant lineages such as Deltaproteobacteria. Metagenome assembled genomes (MAGs) were generated and analyzed from the various wells. Methanogen MAGs were closely related to Methanohalophilus euhalobius, a known methylotrophic methanogen from a high salinity oil environment. Based on metabolic reconstruction of genomes, the Halanaerobium perform glycine betaine fermentation, potentially produced by the methanogens. Industrial introduction of methanol to prevent methane hydrate formation to this environment is likely to be consumed by these methanogens. As such, this subsurface oil population may represent influences from industrial processes.

Abstract Background Methanogenic archaea are of importance to the global C-cycle and to biological methane (CH4) production through anaerobic digestion and pure culture. Here, the individual and combined effects of copper (Cu), zinc (Zn), acetate, and propionate on the metabolism of the autotrophic, hydrogenotrophic methanogen Methanococcus maripaludis S2 were investigated. Cu, Zn, acetate, and propionate may interfere directly and indirectly with the acetyl-CoA synthesis and biological CH4 production. Thus, these compounds can compromise or improve the performance of M. maripaludis, an organism which can be applied as biocatalyst in the carbon dioxide (CO2)-based biological CH4 production (CO2-BMP) process or of methanogenic organisms applied in anaerobic digestion. Results Here, we show that Cu concentration of 1.9 µmol L−1 reduced growth of M. maripaludis, whereas 4.4 and 6.3 µmol L−1 of Cu even further retarded biomass production. However, 1.0 mmol L−1 of Zn enhanced growth, but at Zn concentrations > 2.4 mmol L−1 no growth could be observed. When both, Cu and Zn, were supplemented to the medium, growth and CH4 production could even be observed at the highest tested concentration of Cu (6.3 µmol L−1). Hence, it seems that the addition of 1 mmol L−1 of Zn enhanced the ability of M. maripaludis to counteract the toxic effect of Cu. The physiological effect to rising concentrations of acetate (12.2, 60.9, 121.9 mmol L−1) and/or propionate (10.3, 52.0, 104.1 mmol L−1) was also investigated. When instead of acetate 10.3 mmol L−1 propionate was provided in the growth medium, M. maripaludis could grow without reduction of the specific growth rate (µ) or the specific CH4 productivity (qCH4). A combination of inorganic and/or organic compounds resulted in an increase of µ and qCH4 for Zn/Cu and Zn/acetate beyond the values that were observed if only the individual concentrations of Zn, Cu, acetate were used. Conclusions Our study sheds light on the physiological effect of VFAs and heavy metals on M. maripaludis. Differently from µ and qCH4, MER was not influenced by the presence of these compounds. This indicated that each of these compounds directly interacted with the C-fixation machinery of M. maripaludis. Until now, the uptake of VFAs other than acetate was not considered to enhance growth and CH4 production of methanogens. The finding of propionate uptake by M. maripaludis is important for the interpretation of VFA cycling in anaerobic microenvironments. Due to the importance of methanogens in natural and artificial anaerobic environments, our results help to enhance the understanding the physiological and biotechnological importance with respect to anaerobic digestion, anaerobic wastewater treatment, and CO2-BMP. Finally, we propose a possible mechanism for acetate uptake into M. maripaludis supported by in silico analyses.

Oil reservoirs contain microbial populations that are both autochthonously and allochthonously introduced by industrial development. These microbial populations are greatly influenced by external factors including, but not limited to, salinity and temperature. In this study, we used metagenomics to examine the microbial populations within five wells of the same hydrocarbon reservoir system in the Gulf of Mexico. These elevated salinity (149–181 ppt salinity, 4–5× salinity of seawater) reservoirs have limited taxonomic and functional microbial diversity dominated by methanogens, Halanaerobium and other Firmicutes lineages, and contained less abundant lineages such as Deltaproteobacteria. Metagenome assembled genomes (MAGs) were generated and analyzed from the various wells. Methanogen MAGs were closely related to Methanohalophilus euhalobius , a known methylotrophic methanogen from a high salinity oil environment. Based on metabolic reconstruction of genomes, the Halanaerobium perform glycine betaine fermentation, potentially produced by the methanogens. Industrial introduction of methanol to prevent methane hydrate formation to this environment is likely to be consumed by these methanogens. As such, this subsurface oil population may represent influences from industrial processes. [ABSTRACT FROM AUTHOR]

The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic archaeon Aeropyrum pernix. This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl coenzyme A, two recently discovered enzymes designated phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes, i.e., isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway-- from mevalonate to isopentenyl pyrophosphate--were isolated from the methanogenic archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced into an engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semianaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggests that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production. [ABSTRACT FROM AUTHOR]

The nucleotide binding protein 35 (Nbp35)/cytosolic Fe‐S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe‐S cluster assembly scaffold required for the maturation of Fe‐S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe‐S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal host Methanococcus maripaludis contains a [4Fe‐4S] cluster that can be transferred to a [4Fe‐4S] apoprotein. Deletion of mmp0704 from M. maripaludis does not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe‐4S] cluster transfer protein in methanogenic archaea. [ABSTRACT FROM AUTHOR]

This study delves into the critical role of microbial ecosystems in landfills, which are pivotal for handling municipal solid waste (MSW). Within these landfills, a complex interplay of several microorganisms (aerobic/anaerobic bacteria, archaea or methanotrophs), drives the conversion of complex substrates into simplified compounds and complete mineralization into the water, inorganic salts, and gases, including biofuel methane gas. These landfills have dominant biotic and abiotic environments where various bacterial, archaeal, and fungal groups evolve and interact to decompose substrate by enabling hydrolytic, fermentative, and methanogenic processes. Each landfill consists of diverse bio-geochemical environments with complex microbial populations, ranging from deeply underground anaerobic methanogenic systems to near-surface aerobic systems. These kinds of landfill generate leachates which in turn emerged as a significant risk to the surrounding because generated leachates are rich in toxic organic/inorganic components, heavy metals, minerals, ammonia and xenobiotics. In addition to this, microbial communities in a landfill ecosystem could not be accurately identified using lab microbial-culturing methods alone because most of the landfill's microorganisms cannot grow on a culture medium. Due to these reasons, research on landfills microbiome has flourished which has been characterized by a change from a culture-dependent approach to a more sophisticated use of molecular techniques like Sanger Sequencing and Next-Generation Sequencing (NGS). These sequencing techniques have completely revolutionized the identification and analysis of these diverse microbial communities. This review underscores the significance of microbial functions in waste decomposition, gas management, and heat control in landfills. It further explores how modern sequencing technologies have transformed our approach to studying these complex ecosystems, offering deeper insights into their taxonomic composition and functionality. • MSW in landfill endures either aerobic, anaerobic or semi aerobic biodegradation processes. • Anaerobic waste biodegradation comprises of fermentative, methanogenic etc. processes. • Advanced sequencing and bioinformatics are needed for understanding of the landfill microbiome. [ABSTRACT FROM AUTHOR]