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Methane emissions are mitigated by anaerobic methane-oxidizing archaea, including Methanoperedens. Some Methanoperedens host huge extrachromosomal genetic elements (ECEs) called Borgs that may modulate their activity, yet the broader diversity of Methanoperedens ECEs is understudied. Here we report small enigmatic linear ECEs, circular viruses and unclassified ECEs that are predicted to replicate within Methanoperedens. Linear ECEs have inverted terminal repeats, tandem repeats and coding patterns that are strongly reminiscent of Borgs, but they are only 52-145 kb in length. As they share proteins with Borgs and Methanoperedens, we refer to them as mini-Borgs. Mini-Borgs are genetically diverse and can be assigned to at least five family-level groups. We identify eight families of Methanoperedens viruses, some of which encode multi-haem cytochromes, and circular ECEs encoding transposon-associated TnpB genes with proximal population-heterogeneous CRISPR arrays. These ECEs exchange genetic information with each other and with Methanoperedens, probably impacting their archaeal host activity and evolution., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Methanogenic archaea are main contributors to methane emissions, and have a crucial role in carbon cycling and global warming. Until recently, methanogens were confined to Euryarchaeota, but metagenomic studies revealed the presence of genes encoding the methyl coenzyme M reductase complex in other archaeal clades 1-4 , thereby opening up the premise that methanogenesis is taxonomically more widespread. Nevertheless, laboratory cultivation of these non-euryarchaeal methanogens was lacking to corroborate their potential methanogenic ability and physiology. Here we report the isolation of a thermophilic archaeon LWZ-6 from an oil field. This archaeon belongs to the class Methanosuratincolia (originally affiliated with 'Candidatus Verstraetearchaeota') in the phylum Thermoproteota. Methanosuratincola petrocarbonis LWZ-6 is a strict hydrogen-dependent methylotrophic methanogen. Although previous metagenomic studies speculated on the fermentative potential of Methanosuratincolia members, strain LWZ-6 does not ferment sugars, peptides or amino acids. Its energy metabolism is linked only to methanogenesis, with methanol and monomethylamine as electron acceptors and hydrogen as an electron donor. Comparative (meta)genome analysis confirmed that hydrogen-dependent methylotrophic methanogenesis is a widespread trait among Methanosuratincolia. Our findings confirm that the diversity of methanogens expands beyond the classical Euryarchaeota and imply the importance of hydrogen-dependent methylotrophic methanogenesis in global methane emissions and carbon cycle., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

For several decades, the formation of microbial self-aggregates, known as granules, has been extensively documented in the context of anaerobic digestion. However, current understanding of the underlying microbial-associated mechanisms responsible for this phenomenon remains limited. This study examined morphological and biochemical changes associated with cell aggregation in model co-cultures of the syntrophic propionate oxidizing bacterium Syntrophobacterium fumaroxidans and hydrogenotrophic methanogens, Methanospirillum hungatei or Methanobacterium formicicum. Formerly, we observed that when syntrophs grow for long periods with methanogens, cultures tend to form aggregates visible to the eye. In this study, we maintained syntrophic co-cultures of S. fumaroxidans with either M. hungatei or M. formicicum for a year in a fed-batch growth mode to stimulate aggregation. Millimeter-scale aggregates were observed in both co-cultures within the first 5 months of cultivation. In addition, we detected quorum sensing molecules, specifically N-acyl homoserine lactones, in co-culture supernatants preceding the formation of macro-aggregates (with diameter of more than 20 μm). Comparative transcriptomics revealed higher expression of genes related to signal transduction, polysaccharide secretion and metal transporters in the late-aggregation state co-cultures, compared to the initial ones. This is the first study to report in detail both biochemical and physiological changes associated with the aggregate formation in syntrophic methanogenic co-cultures. KEYPOINTS: • Syntrophic co-cultures formed mm-scale aggregates within 5 months of fed-batch cultivation. • N-acyl homoserine lactones were detected during the formation of aggregates. • Aggregated co-cultures exhibited upregulated expression of adhesins- and polysaccharide-associated genes., (© 2024. The Author(s).)

In rice paddies, soil and plant-derived organic matter are degraded anaerobically to methane (CH 4 ), a powerful greenhouse gas. The highest rate of methane emission occurs during the reproductive stage of the plant when mostly dicarboxylic acids are exudated by the roots. The emission of methane at this stage depends largely on the cooperative interaction between dicarboxylic acid-fermenting bacteria and methanogenic archaea in the rhizosphere. The fermentation of tartrate, one of the major acids exudated, has been scarcely explored in rice paddy soils. In this work, we characterized an anaerobic consortium from rice paddy soil composed of four bacterial strains, whose principal member (LT8) can ferment tartrate, producing H 2 and acetate. Tartrate fermentation was accelerated by co-inoculation with a hydrogenotrophic methanogen. The assembled genome of LT8 possesses a Na + -dependent oxaloacetate decarboxylase and shows that this bacterium likely invests part of the H 2 produced to reduce NAD(P) + to assimilate C from tartrate. The phylogenetic analysis of the 16S rRNA gene, the genome-based classification as well as the average amino acid identity (AAI) indicated that LT8 belongs to a new genus within the Sporomusaceae family. LT8 shares a few common features with its closest relatives, for which tartrate degradation has not been described. LT8 is limited to a few environments but is more common in rice paddy soils, where it might contribute to methane emissions from root exudates.IMPORTANCEThis is the first report of the metabolic characterization of a new anaerobic bacterium able to degrade tartrate, a compound frequently associated with plants, but rare as a microbial metabolite. Tartrate fermentation by this bacterium can be coupled to methanogenesis in the rice rhizosphere where tartrate is mainly produced at the reproductive stage of the plant, when the maximum methane rate emission occurs. The interaction between secondary fermentative bacteria, such as LT8, and methanogens could represent a fundamental step in exploring mitigation strategies for methane emissions from rice fields. Possible strategies could include controlling the activity of these secondary fermentative bacteria or selecting plants whose exudates are more difficult to ferment., Competing Interests: The authors declare no conflict of interest.

Methanogenesis is a critical process in the carbon cycle that is applied industrially in anaerobic digestion and biogas production. While naturally occurring in diverse environments, methanogenesis requires anaerobic and reduced conditions, although varying degrees of oxygen tolerance have been described. Microaeration is suggested as the next step to increase methane production and improve hydrolysis in digestion processes; therefore, a deeper understanding of the methanogenic response to oxygen stress is needed. To explore the drivers of oxygen tolerance in methanogenesis, two parallel enrichments were performed under the addition of H 2 /CO 2 in an environment without reducing agents and in a redox-buffered environment by adding redox mediator 9,10-anthraquinone-2,7-disulfonate disodium. The cellular response to oxidative conditions is mapped using proteomic analysis. The resulting community showed remarkable tolerance to high-redox environments and was unperturbed in its methane production. Next to the expression of pathways to mitigate reactive oxygen species, the higher redox potential environment showed an increased presence of selenocysteine and selenium-associated pathways. By including sulfur-to-selenium mass shifts in a proteomic database search, we provide the first evidence of the dynamic and large-scale incorporation of selenocysteine as a response to oxidative stress in hydrogenotrophic methanogenesis and the presence of a dynamic selenoproteome.

Microplastics (MPs) and Perfluorooctane sulfonate (PFOS) are two hard-biodegradable pollutants widely existing in the waste streams treated by anaerobic digestion. However, their synergistic effect on methanogenic metabolism is still unknown. This study investigated the impact of polyethylene terephthalate (PET) MPs alone and co-existing with PFOS on CO 2 conversion to CH 4 in a thermophilic biogas upgrading system. The results showed that either PET MPs addition alone or coexisting with PFOS improved the ultimate CH 4 percentage and increased CO 2 utilization rate. When Fe 0 was added into the reactors with PET to enhance the interspecies electron transfer, a potential defluorination was observed with a defluorination rate of 15.88 ± 1.53%. Exposure of the reactor to PFOS of 300 μg/L could change the methanogenic pathway, resulting in a newly emerged Methanomassiliicoccus with dominance of 16%. Furthermore, under the exposure of PFOS, the number of predicted genes regulating enzymes in methanogenic steps from CO 2 increased. These results suggest that the co-existence of PET MPs and PFOS will not inhibit the activity of hydrotrophic methanogenes, and a portion of PFOS may be biodegraded during the methanogenesis under Fe 0 regulation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests XIN KONG reports financial support was provided by National Natural Science Foundation of China. XIN KONG reports financial support was provided by Shanxi Province Department of Education., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In situ anoxic bioremediation is a sustainable technology to remediate PAHs contaminated soils. However, the limited degradation rate of PAHs under anoxic conditions has become the primary bottleneck hindering the application of this technology. In this study, coupled low-temperature thermally treatment (<50 °C) and EA biostimulation was used to enhance PAH removal. Anoxic biodegradation of PAHs in soil was explored in microcosms in the absence and presence of added EAs at 3 temperatures (15 °C, 30 °C, and 45 °C). The influence of temperature, EA, and their interaction on the removal of PAHs were identified. A PAH degradation model based on PLSR analysis identified the importance and the positive/negative role of parameters on PAH removal. Soil archaeal and bacterial communities showed similar succession patterns, the impact of temperature was greater than that of EA. Soil microbial community and function were more influenced by temperature than EAs. Close and frequent interactions were observed among soil bacteria, archaea, PAH-degrading genes and methanogenic genes. A total of 15 bacterial OTUs, 1 PAH-degrading gene and 2 methanogenic genes were identified as keystones in the network. Coupled low-temperature thermally treatment and EA stimulation resulted in higher PAH removal efficiencies than EA stimulation alone and low-temperature thermally treatment alone., 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 © 2023 Elsevier B.V. All rights reserved.)

Nanoplastics (NPs) have attracted increasing attention within terrestrial ecosystems. However, our understanding of their impacts on the intricate anaerobic methanogenesis processes occurring in paddy soils microbial communities remains limited with respect to nanoplastics shape, function, and metabolic effects. Herein, we explored the effects of polystyrene nanoplastics (PS-NPs) and microplastics (PS-MPs) on anaerobic methanogenesis in a typical paddy soil. The results show that PS-NPs delayed methane production and the time to reach peak acetate content in incubation process of paddy soils, and the methanogenic rate increased rapidly after 13 days, with a maximum increase of 87.97%. However, PS-MPs had no marked effect on CH 4 , CO 2 and acetate production. In addition, PS-NPs affected soil physicochemical properties by reducing pH and increasing electrical conductivity. Acetoclastic methanogens were enriched and the relative abundance of the genes ackA, pta, ACSS, cdhC, cdhD and cdhE in the acetoclastic pathways were significantly increased under PS-NPs exposure. In addition, PS-MPs had significant effect on the microbial community structure but no effect on methanogenic pathways of the paddy soils. This study provides important insights into the response of key microorganisms, functional genes and methanogenesis pathways to NPs during anaerobic methanogenesis in paddy soils., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Acetate is a major intermediate in the anaerobic digestion of organic waste to produce CH 4 . In methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or syntrophic degradation by acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in the isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB and the mechanisms of their interactions with methanogenic partners are not fully characterized. In this study, the in situ activity and metabolic characteristics of potential SAOB and their interactions with methanogens were elucidated through metagenomics and metatranscriptomics. In addition to the reported SAOB classified in the genera Tepidanaerobacter , Desulfotomaculum , and Thermodesulfovibrio , we identified a number of potential SAOB that are affiliated with Clostridia , Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. The potential SAOB possessing the glycine-mediated acetate oxidation pathway dominates SAOB communities. Moreover, formate appeared to be the main product of the acetate degradation by the most active potential SAOB. We identified the methanogen partner of these potential SAOB in the acetate-fed chemostat as Methanosarcina thermophila . The dominated potential SAOB in each chemostat had similar metabolic characteristics, even though they were in different fatty-acid-fed chemostats. These novel syntrophic lineages are prevalent and may play critical roles in thermophilic methanogenic reactors. This study expands our understanding of the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders and presents novel insights into how they interact with methanogens.IMPORTANCECombining reactor operation with omics provides insights into novel uncultured syntrophic acetate degraders and how they perform in thermophilic anaerobic digesters. This improves our understanding of syntrophic acetate degradation and contributes to the background knowledge necessary to better control and optimize anaerobic digestion processes., Competing Interests: The authors declare no conflict of interest.

Excessive ammonia stresses anaerobic digestion (AD) significantly. Although there has been progress in understanding AD under ammonia exposure, investigations on AD liberated from ammonia exposure are limited. Here, the recovery capability of AD from ammonia stress was evaluated, by examining specific methanogenic activity, energy-conserving capability, microbial community succession, and metabolic pathway reconstruction. The findings demonstrated that ammonia stress relief resulted in < 50% methane recovery, with propionate conversion identified as the critical impediment to AD reactivation. Energy generation could not recovered either. Efforts to mitigate ammonia stress failed to restore acetoclastic methanogens, e.g., Methanothrix soehngenii, and proved futile in awakening propionate oxidizers, e.g., Desulfobulbus. Interestingly, a symbiotic metabolism emerged, prevailing in stress-relieved AD due to its energy-conserving advantage. This study underscores the importance of targeted interventions, including stimulating acetoclastic methanogenesis, propionate oxidation, and energy generation, as priorities for AD recovery following ammonia stress, rather than focusing solely on ammonia level management., 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 © 2023. Published by Elsevier Ltd.)

While wetlands are major sources of biogenic methane (CH 4 ), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH 4 emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site's methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; for Methanosarcinales and Methanobacteriales MAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while for Methanomassiliicoccales , they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats.IMPORTANCEWetlands are the biggest natural source of atmospheric methane (CH 4 ) emissions, yet we have an incomplete understanding of the suite of microbial metabolism that results in CH 4 formation. Specifically, methanogenesis from methylated compounds is excluded from all ecosystem models used to predict wetland contributions to the global CH 4 budget. Though recent studies have shown methylotrophic methanogenesis to be active across wetlands, the broad climatic importance of the metabolism remains critically understudied. Further, some methylotrophic bacteria are known to produce methanogenic by-products like acetate, increasing the complexity of the microbial methylotrophic metabolic network. Prior studies of Stordalen Mire have suggested that methylotrophic methanogenesis is irrelevant in situ and have not emphasized the bacterial capacity for metabolism, both of which we countered in this study. The importance of our findings lies in the significant advancement toward unraveling the broader impact of methylotrophs in wetland methanogenesis and, consequently, their contribution to the terrestrial global carbon cycle., Competing Interests: The authors declare no conflict of interest.

Ruminants are essential for global food security, but these are major sources of the greenhouse gas methane. Methane yield is controlled by the cycling of molecular hydrogen (H2), which is produced during carbohydrate fermentation and is consumed by methanogenic, acetogenic, and respiratory microorganisms. However, we lack a holistic understanding of the mediators and pathways of H2 metabolism and how this varies between ruminants with different methane-emitting phenotypes. Here, we used metagenomic, metatranscriptomic, metabolomics, and biochemical approaches to compare H2 cycling and reductant disposal pathways between low-methane-emitting Holstein and high-methane-emitting Jersey dairy cattle. The Holstein rumen microbiota had a greater capacity for reductant disposal via electron transfer for amino acid synthesis and propionate production, catalyzed by enzymes such as glutamate synthase and lactate dehydrogenase, and expressed uptake [NiFe]-hydrogenases to use H2 to support sulfate and nitrate respiration, leading to enhanced coupling of H2 cycling with less expelled methane. The Jersey rumen microbiome had a greater proportion of reductant disposal via H2 production catalyzed by fermentative hydrogenases encoded by Clostridia, with H2 mainly taken up through methanogenesis via methanogenic [NiFe]-hydrogenases and acetogenesis via [FeFe]-hydrogenases, resulting in enhanced methane and acetate production. Such enhancement of electron incorporation for metabolite synthesis with reduced methanogenesis was further supported by two in vitro measurements of microbiome activities, metabolites, and public global microbiome data of low- and high-methane-emitting beef cattle and sheep. Overall, this study highlights the importance of promoting alternative H2 consumption and reductant disposal pathways for synthesizing host-beneficial metabolites and reducing methane production in ruminants., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The origin of methanogenesis can be traced to the common ancestor of non-DPANN archaea, whereas haloarchaea (or Halobacteria) are believed to have evolved from a methanogenic ancestor through multiple evolutionary events. However, due to the accelerated evolution and compositional bias of proteins adapting to hypersaline habitats, Halobacteria exhibit substantial evolutionary divergence from methanogens, and the identification of the closest methanogen (either Methanonatronarchaeia or other taxa) to Halobacteria remains a subject of debate. Here, we obtained five metagenome-assembled genomes with high completeness from soda-saline lakes on the Ordos Plateau in Inner Mongolia, China, and we proposed the name Candidatus Ordosarchaeia for this novel class. Phylogenetic analyses revealed that Ca. Ordosarchaeia is firmly positioned near the median position between the Methanonatronarchaeia and Halobacteria-Hikarchaeia lineages. Functional predictions supported the transitional status of Ca. Ordosarchaeia with the metabolic potential of nonmethanogenic and aerobic chemoheterotrophy, as did remnants of the gene sequences of methylamine/dimethylamine/trimethylamine metabolism and coenzyme M biosynthesis. Based on the similarity of the methyl-coenzyme M reductase genes mcrBGADC in Methanonatronarchaeia with the phylogenetically distant methanogens, an alternative evolutionary scenario is proposed, in which Methanonatronarchaeia, Ca. Ordosarchaeia, Ca. Hikarchaeia, and Halobacteria share a common ancestor that initially lost mcr genes. However, certain members of Methanonatronarchaeia subsequently acquired mcr genes through horizontal gene transfer from distantly related methanogens. This hypothesis is supported by amalgamated likelihood estimation, phylogenetic analysis, and gene arrangement patterns. Altogether, Ca. Ordosarchaeia genomes clarify the sisterhood of Methanonatronarchaeia with Halobacteria and provide new insights into the evolution from methanogens to haloarchaea., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Using a batch thermophilic anaerobic system established with 60 mL serum bottles, the mechanism on how microbial enrichments obtained from magnetite-amended paddy soil via repeated batch cultivation affected methane production from acetate was investigated. Magnetite-amended enrichments (MAEs) can improve the methane production rate rather than the methane yield. Compared with magnetite-unamended enrichments, the methane production rate in MAE was improved by 50%, concomitant with the pronounced electrochemical response, high electron transfer capacity, and fast acetate degradation. The promoting effects might be ascribed to direct interspecies electron transfer facilitated by magnetite, where magnetite might function as electron conduits to link the acetate oxidizers (Anaerolineaceae and Peptococcaceae) with methanogens (Methanosarcinaceae). The findings demonstrated the potential application of MAE for boosting methanogenic performance during thermophilic anaerobic digestion.

In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane through obligate cross-feeding interactions between SAO bacteria (SAOB) and methanogenic archaea. The SAO pathway is particularly important in engineered environments such as anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of SAOB to the stability of the AD process, little is known about their in situ physiologies due to typically low biomass yields and resistance to isolation. Here, we performed a long-term (300-day) continuous enrichment of a thermophilic (55 °C) SAO community from a municipal AD system using acetate as the sole carbon source. Over 80% of the enriched bioreactor metagenome belonged to a three-member consortium, including an acetate-oxidizing bacterium affiliated with DTU068 encoding for carbon dioxide, hydrogen, and formate production, along with two methanogenic archaea affiliated with Methanothermobacter&#95;A. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. This effort revealed that the two Methanothermobacter&#95;A species differed in their preferred electron donors, with one possessing the ability to grow on formate and the other only consuming hydrogen. A thermodynamic analysis suggested that the presence of the formate-consuming methanogen broadened the environmental conditions where ATP production from SAO was favorable. Collectively, these results highlight how flexibility in electron partitioning during SAO likely governs community structure and fitness through thermodynamic-driven mutualism, shedding valuable insights into the metabolic underpinnings of this key functional group within methanogenic ecosystems., (© 2023. The Author(s).)

Manure management is a significant source of global methane emissions, and there is an increased interest in understanding and predicting emissions. The hydrolysis rate of manure organic matter is critical for understanding and predicting methane emissions. We estimated hydrolysis rate constants of crude protein, fibers, and lipids and used the Arrhenius equation to describe its dependency on temperature. Simultaneously, measurements of methane emission, 13/12 C isotope ratios, and methanogen community were conducted. This was achieved by incubating fresh pig manure without inoculum at 10°C, 15°C, 20°C, and 25°C for 85 days in a lab-scale setup. Hydrolysis of hemicellulose and cellulose increased more with temperature than crude protein, but still, hydrolysis rate of crude protein was highest at all temperatures. Results suggested that crude protein consisted of multiple substrate groups displaying large differences in degradability. Lipids and lignin were not hydrolyzed during incubations. Cumulative methane emissions were 7.13 ± 2.69, 24.6 ± 8.00, 66.7 ± 4.8, and 105.7 ± 7.14 g CH4 kg VS -1 at 10°C, 15°C, 20°C, and 25°C, respectively, and methanogenic community shifted from Methanosphaera toward Methanocorpusculum over time and more quickly at higher temperatures. This study provides important parameter estimates and dependencies on temperature, which is important in mechanistic methane emission models. Further work should focus on characterizing quickly degradable substrate pools in the manure organic matter as they might be the main carbon source of methane emission from manure management., (© 2023 The Authors. Journal of Environmental Quality published by Wiley Periodicals LLC on behalf of American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.)

Methanogenic archaea are major producers of methane, a potent greenhouse gas and biofuel, and are widespread in diverse environments, including the animal gut. The ecophysiology of methanogens is likely impacted by viruses, which remain, however, largely uncharacterized. Here we carried out a global investigation of viruses associated with all current diversity of methanogens by assembling an extensive CRISPR database consisting of 156,000 spacers. We report 282 high-quality (pro)viral and 205 virus-like/plasmid sequences assigned to hosts belonging to ten main orders of methanogenic archaea. Viruses of methanogens can be classified into 87 families, underscoring a still largely undiscovered genetic diversity. Viruses infecting gut-associated archaea provide evidence of convergence in adaptation with viruses infecting gut-associated bacteria. These viruses contain a large repertoire of lysin proteins that cleave archaeal pseudomurein and are enriched in glycan-binding domains (Ig-like/Flg&#95;new) and diversity-generating retroelements. The characterization of this vast repertoire of viruses paves the way towards a better understanding of their role in regulating methanogen communities globally, as well as the development of much-needed genetic tools., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Propionate is a key intermediate in anaerobic digestion processes and often accumulates in association with perturbations, such as elevated levels of ammonia. Under such conditions, syntrophic ammonia-tolerant microorganisms play a key role in propionate degradation. Despite their importance, little is known about these syntrophic microorganisms and their cross-species interactions. Here, we present metagenomes and metatranscriptomic data for novel thermophilic and ammonia-tolerant syntrophic bacteria and the partner methanogens enriched in propionate-fed reactors. A metagenome for a novel bacterium for which we propose the provisional name 'Candidatus Thermosyntrophopropionicum ammoniitolerans' was recovered, together with mapping of its highly expressed methylmalonyl-CoA pathway for syntrophic propionate degradation. Acetate was degraded by a novel thermophilic syntrophic acetate-oxidising candidate bacterium. Electron removal associated with syntrophic propionate and acetate oxidation was mediated by the hydrogen/formate-utilising methanogens Methanoculleus sp. and Methanothermobacter sp., with the latter observed to be critical for efficient propionate degradation. Similar dependence on Methanothermobacter was not seen for acetate degradation. Expression-based analyses indicated use of both H 2 and formate for electron transfer, including cross-species reciprocation with sulphuric compounds and microbial nanotube-mediated interspecies interactions. Batch cultivation demonstrated degradation rates of up to 0.16 g propionate L -1 day -1 at hydrogen partial pressure 4-30 Pa and available energy was around -20 mol -1 propionate. These observations outline the multiple syntrophic interactions required for propionate oxidation and represent a first step in increasing knowledge of acid accumulation in high-ammonia biogas production systems., (© 2023. The Author(s).)

Understanding the source of methane (CH 4 ) is of great significance for improving the anaerobic fermentation efficiency in bioengineering, and for mitigating the emission potential of natural ecosystems. Microbes involved in the process named direct interspecies electron transfer coupling with CO 2 reduction, i.e., electrons released from electroactive bacteria to reduce CO 2 into CH 4 , have attracted considerable attention for wastewater treatment in the past decade. However, how the synergistic effect of microbiota contributes to this anaerobic carbon metabolism accompanied by CH 4 production still remains poorly understood, especial for wastewater with antibiotic exposure. Results show that enhancing lower-abundant acetoclastic methanogens and acetogenic bacteria, rather than electroactive bacteria, contributed to CH 4 production, based on a metagenome-assembled genomes network analysis. Natural and artificial isotope tracing of CH 4 further confirmed that CH 4 mainly originated from acetoclastic methanogenesis. These findings reveal the contribution of direct acetate cleavage (acetoclastic methanogenesis) and provide insightsfor further regulation of methanogenic strategies., 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 © 2023 Elsevier Ltd. All rights reserved.)

Hydrogen (H 2 ) is the primary electron donor for methane formation in ruminants, but the H 2 -producing organisms involved are largely uncharacterized. This work integrated studies of microbial physiology and genomics to characterize rumen bacterial isolate NK3A20 of the family Lachnospiraceae . Isolate NK3A20 was the first recognized isolate of the NK3A20 group, which is among the ten most abundant bacterial genera in 16S rRNA gene surveys of rumen microbiota. NK3A20 produced acetate, butyrate, H 2 , and formate from glucose. The end product ratios varied when grown with different substrates and at different H 2 partial pressures. NK3A20 produced butyrate as a major product using glucose or under high H 2 partial pressures and switched to mainly acetate in the presence of galacturonic acid (an oxidized sugar) or in coculture with a methanogen. Growth with galacturonic acid was faster at elevated H 2 concentrations, while elevated H 2 slowed growth with glucose. Genome analyses revealed the presence of multiple hydrogenases including a membrane-bound Ech hydrogenase, an electron bifurcating butyryl-CoA dehydrogenase (Bcd-Etf), and an Rnf complex that may be involved in modulating the observed metabolic pathway changes, providing insight into H 2 formation in the rumen. IMPORTANCE The genus-level NK3A20 group is one of the ten most abundant genera of rumen bacteria. Like most of the rumen bacteria that produce the hydrogen that is converted to methane in the rumen, it is understudied, without any previously characterized isolates. We investigated isolate NK3A20, a cultured member of this genus, and showed that it modulates hydrogen production in response to its growth substrates and the hydrogen concentration in its environment. Low-hydrogen concentrations stimulated hydrogen formation, while high concentrations inhibited its formation and shifted the fermentation to more reduced organic acid products. We found that growth on uronic acids, components of certain plant polymers, resulted in low hydrogen yields compared to glucose, which could aid in the selection of low-methane feeds. A better understanding of the major genera that produce hydrogen in the rumen is part of developing strategies to mitigate biogenic methane emitted by livestock agriculture., Competing Interests: The authors declare no conflict of interest.

It is known that the presence of sulfate decreases the methane yield in the anaerobic digestion systems. Sulfate-reducing bacteria can convert sulfate to hydrogen sulfide competing with methanogens for substrates such as H 2 and acetate. The present work aims to elucidate the microbial interactions in biogas production and assess the effectiveness of electron-conductive materials in restoring methane production after exposure to high sulfate concentrations. The addition of magnetite led to a higher methane content in the biogas and a sharp decrease in the level of hydrogen sulfide, indicating its beneficial effects. Furthermore, the rate of volatile fatty acid consumption increased, especially for butyrate, propionate, and acetate. Genome-centric metagenomics was performed to explore the main microbial interactions. The interaction between methanogens and sulfate-reducing bacteria was found to be both competitive and cooperative, depending on the methanogenic class. Microbial species assigned to the Methanosarcina genus increased in relative abundance after magnetite addition together with the butyrate oxidizing syntrophic partners, in particular belonging to the Syntrophomonas genus. Additionally, Ruminococcus sp. DTU98 and other species assigned to the Chloroflexi phylum were positively correlated to the presence of sulfate-reducing bacteria, suggesting DIET-based interactions. In conclusion, this study provides new insights into the application of magnetite to enhance the anaerobic digestion performance by removing hydrogen sulfide, fostering DIET-based syntrophic microbial interactions, and unraveling the intricate interplay of competitive and cooperative interactions between methanogens and sulfate-reducing bacteria, influenced by the specific methanogenic group.

Recent studies in aquatic environments have indicated that microbial methane production is not limited to strictly anoxic conditions and is widespread in the oxic water column. Based on recent investigations proposing linkage between the microbial turnover of methylphosphonate (MPn) and the widespread methane oversaturation in surface waters, we conducted an MPn/ 13 C-MPn tracer approach that combines liquid chromatography-mass spectrometry and gas chromatography-stable isotope ratio mass spectrometry to assess concentrations of the MPn tracer and its contribution to oxic methane formation. In our study, conducted during summer 2020 in the Baltic Sea, we show that MPn is a potent methanogenic substrate in the surface water. However, we found that MPn was produced within the surface and subthermocline water bodies and that its turnover was not limited to the phosphorus-stressed and cyanobacteria-rich surface water. However, our study revealed that most of the MPn was probably degraded via alternative pathways, not releasing methane. Our assessment indicates that the contribution of the MPn degradation pathway only contributed marginally to oxic methane production at the study site in the Baltic Sea and that a variety of methanogenic pathways are probably responsible for the surface-water methane enrichments.

Large amounts of carbon sequestered in permafrost on the Tibetan Plateau (TP) are becoming vulnerable to microbial decomposition in a warming world. However, knowledge about how the responsible microbial community responds to warming-induced permafrost thaw on the TP is still limited. This study aimed to conduct a comprehensive comparison of the microbial communities and their functional potential in the active layer of thawing permafrost on the TP. We found that the microbial communities were diverse and varied across soil profiles. The microbial diversity declined and the relative abundance of Chloroflexi, Bacteroidetes, Euryarchaeota, and Bathyarchaeota significantly increased with permafrost thawing. Moreover, warming reduced the similarity and stability of active layer microbial communities. The high-throughput qPCR results showed that the abundance of functional genes involved in liable carbon degradation and methanogenesis increased with permafrost thawing. Notably, the significantly increased mcrA gene abundance and the higher methanogens to methanotrophs ratio implied enhanced methanogenic activities during permafrost thawing. Overall, the composition and functional potentials of the active layer microbial community in the Tibetan permafrost region are susceptible to warming. These changes in the responsible microbial community may accelerate carbon degradation, particularly in the methane releases from alpine permafrost ecosystems on the TP., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Methane production by microbial fermentation of municipal waste is a challenge for better yield processes. This work describes the characterization of a hydrogenotrophic methanogen microbial community used in a bioaugmentation procedure to improve the methane yield in a thermophilic anaerobic process, digesting the organic fraction of municipal solid waste. The performance of the bioaugmentation was assessed in terms of methane production and changes in the microbial community structure. The results showed that bioaugmentation slightly improved the cumulative methane yield (+ 4%) in comparison to the control, and its use led to an acceleration of the methanogenesis stage. We observed associated significant changes in the relative abundance of taxa and their interactions, using high throughput DNA sequencing of V3-16S rRNA gene libraries, where the abundance of the archaeal hydrogenotrophic genus Methanoculleus (class Methanomicrobia, phylum Euryarchaeota) and the bacterial order MBA08 (class Clostridia, phylum Firmicutes) were dominant. The relevant predicted metabolic pathways agreed with substrate degradation and the anaerobic methanogenic process. The purpose of the study was to evaluate the effect of the addition of hydrogenotrophic methanogens in the generation of methane, while treating organic waste through anaerobic digestion., (© 2023. The Author(s).)

Anaerobic digestion (AD) is a promising method to treat organic matter. However, AD performance was limited by the inefficient electron transfer and metabolism imbalance between acid-producing bacteria and methanogens. In this study, a novel binary electroactive material (Fe 3 O 4 @biochar) with pseudocapacitance (1.4 F/g) and conductance (10.2 μS/cm) was exploited to store-release electrons as well as enhance the direct electron transfer between acid-producing bacteria and methanogens during the AD process. The mechanism of pseudocapacitance/conductance on mediating interspecies electron transfer was deeply studied at each stage of AD. In the hydrolysis acidification stage, the pseudocapacitance of Fe 3 O 4 @biochar acting as electron acceptors proceeded NADH/NAD + transformation of bacteria to promote ATP synthesis by 21% which supported energy for organics decomposition. In the methanogenesis stage, the conductance of Fe 3 O 4 @biochar helped the microbes establish direct interspecies electron transfer (DIET) to increase the coenzyme F 420 content by 66% and then improve methane production by 13%. In the complete AD experiment, electrons generated from acid-producing bacteria were rapidly transported to methanogens via conductors. Excess electrons were buffered by the pseudocapacitor and then gradually released to methanogens which alleviated the drastic drop in pH. These findings provided a strategy to enhance the electron transfer in anaerobic treatment as well as guided the design of electroactive materials.

Bathyarchaeota, known as key participants of global elements cycling, is highly abundant and diverse in the sedimentary environments. Bathyarchaeota has been the research spotlight on sedimentary microbiology; however, its distribution in arable soils is far from understanding. Paddy soil is a habitat similar to freshwater sediments, while the distribution and composition of Bathyarchaeota in paddy soils have largely been overlooked. In this study, we collected 342 in situ paddy soil sequencing data worldwide to illuminate the distribution patterns of Bathyarchaeota and explore their potential ecological functions in paddy soils. The results showed that Bathyarchaeota is the dominant archaeal lineage, and Bathy-6 is the most predominant subgroup in paddy soils. Based on random forest analysis and construction of a multivariate regression tree, the mean annual precipitation and mean annual temperature are identified as the factors significantly influencing the abundance and composition of Bathyarchaeota in paddy soils. Bathy-6 was abundant in temperate environments, while other subgroups were more abundant in sites with higher rainfall. There are highly frequent associations between Bathyarchaeota and methanogens and ammonia-oxidizing archaea. The interactions between Bathyarchaeota and microorganisms involved in carbon and nitrogen metabolism imply a potential syntrophy between these microorganisms, suggesting that members of Bathyarchaeota could be important participants of geochemical cycle in paddy soils. These results shed light on the ecological lifestyle of Bathyarchaeota in paddy soils, and provide some baseline for further understanding Bathyarchaeota in arable soils. IMPORTANCE Bathyarchaeota, the dominant archaeal lineage in sedimentary environments, has been the spotlight of microbial research due to its vital role in carbon cycling. Although Bathyarchaeota has been also detected in paddy soils worldwide, its distribution in this environment has not yet been investigated. In this study, we conducted a global scale meta-analysis and found that Bathyarchaeota is also the dominant archaeal lineage in paddy soils with significant regional abundance differences. Bathy-6 is the most predominant subgroup in paddy soils, which differs from sediments. Furthermore, Bathyarchaeota are highly associated with methanogens and ammonia-oxidizing archaea, suggesting that they may be involved in the carbon and nitrogen cycle in paddy soil. These interactions provide insight into the ecological functions of Bathyarchaeota in paddy soils, which will be the foundation of future studies regarding the geochemical cycle in arable soils and global climate change., Competing Interests: The authors declare no conflict of interest.

The control of pH is effective for inhibiting methanogenesis in the chain elongation fermentation (CEF) system. However, obscure conclusions exist especially with regard to the underlying mechanism. This study comprehensively explored the responses of methanogenesis in granular sludge at various pH levels, ranging from 4.0 to 10.0, from multiple aspects including methane production, methanogenesis pathway, microbial community structure, energy metabolism and electron transport. Results demonstrated that compared with that at pH 7.0, pH at 4.0, 5.5, 8.5 and 10.0 triggered a 100%, 71.7%, 23.8% and 92.1% suppression on methanogenesis by the end of 3 cycles lasting 21 days. This might be explained by the remarkably inhibited metabolic pathways and intracellular regulations. To be more specific, extreme pH conditions decreased the abundance of the acetoclastic methanogens. However, obligate hydrogenotrophic and facultative acetolactic/hydrogenotrophic methanogens were significantly enriched by 16.9%-19.5 fold. pH stress reduced the gene abundance and/or activity of most enzymes involved in methanogenesis such as acetate kinase (by 81.1%-93.1%), formylmethanofuran dehydrogenase (by 10.9%-54.0%) and tetrahydromethanopterin S-methyltransferase (by 9.3%-41.5%). Additionally, pH stress suppressed electron transport via improper electron carriers and decreased electron amount as evidenced by 46.3%-70.4% reduced coenzyme F 420 content and diminished abundance of CO dehydrogenase (by 15.5%-70.5%) and NADH:ubiquinone reductase (by 20.2%-94.5%). pH stress also regulated energy metabolism with inhibited ATP synthesis (e.g., ATP citrate synthase level reduced by 20.1%-95.3%). Interestingly, the protein and carbohydrate content secreted in EPS failed to show consistent responses to acidic and alkaline conditions. Specifically, when compared with pH 7.0, the acidic condition remarkably reduced the levels of total EPS and EPS protein while both levels were enhanced in the alkaline condition. However, the EPS carbohydrate content at pH 4.0 and 10.0 both decreased. This study is expected to promote the understanding of the pH control-induced methanogenesis inhibition in the CEF system., 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 © 2023 Elsevier B.V. All rights reserved.)

Co-landfill of incineration slag and municipal solid waste (MSW) is a main method for disposal of slag, and it has the potential of promoting methane (CH 4 ) production and accelerating landfill stabilization. Four simulated MSW landfill columns loaded with different amount of slag (A, 0%; B, 5%; C, 10%; D, 20%) were established, and the CH 4 production characteristics and methanogenic mechanisms were investigated. The maximum CH 4 concentration in columns A, B, C and D was 10.8%, 23.3%, 36.3% and 34.3%, respectively. Leachate pH and refuse pH were positively correlated with CH 4 concentration. Methanosarcina was the dominant genus with abundance of 35.1%∼75.2% and it was positively correlated with CH 4 concentration. CO 2 -reducing and acetoclastic methanogenesis were the main types of methanogenesis pathway, and the methanogenesis functional abundance increased with slag proportion during stable methanogenesis process. This research can help understanding the impact of slag on CH 4 production characteristics and microbiological mechanisms in landfills., 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 © 2023 Elsevier Ltd. All rights reserved.)

Investigating the effect of butyric acid concentration on anaerobic digestion systems in complex systems is important for the efficient degradation of butyric acid and improving the efficiency of anaerobic digestion. In this study, different loadings of butyric acid with 2.8, 3.2, and 3.6 g/(L·d) were added to the anaerobic reactor. At a high organic loading rate of 3.6 g/(L·d), methane was efficiently produced with VBP (Volumetric Biogas Production) of 1.50 L/(L·d) and biogas content between 65% and 75%. VFAs concentration remained below 2000 mg/L. Metagenome sequencing revealed changes in the functional flora within different stages. Methanosarcina, Syntrophomonas, and Lentimicrobium were the main and functional microorganisms. That the relative abundance of methanogens exceeded 35% and methanogenic metabolic pathways were increased indicated the methanogenic capacity of the system significantly improved. The presence of a large number of hydrolytic acid-producing bacteria also indicated the importance of the hydrolytic acid-producing stage in the system., 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 © 2023 Elsevier Ltd. All rights reserved.)

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria , is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria , previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade " Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that " Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of " Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate " Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that " Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other " Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny " Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis., Competing Interests: The authors declare no conflict of interest.

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 possess diverse metabolic characteristics and are an ecologically and biotechnologically important group of anaerobic microorganisms. Although the scientific and biotechnological value of methanogens is evident with regard to their methane-producing physiology, little is known about their amino acid excretion, and virtually nothing is known about the lipidome at different substrate concentrations and temperatures on a quantitative comparative basis. Here, we present the lipidome and a comprehensive quantitative analysis of proteinogenic amino acid excretion as well as methane, water, and biomass production of the three autotrophic, hydrogenotrophic methanogens Methanothermobacter marburgensis, Methanothermococcus okinawensis, and Methanocaldococcus villosus under varying temperatures and nutrient supplies. The patterns and rates of production of excreted amino acids and the lipidome are unique for each tested methanogen and can be modulated by varying the incubation temperature and substrate concentration, respectively. Furthermore, the temperature had a significant influence on the lipidomes of the different archaea. The water production rate was much higher, as anticipated from the rate of methane production for all studied methanogens. Our results demonstrate the need for quantitative comparative physiological studies connecting intracellular and extracellular constraints of organisms to holistically investigate microbial responses to environmental conditions. IMPORTANCE Biological methane production by methanogenic archaea has been well studied for biotechnological purposes. This study reveals that methanogenic archaea actively modulate their lipid inventory and proteinogenic amino acid excretion pattern in response to environmental changes and the possible utilization of methanogenic archaea as microbial cell factories for the targeted production of lipids and amino acids.

Complex interactions exist among microorganisms in a community to carry out ecological processes and adapt to changing environments. Here, we constructed a quad-culture consisting of a cellulolytic bacterium (Ruminiclostridium cellulolyticum), a hydrogenotrophic methanogen (Methanospirillum hungatei), an acetoclastic methanogen (Methanosaeta concilii), and a sulfate-reducing bacterium (Desulfovibrio vulgaris). The four microorganisms in the quad-culture cooperated via cross-feeding to produce methane using cellulose as the only carbon source and electron donor. The community metabolism of the quad-culture was compared with those of the R. cellulolyticum-containing tri-cultures, bi-cultures, and mono-culture. Methane production was higher in the quad-culture than the sum of the increases in the tri-cultures, which was attributed to a positive synergy of four species. In contrast, cellulose degradation by the quad-culture was lower than the additive effects of the tri-cultures which represented a negative synergy. The community metabolism of the quad-culture was compared between a control condition and a treatment condition with sulfate addition using metaproteomics and metabolic profiling. Sulfate addition enhanced sulfate reduction and decreased methane and CO 2 productions. The cross-feeding fluxes in the quad-culture in the two conditions were modeled using a community stoichiometric model. Sulfate addition strengthened metabolic handoffs from R. cellulolyticum to M. concilii and D. vulgaris and intensified substrate competition between M. hungatei and D. vulgaris. Overall, this study uncovered emergent properties of higher-order microbial interactions using a four-species synthetic community. IMPORTANCE A synthetic community was designed using four microbial species that together performed distinct key metabolic processes in the anaerobic degradation of cellulose to methane and CO 2 . The microorganisms exhibited expected interactions, such as cross-feeding of acetate from a cellulolytic bacterium to an acetoclastic methanogen and competition of H 2 between a sulfate reducing bacterium and a hydrogenotrophic methanogen. This validated our rational design of the interactions between microorganisms based on their metabolic roles. More interestingly, we also found positive and negative synergies as emergent properties of high-order microbial interactions among three or more microorganisms in cocultures. These microbial interactions can be quantitatively measured by adding and removing specific members. A community stoichiometric model was constructed to represent the fluxes in the community metabolic network. This study paved the way toward a more predictive understanding of the impact of environmental perturbations on microbial interactions sustaining geochemically significant processes in natural systems.

Methyl-based methanogenesis is one of three broad categories of archaeal anaerobic methanogenesis, including both the methyl dismutation (methylotrophic) pathway and the methyl-reducing (also known as hydrogen-dependent methylotrophic) pathway. Methyl-based methanogenesis is increasingly recognized as an important source of methane in a variety of environments. Here, we provide an overview of methyl-based methanogenesis research, including the conditions under which methyl-based methanogenesis can be a dominant source of methane emissions, experimental methods for distinguishing different pathways of methane production, molecular details of the biochemical pathways involved, and the genes and organisms involved in these processes. We also identify the current gaps in knowledge and present a genomic and metagenomic survey of methyl-based methanogenesis genes, highlighting the diversity of methyl-based methanogens at multiple taxonomic levels and the widespread distribution of known methyl-based methanogenesis genes and families across different environments.

Phenanthrene (PHE) as a typical polycyclic aromatic hydrocarbon (PAH) is prevalent and harmful to organisms in petroleum-polluted sites. The effects of PHE concentration levels on performance, microbial community and functions in methanogenic system were comprehensively investigated by an operation of UASB reactor (198 days) and a series of batch tests. The results found that PHE was prone to accumulate in reactor by sludge adsorption (Final concentration = 12.53 mg/g TS Sludge), which posed significant influences on methanogenic system. The removal of chemical oxygen demand (COD), NH 4 + -N and volatile fatty acids (VFAs) in reactor were reduced with PHE accumulation. Meanwhile, microbes with higher ATPase secrete more EPS activity to self-protect against PHE toxicity. Sequencing analysis showed that PHE interfered significantly diversity and structure of microbial community. For bacteria, PHE was toxic to Bacteroidetes and Latescibacteria, while syntrophs (f&#95;Syntrophaceae, Syntrophorhabdus, etc.) involved in VFAs oxidation and aromatic organics degradation were tolerant of PHE stress. For archaea, acetoclastic methanogens (Methanosaeta) abundance was continuously diminished by 45.1% under long-term PHE exposure. Further functions analysis suggested that microbial community accelerated amino acid metabolism, energy metabolism and xenobiotics biodegradation & metabolism to satisfy physiological demanding under PHE stress. Combining batch tests of methanogenic metabolism proved that acetoclastic methanogenesis was negatively affected by PHE due to inhibition of functional enzymes (acetate kinase, phosphate acetyltransferase, etc.) expression. These findings may provide the basis for enhancing bioremediation of PAH pollution in anaerobic environment., 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 © 2022. Published by Elsevier Ltd.)

Thermodynamic characterization of the relative stabilities of chemical compounds is a pillar of conceptual models in various fields of geosciences. Analogous models applied to genomes can yield new information about the relationship between genomes and their geochemical environments. In this perspective article, we present a chemical and thermodynamic analysis of prokaryotic lineages that have been the target of previous phylogenomic studies of evolutionary adaptation to varying redox conditions. The thermodynamic model development begins by quantifying the effects of hydrogen activity (aH 2 ) and temperature on the relative stabilities of organic compounds with different carbon oxidation state. When applied to proteins instead of metabolites, the same techniques can be used to identify combinations of aH 2 and temperature at which reference proteomes for Class I or Class II methanogens are relatively stable. The calculated aH 2 values are compatible with reported measurements for habitats of methanogens ranging from highly reducing submarine hydrothermal systems to less reducing environments including methanogenic sediments. In contrast to the transition between the two classes of methanogenic archaea, that between basal and terrestrial groups of Thaumarchaeota (denoting the origin of ammonia-oxidizing archaea) occurs at a less-reducing redox boundary. These examples reveal the consequences of energy minimization driving evolution and show how geochemical calculations involving biomolecules can be used to quantify and better understand the coevolution of the geosphere and biosphere., (© 2022 John Wiley & Sons Ltd.)

Rice straw (RS) has been recognized as a sustainable renewable energy resource for converting into sugars and volatile fatty acids (acetate, propionate, and butyrate) and subsequently to produce biogas. Enhanced production of these intermediates from RS by the different combinations of two consortia was investigated. Anaerobic microbial cocktails of fungi, bacteria, and methanogens were evaluated for performance and stability in the anaerobic digestion of untreated RS. The best-defined anaerobic microbial cocktail for high RS degradation and methane production, consisting of anaerobic bacteria (mainly Proteiniphilum acetatigenes, Pyramidobacter piscolens, and Mesotoga prima) and anaerobic lignocellulolytic/fermentative fungi (uncultured Neocallimastigales, Orpinomyces, Anaeromyces, and Feramyces sp.) at a copy number ratio of 10 3 -10 5 copies/mL, including hydrogenotrophic and acetoclastic methanogens (Methanosarcina mazei, Methanoculleus marisnigri, Methanofollis liminatans, Methanoculleus bourgensis, and Methanosaeta harundinacea) concentration of 10 6 copies/mL, was successfully constructed. The system performance was 80% VS (volatile solids) RS degradation, 34 mL/day methane production rate, 318 mL/g VS added methane yield, and a pH range of 6.90-7.70 within a short time of 14 days. A defined microbial cocktail has been proven as a potential alternative process for lignocellulose hydrolysis and methane production., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

An anaerobic membrane bioreactor (AnMBR) with media is an emerging carbon-neutral biotechnology for low-strength wastewater (LSWW) treatment and methane recovery. Understanding metabolic dynamics among methanogens and syntrophic bacteria is important in optimizing the design and operation of AnMBR. However, little is known about it, especially in media-attached microbial communities. This study explored metabolic dynamics to compare media-attached and suspended conditions. Accordingly, metagenomes and metatranscriptomes from AnMBRs with polymeric media and fed with different influent concentrations (350 and 700 mg-COD/L) were analyzed. Metabolic dynamics were profoundly influenced by the different growth habitats and influent conditions, although the applied influent concentrations are within the range of typical LSWW. Metabolic dynamics prediction results suggest that media-attached-growth habitats may have provided a more favorable microenvironment for methanogens to grow and produce methane, especially under low influent conditions. These findings provide significant implications for optimizing floating media design and operation of AnMBR-producing methane from LSWW., 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 © 2022. Published by Elsevier Ltd.)

High solid anaerobic digestion (AD) has been considered as a promising and sustainable technology for treating kitchen waste. To enhance AD of kitchen waste, alkali pretreatment and bentonite addition treatment (AP/Be) was performed on kitchen waste, and microbial community was investigated at different total solids (TS) content (10%, 13%, 19%, 22% and 25%). The results indicated that after AP/Be treatment, methane yield was as high as 198 mL CH 4 /g volatile solid (VS), which increased by 236% as the control. Moreover, microbial community analysis revealed that AP/Be treatment enriched bacterial microbial diversity. At TS of 10%, AP/Be treatment enhanced the hydrogenotrophic methanogens (Methanobacterium) significantly. In addition, the dominant methanogenic pathways changed at different TS content. These results demonstrated AP/Be treatment had a positive effect on methanogenesis during kitchen waste anaerobic digestion process. This study threw new insights towards enhancing kitchen waste anaerobic digestion, as well as the microbial mechanism., 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 © 2022 Elsevier Ltd. All rights reserved.)

Archaea represents the third domain of life, displaying a closer relationship with eukaryotes than bacteria. These microorganisms are valuable model systems for molecular biology and biotechnology. In fact, nowadays, methanogens, halophiles, thermophilic euryarchaeota, and crenarchaeota are the four groups of archaea for which genetic systems have been well established, making them suitable as model systems and allowing for the increasing study of archaeal genes' functions. Furthermore, thermophiles are used to explore several aspects of archaeal biology, such as stress responses, DNA replication and repair, transcription, translation and its regulation mechanisms, CRISPR systems, and carbon and energy metabolism. Extremophilic archaea also represent a valuable source of new biomolecules for biological and biotechnological applications, and there is growing interest in the development of engineered strains. In this review, we report on some of the most important aspects of the use of archaea as a model system for genetic evolution, the development of genetic tools, and their application for the elucidation of the basal molecular mechanisms in this domain of life. Furthermore, an overview on the discovery of new enzymes of biotechnological interest from archaea thriving in extreme environments is reported.

Inoculation with microorganisms is an effective strategy for improving traditional composting processes. This study explored the effects of inoculation with lignocellulose-degrading microorganisms (LDM) on the degradation of organic matter (OM), methane (CH 4 ) emissions, and the microbial community (bacteria and methanogens) during composting. The results showed that LDM accelerated the degradation of OM (including the lignocellulose fraction) and increased the CH 4 releases in the later thermophilic and cooling stages during composting. At the ending of composting, LDM increased the CH 4 emissions by 38.6% compared with the control. Moreover, LDM significantly increased the abundances of members of the bacterial and methanogenic community during the later thermophilic period (P < 0.05). In addition, LDM promoted the growth and activity of major bacterial genera (e.g., Ureibacillus) with the ability to degrade macromolecular OM, as well as affecting key methanogens (e.g., Methanocorpusculum) in the composting system. Network analysis and variance partitioning analysis indicated that OM and temperature were the main factors that affected the bacterial and methanogen community structures. Structural equation modeling demonstrated that the higher CH 4 emissions under LDM were related to the growth of methanogens, which was facilitated by the anaerobic environment produced by large amounts of CO 2 . Thus, aerobic conditions should be improved during the end of the thermophilic and cooling composting period when inoculating with lignocellulose-degrading microorganisms in order to reduce CH 4 emissions., 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 © 2022 Elsevier Ltd. All rights reserved.)

Integration of methanogens with semiconductors is an effective approach to sustainable solar-driven methanogenesis. However, the H 2 production rate by semiconductors largely exceeds that of methanogen metabolism, resulting in abundant H 2 as side product. Here, we report that binary metallic active sites (namely, NiCu alloys) are incorporated into the interface between CdS semiconductors and Methanosarcina barkeri. The self-assembled Methanosarcina barkeri-NiCu@CdS exhibits nearly 100% CH 4 selectivity with a quantum yield of 12.41 ± 0.16% under light illumination, which not only exceeds the reported biotic-abiotic hybrid systems but also is superior to most photocatalytic systems. Further investigation reveal that the Ni-Cu-Cu hollow sites in NiCu alloys can directly supply hydrogen atoms and electrons through photocatalysis to the Methanosarcina barkeri for methanogenesis via both extracellular and intracellular hydrogen cycles, effectively turning down the H 2 production. This work provides important insights into the biotic-abiotic hybrid interface, and offers an avenue for engineering the methanogenesis process., (© 2022. The Author(s).)

Ruminants are important for global food security but emit the greenhouse gas methane. Rumen microorganisms break down complex carbohydrates to produce volatile fatty acids and molecular hydrogen. This hydrogen is mainly converted into methane by archaea, but can also be used by hydrogenotrophic acetogenic and respiratory bacteria to produce useful metabolites. A better mechanistic understanding is needed on how dietary carbohydrates influence hydrogen metabolism and methanogenesis. We profiled the composition, metabolic pathways, and activities of rumen microbiota in 24 beef cattle adapted to either fiber-rich or starch-rich diets. The fiber-rich diet selected for fibrolytic bacteria and methanogens resulting in increased fiber utilization, while the starch-rich diet selected for amylolytic bacteria and lactate utilizers, allowing the maintenance of a healthy rumen and decreasing methane production (p &lt; 0.05). Furthermore, the fiber-rich diet enriched for hydrogenotrophic methanogens and acetogens leading to increased electron-bifurcating [FeFe]-hydrogenases, methanogenic [NiFe]- and [Fe]-hydrogenases and acetyl-CoA synthase, with lower dissolved hydrogen (42%, p &lt; 0.001). In contrast, the starch-rich diet enriched for respiratory hydrogenotrophs with greater hydrogen-producing group B [FeFe]-hydrogenases and respiratory group 1d [NiFe]-hydrogenases. Parallel in vitro experiments showed that the fiber-rich selected microbiome enhanced acetate and butyrate production while decreasing methane production (p &lt; 0.05), suggesting that the enriched hydrogenotrophic acetogens converted some hydrogen that would otherwise be used by methanogenesis. These insights into hydrogen metabolism and methanogenesis improve understanding of energy harvesting strategies, healthy rumen maintenance, and methane mitigation in ruminants., (© 2022. The Author(s).)

Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5'-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5'-phosphate (α-RP) or α-adenylyl-5'-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT., (© 2022. The Author(s).)

To investigate the biogas generation characteristics of the organic matter in lignite, methanol extraction was conducted to obtain the soluble fraction and the residual of lignite, which were subsequently taken as the sole carbon source for biogas production by a methanogenic consortium. Afterward, the composition of compounds before and after the fermentation was characterized by UV-Vis, GC-MS, and HPLC-MS analysis. The results indicated that the methanogenic microorganisms could produce H2 and CO2 without accumulating CH4 by utilizing the extract, and the methane production of the residue was 18% larger than that of raw lignite, reaching 1.03 mmol/g. Moreover, the organic compounds in the methanol extract were degraded and their molecular weight was reduced. Compounds such as 1, 6-dimethyl-4-(2-methylethyl) naphthalene, 7-butyl-1-hexylnaphthalene, simonellite, and retene were completely degraded by microorganisms. In addition, both aromatic and non-aromatic metabolites produced in the biodegradation were detected, some of which may have a negative effect on the methanogenesis process. These results revealed the complexity of the interaction between coal and organism from another point of view., Competing Interests: The authors have declared that no competing interests exist.

Methane produced by methanogenic archaea has an important influence on Earth's changing climate. Methanogenic archaea are phylogenetically diverse and widespread in anoxic environments. These microorganisms can be divided into two subgroups based on whether or not they use b-type cytochromes for energy conservation. Methanogens with b-type cytochromes have a wider substrate range and higher growth yields than those without them. To date, methanogens with b-type cytochromes were found exclusively in the phylum "Ca. Halobacteriota" (formerly part of the phylum Euryarchaeota). Here, we present the discovery of metagenome-assembled genomes harboring methyl-coenzyme M reductase genes reconstructed from mesophilic anoxic sediments, together with the previously reported thermophilic "Ca. Methylarchaeum tengchongensis", representing a novel archaeal order, designated the "Ca. Methylarchaeales", of the phylum Thermoproteota (formerly the TACK superphylum). These microorganisms contain genes required for methyl-reducing methanogenesis and the Wood-Ljundahl pathway. Importantly, the genus "Ca. Methanotowutia" of the "Ca. Methylarchaeales" encode a cytochrome b-containing heterodisulfide reductase (HdrDE) and methanophenazine-reducing hydrogenase complex that have similar gene arrangements to those found in methanogenic Methanosarcinales. Our results indicate that members of the "Ca. Methylarchaeales" are methanogens with cytochromes and can conserve energy via membrane-bound electron transport chains. Phylogenetic and amalgamated likelihood estimation analyses indicate that methanogens with cytochrome b-containing electron transfer complexes likely evolved before diversification of Thermoproteota or "Ca. Halobacteriota" in the early Archean Eon. Surveys of public sequence databases suggest that members of the lineage are globally distributed in anoxic sediments and may be important players in the methane cycle., (© 2022. The Author(s).)

Anaerobic digestion (AD) relies on myriads of functions performed by complex microbial communities in customized settings, thus, a comprehensive investigation on the AD microbiome is central to the fine-tuned control. Most current AD microbiome studies are based on relative abundance, which hinders the interpretation of microbes' dynamics and inter-sample comparisons. Here, we developed an absolute quantification (AQ) approach that integrated cellular spike-ins with metagenomic sequencing to elucidate microbial community variations and population dynamics in four anaerobic digesters. Using this method, 253 microbes were defined as decaying populations with decay rates ranging from -0.05 to -5.85 d -1 , wherein, a population from Flavobacteriaceae family decayed at the highest rates of -3.87 to -5.85 d -1 in four digesters. Meanwhile, 25 microbes demonstrated the growing trend in the AD processes with growth rates ranging from 0.11 to 1.77 d -1 , and genome-centric analysis assigned some of the populations to the functional niches of hydrolysis, short-chain fatty acids metabolism, and methane generation. Additionally, we observed that the specific activity of methanogens was lower in the prolonged digestion stage, and redundancy analysis revealed that the feedstock composition and the digestion duration were the two key parameters in governing the AD microbial compositions., 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 © 2022. Published by Elsevier Ltd.)

A shift from the acetoclastic to the hydrogenotrophic pathway in methanogenesis under ammonia inhibition is a common observation in anaerobic digestion. However, there are still considerable knowledge gaps concerning the differential ammonia tolerance of acetoclastic and hydrogenotrophic methanogens (AMs and HMs), their responses to different ammonia species (NH 4 + , NH 3 ), and their recoverability after ammonia inhibition. With the successful enrichment of mesophilic AMs and HMs cultures, this study aimed at addressing the above knowledge gaps through batch inhibition/recovery tests and kinetic modeling under varying total ammonia (TAN, 0.2-10 g N/L) and pH (7.0-8.5) conditions. The results showed that the tolerance level of HMs to free ammonia (FAN, IC 50 =1345 mg N/L) and NH 4 + (IC 50 =6050 mg N/L) was nearly 11 times and 3 times those of AMs (NH 3 , IC 50 =123 mg N/L; NH 4 + , IC 50 =2133 mg N/L), respectively. Consistent with general belief, the AMs were more impacted by FAN. However, the HMs were more adversely affected by NH 4 + when the pH was ≤8.0. A low TAN (1.0-4.0 g N/L) could cause irreversible inhibition of the AMs due to significant cell death, whereas the activity of HMs could be fully or even over recovered from severe ammonia stress (FAN≤ 0.9 g N/L or TAN≤10 g N/L; pH ≤8.0). The different tolerance responses of AMs and HMs might be associated with the cell morphology, multiple energy-converting systems, and Gibbs free energy from substrate-level phosphorylation., 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 © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We investigated the impact of oxygen on a strictly anaerobic, methanogenic benzene-degrading enrichment culture derived decades ago from oil-contaminated sediment. The culture includes a benzene fermenter from Deltaproteobacteria candidate clade Sva0485 (referred to as ORM2) and methanogenic archaea. A one-time injection of 0.1 mL air , simulating a small leak into 30 mL batch culture bottle, had no measurable impact on benzene degradation rates, although retrospectively, a tiny enrichment of aerobic taxa was detected. A subsequent 100 times larger injection of air stalled methanogenesis and caused drastic perturbation of the microbial community. A benzene-degrading Pseudomonas became highly enriched and consumed all available oxygen. Anaerobic benzene-degrading ORM2 cell numbers plummeted during this time; re-growth and associated recovery of methanogenic benzene degradation took almost 1 year. These results highlight the oxygen sensitivity of this methanogenic culture and confirm that the mechanism for anaerobic biotransformation of benzene is independent of oxygen, fundamentally different from established aerobic pathways, and is carried out by distinct microbial communities. The study also highlights the importance of including microbial decay in characterizing and modeling mixed microbial communities.

Background: Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H 2 ) + carbon dioxide and from H 2 + methanol or methylamines. The methanogenic substrates are provided by non-methanogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbiome and to determine the fate of H 2 that accumulates less than expected under inhibited methanogenesis., Results: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methanosphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H 2 concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H 2 -producing bacteria to regulate the amount of H 2 production. No previously reported alternative H 2 sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway., Conclusion: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H 2 metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. Video Abstract., (© 2022. The Author(s).)