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UNLABELLED: Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural research studies have generated detailed insights into MCR function in vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this has not been categorically tested. In this study, to gain a more direct understanding of MCRs control on the growth of Methanosarcina acetivorans, we generate a strain with an inducible mcr operon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate-limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis of M. acetivorans experiencing MCR limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, the mcr operon is not transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is a wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors such as anabolic reactions might be rate-limiting for methanogenic growth. IMPORTANCE: Methane is a potent greenhouse gas that has contributed to ca. 25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. Methyl-coenzyme M reductase (MCR) catalyzes methane formatio in methanogens. Even though MCR comprises ca. 10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. In this study, we show that Methanosarcina acetivorans cells grown in substrate-replicate batch cultures produce more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher-throughput manner than isolation and biochemical characterization of pure protein.

Reservoirs are an important source of methane (CH4) emissions, but the relative contribution of CH4 ebullition and diffusion fluxes to total fluxes has received little attention in the past. In this study, we systematically monitored the CH4 fluxes of nine cascade reservoirs (Dahejia, Jishixia, Huangfeng, Suzhi, Kangyang, Zhiganglaka, Lijiaxia, Nina, and Longyangxia) in the upper reaches of the Yellow River in the dry (May 2023) and wet seasons (August 2023) using the static chamber gas chromatography and headspace equilibrium methods. We also simultaneously measured environmental physicochemical properties as well as the abundance of methanogens and methanotrophs in sediments. The results showed the following: (1) All reservoirs were sources of CH4 emissions, with an average diffusion flux of 0.08 ± 0.05 mg m−2 h−1 and ebullition flux of 0.38 ± 0.41 mg m−2 h−1. Ebullition flux accounted for 78.01 ± 7.85% of total flux. (2) Spatially, both CH4 diffusion and ebullition fluxes increased from upstream to downstream. Temporally, CH4 diffusion flux in the wet season (0.09 ± 0.06 mg m−2 h−1) was slightly higher than that in the dry season (0.08 ± 0.04 mg m−2 h−1), but CH4 ebullition flux in the dry season (0.38 ± 0.48 mg m−2 h−1) was higher than that in the wet season (0.32 ± 0.2 mg m−2 h−1). (3) qPCR showed that methanogens (mcrA gene) were more abundant in the wet season (5.43 ± 3.94 × 105 copies g−1) than that in the dry season (3.74 ± 1.34 × 105 copies g−1). Methanotrophs (pmoA gene) also showed a similar trend with more abundance found in the wet season (7 ± 2.61 × 105 copies g−1) than in the dry season (1.47 ± 0.92 × 105 copies g−1. (4) Structural equation modeling revealed that the ratio of mcrA/pmoA genes, water N/P, and reservoir age were key factors affecting CH4 ebullition flux. Variation partitioning further indicated that the ratio of mcrA/pmoA genes was the main factor causing the spatial variation in CH4 ebullition flux, explaining 35.69% of its variation. This study not only reveals the characteristics and influencing factors of CH4 emissions from cascade reservoirs on the Qinghai Plateau but also provides a scientific basis for calculating fluxes and developing global CH4 reduction strategies for reservoirs. [ABSTRACT FROM AUTHOR]

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]

Methyl‐coenzyme M reductase (MCR) is a multi‐subunit (α2β2γ2) enzyme responsible for methane formation via its unique F430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high‐resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin‐like domain assembled into an α + β barrel‐like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas. [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.

Methyl‐coenzyme M reductase (MCR) is a multi‐subunit (α2β2γ2) enzyme responsible for methane formation via its unique F430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high‐resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin‐like domain assembled into an α + β barrel‐like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.

The efficiency of anaerobic night soil (human excreta) biodegradation depends on the characteristics of the inoculum used. This anaerobic microbial inoculum (AMI) contains a microbial consortium, including rate-limiting methanogens whose growth depends on various physicochemical and biological parameters necessitating the inoculum characterisation essential for optimum night soil (NS) biodegradation. The inoculum contained 24.567 g/L (±0.07) total solid, 5.022 (±0.57) g/L total dissolved solid, 18.148 (±0.0.058) g/L volatile solid (VS), and 7.0±0.2 pH. The Gas chromatography analysis confirmed the presence of a small amount of volatile fatty acid (VFA), (≤ 4 mM except propionate) showing rapid conversion of VFA to methane, and 55% methane. Further, the presence of methanosaetaceae group of methanogens was detected through Taqman probe-based real-time PCR. The inoculum performance was evaluated for NS biodegradation in semicontinuous mode of feeding in four 2 L anaerobic biodigesters (2D HRT, 4D HRT, 5D HRT, and 10D HRT) with varying hydraulic retention time (HRT) (2, 5, 8, and 10 days, respectively) at mesophilic temperature (35°C). After 5 HRT, VS reduction for 2D HRT and 5D HRT was 45-50% and >45-60%, respectively. The methane content was 45-50% and 50-65% methane, respectively. The % VS reduction and methane content showed the overfeeding of NS in biodigesters 2D HRT and 5D HRT while 8D HRT showed towards optimum and 10D HRT optimum with improved VS reduction and methane content.

Methanogenic and methanotrophic microbes together determine the net methane flux from rice fields. Despite much research on them as separate communities, there has been little study of combined community patterns, and how these vary between the rhizoplane (root surface), rhizosphere (soil surrounding the root) and bulk soil around rice plants, especially at larger spatial scale. We collected samples from 32 geographically scattered rice fields in east central China, amplicon targeting the mcrA gene for methanogenesis and pmoA gene for methanotrophy by using high‐throughput sequencing. Distinct communities of both methanogens and methanotrophs occurred in each of the three compartments, and predominantly positive links were found between methanogens and methanotrophs in all compartments indicating cross‐feeding or consortia relationships. Methanogens were acting as the network hub in the bulk soil, and methanotrophs in rhizoplane. Network complexity and stability was greater in the rhizosphere than rhizoplane and bulk soil, with no network hubs detected, suggesting the strongest effect of homeostatic influence by plant occurred in the rhizosphere. The proportion of determinism (homogeneous selection) and distance–decay relation (DDR) in rhizoplane was consistently lower than that in the rhizosphere for both communities, indicating weaker phylogenetic clustering in rice root surface. Our results have provided a better understanding of CH4 oxidation and emission in rice paddy fields and future agriculture management could take into consideration of the subtle variation among different soil compartments and interactions within methanogenic and methanotrophic communities. [ABSTRACT FROM AUTHOR]

The efficiency of anaerobic night soil (human excreta) biodegradation depends on the characteristics of the inoculum used. This anaerobic microbial inoculum (AMI) contains a microbial consortium, including rate-limiting methanogens whose growth depends on various physicochemical and biological parameters necessitating the inoculum characterisation essential for optimum night soil (NS) biodegradation. The inoculum contained 24.567 g/L (±0.07) total solid, 5.022 (±0.57) g/L total dissolved solid, 18.148 (±0.0.058) g/L volatile solid (VS), and 7.0±0.2 pH. The Gas chromatography analysis confirmed the presence of a small amount of volatile fatty acid (VFA), (≤ 4 mM except propionate) showing rapid conversion of VFA to methane, and 55% methane. Further, the presence of methanosaetaceae group of methanogens was detected through Taqman probe-based real-time PCR. The inoculum performance was evaluated for NS biodegradation in semicontinuous mode of feeding in four 2 L anaerobic biodigesters (2D HRT, 4D HRT, 5D HRT, and 10D HRT) with varying hydraulic retention time (HRT) (2, 5, 8, and 10 days, respectively) at mesophilic temperature (35°C). After 5 HRT, VS reduction for 2D HRT and 5D HRT was <40% while for 8D HRT and 10D HRT was >45-50% and >45-60%, respectively. The methane content was <20% in biogas for 2 and 5 days HRT while for 8 and 10 days >45-50% and 50-65% methane, respectively. The % VS reduction and methane content showed the overfeeding of NS in biodigesters 2D HRT and 5D HRT while 8D HRT showed towards optimum and 10D HRT optimum with improved VS reduction and methane content. [ABSTRACT FROM AUTHOR]

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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. These references provide a comprehensive overview of current knowledge and research on archaea, making them a valuable resource for library patrons conducting research on this topic. [Extracted from the article]

Abstract Anaerobic microbial corrosion of iron‐containing metals causes extensive economic damage. Some microbes are capable of direct metal‐to‐microbe electron transfer (electrobiocorrosion), but the prevalence of electrobiocorrosion among diverse methanogens and acetogens is poorly understood because of a lack of tools for their genetic manipulation. Previous studies have suggested that respiration with 316L stainless steel as the electron donor is indicative of electrobiocorrosion, because, unlike pure Fe0, 316L stainless steel does not abiotically generate H2 as an intermediary electron carrier. Here, we report that all of the methanogens (Methanosarcina vacuolata, Methanothrix soehngenii, and Methanobacterium strain IM1) and acetogens (Sporomusa ovata and Clostridium ljungdahlii) evaluated respired with pure Fe0 as the electron donor, but only M. vacuolata, Mx. soehngenii, and S. ovata were capable of stainless steel electrobiocorrosion. The electrobiocorrosive methanogens required acetate as an additional energy source in order to produce methane from stainless steel. Cocultures of S. ovata and Mx. soehngenii demonstrated how acetogens can provide acetate to methanogens during corrosion. Not only was Methanobacterium strain IM1 not capable of electrobiocorrosion, but it also did not accept electrons from Geobacter metallireducens, an effective electron‐donating partner for direct interspecies electron transfer to all methanogens that can directly accept electrons from Fe0. The finding that M. vacuolata, Mx. soehngenii, and S. ovata are capable of electrobiocorrosion, despite a lack of the outer‐surface c‐type cytochromes previously found to be important in other electrobiocorrosive microbes, demonstrates that there are multiple microbial strategies for making electrical contact with Fe0.

Biomass from agriculture, forestry, and urban wastes is a potential renewable organic resource for energy generation. Many investigations have demonstrated that anaerobic fungi and methanogens could be co-cultured to degrade lignocellulose for methane generation. Thus, this study aimed to evaluate the effect of natural anaerobic fungi-methanogens co-culture on the methane production and lignocellulosic degradation of wastes from rice, corn and sugarcane. Hu sheep rumen digesta was used to develop a natural anaerobic fungi-methanogen co-culture. The substrates were rice straw (RS), rich husk (RH), corn stover (CS), corn cobs (CC), and sugarcane baggage (SB). Production of total gas and methane, metabolization rate of reducing sugar, glucose, and xylose, digestibility of hemicellulose and cellulose, activity of carboxymethylcellulase and xylanase, and concentrations of total acid and acetate were highest (P < 0.05) in CC, moderate (P < 0.05) in RS and CS, and lowest (P < 0.05) in SB and RH. The pH, lactate and ethanol were lowest (P < 0.05) in CC, moderate (P < 0.05) in RS and CS, and lowest (P < 0.05) SB and RH. Formate was lowest (P < 0.05) in CC, RS and CS, moderate (P < 0.05) in SB, and lowest (P < 0.05) in RH. Therefore, this study indicated that the potential of methane production and lignocellulosic degradation by natural anaerobic fungi-methanogens co-culture were highest in CC, moderate in RS and CS, and lowest in SB and RH. [ABSTRACT FROM AUTHOR]

The unprecedented population and anthropogenic activity rise have challenged the future look up for shifts in global temperature and climate patterns. Anthropogenic activities such as land fillings, building dams, wetlands converting to lands, combustion of biomass, deforestation, mining, and the gas and coal industries have directly or indirectly increased catastrophic methane (CH4) emissions at an alarming rate. Methane is 25 times more potent trapping heat when compared to carbon dioxide (CO2) in the atmosphere. A rise in atmospheric methane, on a 20-year time scale, has an impact of 80 times greater than that of CO2. With increased population growth, waste generation is rising and is predicted to reach 6 Mt by 2025. CH4 emitted from landfills is a significant source that accounts for 40% of overall global methane emissions. Various mitigation and emissions reduction strategies could significantly reduce the global CH4 burden at a cost comparable to the parallel and necessary CO2 reduction measures, reversing the CH4 burden to pathways that achieve the goals of the Paris Agreement. CH4 mitigation directly benefits climate change, has collateral impacts on the economy, human health, and agriculture, and considerably supports CO2 mitigation. Utilizing the CO2 from the environment, methanogens produce methane and lower their carbon footprint. NGOs and the general public should act on time to overcome atmospheric methane emissions by utilizing the raw source for producing carbon–neutral fuel. However, more research potential is required for green energy production and to consider investigating the untapped potential of methanogens for dependable energy generation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Microbial populations in the rumen play an essential role in the degradation of Cellulosic dietary components and in providing nutrients to the host animal. Objective This study aims to detect the effect of pomegranate seed pulp (PSP) on rumen fermentation, digestibility and methanogens and the protozoa population (by competitive polymerase chain reaction [PCR]) of the camel and goat rumen fluid. Materials and methods PSP was added to the experimental treatments and replaced by wheat bran (0%, 5% and 10%). Rumen fluid was collected from three goats and two camels according to the similarity of sex, breed, origin and time and used for three gas production studies. DNA extraction was performed by the RBB + c method, the ImageJ programme calculated band intensities (target and competing DNA), and line gradients were plotted based on the number of copies and intensity. Results Our result showed that diets did not significantly affect the methanogen and protozoa population. Animal species affected microbial populations so that both populations in camels were less than goats. The production of gas and volatile fatty acids was not affected by diets. These two parameters and NH3 concentration and methane production in goats were higher than in camel. The pH of digested dry matter and microbial protein in camels was higher than in goats. Conclusions Therefore, the competitive PCR technique is an effective method for enumerating rumen microbiota. This supplementation can be considered a strategy to achieve performance and environmental benefits.

Reservoirs are an important source of methane (CH4) emissions, but the relative contribution of CH4 ebullition and diffusion fluxes to total fluxes has received little attention in the past. In this study, we systematically monitored the CH4 fluxes of nine cascade reservoirs (Dahejia, Jishixia, Huangfeng, Suzhi, Kangyang, Zhiganglaka, Lijiaxia, Nina, and Longyangxia) in the upper reaches of the Yellow River in the dry (May 2023) and wet seasons (August 2023) using the static chamber gas chromatography and headspace equilibrium methods. We also simultaneously measured environmental physicochemical properties as well as the abundance of methanogens and methanotrophs in sediments. The results showed the following: (1) All reservoirs were sources of CH4 emissions, with an average diffusion flux of 0.08 ± 0.05 mg m−2 h−1 and ebullition flux of 0.38 ± 0.41 mg m−2 h−1. Ebullition flux accounted for 78.01 ± 7.85% of total flux. (2) Spatially, both CH4 diffusion and ebullition fluxes increased from upstream to downstream. Temporally, CH4 diffusion flux in the wet season (0.09 ± 0.06 mg m−2 h−1) was slightly higher than that in the dry season (0.08 ± 0.04 mg m−2 h−1), but CH4 ebullition flux in the dry season (0.38 ± 0.48 mg m−2 h−1) was higher than that in the wet season (0.32 ± 0.2 mg m−2 h−1). (3) qPCR showed that methanogens (mcrA gene) were more abundant in the wet season (5.43 ± 3.94 × 105 copies g−1) than that in the dry season (3.74 ± 1.34 × 105 copies g−1). Methanotrophs (pmoA gene) also showed a similar trend with more abundance found in the wet season (7 ± 2.61 × 105 copies g−1) than in the dry season (1.47 ± 0.92 × 105 copies g−1. (4) Structural equation modeling revealed that the ratio of mcrA/pmoA genes, water N/P, and reservoir age were key factors affecting CH4 ebullition flux. Variation partitioning further indicated that the ratio of mcrA/pmoA genes was the main factor causing the spatial variation in CH4 ebullition flux, explaining 35.69% of its variation. This study not only reveals the characteristics and influencing factors of CH4 emissions from cascade reservoirs on the Qinghai Plateau but also provides a scientific basis for calculating fluxes and developing global CH4 reduction strategies for reservoirs.

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]

Background: Microbial populations in the rumen play an essential role in the degradation of Cellulosic dietary components and in providing nutrients to the host animal. Objective: This study aims to detect the effect of pomegranate seed pulp (PSP) on rumen fermentation, digestibility and methanogens and the protozoa population (by competitive polymerase chain reaction [PCR]) of the camel and goat rumen fluid. Materials and methods: PSP was added to the experimental treatments and replaced by wheat bran (0%, 5% and 10%). Rumen fluid was collected from three goats and two camels according to the similarity of sex, breed, origin and time and used for three gas production studies. DNA extraction was performed by the RBB + c method, the ImageJ programme calculated band intensities (target and competing DNA), and line gradients were plotted based on the number of copies and intensity. Results: Our result showed that diets did not significantly affect the methanogen and protozoa population. Animal species affected microbial populations so that both populations in camels were less than goats. The production of gas and volatile fatty acids was not affected by diets. These two parameters and NH3 concentration and methane production in goats were higher than in camel. The pH of digested dry matter and microbial protein in camels was higher than in goats. Conclusions: Therefore, the competitive PCR technique is an effective method for enumerating rumen microbiota. This supplementation can be considered a strategy to achieve performance and environmental benefits. [ABSTRACT FROM AUTHOR]

Abstract Background Hydrogen gas (H2) is a common product of carbohydrate fermentation in the human gut microbiome and its accumulation can modulate fermentation. Concentrations of colonic H2 vary between individuals, raising the possibility that H2 concentration may be an important factor differentiating individual microbiomes and their metabolites. Butyrate-producing bacteria (butyrogens) in the human gut usually produce some combination of butyrate, lactate, formate, acetate, and H2 in branched fermentation pathways to manage reducing power generated during the oxidation of glucose to acetate and carbon dioxide. We predicted that a high concentration of intestinal H2 would favor the production of butyrate, lactate, and formate by the butyrogens at the expense of acetate, H2, and CO2. Regulation of butyrate production in the human gut is of particular interest due to its role as a mediator of colonic health through anti-inflammatory and anti-carcinogenic properties. Results For butyrogens that contained a hydrogenase, growth under a high H2 atmosphere or in the presence of the hydrogenase inhibitor CO stimulated production of organic fermentation products that accommodate reducing power generated during glycolysis, specifically butyrate, lactate, and formate. Also as expected, production of fermentation products in cultures of Faecalibacterium prausnitzii strain A2-165, which does not contain a hydrogenase, was unaffected by H2 or CO. In a synthetic gut microbial community, addition of the H2-consuming human gut methanogen Methanobrevibacter smithii decreased butyrate production alongside H2 concentration. Consistent with this observation, M. smithii metabolic activity in a large human cohort was associated with decreased fecal butyrate, but only during consumption of a resistant starch dietary supplement, suggesting the effect may be most prominent when H2 production in the gut is especially high. Addition of M. smithii to the synthetic communities also facilitated the growth of E. rectale, resulting in decreased relative competitive fitness of F. prausnitzii. Conclusions H2 is a regulator of fermentation in the human gut microbiome. In particular, high H2 concentration stimulates production of the anti-inflammatory metabolite butyrate. By consuming H2, gut methanogenesis can decrease butyrate production. These shifts in butyrate production may also impact the competitive fitness of butyrate producers in the gut microbiome. Video Abstract

ABSTRACTMethanogens, reductive acetogens and sulfate-reducing bacteria play an important role in disposing of hydrogen in gut ecosystems. However, how they interact with each other remains largely unknown. This in vitro study cocultured Blautia hydrogenotrophica (reductive acetogen), Desulfovibrio piger (sulfate reducer) and Methanobrevibacter smithii (methanogen). Results revealed that these three species coexisted and did not compete for hydrogen in the early phase of incubations. Sulfate reduction was not affected by B. hydrogenotrophica and M. smithii. D. piger inhibited the growth of B. hydrogenotrophica and M. smithii after 10 h incubations, and the inhibition on M. smithii was associated with increased sulfide concentration. Remarkably, M. smithii growth lag phase was shortened by coculturing with B. hydrogenotrophica and D. piger. Formate was rapidly used by M. smithii under high acetate concentration. Overall, these findings indicated that the interactions of the hydrogenotrophic microbes are condition-dependent, suggesting their interactions may vary in gut ecosystems.

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 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]

Biogas is produced by a consortium of bacteria and archaea. We studied how the microbiome of poultry litter digestate was affected by time and treatments that enhanced biogas production. The microbiome was analyzed at six, 23, and 42 weeks of incubation. Starting at week seven, the digesters underwent four treatments: control, microaeration with 6 mL air L−1 digestate per day, treatment with a 1000 Hz sine wave, or treatment with the sound wave and microaeration. Both microaeration and sound enhanced biogas production relative to the control, while their combination was not as effective as microaeration alone. At week six, over 80% of the microbiome of the four digesters was composed of the three phyla Actinobacteria, Proteobacteria, and Firmicutes, with less than 10% Euryarchaeota and Bacteroidetes. At week 23, the digester microbiomes were more diverse with the phyla Spirochaetes, Synergistetes, and Verrucomicrobia increasing in proportion and the abundance of Actinobacteria decreasing. At week 42, Firmicutes, Bacteroidetes, Euryarchaeota, and Actinobacteria were the most dominant phyla, comprising 27.8%, 21.4%, 17.6%, and 12.3% of the microbiome. Other than the relative proportions of Firmicutes being increased and proportions of Bacteroidetes being decreased by the treatments, no systematic shifts in the microbiomes were observed due to treatment. Rather, microbial diversity was enhanced relative to the control. Given that both air and sound treatment increased biogas production, it is likely that they improved poultry litter breakdown to promote microbial growth. [ABSTRACT FROM AUTHOR]

An increased frequency of droughts due to anthropogenic climate change can lead to considerable stress for soil microorganisms and their functioning within northern peatlands. A better understanding of the diversity and relative abundance of methane producing and oxidizing taxa, and their functional genes, can help predict the functional potential of peatlands and how the microorganisms respond to disturbances such as drought. To address knowledge gaps in the understanding of how functional genetic diversity shifts under drought conditions, we investigated a hemi boreal mire in Southern Sweden. Environmental parameters, including soil and air temperature, precipitation, and water table depth, as well as methane flux data were collected during the summer of 2017 under typical growing conditions, and in 2018 during a drought. In addition, the diversity and composition of genes encoding for methane metabolism were determined using the captured metagenomics technique. During drought we observed a substantial increase in air and soil temperature, reduced precipitation, and a lower water table depth. Taxonomic and functional gene composition significantly changed during the drought, while diversity indices, such as alpha and beta diversity, remained similar. These results indicate that methane producing and oxidizing microbial communities, and their functional genes, displayed a resilience to drought with specific genera having the ability to outcompete others under stress. Furthermore, our results show that although methane emissions are substantially reduced during drought, we can expect to see a shift toward more resilient methanogens and methanotrophs under future climate conditions. Plain Language Summary: Droughts and heat waves are increasing due to climate change. This can lead to considerable stress on soil microorganisms in northern wetlands which emit strong greenhouse gases such as methane. A better understanding of these methane producing and oxidizing microorganisms can help us predict how the community responds to droughts, and thus, how much greenhouse gases may be emitted in the future contributing further to climate change. Key Points: Taxonomic and functional gene composition significantly changed during the droughtMethane fluxes significantly reduced during drought but not in all ecotypesSpecialist genera respond to drought stronger than others [ABSTRACT FROM AUTHOR]

The Fe protein of nitrogenase reduces two C1 substrates, CO2 and CO, under ambient conditions when its [Fe4S4] cluster adopts the all-ferrous [Fe4S4]0 state. Here, we show disparate reactivities of the nifH- and vnf-encoded Fe proteins from Methanosarcina acetivorans (designated MaNifH and MaVnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO2 and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that MaVnfH adopts the all-ferrous state at a more positive reduction potential than MaNifH, which could account for the weaker reactivity of the MaVnfH toward C1 substrates than MaNifH. More importantly, MaVnfH already displays the g = 16.4 parallel-mode EPR signal that is characteristic of the all-ferrous [Fe4S4]0 cluster at a reduction potential of -0.44 V, and the signal reaches 50% maximum intensity at a reduction potential of -0.59 V, suggesting the possibility of this Fe protein to access the all-ferrous [Fe4S4]0 state under physiological conditions. These results bear significant relevance to the long-lasting debate of whether the Fe protein can utilize the [Fe4S4]0/2+ redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes.

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.

Amazonian rainforest is undergoing increasing rates of deforestation, driven primarily by cattle pasture expansion. Forest-to-pasture conversion has been associated with increases in soil methane (CH4) emission. To better understand the drivers of this change, we measured soil CH4 flux, environmental conditions, and belowground microbial community structure across primary forests, cattle pastures, and secondary forests in two Amazonian regions. We show that pasture soils emit high levels of CH4 (mean: 3454.6 ± 9482.3 μg CH4 m-2 d-1), consistent with previous reports, while forest soils on average emit CH4 at modest rates (mean: 9.8 ± 120.5 μg CH4 m-2 d-1), but often act as CH4 sinks. We report that secondary forest soils tend to consume CH4 (mean: -10.2 ± 35.7 μg CH4 m-2 d-1), demonstrating that pasture CH4 emissions can be reversed. We apply a novel computational approach to identify microbial community attributes associated with flux independent of soil chemistry. While this revealed taxa known to produce or consume CH4 directly (i.e. methanogens and methanotrophs, respectively), the vast majority of identified taxa are not known to cycle CH4. Each land use type had a unique subset of taxa associated with CH4 flux, suggesting that land use change alters CH4 cycling through shifts in microbial community composition. Taken together, we show that microbial composition is crucial for understanding the observed CH4 dynamics and that microorganisms provide explanatory power that cannot be captured by environmental variables.

ABSTRACT Eutrophication increases the input of labile, algae-derived, organic matter (OM) into lake sediments. This potentially increases methane (CH4) emissions from sediment to water through increased methane production rates and decreased methane oxidation efficiency in sediments. However, the effect of OM lability on the structure of methane oxidizing (methanotrophic) and methane producing (methanogenic) microbial communities in lake sediments is still understudied. We studied the vertical profiles of the sediment and porewater geochemistry and the microbial communities (16S rRNA gene amplicon sequencing) at five profundal stations of an oligo-mesotrophic, boreal lake (Lake Pääjärvi, Finland), varying in surface sediment OM sources (assessed via sediment C:N ratio). Porewater profiles of methane, dissolved inorganic carbon (DIC), acetate, iron, and sulfur suggested that sites with more autochthonous OM showed higher overall OM lability, which increased remineralization rates, leading to increased electron acceptor (EA) consumption and methane emissions from sediment to water. When OM lability increased, the abundance of anaerobic nitrite-reducing methanotrophs (Candidatus Methylomirabilis) relative to aerobic methanotrophs (Methylococcales) in the methane oxidation layer of sediment surface decreased, suggesting that Methylococcales were more competitive than Ca. Methylomirabilis under decreasing redox conditions and increasing methane availability due to their more diverse metabolism (fermentation and anaerobic respiration) and lower affinity for methane. Furthermore, when OM lability increased, the abundance of methanotrophic community in the sediment surface layer, especially Ca. Methylomirabilis, relative to the methanogenic community decreased. We conclude that increasing input of labile OM, subsequently affecting the redox zonation of sediments, significantly modifies the methane producing and consuming microbial community of lake sediments. Importance Lakes are important natural emitters of the greenhouse gas methane (CH4). It has been shown that eutrophication, via increasing the input of labile organic matter (OM) into lake sediments and subsequently affecting the redox conditions, increases methane emissions from lake sediments through increased sediment methane production rates and decreased methane oxidation efficiency. However, the effect of organic matter lability on the structure of the methane-related microbial communities of lake sediments is not known. In this study, we show that, besides the activity, also the structure of lake sediment methane producing and consuming microbial community is significantly affected by changes in the sediment organic matter lability.

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.

Methanogenesis is a unique energy metabolism carried out by members of the domain Archaea. Unlike most other methanogens, which reduce CO2 to methane with hydrogen as the electron donor, Methanosarcina acetivorans is able to grow on methylated compounds, on acetate, and on carbon monoxide (CO). These substrates are metabolized via distinct yet overlapping pathways. For the use of any single methanogenic substrate, the membrane-integral, energy-converting N5-methyl-tetrahydrosarcinapterin (H4SPT):coenzyme M (HS-CoM) methyltransferase (Mtr) is required. It was proposed that M. acetivorans can bypass the methyl transfer catalyzed by Mtr via cytoplasmic activities. To address this issue, conversion of different energy substrates by an mtr deletion mutant was analyzed. No significant methyl transfer from H4SPT to HS-CoM could be detected with CO as the electron donor. In contrast, formation of methane and CO2 in the presence of methanol or trimethylamine was indicative of an Mtr bypass in the oxidative direction. As methane thiol and dimethyl sulfide were transiently produced during methylotrophic methanogenesis in the mtr mutant, involvement in this process of methyl sulfide-dependent methyltransferases (Mts) was analyzed in a strain lacking both the Mts system and Mtr. It could be unequivocally demonstrated that the Mts system is not involved in bypassing Mtr, thereby ruling out previous proposals. Conversion of [13C]methanol indicated that in the absence of Mtr M. acetivorans provides the reducing equivalents for methyl-S-CoM reduction to methane by oxidizing (an) intracellular compound(s) to CO2 rather than disproportioning the source of methyl groups. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans. [ABSTRACT FROM AUTHOR]

Background: Hydrogen gas (H2) is a common product of carbohydrate fermentation in the human gut microbiome and its accumulation can modulate fermentation. Concentrations of colonic H2 vary between individuals, raising the possibility that H2 concentration may be an important factor differentiating individual microbiomes and their metabolites. Butyrate-producing bacteria (butyrogens) in the human gut usually produce some combination of butyrate, lactate, formate, acetate, and H2 in branched fermentation pathways to manage reducing power generated during the oxidation of glucose to acetate and carbon dioxide. We predicted that a high concentration of intestinal H2 would favor the production of butyrate, lactate, and formate by the butyrogens at the expense of acetate, H2, and CO2. Regulation of butyrate production in the human gut is of particular interest due to its role as a mediator of colonic health through anti-inflammatory and anti-carcinogenic properties. Results: For butyrogens that contained a hydrogenase, growth under a high H2 atmosphere or in the presence of the hydrogenase inhibitor CO stimulated production of organic fermentation products that accommodate reducing power generated during glycolysis, specifically butyrate, lactate, and formate. Also as expected, production of fermentation products in cultures of Faecalibacterium prausnitzii strain A2-165, which does not contain a hydrogenase, was unaffected by H2 or CO. In a synthetic gut microbial community, addition of the H2-consuming human gut methanogen Methanobrevibacter smithii decreased butyrate production alongside H2 concentration. Consistent with this observation, M. smithii metabolic activity in a large human cohort was associated with decreased fecal butyrate, but only during consumption of a resistant starch dietary supplement, suggesting the effect may be most prominent when H2 production in the gut is especially high. Addition of M. smithii to the synthetic communities also facilitated the growth of E. rectale, resulting in decreased relative competitive fitness of F. prausnitzii. Conclusions: H2 is a regulator of fermentation in the human gut microbiome. In particular, high H2 concentration stimulates production of the anti-inflammatory metabolite butyrate. By consuming H2, gut methanogenesis can decrease butyrate production. These shifts in butyrate production may also impact the competitive fitness of butyrate producers in the gut microbiome. 3hTM6nso89x7h1g_xY7TKk Video Abstract [ABSTRACT FROM AUTHOR]

Methane (CH4) is produced by a number of natural processes that add to the global CH4 budget in various ways. A change in the planet's climate can be influenced by CH4 if there is a surplus or deficit in the CH4 budget. Major contributors to atmospheric CH4 levels include wetlands, paddies, animals, industrial facilities, and fossil fuels. CH4 is emitted from wetland and rice field ecosystems due in large part to the activity of methanogen microbes. CH4 emission is affected by several variables, including the level of the water table, the average temperature, and the composition of the local vegetation. Understanding the temperature response of microbial methanogenesis in anaerobic soils is crucial for predicting the feedback between this potent greenhouse gas and climate change. It was the bacterial and/or archaeal community structures that determined the methanogenic function of the soil, which in turn was determined by the incubation temperature, albeit to a large extent on an individual basis for each soil. Different taxonomic community structures in the various soils and at various temperatures indicated that there was quite a bit of functional redundancy between them. [ABSTRACT FROM AUTHOR]

Wetlands are an important source of atmospheric methane (CH4) and are sensitive to global climate change. Alpine swamp meadows, accounting for ~50% of the natural wetlands on the Qinghai-Tibet Plateau, were considered one of the most important ecosystems. Methanogens are important functional microbes that perform the methane producing process. However, the response of methanogenic community and the main pathways of CH4 production to temperature rise remains unknown in alpine swamp meadow at different water level in permafrost wetlands. In this study, we investigated the response of soil CH4 production and the shift of methanogenic community to temperature rise in the alpine swamp meadow soil samples with different water levels collected from the Qinghai-Tibet Plateau through anaerobic incubation at 5°C, 15°C and 25°C. The results showed that the CH4 contents increased with increasing incubation temperature, and were 5-10 times higher at the high water level sites (GHM1 and GHM2) than that at the low water level site (GHM3). For the high water level sites (GHM1 and GHM2), the change of incubation temperatures had little effect on the methanogenic community structure. Methanotrichaceae (32.44-65.46%), Methanobacteriaceae (19.30-58.86%) and Methanosarcinaceae (3.22-21.24%) were the dominant methanogen groups, with the abundance of Methanotrichaceae and Methanosarcinaceae having a significant positive correlation with CH4 production (p < 0.01). For the low water level site (GHM3), the methanogenic community structure changed greatly at 25°C. The Methanobacteriaceae (59.65-77.33%) was the dominant methanogen group at 5°C and 15°C; In contrast, the Methanosarcinaceae (69.29%) dominated at 25°C, and its abundance showed a significant positive correlation with CH4 production (p < 0.05). Collectively, these findings enhance the understanding of methanogenic community structures and CH4 production in permafrost wetlands with different water levels during the warming process. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

With global warming, the dramatic retreat of glaciers in the Tibetan Plateau (TP) might accelerate release of stored methane (CH4) into the atmosphere; thus, this region might become a new source of CH4. CH4-metabolic microbial communities can produce or consume CH4 in the environment, which is critical for evaluating the CH4 budget of glaciers. However, studies on the influence of CH4-metabolic microbial communities on the CH4 budget during glacier retreat in the TP remain scarce. In this work, ice samples were collected at the terminus of the Guliya Ice Cap in the northwestern TP. The community composition of CH4-metabolic microorganisms, including methanogens and methanotrophs, was determined using genomic analysis, and the metabolic rates of the two microorganisms were further estimated. The abundance of methanotrophs in Guliya was one order of magnitude higher than that of methanogens. The CH4 consumption flux by the combined action of the two microorganisms was ca. 1.42 × 103 pmol·mL−1·d−1, suggesting that CH4 metabolic microbial communities in the glacier might be an important CH4 sink, and can reduce subglacial CH4 emission during glacier retreat. This is important for predicting the CH4 budget in glaciers on the TP and corresponding climate impacts during glacier retreat. [ABSTRACT FROM AUTHOR]

Abstract Anaerobic fungus–methanogen co-cultures from rumen liquids and faeces can degrade lignocellulose efficiently. In this study, 31 fungus–methanogen co-cultures were first obtained from the rumen of yaks grazing in Qinghai Province, China, using the Hungate roll-tube technique. The fungi were identified according to morphological characteristics and internal transcribed spacer (ITS) sequences. The methanogens associated with each fungus were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rRNA gene sequencing. They were five co-culture types: Neocallimastix frontalis + Methanobrevibacter ruminantium, Neocallimastix frontalis + Methanobrevibacter gottschalkii, Orpinomyces joyonii + Methanobrevibacter ruminantium, Caecomyces communis + Methanobrevibacter ruminantium, and Caecomyces communis + Methanobrevibacter millerae. Among the 31 co-cultures, during the 5-day incubation, the N. frontalis + M. gottschalkii co-culture YakQH5 degraded 59.0%–68.1% of the dry matter (DM) and 49.5%–59.7% of the neutral detergent fiber (NDF) of wheat straw, corn stalk, rice straw, oat straw and sorghum straw to produce CH4 (3.0–4.6 mmol/g DM) and acetate (7.3–8.6 mmol/g DM) as end-products. Ferulic acid (FA) released at 4.8 mg/g DM on corn stalk and p-coumaric acid (PCA) released at 11.7 mg/g DM on sorghum straw showed the highest values, with the following peak values of enzyme activities: xylanase at 12,910 mU/mL on wheat straw, ferulic acid esterase (FAE) at 10.5 mU/mL on corn stalk, and p-coumaric acid esterase (CAE) at 20.5 mU/mL on sorghum straw. The N. frontalis + M. gottschalkii co-culture YakQH5 from Qinghai yaks represents a new efficient combination for lignocellulose biodegradation, performing better than previously reported fungus–methanogen co-cultures from the digestive tract of ruminants.

Storing manure is an essential aspect of nutrient management on dairy farms. It presents the opportunity to use manure efficiently as a fertilizer in crop and pasture production. Typically, the manure storages are constructed as earthen, concrete, or steel-based structures. However, storing manure can potentially emit aerial pollutants to the atmosphere, including nitrogen and greenhouse gases, through microbial and physicochemical processes. We have characterized the composition of the microbiome in two manure storage structures, a clay-lined earthen pit and an aboveground concrete storage tank, on commercial dairy farms, to discern the nitrogen transformation processes, and thereby, inform the development of mitigation practices to preserve the value of manure. First, we analyzed the 16S rRNA-V4 amplicons generated from manure samples collected from several locations and depths (0.3, 1.2, and 2.1–2.75 m below the surface) of the storages, identifying a set of Amplicon Sequence Variant (ASVs) and quantifying their abundances. Then, we inferred the respective metabolic capabilities. These results showed that the manure microbiome composition was more complex and exhibited more location-to-location variation in the earthen pit than in the concrete tank. Further, the inlet and a location with hard surface crust in the earthen pit had unique consortia. The microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to gaseous compounds. However, the microbial conversion of nitrate to gaseous N2, NO, and N2O via denitrification and to stable ammonia via dissimilatory nitrite reduction seemed possible; minor quantities of nitrate was present in manure, potentially originating from oxidative processes occurring on the barn floor. The nitrate-transformation linked ASVs were more prevalent at the near-surface locations and all depths of the inlet. Anammox bacteria and archaeal or bacterial autotrophic nitrifiers were not detected in either storage. Hydrogenotrophic Methanocorpusculum species were the primary methanogens or methane producers, exhibiting higher abundance in the earthen pit. These findings suggested that microbial activities were not the main drivers for nitrogen loss from manure storage, and commonly reported losses are associated with the physicochemical processes. Finally, the microbiomes of stored manure had the potential to emit greenhouse gases such as NO, N2O, and methane. [ABSTRACT FROM AUTHOR]

The question of whether environmental heat energy could be utilized as a source of energy for biological metabolism is the center of this exploratory research. In 1979, this author postulated a hypothesis for the existence of thermotrophs that could isothermally utilize environmental heat energy as a source of their energy on Earth. According to this hypothesis, the thermotrophs could be the first primitive forms of life in the early Earth environment. The chemotrophs and phototrophs that we currently are all well familiar with might have been evolved somehow from the primitive thermotrophs. Furthermore, all the organisms currently regarded as the "chemotrophs" and "phototrophs" could actually be the mixed trophy types containing thermotrophic features: thermo-chemotrophs and thermo-phototrophs. Energetic analysis with the thermodynamic first law indicated that the anaerobic acetate-utilizing methanogenic archaea Methanosarcina could be a "living fossil specimen" of the thermotrophs. Experiments with enriched acetate-utilizing methanogen, including Methanosarcina, has, for the first time, demonstrated that their anaerobic metabolism was indeed associated with isothermal environmental heat utilization, resulting in their liquid culture temperature to change (decrease) by about -0.10 oC and sometimes drop by as much as -0.45 oC observed in the experiments. The mean temperature change (drop) was -0.25 ±0.06 oC. [ABSTRACT FROM AUTHOR]

Direct interspecies electron transfer (DIET), which does not involve mediation by electron carriers, is realized by the addition of conductive materials to an anaerobic digester, which then activates syntrophism between acetogenic and methanogenic microorganisms. This study aimed to investigate the effect of the addition of two conductive materials, zero-valent iron (ZVI) and magnetite, on the methane production and microbial consortium via DIET in the anaerobic digestion of food wastewater. The operation of a batch reactor for food wastewater without the addition of the conductive materials yielded a biochemical methane potential (Bu), maximum methane production rate (Rm), and lag phase time (λ) of 0.380 Nm3 kg−1-VSadded, 15.73 mL day−1, and 0.541 days, respectively. Upon the addition of 1.5% ZVI, Bu and Rm increased significantly to 0.434 Nm3 kg−1-VSadded and 19.63 mL day−1, respectively, and λ was shortened to 0.065 days. Simultaneously, Methanomicrobiales increased from 26.60% to 46.90% and Methanosarcinales decreased from 14.20% to 1.50% as the ZVI input increased from 0% to 1.50%. Magnetite, at an input concentration of 1.00%, significantly increased the Bu and Rm to 0.431 Nm3 kg−1-VSadded and 18.44 mL day−1, respectively. However, although magnetite improves the efficiency of methanogenesis via DIET, the effect thereof on the methanogen community remains unclear. [ABSTRACT FROM AUTHOR]

Constructed wetlands (CWs) are artificial systems that use natural processes to treat wastewater containing organic pollutants. This approach has been widely applied in both developing and developed countries worldwide, providing a cost-effective method for industrial wastewater treatment and the improvement of environmental water quality. However, due to the large organic carbon inputs, CWs is produced in varying amounts of CH4 and have the potential to become an important contributor to global climate change. Subsequently, research on the mitigation of CH4 emissions by CWs is key to achieving sustainable, low-carbon dependency wastewater treatment systems. This review evaluates the current research on CH4 emissions from CWs through bibliometric analysis, summarizing the reported mechanisms of CH4 generation, transfer and oxidation in CWs. Furthermore, the important environmental factors driving CH4 generation in CW systems are summarized, including: temperature, water table position, oxidation reduction potential, and the effects of CW characteristics such as wetland type, plant species composition, substrate type, CW-coupled microbial fuel cell, oxygen supply, available carbon source, and salinity. This review provides guidance and novel perspectives for sustainable and effective CW management, as well as for future studies on CH4 reduction in CWs. [ABSTRACT FROM AUTHOR]

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.

Nitrogenase iron (Fe) proteins reduce CO2 to CO and/or hydrocarbons under ambient conditions. Here, we report a 2.4-Å crystal structure of the Fe protein from Methanosarcina acetivorans (MaNifH), which is generated in the presence of a reductant, dithionite, and an alternative CO2 source, bicarbonate. Structural analysis of this methanogen Fe protein species suggests that CO2 is possibly captured in an unactivated, linear conformation near the [Fe4S4] cluster of MaNifH by a conserved arginine (Arg) pair in a concerted and, possibly, asymmetric manner. Density functional theory calculations and mutational analyses provide further support for the capture of CO2 on MaNifH while suggesting a possible role of Arg in the initial coordination of CO2 via hydrogen bonding and electrostatic interactions. These results provide a useful framework for further mechanistic investigations of CO2 activation by a surface-exposed [Fe4S4] cluster, which may facilitate future development of FeS catalysts for ambient conversion of CO2 into valuable chemical commodities.IMPORTANCE This work reports the crystal structure of a previously uncharacterized Fe protein from a methanogenic organism, which provides important insights into the structural properties of the less-characterized, yet highly interesting archaeal nitrogenase enzymes. Moreover, the structure-derived implications for CO2 capture by a surface-exposed [Fe4S4] cluster point to the possibility of developing novel strategies for CO2 sequestration while providing the initial insights into the unique mechanism of FeS-based CO2 activation.

BackgroundAs 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.ResultsHere, 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.ConclusionsThe findings provide an ecological and evolutionary framework for Woesearchaeota at a global scale and indicate their potential ecological roles, especially in methanogenesis.

Chlorine-containing organic compounds are important industrial solvents but are severely toxic to humans and the environment. Because of their stability and dense non-aqueous phase, they barely biodegrade when released into soil and groundwater systems and may significantly impact the soil environment. One bioremediation approach, biostimulation, adds rate-limiting nutrients to the soil to promote biodegradation processes, but the link remains unclear between stimulated microbial communities and nutrient inputs in anaerobic environments. This study evaluated changes to soil microbial communities in 1,2-dichlorobenzene (1,2-DCB)-contaminated soil under diverse carbon (C) and nutrient conditions. The experiments used anaerobic microcosms that were amended with various C and nutrient sources, and the analysis employed real-time PCR and next-generation sequencing. The results reveal that methanogens may have high resistance to 1,2-DCB in oligotrophic conditions. However, bacteria such as Pseudomonas, Sphingomonas, and some uncultured genera in the Xanthomonadaceae, Pseudomonadaceae, and Bacillales families can resist high 1,2-DCB concentrations when N and P sources are available. These results indicate that external N and P sources are important for stabilizing soil microbial communities and their processes in contaminant sites. [ABSTRACT FROM AUTHOR]