Methanothermobacter thermautotrophicus and Alternative Methanogens: Archaea-Based Production.

Methanogenic archaea convert bacterial fermentation intermediates from the decomposition of organic material into methane. This process has relevance in the global carbon cycle and finds application in anthropogenic processes, such as wastewater treatment and anaerobic digestion. Furthermore, methanogenic archaea that utilize hydrogen and carbon dioxide as substrates are being employed as biocatalysts for the biomethanation step of power-to-gas technology. This technology converts hydrogen from water electrolysis and carbon dioxide into renewable natural gas (i.e., methane). The application of methanogenic archaea in bioproduction beyond methane has been demonstrated in only a few instances and is limited to mesophilic species for which genetic engineering tools are available. In this chapter, we discuss recent developments for those existing genetically tractable systems and the inclusion of novel genetic tools for thermophilic methanogenic species. We then give an overview of recombinant bioproduction with mesophilic methanogenic archaea and thermophilic non-methanogenic microbes. This is the basis for discussing putative products with thermophilic methanogenic archaea, specifically the species Methanothermobacter thermautotrophicus. We give estimates of potential conversion efficiencies for those putative products based on a genome-scale metabolic model for M. thermautotrophicus.

Fluorescently probing anaerobic digester sludge: Measuring single-cell anabolic activity in methanogens (Methanosarcina and Methanothermobacter) with deuterium-labeled Raman analysis.

This study addresses the challenge of obtaining in situ information on substrate utilization rates for individual microbial species in complex microbial communities such as anaerobic digester sludge. To overcome this hurdle, a novel approach combining doubly-labelled deuterium, fluorescence in situ hybridization (FISH) and Raman microspectroscopy was developed. The method enables quantitative determination of anabolic heavy hydrogen incorporation into FISH-targeted, exemplified by methanogenic cells from the genera Methanosarcina and Methanothermobacter. The deuterium incorporation rates ascertained by Raman red-shifting of C-Hx vibrational region to C-Dx vibrations, quantified through Raman peak area ratios, were compared for different carbon sources. Methanosarcina exhibited highest kinetic rates with acetate and propionate, while Methanothermobacter demonstrated faster incorporation under acetate and methanol supplementation. This groundbreaking study demonstrates the feasibility of obtaining quantitative metabolic rate information at a single-cell level using deuterium, FISH probes, and Raman microspectroscopy.

Hydrochemical gradients driving extremophile distribution in saline and brine groundwater of southern Poland.

Extreme environments, such as highly saline ecosystems, are characterised by a limited presence of microbial communities capable of tolerating and thriving under these conditions. To better understand the limits of life and its chemical and microbiological drivers, highly saline and brine groundwaters of Na-Cl and Na-Ca-Cl types with notably diverse SO4 contents were sampled in water intakes and springs from sedimentary aquifers located in the Outer Carpathians and the Carpathian Foredeep basin and its basement in Poland. Chemical and microbiological methods were used to identify the composition of groundwaters, determine microbial diversity, and indicate processes controlling their distribution using multivariate statistical analyses. DNA sequencing targeting V3-V4 and V4-V5 gene regions revealed a predominance of Proteobacteriota, Methanobacteria, Methanomicrobia, and Nanoarchaea in most of the water samples, irrespective of their geological context. Despite the sample-size constraint, redundancy analysis employing a compositional approach to hydrochemical predictors identified Cl/SO4 and Cl/HCO3 ratios, and specific electrical conductivity, as key gradients shaping microbial communities, depending on the analysed gene regions. Analysis of functional groups revealed that methanogenesis, sulphate oxidation and reduction, and the nitrogen cycle define and distinguish the halotolerant communities in the samples. These communities are characterised by an inverse relationship between methanogens and sulphur-cycling microorganisms.

The origin of methyl group in methanogen-mediated mercury methylation: From the Wolfe cycle.

Methylmercury (MeHg) is a bioaccumulating neurotoxin mainly produced by anaerobic microorganisms, with methanogen being one of the important methylators. A critical aspect for understanding the mechanism for microbial mercury (Hg) methylation is the origin of the methyl group. However, the origin of methyl group in methanogen-mediated Hg methylation remains unclear. This study aims to identify the source of methyl group for MeHg synthesis in methanogens. Our study revealed that Hg methylation in Methanospirillum hungatei JF-1 is closely related to methanogenesis process, according to the results of proteomic study and substrate limitation study. Next, we proved that nearly all methyl group in MeHg derives from the Wolfe cycle in this species, rather than the previously demonstrated acetyl-coenzyme A pathway, based on the results of 13C labeling study. We then proposed the Wolfe cycle-dependent Hg methylation mechanism in this species. Further genome analyses and 13C labeling experiments indicated that the involvement of the Wolfe cycle in Hg methylation is probably a universal feature among Hg-methylating methanogens. These findings reveal a unique Hg methylation mechanism in methanogens. Our study broadens the carbon substrates and controlling factors for MeHg synthesis in the environment, which can inform the prediction of MeHg production potential and remediation strategies for MeHg contamination.

Beyond bacteria: Role of non-bacterial gut microbiota species in inflammatory bowel disease and colorectal cancer progression.

This letter emphasizes the need to expand discussions on gut microbiome's role in inflammatory bowel disease (IBD) and colorectal cancer (CRC) by including the often-overlooked non-bacterial components of the human gut flora. It highlights how viral, fungal and archaeal inhabitants of the gut respond towards gut dys-biosis and contribute to disease progression. Viruses such as bacteriophages target certain bacterial species and modulate the immune system. Other viruses found associated include Epstein-Barr virus, human papillomavirus, John Cunningham virus, cytomegalovirus, and human herpes simplex virus type 6. Fungi such as Candida albicans and Malassezia contribute by forming tissue-invasive filaments and producing inflammatory cytokines, respectively. Archaea, mainly metha-nogens are also found altering the microbial fermentation pathways. This corres-pondence, thus underscores the significance of considering the pathological and physiological mechanisms of the entire spectrum of the gut microbiota to develop effective therapeutic interventions for both IBD and CRC.

Stand age-related effects of mangrove on archaeal methanogenesis in sediments: Community assembly and co-occurrence patterns.

Mangrove sediment is a key source of methane emissions; however, archaea community structure dynamics and methanogenesis activities during long-term mangrove restoration remain unclear. In this study, microcosm incubations revealed a substantial reduction in microbial-mediated methane production potential from mangrove sediments with increasing stand age; methane production rates decreased from 0.42 ng g-1 d-1 in 6-year-old stands to 0.23 ng g-1 d-1 in 64-year-old stands. High-throughput sequencing revealed a reduction in community diversity because of specific microorganism colonization and species loss, notably a decline in the relative abundance of Bathyarchaeia in sediments of 64-year-old stands. In addition, mangrove sediments, especially those in older stands (20- and 64-year-old), had more complex and stable co-occurrence microbial networks than mudflats. Furthermore, archaea community assembly in older stands was dominated by stochastic processes wherein dispersal limitation was prominent, and that in younger stands (6- and 12-year-old) was driven by deterministic processes. The proportion of dispersal limitation of Bathyarchaeia and traditional methanogens in sediment decreased with an increase in stand age. Quantitative polymerase chain reaction analysis confirmed a decrease in Bathyarchaeia (from 3.50 to 0.54 copies g-1) and mcrA gene (from 3.83 to 0.25 copies g-1) abundance in mangrove sediments with an increase in stand age. These findings demonstrate the critical role of Bathyarchaeia in methanogenesis; the decline in microbial interactions and abundance, and the reduced proportion of dispersal limitation of Bathyarchaeia and traditional methanogens collectively contributed to the mitigation of microbial-mediated methane production potential in older mangrove stands.

The biological mechanism of a lower carbon/nitrogen ratio increases methane emissions during vegetable waste composting.

The initial carbon/nitrogen (C/N) ratio is one of the most important factors impacting composting processes, such as methane (CH4) emissions. However, the effects of the C/N ratio on CH4 emissions and the associated biological mechanisms during vegetable waste composting are largely unknown. In this study, a lab-scale experiment was conducted to investigate the effects of different C/N ratios on CH4 emissions and the mechanisms associated with methane-metabolizing microorganisms (methanogens and methanotrophs) during capsicum straw composting. The initial C/N ratios were set to 18, 30 and 50 to simulate the low (L), medium (M) and high (H) C/N ratios, respectively. The results showed that CH4 emissions were mainly concentrated in the thermophilic phase and that the cumulative CH4 emissions were significantly greater in the L treatment than in the M and H treatments by 10.8 and 15.4 times, respectively. During the methanogenic process, the relative abundance of the dominant genus Methanoculleus (47.59 % ~ 76.92 %) was higher than in the L treatment than in the M and H treatments at the thermophilic and maturation stages, and the Chao1 index and the mcrA gene abundance followed the order of L > M > H at each composting stage. During the methanotrophic process, the dominant genus unclassified_d_bacteria (51.3 % ~ 91.87 %), Chao1 index, pmoA gene abundance and CO2 emissions were in the order of L > M > H at each composting stage. This pattern suggests that a lower C/N ratio simultaneously enhanced CH4 production and oxidation. A structural equation model further revealed that the methanogenic community, which was driven directly by the relative contents of hemicellulose and cellulose in the substrates, as indicated by the C/N ratio, made greater contributions to CH4 emissions than did the methanotrophic community. In conclusion, a lower C/N ratio increased CH4 emissions mainly by regulating the population and composition of methanogen community.

Transcriptional response of Methanosarcina acetivorans to repression of the energy-conserving methanophenazine: CoM-CoB heterodisulfide reductase enzyme HdrED.

Methane-producing archaea are key organisms in the anaerobic carbon cycle. These organisms, also called methanogens, grow by converting substrate to methane gas in a process called methanogenesis. Previous research showed that the reduction of the terminal electron acceptor is the rate-limiting step in methanogenesis by Methanosarcina acetivorans. In order to gain insight into how the cells sense and respond to the availability of the terminal electron acceptor, we designed an experiment to deplete cells of the essential terminal oxidase enzyme, HdrED. We found that the depletion of HdrED in vivo results in a higher abundance of transcripts for methyltransferases (mtaC2, mtaB3, mtaC3), coenzyme B biosynthesis, C1 metabolism, and pyrimidine compounds. In most cases, these changes were distinct from transcript abundance changes observed during the transition from exponential growth to stationary phase cultures. These data implicate the methylotrophic methanogenesis regulator MsrC (MA4383) in CoM-S-S-CoB heterodisulfide sensing and indicate cells have a specific mechanism to sense intracellular ratio of CoM-S-S-CoB, coenzyme M, and coenzyme B thiols and further suggest transcripts encoding translation and methanogenesis functions are controlled by feed-forward regulation depending on substrate availability.IMPORTANCEMethanosarcina is an emerging model archaeon and synthetic biology platform for the production of renewable energy and sustainable chemicals to reduce dependence on petroleum. Research into metabolic networks and gene regulation in this organism and other methanogens will inform genome-scale metabolic modeling and microbial function prediction in uncultured or non-model anaerobes and archaea. This study suggests methanogens use unknown mechanisms to efficiently couple methanogenesis to gene regulation via CoM-S-S-CoB and ATP availability.

Methanobrevibacter smithii cell variants in human physiology and pathology: A review.

Methanobrevibacter smithii (M. smithii), initially isolated from human feces, has been recognised as a distinct taxon within the Archaea domain following comprehensive phenotypic, genetic, and genomic analyses confirming its uniqueness among methanogens. Its diversity, encompassing 15 genotypes, mirrors that of biotic and host-associated ecosystems in which M. smithii plays a crucial role in detoxifying hydrogen from bacterial fermentations, converting it into mechanically expelled gaseous methane. In microbiota in contact with host epithelial mucosae, M. smithii centres metabolism-driven microbial networks with Bacteroides, Prevotella, Ruminococcus, Veillonella, Enterococcus, Escherichia, Enterobacter, Klebsiella, whereas symbiotic association with the nanoarchaea Candidatus Nanopusillus phoceensis determines small and large cell variants of M. smithii. The former translocate with bacteria to induce detectable inflammatory and serological responses and are co-cultured from blood, urine, and tissular abscesses with bacteria, prototyping M. smithii as a model organism for pathogenicity by association. The sources, mechanisms and dynamics of in utero and lifespan M. smithii acquisition, its diversity, and its susceptibility to molecules of environmental, veterinary, and medical interest still have to be deeply investigated, as only four strains of M. smithii are available in microbial collections, despite the pivotal role this neglected microorganism plays in microbiota physiology and pathologies.

Phenotypic traits related to methane emissions from Holstein dairy cows challenged by low or high forage proportion.

Limited literature is available identifying phenotypical traits related to enteric methane (CH4) production from dairy cows, despite its relevance in relation to breeding for animals with a low CH4 yield (g/kg DMI), and the derived consequences hereof. This study aimed to investigate the relationships between CH4 yield and different animal phenotypes when 16 2nd parity dairy cows, fitted with a ruminal cannula, were fed 2 diets differing in forage:concentrate ratio in a crossover design. The diets had either a low forage proportion (35% on DM basis, F35) or a high forage proportion (63% on DM basis, F63). Gas exchange was measured by means of indirect calorimetry. Spot samples of feces were collected, and indigestible NDF (INDF) was used as an internal marker to determine total-tract digestibility. In addition, ruminal evacuations, monitoring of chewing activity, determination of ruminal VFA concentration, analysis of relative abundance of methanogens, and measurement of liquid passage rate were performed. Statistical differences were analyzed by a linear mixed model with diet, days in milk, and period as fixed effects, and cow as random effect. The random cow estimates (RCE) were extracted from the model to get the Pearson correlations (r) between RCE of CH4 yield with RCE of all other variables measured, to identify possible phenotypes related to CH4 yield. Significant correlations were observed between RCE of CH4 yield and RCE of OM digestibility (r = 0.63) and ruminal concentration of valeric acid (r = -0.61), acetic acid (r = 0.54), ammonium (r = 0.55), and lactic acid (r = ‒0.53). Additionally, tendencies were observed for correlations between RCE of CH4 yield and RCE of H2 yield in g/kg DM (r = 0.47, P = 0.07), and ruminal isobutyric acid concentration (r = 0.43, P = 0.09). No correlations were observed between RCE of CH4 yield and RCE of ruminal pool sizes, milk data, urinary measurements, or chewing activity. Cows had a lower DMI and ECM, when they were fed F63 compared with F35. Cows fed F63 had higher NDF digestibility, CH4 emissions (g/d, g/kg of DMI, and g/kg of ECM), ruminal concentration of acetic acid, ruminal pH, degradation rate of digestible NDF (DNDF, %/h), and longer rumen retention time (h). Also, rumination and total chewing time (min/kg DMI) were higher for cows fed F63. The results in the present study emphasize the positive relation between cow's ability to digest OM and their CH4 emissions. The derived consequences of breeding for lower CH4 emission might be cows with lower ability to digest OM, but more studies are warranted for further documentation of this relationship.

Carbon dioxide capture, sequestration, and utilization models for carbon management and transformation.

The elevated level of carbon dioxide in the atmosphere has become a pressing concern for environmental health due to its contribution to climate change and global warming. Simultaneously, the energy crisis is a significant issue for both developed and developing nations. In response to these challenges, carbon capture, sequestration, and utilization (CCSU) have emerged as promising solutions within the carbon-neutral bioenergy sector. Numerous technologies are available for CCSU including physical, chemical, and biological routes. The aim of this study is to explore the potential of CCSU technologies, specifically focusing on the use of microorganisms based on their well-established metabolic part. By investigating these biological pathways, we aim to develop sustainable strategies for climate management and biofuel production. One of the key novelties of this study lies in the utilization of microorganisms for CO2 fixation and conversion, offering a renewable and efficient method for addressing carbon emissions. Algae, with its high growth rate and lipid contents, exhibits CO2 fixation capabilities during photosynthesis. Similarly, methanogens have shown efficiency in converting CO2 to methane by methanogenesis, offering a viable pathway for carbon sequestration and energy production. In conclusion, our study highlights the importance of exploring biological pathways, which significantly reduce carbon emissions and move towards a more environmentally friendly future. The output of this review highlights the significant potential of CCSU models for future sustainability. Furthermore, this review has been intensified in the current agenda for reduction of CO2 at considerable extends with biofuel upgrading by the microbial-shift reaction.

Editing microbes to mitigate enteric methane emissions in livestock.

Livestock production significantly contributes to greenhouse gas (GHG) emissions particularly methane (CH4) emissions thereby influencing climate change. To address this issue further, it is crucial to establish strategies that simultaneously increase ruminant productivity while minimizing GHG emissions, particularly from cattle, sheep, and goats. Recent advancements have revealed the potential for modulating the rumen microbial ecosystem through genetic selection to reduce methane (CH4) production, and by microbial genome editing including CRISPR/Cas9, TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), RNA interference (RNAi), Pime editing, Base editing and double-stranded break-free (DSB-free). These technologies enable precise genetic modifications, offering opportunities to enhance traits that reduce environmental impact and optimize metabolic pathways. Additionally, various nutrition-related measures have shown promise in mitigating methane emissions to varying extents. This review aims to present a future-oriented viewpoint on reducing methane emissions from ruminants by leveraging CRISPR/Cas9 technology to engineer the microbial consortia within the rumen. The ultimate objective is to develop sustainable livestock production methods that effectively decrease methane emissions, while maintaining animal health and productivity.

The role of monoammonium glycyrrhizinate as a methane inhibitor to limit the rumen methane emissions of Karakul sheep.

Methane (CH4) from ruminant production systems produces greenhouse gases that contribute to global warming. Our goal was to determine whether monoammonium glycyrrhizinate could inhibit CH4 emissions over the long term without affecting animal performance and immune indices in Karakul sheep. This study aimed to assess the effects of medium-term (60 days) addition of monoammonium glycyrrhizinate on growth performance, apparent digestibility, CH4 emissions, methanogens, fibre-degrading bacteria and blood characteristics in Karakul sheep. Twelve male Karakul sheep (40.1 ± 3.59 kg) with fistula were randomly divided into two groups (n = 6): the Control group received a basal diet + the same volume of distilled water (30 ml) and the Treatment group received a basal diet + 8.75 g/kg monoammonium glycyrrhizinate injected via fistula. The adaptation stage was 15 days, and the measurement stage was 60 days. The sampling during the measurement stage was divided into two stages, stage I (1 ∼ 30 d) and stage II (31 ∼ 60 d). The results showed that monoammonium glycyrrhizinate significantly reduced the relative abundance of Bacteroides caccae, daily CH4 emission and protozoa population, significantly increased the relative abundance of Lachnospiraceae bacterium AD3010, Lachnospiraceae bacterium FE2018, Lachnospiraceae bacterium NK3A20, Lachnospiraceae bacterium NK4A179 and Lachnospiraceae bacterium V9D3004 in stage I (P < 0.05); significantly increased the relative abundance of Lachnospiraceae bacterium AD3010, but significantly decreased the relative abundance of Lachnospiraceae bacterium NK4A179 and Lachnospiraceae bacterium C6A11 in stage II (P < 0.05). Therefore, monoammonium glycyrrhizinate could be used as a CH4 inhibitor to limit the rumen CH4 emissions of Karakul sheep in short-term period (30 days) without affecting the growth performance, fibre digestibility and blood parameters.

A promoter-RBS library for fine-tuning gene expression in Methanosarcina acetivorans.

Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter-RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the ß-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter-RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter-RBS combinations. Our promoter-RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds. IMPORTANCE: Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for M. acetivorans. Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.

Metagenomes, metagenome-assembled genomes, and metatranscriptomes from a chlorinated ethene-dechlorinating culture amended with biochar pyrolyzed at different temperatures.

We investigated the effects of biochar and pyrolysis temperature on a chlorinated ethene-dechlorinating anaerobic consortium. Sequencing of nucleic acids from suspended and biochar-attached cells yielded 9 metagenomes, 122 metagenome-assembled genomes, and 18 metatranscriptomes that provide insights into the structure, function, activity, and interactions of the dehalogenating consortium with biochar.

Role of methanogenesis and methanotrophy in CH4 fluxes from rice paddies under elevated CO2 concentration and elevated temperature.

The differential responses of methanogenesis and methanotrophy to elevated carbon dioxide concentrations ([CO2]) (e[CO2]) and elevated temperature ([T]) (e[T]) may lead to dramatic changes in the response of CH4 emissions from rice paddies to global warming. In this study, we systematically investigated the responses and mechanisms of CH4 flux from rice paddies to e[CO2] and e[T] based on the production and oxidation of CH4. The CH4 flux, soil properties, and soil methanogenesis and methanotrophy were observed under CK (ambient [CO2] + ambient [T]), EC (e[CO2] by 200 µmol mol-1 + ambient [T]), ET (ambient [CO2] + e[T] by 2 °C), and ECT (e[CO2] by 200 µmol mol-1 + e[T] by 2 °C) treatments. The results revealed that EC, ET, and ECT significantly increased the cumulative amount of CH4 (CAC) in the rice paddies by 10.63, 15.20, and 11.77 kg ha-1, respectively, compared with CK. ECT increased the CAC in the rice paddies by 1.14 kg ha-1 compared with EC. Moreover, EC, ET, and ECT significantly enhanced the methane production potential (MPP) and methane oxidation potential (MOP) and tended to increase the mcrA gene abundance of the methanogens. EC tended to prompt the pmoA gene abundance of the methanotrophs, but the effect of ET on the pmoA gene abundance was less consistent across the growth stages. ECT significantly decreased the relative abundances of Methanosarcina and Methylocystis (Type II) by 4.9 % and 14.2 %, respectively, while it increased the relative abundance of Methylosarcina (Type I) by 24.0 % compared with CK. Overall, the increased MPP/MOP, mcrA/pmoA, and microbial biomass carbon under climate change increased the CH4 flux from the rice paddies. The contribution of e[CO2] to the CH4 flux was significantly enhanced by e[T], which could further exacerbate the risk of global climate change induced by e[CO2].

Methylotrophic substrates stimulated higher methane production than competitive substrates in mangrove sediments.

Although mangrove forests can uptake atmospheric CO2 and store carbon as organic matter called "blue carbon", it is also an important natural source of greenhouse gas methane. Methanogens are major contributors to methane and play important roles in the global carbon cycle. However, our understanding of the key microbes and metabolic pathways responsible for methanogenesis under specific substrates in mangrove sediments is still very limited. Here, we set an anaerobic incubation to evaluate the responses of methanogens in mangrove sediments from South China to the addition of diverse methanogenic substrates (H2/CO2, acetate, trimethylamine (TMA), and methanethiol (MT)) and further investigated the dynamics of the whole microbial community. Our results showed that diverse substrates stimulated methanogenic activities at different times. The stimulation of methanogenesis was more pronounced at early and late periods by the addition of methylotrophic substrates TMA and MT, respectively. The amplicon sequencing analysis showed that genus Methanococcoides was mainly responsible for TMA-utilized methanogenesis in mangrove sediment, while the multitrophic Methanococcus was most abundant in H2/CO2 and MT treatments. Apart from that, the bacteria enrichments of Syntrophotalea, Clostridium_sensu_stricto_12, Fusibacter in MT treatments might also be associated with the stimulation of methane production. In addition, the metagenomic analysis suggested that Methanosarcinaceae was also one of the key methanogens in MT treatments with different genomic information compared to that in TMA treatments. Finally, the total relative abundances of methanogenesis-related genes were also highest in TMA and MT treatments. These results will help advance our understanding of the contributions of different methanogenesis pathways and methanogens to methane emissions in mangrove sediments.

Experimental simulation of H2 coinjection via a high-pressure reactor with natural gas in a low-salinity deep aquifer used for current underground gas storage.

If dihydrogen (H2) becomes a major part of the energy mix, massive storage in underground gas storage (UGS), such as in deep aquifers, will be needed. The development of H2 requires a growing share of H2 in natural gas (and its current infrastructure), which is expected to reach approximately 2% in Europe. The impact of H2 in aquifers is uncertain, mainly because its behavior is site dependent. The main concern is the consequences of its consumption by autochthonous microorganisms, which, in addition to energy loss, could lead to reservoir souring and alter the petrological properties of the aquifer. In this work, the coinjection of 2% H2 in a natural gas blend in a low-salinity deep aquifer was simulated in a three-phase (aquifer rock, formation water, and natural gas/H2 mix) high-pressure reactor for 3 months with autochthonous microorganisms using a protocol described in a previous study. This protocol was improved by the addition of protocol coupling experimental measures and modeling to calculate the pH and redox potential of the reactor. Modeling was performed to better analyze the experimental data. As in previous experiments, sulfate reduction was the first reaction to occur, and sulfate was quickly consumed. Then, formate production, acetogenesis, and methanogenesis occurred. Overall, H2 consumption was mainly caused by methanogenesis. Contrary to previous experiments simulating H2 injection in aquifers of higher salinity using the same protocol, microbial H2 consumption remained limited, probably because of nutrient depletion. Although calcite dissolution and iron sulfide mineral precipitation likely occurred, no notable evolution of the rock phase was observed after the experiment. Overall, our results suggested that H2 can be stable in this aquifer after an initial loss. More generally, aquifers with low salinity and especially low electron acceptor availability should be favored for H2 costorage with natural gas.

Effect of biochar addition on the anaerobic digestion of food waste: microbial community structure and methanogenic pathways.

This study assessed the effects of the addition of biochar prepared at 700 °C with different dosages on the anaerobic digestion of food waste. The biochar addition at a concentration of 10.0 g/L increased the cumulative methane yield by 128%, and daily methane production was also significantly promoted. The addition of biochar derived from poplar sawdust significantly increased the relative abundance of dominant bacteria for anaerobic digestion by 85.54-2530% and promoted the degradation of refractory organic matter and the transfer of materials between the hydrolysis and acid production stages. Further analysis has demonstrated that Bathyarchaeia and hydrogenotrophic methanogens were enriched by the biochar addition. Meanwhile, the relative abundances of functional genes, including C5-branched dibasic acid metabolism, and pyruvate metabolism, were increased by 11.38-26.27%. The relative abundances of genes related to major amino acid metabolism, including histidine metabolism, lysine biosynthesis, and phenylalanine, tyrosine, and tryptophan biosynthesis, were increased by 11.96-15.71%. Furthermore, the relative abundances of genes involved in major replication and repair were increased by 14.76-22.76%, and the major folding, sorting, degradation, and translation were increased by 14.47-19.95%, respectively. The relative abundances of genes related to major membrane transport and cell motility were increased by 10.02 and 83.09%, respectively.

Genome reduction in novel, obligately methyl-reducing Methanosarcinales isolated from arthropod guts (Methanolapillus gen. nov. and Methanimicrococcus).

Recent metagenomic studies have identified numerous lineages of hydrogen-dependent, obligately methyl-reducing methanogens. Yet, only a few representatives have been isolated in pure culture. Here, we describe six new species with this capability in the family Methanosarcinaceae (order Methanosarcinales), which makes up a substantial fraction of the methanogenic community in arthropod guts. Phylogenomic analysis placed the isolates from cockroach hindguts into the genus Methanimicrococcus (M. hacksteinii, M. hongohii, and M. stummii) and the isolates from millipede hindguts into a new genus, Methanolapillus (M. africanus, M. millepedarum, and M. ohkumae). Members of this intestinal clade, which includes also uncultured representatives from termites and vertebrates, have substantially smaller genomes (1.6-2.2 Mbp) than other Methanosarcinales. Genome reduction was accompanied by the loss of the upper part of the Wood-Ljungdahl pathway, several energy-converting membrane complexes (Fpo, Ech, and Rnf), and various biosynthetic pathways. However, genes involved in the protection against reactive oxygen species (catalase and superoxide reductase) were conserved in all genomes, including cytochrome bd (CydAB), a high-affinity terminal oxidase that may confer the capacity for microaerobic respiration. Since host-associated Methanosarcinales are nested within omnivorous lineages, we conclude that the specialization on methyl groups is an adaptation to the intestinal environment.