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.

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].

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.

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 β-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.
OBJECTIVE: 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.

This study presents a bidirectional flow tidal bioreactor designed to enhance H2-driven CO2 biomethanation. The bioreactor alternated biofilms between immersion in nutrient solution and exposure to H2/CO2, creating alternating dry and wet states. This tidal operation minimized liquid film thickness during dry periods and ensured uniform nutrient distribution during wet periods. Bidirectional H2/CO2 supply was used to reduce biofilm thickness heterogeneity across the reactor height. CO2 biomethanation remained stable with an empty bed residence time of 9.7 min, achieving a methane (CH4) formation rate of 26.8 Nm3 CH4/(m3·d). The product gas contained 95.0 ± 2.5 % CH4, with a H2/CO2 conversion efficiency of 90.8 %. Tidal operation mitigated the buildup of dissolved and suspended organics, such as organic acids and detached biofilms. Dominant bacteria in biofilms included fermentative species like Petrimonas and H2-utilizing homoacetogens like Sporomusa. Enriched hydrogenotrophic methanogens, particularly Methanobacterium, were observed. Overall, this study highlights the bioreactor\'s effectiveness in improving CO2 biomethanation.

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.

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, M. stummii) and the isolates from millipede hindguts into a new genus, Methanolapillus (M. africanus, M. millepedarum, 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, 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.

Two protocols involving batch cultures were used to investigate the bioaugmentation of methane production by Pecoramyces ruminantium, and Methanobrevibacter thaueri. Protocol I examined the effect of altering the proportion of the microbial constituents in inoculum on alfalfa stalk fermentations and showed a 25 % improvement in dry matter loss in cultures where the inoculum contained just 30 % of co-culture and 70 % of fungal monoculture. Protocol II involved consecutive cultures and alternating inoculations. This protocol resulted in 17-22 mL/g DM methane production with co-cultures a 30 % increase in methane relative to the fungal monoculture. Both protocols indicate that the co-culture rapidly dominated and was more resilient than the monoculture. Synergistic interaction between fungus and methanogen, promoted more efficient lignocellulose degradation and higher methane yield. This study highlighted the potential of microbial co-cultures for enhancing methane production from lignocellulosic biomass, offering a promising bioaugmentation strategy for improving biogas yields and waste valorization.

With the increasing prominence of the global energy problem, socioeconomic activities have been seriously affected. Biofuels, as a renewable source of energy, are of great significance in promoting sustainable development. In this study, batch anaerobic digestion (AD) of frass (swine manure after bioconversion by black soldier fly larvae) and co-digestion with corn straw after the addition of iron oxide (Fe3O4) nanoparticles is investigated, as well as the start-up period without inoculation. The biochemical methane potential of pure frass was obtained using blank 1 group and after the addition of various sizes of Fe3O4 nanoparticles for 30 days period, and similarly, the digestion of frass with straw (blank 2) and after the addition of various sizes of Fe3O4 nanoparticles for 61 days period. The results showed that the average gas production was 209.43 mL/gVS, 197.68 mL/gVS, 151.85 mL/gVS, and 238.15 mL/gVS for the blank, ~176 nm, ~164 nm, and ~184 nm, respectively. The average gas production of frass with straw (blank 2) was 261.64 mL/gVS, 259.62 mL/gVS, 241.51 mL/gVS, and 285.98 mL/gVS for blank 2, ~176 nm, ~164 nm, and ~184 nm, respectively. Meanwhile, the accumulated methane production of the ~184 nm group was 2312.98 mL and 10,952.96 mL, respectively, which significantly increased the biogas production compared to the other groups. The methanogenic results of the frass (30 days) indicated that Methanocorpusculum, Methanosarcina, and Methanomassiliicoccus are the important methanogenic species in the AD reactor, while the microbial diversity of the ~184 nm group was optimal, which may be the reason for the high gas production of ~184 nm.

Conductive materials (CM) enhance methanogenesis, but there is no clear correlation between conductivity and faster methane production (MP) rates. We investigated if MP by pure cultures of methanogens (Methanobacterium formicicum, Methanospirillum hungatei, Methanothrix harundinacea and Methanosarcina barkeri) is affected by CM (activated carbon (AC), magnetite), and other sustainable alternatives (sand and glass beads, without conductivity, and zeolites (Zeo)). The significant impact of the materials was on M. formicicum as MP was significantly accelerated by non-CM (e.g., sand reduced the lag phase (LP) duration by 48 %), Zeo and AC (LP reduction in 71% and 75 %, respectively). Conductivity was not correlated with LP reduction. Instead, silicon content in the materials was inversely correlated with the time required for complete MP, and silicon per se stimulated M. formicicum\'s activity. These findings highlight the potential of using non-CM silicon-containing materials in anaerobic digesters to accelerate methanogenesis.