Search Results (34)

Inspired by the active site of methane monooxygenase, we designed a Cu₂O cluster anchored in the six-membered nitrogen cavity of a C₂N monolayer (Cu₂O@C₂N) as a stable and efficient enzyme-like catalyst. Density functional theory (DFT) calculations reveal that the bridged Cu-O-Cu structure within C₂N exhibits strong electronic coupling, which is favorable for methanol formation. Two competing mechanisms—the concerted and radical-rebound pathways—were systematically investigated, with the former being energetically preferred due to lower energy barriers and more stable intermediate states. Furthermore, strain engineering was employed to tune the geometric and electronic structure of the Cu-O-Cu site. Biaxial strain modulates the Cu-O-Cu bond angle, adsorption properties, and d-band center alignment, thereby selectively enhancing the concerted pathway. A volcano-like trend was observed between the applied strain and the methanol formation barrier, with 1% tensile strain yielding the overall energy barrier to methanol formation (ΔG_overall) as low as 1.31 eV. N₂O effectively regenerated the active site and demonstrated strain-responsive kinetics. The electronic descriptor Δε (ε_d − ε_p) captured the structure–activity relationship, confirming the role of strain in regulating catalytic performance. This work highlights the synergy between geometric confinement and mechanical modulation, offering a rational design strategy for advanced C1 activation catalysts. Full article

The effects of fallow season water and straw management on methane (CH₄) emissions during the fallow season and the subsequent rice-growing season are rarely reported, and the underlying microbial mechanisms remain unclear. A field experiment was conducted with four treatments: (1) fields flooded in both the fallow and rice seasons (FF), (2) fields drained in the fallow season and flooded in the rice season (DF), (3) FF with straw retention (FFS), and (4) DF with straw retention (DFS). The CH₄ emissions in fields under different water and straw treatments were monitored using the static closed chamber method. Methanogenic and methanotrophic communities in these fields were examined using terminal restriction fragment length polymorphism (T-RFLP) analysis based on the mcrA gene and pmoA gene encoding methyl coenzyme M reductase and particulate methane monooxygenase, respectively. The results showed that CH₄ emissions were significantly affected by water management, straw retention, season, and their interactions. Over 80% of CH₄ emissions occurred during the rice season. Field drainage during the fallow season reduced CH₄ emissions by 47.0% and 53.8% with and without straw during the rice season, respectively. Water management altered the abundance and composition of methanogens and methanotrophs, whereas the effects of straw retention were less pronounced. The quantitative polymerase chain reaction (qPCR) assay revealed that field drainage in the fallow season decreased the mcrA gene abundance by 30.0% and 23.2% with and without straw in rice season, respectively, and increased the pmoA gene abundance by 108.9% and 213.7% with and without straw in rice season, respectively. CH₄ flux was significantly positively associated with mcrA gene copy number and the ratio of mcrA to pmoA gene copy number, whereas it was significantly negatively correlated with the pmoA gene copy number. Results indicated that fallow drainage greatly decreased CH₄ emission not only during the fallow season but also during the subsequent rice season by altering the community composition of methanogens and methanotrophs. These findings provide scientific insight into the role of water and straw management in controlling CH₄ emissions through microbial community dynamics. Full article

This study extensively analyzed the bacterial information of biofilms and activated sludge in oxic reactors of full-scale moving bed biofilm reactor-integrated fixed-film activated sludge (MBBR-IFAS) systems. The bacterial communities of biofilms and activated sludge differed statistically (R = 0.624, p < 0.01). The denitrifying genera Ignavibacterium, Phaeodactylibacter, Terrimonas, and Arcobacter were more abundant in activated sludge (p < 0.05), while comammox Nitrospira was more abundant in biofilms (p < 0.05), with an average relative abundance of 8.13%. Nitrospira and Nitrosomonas had weak co-occurrence relationships with other genera in the MBBR-IFAS systems. Potential function analysis revealed no differences in pathways at levels 1 and 2 based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) between biofilms and activated sludge. However, in terms of pathways at level 3, biofilms had more potential in 26 pathways, including various organic biodegradation and membrane and signal transportation pathways. In comparison, activated sludge had more potential in only five pathways, including glycan biosynthesis and metabolism. With respect to nitrogen metabolism, biofilms had greater potential for nitrification (ammonia oxidation) (M00528), and complete nitrification (comammox) (M00804) concretely accounted for methane/ammonia monooxygenase (K10944, K10945, and K10946) and hydroxylamine dehydrogenase (K10535). This study provides a theoretical basis for MBBR-IFAS systems from the perspective of microorganisms. Full article

(1) Background: Particulate methane monooxygenase (pMMO) has a strong dependence on the natural electron transfer path and is prone to denaturation, which results in its redox activity centers being unable to transfer electrons with bare electrodes directly and making it challenging to observe an electrochemical response; (2) Methods: Using methanobactin (Mb) as the electron transporter between gold electrodes and pMMO, a bionic interface with high biocompatibility and stability was created. The Mb-AuNPs-modified functionalized gold net electrode as a working electrode, the kinetic behaviors of pMMO bioelectrocatalysis, and the effect of Mb on pMMO were analyzed. The CV tests were performed at different scanning rates to obtain electrochemical kinetics parameters. (3) Results: The values of the electron transfer coefficient (α) and electron transfer rate constant (k_s) are relatively large in test environments containing only CH₄ or O₂. In contrast, in the test environment containing both CH₄ and O₂, the bioelectrocatalysis of pMMO is a two-electron transfer process with a relatively small α and k_s; (4) Conclusions: It was inferred that Mb formed the complex with pMMO. More importantly, Mb not only played a role in electron transfer but also in stabilizing the enzyme structure of pMMO and maintaining a specific redox state. Furthermore, the continuous catalytic oxidation of natural substrate methane was realized. Full article

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains—two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus. Full article

Over the past decade, copper (Cu) has been recognized as a crucial metal in the differential expression of soluble (sMMO) and particulate (pMMO) forms of methane monooxygenase (MMO) through a mechanism referred to as the “Cu switch”. In this study, we used Methylosinus trichosporium OB3b as a model bacterium to investigate the range of Cu concentrations that trigger the expression of sMMO to pMMO and its effect on growth and methane oxidation. The Cu switch was found to be regulated within Cu concentrations from 3 to 5 µM, with a strict increase in the methane consumption rates from 3.09 to 3.85 µM occurring on the 6th day. Our findings indicate that there was a decrease in the fold changes in the expression of methanobactin (Mbn) synthesis gene (mbnA) with a higher Cu concentration, whereas the Ton-B siderophore receptor gene (mbnT) showed upregulation at all Cu concentrations. Furthermore, the upregulation of the di-heme enzyme at concentrations above 5 µM Cu may play a crucial role in the copper switch by increasing oxygen consumption; however, the role has yet not been elucidated. We developed a quantitative assay based on the naphthalene–Molisch principle to distinguish between the sMMO- and pMMO-expressing cells, which coincided with the regulation profile of the sMMO and pMMO genes. At 0 and 3 µM Cu, the naphthol concentration was higher (8.1 and 4.2 µM, respectively) and gradually decreased to 0 µM naphthol when pMMO was expressed and acted as the sole methane oxidizer at concentrations above 5 µM Cu. Using physical protein–protein interaction, we identified seven transporters, three cell wall biosynthesis or degradation proteins, Cu resistance operon proteins, and 18 hypothetical proteins that may be involved in Cu toxicity and homeostasis. These findings shed light on the key regulatory genes of the Cu switch that will have potential implications for bioremediation and biotechnology applications. Full article

Methane, a potent greenhouse gas, has gained significant attention due to its environmental impact and economic potential. Chemical industries have focused on specialized catalytic systems, like zeolites, to convert methane into methanol. However, inherent limitations in selectivity, irreversibility, and pore blockages result in high costs and energy requirements, thus hindering their commercial viability and profitability. In contrast, biological methane conversion using methanotrophs has emerged as a promising alternative, offering higher conversion rates, self-renewability, improved selectivity, and economically feasible upstream processes. Nevertheless, biological methane oxidation encounters challenges including the difficulty in cultivating methanotrophs and their slow growth rates, which hinder large-scale bioprocessing. Another highlighted limitation is the limited mass transfer of methane into liquid in bioreactors. Practical strategies to enhance methane oxidation in biological systems, including optimizing reactor design to improve mass transfer, altering metal concentrations, genetic engineering of methane monooxygenases, enzyme encapsulation, and utilizing microbial consortia are discussed. By addressing the limitations of chemical approaches and highlighting the potential of biological methods, the review concluded that the utilization of genetically engineered methanotrophic biofilms on beads within a biotrickling reactor, along with enhanced aeration rates, will likely enhance methane oxidation and subsequent methane conversion rates. Full article

Waste valorization is an important strategy to reduce environmental pollution and dependency on petroleum-based fuels. In this regard, utilization of food waste as a versatile and low-cost resource is important. Several advanced catalytic methods for the valorization of food waste have been widely investigated for the production of liquid biofuels. Along this line, chemical catalysts have been explored for the synthesis of liquid biofuels. Chemo-catalysis is mainly metal based, which requires harsh process conditions. Alternatively, biocatalysts are currently being investigated as a result of several advantages such as mild reaction conditions, recyclability, selectivity and biodegradability. In this work, recent biocatalytic technologies for the preparation of liquid biofuels through food waste valorization are discussed thoroughly. Lipases are employed for the synthesis of biodiesel and the upgradation of bio-oil, whereas methane mono-oxygenases could be explored for the production of methanol via the oxidation of methane generated from food wastes. Industrial production of ethanol from food waste using bioconversion technologies is a success story. To date, there has been no specific report on the use of food waste for propanol preparation using enzymes. The ABE process (Acetone–Butanol–Ethanol) (using suitable microorganisms) is used for butanol preparation, where the vacuum stripping system is integrated to remove butanol from the broth and circumvent inhibition. The synthesis of hydrocarbon fuels from fatty acids and triglycerides can be carried out using enzymes, such as carboxylic acid reductase and fatty acid photodecarboxylase (an algal photoenzyme). Both carboxylic acid reductase and fatty acid photodecarboxylase have not yet been applied in the direct valorization of food wastes. Furthermore, limitations of the reported methods, societal and economic aspects and a fresh perspective on the subject, along with important examples, are described. Full article

Interactions between metals and microbes are critical in geomicrobiology and vital in microbial ecophysiological processes. Methane-oxidizing bacteria (MOB) and ammonia-oxidizing microorganisms (AOM) are key members in aerobic environments to start the C and N cycles. Ammonia and methane are firstly oxidized by copper-binding metalloproteins, monooxygenases, and diverse iron and copper-containing enzymes that contribute to electron transportation in the energy gain pathway, which is evolutionally connected between MOB and AOM. In this review, we summarized recently updated insight into the diverse physiological pathway of aerobic ammonia and methane oxidation of different MOB and AOM groups and compared the metabolic diversity mediated by different metalloenzymes. The elevation of iron and copper concentrations in ecosystems would be critical in the activity and growth of MOB and AOM, the outcome of which can eventually influence the global C and N cycles. Therefore, we also described the impact of various concentrations of metal compounds on the physiology of MOB and AOM. This review study could give a fundamental strategy to control MOB and AOM in diverse ecosystems because they are significantly related to climate change, eutrophication, and the remediation of contaminated sites for detoxifying pollutants. Full article

Inspired by the advantages of bi-atom catalysts and recent exciting progresses of nanozymes, by means of density functional theory (DFT) computations, we explored the potential of metal dimers embedded in phthalocyanine monolayers (M₂-Pc), which mimics the binuclear centers of methane monooxygenase, as catalysts for methane conversion using H₂O₂ as an oxidant. In total, 26 transition metal (from group IB to VIIIB) and four main group metal (M = Al, Ga, Sn and Bi) dimers were considered, and two methane conversion routes, namely *O-assisted and *OH-assisted mechanisms were systematically studied. The results show that methane conversion proceeds via an *OH-assisted mechanism on the Ti₂-Pc, Zr₂-Pc and Ta₂-Pc, a combination of *O- and *OH-assisted mechanism on the surface of Sc₂-Pc, respectively. Our theoretical work may provide impetus to developing new catalysts for methane conversion and help stimulate further studies on metal dimer catalysts for other catalytic reactions. Full article

Particulate methane monooxygenase (pMMO), a membrane-bound enzyme having three subunits (α, β, and γ) and copper-containing centers, is found in most of the methanotrophs that selectively catalyze the oxidation of methane into methanol. Active sites in the pMMO of Methylosinus trichosporium OB3b were determined by docking the modeled structure with ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene. The docking energy between the modeled pMMO structure and ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene was −5.2, −5.7, −4.2, and −3.8 kcal/mol, respectively, suggesting the existence of more than one active site within the monomeric subunits due to the presence of multiple binding sites within the pMMO monomer. The evaluation of tunnels and cavities of the active sites and the docking results showed that each active site is specific to the radius of the substrate. To increase the catalysis rates of methane in the pMMO of M. trichosporium OB3b, selected amino acid residues interacting at the binding site of ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene were mutated. Based on screening the strain energy, docking energy, and physiochemical properties, five mutants were downselected, B:Leu31Ser, B:Phe96Gly, B:Phe92Thr, B:Trp106Ala, and B:Tyr110Phe, which showed the docking energy of −6.3, −6.7, −6.3, −6.5, and −6.5 kcal/mol, respectively, as compared to the wild type (−5.2 kcal/mol) with ethylbenzene. These results suggest that these five mutants would likely increase methane oxidation rates compared to wild-type pMMO. Full article

Rokubacteria is a phylogenetic clade of as-yet-uncultivated prokaryotes, which are detected in diverse terrestrial habitats and are commonly addressed as members of the rare biosphere. This clade was originally described as a candidate phylum; however, based on the results of comparative genome analysis, was later defined as the order-level lineage, Rokubacteriales, within the phylum Methylomirabilota. The physiology and lifestyles of these bacteria are poorly understood. A dataset of 16S rRNA gene reads retrieved from four boreal raised bogs and six eutrophic fens was examined for the presence of the Rokubacteriales; the latter were detected exclusively in fens. Their relative abundance varied between 0.2 and 4% of all bacteria and was positively correlated with pH, total nitrogen content, and availability of Ca and Mg. To test an earlier published hypothesis regarding the presence of methanotrophic capabilities in Rokubacteria, peat samples were incubated with 10% methane for four weeks. No response to methane availability was detected for the Rokubacteriales, while clear a increase in relative abundance was observed for the conventional Methylococcales methanotrophs. The search for methane monooxygenase encoding genes in 60 currently available Rokubacteriales metagenomes yielded negative results, although copper-containing monooxygenases were encoded by some members of this order. This study suggests that peat-inhabiting Rokubacteriales are neutrophilic non-methanotrophic bacteria that colonize nitrogen-rich wetlands. Full article

Methanotrophic verrucomicrobia of the order Methylacidiphilales are known as extremely acidophilic, thermophilic or mesophilic bacteria that inhabit acidic geothermal ecosystems. The occurrence of verrucomicrobial methanotrophs in other types of acidic environments remains an open question. Notably, Methylacidiphilales-affiliated 16S rRNA gene sequences are commonly retrieved from acidic (pH 3.5–5.5) peatlands. In this study, we compared the patterns of verrucomicrobial diversity in four acidic raised bogs and six neutral fens located in European North Russia. Methylacidiphilales-like 16S rRNA gene reads displaying 83–86% similarity to 16S rRNA gene sequences of currently described verrucomicrobial methanotrophs were recovered exclusively from raised bogs. Laboratory incubation of peat samples with 10% methane for 3 weeks resulted in the pronounced increase of a relative abundance of alphaproteobacterial methanotrophs, while no response was detected for Methylacidiphilales-affiliated bacteria. Three metagenome-assembled genomes (MAGs) of peat-inhabiting Methylacidiphilales bacteria were reconstructed and examined for the presence of genes encoding methane monooxygenase enzymes and autotrophic carbon fixation pathways. None of these genomic determinants were detected in assembled MAGs. Metabolic reconstructions predicted a heterotrophic metabolism, with a potential to hydrolyze several plant-derived polysaccharides. As suggested by our analysis, peat-inhabiting representatives of the Methylacidiphilales are acidophilic aerobic heterotrophs, which comprise a sister family of the methanotrophic Methylacidiphilaceae. Full article

The genus Gemmobacter grows phototrophically, aerobically, or anaerobically, and utilizes methylated amine. Here, we present two high-quality complete genomes of the strains con4 and con5^T isolated from a culture of Anabaena. The strains possess sMMO (soluble methane monooxygenase)-oxidizing alkanes to carbon dioxide. Functional genes for methane-oxidation (prmAC, mimBD, adh, gfa, fdh) were identified. The genome of strain con5^T contains nirB, nirK, nirQ, norB, norC, and norG genes involved in dissimilatory nitrate reduction. The presence of nitrite reductase gene (nirK) and the nitric-oxide reductase gene (norB) indicates that it could potentially use nitrite as an electron acceptor in anoxic environments. Taxonomic investigations were also performed on two strains through polyphasic methods, proposing two isolates as a novel species of the genus Gemmobacter. The findings obtained through the whole genome analyses provide genome-based evidence of complete oxidation of methane to carbon dioxide. This study provides a genetic blueprint of Gemmobacter fulva con5^T and its biochemical characteristics, which help us to understand the evolutionary biology of the genus Gemmobacter. Full article

A combination of physicochemical and radiotracer analysis, high-throughput sequencing of the 16S rRNA, and particulate methane monooxygenase subunit A (pmoA) genes was used to link a microbial community profile with methane, sulfur, and nitrogen cycling processes. The objects of study were surface sediments sampled at five stations in the northern part of the Barents Sea. The methane content in the upper layers (0–5 cm) ranged from 0.2 to 2.4 µM and increased with depth (16–19 cm) to 9.5 µM. The rate of methane oxidation in the oxic upper layers varied from 2 to 23 nmol CH₄ L⁻¹ day⁻¹ and decreased to 0.3 nmol L⁻¹ day⁻¹ in the anoxic zone at a depth of 16–19 cm. Sulfate reduction rates were much higher, from 0.3 to 2.8 µmol L⁻¹ day⁻¹. In the surface sediments, ammonia-oxidizing Nitrosopumilaceae were abundant; the subsequent oxidation of nitrite to nitrate can be carried out by Nitrospira sp. Aerobic methane oxidation could be performed by uncultured deep-sea cluster 3 of gamma-proteobacterial methanotrophs. Undetectable low levels of methanogenesis were consistent with a near complete absence of methanogens. Anaerobic methane oxidation in the deeper sediments was likely performed by ANME-2a-2b and ANME-2c archaea in consortium with sulfate-reducing Desulfobacterota. Sulfide can be oxidized by nitrate-reducing Sulfurovum sp. Thus, the sulfur cycle was linked with the anaerobic oxidation of methane and the nitrogen cycle, which included the oxidation of ammonium to nitrate in the oxic zone and denitrification coupled to the oxidation of sulfide in the deeper sediments. Methane concentrations and rates of microbial biogeochemical processes in sediments in the northern part of the Barents Sea were noticeably higher than in oligotrophic areas of the Arctic Ocean, indicating that an increase in methane concentration significantly activates microbial processes. Full article