Aerobic Methanotrophs Research Articles

Biofilm Formation on Excavation Damaged Zone Fractures in Deep Neogene Sedimentary Rock.

Deep underground galleries are used to access the deep biosphere in addition to mining and other engineering applications, such as geological disposal of radioactive waste. Fracture networks developed in the excavation damaged zone (EDZ) are concerned with accelerating mass transport, where microbial colonization might be possible due to the availability of space and nutrients. In this study, microbial biofilms at EDZ fractures were investigated by drilling from a 350-m-deep gallery and subsequent borehole logging at the Horonobe Underground Research Laboratory (URL). By using microscopic and spectroscopic techniques, the dense colonization of microbial cells was demonstrated at the surfaces of the EDZ fractures with high hydraulic conductivity. 16S rRNA gene sequence analysis revealed the dominance of gammaproteobacterial lineages, the cultivated members of which are aerobic methanotrophs. The near-complete genomes from Horonobe groundwater, affiliated with the methanotrophic lineages, were fully equipped with genes involved in aerobic methanotrophy. Although the mediation of aerobic methanotrophy remains to be demonstrated, microbial O2 production was supported by the presence of genes in the near-complete genomes, such as catalase and superoxide dismutase that produce O2 from reactive oxygen species and a nitric oxide reductase gene with the substitutions of amino acids in motifs. It is concluded that the EDZ fractures provide energetically favorable subsurface habitats for microorganisms.

Glacier melting promotes methane emission via increased methanogenic activity in the foreland alpine meadow

Annual glacier melting alters hydrothermal conditions of the foreland alpine meadows, and causes significant fluctuations in methane (CH4) flux. Previously we found that Tibetan glacier foreland alpine meadow shifts to CH4 source from sink during the melting season, but the potential mechanisms remain unclear. This study, via combination of in-situ measurement of seasonal CH4 flux and survey of microbial species that may involve in CH4 metabolism, explores the causes of glacier melting on CH4 flux in a glacier foreland alpine meadow on Tibetan Plateau. We determined a pronounced CH4 emission (13.95 μg·m−2·h−1) in August (melting season) but CH4 uptake in June (−3.76 μg·m−2·h−1) and October (−17.77 μg·m−2·h−1), and 1.4-fold higher soil moisture in August than the other two months. This showed a direct correlation of CH4 flux with glacier melting increased soil water. Additionally, glacier melting caused more CH4 fluxes increase in hollows than in hummocks. Amplicon sequencing determined 126-fold higher abundance of mcrA, the methanogenic marker gene, in August than in June and October, and a higher relative abundance of a fungal phylum Mortierellomycota and syntrophic bacteria that convert the fatty acids, the degradation intermediates of organic complexes to CO2 and acetate, the methanogenic substrates like in August. However, no seasonal variation of pmoA, the marker gene of aerobic methanotrophs, was observed. It appears that glacier melting promotes the CH4 producing but not the consuming microorganisms, thus leading to increased CH4 emission. The findings of this work indicate that global warming resulted glacier melting would increase global CH4 emissions, and in turn worsens global warming, so an alarming positive feedback loop.

Treatment of Anaerobic Digester Liquids via Membrane Biofilm Reactors: Simultaneous Aerobic Methanotrophy and Nitrogen Removal.

Anaerobic digestion (AD) produces useful biogas and waste streams with high levels of dissolved methane (CH4) and ammonium (NH4+), among other nutrients. Membrane biofilm reactors (MBfRs), which support dissolved methane oxidation in the same reactor as simultaneous nitrification and denitrification (ME-SND), are a potential bubble-less treatment method. Here, we demonstrate ME-SND taking place in single-stage, AD digestate liquid-fed MBfRs, where oxygen (O2) and supplemental CH4 were delivered via pressurized membranes. The effects of two O2 pressures, leading to different O2 fluxes, on CH4 and N removal were examined. MBfRs achieved up to 98% and 67% CH4 and N removal efficiencies, respectively. The maximum N removal rates ranged from 57 to 94 mg N L-1 d-1, with higher overall rates observed in reactors with lower O2 pressures. The higher-O2-flux condition showed NO2- as a partial nitrification endpoint, with a lower total N removal rate due to low N2 gas production compared to lower-O2-pressure reactors, which favored complete nitrification and denitrification. Membrane biofilm 16S rRNA amplicon sequencing showed an abundance of aerobic methanotrophs (especially Methylobacter, Methylomonas, and Methylotenera) and enrichment of nitrifiers (especially Nitrosomonas and Nitrospira) and anammox bacteria (especially Ca. Annamoxoglobus and Ca. Brocadia) in high-O2 and low-O2 reactors, respectively. Supplementation of the influent with nitrite supported evidence that anammox bacteria in the low-O2 condition were nitrite-limited. This work highlights coupling of aerobic methanotrophy and nitrogen removal in AD digestate-fed reactors, demonstrating the potential application of ME-SND in MBfRs for the treatment of AD's residual liquids and wastewater. Sensor-based tuning of membrane O2 pressure holds promise for the optimization of bubble-less treatment of excess CH4 and NH4+ in wastewater.

The effect of substrate concentration on the methane-driven interaction network

Methane, the primary substrate for aerobic methanotrophs, regulates the rate of methanotrophic activity and shapes the composition of the methane-oxidizing community. Given that methane-derived carbon may fuel the food web in the soil, methane availability can potentially be a key determinant, structuring the network of the interacting methane-oxidizing community. Here, we determined the response of the methane-driven interaction network to different methane concentrations (∼1.5 %v/v, 3 %v/v, and 7 %v/v), indicative of different levels of energy flow through the soil food web, using a stable isotope probing approach with 13C-methane coupled to a co-occurrence network analysis in a microcosm study. The accumulated 13C-atom fraction in the total carbon content increased from 1.08 % (background level) to an average of 7.2 % in the incubation under 7 %v/v methane, indicating that the carbon-flow via the methanotrophs can significantly contribute to the total carbon in the rice paddy soil. The 13C-enriched 16 S rRNA gene sequencing analysis revealed the predominance of gammaproteobacterial methanotrophs and Methylocystis. The composition of the actively growing (13C-labelled) bacterial community was dissimilar in the incubation under ∼3 %v/v than under 1.5 %v/v and 7 %v/v methane. This was also reflected in the co-occurrence network analysis, where the topological properties indicated a more complex and connected network in the incubation under 3 %v/v methane. It thus appears that moderate methane concentrations fostered closer associations among members of the methane-oxidizing community. Overall, our research findings showed that the methanotrophs can contribute to the total soil carbon, and methane concentrations not only shifted the bacterial community, including the methanotrophic composition, but also affected bacterial interactions.

Heterologous Biosynthesis of Methanobactin from Methylocystis sp. Strain SB2 in Methylosinus trichosporium OB3b.

Aerobic methanotrophs, or methane-consuming microbes, are strongly dependent on copper for their activity. To satisfy this requirement, some methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin (MB). In addition to playing a critical role in methanotrophy, MB has also been shown to have great promise in treating copper-related human diseases, perhaps most significantly Wilson's disease. In this congenital disorder, copper builds up in the liver, leading to irreversible damage and, in severe cases, complete organ failure. Remarkably, MB has been shown to reverse such damage in animal models, and there is a great deal of interest in upscaling MB production for expanded clinical trials. Such efforts, however, are currently hampered as (1) the natural rate of MB production rate by methanotrophs is low, (2) the use of methane as a substrate for MB production is problematic as it is explosive in air, (3) there is limited understanding of the entire pathway of MB biosynthesis, and (4) the most attractive form of MB is produced by Methylocystis sp. strain SB2, a methanotroph that is genetically intractable. Herein, we report heterologous biosynthesis of MB from Methylocystis sp. strain SB2 in an alternative methanotroph, Methylosinus trichosporium OB3b, not only on methane but also on methanol. As a result, the strategy described herein not only facilitates enhanced MB production but also provides opportunities to construct various mutants to delineate the entire pathway of MB biosynthesis, as well as the creation of modified forms of MB that may have enhanced therapeutic value.

Diverse and unconventional methanogens, methanotrophs, and methylotrophs in metagenome-assembled genomes from subsurface sediments of the Slate River floodplain, Crested Butte, CO, USA.

We use metagenome-assembled genomes (MAGs) to understand single-carbon (C1) compound-cycling-particularly methane-cycling-microorganisms in montane riparian floodplain sediments. We generated 1,233 MAGs (>50% completeness and <10% contamination) from 50- to 150-cm depth below the sediment surface capturing the transition between oxic, unsaturated sediments and anoxic, saturated sediments in the Slate River (SR) floodplain (Crested Butte, CO, USA). We recovered genomes of putative methanogens, methanotrophs, and methylotrophs (n = 57). Methanogens, found only in deep, anoxic depths at SR, originate from three different clades (Methanoregulaceae, Methanotrichaceae, and Methanomassiliicoccales), each with a different methanogenesis pathway; putative methanotrophic MAGs originate from within the Archaea (Candidatus Methanoperedens) in anoxic depths and uncultured bacteria (Ca. Binatia) in oxic depths. Genomes for canonical aerobic methanotrophs were not recovered. Ca. Methanoperedens were exceptionally abundant (~1,400× coverage, >50% abundance in the MAG library) in one sample that also contained aceticlastic methanogens, indicating a potential C1/methane-cycling hotspot. Ca. Methylomirabilis MAGs from SR encode pathways for methylotrophy but do not harbor methane monooxygenase or nitrogen reduction genes. Comparative genomic analysis supports that one clade within the Ca. Methylomirabilis genus is not methanotrophic. The genetic potential for methylotrophy was widespread, with over 10% and 19% of SR MAGs encoding a methanol dehydrogenase or substrate-specific methyltransferase, respectively. MAGs from uncultured Thermoplasmata archaea in the Ca. Gimiplasmatales (UBA10834) contain pathways that may allow for anaerobic methylotrophic acetogenesis. Overall, MAGs from SR floodplain sediments reveal a potential for methane production and consumption in the system and a robust potential for methylotrophy.IMPORTANCEThe cycling of carbon by microorganisms in subsurface environments is of particular relevance in the face of global climate change. Riparian floodplain sediments contain high organic carbon that can be degraded into C1 compounds such as methane, methanol, and methylamines, the fate of which depends on the microbial metabolisms present as well as the hydrological conditions and availability of oxygen. In the present study, we generated over 1,000 MAGs from subsurface sediments from a montane river floodplain and recovered genomes for microorganisms that are capable of producing and consuming methane and other C1 compounds, highlighting a robust potential for C1 cycling in subsurface sediments both with and without oxygen. Archaea from the Ca. Methanoperedens genus were exceptionally abundant in one sample, indicating a potential C1/methane-cycling hotspot in the Slate River floodplain system.

Methane-Driven Perchlorate Reduction by a Microbial Consortium.

The phenomenon of methane oxidation linked to perchlorate reduction has been reported in multiple studies; yet, the underlying microbial mechanisms remain unclear. Here, we enriched suspended cultures by performing methane-driven perchlorate reduction under oxygen-limiting conditions in a membrane bioreactor (MBR). Batch test results proved that perchlorate reduction was coupled to methane oxidation, in which acetate was predicted as the potential intermediate and oxygen played an essential role in activating methane. By combining DNA-based stable isotope probing incubation and high-throughput sequencing analyses of 16S rRNA gene and functional genes (pmoA, pcrA, and narG), we found that synergistic interactions between aerobic methanotrophs (Methylococcus and Methylocystis) and perchlorate-reducing bacteria (PRB; Denitratisoma and Dechloromonas) played active roles in mediating methane-driven perchlorate reduction. This partnership was further demonstrated by coculture experiments in which the aerobic methanotroph could produce acetate to support PRB to complete perchlorate reduction. Our findings advance the understanding of the methane-driven perchlorate reduction process and have implications for similar microbial consortia linking methane and chlorine biogeochemical cycles in natural environments.

Survival strategies of aerobic methanotrophs under hypoxia in methanogenic lake sediments

BackgroundMicrobial methane oxidation, methanotrophy, plays a crucial role in mitigating the release of the potent greenhouse gas methane from aquatic systems. While aerobic methanotrophy is a well-established process in oxygen-rich environments, emerging evidence suggests their activity in hypoxic conditions. However, the adaptability of these methanotrophs to such environments has remained poorly understood. Here, we explored the genetic adaptability of aerobic methanotrophs to hypoxia in the methanogenic sediments of Lake Kinneret (LK). These LK methanogenic sediments, situated below the oxidic and sulfidic zones, were previously characterized by methane oxidation coupled with iron reduction via the involvement of aerobic methanotrophs.ResultsIn order to explore the adaptation of the methanotrophs to hypoxia, we conducted two experiments using LK sediments as inoculum: (i) an aerobic "classical" methanotrophic enrichment with ambient air employing DNA stable isotope probing (DNA-SIP) and (ii) hypoxic methanotrophic enrichment with repeated spiking of 1% oxygen. Analysis of 16S rRNA gene amplicons revealed the enrichment of Methylococcales methanotrophs, being up to a third of the enriched community. Methylobacter, Methylogaea, and Methylomonas were prominent in the aerobic experiment, while hypoxic conditions enriched primarily Methylomonas. Using metagenomics sequencing of DNA extracted from these experiments, we curated five Methylococcales metagenome-assembled genomes (MAGs) and evaluated the genetic basis for their survival in hypoxic environments. A comparative analysis with an additional 62 Methylococcales genomes from various environments highlighted several core genetic adaptations to hypoxia found in most examined Methylococcales genomes, including high-affinity cytochrome oxidases, oxygen-binding proteins, fermentation-based methane oxidation, motility, and glycogen use. We also found that some Methylococcales, including LK Methylococcales, may denitrify, while metals and humic substances may also serve as electron acceptors alternative to oxygen. Outer membrane multi-heme cytochromes and riboflavin were identified as potential mediators for the utilization of metals and humic material. These diverse mechanisms suggest the ability of methanotrophs to thrive in ecological niches previously thought inhospitable for their growth.ConclusionsOur study sheds light on the ability of enriched Methylococcales methanotrophs from methanogenic LK sediments to survive under hypoxia. Genomic analysis revealed a spectrum of genetic capabilities, potentially enabling these methanotrophs to function. The identified mechanisms, such as those enabling the use of alternative electron acceptors, expand our understanding of methanotroph resilience in diverse ecological settings. These findings contribute to the broader knowledge of microbial methane oxidation and have implications for understanding and potential contribution methanotrophs may have in mitigating methane emissions in various environmental conditions.

Diversity, Methane Oxidation Activity, and Metabolic Potential of Microbial Communities in Terrestrial Mud Volcanos of the Taman Peninsula.

Microbial communities of terrestrial mud volcanoes are involved in aerobic and anaerobic methane oxidation, but the biological mechanisms of these processes are still understudied. We have investigated the taxonomic composition, rates of methane oxidation, and metabolic potential of microbial communities in five mud volcanoes of the Taman Peninsula, Russia. Methane oxidation rates measured by the radiotracer technique varied from 2.0 to 460 nmol CH4 cm-3 day-1 in different mud samples. This is the first measurement of high activity of microbial methane oxidation in terrestrial mud volcanos. 16S rRNA gene amplicon sequencing has shown that Bacteria accounted for 65-99% of prokaryotic diversity in all samples. The most abundant phyla were Pseudomonadota, Desulfobacterota, and Halobacterota. A total of 32 prokaryotic genera, which include methanotrophs, sulfur or iron reducers, and facultative anaerobes with broad metabolic capabilities, were detected in relative abundance >5%. The most highly represented genus of aerobic methanotrophs was Methyloprofundus reaching 36%. The most numerous group of anaerobic methanotrophs was ANME-2a-b (Ca. Methanocomedenaceae), identified in 60% of the samples and attaining relative abundance of 54%. The analysis of the metagenome-assembled genomes of a community with high methane oxidation rate indicates the importance of CO2 fixation, Fe(III) and nitrate reduction, and sulfide oxidation. This study expands current knowledge on the occurrence, distribution, and activity of microorganisms associated with methane cycle in terrestrial mud volcanoes.

Detection of Thermoacidophiles from Yellowstone Hot Spring

Aerobic methanotrophic bacteria maintain an unrivalled capability of utilizing methane as their sole carbon and energy source and have been retrieved from various environments. Phylogenetically, true aerobic thermoacidophilic methane oxidizers capable of growing below pH 3 have hitherto been associated only with the phylum Verrucomicrobia. In this report, the initial detection of a moderately thermoacidophilic Methylococcus-like Type Ib methanotroph of the class Gammaproteobacteria from an acidic thermal spring (50oC and pH 2.8) in the Yellowstone National Park, USA is presented. The isolate, termed YT-MC, was identified in a methane enrichment (55°C), which may represent a novel strain in the family Methylococcaceae Type Ib. The existence of this bacterium in the enrichments was demonstrated by the detection of pmoA gene, Southern blotting technique, phase-contrast, and electron microscopy. The coccus-typed cells showed tubular membranes instead of intracytoplasmic membrane systems (ICM). The soluble methane monooxygenase (sMMO) was not detected by PCR, indicating that the biotransformation of methane to methanol is oxidized by the particulate methane monooxygenase (pMMO). Moreover, YT-MC performs in a formerly undiscovered active biological methane sink in geothermal acidic environments, magnifying our knowledge of its ecological role in methane cycling, diversity, and coexistence of aerobic methanotrophy. Furthermore, the present study also reports the isolation and identification of an alphaproteobacterial heterotroph (strain YT-AC) and a verrucomicrobial methanotroph (strain YT-VM) from the same environment. Bangladesh J Microbiol, Volume 40, Number 2, December 2023, pp 86-94

Reconstructing the palaeoecology of a middle Permian alkaline lake using molecular fossils, case study of the Lucaogou Formation in the Junggar Basin, NW China

The lacustrine Lucaogou Formation was deposited during the middle Permian in the Junggar Basin, northwestern China. The depositional environment was an alkaline, brackish to saline lake. The biological diversity of the Lucaogou Formation was reconstructed using biologically informative molecules such as n-alkanes, steranes, and hopanes, and the δ13C composition of organic matter. C30 4α-methyl-24-ethylsterane, C31 lanostane, and abundant C30 − C31 3β-methylhopanes were detected in the organic matter, suggesting the presence of type Ⅰ aerobic methanotrophs including the Methylococcaceae. C30 − C31 2α-methylhopanes and 7- and 8-methylheptadecanes are suggestive of the presence of cyanobacteria. Cyanobacteria and green algae are proposed to have made the dominant contribution to the organic matter. The presence of more 13C-enriched hopanoids in the lower unit compared to the upper unit of the Lucaogou Formation suggest a greater contribution of cyanobacteria to the organic matter in the lower unit. The high abundances of C28 and C29 steranes relative to C27 steranes are likely due to green algae from the Chlorophyta, including the class Chlorophyceae. Dinosteranes that are diagnostic for dinoflagellates were not detected. The low concentrations of C40 isorenieratane, renieratane, β-isorenieratane, renierapurpurane, and β-renierapurpurane that are indicative of photosynthetic green and purple sulfur bacteria indicate a minor contribution from Chlorobiaceae and Chromatiaceae to the organic matter. It is inferred that low to moderate amounts of ciliates grew at or below the relatively unstable chemocline in the lake. The presence of C19-norisopimarane, 8β(H)-labdane, and 4β(H)-19-norisopimarane suggests the presence of coniferous plants growing around the lake, probably including Pinaceae, Araucariaceae, Cupressaceae.

Solid phase speciation controls copper mobilisation from marine sediments by methanobactin

Although marine environments represent huge reservoirs of the potent greenhouse gas methane, they currently contribute little to global net methane emissions. Most of the methane is oxidized by methanotrophs, minimizing escape to the atmosphere. Aerobic methanotrophs oxidize methane mostly via the copper (Cu)-bearing enzyme particulate methane monooxygenase (pMMO). Therefore, aerobic methane oxidation depends on sufficient Cu acquisition by methanotrophs. Because they require both oxygen and methane, aerobic methanotrophs reside at oxic-anoxic interfaces, often close to sulphidic zones where Cu bioavailability can be limited by poorly soluble Cu sulphide mineral phases. Under Cu-limiting conditions, certain aerobic methanotrophs exude Cu-binding ligands termed chalkophores, such as methanobactin (mb) exuded by Methylosinus trichosporium OB3b. Our main objective was to establish whether chalkophores can mobilise Cu from Cu sulphide-bearing marine sediments to enhance Cu bioavailability.Through a series of kinetic batch experiments, we investigated Cu mobilisation by mb from a set of well-characterized sulphidic marine sediments differing in sediment properties, including Cu content and phase distribution. Characterization of solid-phase Cu speciation included X-ray absorption spectroscopy and a targeted sequential extraction. Furthermore, in batch experiments, we investigated to what extent adsorption of metal-free mb and Cu-mb complexes to marine sediments constrains Cu mobilisation.Our results are the first to show that both solid phase Cu speciation and chalkophore adsorption can constrain methanotrophic Cu acquisition from marine sediments. Only for certain sediments did mb addition enhance dissolved Cu concentrations. Cu mobilisation by mb was not correlated to the total Cu content of the sediment, but was controlled by solid-phase Cu speciation. Cu was only mobilised from sediments containing a mono-Cu-sulphide (CuSx) phase. We also show that mb adsorption to sediments limits Cu acquisition by mb to less compact (surface) sediments. Therefore, in sulphidic sediments, mb-mediated Cu acquisition is presumably constrained to surface-sediment interfaces containing mono-Cu-sulphide phases.

Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields

Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH4) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH4 oxidation couples with denitrification in hypoxic paddy fields remains virtually unknown. Here we combine a ~3300 km field study across main rice-producing areas of China and 13CH4-DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH4 oxidation in supporting denitrification. Our results reveal positive relationships between CH4 oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH4 and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, 13CH4-DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating 13C, which are mostly belonged to Rubrivivax, Magnetospirillum, and Bradyrhizobium. Combined analyses of 13C-metagenome-assembled genomes and 13C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH4 oxidation, for the coupling of CH4 oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH4 oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.

Methylomonas defluvii sp. nov., a type I methane-oxidizing bacterium from a secondary sedimentation tank of a wastewater treatment plant.

An aerobic methanotroph was isolated from a secondary sedimentation tank of a wastewater treatment plant and designated strain OY6T. Cells of OY6T were Gram-stain-negative, pink-pigmented, motile rods and contained an intracytoplasmic membrane structure typical of type I methanotrophs. OY6T could grow at a pH range of 4.5-7.5 (optimum pH 6.5) and at temperatures ranging from 20 °C to 37 °C (optimum 30 °C). The major cellular fatty acids were C14 : 0, C16 : 1ω7c/C16 : 1ω6c and C16 : 1ω5c; the predominant respiratory quinone was MQ-8. The genome size was 5.41 Mbp with a DNA G+C content of 51.7 mol%. OY6T represents a member of the family Methylococcaceae of the class Gammaproteobacteria and displayed 95.74-99.64 % 16S rRNA gene sequence similarity to the type strains of species of the genus Methylomonas. Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) confirmed that OY6T should be classified as representing a novel species. The most closely related type strain was Methylomonas fluvii EbBT, with 16S rRNA gene sequence similarity, ANI by blast (ANIb), ANI by MUMmer (ANIm) and dDDH values of 99.64, 90.46, 91.92 and 44.5 %, respectively. OY6T possessed genes encoding both the particulate methane monooxygenase enzyme and the soluble methane monooxygenase enzyme. It grew only on methane or methanol as carbon sources. On the basis of phenotypic, genetic and phylogenetic data, strain OY6T represents a novel species within the genus Methylomonas for which the name Methylomonas defluvii sp. nov. is proposed, with strain OY6T (=GDMCC 1.4114T=KCTC 8159T=LMG 33371T) as the type strain.

Different oxygen affinities of methanotrophs and Comammox Nitrospira inform an electrically induced symbiosis for nitrogen loss

Aerobic methanotrophs establish a symbiotic association with denitrifiers to facilitate the process of aerobic methane oxidation coupled with denitrification (AME-D). However, the symbiosis has been frequently observed in hypoxic conditions continuing to pose an enigma. The present study has firstly characterized an electrically induced symbiosis primarily governed by Methylosarcina and Hyphomicrobium for the AME-D process in a hypoxic niche caused by Comammox Nitrospira. The kinetic analysis revealed that Comammox Nitrospira exhibited a higher apparent oxygen affinity compared to Methylosarcina. While the coexistence of comammox and AME-D resulted in an increase in methane oxidation and nitrogen loss rates, from 0.82 ± 0.10 to 1.72 ± 0.09 mmol CH4 d−1 and from 0.59 ± 0.04 to 1.30 ± 0.15 mmol N2 d−1, respectively. Furthermore, the constructed microbial fuel cells demonstrated a pronounced dependence of the biocurrents on AME-D due to oxygen competition, suggesting the involvement of direct interspecies electron transfer in the AME-D process under hypoxic conditions. Metagenomic and metatranscriptomic analysis revealed that Methylosarcina efficiently oxidized methane to formaldehyde, subsequently generating abundant NAD(P)H for nitrate reduction by Hyphomicrobium through the dissimilatory RuMP pathway, leading to CO2 production. This study challenges the conventional understanding of survival mechanism employed by AME-D symbionts, thereby contributing to the characterization responsible for limiting methane emissions and promoting nitrogen removal in hypoxic regions.

Draft genomes of three aerobic methanotrophs from a temperate eutrophic fishpond.

Here we introduce draft genomes of three methanotrophs belonging to the Methylococcaceae, a family of typically fast-growing methane oxidizers. Methylobacter sp. Wu1, Methylomicrobium sp. Wu6, and Methylobacter sp. Wu8 were cultured from the top sediment collected from a shore of a eutrophic fishpond in the southern Czech Republic.

Coupling Partial Nitritation, Anammox and n-DAMO in a membrane aerated biofilm reactor for simultaneous dissolved methane and nitrogen removal

Anaerobic technologies with downstream autotrophic nitrogen removal have been proposed to enhance bioenergy recovery and transform a wastewater treatment plant from an energy consumer to an energy exporter. However, approximately 20–50 % of the produced methane is dissolved in the anaerobically treated effluent and is easily stripped into the atmosphere in the downstream aerobic process, contributing to the release of greenhouse gas emissions. This study aims to develop a solution to beneficially utilize dissolved methane to support high-level nitrogen removal from anaerobically treated mainstream wastewater. A novel technology, integrating Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction (i.e. PNAM) was demonstrated in a membrane-aerated biofilm reactor (MABR). With the feeding of ∼50 mg NH4+-N/L and ∼20 mg/L dissolved methane at a hydraulic retention time of 15 h, around 90 % of nitrogen and ∼100 % of dissolved methane can be removed together in the MABR. Microbial community characterization revealed that ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation microorganisms (n-DAMO bacteria and archaea) and aerobic methanotrophs co-existed in the established biofilm. Batch tests confirmed the active microbial pathways and showed that AOB, anammox bacteria and n-DAMO microbes were jointly responsible for the nitrogen removal, and dissolved methane was mainly removed by the n-DAMO process, with aerobic methane oxidation making a minor contribution. In addition, the established system was robust against dynamic changes in influent composition. The study provides a promising technology for the simultaneous removal of dissolved methane and nitrogen from domestic wastewater, which can support the transformation of wastewater treatment from an energy- and carbon-intensive process, to one that is energy- and carbon-neutral.

Vertical variations of bacteriohopanepolyols near the Changjiang Estuary waters: Implications for marine hypoxia and bacterial activities

Bacteriohopanepolyols (BHPs) are widespread in bacteria and are used as lipid biomarkers for specific bacterial activities and environmental changes. However, deposition and degradation dynamics of BHPs are still poorly constrained in marine settings, thereby affecting their reliability as biomarkers. Here, we investigated BHPs in the water columns and sediments near the Changjiang Estuary to evaluate their origins, preservation and responses to seawater hypoxia and bacterial activities. Results indicated that bacteriohopanetetrol stereoisomer (BHT-isomer) in this area was predominantly BHT-x produced by Ca. Scalindua, present in hypoxic bottom seawater. 2-Methyl BHT and nitrogen-fixing cyanobacteria showed similar vertical distribution in the water column, as did amnio-BHPs (aminotetrol and aminopentol) and the aerobic methane oxidation (AMO) gene pmoA. 2-Methyl BHT, aminotetrol and aminopentol were thought to primarily derive from cyanobacteria and aerobic methanotrophs in seawater, respectively, despite other potential sources. Depth profiles suggest that in-situ production in sediments is not a major contribution to sedimentary BHPs, compared to the export and deposition from the water column. The abundances of unsaturated BHT and amino-BHPs in surface sediments were decreased compared with those in the water column, indicating that unsaturated BHT and amino-BHPs appear to undergo a certain degree of early diagenetic degradation. BHT-isomer and 2-Methyl BHT preserved preferentially over amino-BHPs. Additionally, BHT-isomer significantly correlated with anammox functional gene amx-16S rRNA, and 2-Methyl BHT significantly correlated with nitrogen-fixing functional gene nifH. This indicated that BHT-isomer and 2-Methyl BHT are suitable as biomarkers for anammox and cyanobacterial nitrogen fixation. The BHT-isomer ratio accurately reflects the situation in the water column and appears to be a very appropriate proxy for hypoxia. Conversely, sedimentary aminotetrol and aminopentol underestimate the importance of aerobic methane oxidation in seawater, as these are more rapidly degraded upon deposition to the sea floor.

Novel sterol binding domains in bacteria.

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

Novel sterol binding domains in bacteria

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.