Anaerobic Methanotrophs Research Articles

Tidal control on aerobic methane oxidation and mitigation of methane emissions from coastal mangrove sediments

Mangrove forests represent important sources of methane, partly thwarting their ecosystem function as an efficient atmospheric carbon dioxide sink. Many studies have focused on the spatial and temporal variability of methane emissions from mangrove ecosystems, yet little is known about the microbial and physical controls on the release of biogenic methane from tidally influenced mangrove sediments. Here, we show that aerobic methane oxidation is a key microbial process that effectively reduces methane emissions from mangrove sediments. We further demonstrate clear links between the tidal cycle and fluctuations in methane fluxes, with contrasting methane emission rates under different tidal amplitudes. Our data suggest that both the microbial methane oxidation activity and pressure-induced advective transport modulated methane fluxes in the mangrove sediments. Methane oxidation activity is limited by the availability of oxygen in the surface sediments, which in turn is controlled by tidal dynamics, further highlighting the interactive physico-biogeochemical controls on biological methane fluxes. Although we found some molecular evidence for anaerobic methanotrophs in the deeper sediments, anaerobic methane oxidation seems to play only a minor role in the mangrove sediments, with potential rates being two orders of magnitude lower than those of aerobic methane oxidation. Our findings confirmed the importance of surface sediments as biological barrier for methane. Specifically, when sediments were exposed to the air, methane consumption increased by ∼227%, and the methane flux was reduced by ∼62%, compared to inundated conditions. Our data demonstrate how tides can orchestrate the daily rhythm of methane consumption and production within mangrove sediments, thus explaining the temporal variability of methane emissions in the tidally influenced coastal mangrove systems.

Chemolithoautotrophic bacteria flourish at dark water-ice interfaces of an emerged Arctic cold seep.

Below their ice shells, icy moons may offer a source of chemical energy that could support microbial life in the absence of light. In the Arctic, past and present glacial retreat leads to isostatic uplift of sediments through which cold and methane-saturated groundwater travels. This fluid reaches the surface and freezes as hill-shaped icings during winter, producing dark ice-water interfaces above water ponds containing chemical energy sources. In one such system characterized by elevated methane concentrations - the Lagoon Pingo in Adventdalen, Svalbard (~10mg/L CH4, <0.3mg/L O2, -0.25°C, pH7.9), we studied amplicons of the bacterial and archaeal (microbial) 16S rRNA gene and transcripts in the water pond and overlaying ice. We show that active chemolithoautotrophic sulfur-oxidizing microorganisms (Sulfurimonas, Thiomicrorhabdus) dominated a niche at the bottom of the ice in contact with the anoxic water reservoir. There, the growing ice offers surfaces interfacing with water, and hosts favorable physico-chemical conditions for sulfide oxidation. Detection of anaerobic methanotrophs further suggests that the ice led to a steady-state dark and cold methane sink under the ice throughout winter, in two steps: first methane is oxidized to carbon dioxide and sulfates concomitantly reduced to sulfides by the activity of ANME-1a and SEEP-SRB1 consortia, in a second time energy from sulfides is used by sulfur- oxidizing microorganisms to fix carbon dioxide into organic carbon. Our results underline ice- covered and dark ecosystems as a hitherto overlooked oasis of microbial life and emphasize the need to study microbial communities in icy habitats.

Research progress on the effects of elevated atmospheric CO2 concentration on CH4 emission and related microbial processes in paddy fields.

Paddy fields are recognized as significant sources of methane (CH4) emissions, playing a pivotal role in global climate change. Elevated atmospheric carbon dioxide (CO2) concentrations (e[CO2]) exert a profound influence on the carbon cycling of paddy fields. Understanding the effects of e[CO2] on CH4 emissions, as well as the underlying microbial processes, is crucial for enhancing carbon sequestration and reducing emissions in paddy fields. We reviewed the impacts of e[CO2] on CH4 emission in paddy fields, focusing on the activity, abundance, community structure, and diversity of carbon-cycling-related microbes. We also delineated the roles of various microbial processes in mitigating CH4 emissions under e[CO2], as well as the primary environmental determinants. Overall, the type of e[CO2] experimental platforms, duration of fumigation, concentration gradients, and the methods of CO2 enrichment all influence CH4 emissions from paddy fields. e[CO2] initially stimulates CH4 emissions, which may decrease over time, indicating an adaptability of the methane-emitting microbial community to e[CO2]. This response exhibits a trend of initial attenuation followed by an intensification of the positive effects on CH4 emissions. Experiments with abrupt increase of CO2 concentration might overestimate CH4 emissions. The impact of e[CO2] on microbial processes is predominantly characterized by enhanced activities and abundance of methanogens, aerobic and anaerobic methanotrophs. It significantly alters the community composition and diversity of methanotrophs, with minimal effects on methanogens and anaerobic methanotrophic communities. Finally, we outlined future research directions: 1) Integrated investigations into the effects of e[CO2] on CH4 emissions, methanogenesis, and both aerobic and anaerobic methanotrophs in paddy fields could elucidate the mechanisms underlying the impacts of climate change on CH4 emissions; 2) Long-term studies are essential to understand the mechanisms of e[CO2] on CH4 emissions and associated microbial processes more accurately and realistically; 3) Multi-scale (temporal and spatial), multi-factorial (CO2 concentration, temperature, atmospheric nitrogen deposition, and water management practices), and multi-methodological (observational, data, and model integration) research is necessary to effectively reduce the uncertainties in assessing the response of CH4 emissions in paddy fields and related microbial processes to e[CO2] under future climate change scenarios.

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.

Carbonate chimneys at the highly productive point Dume methane seep: Fine-scale mineralogical, geochemical, and microbiological heterogeneity reflects dynamic and long-lived methane-metabolizing habitats.

Methane is a potent greenhouse gas that enters the marine system in large quantities at seafloor methane seeps. At a newly discovered seep site off the coast of Point Dume, CA, ~ meter-scale carbonate chimneys host microbial communities that exhibit the highest methane-oxidizing potential recorded to date. Here, we provide a detailed assessment of chimney geobiology through correlative mineralogical, geochemical, and microbiological studies of seven chimney samples in order to clarify the longevity and heterogeneity of these highly productive systems. U-Th dating indicated that a methane-driven carbonate precipitating system at Point Dume has existed for ~20 Kyr, while millimeter-scale variations in carbon and calcium isotopic values, elemental abundances, and carbonate polymorphs revealed changes in carbon source, precipitation rates, and diagenetic processes throughout the chimneys' lifespan. Microbial community analyses revealed diverse modern communities with prominent anaerobic methanotrophs, sulfate-reducing bacteria, and Anaerolineaceae; communities were more similar within a given chimney wall transect than in similar horizons of distinct structures. The chimneys represent long-lived repositories of methane-oxidizing communities and provide a window into how carbon can be transformed, sequestered, and altered over millennia at the Point Dume methane seep.

Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F430 Modifications.

Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F430, a nickel-containing tetrahydrocorphin. Several modified versions of F430 have been discovered, including the 172-methylthio-F430 (mtF430) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F430 compared to 172-thioether coenzyme F430 variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 172 methylthio modification in ANME-1 MCR. Modifications at the 172 position disrupt the canonical substrate positioning in M. acetivorans MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F430 variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH3-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme's organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.

Integrated geochemical and microbiological assessments of Astroni lakes reveals Campi Flegrei unrest signatures

Astroni volcano in the Campi Flegrei caldera (southern Italy) is a 2 km wide, densely vegetated tuff ring and hosting several volcanic structures, including tuff cones, scoriae cones, lava domes, and three small lakes. Geochemical data of waters and dissolved gases from the lakes, coupled with microbiological analyses on lake water and sediments, were used to shed light on the possible relationship between the lakes and the hydrothermal fluid circulation system as suggested by previous geophysical surveys. Water chemistry was dominated by solutes, mainly Na+ and HCO3−, deriving from fluids and CO2-rich gases typically found in discharges located at the periphery of hydrothermal-volcanic systems. Lago Grande (LG) lake showed an anoxic hypolimnion with abundant non-atmospheric dissolved gases, consisting of biogenic CH4 and CO2, the latter having a twofold origin, biogenic and hydrothermal. The occurrence of anaerobic methanotrophs coupled with the lack of hydrogenotrophic methanogenic archaea along the whole vertical profile of LG suggested that CH4 was mostly produced from degradation of abundant terrestrial organic matter within the deep lake sediments, and then consumed during its diffusion through the lake. Notwithstanding, the output rate of CH4 from LG surface was anomalously high relative to those commonly measured in lakes. Carbon dioxide from the hydrothermal source and produced by CH4 oxidation was partially fixed in the lake via the acetyl-CoA pathway. Accordingly, the CO2 fluxes from the LG surface were relatively low, in the range of those measured in volcanic lakes dominated by biogenic CO2. The dependence of the chemistry of the Astroni lakes on inputs from the Campi Flegrei hydrothermal system, besides on biogeochemical processes, offers a possible explanation for the anomalous increase of the LG water level occurred in the last years, which was not consistent with the recorded local rainfall but likely caused by an increasing hydraulic pressure related to the enhanced hydrothermal activity recorded at Campi Flegrei in the last decades. According to this hypothesis, the future evolution of the current volcanic unrest may govern the fate of the lake water level with important implications for the functioning of the precious Astroni ecosystem.

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.

Impact of Concurrent aerobic-anaerobic Methanotrophy on Methane Emission from Marine Sediments in Gas Hydrate Area.

Microbial methane oxidation has a significant impact on the methane flux from marine gas hydrate areas. However, the environmental fate of methane remains poorly constrained. We quantified the relative contributions of aerobic and anaerobic methanotrophs to methane consumption in sediments of the gas hydrate-bearing Sakata Knoll, Japan, by in situ geochemical and microbiological analyses coupled with 13C-tracer incubation experiments. The anaerobic ANME-1 and ANME-2 species contributed to the oxidation of 33.2 and 1.4% methane fluxes at 0-10 and 10-22 cm below the seafloor (bsf), respectively. Although the aerobic Methylococcaceae species consumed only 0.9% methane flux in the oxygen depleted 0.0-0.5 cmbsf zone, their metabolic activity was sustained down to 6 cmbsf (based on rRNA and lipid biosyntheses), increasing their contribution to 10.3%. Our study emphasizes that the co-occurrence of aerobic and anaerobic methanotrophy at the redox transition zone is an important determinant of methane flux.

Vertical and temporal variations in activity, abundance, and composition of nitrite-driven anaerobic methanotrophs in a paddy field

Paddy soil is an important source of methane (CH4) emissions, and the nitrite-driven anaerobic oxidation of methane (AOM) mediated by Candidatus Methylomirabilis bacteria is a crucial process for CH4 mitigation. However, the vertical and temporal fluctuations in nitrite-driven AOM activity, as well as the abundance and composition of Methylomirabilis bacteria in paddy soil, remain poorly understood. We investigated the potential rate of nitrite-driven AOM and the composition of Methylomirabilis bacteria at varying depths (0–40 cm, at 10 cm intervals) within a paddy soil during different growth stages of rice, including tillering, jointing, flowering, and milky. The potential rate of nitrite-driven AOM ranged from 0.24 to 20.6 nmol CO2 g−1 (dry soil) d−1, and the abundance of Methylomirabilis bacterial 16S rRNA genes varied from 5.8 × 106 to 4.4 × 107 copies g−1 dry soil. The 10–20 cm soil depth showed higher potential activity and bacterial abundance compared to other depths, and the tillering and jointing stages exhibited greater activity and abundance. The composition of Methylomirabilis bacteria showed significant variation across different soil depths but remained stable throughout the growth stages of rice. Soil organic carbon content was positively correlated with potential nitrite-driven AOM rate, and nitrite content was negatively correlated with the abundance of Methylomirabilis bacteria. Soil organic carbon and ammonium content had a significant impact on the composition of Methylomirabilis bacteria. The results underscore the substantial influence of soil depth and the growth stage of rice on nitrite-driven AOM in paddy ecosystems.

Mechanisms of extracellular electron transfer in anaerobic methanotrophic archaea

Anaerobic methanotrophic (ANME) archaea are environmentally important, uncultivated microorganisms that oxidize the potent greenhouse gas methane. During methane oxidation, ANME archaea engage in extracellular electron transfer (EET) with other microbes, metal oxides, and electrodes through unclear mechanisms. Here, we cultivate ANME-2d archaea (‘Ca. Methanoperedens’) in bioelectrochemical systems and observe strong methane-dependent current (91–93% of total current) associated with high enrichment of ‘Ca. Methanoperedens’ on the anode (up to 82% of the community), as determined by metagenomics and transmission electron microscopy. Electrochemical and metatranscriptomic analyses suggest that the EET mechanism is similar at various electrode potentials, with the possible involvement of an uncharacterized short-range electron transport protein complex and OmcZ nanowires.

Efficient perchlorate reduction in microaerobic environment facilitated by partner methane oxidizers

The conventional perchlorate (ClO4−) reduction typically necessitates anaerobic conditions. However, in this study, we observed efficient ClO4− reduction using CH4 as the electron donor in a microaerobic environment. The maximum ClO4− removal flux of 2.18 g/m2·d was achieved in CH4-based biofilm. The kinetics of ClO4− reduction showed significant differences, with trace oxygen increasing the reduction rate of ClO4−, whereas oxygen levels exceeding 2 mg/L decelerated the ClO4− reduction. In the absence of exogenous oxygen, anaerobic methanotrophic (ANME) archaea contribute more than 80% electrons through the reverse methanogenesis pathway for ClO4− reduction. Simultaneously, microorganisms activate CH4 by utilizing oxygen generated from chlorite (ClO2−) disproportionation. In the presence of exogenous oxygen, methane oxidizers predominantly consume oxygen to drive the aerobic oxidation of methane. It is indicated that methane oxidizers and perchlorate reducing bacteria can form aggregates to resist external oxygen shocks and achieve efficient ClO4− reduction under microaerobic condition. These findings provide new insights into biological CH4 mitigation and ClO4− removal in hypoxic environment.

Variations of activity and community structure of nitrite-driven anaerobic methanotrophs in soils between native and invasive species in China's coastal wetlands

Nitrite-driven anaerobic oxidation of methane (nitrite-driven AOM), mediated by ‘Candidatus Methylomirabilis oxyfera’ (M. oxyfera)-related bacteria, is a newly-discovered CH4 consumption process in coastal wetlands. Although Spartina alterniflora invasion significantly affects CH4 emissions from coastal wetlands, its impact on the nitrite-driven AOM process and the underlying mechanisms remain unknown. Here, we examined nitrite-driven AOM activity and M. oxyfera-related bacterial community in four coastal wetlands along the southeastern coast of China, under invasive Spartina alterniflora and native plants, including Kandelia candel, Avicennia marina or Phragmites australis. Linear mixed-effects models indicated that the Spartina alterniflora invasion stimulated the overall nitrite-driven AOM activity by an average of 61.5% in coastal wetlands (p < 0.05), but had no impact on the M. oxyfera-related bacterial abundance (p > 0.05). The nitrite-driven AOM activity was 7.1 times higher under Spartina alterniflora than under native species in Yueqing Bay (p < 0.05), and was 34.7%, 8.9% and 15.1% higher under Spartina alterniflora than under native species in Hengsha Island, Jiulong River and Zhanjiang, respectively (p > 0.05). Spartina alterniflora invasion increased the bacterial abundance in Yueqing Bay and Jiulong River Estuary by 6.8 and 7.6 times, respectively, while decreased the abundance by 34.4% and 51.4%, respectively, in Hengsha Island and Zhanjiang (p > 0.05). The partial least squares path model indicated an indirect impact of Spartina alterniflora invasion on the nitrite-driven AOM activity through its effect on soil properties, primarily including dissolved organic carbon content and nitrate content. The Spartina alterniflora invasion did not greatly alter M. oxyfera-related bacterial community. Overall, we shed new light on the potential impact of Spartina alterniflora invasion on CH4 cycling in coastal wetlands.

Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress.

Climate change-driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-β-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-β-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress.

Recent findings in methanotrophs: genetics, molecular ecology, and biopotential.

The potential consequences for mankind could be disastrous due to global warming, which arises from an increase in the average temperature on Earth. The elevation in temperature primarily stems from the escalation in the concentration of greenhouse gases (GHG) such as CO2, CH4, and N2O within the atmosphere. Among these gases, methane (CH4) is particularly significant in driving alterations to the worldwide climate. Methanotrophic bacteria possess the distinctive ability to employ methane as both as source of carbon and energy. These bacteria show great potential as exceptional biocatalysts in advancing C1 bioconversion technology. The present review describes recent findings in methanotrophs including aerobic and anaerobic methanotroph bacteria, phenotypic characteristics, biotechnological potential, their physiology, ecology, and native multi-carbon utilizing pathways, and their molecular biology. The existing understanding of methanogenesis and methanotrophy in soil, as well as anaerobic methane oxidation and methanotrophy in temperate and extreme environments, is also covered in this discussion. New types of methanogens and communities of methanotrophic bacteria have been identified from various ecosystems and thoroughly examined for a range of biotechnological uses. Grasping the processes of methanogenesis and methanotrophy holds significant importance in the development of innovative agricultural techniques and industrial procedures that contribute to a more favorable equilibrium of GHG. This current review centers on the diversity of emerging methanogen and methanotroph species and their effects on the environment. By amalgamating advanced genetic analysis with ecological insights, this study pioneers a holistic approach to unraveling the biopotential of methanotrophs, offering unprecedented avenues for biotechnological applications. KEY POINTS: • The physiology of methanotrophic bacteria is fundamentally determined. • Native multi-carbon utilizing pathways in methanotrophic bacteria are summarized. • The genes responsible for encoding methane monooxygenase are discussed.

Climatic factors and fertilization rates co-regulate anaerobic methane oxidation driven by multiple electron acceptors in Chinese paddy fields

Paddy fields constitute a substantial anthropogenic reservoir of methane, with their inundation management fostering an ideal habitat for anaerobic methane oxidation. Within this context, a novel clade of anaerobic methanotrophic (ANME) archaea, known as ANME-2d, has been identified as capable of catalyzing anaerobic methane oxidation in conjunction with nitrate and metal oxide reduction processes within paddy fields. Nevertheless, our comprehension of the mechanisms governing anaerobic methane oxidation and its pivotal role in regulating methane emissions within rice paddies remains limited. This study quantified the rates of nitrate- and iron (III)-driven anaerobic methane oxidation through 13C-labeled stable isotope tracing experiments in Chinese paddy fields spanning diverse climate zones. Additionally, it investigated the ANME-2d archaeal community using quantitative polymerase chain reaction and high-throughput sequencing techniques. The nitrate-driven anaerobic methane oxidation contributed 10.9% to methane emission reduction. This contribution is equal to the previously identified contribution of nitrite-driven anaerobic methane oxidation (11.2%) mediated via NC10 bacteria, but played more important roles than iron-driven one (4.1%). The rates of nitrate- and nitrite-driven anaerobic methane oxidation differed significantly among climate zones and showed positive correlation with the mean annual temperature. Furthermore, their rates were more sensitive to temperature increases at higher and lower latitudes, respectively, under both representative concentration pathways 2.6 and 8.5. The rate of anaerobic methane oxidation driven by nitrate exhibited a positive correlation with nitrogen fertilization rate but displayed a negative correlation with phosphorus fertilization rate. Conversely, the rate of anaerobic methane oxidation driven by iron showed no significant correlation with either nitrogen or phosphorus fertilization rates. This study underscores the great potential of anaerobic methane oxidation in mitigating global warming, particularly under the conditions of future climate change and elevated nitrogen loading. These findings underline the necessity of incorporating anaerobic methane oxidation as a crucial parameter in methane emission prediction models.

Livestock dung rather than biochar enhances the anaerobic methane oxidation in grassland soils

The terrestrial anaerobic methane oxidation (AOM) coupled with denitrification is considered to be an important link in the “cryptic cycle of methane”. However, it remains uncertain how land use activity such as biochar and livestock dung amendments regulate the AOM in grassland. Here, we incubated soils with biochar and dung amendments in microcosms to monitor the AOM activity and quantified the maker genes of anaerobic methanotrophs and their potential syntrophs. Dung enhanced the AOM mediated by Candidatus Methylomirabilis oxyfera and stimulated denitrifying bacteria and anammox growths as well. The biochar amendment inhibited AOM due to the trapping of NO3− and NO2−. Our study raised the possibility that anthropogenic activity can regulate AOM through porosity alteration and substrate limitation.

Electron Acceptor Addition to Stimulate Anaerobic Methanotrophy in Oil Sands End Pit Lakes

A major question for the Alberta oilsands extraction industry is how to return tailings ponds to their original ecosystem functions at the end of their lifespan. This question is one of the most pressing in the oil industry, and while many potential solutions are being trialed, none are without issue. One solution is the creation of end pit lakes (EPLs), formed by filling a mine pit or tailings pond with fluid tailings and covering this with a freshwater cap. Over time, tailings settle to the bottom of the lake and become sequestered. As of 2018, there were 23 plans to pursue EPLs in Albertan oilsands mining operations (COSIA 2021), so research in oilsands reclamation via EPLs is essential. Alberta has only one full-scale EPL formed using tailings, Base Mine Lake (BML). It has been under reclamation since 2011. Although BML water quality is gradually improving over time, one persistent problem is the production of methane bubbles from the entrenched hydrocarbons. Ebullition of these bubbles carries contaminants into the water column. The potential to limit methane production biologically by stimulating methanotroph metabolism, is therefore of interest. Methanotrophs are microbes that consume methane. These methanotrophs can be aerobic, in the water column, or anaerobic below the sediment interface, where oxygen is depleted. Anaerobic oxidizers of methane, or AOM, are of particular interest due to methane only being produced in anaerobic conditions. However, AOM are not well-studied and there are many gaps in our knowledge about them. Base Mine Lake presents an important opportunity to document the presence of AOM in oilsands ecosystems, and identify if AOM can remove methane before it enters the water column. Methane is probably not the limiting factor for AOM in BML- instead, the electron acceptors used in the absence of oxygen are scarce. If microbiological methane removal were to increase after supplementing electron acceptors such as nitrate, sulfate, or iron, then this could be applied to the reclamation of artificial end pit lake systems. Our research aims to illustrate if AOM are present in BML, and if we can stimulate their metabolism via electron acceptor amendment. We performed amplicon and metagenomic sequencing across 5 sediment cores from BML. These cores penetrate over two meters below the water-sediment interface, up to 14 meters below the water’s surface. We designed anaerobic, 13C-isotope-spiked incubations for this sediment to track if any CH4 to CO2 conversion was underway. By tagging methane molecules with 13C, we are able to follow the biogeochemical transformations and demonstrate if any 13CH4 is converted to 13CO2, which would indicate methanotrophic activity. Additionally, microbes who take up 13C produce heavier DNA, possibly allowing us to identify the DNA of the players at work. Additionally, Illumina sequencing was done on all incubations to compare how electron acceptor addition prompted community shifts. In the incubation experiments, 13CO2 is being produced in several core samples incubated without O2. This finding indicates that methanotrophs are, in some capacity, active in anoxic conditions. When comparing different amendments of electron acceptors, there did appear to be a significant amount (p = 0.027) of 13CO2 produced in the sulfate-added 13CH4 treatment (Fig. 1). Other amendments had encouraging results, but were not statistically significant. Ongoing trials will provide more illuminating results via larger sample sizes, which will be completed by the end of the summer. Next we will characterize communities in the post-incubation sediment, using both amplicon sequencing and DNA stable isotope probing. This will suggest not only the identity of the organisms at play, but will also illuminate which electron acceptors were most beneficial to which groups. This analysis is complex and underway. Overall, the study of methanotrophy in artificial ecosystems with high emissions is an area of great interest, due to the high potential for amendment and improvement of the ecosystems using methanotrophs, as well as the ongoing discovery on AOM in artificial ecosystems. Oilsands end pit lakes are of interest, as an issue of cultural and political emphasis.

The presence of benzene ring activating CoA ligases for aromatics degradation in the ANaerobic MEthanotrophic (ANME) archaea.

Petroleum-source and black carbon-source aromatic compounds are present in the cold seep environments, where ANaerobic MEthanotrophic (ANME) archaea as the dominant microbial community mediates the anaerobic oxidation of methane to produce inorganic and organic carbon. Here, by predicting the aromatics catabolic pathways in ANME metagenome-assembled genomes, we provide genomic and biochemical evidences that ANME have the potential of metabolizing aromatics via the strategy of CoA activation of the benzene ring using phenylacetic acid and benzoate as the substrates. Two ring-activating enzymes phenylacetate-CoA ligase (PaaKANME) and benzoate-CoA ligase (BadAANME) are able to convert phenylacetate to phenylacetyl-CoA and benzoate to benzoyl-CoA in vitro, respectively. They are mesophilic, alkali resistance, and with broad substrate spectra showing different affinity with various substrates. An exploration of the relative gene abundance in ANME genomes and cold seep environments indicates that about 50% of ANME genomes contain PCL genes, and various bacteria and archaea contain PCL and BCL genes. The results provide evidences for the capability of heterotrophic metabolism of aromatic compounds by ANME. This has not only enhanced our understanding of the nutrient range of ANME but also helped to explore the additional ecological and biogeochemical significance of this ubiquitous sedimentary archaea in the carbon flow in the cold seep environments. IMPORTANCE ANaerobic MEthanotrophic (ANME) archaea is the dominant microbial community mediating the anaerobic oxidation of methane in the cold seep environments, where aromatic compounds are present. Then it is hypothesized that ANME may be involved in the metabolism of aromatics. Here, we provide genomic and biochemical evidences for the heterotrophic metabolism of aromatic compounds by ANME, enhancing our understanding of their nutrient range and also shedding light on the ecological and biogeochemical significance of these ubiquitous sedimentary archaea in carbon flow within cold seep environments. Overall, this study offers valuable insights into the metabolic capabilities of ANME and their potential contributions to the global carbon cycle.

Physiological potential and evolutionary trajectories of syntrophic sulfate-reducing bacterial partners of anaerobic methanotrophic archaea.

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it.