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Recent observations have shown the atmospheric greenhouse gas methane (CH 4 ) is consumed by microorganisms (methanotrophs) in caves at rates comparable to CH 4 oxidation in surface soils. Caves are abundant in karst landscapes that comprise 14% of Earth’s land surface area, and therefore may represent a potentially important, but overlooked, CH 4 sink.
The search for methanotrophs as plant-growth-promoting rhizobacteria (PGPR) presents an important contribution to mitigating the impact of global warming by restoring the natural soil potential for consuming methane while benefiting plants during droughts. Our in silico simulations suggest that water, produced as a byproduct of methane oxidation, can satisfy the cell growth requirement. In addition to water, methanotrophs can produce metabolites that stimulate plant growth. Considering this, we proposed that applying methanotrophs as PGPR can alleviate the effect of droughts on crops, while stimulating atmospheric methane consumption. In this work, we isolated a series of methanotrophic communities from the rhizospheres of different crops, including Italian sweet pepper and zucchini, using an atmosphere enriched with pure methane gas, to determine their potential for alleviating drought stress in wheat plants. Subsequently, 23 strains of nonmethanotrophic bacteria present in the methanotrophic communities were isolated and characterized. We then analyzed the contribution of the methane-consuming consortia to the improvement of plant growth under drought conditions, showing that some communities contributed to increases in the wheat plants’ lengths and weights, with statistically significant differences according to ANOVA models. Furthermore, we found that the presence of methane gas can further stimulate the plant–microbe interactions, resulting in larger plants and higher drought tolerance.
Aquatic environments account for half of global CH 4 emissions, with freshwater wetlands being the most significant contributors. These CH 4 fluxes can be partially offset by aerobic CH 4 oxidation driven by methanotrophs. Additionally, some methanotrophs can convert CH 4 into polyhydroxyalkanoate (PHA), an energy storage molecule as well as a promising bioplastic polymer. In this study, we investigate how PHA-accumulating methanotrophic communities enriched from wetlands were shaped by varying resource availability (i.e., C and N concentrations) at a fixed C/N ratio. Cell yields, PHA accumulation, and community composition were evaluated in high (20% CH 4 and 10 mM NH 4 + ) and low resource (0.2% CH 4 and 0.1 mM NH 4 + ) conditions simulating engineered and environmental settings, respectively. High resource availability decreased C-based cell yields, while N-based cell yields remained stable, suggesting nutrient exchange patterns differed between methanotrophic communities at different resource concentrations. PHA accumulation was only observed in high resource enrichments, producing approximately 12.6% ± 2.4% (m/m) PHA, while PHA in low resource enrichments remained below detection. High resource enrichments were dominated by Methylocystis methanotrophs, while low resource enrichments remained significantly more diverse and contained only a minor population of methanotrophs. This study demonstrates that resource concentration shapes PHA-accumulating methanotrophic communities. Together, this provides useful information to leverage such communities in engineering settings as well as to begin understanding their role in the environment.
Abstract Aerobic methanotrophy is strongly controlled by copper, and methanotrophs are known to use different mechanisms for copper uptake. Some methanotrophs secrete a modified polypeptide—methanobactin—while others utilize a surface-bound protein (MopE) and a secreted form of it (MopE*) for copper collection. As different methanotrophs have different means of sequestering copper, competition for copper significantly impacts methanotrophic activity. Herein, we show that Methylomicrobium album BG8, Methylocystis sp. strain Rockwell, and Methylococcus capsulatus Bath, all lacking genes for methanobactin biosynthesis, are not limited for copper by multiple forms of methanobactin. Interestingly, Mm. album BG8 and Methylocystis sp. strain Rockwell were found to have genes similar to mbnT that encodes for a TonB-dependent transporter required for methanobactin uptake. Data indicate that these methanotrophs “steal” methanobactin and such “theft” enhances the ability of these strains to degrade methylmercury, a potent neurotoxin. Further, when mbnT was deleted in Mm. album BG8, methylmercury degradation in the presence of methanobactin was indistinguishable from when MB was not added. Mc. capsulatus Bath lacks anything similar to mbnT and was unable to degrade methylmercury either in the presence or absence of methanobactin. Rather, Mc. capsulatus Bath appears to rely on MopE/MopE* for copper collection. Finally, not only does Mm. album BG8 steal methanobactin, it synthesizes a novel chalkophore, suggesting that some methanotrophs utilize both competition and cheating strategies for copper collection. Through a better understanding of these strategies, methanotrophic communities may be more effectively manipulated to reduce methane emissions and also enhance mercury detoxification in situ.
Hydrocarbon gas emissions from active, inactive, and improperly sealed or abandoned oil/gas wells significantly contribute to anthropogenically emitted greenhouse gases, predominantly in the form of methane (CH 4 ). We explored the extent of hydrocarbon gas emissions from 20 active, inactive, plugged and abandoned oil/gas wells in Indiana (USA), where it is estimated that there are more than 80,000 well sites throughout the state. After this initial survey, using a static flux tent, we quantified fugitive CH4 emissions from an active gas well to approximately 2 L h -1 . To evaluate the potential for microbial mitigation of hydrocarbon emissions to the atmosphere, we conducted laboratory microcosm experiments to quantify the CH 4 oxidizing potential of soils collected from sites with varying distances to the leaking gas well. Soils in close proximity to the well (0.5 m) efficiently consumed nearly all (97 %) of the added CH 4 , while only 14 % of added CH 4 was consumed by soils that were more distant from the well (20 m). These results suggest that fugitive CH 4 emissions enrich methanotrophic bacteria in soils immediately adjacent to the well. Consistent with this view, we found that prolonged exposure of soils to elevated concentrations of CH 4 enhanced the methanotrophic activity. Together, these findings prompted us to design a “methanotrophic soil mound” to assess the feasibility of mitigating point sources of CH 4 by harnessing the natural methanotrophic capacity of soil microbial communities. We found that a methanotrophic soil mound from a landfill could sustainably mitigate the CH 4 emission from the artificial source, providing a promising low-cost solution to ameliorate fugitive CH 4 emissions from abandoned oil and gas wells to the atmosphere. Finally, the effectiveness of microbe-based remediation is limited in cold climates and arid environments.
At marine methane seeps, vast quantities of methane move through the shallow subseafloor, where it is largely consumed by microbial communities. This process plays an important role in global methane dynamics, but we have yet to identify all of the methane sinks in the deep sea. Here, we conducted a continental-scale survey of seven geologically diverse seafloor seeps and found that carbonate rocks from all sites host methane-oxidizing microbial communities with substantial methanotrophic potential. In laboratory-based mesocosm incubations, chimney-like carbonates from the newly described Point Dume seep off the coast of Southern California exhibited the highest rates of anaerobic methane oxidation measured to date. After a thorough analysis of physicochemical, electrical, and biological factors, we attribute this substantial metabolic activity largely to higher cell density, mineral composition, kinetic parameters including an elevated V max , and the presence of specific microbial lineages. Our data also suggest that other features, such as electrical conductance, rock particle size, and microbial community alpha diversity, may influence a sample’s methanotrophic potential, but these factors did not demonstrate clear patterns with respect to methane oxidation rates. Based on the apparent pervasiveness within seep carbonates of microbial communities capable of performing anaerobic oxidation of methane, as well as the frequent occurrence of carbonates at seeps, we suggest that rock-hosted methanotrophy may be an important contributor to marine methane consumption.
We report Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b (“Candidatus Methanocomedenaceae”) and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides, Anaerolineales, and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.
According to report, temperature of growth of thermotolerant, methane-oxidizing bacterium Methylococcus capsulatus (Bath) affects both proportion of monounsaturated fatty acids and cis/trans ratio of these acids in cell membrane. Because suboptimum growth temperature is potential stress factor, it may be possible to use such cis/trans ratios as indices of stresses upon methane-oxidizing microbial communities. Research in microbiology of methanotrophs increasing because of possible commercial exploitation of these organisms as biocatalysts or as sources of useful polymers; knowledge of effect of temperature on ability of methanotrophs to utilize methane useful in optimization of conditions of growth.
In this study, we aimed to investigate, through high-resolution metagenomics and metatranscriptomics, the composition and the trajectories of microbial communities originating from a natural sample, fed exclusively with methane, over 14 weeks of laboratory incubation. This study builds on our prior data, suggesting that multiple functional guilds feed on methane, likely through guild-to-guild carbon transfer, and potentially through intraguild and intraspecies interactions. We observed that, under two simulated dioxygen partial pressures—low versus high—community trajectories were different, with considerable variability among the replicates. In all microcosms, four major functional guilds were prominently present, representing Methylococcaceae (the true methanotrophs), Methylophilaceae (the nonmethanotrophic methylotrophs), Burkholderiales, and Bacteroidetes. Additional functional guilds were detected in multiple samples, such as members of Opitutae, as well as the predatory species, suggesting additional complexity for methane-oxidizing communities. Metatranscriptomic analysis suggested simultaneous expression of the two alternative types of methanol dehydrogenases in both Methylococcaceae and Methylophilaceae, while high expression of the oxidative/nitrosative stress response genes suggested competition for dioxygen among the community members. The transcriptomic analysis further suggested that Burkholderiales likely feed on acetate that is produced by Methylococcaceae under hypoxic conditions, while Bacteroidetes likely feed on biopolymers produced by both Methylococcaceae and Methylophilaceae.
The use of lipid biomarkers and their carbon isotopic compositions are valuable tools for establishing links to ancient microbial ecosystems. As witnessed by the stromatolite record, benthic microbial mats grew in shallow water lagoonal environments where microorganisms had virtually no competition apart from the harsh conditions of hypersalinity, desiccation and intense light. Today, the modern counterparts of these microbial ecosystems find appropriate niches in only a few places where extremes eliminate eukaryotic grazers. Answers to many outstanding questions about the evolution of microorganisms and their environments on early Earth are best answered through study of these extant analogs. Lipids associated with various groups of bacteria can be valuable biomarkers for identification of specific groups of microorganisms both in ancient organic-rich sedimentary rocks (geolipids) and contemporary microbial communities (membrane lipids). Use of compound specific isotope analysis adds additional refinement to the identification of biomarker source, so that it is possible to take advantage of the 3C-depletions associated with various functional groups of organisms (i.e. autotrophs, heterotrophs, methanotrophs, methanogens) responsible for the cycling of carbon within a microbial community. Our recent work has focused on a set of hypersaline evaporation ponds at Guerrero Negro, Baja California Sur, Mexico which support the abundant growth of Microcoleus-dominated microbial mats. Specific biomarkers for diatoms, cyanobacteria, archaea, green nonsulfur (GNS), sulfate reducing, and methanotrophic bacteria have been identified. Analyses of the ester-bound fatty acids indicate a highly diverse microbial community, dominated by photosynthetic organisms at the surface.
The main goal of this project was to follow up on prior discovery of the communal nature of methane utilization by the aerobic methanotrophic bacteria, resulting in carbon transfer along a food chain. This goal was approached via manipulation of specially designed, synthetic bacterial communities, fed exclusively by methane, under a variety of simulated environmental conditions. Using (meta)genomics, metatranscriptomics, stable isotope probing and mutant generation, we obtained solid evidence for the nature of the core microbiota involved in collaborative methane consumption, the major players (true methanotrophs) being the members of the family Methylococcaceae, their major partners (non-methanotrophic methylotrophs) being members of the family Methylophilaceae, and minor but persistent partners in the communities being members of the orders Burkholderiales and Bacteroidetes. Metabolic reconstruction from single genomes and metagenome-assembled genomes (MAGs), along with metatranscriptomics results delineated major pathways for carbon metabolism, suggesting that the methanotrophs share different organic compounds with different functional guilds: methanol with Methylophilaceae, acetate with Burkholderiales, and polymeric substances with Bacteroidetes. Overall, the results of this project expand our understanding of microbial metabolism of methane and, thus, provide basis for strategies for microbial methane mitigation, with obvious benefits to the society of the United States and worldwide.
Peat mosses of the genus Sphagnum are ecosystem engineers that frequently predominate over photosynthetic production in boreal peatlands. Sphagnum spp. host diverse microbial communities capable of nitrogen fixation (diazotrophy) and methane oxidation (methanotrophy), thereby potentially supporting plant growth under severely nutrient-limited conditions. Moreover, diazotrophic methanotrophs represent a possible “missing link” between the carbon and nitrogen cycles, but the functional contributions of the Sphagnum-associated microbiome remain in question. A combination of metagenomics, metatranscriptomics, and dual-isotope incorporation assays was applied to investigate Sphagnum microbiome community composition across the North American continent and provide empirical evidence for diazotrophic methanotrophy in Sphagnum-dominated ecosystems. Remarkably consistent prokaryotic communities were detected in over 250 Sphagnum SSU rRNA libraries from peatlands across the United States (5 states, 17 bog/fen sites, 18 Sphagnum species), with 12 genera of the core microbiome comprising 60% of the relative microbial abundance. Additionally, nitrogenase (nifH) and SSU rRNA gene amplicon analysis revealed that nitrogen-fixing populations made up nearly 15% of the prokaryotic communities, predominated by Nostocales cyanobacteria and Rhizobiales methanotrophs. While cyanobacteria comprised the vast majority (>95%) of diazotrophs detected in amplicon and metagenome analyses, obligate methanotrophs of the genus Methyloferula (order Rhizobiales) accounted for one-quarter of transcribed nifH genes. Furthermore, in dual-isotope tracer experiments, members of the Rhizobiales showed substantial incorporation of 13 CH 4 and 15 N 2 isotopes into their rRNA. Our study characterizes the core Sphagnum microbiome across large spatial scales and indicates that diazotrophic methanotrophs, here defined as obligate methanotrophs of the rare biosphere (Methyloferula spp. of the Rhizobiales) that also carry out diazotrophy, play a keystone role in coupling of the carbon and nitrogen cycles in nutrient-poor peatlands.
Remediation of toxic chlorinated ethenes via microbial reductive dechlorination can lead to ethene formation; however, the process stalls in acidic groundwater, leading to the accumulation of carcinogenic vinyl chloride (VC). This study explored the feasibility of cometabolic VC degradation by moderately acidophilic methanotrophs. Furthermore, two novel isolates, Methylomonas sp. strain JS1 and Methylocystis sp. strain MJC1, were obtained from distinct alpine peat bogs located in South Korea. Both isolates cometabolized VC with CH 4 as the primary substrate under oxic conditions at pH at or below 5.5. VC cometabolism in axenic cultures occurred in the presence (10 μM) or absence (<0.01 μM) of copper, suggesting that VC removal had little dependence on copper availability, which regulates expression and activity of soluble and particulate methane monooxygenases in methanotrophs. The model neutrophilic methanotroph Methylosinus trichosporium strain OB3b also grew and cometabolized VC at pH 5.0 regardless of copper availability. Bioaugmentation of acidic peat soil slurries with methanotroph isolates demonstrated enhanced VC degradation and VC consumption below the maximum concentration level of 2 μg L –1 . Community profiling of the microcosms suggested species-specific differences, indicating that robust bioaugmentation with methanotroph cultures requires further research.
Methane is a potent greenhouse gas contributing to global warming. Microorganisms largely drive the biogeochemical cycling of methane, yet little is known about viral contributions to methane metabolism (MM). We analyzed 982 publicly available metagenomes from host-associated and environmental habitats containing microbial MM genes, expanding the known MM auxiliary metabolic genes (AMGs) from three to 24, including seven genes exclusive to MM pathways. These AMGs are recovered on 911 viral contigs predicted to infect 14 prokaryotic phyla including Halobacteriota, Methanobacteriota, and Thermoproteota. Of those 24, most were encoded by viruses from rumen (16/24), with substantially fewer by viruses from environmental habitats (0–7/24). To search for additional MM AMGs from an environmental habitat, we generate metagenomes from methane-rich sediments in Vrana Lake, Croatia. Therein, we find diverse viral communities, with most viruses predicted to infect methanogens and methanotrophs and some encoding 13 AMGs that can modulate host metabolisms. However, none of these AMGs directly participate in MM pathways. Together these findings suggest that the extent to which viruses use AMGs to modulate host metabolic processes (e.g., MM) varies depending on the ecological properties of the habitat in which they dwell and is not always predictable by habitat biogeochemical properties.
Metabolic interactions within a microbial community play a key role in determining the structure, function, and composition of the community. However, due to the complexity and intractability of natural microbiomes, limited knowledge is available on interspecies interactions within a community. In this work, using a binary synthetic microbiome, a methanotroph-photoautotroph (M-P) coculture, as the model system, we examined different genome-scale metabolic modeling (GEM) approaches to gain a better understanding of the metabolic interactions within the coculture, how they contribute to the enhanced growth observed in the coculture, and how they evolve over time. Using batch growth data of the model M-P coculture, we compared three GEM approaches for microbial communities. Two of the methods are existing approaches: SteadyCom, a steady state GEM, and dynamic flux balance analysis (DFBA) Lab, a dynamic GEM. We also proposed an improved dynamic GEM approach, DynamiCom, for the M-P coculture. SteadyCom can predict the metabolic interactions within the coculture but not their dynamic evolutions; DFBA Lab can predict the dynamics of the coculture but cannot identify interspecies interactions. DynamiCom was able to identify the cross-fed metabolite within the coculture, as well as predict the evolution of the interspecies interactions over time. A new dynamic GEM approach, DynamiCom, was developed for a model M-P coculture. Constrained by the predictions from a validated kinetic model, DynamiCom consistently predicted the top metabolites being exchanged in the M-P coculture, as well as the establishment of the mutualistic N-exchange between the methanotroph and cyanobacteria. The interspecies interactions and their dynamic evolution predicted by DynamiCom are supported by ample evidence in the literature on methanotroph, cyanobacteria, and other cyanobacteria-heterotroph cocultures.
Lipid biomarkers and compound specific isotopic abundances are powerful tools for studies of contemporary microbial ecosystems. Knowledge of the relationship of biomarkers to microbial physiology and community structure creates important links for understanding the nature of early organisms and paleoenvironments. Our recent work has focused on the hypersaline microbial mats in evaporation ponds at Guerrero Negro, Baja California Sur, Mexico. Specific biomarkers for diatoms, cyanobacteria, archaea, green nonsulfur (GNS), sulfate reducing, sulfur oxidizing and methanotrophic bacteria have been identified. Analyses of the ester-bound fatty acids indicate a highly diverse microbial community, dominated by photosynthetic organisms at the surface. The delta C-13 of cyanobacterial biomarkers such as the monomethylalkanes and hopanoids are consistent with the delta C-13 measured for bulk mat (-10%o), while a GNS biomarker, wax esters (WXE), suggests a more depleted delta C-13 for GNS biomass (-16%o). This isotopic relationship is different than that observed in mats at Octopus Spring, Yellowstone National Park (YSNP) where GNS appear to grow photoheterotrophic ally. WXE abundance, while relatively low, is most pronounced in an anaerobic zone just below the cyanobacterial layer. The WXE isotope composition at GN suggests that these bacteria utilize photoautotrophy incorporating dissolved inorganic carbon (DIC) via the 3-hydroxypropionate pathway using H2S or H2.
Seasonal floodplains in the Amazon basin are important sources of methane (CH 4 ), while upland forests are known for their sink capacity. Climate change effects, including shifts in rainfall patterns and rising temperatures, may alter the functionality of soil microbial communities, leading to uncertain changes in CH 4 cycling dynamics. To investigate the microbial feedback under climate change scenarios, we performed a microcosm experiment using soils from two floodplains (i.e., Amazonas and Tapajós rivers) and one upland forest. We employed a two-factorial experimental design comprising flooding (with non-flooded control) and temperature (at 27 °C and 30 °C, representing a 3 °C increase) as variables. We assessed prokaryotic community dynamics over 30 days using 16S rRNA gene sequencing and qPCR. These data were integrated with chemical properties, CH 4 fluxes, and isotopic values and signatures. In the floodplains, temperature changes did not significantly affect the overall microbial composition and CH 4 fluxes. CH 4 emissions and uptake in response to flooding and non-flooding conditions, respectively, were observed in the floodplain soils. By contrast, in the upland forest, the higher temperature caused a sink-to-source shift under flooding conditions and reduced CH 4 sink capability under dry conditions. The upland soil microbial communities also changed in response to increased temperature, with a higher percentage of specialist microbes observed. Floodplains showed higher total and relative abundances of methanogenic and methanotrophic microbes compared to forest soils. Isotopic data from some flooded samples from the Amazonas river floodplain indicated CH 4 oxidation metabolism. This floodplain also showed a high relative abundance of aerobic and anaerobic CH 4 oxidizing Bacteria and Archaea. Taken together, our data indicate that CH 4 cycle dynamics and microbial communities in Amazonian floodplain and upland forest soils may respond differently to climate change effects. We also highlight the potential role of CH 4 oxidation pathways in mitigating CH 4 emissions in Amazonian floodplains.
Copper plays a critical role in controlling greenhouse gas emissions as it is a key component of the particulate methane monooxygenase and nitrous oxide reductase. Some methanotrophs excrete methanobactin (MB) that has an extremely high copper affinity. As a result, MB may limit the ability of other microbes to gather copper, thereby decreasing their activity as well as impacting microbial community composition. Here, we show using forest soil microcosms that multiple forms of MB; MB from Methylosinus trichosporium OB3b (MB-OB3b) and MB from Methylocystis sp. strain SB2 (MB-SB2) increased nitrous oxide (N 2 O) production as well caused significant shifts in microbial community composition. Such effects, however, were mediated by the amount of copper in the soils, with low-copper soil microcosms showing the strongest response to MB. Furthermore, MB-SB2 had a stronger effect, likely due to its higher affinity for copper. The presence of either form of MB also inhibited nitrite reduction and generally increased the presence of genes encoding for the iron-containing nitrite reductase (nirS) over the copper-dependent nitrite reductase (nirK). Lastly, these data indicate the methanotrophic-mediated production of MB can significantly impact multiple steps of denitrification, as well as have broad effects on microbial community composition of forest soils.