Survival strategies of aerobic methanotrophs under hypoxia in methanogenic lake sediments.

BACKGROUND: Microbial 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. RESULTS: In 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. CONCLUSIONS: Our 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.

Climate change induces shifts in coastal Baltic Sea surface water microorganism stress and photosynthesis gene expression.

The world's oceans are challenged by climate change linked warming with typically highly populated coastal areas being particularly susceptible to these effects. Many studies of climate change on the marine environment use large, short-term temperature manipulations that neglect factors such as long-term adaptation and seasonal cycles. In this study, a Baltic Sea 'heated' bay influenced by thermal discharge since the 1970s from a nuclear reactor (in relation to an unaffected nearby 'control' bay) was used to investigate how elevated temperature impacts surface water microbial communities and activities. 16S rRNA gene amplicon based microbial diversity and population structure showed no difference in alpha diversity in surface water microbial communities, while the beta diversity showed a dissimilarity between the bays. Amplicon sequencing variant relative abundances between the bays showed statistically higher values for, e.g., Ilumatobacteraceae and Burkholderiaceae in the heated and control bays, respectively. RNA transcript-derived activities followed a similar pattern in alpha and beta diversity with no effect on Shannon's H diversity but a significant difference in the beta diversity between the bays. The RNA data further showed more elevated transcript counts assigned to stress related genes in the heated bay that included heat shock protein genes dnaKJ, the co-chaperonin groS, and the nucleotide exchange factor heat shock protein grpE. The RNA data also showed elevated oxidative phosphorylation transcripts in the heated (e.g., atpHG) compared to control (e.g., atpAEFB) bay. Furthermore, genes related to photosynthesis had generally higher transcript numbers in the control bay, such as photosystem I (psaAC) and II genes (psbABCEH). These increased stress gene responses in the heated bay will likely have additional cascading effects on marine carbon cycling and ecosystem services.

State-of the-Art Constraint-Based Modeling of Microbial Metabolism: From Basics to Context-Specific Models with a Focus on Methanotrophs.

Methanotrophy is the ability of an organism to capture and utilize the greenhouse gas, methane, as a source of energy-rich carbon. Over the years, significant progress has been made in understanding of mechanisms for methane utilization, mostly in bacterial systems, including the key metabolic pathways, regulation and the impact of various factors (iron, copper, calcium, lanthanum, and tungsten) on cell growth and methane bioconversion. The implementation of -omics approaches provided vast amount of heterogeneous data that require the adaptation or development of computational tools for a system-wide interrogative analysis of methanotrophy. The genome-scale mathematical modeling of its metabolism has been envisioned as one of the most productive strategies for the integration of muti-scale data to better understand methane metabolism and enable its biotechnological implementation. Herein, we provide an overview of various computational strategies implemented for methanotrophic systems. We highlight functional capabilities as well as limitations of the most popular web resources for the reconstruction, modification and optimization of the genome-scale metabolic models for methane-utilizing bacteria.

MmoD regulates soluble methane monooxygenase and methanobactin production in Methylosinus trichosporium OB3b.

IMPORTANCE: Aerobic methanotrophs play a critical role in the global carbon cycle, particularly in controlling net emissions of methane to the atmosphere. As methane is a much more potent greenhouse gas than carbon dioxide, there is increasing interest in utilizing these microbes to mitigate future climate change by increasing their ability to consume methane. Any such efforts, however, require a detailed understanding of how to manipulate methanotrophic activity. Herein, we show that methanotrophic activity is strongly controlled by MmoD, i.e., MmoD regulates methanotrophy through the post-transcriptional regulation of the soluble methane monooxygenase and controls the ability of methanotrophs to collect copper. Such data are likely to prove quite useful in future strategies to enhance the use of methanotrophs to not only reduce methane emissions but also remove methane from the atmosphere.

Application of nitrogen-rich sunflower husks biochar promotes methane oxidation and increases abundance of Methylobacter in nitrogen-poor soil.

The area of sunflower crops is steadily increasing. A beneficial way of managing sunflower waste biomass could be its use as a feedstock for biochar production. Biochar is currently being considered as an additive for improving soil parameters, including the ability to oxidise methane (CH4) - one of the key greenhouse gases (GHG). Despite the high production of sunflower husk, there is still insufficient information on the impact of sunflower husk biochar on the soil environment, especially on the methanotrophy process. To fill this knowledge gap, an experiment was designed to evaluate the effects of addition of sunflower husk biochar (produced at 450-550 °C) at a wide range of doses (1-100 Mg ha-1) to Haplic Luvisol. In the presented study, the CH4 oxidation potential of soil with and without sunflower husk biochar was investigated at 60 and 100% water holding capacity (WHC), and with the addition of 1% CH4 (v/v). The comprehensive study included GHG exchange (CH4 and CO2), physicochemical properties of soil (pH, soil organic carbon (SOC), dissolved organic carbon (DOC), nitrate nitrogen (NO3--N), WHC), and the structure of soil microbial communities. That study showed that even low biochar doses (5 and 10 Mg ha-1) were sufficient to enhance pH, SOC, DOC and NO3--N content. Importantly, sunflower husk biochar was significant source of NO3--N, which soil concentration increased from 9.40 ± 0.09 mg NO3--N kg-1 for the control to even 19.40 ± 0.26 mg NO3--N kg-1 (for 100 Mg ha-1). Significant improvement of WHC (by 11.0-12.4%) was observed after biochar addition at doses of 60 Mg ha-1 and higher. At 60% WHC, application of biochar at a dose of 40 Mg ha-1 brought significant improvements in CH4 oxidation rate, which was 4.89 ± 0.37 mg CH4-C kg-1 d-1. Higher biochar doses were correlated with further improvement of CH4 oxidation rates, which at 100 Mg ha-1 was seventeen-fold higher (8.36 ± 0.84 mg CH4-C kg-1 d-1) than in the biochar-free control (0.48 ± 0.28 mg CH4-C kg-1 d-1). CO2 emissions were not proportional to biochar doses and only grew circa (ca.) twofold from 3.16 to 6.90 mg CO2-C kg-1 d-1 at 100 Mg ha-1. Above 60 Mg ha-1, the diversity of methanotrophic communities increased, with Methylobacter becoming the most abundant genus, which was as high as 7.45%. This is the first, such advanced and multifaceted study of the wide range of sunflower husk biochar doses on Haplic Luvisol. The positive correlation between soil conditions, methanotroph abundance and CH4 oxidation confirmed the multifaceted, positive effect of sunflower husk biochar on Haplic Luvisol. Sunflower husk biochar can be successfully used for Haplic Luvisol supplementation. This additive facilitates soil protection against degradation and has the potential to mitigate GHG emissions.

Microbial activity contributes to spatial heterogeneity of wetland methane fluxes.

The emission of methane from wetlands is spatially heterogeneous, as concurrently measured surface fluxes can vary by orders of magnitude within the span of a few meters. Despite extensive study and the climatic significance of these emissions, it remains unclear what drives large, within-site variations. While geophysical factors (e.g., soil temperature) are known to correlate with methane (CH4) flux, measurable variance in these parameters often declines as spatial and temporal scales become finer. As methane emitted from wetlands is the direct, net product of microbial metabolisms which both produce and degrade CH4, it stands to reason that characterizing the spatial variability of microbial communities within a wetland-both horizontally and vertically-may help explain observed variances in flux. To that end, we surveyed microbial communities to a depth of 1 m across an ombrotrophic peat bog in Maine, USA using amplicon sequencing and gene expression techniques. Surface methane fluxes and geophysical factors were concurrently measured. Across the first meter of peat at the site, we observed significant changes in the abundance and composition of methanogenic taxa at every depth sampled, with variance in methanogen abundance explaining 70% of flux heterogeneity at a subset of plots. Among measured environmental factors, only peat depth emerged as correlated with flux, and had significant impact on the abundance and composition of methane-cycling communities. These conclusions suggest that a heightened awareness of how microbial communities are structured and spatially distributed within wetlands could offer improved insights into predicting CH4 flux dynamics. IMPORTANCE Globally, wetlands are one of the largest sources of methane (CH4), a greenhouse gas with a warming impact significantly greater than CO2. Methane produced in wetlands is the byproduct of a group of microorganisms which convert organic carbon into CH4. Despite our knowledge of how this process works, it is still unclear what drives dramatic, localized (<10 m) variance in emission rates from the surface of wetlands. While environmental conditions, like soil temperature or water table depth, correlate with methane flux when variance in these factors is large (e.g., spring vs fall), the explanatory power of these variables decline when spatial and temporal scales become smaller. As methane fluxes are the direct product of microbial activity, we profiled how the microbial community varied, both horizontally and vertically, across a peat bog in Maine, USA, finding that variance in microbial communities was likely contributing to much of the observed variance in flux.

Aerobic methanotrophy increases the net iron reduction in methanogenic lake sediments.

In methane (CH4) generating sediments, methane oxidation coupled with iron reduction was suggested to be catalyzed by archaea and bacterial methanotrophs of the order Methylococcales. However, the co-existence of these aerobic and anaerobic microbes, the link between the processes, and the oxygen requirement for the bacterial methanotrophs have remained unclear. Here, we show how stimulation of aerobic methane oxidation at an energetically low experimental environment influences net iron reduction, accompanied by distinct microbial community changes and lipid biomarker patterns. We performed incubation experiments (between 30 and 120 days long) with methane generating lake sediments amended with 13C-labeled methane, following the additions of hematite and different oxygen levels in nitrogen headspace, and monitored methane turnover by 13C-DIC measurements. Increasing oxygen exposure (up to 1%) promoted aerobic methanotrophy, considerable net iron reduction, and the increase of microbes, such as Methylomonas, Geobacter, and Desulfuromonas, with the latter two being likely candidates for iron recycling. Amendments of 13C-labeled methanol as a potential substrate for the methanotrophs under hypoxia instead of methane indicate that this substrate primarily fuels methylotrophic methanogenesis, identified by high methane concentrations, strongly positive δ13CDIC values, and archaeal lipid stable isotope data. In contrast, the inhibition of methanogenesis by 2-bromoethanesulfonate (BES) led to increased methanol turnover, as suggested by similar 13C enrichment in DIC and high amounts of newly produced bacterial fatty acids, probably derived from heterotrophic bacteria. Our experiments show a complex link between aerobic methanotrophy and iron reduction, which indicates iron recycling as a survival mechanism for microbes under hypoxia.

Methane cycling in the carbonate critical zone.

The carbonate critical zone (CZ) is characterized by extensive groundwater-surface water exchange that leads to highly variable redox states of groundwater. Changes in redox condition may cause either production or consumption of methane (CH4), thereby providing an atmospheric source or sink of this important greenhouse gas. To assess how groundwater-surface water exchange affects redox state and CH4 cycling in the carbonate CZ, we measured CH4 concentrations and 13C isotopes in water from streams, spring systems, and wells in north-central Florida. Sampled groundwater has subsurface residence times ranging from hours at a stream sink-rise system, to months following a flood recharge event into a spring vent, to decades at springs with limited point recharge. Concentrations of CH4 ranged from 0.002 to 89 µM, with an inverse relationship in springs between subsurface residence time and CH4 concentration. Where residence time is short, low CH4 concentrations result from methanotrophy linked to elevated dissolved oxygen (DO) concentrations. Following flooding, methanotrophy occurs soon after recharge and is followed by methanogenesis as groundwater becomes increasingly reducing. Groundwater extracted from wells had CH4 concentrations greater than spring water indicating CH4 is lost during flow to spring vents. CH4 concentrations covary with δ13C-CH4 values, which supports both methanogenesis and methanotrophy with changing residence times. Mean fluxes of CH4 ranged from -0.05 to 1.0 mg m-2 d-1 at spring vents, with negative values caused by CH4 uptake in water undersaturated with respect to atmospheric concentration. Most springs are dominated by methanotrophy, limiting atmospheric evasion of CH4 produced in the carbonate CZ. We estimate CH4 emissions to be 12.6 × 10-6 Tg a-1 across all Florida springs or about two orders of magnitude less than emissions from Floridan aquifer groundwater abstraction (3041 × 10-6 Tg a-1). Although CH4 is produced in the carbonate CZ, natural attenuation limits its effects on the global carbon cycle.

Polyhydroxybutyrate Production from Methane and Carbon Dioxide by a Syntrophic Consortium of Methanotrophs with Oxygenic Photogranules without an External Oxygen Supply.

Here, a syntrophic process was developed to produce polyhydroxy-ß-butyrate (PHB) from a gas stream containing CH4 and CO2 without an external oxygen supply using a combination of methanotrophs with the community of oxygenic photogranules (OPGs). The co-culture features of Methylomonas sp. DH-1 and Methylosinus trichosporium OB3b were evaluated under carbon-rich and carbon-lean conditions. The critical role of O2 in the syntrophy was confirmed through the sequencing of 16S rRNA gene fragments. Based on their carbon consumption rates and the adaptation to a poor environment, M. trichosporium OB3b with OPGs was selected for methane conversion and PHB production. Nitrogen limitation stimulated PHB accumulation in the methanotroph but hindered the growth of the syntrophic consortium. At 2.9 mM of the nitrogen source, 1.13 g/L of biomass and 83.0 mg/L of PHB could be obtained from simulated biogas. These results demonstrate that syntrophy has the potential to convert greenhouse gases into valuable products efficiently.

Climate warming has direct and indirect effects on microbes associated with carbon cycling in northern lakes.

Northern lakes disproportionately influence the global carbon cycle, and may do so more in the future depending on how their microbial communities respond to climate warming. Microbial communities can change because of the direct effects of climate warming on their metabolism and the indirect effects of climate warming on groundwater connectivity from thawing of surrounding permafrost, especially at lower landscape positions. Here we used shotgun metagenomics to compare the taxonomic and functional gene composition of sediment microbes in 19 peatland lakes across a 1600-km permafrost transect in boreal western Canada. We found microbes responded differently to the loss of regional permafrost cover than to increases in local groundwater connectivity. These results suggest that both the direct and indirect effects of climate warming, which were respectively associated with loss of permafrost and subsequent changes in groundwater connectivity interact to change microbial composition and function. Archaeal methanogens and genes involved in all major methanogenesis pathways were more abundant in warmer regions with less permafrost, but higher groundwater connectivity partly offset these effects. Bacterial community composition and methanotrophy genes did not vary with regional permafrost cover, and the latter changed similarly to methanogenesis with groundwater connectivity. Finally, we found an increase in sugar utilization genes in regions with less permafrost, which may further fuel methanogenesis. These results provide the microbial mechanism for observed increases in methane emissions associated with loss of permafrost cover in this region and suggest that future emissions will primarily be controlled by archaeal methanogens over methanotrophic bacteria as northern lakes warm. Our study more generally suggests that future predictions of aquatic carbon cycling will be improved by considering how climate warming exerts both direct effects associated with regional-scale permafrost thaw and indirect effects associated with local hydrology.

Recent advances in constructed wetlands methane reduction: Mechanisms and methods.

Constructed wetlands (CWs) are artificial systems that use natural processes to treat wastewater containing organic pollutants. This approach has been widely applied in both developing and developed countries worldwide, providing a cost-effective method for industrial wastewater treatment and the improvement of environmental water quality. However, due to the large organic carbon inputs, CWs is produced in varying amounts of CH4 and have the potential to become an important contributor to global climate change. Subsequently, research on the mitigation of CH4 emissions by CWs is key to achieving sustainable, low-carbon dependency wastewater treatment systems. This review evaluates the current research on CH4 emissions from CWs through bibliometric analysis, summarizing the reported mechanisms of CH4 generation, transfer and oxidation in CWs. Furthermore, the important environmental factors driving CH4 generation in CW systems are summarized, including: temperature, water table position, oxidation reduction potential, and the effects of CW characteristics such as wetland type, plant species composition, substrate type, CW-coupled microbial fuel cell, oxygen supply, available carbon source, and salinity. This review provides guidance and novel perspectives for sustainable and effective CW management, as well as for future studies on CH4 reduction in CWs.

Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments.

Verrucomicrobia are a group of microorganisms that have been proposed to be deeply rooted in the Tree of Life. Some are methanotrophs that oxidize the potent greenhouse gas methane and are thus important in decreasing atmospheric concentrations of the gas, potentially ameliorating climate change. They are widespread in various environments including soil and fresh or marine waters. Recently, a clade of extremely acidophilic Verrucomicrobia, flourishing at pH < 3, were described from high-temperature geothermal ecosystems. This novel group could be of interest for studies about the emergence of life on Earth and to astrobiologists as homologs for possible extraterrestrial life. In this paper, we describe predicted mechanisms for survival of this clade at low pH and suggest its possible evolutionary trajectory from an inferred neutrophilic ancestor. Extreme acidophiles are defined as organisms that thrive in extremely low pH environments (≤ pH 3). Many are polyextremophiles facing high temperatures and high salt as well as low pH. They are important to study for both providing fundamental insights into biological mechanisms of survival and evolution in such extreme environments and for understanding their roles in biotechnological applications such as industrial mineral recovery (bioleaching) and mitigation of acid mine drainage. They are also, potentially, a rich source of novel genes and pathways for the genetic engineering of microbial strains. Acidophiles of the Verrucomicrobia phylum are unique as they are the only known aerobic methanotrophs that can grow optimally under acidic (pH 2-3) and moderately thermophilic conditions (50-60°C). Three moderately thermophilic genera, namely Methylacidiphilum, Methylacidimicrobium, and Ca. Methylacidithermus, have been described in geothermal environments. Most of the investigations of these organisms have focused on their methane oxidizing capabilities (methanotrophy) and use of lanthanides as a protein cofactor, with no extensive study that sheds light on the mechanisms that they use to flourish at extremely low pH. In this paper, we extend the phylogenetic description of this group of acidophiles using whole genome information and we identify several mechanisms, potentially involved in acid resistance, including "first line of defense" mechanisms that impede the entry of protons into the cell. These include the presence of membrane-associated hopanoids, multiple copies of the outer membrane protein (Slp), and inner membrane potassium channels (kup, kdp) that generate a reversed membrane potential repelling the intrusion of protons. Acidophilic Verrucomicrobia also display a wide array of proteins potentially involved in the "second line of defense" where protons that evaded the first line of defense and entered the cell are expelled or neutralized, such as the glutamate decarboxylation (gadAB) and phosphate-uptake systems. An exclusive N-type ATPase F0-F1 was identified only in acidophiles of Verrucomicrobia and is predicted to be a specific adaptation in these organisms. Phylogenetic analyses suggest that many predicted mechanisms are evolutionarily conserved and most likely entered the acidophilic lineage of Verrucomicrobia by vertical descent from a common ancestor. However, it is likely that some defense mechanisms such as gadA and kup entered the acidophilic Verrucomicrobia lineage by horizontal gene transfer.

Prospecting the significance of methane-utilizing bacteria in agriculture.

Microorganisms act as both the source and sink of methane, a potent greenhouse gas, thus making a significant contribution to the environment as an important driver of climate change. The rhizosphere and phyllosphere of plants growing in natural (mangroves) and artificial wetlands (flooded agricultural ecosystems) harbor methane-utilizing bacteria that oxidize methane at the source and reduce its net flux. For several decades, microorganisms have been used as biofertilizers to promote plant growth. However, now their role in reducing net methane flux, especially from flooded agricultural ecosystems is gaining momentum globally. Research in this context has mainly focused on taxonomic aspects related to methanotrophy among diverse bacterial genera, and environmental factors that govern methane utilization in natural and artificial wetland ecosystems. In the last few decades, concerted efforts have been made to develop multifunctional microbial inoculants that can oxidize methane and alleviate greenhouse gas emissions, as well as promote plant growth. In this context, combinations of taxonomic groups commonly found in rice paddies and those used as biofertilizers are being explored. This review deals with methanotrophy among diverse bacterial domains, factors influencing methane-utilizing ability, and explores the potential of novel methane-utilizing microbial consortia with plant growth-promoting traits in flooded ecosystems.

Sulfur and methane oxidation by a single microorganism.

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic-anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, 'Methylovirgula thiovorans' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox-rDsr pathway and the S4I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic-anoxic interface environments.

Diversity and Evolution of Methane-Related Pathways in Archaea.

Methane is one of the most important greenhouse gases on Earth and holds an important place in the global carbon cycle. Archaea are the only organisms that use methanogenesis to produce energy and rely on the methyl-coenzyme M reductase complex (Mcr). Over the last decade, new results have significantly reshaped our view of the diversity of methane-related pathways in the Archaea. Many new lineages that synthesize or use methane have been identified across the whole archaeal tree, leading to a greatly expanded diversity of substrates and mechanisms. In this review, we present the state of the art of these advances and how they challenge established scenarios of the origin and evolution of methanogenesis, and we discuss the potential trajectories that may have led to this strikingly wide range of metabolisms.

Maintaining grass coverage increases methane uptake in Amazonian pastures, with a reduction of methanogenic archaea in the rhizosphere.

Cattle ranching is the largest driver of deforestation in the Brazilian Amazon. The rainforest-to-pasture conversion affects the methane cycle in upland soils, changing it from sink to source of atmospheric methane. However, it remains unknown if management practices could reduce the impact of land-use on methane cycling. In this work, we evaluated how pasture management can regulate the soil methane cycle either by maintaining continuous grass coverage on pasture soils, or by liming the soil to amend acidity. Methane fluxes from forest and pasture soils were evaluated in moisture-controlled greenhouse experiments with and without grass cover (Urochloa brizantha cv. Marandu) or liming. We also assessed changes in the soil microbial community structure of both bare (bulk) and rhizospheric pasture soils through high throughput sequencing of the 16S rRNA gene, and quantified the methane cycling microbiota by their respective marker genes related to methane generation (mcrA) or oxidation (pmoA). The experiments used soils from eastern and western Amazonia, and concurrent field studies allowed us to confirm greenhouse data. The presence of a grass cover not only increased methane uptake by up to 35% in pasture soils, but also reduced the abundance of the methane-producing community. In the grass rhizosphere this reduction was up to 10-fold. Methane-producing archaea belonged to the genera Methanosarcina sp., Methanocella sp., Methanobacterium sp., and Rice Cluster I. Further, we showed that soil liming to increasing pH compromised the capacity of forest and pasture soils to be a sink for methane, and instead converted formerly methane-consuming forest soils to become methane sources in only 40-80 days. Liming reduced the relative abundance of Beijerinckiacea family in forest soils, which account for many known methanotrophs. Our results demonstrate that pasture management that maintains grass coverage can mitigate soil methane emissions, compared to bare (bulk) pasture soil.

In Vivo Evidence of Single 13C and 15N Isotope-Labeled Methanotrophic Nitrogen-Fixing Bacterial Cells in Rice Roots.

Methane-oxidizing bacteria (methanotrophs) play an ecological role in methane and nitrogen fluxes because they are capable of nitrogen fixation and methane oxidation, as indicated by genomic and cultivation-dependent studies. However, the chemical relationships between methanotrophy and diazotrophy and aerobic and anaerobic reactions, respectively, in methanotrophs remain unclear. No study has demonstrated the cooccurrence of both bioactivities in a single methanotroph bacterium in its natural environment. Here, we demonstrate that both bioactivities in type II methanotrophs occur at the single-cell level in the root tissues of paddy rice (Oryza sativa L. cv. Nipponbare). We first verified that difluoromethane, an inhibitor of methane monooxygenase, affected methane oxidation in rice roots. The results indicated that methane assimilation in the roots mostly occurred due to oxygen-dependent processes. Moreover, the results indicated that methane oxidation-dependent and methane oxidation-independent nitrogen fixation concurrently occurred in bulk root tissues. Subsequently, we performed fluorescence in situ hybridization and NanoSIMS analyses, which revealed that single cells of type II methanotrophs (involving six amplicon sequence variants) in paddy rice roots simultaneously and logarithmically fixed stable isotope gases 15N2 and 13CH4 during incubation periods of 0, 23, and 42 h, providing in vivo functional evidence of nitrogen fixation in methanotrophic cells. Furthermore, 15N enrichment in type II methanotrophs at 42 h varied among cells with an increase in 13C accumulation, suggesting that either the release of fixed nitrogen into root systems or methanotroph metabolic specialization is dependent on different microenvironmental niches in the root. IMPORTANCE Atmospheric methane concentrations have been continually increasing, causing methane to become a considerable environmental concern. Methanotrophy may be the key to regulating methane fluxes. Although research suggests that type II methanotrophs are involved in methane oxidation aerobically and nitrogen fixation anaerobically, direct evidence of simultaneous aerobic and anaerobic bioreactions of methanotrophs in situ is still lacking. In this study, a single-cell isotope analysis was performed to demonstrate these in vivo parallel functions of type II methanotrophs in the root tissues of paddy rice (Oryza sativa L. cv. Nipponbare). The results of this study indicated that methanotrophs might provide fixed nitrogen to root systems or depend on cells present in the spatially localized niche of the root tissue. Furthermore, our results suggested that single type II methanotrophic cells performed simultaneous methane oxidation and nitrogen fixation in vivo. Under natural conditions, however, nitrogen accumulation varied at the single-cell level.

Positron-emitting radiotracers spatially resolve unexpected biogeochemical relationships linked with methane oxidation in Arctic soils.

Arctic soils are marked by cryoturbic features, which impact soil-atmosphere methane (CH4 ) dynamics vital to global climate regulation. Cryoturbic diapirism alters C/N chemistry within frost boils by introducing soluble organic carbon and nutrients, potentially influencing microbial CH4 oxidation. CH4 oxidation in soils, however, requires a spatio-temporal convergence of ecological factors to occur. Spatial delineation of microbial activity with respect to these key microbial and biogeochemical factors at relevant scales is experimentally challenging in inherently complex and heterogeneous natural soil matrices. This work aims to overcome this barrier by spatially linking microbial CH4 oxidation with C/N chemistry and metagenomic characteristics. This is achieved by using positron-emitting radiotracers to visualize millimeter-scale active CH4 uptake areas in Arctic soils with and without diapirism. X-ray absorption spectroscopic speciation of active and inactive areas shows CH4 uptake spatially associates with greater proportions of inorganic N in diapiric frost boils. Metagenomic analyses reveal Ralstonia pickettii associates with CH4 uptake across soils along with pertinent CH4 and inorganic N metabolism associated genes. This study highlights the critical relationship between CH4 and N cycles in Arctic soils, with potential implications for better understanding future climate. Furthermore, our experimental framework presents a novel, widely applicable strategy for unraveling ecological relationships underlying greenhouse gas dynamics under global change.

Variable Inhibition of Nitrous Oxide Reduction in Denitrifying Bacteria by Different Forms of Methanobactin.

Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when copper is limiting (ambient concentrations less than 1 µM), some methanotrophs produce and secret a small modified peptide (less than 1,300 Da) termed methanobactin (MB) that binds copper with high affinity. As MB is secreted into the environment, other microbes that require copper for their metabolism may be inhibited as MB may make copper unavailable; e.g., inhibition of denitrifiers as complete conversion nitrate to dinitrogen involves multiple enzymes, some of which are copper-dependent. Of key concern is inhibition of the copper-dependent nitrous oxide reductase (NosZ), the only known enzyme capable of converting nitrous oxide (N2O) to dinitrogen. Herein, we show that different forms of MB differentially affect copper uptake and N2O reduction by Pseudomonas stutzeri strain DCP-Ps1 (that expresses clade I NosZ) and Dechloromonas aromatica strain RCB (that expresses clade II NosZ). Specifically, in the presence of MB from Methylocystis sp. strain SB2 (SB2-MB), copper uptake and nosZ expression were more significantly reduced than in the presence of MB from Methylosinus trichosporium OB3b (OB3b-MB). Further, N2O accumulation increased more significantly for both P. stutzeri strain DCP-Ps1 and D. aromatica strain RCB in the presence of SB2-MB versus OB3b-MB. These data illustrate that copper competition between methanotrophs and denitrifying bacteria can be significant and that the extent of such competition is dependent on the form of MB that methanotrophs produce. IMPORTANCE Herein, it was demonstrated that the different forms of methanobactin differentially enhance N2O emissions from Pseudomonas stutzeri strain DCP-Ps1 (harboring clade I nitrous oxide reductase) and Dechloromonas aromatica strain RCB (harboring clade II nitrous oxide reductase). This work contributes to our understanding of how aerobic methanotrophs compete with denitrifiers for the copper uptake and also suggests how MBs prevent copper collection by denitrifiers, thus downregulating expression of nitrous oxide reductase. This study provides critical information for enhanced understanding of microbe-microbe interactions that are important for the development of better predictive models of net greenhouse gas emissions (i.e., methane and nitrous oxide) that are significantly controlled by microbial activity.

Recent advances toward the bioconversion of methane and methanol in synthetic methylotrophs.

Abundant natural gas reserves, along with increased biogas production, have prompted recent interest in harnessing methane as an industrial feedstock for the production of liquid fuels and chemicals. Methane can either be used directly for fermentation or first oxidized to methanol via biological or chemical means. Methanol is advantageous due to its liquid state under normal conditions. Methylotrophy, defined as the ability of microorganisms to utilize reduced one-carbon compounds like methane and methanol as sole carbon and energy sources for growth, is widespread in bacterial communities. However, native methylotrophs lack the extensive and well-characterized synthetic biology toolbox of platform microorganisms like Escherichia coli, which results in slow and inefficient design-build-test cycles. If a heterologous production pathway can be engineered, the slow growth and uptake rates of native methylotrophs generally limit their industrial potential. Therefore, much focus has been placed on engineering synthetic methylotrophs, or non-methylotrophic platform microorganisms, like E. coli, that have been engineered with synthetic methanol utilization pathways. These platform hosts allow for rapid design-build-test cycles and are well-suited for industrial application at the current time. In this review, recent progress made toward synthetic methylotrophy (including methanotrophy) is discussed. Specifically, the importance of amino acid metabolism and alternative one-carbon assimilation pathways are detailed. A recent study that has achieved methane bioconversion to liquid chemicals in a synthetic E. coli methanotroph is also briefly discussed. We also discuss strategies for the way forward in order to realize the industrial potential of synthetic methanotrophs and methylotrophs.