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| 15 N incorporation from 15 N 2 into roots (A) and the N 2 fixation rate (B) of wetland-grown macrophytes in the presence and absence of 5% CH 4 . Air and 15 N 2 , 15 N concentrations before and after root exposure to a gas mixture of 32% (v/v) 15 N 2 and 5% (v/v) O 2 in argon balance, respectively. +CH 4 , addition of 5% (v/v) CH 4 to the gas phase for 48 h. Bars bearing the same letter (a or b) within a panel do not differ significantly according to Tukey's test for pairwise mean comparison at α = 0.05. ST: Scirpus triqueter; TA: Typha angustifolia.
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Root-associated aerobic methanotroph plays an important role in reducing methane emissions from wetlands. In this study, we examined the activity of methane-dependent nitrogen fixation and active nitrogen-fixing bacterial communities on the roots of Typha angustifolia and Scirpus triqueter using a 15N-N2 feeding experiment and a cDNA-based clone li...
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... estimate the ability of methanotrophs that inhabit the root systems of macrophytes grown in wetlands to fix nitrogen, the roots of S. triqueter and T. angustifolia were exposed to 15 N-labeled N 2 gas in the presence and absence of CH 4 1 https://www.r-project.org/ (Figure 2A and Supplementary Table 2). In the absence of CH 4 , the 15 N atom percentage of S. triqueter roots that had been incubated was significantly higher than that of T. angustifolia. ...
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... the absence of CH 4 , the 15 N atom percentage of S. triqueter roots that had been incubated was significantly higher than that of T. angustifolia. In contrast, the presence of CH 4 significantly enhanced the concentration of 15 N in the roots of both plants (Figure 2A). In particular, the 15 N concentration of S. triqueter roots increased by more than threefold, which was significantly higher than that of T. angustifolia. ...
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... CH 4 -dependent nitrogen fixation of the S. triqueter roots was markedly higher than that of T. angustifolia. The rate of 15 N-labeled N 2 assimilation was calculated on the basis of the total root N content, dry weight and concentration of 15 N ( Figure 2B and Supplementary Table 2). ...
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... (9.8-10.6%), Gallionellales (6.5-8.3%), and Rhodospirillales (4.3-5.4%) were minor in both libraries at the order level ( Supplementary Figure 2A,B). At the family level, the diazotrophic composition clearly differed between both plant species. ...
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... differed in both plants. In addition, Beijerinckiaceae (5.4%) and Azonexaceae (4.5%) were only detected in T. angustifolia (Supplementary Figure 2C). ...
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... this study, we present direct evidence that shows that CH 4 -dependent 15 N 2 fixation occurred in the roots of two different emergent macrophytes (S. triqueter and T. angustifolia) grown in a natural wetland (Figure 2A). CH 4 -stimulated N 2 fixation occurred in an N-sufficient environment, which combined two different processes of research on N 2 fixation and methanotrophs in eutrophic wetlands. ...
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... contrast, T. angustifolia is primarily distributed in deep waters in wetlands ( Cui et al., 2020) where the root system has relatively oxic conditions because of the large amount of O 2 diffusion ( Clevering et al., 1996). This could explain why the root-associated Methylosinus of S. triqueter was found in abundance, and the plant had a greater ability to fix nitrogen compared with T. angustifolia (Figures 2-4). Moreover, other minor methanotrophs, such as Methylocella, Methylocystis and Methylobacter, may also contribute to CH 4 oxidation-dependent nitrogen fixation in this study. ...
Citations
... Another study also revealed that methanotroph inoculation on rice fields reduced CH 4 emissions by 60% and increased the rice yield by 35% [26]. The augmentation of rice yields, known as several methanotrophs, can increase the soil N availability, enhancing photosynthate allocation to grains [26,31,32]. However, the studies above examined the potential of CH 4 emission reductions by inoculating methanotrophs on rice fields under the difference of fertilizer application dosages or fertilizer types, while fields amended with external organic carbon or higher dissolved organic carbon remains uncertain with methanotroph inoculation. ...
... These strains are affiliated with type II methanotrophs, which adapt to a fluctuating environment and high CH 4 concentration, indicating the potential for reducing GHG emission based on methanotroph inoculation in rice fields of the VMD. Although the efficacy of methanotroph inoculation in reducing GHG emissions and promoting grain yield has been observed by several strains in previous studies [26,31,32], the CH 4 oxidation potential of methanotrophic strains isolated in the VMD's rice fields has not been examined. In addition, the utilization of BDE for rice fields has recently been popularized in the VMD, particularly in small-scale livestock farming. ...
... Another study also revealed that methanotroph inoculation on rice fields lowered CH 4 emissions by 60% and augmented rice yield by 35% [26]. The augmentation of rice yields, known as several methanotrophs, can increase the soil N availability, enhancing photosynthate allocation to grains [26,31,32]. However, these studies examined differences in chemical fertilizer dosages or types of methanotrophs. ...
Biogas digestive effluent (BDE) is a nutrient-enriched source that can be utilized as an organic fertilizer for rice cultivation without synthetic fertilizer (SF) application. However, a primary concern is the stimulation of methane (CH4) emissions due to the enrichment of the labile organic carbon, a favorite substrate of methanogenic archaea. Methanotrophs potentially reduce greenhouse gas (GHG) emissions from rice fields owing to metabolizing CH4 as a carbon source and energy. We therefore examined the effect of the application of methanotroph-inoculated BDE to the rice cultivated paddy soil on GHG emissions and rice productivity under a pot experiment. Methanotrophs (Methylosinus sp. and Methylocystis sp.), isolated from the Vietnamese Mekong Delta’s rice fields, were separately inoculated to the heated BDE, followed by a 5-day preincubation. Methanotroph-inoculated BDE was supplied to rice cultivation to substitute SF at 50% or 100% in terms of nitrogen amount. The results showed that the total CH4 emissions increased ~34% with the application of BDE. CH4 emissions were significantly reduced by ~17–21% and ~28–44% under the application of methanotroph-inoculated BDE at 100% and 50%, respectively. The reduction in CH4 was commensurate with the augmentation of pmoA transcript copy number under methanotroph-inoculated BDE. In addition, methanotroph-inoculated BDE application did not increase nitrous oxide (N2O) emissions and adversely affect rice growth and grain productivity. This study highlighted the BDE-recirculated feasibility for a lower CH4 emission rice production based on methanotrophs where high CH4-emitting fields were confirmed.
... Type II methylotrophs exhibit a remarkable trait of expressing nitrogenase (encoded by nifH) to harness atmospheric N 2 as a nitrogen source, a pivotal adaptation in their ecological niche (14,73). Auman et al. investigated this trait by assessing type II NifH amplification and acetylene reduction activity, confirming N 2 -fixing capability in Methylocystis spp. ...
A comprehensive pangenomic approach was employed to analyze the genomes of 75 type II methylotrophs spanning various genera. Our investigation revealed 256 exact core gene families shared by all 75 organisms, emphasizing their crucial role in the survival and adaptability of these organisms. Additionally, we predicted the functionality of 12 hypothetical proteins. The analysis unveiled a diverse array of genes associated with key metabolic pathways, including methane, serine, glyoxylate, and ethylmalonyl-CoA (EMC) metabolic pathways. While all selected organisms possessed essential genes for the serine pathway, Methylooceanibacter marginalis lacked serine hydroxymethyltransferase (SHMT), and Methylobacterium variabile exhibited both isozymes of SHMT, suggesting its potential to utilize a broader range of carbon sources. Notably, Methylobrevis sp. displayed a unique serine-glyoxylate transaminase isozyme not found in other organisms. Only nine organisms featured anaplerotic enzymes (isocitrate lyase and malate synthase) for the glyoxylate pathway, with the rest following the EMC pathway. Methylovirgula sp. 4MZ18 stood out by acquiring genes from both glyoxylate and EMC pathways, and Methylocapsa sp. S129 featured an A-form malate synthase, unlike the G-form found in the remaining organisms. Our findings also revealed distinct phylogenetic relationships and clustering patterns among type II methylotrophs, leading to the proposal of a separate genus for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129. This pangenomic study unveils remarkable metabolic diversity, unique gene characteristics, and distinct clustering patterns of type II methylotrophs, providing valuable insights for future carbon sequestration and biotechnological applications.
IMPORTANCE
Methylotrophs have played a significant role in methane-based product production for many years. However, a comprehensive investigation into the diverse genetic architectures across different genera of methylotrophs has been lacking. This study fills this knowledge gap by enhancing our understanding of core hypothetical proteins and unique enzymes involved in methane oxidation, serine, glyoxylate, and ethylmalonyl-CoA pathways. These findings provide a valuable reference for researchers working with other methylotrophic species. Furthermore, this study not only unveils distinctive gene characteristics and phylogenetic relationships but also suggests a reclassification for Methylovirgula sp. 4M-Z18 and Methylocapsa sp. S129 into separate genera due to their unique attributes within their respective genus. Leveraging the synergies among various methylotrophic organisms, the scientific community can potentially optimize metabolite production, increasing the yield of desired end products and overall productivity.
... Oxygen deficiency is presumed to be as a key constraint on methanotrophic BNF in the bulk soil, as aerobic methane oxidation (AMO) depends on oxygen. Unfortunately, most previous researches on methanotrophic BNF by MOB were focused on soil-free settings with a plenty of oxygen (>2%, v/v) in the headspace [15][16][17][18], whereas methanotrophic BNF by soil MOB suffering from strong oxygen constraint has as yet been unobserved. The widespread distribution of MOB in bulk soils necessitates a further exploration on whether MOB are able to perform methanotrophic BNF under hypoxia. ...
... The 15 N and 13 C abundances in the freeze-dried soil were measured by a stable-isotope ratio mass spectrometer (Isoprime-100, Elementar). The BNF rates were calculated using the formula (SW × TN/100 × ( 15 N c1 -15 N c2 )/ 15 N g × 100/MW/t) as previously described [17], where SW is the dried soil weight (1.00 g/reactor), TN is the average nitrogen content (%, w/w), and MW is the molecular weight (30) of 15 N 2 . The 15 N c1 and 15 N c2 represent the respective final and initial 15 N concentrations (atom% excess) in the soils, respectively. ...
Biological nitrogen fixation (BNF) by methanotrophic bacteria has been shown to play an important role in maintaining fertility. However, this process is still limited to aerobic methane oxidation with sufficient oxygen. It has remained unknown whether and how methanotrophic BNF proceeds in hypoxic environments. Herein, we incubated paddy soils with a ferrihydrite-containing mineral salt medium to enrich methanotrophic bacteria in the presence of methane (20%, v/v) under oxygen constraints (0.27%, v/v). The resulting microcosms showed that ferrihydrite-dependent aerobic methane oxidation significantly contributed (81%) to total BNF, increasing the 15N fixation rate by 13-fold from 0.02 to 0.28 μmol 15N2 (g dry weight soil) -1 d−1. BNF was reduced by 97% when ferrihydrite was omitted, demonstrating the involvement of ferrihydrite in methanotrophic BNF. DNA stable-isotope probing indicated that Methylocystis, Methylophilaceae, and Methylomicrobium were the dominant methanotrophs/methylotrophs that assimilated labeled isotopes (13C or 15N) into biomass. Metagenomic binning combined with electrochemical analysis suggested that Methylocystis and Methylophilaceae had the potential to perform methane-induced BNF and likely utilized riboflavin and c-type cytochromes as electron carriers for ferrihydrite reduction. It was concluded that ferrihydrite mediated methanotrophic BNF by methanotrophs/methylotrophs solely or in conjunction with iron-reducing bacteria. Overall, this study revealed a previously overlooked yet pronounced coupling of iron-dependent aerobic methane oxidation to BNF and improves our understanding of methanotrophic BNF in hypoxic zones.
... We also recovered a medium-quality MAG affiliated with Gemmatimonadota that was predicted to fix N 2 and oxidize CH 4 in the G27 groundwater sample (reducing aquifer); Gemmatimonadota has been identified as a fourth phylum potentially capable of aerobic methanotrophy [105]. Besides methanotrophs, diazotrophs found in the current study, such as Rhizobium and Bradyrhizobium, can utilize the methanol produced by methanotrophs [106]. Together, these findings may explain the correlation between N 2 fixation and CH 4 oxidation in groundwater (Fig. 4A, B) [106]. ...
... Besides methanotrophs, diazotrophs found in the current study, such as Rhizobium and Bradyrhizobium, can utilize the methanol produced by methanotrophs [106]. Together, these findings may explain the correlation between N 2 fixation and CH 4 oxidation in groundwater (Fig. 4A, B) [106]. The positive influence of organic matter (OM) on N 2 fixation was probably due to the supplementation of energy for heterotrophic diazotrophs [107]. ...
Biological nitrogen fixation (BNF), the conversion of N2 into bioavailable nitrogen (N), is the main process for replenishing N loss in the biosphere. However, BNF in groundwater systems remains poorly understood. In this study, we examined the activity, abundance, and community composition of diazotrophs in groundwater in the Hetao Plain of Inner Mongolia using 15N tracing methods, reverse transcription qPCR (RT-qPCR), and metagenomic/metatranscriptomic analyses. 15N2 tracing incubation of near in situ groundwater (9.5-585.4 nmol N L-1 h-1) and N2-fixer enrichment and isolates (13.2-1728.4 nmol N g-1 h-1, as directly verified by single-cell resonance Raman spectroscopy), suggested that BNF is a non-negligible source of N in groundwater in this region. The expression of nifH genes ranged from 3.4 × 103 to 1.2 × 106 copies L-1 and was tightly correlated with dissolved oxygen (DO), Fe(II), and NH4+. Diazotrophs in groundwater were chiefly aerobes or facultative anaerobes, dominated by Stutzerimonas, Pseudomonas, Paraburkholderia, Klebsiella, Rhodopseudomonas, Azoarcus, and additional uncultured populations. Active diazotrophs, which prefer reducing conditions, were more metabolically diverse and potentially associated with nitrification, sulfur/arsenic mobilization, Fe(II) transport, and CH4 oxidation. Our results highlight the importance of diazotrophs in subsurface geochemical cycles.
... Methanotrophs (Methylocystis, Methylosinus, and Methylocella) have been shown to fix N 2 in maize stems and rice phyllosphere [41,91]. Macrophytes grown in natural wetlands and mosses are colonized by methanotrophs with high methane oxidation and nitrogen fixation potentials [92,93]. The high methane oxidation potential is also evident for bark-dwelling diazotrophic Methylomanas spp. ...
The importance of biological nitrogen fixation (BNF) in securing food production for the growing world population with minimal environmental cost has been increasingly acknowledged. Leaf surfaces are one of the biggest microbial habitats on Earth, harboring diverse free-living N2-fixers. These microbes inhabit the epiphytic and endophytic phyllosphere and contribute significantly to plant N supply and growth. Here, we summarize the contribution of phyllosphere-BNF to global N cycling, evaluate the diversity of leaf-associated N2-fixers across plant hosts and ecosystems, illustrate the ecological adaptation of N2-fixers to the phyllosphere, and identify the environmental factors driving BNF. Finally, we discuss potential BNF engineering strategies to improve the nitrogen uptake in plant leaves and thus sustainable food production.
Keywords
... In addition, previous studies have demonstrated the presence and activity of MOB in biofilms from freshwater canals (Pelsma et al., 2021), but it is still unknown whether MOB are associated with filamentous algae and what their contribution to reducing dissolved CH 4 in the water column might be. Furthermore, MOB are present and active on both above and below ground parts of aquatic macrophytes (Cui et al., 2022(Cui et al., , 2020Faußer et al., 2012) and it has been demonstrated that MOB associated with submerged aquatic macrophytes can also reduce CH 4 emission (Sorrell et al., 2002;Yoshida et al., 2014), but the range of CH 4 -oxidation potential differs among species and the studied tissues (Heilman and Carlton, 2001). However, it is still not clear how different nutrient loading and warming treatments can influence MOB presence and activity associated with submerged aquatic plants and their contribution to oxidizing the produced CH 4 . ...
Shallow lakes produce and emit substantial amounts of methane (CH4). Part of the CH4 produced in lakes is consumed by methane-oxidizing bacteria (MOB) present in the sediment and water column, thus reducing the overall CH4 emissions. However, the role of aquatic plants as habitat for CH4 oxidation by MOB is poorly understood. In this study, we compared CH4 oxidation rates and MOB abundance associated with different types of aquatic plants (periphyton, filamentous algae, and both above-ground macrophytes and their rhizosphere). The plants were collected from shallow lake mesocosms exposed to experimental nutrient enrichment and warming treatments for 17 years prior to this study. Incubations of all sampled plants showed CH4 oxidation, with above-ground macrophyte tissue and filamentous algae having the highest rates of up to 0.25 µmol CH4 h⁻¹ g⁻¹ dw. Oxidation rates associated with macrophytes were species dependent, with consumption rates on rhizospheres of Potamogeton crispus higher than those on Elodea canadensis. The increase in nutrients and dissolved CH4 in the water tended to increase MOB abundance and activity for all plant types, while no effect of long term warming was detectable. Our results showed that MOB associated with periphyton, filamentous algae and macrophytes oxidize CH4 in shallow lakes at different rates across species or plant types. We also found that high macrophyte biomass is associated with reduced CH4 concentration in the water. This study shows that CH4 oxidation occurs on many plant surfaces but that oxidation rates alone cannot explain the reduced CH4 emissions at higher plant biomass.
As an essential element for plants, animals, and humans, selenium (Se) has been shown to participate in microbial methane oxidation. We studied the growth response and rhizosphere methane oxidation of an economic crop (prickly pear, Rosa roxburghii Tratt) through three treatments (Se0.6 mg/kg, Se2.0 mg/kg, and Se10 mg/kg) and a control (Se0 mg/kg) in a two-month pot experiment. The results showed that the height, total biomass, root biomass, and leaf biomass of prickly pear were significantly increased in the Se0.6 and Se2.0 treatments. The root-to-shoot ratio of prickly pear reached a maximum value in the Se2 treatment. The leaf carotenoid contents significantly increased in the three treatments. Antioxidant activities significantly increased in the Se0.6 and Se2 treatments. Low Se contents (0.6, 2 mg/kg) promoted root growth, including dry weight, length, surface area, volume, and root activity. There was a significant linear relationship between root and aboveground Se contents. The Se translocation factor increased as the soil Se content increased, ranging from 0.173 to 0.288. The application of Se can improve the state of rhizosphere soil’s organic C and soil nutrients (N, P, and K). Se significantly promoted the methane oxidation rate in rhizosphere soils, and the Se10 treatment showed the highest methane oxidation rate. The soil Se gradients led to differentiation in the growth, rhizosphere soil properties, and methane oxidation capacity of prickly pear. The root Se content and Se translocation factor were significantly positively correlated with the methane oxidation rate. Prickly pear can accumulate Se when grown in Se-enriched soil. The 2 mg/kg Se soil treatment enhanced growth and methane oxidation in the rhizosphere soil of prickly pear.
Methanotrophs oxidize methane (CH 4 ) and greatly help in mitigating greenhouse effect. Increased temperatures due to global climate change can facilitate lake salinization, particularly in the regions with cold semiarid climate. However, the effects of salinity on the CH 4 oxidation activity and diversity and composition of methanotrophic community in the sediment of natural lakes at a regional scale are still unclear. Therefore, we collected lake sediment samples from 13 sites in Mongolian Plateau; CH 4 oxidation activities of methanotrophs were investigated, and the diversity and abundance of methanotrophs were analyzed using real-time quantitative polymerase chain reaction and high throughput sequencing approach. The results revealed that the diversity of methanotrophic community decreased with increasing salinity, and community structure of methanotrophs was clearly different between the hypersaline sediment samples (HRS; salinity > 0.69%) and hyposaline sediment samples (HOS; salinity < 0.69%). Types II and I methanotrophs were predominant in HRS and HOS, respectively. Salinity was significantly positively correlated with the relative abundance of Methylosinus and negatively correlated with that of Methylococcus . In addition, CH 4 oxidation rate and pmoA gene abundance decreased with increasing salinity, and salinity directly and indirectly affected CH 4 oxidation rate via regulating the community diversity. Moreover, high salinity decreased cooperative association among methanotrophs and number of key methanotrophic species ( Methylosinus and Methylococcus , e.g). These results suggested that salinity is a major driver of CH 4 oxidation in lake sediments and acts by regulating the diversity of methanotrophic community and accociation among the methanotrophic species.
