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Growth performance of wild-type methanotrophs at 500 ppm methane. (A-G) Growth curves of M. buryatense 5GB1C (A), M. alcaliphilum 20Z (B), Methylosinus sp. LW4 (C), Methylocystis sp. LW5 (D), Methylomonas sp. LW13 (E), and Methylomicrobium lacus LW14 (F) (n = 3). A, B, E, and F = gammaproteobacteria; C and D = alphaproteobacteria. (G) Daily OD 600 increase of the seven methanotrophs during a 14-d growth period (n = 3), which were determined based on the slopes of the linear region of growth curves. **P < 0.01, which was determined by the unpaired t test. Error bars represent SDs.
Source publication
The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO2 and biomass, a value-added...
Contexts in source publication
Context 1
... 5GB1C, Methylomicrobium (previously Methylosarcina) lacus LW14, Methylosinus sp. LW4, and Methylocystis sp. LW5 were able to grow at 500 ppm methane. However, M. lacus LW14 did not show sustained growth after 10 d at 500 ppm methane. Little or no growth was observed for Methylomonas LW13 or Methylotuvimicrobium alcaliphilum 20Z incubated for 14 d (Fig. 1). Additionally, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, two broadly studied methanotrophs, had previously been shown to not grow or grow poorly at 1,000 ppm or lower concentrations (18), and thus, their growth analyses were not repeated here. In this screen, the four methanotrophs capable of growing at 500 ...
Context 2
... Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, two broadly studied methanotrophs, had previously been shown to not grow or grow poorly at 1,000 ppm or lower concentrations (18), and thus, their growth analyses were not repeated here. In this screen, the four methanotrophs capable of growing at 500 ppm methane show generally linear growth curves ( Fig. 1 A, C, D, and F), likely because the headspace in each bottle was refreshed with 500 ppm methane once a day and the daily biomass growth was capped by this restricted supply of methane. Of these four methanotrophs, M. buryatense 5GB1C exhibited the fastest growth and highest optical density values at the end of cultivation at 500 ppm; hence, we focused on this strain in the following analyses. ...
Context 3
... which changes the methanol oxidation system induced, can improve the growth rate of M. buryatense 5GB1C by 10% in the presence of sufficient methane (21), but the impact on growth at 500 ppm methane was unknown. We tested the addition of lanthanum, but it did not stimulate growth at 500 ppm methane under the conditions tested (SI Appendix, Fig. ...
Citations
... 7 Due to the soil microbiota in anaerobiosis transforms NO 3 into N 2 O which favors global warming with negative consequences for life on Earth, 8 including able to oxide methane (CH 4) a promising biological tool against global warming. 9 An alternative solution is to regulate or reduce the dose of NH 4 NO 3 without compromising the healthy growth of P. vulgaris, by inoculating the seed with Methylobacterium symbioticum, a genus and species of endophyte that, in addition to colonizing the leaves of P. vulgaris, 10,11 also invades the roots of the legume in order to optimize the uptake of the reduced NH 4 NO 3 . 12,13 Since foliar inoculation with P. vulgaris would entail a greater risk of a positive response, 13 the objective of this work was to analyze the effect of M. symbioticum on P. vulgaris seeds plus NH 4 NO 3 at 50%. ...
... Although instead of inoculating the foliar part of P. vulgaris, 1 the seeds were treated, which implies that M. symbioticum has the ability to invade not only the aerial part of the plant 2 it also responds to the organic products of germination 4-6 in addition to being a way to enter the conduction system of P. vulgaris. 8 M. symbioticum was recovered from the leaves of P. vulgaris, which increases the possibilities of applying M. symbioticum to support the healthy growth of P. vulgaris based on a dose that is uptake effectively, 13,14 at the same time minimizing N 2 O generation 6,9 and other negative impacts to the soil such as the loss of organic matter and / or the contamination of surface water or aquifers, 15,18 by excess of NH 4 NO 3 not uptake by P. vulgaris. 19 Table 3 shows the phenology at pre-flowering stage of P. vulgaris with M. symbioticum 3 and 50% NH 4 NO 3 where it reached 59.06 cm of plant height (PH) and 14.46 cm of root length (RL), with not statical difference registered with M. symbioticum isolates 1, 2 and with the mixture of the 3 whose numerical values were statistically different compared to 21.66 cm of PH and 7.75 cm of RL of P. vulgaris not inoculated with 100% NH 4 NO 3 or relative control (RC). ...
The healthy growth of Phaseolus vulgaris relies on fertilization of nitrogen with
NH4NO3. However, excessive application leads to environmental degradation, including
loss of soil fertility and, green gas emissions such as N2O. As an ecological alternative,
this study investigates whether reduce the dose to 50% and inoculate P. vulgaris with
Methylobacterium symbioticum, an endophytic bacterium can promotes plant growth. The
objective of this research was to analyze the effect of M. symbioticum on the growth of P.
vulgaris plus NH4NO3 at 50%. Three isolates of M. symbioticum, obtained from P. vulgaris
leaves, were individually and jointly inoculated into bean seeds. A randomized block design
was used with two controls (uninoculated plants with either 100% NH₄NO₃ or water only)
and four treatments (three individual isolates and one combined treatment) under 50%
NH₄NO₃ fertilization. Growth performance was assessed through germination rate, plant
height, root lenght, and biomass at seedling and pre-flowering stages. Data were analyzed
using ANOVA and Tukey’s HSD (p < 0.05).
The results showed a positive effect of M. symbioticum on the germination of P. vulgaris
compared to uninoculated P. vulgaris and 100% NH4NO3. Likewise, a positive effect
was observed on the phenology and biomass of P. vulgaris in the different isolates of M.
symbioticum with 50% NH4NO3. There was evidence that although M. symbioticum is
an endophyte of leaves of domestic plants, it can invade the root tissue of P. vulgaris.
These findings suggest that inoculation with M. symbioticum allows for reduced nitrogen
fertilization without compromising plant health, potentially mitigating environmental harm
such as N₂O release and water contamination.
Keywords: soil, legume seeds, greenhouse gases, foliar endophyte, radical colonization,
plant health
... Agricultural sources were the largest source of anthropogenic CH 4 emissions, with bottom-up estimates of 211 (204-216)Tg y −1 in 2020(Jackson et al., 2024). Within the agricultural sector, enteric fermentation and manure management together contributed 147 (143-149) Tg y −1 , and rice cultivation contributed 32(29)(30)(31)(32)(33)(34)(35)(36)(37) Tg y −1 in 2020(Jackson et al., 2024). Here, we focus on reviewing recent efforts to mitigate CH 4 from rice production.According to our current knowledge, the metabolic processes ofCH 4 generation and oxidation can be summarized in Figure 2. In flooded rice soils, CH 4 is the final product of the reductive process under anaerobic conditions and is produced by microbial methanogens (Minamikawa et al., 2006). ...
Anthropogenic global warming is closing to the red line of the irreversible impacts on climate change. Soil microorganisms have multiple functions with great potential for greenhouse gas mitigation; however, these technologies have not been effectively applied on a large scale. This minireview aimed to awaken people's awareness of the importance of soil microorganisms in greenhouse gas mitigation. In this paper, we introduced the current statute of global warming and reviewed recent highlights in microbe-based solutions to reduce nitrous oxide and methane emissions. In addition, we discussed the effects of soil management strategy on carbon sequestration in soil. Finally, we presented some novel approaches to the greenhouse gas mitigation and our vision for the development of new biotechnologies to further unlock the microbial ability of greenhouse gas mitigation. This minireview is also a call for immediate and decisive emergency action to curb global warming.
... CH₄ concentrations were highest under cold (43%) conditions due to cold-seep habitats, gas hydrates, and methane bubbles releasing methane-rich fluids (Peketi et al. 2021). Heat conditions significantly impacted methanogenesis, with extreme heat could increase methanogen activity and leading to higher CH₄ production ( Figure 2b); however, heat probably inhibits methane consumption by methanotrophic bacteria (He et al. 2023). ...
River riparian basins play a crucial role in mitigating greenhouse gas (GHG) emissions through carbon sequestration and nitrogen sinks. However, increased ecological stresses led to the release of CO 2 , CH 4 and N 2 O. This study aimed to investigate how extreme temperatures, water levels, moisture content, land use changes and soil composition influence GHG emissions in the riparian corridor and to recommend mitigation techniques. It was carried out at the Yangtze River Riparian zone, China, using soil column testing. It used soil column testing. The results showed that extreme temperatures caused the highest emissions of CO₂ (29–45%), CH₄ (24–43%) and N₂O (27–33%). This was due to increased soil temperatures and accelerated organic carbon/nitrogen decomposition. Conversely, control and wet–dry cycles absorbed CO 2 (1–3%), CH 4 (3–10%) and N 2 O (1–21%) by improving soil aeration, increased oxygen availability, soil structure, stable water table and low temperature change. Grasses in riparian areas also improved carbon sinks. Highest water levels had lowest gas concentrations and emissions due to low oxygen level. Adaptive wet‐dry cycles, grass cover and better water table management can restore riparian areas, maintain soil moisture, balance soil carbon/nitrogen levels and mitigate climate change by improving soil quality. Dissolved organic matter fluorescence (DOMFluor) components are essential for soil carbon dynamics, aquatic biome safety, nutrient cycling and ecological balance in riparian zones. The study recommends implementing restoration practices, managing soil moisture, afforestation, regulating temperature and monitoring water tables to mitigate GHG emissions and address climate change. Future policies should focus on promoting resilient land use and ecosystems.
... Adding cover soils to landfill sites is an effective technique for enhancing CH4 removal since this provides soil for methanotrophic microbes to flourish (Nisbet-Jones et al. 2022). The anaerobic digestion of various organic materials and wastes can exploit methanogenesis for CH4 harvesting, avoiding the release of gasses such as N2O and CH4 that occur when these materials are applied to soils He et al. (2023) identified the methanotroph Methylotuvimicrobium buryatense as a good candidate for CH4 removal at emission sites such as anaerobic digestors, gas wells and landfills since this species demonstrates rapid CH4 uptake, even at low CH4 concentrations. ...
Global warming refers to the long-term increase in the Earth's surface temperature, which can lead to significant and widespread impacts on the planet. These effects include rising sea levels, changes in precipitation patterns, and more frequent and severe natural disasters. Atmospheric levels of methane (CH4) have risen by over 150% since the pre-industrial era, with agriculture and livestock production being major contributors. This review aims to explore the effect of climate change on methanotrophic microorganisms and the potential of managing microbial communities to reduce CH4 emissions, thereby mitigating CH4 emissions as part of broader climate change strategies. While methanogens are CH4-producing bacteria, methanotrophic bacteria can utilise CH4 as a source of energy source and can consume large amounts of CH4 directly from both the atmosphere and soils. Many factors influence the balance of microbes acting as a sink or consumers of greenhouse gasses including changes in terrestrial and marine environments. Temperature, CO2 levels and precipitation have all been shown to have a profound effect on the ecology of methanogens, driving positive feedback which exacerbates the rate of climate change. Reducing CH4 emissions is an important aspect of mitigating the impacts of climate change, and it may be theoretically possible to mitigate a considerable portion of global CH4 emissions by managing microbial communities in various environments by reviewing land use and farming management practices. Examples of these practices include the reforestation of native woodlands, altering the management of organic materials in rice paddies and the reduction of CH4 release from ruminant livestock utilizing additives to animal feeds such as red seaweed or hormonal products such as bovine somatotropin. Although efforts to mitigate CH4-induced climate change effects are ongoing, further research is required to better elucidate the mechanisms involved in methanogenesis and the potential for reducing CH4 emissions through targeted interventions.
... Alternatively, methane removal in bioreactors containing methanotrophic bacteria [82,83] is an attractive option, as is encouraging uptake from soil and trees [66] This costs little energy but unfortunately methanotrophy is very slow for mixing ratios found in cattle barns. Nevertheless, it is possible that effective bioremoval methanotrophs can be found. ...
This review summarizes the rapid advances in direct practical methods to quantify and reduce agricultural methane emissions worldwide. Major tasks are location, identification, quantification and distinction between different specific sources (often multiple emitters such as manure pools, animal housing, biodigesters and landfills are co-located). Emission reduction, facilitated by developing methodologies for locating hot spots, is the least-cost choice for action, especially from manure stores, biodigesters and from controlling biomass burning. Agricultural methane can also be used to generate electricity or, in appropriate circumstances, can be destroyed by oxidation. It may be possible to cut North American, East Asian and European emissions sharply and rapidly. In Africa and South Asia, emissions from crop waste and food waste in landfills, also a source of air pollution, can be sharply and quickly reduced. Globally, cutting total annual agricultural and waste emissions by a third would demand reductions of very approximately 75 Tg yr⁻¹. Apportioned by source type, notional cuts might be 30–40 Tg yr⁻¹ from livestock and manure, 5-10 Tg yr⁻¹ from rice cultivation and 20 Tg yr⁻¹ or more from specifically agricultural waste.
... Unfortunately, most research has focused on high methane concentrations (10000 -60000 ppmv), and studies with low methane concentrations are scarcely studied in the literature, leaving a critical knowledge gap for developing practical methane mitigation options. A PNAS study (He et al., 2023) reported a strain of methanotrophs that thrive on low methane concentrations (10 ppm or lower) in the head space. However, this is not directly beneficial to our efforts for the following reasons: 1) It is a batch study without soil, and thus mass transfer is likely to be larger than in soil columns where the water occupies the small pores and thus one could encounter a reduction of diffusion by at least an order of magnitude. ...
... We selected Methylotuvimicrobium alcaliphilum 20Z R , as over the years, the Methylotuvimicrobium spp. has become a testable microbial platform for methane capturing and valorization (16)(17)(18)(19)(20). The metabolic flux balance models of the whole genome have been generated and manually curated (21-23), allowing for a direct comparison between expert-curated and automated optimization of GSMs for non-model microbes. ...
Context-specific genome-scale model (CS-GSM) reconstruction is becoming an efficient strategy for integrating and cross-comparing experimental multi-scale data to explore the relationship between cellular genotypes, facilitating fundamental or applied research discoveries. However, the application of CS modeling for non-conventional microbes is still challenging. Here, we present a graphical user interface that integrates COBRApy, EscherPy, and RIPTiDe, Python-based tools within the BioUML platform, and streamlines the reconstruction and interrogation of the CS genome-scale metabolic frameworks via Jupyter Notebook. The approach was tested using -omics data collected for Methylotuvimicrobium alcaliphilum 20ZR, a prominent microbial chassis for methane capturing and valorization. We optimized the previously reconstructed whole genome-scale metabolic network by adjusting the flux distribution using gene expression data. The outputs of the automatically reconstructed CS metabolic network were comparable to manually optimized iIA409 models for Ca-growth conditions. However, the CS model questions the reversibility of the phosphoketolase pathway and suggests higher flux via primary oxidation pathways. The model also highlighted unresolved carbon partitioning between assimilatory and catabolic pathways at the formaldehyde-formate node. Only a very few genes and only one enzyme with a predicted function in C1 metabolism, a homolog of the formaldehyde oxidation enzyme (fae1-2), showed a significant change in expression in La-growth conditions. The CS-GSM predictions agreed with the experimental measurements under the assumption that the Fae1-2 is a part of the tetrahydrofolate-linked pathway. The cellular roles of the tungsten (W)-dependent formate dehydrogenase (fdhAB) and fae homologs (fae1-2 and fae3) were investigated via mutagenesis. The phenotype of the fdhAB mutant followed the model prediction. Furthermore, a more significant reduction of the biomass yield was observed during growth in La-supplemented media, confirming a higher flux through formate. M. alcaliphilum 20ZR mutants lacking fae1-2 did not display any significant defects in methane or methanol-dependent growth. However, contrary to fae1, the fae1-2 homolog failed to restore the formaldehyde-activating enzyme function in complementation tests. Overall, the presented data suggest that the developed computational workflow supports the reconstruction and validation of CS-GSM networks of non-model microbes.
IMPORTANCE
The interrogation of various types of data is a routine strategy to explore the relationship between genotype and phenotype. An efficient approach for integrating and cross-comparing experimental multi-scale data in the context of whole-genome-based metabolic network reconstruction becomes a powerful tool that facilitates fundamental and applied research discoveries. The present study describes the reconstruction of a context-specific (CS) model for the methane-utilizing bacterium, Methylotuvimicrobium alcaliphilum 20ZR. M. alcaliphilum 20ZR is becoming an attractive microbial platform for the production of biofuels, chemicals, pharmaceuticals, and bio-sorbents for capturing atmospheric methane. We demonstrate that this pipeline can help reconstruct metabolic models that are similar to manually curated networks. Furthermore, the model is able to highlight previously overlooked pathways, thus advancing fundamental knowledge of non-model microbial systems or promoting their development toward biotechnological or environmental implementations.
... These include sealing off disused wells to prevent leaks and implementing microbiological techniques alongside dietary modifications to lower the CH 4 cattle produce [6,7]. One promising approach to mitigating CH 4 emissions is through the use of methanotrophs, organisms that consume CH 4 and act as an effective natural CH 4 sink [8]. Aerobic methanotrophs have been well studied and classified into two types, I and II, primarily based upon their CH 4 assimilation route. ...
... A previous report demonstrated that type I methanotrophs have strong CH 4 biotransformation potential in paddy fields (Zheng et al., 2024). Ecosystems like paddy fields typically contain 500 ppm CH 4 , and the ability of type 1 methanotrophs to utilize such low levels of CH 4 has been reported recently (He et al., 2023). M. capsulatus cells in the biostimulant formulation are thus possibly acting as crucial biological filters to alleviate CH 4 emissions from paddy fields. ...
Introduction
More than half of the world’s population consumes rice as their primary food. The majority of rice production is concentrated in Asia, with the top 10 rice-growing countries accounting for 84% of the world’s total rice cultivation. However, rice production is also strongly linked to environmental changes. Among all the global sources of greenhouse gas (GHG) emissions, paddy cultivation stands out as a significant contributor to global methane (CH4) and nitrous oxide (N2O) emissions. This contribution is expected to increase further with the projected increase of 28% in global rice output by 2050. Hence, modifications to rice management practices are necessary both to increase yield and mitigate GHG emissions.
Methods
We investigated the effect of seedling treatment, soil application, and foliar application of a methane-derived microbial biostimulant on grain yield and GHG emissions from rice fields over three seasons under 100% fertilizer conditions. Further, microbial biostimulant was also tested under 75% nitrogen (N) levels to demonstrate its effect on grain yield. To understand the mechanism of action of microbial biostimulant on crop physiology and yield, a series of physiological, transcript, and metabolite analyses were also performed.
Results
Our three-season open-field studies demonstrated a significant enhancement of grain yield, up to 39%, with a simultaneous reduction in CH4 (31%–60%) and N2O (34%–50%) emissions with the use of methane-derived microbial biostimulant. Under 75% N levels, a 34% increase in grain yield was observed with microbial biostimulant application. Based on the physiological, transcript, and metabolite analyses data, we were further able to outline the potential mechanisms for the diverse synergistic effects of methane-derived microbial biostimulant on paddy, including indole-3-acetic acid production, modulation of photosynthesis, tillering, and panicle development, ultimately translating to superior yield.
Conclusion
The reduction in GHG emission and enhanced yield observed under both recommended and reduced N conditions demonstrated that the methane-derived biostimulant can play a unique and necessary role in the paddy ecosystem. The consistent improvements seen across different field trials established that the methane-derived microbial biostimulant could be a scalable solution to intensify rice productivity with a lower GHG footprint, thus creating a win–win–win solution for farmers, customers, and the environment.
... Overexpression of CAs improved M. capsulatus growth kinetics and CH 4 conversion efficiencies. Our results advance the understanding of inorganic carbon metabolism in methanotrophic bacteria and highlight a genetic engineering strategy that leverages CAs to mitigate both CH 4 and CO 2 GHGs. ...
Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH4 and CO2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO2-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO2 evolution from CH4-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.
IMPORTANCE
Methanotrophs transform CH4 into CO2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO2. We developed an engineered strain with improved CO2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH4 conversion bioprocesses.
