Composition for reducing methane emissions in rice paddies

Abstract

The present invention relates to a composition comprising methanotrophic bacteria, which can reduce methane emissions in rice farming, promote rice growth, increase rice yield, and reduce nitrogen fertilization, thereby improving agricultural productivity. Specifically, the present invention relates to a composition for reducing methane emissions in rice paddies and for promoting rice growth, comprising methanotrophic bacteria.

Description

Composition for reducing methane emissions in rice paddies

[0001] The present invention relates to a composition for reducing methane emissions in rice paddies.

[0002] Rice, a staple food for more than half the world's population, is crucial for food security and human nutrition. Global rice consumption has increased significantly, from 157 million tons in 1960 to 520 million tons in 2022. It is projected that rice consumption will increase by approximately 6% by 2030. While rice production has significantly contributed to global food security, it is also a major source of the greenhouse gas methane. Methane has a global warming potential (GWP) approximately 84 times greater than CO2, making it a major contributor to climate change. It is estimated that producing 1 kg of rice releases approximately 100 g of methane into the atmosphere. As the global population is expected to grow, demand for rice production is expected to increase, significantly increasing methane emissions.

[0003] Traditional methane reduction strategies in rice farming have primarily focused on water management, fertilizer management, and cultivation practices. Recent attempts have focused on optimizing plant carbon allocation, regulating root exudate composition, and modifying root architecture to reduce methane emissions. However, these efforts inevitably raise the issue of genetically modified organisms (LMOs).

[0004] With the rapid advancement of microbiome technology, microbial inoculants are considered promising tools in sustainable agricultural systems. Microbial inoculants enhance crop yields by improving nutrient availability to plants and alleviating abiotic and biotic stresses (e.g., drought, salinity, and disease).

[0005] This invention demonstrates that methanotrophs, which utilize methane as their sole carbon source, have the potential to reduce methane emissions in rice farming. Furthermore, some methanotrophs also possess the ability to fix atmospheric nitrogen, suggesting they may play a crucial ecological role in regulating methane and nitrogen metabolism in rice fields. The supply of nitrogen to rice by methanotrophs could reduce nitrogen fertilizer use, thereby improving agricultural economics and reducing environmental pollution. However, research on the isolation and application of methanotrophs as bioinoculants to mitigate methane emissions and provide nitrogen sources in rice farming has been limited.

[0006] The Haber-Bosch process used to manufacture nitrogen fertilizers is a prime example of environmental pollution. Reducing nitrogen fertilizer use is crucial for environmental conservation, as nitrogen fertilizers are converted to nitrous oxide (N2O), a potent greenhouse gas, in rice paddies. Nitrous oxide has a global warming potential (GWP) approximately 350 times greater than CO2.

[0007] Methanotrophs can utilize methane as both an energy and carbon source, making them a promising solution for mitigating methane emissions from rice paddies while potentially increasing rice yields. This invention suggests the potential use of methanotrophs as bioinoculants for rice, improving rice productivity while reducing methane emissions.

[0008] The present invention aims to provide a composition and method capable of effectively reducing methane emissions from rice.

[0009] 1. A composition for reducing methane emissions from a paddy field containing methanotrophic bacteria.

[0010] 2. In the above 1, the methanotrophic bacteria is a composition for reducing methane emissions from rice fields derived from the root zone of rice.

[0011] 3. A composition for reducing methane emissions in a paddy field, wherein the methanotrophic bacteria in the above 1 is a bacterium of the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylrosaricna or Methylacidiphyllum.

[0012] 4. A composition for reducing methane emissions in a paddy field, further comprising methylotrophic bacteria in the above 1.

[0013] 5. In the above 4, the methylotrophic bacteria is a composition for reducing methane emissions in rice fields derived from the root zone of rice.

[0014] 6. In the above 4, the methylotrophic bacteria is a composition for reducing methane emissions in a paddy field, which is a Methylophilus genus bacteria.

[0015] 7. A method for reducing methane emissions from rice, comprising a step of treating the root zone of rice with a composition of any one of items 1 to 6 above.

[0016] 8. A composition for promoting rice growth containing methanotrophic bacteria.

[0017] 9. In the above 8, the methanotrophic bacteria is a composition for promoting the growth of rice derived from the root zone of rice.

[0018] 10. A composition for promoting rice growth, wherein the methanotrophic bacteria in the above 8 is a bacterium belonging to the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylrosaricna or Methylacidiphyllum.

[0019] 11. A composition for promoting rice growth, further comprising methylotrophic bacteria in the above 8.

[0020] 12. In the above 11, the methylotrophic bacteria is a composition for promoting the growth of rice derived from the root zone of rice.

[0021] 13. In the above 11, the methylotrophic bacteria is a composition for promoting rice growth, which is a bacterium of the genus Methylophilus.

[0022] 14. A composition for promoting rice growth, comprising a step of treating the root zone of a bone with a composition of any one of the above 8 to 13.

[0023] The present invention has an excellent effect in reducing methane emissions from rice farming.

[0024] The present invention can improve the growth of rice.

[0025] The present invention can improve the yield of rice.

[0026] Figure 1. Culture test of methanotrophic isolates NCN8 and UCBe9 using methane as the sole carbon source. A) Cell growth (OD600) and B) changes in culture medium pH.

[0027] Figure 2. Nitrogen fixation ability tests of methanotrophic isolates NCN8 and UCBe9. A) Gel electrophoresis images of PCR-amplified nif-H and nif-D genes. B) Growth of NCN8 and UCBe9 in nitrogen-free medium.

[0028] Figure 3. SEM images of NCN8 and UCBe9 cells showing two morphologically distinct cell types.

[0029] Figure 4. A) Circular chromosome map including the complete genome of Methylocystis species and two plasmids, and genomic comparisons with closely related species. The innermost ring indicates GC skew (green -, purple +) and GC content (black). Rings and colors in the legend indicate closely related strains used for comparison with Methylocystis species in NCN8. B) Circular chromosome map comparing the complete genome of Methylophilus species in NCN8 with closely related species.

[0030] Figure 5. Effect of methanotroph-based bioinoculation on methane emissions. A) Methane production in three experimental groups (control, NCN8, and UCBe9). B) Cumulative methane emissions.

[0031] Figure 6. Effect of NCN8 bioinoculation on methane emissions. A) Methane production in three experimental groups (control, LOW, and HIGH). B) Cumulative methane emissions.

[0032] Fig. 7. Effect of methanotroph-based bioinoculants on plant growth. A) Photographs of rice plants before harvest (125 days after transplanting) in three experimental groups (control, NCN8, and UCBe9). B) Rice root weight (g) in the three experimental groups. C) Photographs of rice roots in the three experimental groups.

[0033] Figure 8. Relative abundance of bacterial populations (A) and archaeal populations (B) at the phylum level in three experimental groups (control, NCN8, and UCBe9). Relative abundance of the genera Methylocystis and Methylophilus between the control, NCN8, and UCBe9 (C).

[0034] The present invention is described in detail below.

[0035]

[0036] The present invention relates to a composition for reducing methane emissions in a paddy field, comprising methanotrophic bacteria.

[0037] The composition of the present invention can be applied to rice, metabolizing methane produced by methanogens in an anaerobic environment of a paddy field, thereby reducing methane production.

[0038] Methanotrophic bacteria may be capable of growing and operating in the environment around rice roots.

[0039] For example, it may be capable of growth under anaerobic or partially aerobic conditions. This may be due to, for example, the ability to utilize oxygen-substituting electron acceptors under anaerobic conditions.

[0040] Methanotrophs may originate from the rhizosphere of rice.

[0041] Methanotrophic bacteria can be aerobic or anaerobic strains.

[0042] The methanotrophic bacteria may be, for example, strains from the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylrosaricna, or Methylacidiphilum. For example, strains from the genus Methylocystis may be Methylocystis fabus, Methylocystis rosea, or Methylocystis econoides.

[0043] The composition of the present invention may further comprise methylotrophic bacteria.

[0044] Methylotrophic bacteria can promote the growth of methanotrophic bacteria, further increasing the effect of reducing methane emissions.

[0045] Methylotrophic bacteria may originate from the rhizosphere of rice.

[0046] The methylotrophic bacteria can be, for example, members of the genus Methylophilus. For example, Methylophilus sp. DW102.

[0047] The bacteria used in the composition of the present invention may be, for example, a consortium of methanotrophic bacteria and methylotrophic bacteria. These may be, for example, bacteria of the genus Methylocystis and bacteria of the genus Methylophilus.

[0048] The composition of the present invention can be used as a formulation for treating paddy soil. For example, the formulation can be in various forms, such as liquid, powder, granule, or tablet, and may further include additional components suitable for treating paddy soil, such as carriers, excipients, stabilizers, and preservatives.

[0049]

[0050] In addition, the present invention relates to a method for reducing methane emissions in a field.

[0051] The method of the present invention comprises a step of treating the aforementioned methane emission reduction composition to the rhizosphere of rice.

[0052] The composition can be applied in various ways, such as applying to a seedling bed, applying when transplanting rice seedlings, applying by mixing with irrigation water, applying to the soil before tillage, applying to the surface of a paddy field, or applying by irrigation to the soil around rice after transplanting.

[0053]

[0054] In addition, the present invention relates to a composition for promoting the growth of rice, which comprises methanotrophic bacteria.

[0055] Methanotrophic bacteria may be an example of this.

[0056] The composition of the present invention may further comprise methylotrophic bacteria, which may be as exemplified above.

[0057] The composition of the present invention can be applied to fields.

[0058] The bacteria used in the composition of the present invention may be, for example, a consortium of methanotrophic bacteria and methylotrophic bacteria. These may be, for example, bacteria of the genus Methylocystis and bacteria of the genus Methylophilus.

[0059] The composition of the present invention, when applied to rice fields, reproduces in the root zone of rice plants, oxidizing methane produced by methanogenic bacteria in the soil, thereby reducing methane emissions into the atmosphere. Furthermore, intermediate metabolites produced during the methane oxidation process promote rice growth, and, depending on their nitrogen fixation capacity, further promote rice growth.

[0060] The composition of the present invention can be used in the form of a fertilizer, fertilizer additive, etc.

[0061] The composition of the present invention may further include conventional components included in fertilizers.

[0062]

[0063] In addition, the present invention relates to a method for promoting the growth of rice.

[0064] The method of the present invention comprises a step of treating the composition to the root zone of rice.

[0065] The composition can be applied in various ways, such as applying to a seedling bed, applying when transplanting rice seedlings, applying by mixing with irrigation water, applying to the soil before tillage, applying to the surface of a paddy field, or applying by irrigation to the soil around rice after transplanting.

[0066]

[0067] The present invention will be described in more detail with reference to the following examples.

[0068]

[0069] Example

[0070] method

[0071] Enrichment and separation of methanotrophic consortia

[0072] Rice (Oryza sativa L. ssp. japonica var. Saeilmi) was uprooted from a paddy field in Sacheon, Korea, separated, and washed to remove any attached soil. After washing, the roots were cut into small pieces in an aseptic environment and mixed with sterile nitrate mineral salts medium (NMS-Cu; ATCC medium 1306) supplemented with 10 mM CuCl2 and urea mineral salts medium (UMS-Cu) in which nitrate was replaced with urea. A small aliquot of the mixture was pipetted and mixed with fresh NMS-Cu or UMS-Cu medium at a ratio of 1:10, respectively, and cultured in serum bottles filled with a mixture of methane and air at a ratio of 20:80 (v / v) at 30°C for 1 week. A portion of this culture was then mixed with fresh NMS-Cu or UMS-Cu medium and cultured again under the aforementioned conditions. This procedure was repeated six times for the enrichment of methanotrophic bacteria.

[0073] The concentrated culture broth was filtered through a 0.2 μm pore size polycarbonate membrane (Sterlitech PCT027630), and the membrane filters with the concentrated culture attached were transferred to petri dishes containing 30 mL of fresh NMS-Cu or UMS-Cu medium, so that the polycarbonate membrane floated on the medium. The dishes were then placed in a sealed chamber containing a mixture of methane and air (50:50) at 30°C. The membranes were observed regularly, and the chamber was replaced with a new methane-air mixture every two days. After two weeks of culture, pinkish colonies were observed. The colonies were then directly transferred to fresh liquid NMS-Cu or UMS-Cu medium and cultured in serum bottles at 30°C with shaking at 180 rpm for one week. The isolated colonies were named NCN for colonies isolated using NMS-Cu medium and UCB for colonies isolated using UMS-Cu medium.

[0074] Analysis of growth and morphological characteristics

[0075] Methanotrophic growth experiments were performed in 120-ml serum bottles containing 30 ml of NMS-Cu or UMS-Cu medium. The bottles were sealed with butyl rubber stoppers and filled with a mixture of 20% (v / v) methane and 80% (v / v) air. The same methane:air mixture was used in all growth experiments. Two methanotrophic isolates (NCN8 and UCB9) were cultured in a shaking incubator at 30°C and 180 rpm, and growth rates were measured. Growth was monitored by optical density (OD600) using an Ultrospec 10 cell density meter (Amersham Biosciences).

[0076] To investigate the nitrogen fixation ability of the above methanotrophic isolates, their ability to grow in nitrogen-free media was tested. A nitrogen-free medium was created by removing the nitrogen source KNO3 from nitrate mineral salts medium (NMS-Cu) supplemented with 10 mM CuCl2. The methanotrophic isolates were first cultured in NMS-Cu or UMS-Cu, then centrifuged at 3,500 rpm at 4°C to collect the cells, suspended in nitrogen-free media without a nitrogen source, and centrifuged again to collect the cells, inoculate them into nitrogen-free media, and cultured according to the above culture method. The cell concentration (OD600) was analyzed every 12 hours during 48 h of cultivation. In addition, the presence of key nitrogen fixation genes such as nifH and nifD was confirmed by PCR.

[0077] To observe the exact morphology of the isolated strains using an electron microscope, the samples were pretreated and observed using a scanning electron microscope (SEM) (Zeiss model EVO-MA-15 SEM).

[0078] Genome feature analysis

[0079] High-molecular-weight genomic DNA was extracted from NCN8 using the Wizard® HMW DNA Extraction Kit from Promega according to the manufacturer's instructions. The genome was sequenced on the PacBio Sequel II e sequencing platform (Pacific Biosciences, USA) using the Sequel II Sequencing Kit 2.0. The assembled genome was annotated using Prokka version 1.14.6. Circular chromosome maps of the two complete genomes and two circular plasmids were generated using the Proksee tool. To further confirm the taxonomic position, in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI) and average amino acid identity (AAI) were calculated. isDDH, ANI, and AAI values ​​were also calculated for closely related species using the Type (Strain) Genome Server, the OrthoANIu algorithm, and the EzAAI tool from EZBioCloud.

[0080] Rice pot experiment

[0081] To test the effectiveness of methanotrophs as a bioinoculant in rice cultivation in a greenhouse environment, a rice pot experiment was conducted. Approximately 400 kg of soil was collected from a paddy field (Sacheon, Korea), naturally dried, and sieved (to a size of less than 2 mm). The soil was transferred to a Wagner pot (diameter 24 cm x height 30 cm) and used at a concentration of 1.2 g / cm. 3 filled with a bulk density of .

[0082] The isolates of NCN8 and UCBe9 were cultured on NMS-Cu and UMS-Cu media, respectively, washed with phosphate-buffered saline (PBS), and the final cell concentration was 5.1 x10 7CFU / ml. In the first year, 5.1 x10 of the roots of 3-week-old rice seedlings (Saeil-mi variety; Oryza sativa L. ssp. japonica var., Saeil-mi) were inoculated. 7 The seedlings were soaked in a suspension of UCBe9 or NCN8 at a concentration of 10 CFU / ml for 5 hours, and the roots of the control seedlings were soaked in PBS buffer for the same time. Then, the seedlings were transplanted into Wagner pots containing soil. Immediately after transplanting, 30 ml of a suspension of NCN8 or UCBe9 was applied near the roots at a density of 1.5 x10 9 After inoculation at a concentration of 10 CFU / pot, the same amount of isolates was inoculated in the same manner every two weeks until harvest. For the control group, 30 ml of PBS buffer was injected instead of the suspension of isolates.

[0083] In the second year, the roots of 3-week-old rice seedlings (Yeonghojinmi variety; Oryza sativa L. ssp. japonica var., Younghojin-mi) were injected into the soil at a low concentration (7.7 x10 7 CFU / ml) and high concentration (7.7 x10 8 The seedlings were soaked in a suspension of NCN8 culture (10 CFU / ml) for approximately 24 hours, while the control seedlings were soaked in PBS buffer for the same period. Next, the seedlings were transplanted into Wagner pots containing soil. Immediately after transplanting, approximately 100 mL of low- and high-concentration NCN8 suspensions were injected near the roots. Low- and high-concentration NCN8 were injected once a month until harvest.

[0084] A standard composition of chemical fertilizer consisting of urea (55 kg N / ha), molten superphosphate (45 kg P2O5 / ha), and potassium chloride (40 kg K2O / ha) was added to the planting soil one day before transplanting. An additional 22 kg N / ha was applied at the tillage stage, approximately 2 weeks after transplanting, and 33 kg N / ha and 18 kg K2O / ha were added at the panicle flowering stage, approximately 6–7 weeks after transplanting. The soil was flooded to a depth of 5–10 cm until rice harvest. All experiments were conducted with three replicates per treatment, and rice was harvested in mid-October.

[0085] Harvested rice was naturally dried and then separated into grains and stems. Rice growth indices, including number of ears per plant, number of grains per ear, ripening rate (%), and 1,000-grain weight, were measured according to Korean standards established by the Rural Development Administration.

[0086] CH4 emission measurement

[0087] Methane emissions were measured using the closed-chamber method. Every Wednesday at approximately 4:00 PM, the pots were covered with a fan-equipped cylindrical transparent acrylic chamber (100 cm high x 24 cm diameter), and gas samples were taken inside the chamber at 0 and 30 min using a 50-ml sealed syringe. The collected gas was immediately transferred to a 20-ml glass vial from which air had been removed. The gas samples were then analyzed using a gas chromatograph (GC-2010, Shimadzu, Japan) equipped with a flame ionization detector (FID) and a Porapak NQ column (Q 80-100 mesh).

[0088] Methane emission rates were calculated using the formula described below.

[0089] Methane emission rate (mg / m) 2 / h) = △C / △tx (V / A) x ρ x (273 / T)

[0090] Here △C(m 3 / m 3 ) is the increased gas concentration inside the chamber headspace, and t represents the time (0.5 h) when the chamber is closed. V(m 3 ) and A(m 2 ) represent the headspace volume and surface area of ​​the chamber, respectively. ρ (mg / cm 3 ) is the density of methane gas at standard conditions. T(K) is the absolute temperature of the inner chamber during gas sampling.

[0091] Microbiological analysis

[0092] After harvest, soil samples from each pot were collected to a depth of 10 cm using an auger, targeting the root zone to collect soil attached to the roots. The samples were then immediately frozen at -80°C for later DNA extraction and microbial community analysis.

[0093] Computational and statistical analysis

[0094] Statistical analysis of variance (ANOVA) of the data was performed using the SAS computer program (SAS Institute, Cary, North Carolina, USA). Duncan's multiple range test was used to compare means across groups at a significance level of 5%. Data visualization and plotting were performed using the seaborn v.0.13.2 Python package.

[0095]

[0096] result

[0097] Isolation and characterization of NCN8 and UCBe9

[0098] Methanotrophs are known to exist in the roots and rhizosphere of rice plants. In this study, aerobic methanotrophs were isolated from rice root systems through a series of subcultures and repeated re-cultivation processes on agar plates. Colonies were then transferred to polycarbonate membranes to minimize contamination with heterotrophic bacteria that may be present on agar plates.

[0099] The growth of two promising isolates (NCN8 and UCBe9) obtained from polycarbonate membranes was compared at 30°C. Within 36 h, each strain showed a growth rate of 0.1052 h. -1 and 0.0967 h -1 It grew to an OD600 value of 2 or more at a specific growth rate of (Fig. 1) (Table 1).

[0100]

[0101] Growth rates of two isolates, NCN8 and UCBe9

[0102] Temperature (°C)μ max (h-1 )Td (h)NCN8300.1052±0.00416.6±0.25UCBe9300.0967±0.00587.2±0.44

[0103] Data are expressed as mean ± standard deviation (SD). μ max , Maximum growth rate. Td, doubling time. Nitrogen fixation capacity

[0104] The genomes of NCN8 and UCBe9 contain the major structural nitrogenase genes (nifH and nifD), as shown in Figure 2A, suggesting the possibility of nitrogen fixation. Table 2 lists the PCR primers used to confirm the nitrogenase genes (nifH and nifD). However, when cultured in a nitrogen-free medium using methane as the sole carbon source, only NCN8 exhibited the ability to fix atmospheric nitrogen for growth. NCN8 grew rapidly from an OD of 0.2 to an OD of 0.7 within 24 h (Figure 2B). These differentiated growth phenotypes, along with their distinct morphological characteristics, suggest that NCN8 and UCBe9 are distinct microorganisms. In particular, the excellent atmospheric nitrogen fixation ability of NCN8 will reduce the use of nitrogen fertilizer in rice fields and have positive consequences for rice growth. The genetic characteristics of NCN8 were subsequently identified through whole-genome sequencing.

[0105]

[0106] PCR primers used to amplify nitrogenase genes (nifH and nifD)

[0107] Primer name Product Sequence ReferencenifH-FnifHTAYGGNAARGGNGGNATYGGNAARTC (SEQ ID NO: 1) Boulygina et al., (2002)nifH-R2TCNGGNGARATGATGGC (SEQ ID NO: 2)nifD-fnifDGYGGYTGCGCCTAYGCCGG (SEQ ID NO: 3) Dedysh et al., (2004)nifD-rTCCCANGARTGCATCTGRCGGA (SEQ ID NO: 4)

[0108] SEM: Both NCN8 and UCBe9 appeared to be composed of two bacterial species, as two morphologically distinct cell types were detected under scanning electron microscopy (Figs. 3A and 3B). In NCN8, one type of cell appeared as a rod-shaped bacillus, measuring approximately 2.4–2.9 x 0.8–1 micron. The second type appeared as a curved coccus with a rough surface, measuring approximately 1.3–1.5 x 0.8–1 micron (Fig. 3A). Similarly, UCBe9 also showed two different cell types, one type appeared as a coccus measuring approximately 0.8 micron in diameter, and the second type appeared as a bacillus measuring approximately 1.5 x 0.45 micron (Fig. 3B). Furthermore, genome sequencing of the NCN8 culture revealed two genomic DNA fragments closely related to the methanotroph Methylocystis parvus, and the other was identified as belonging to the genus Methylophilus. Therefore, based on existing literature, the rod-shaped bacilli were assumed to be Methylophilus species, and the other curved bacilli with a rough surface were assumed to be Methylocystis species.

[0109] Genome Analysis: Analysis of the NCN8 genome revealed two circular chromosomes and two circular plasmids. The larger chromosome, measuring 4.2 Mbp, belongs to the genus Methylocystis. The other chromosome, measuring 3.04 Mbp, belongs to the genus Methylophilus. The two plasmids, measuring 162 Kbp and 87 Kbp, both belong to Methylocystis species. Genome features such as GC content, tRNAs, rRNAs, gene and protein counts were calculated using Prokka (Table 3). The genomes of NCN8, Methylocystis (Fig. 4A) and Methylophilus (Fig. 4B), were visualized and compared with closely related species, as shown in Fig. 3.

[0110]

[0111] Two circular chromosome genome features of NCN8

[0112] TraitsMethylophilussp.Methylocystissp.Genome size3.04 Mbp4.22 MbpCircularYesYesGC%51.3364.15tRNA4552rRNA99Proteins2837406616S33pmoAs-2Integrated plasmids-2Genbank Accession NoCP173101CP172975

[0113] SUBIDBioProjectBioSampleLocalidAccessionOrganismSUB14831851PRJNA1180736SAMN44527556Methylocystis_NCN8CP172975Methylocystis sp. NCN8SUB14831851PRJNA1180736SAMN44527556pNCN8_1CP172976Methylocystis sp. NCN8SUB14831851PRJNA1180736SAMN44527556pNCN8_2CP172977Methylocystis sp. NCN8SUB14831932PRJNA1180737SAMN44527557Methylophilus_NCN8CP173101Methylophilus sp. NCN8

[0114] Genome-based comparisons of the Methylocystis and Methylophilus species present in NCN8 with closely related species were performed to calculate the average nucleotide identity (ANI), in silico DNA-DNA hybridization (DDH), and average amino-acid identity (AAI). The ANI, AAI, and DDH values ​​between the Methylocystis strain and its closest relative, Methylocystis parvus OBBP, were 82.23%, 85.04%, and 35.5%, respectively, which were lower than the threshold values ​​(95% for ANI or AAI and 70% for DDH) (Table 4). Therefore, we proposed that the Methylocystis strain in NCN8 represents a new species of the genus Methylocystis in the family Methylocystidae. Similarly, when the genome of the Methylophilus species in NCN8 was compared with that of closely related Methylophilus species, it showed the highest similarity with Methylophilus sp. DW102, with ANI, AAI, and DDH values ​​of 97.29%, 98.1%, and 82.3%, respectively (Table 5). It was very difficult to further isolate methanotrophs alone from NCN8, a consortium of methanotrophs and methylotrophs, and the growth rate of the isolated methanotrophs was severely inhibited. To maximize methane reduction, it is ideal to use methanotrophs with high growth rates that can rapidly metabolize methane. Therefore, for methane reduction in rice paddies, it may be best to utilize a consortium of methanotrophs and methylotrophs rather than pure methanotrophs. Methylotrophs can metabolize the excess methanol produced during methane oxidation, reducing methanol toxicity and promoting methanotroph growth. Furthermore, the exchange of essential nutrients between Methylocystis and Methylophilus can enhance the overall growth performance of this consortium.

[0115]

[0116] Genome comparison of NCN8 with other closely related species of the genus Methylocystis and Methylophilus. isDDH: in silico DNA-DNA hybridization; ANI: average nucleotide identity; AAI: average amino acid identity.

[0117] Rice isolate (NCN8)Closely related membersisDDH (%)ANI (%)AAI (%)Methylocystissp.Methylocystis parvusOBBP35.5 [26.5-42.6]83.2385.04Methylocystis iwaonisJCM 34278T26.7 [25.3-28.3]81.5781.71Methylocystis echinoidesLMG 2719826.3 [24.5-28.1]80.9480.07Methylocystis_NCN8 (Rumen isolate)26.2 [23.8-28.5]80.6580.55Methylocystis roseaSV9819.1 [17.8-21.6]77.3574.58Methylosinus trichosporiumOB3b17.9 [16.0-21.5]75.9669.18Methylophilussp.Methylophilus sp. DW10282.3 [76.7-85.9]97.2998.10Methylophilus methylotrophus D2231.2 [22.8-38.0]79.6485.92Methylophilus sp. TWE230.6 [23.0-36.7]79.6485.68Methylophilus medardicus MMS-M-5124.6 [20.1-28.4]76.9083.73

[0118] In rice paddies, methane emissions are an ecological balance between two metabolic processes: methane production by methanogens and methane oxidation by methanotrophs. Persistent submersion of rice creates an anaerobic environment favorable to methanogens, consuming soil organic matter as a carbon source and releasing methane gas into the atmosphere. Methanotrophs, on the other hand, are aerobic bacteria that oxidize methane as a carbon source. Therefore, significantly increasing the population of methanotrophs and thereby oxidizing more methane can reduce methane emissions from rice paddies. The simplest and most direct method is to directly inject methanotroph-based bioinoculants into rice and rice paddies.

[0119] Inoculation of rice pots with the isolated methanotrophs NCN8 and UCBe9 in the first year resulted in a 15.49% and 16.41% reduction in cumulative methane emissions, respectively, compared to the control group (Fig. 5). Considering the impact on grain yield, the NCN8 and UCBe9 bioinoculants significantly reduced methane intensity (kg methane / ton rice) by 34.64% and 35.94%, respectively (Table 6).

[0120]

[0121] The Effects of Methanotrophic Bioinoculation on Cumulative Methane Emissions and Methane Intensity

[0122] ItemsTreatments 1 SEM 2 p valueControlNCN8UCBe9Methane emission(kg CH4 / ha)1,223.58±66.571,034.05±63.501,022.79±86.4938.770.026*Methane intensity(kg CH4 / ton rice)250.36±18.49163.63±18.37160.39±26.1615.970.003*

[0123] Data are expressed as mean ± standard deviation (SD). 1Treatments: CON, 30ml of PBS buffer; NCN8, inoculation of NCN8 strain (1.5 x 10 9 cell / pot); UCBe9, inoculation of UCBe9 strain (1.5 x 10 9 cell / pot). 2 SEM, standard error of the mean. *p value <0.05. In the second year, a bioinoculant experiment was conducted with only NCN8, which showed relatively greater methane reduction and plant growth promotion effects, at two concentrations: high (HIGH) and low (LOW). Methane production was measured for up to 110 days after transplanting rice seedlings (Fig. 6A). Methane emissions from all rice plants treated with LOW and HIGH NCN8 were significantly reduced by more than 15% and 27%, respectively, compared to the untreated control group (Fig. 6B). The cumulative methane emissions for 110 days in the control, LOW, and HIGH groups were 1,823, 1,539, and 1,325 kg CH4 / ha, respectively (Table 7).

[0124] Most importantly, due to the effects of global warming, the average temperature in the second year was higher than in the first year, and the number of days exceeding 40℃ was higher (Figs. 5A and 6A). Consequently, methane production in the control group tended to be higher in the second year, and the reduction in methane production was also greater in the second year when treated with a methanotrophic bioinoculant (Figs. 5B and 6B). Therefore, as global warming intensifies, the methane reduction effect in rice paddies by methanotrophic bioinoculants will become even more dramatic.

[0125]

[0126] Effect of the methanotroph-based bioinoculant NCN8 on cumulative methane emissions

[0127] ItemsTreatments 1 SEM 2p valueControlLOWHIGHMethane emission (kg CH4 / ha)1,823±331,539±1281,325±17785.10.023*

[0128] In the first year, most measured growth indices were improved when rice plants were inoculated with NCN8 and UCBe9 bioinoculants compared to the negative control (Fig. 7A) (Table 8). Root length and weight, ear length and weight, number of tillers, number of ears per plant, and grain yield were significantly improved when NCN8 was inoculated. In particular, root weight more than doubled when NCN8 was inoculated (19.83 + 1.66) compared to the negative control (9.70 + 1.79), as shown in Figs. 7B and 7C. The significant increase in rice growth by NCN8 and UCBe9 inoculation suggests that these bioinoculants may be involved in the production of rooting hormones such as indole-3-acetic acid (IAA). IAA-producing bacteria are known to stimulate root proliferation and increase root surface area and volume. Above all, when NCN8 and UCBe9 were inoculated, grain yield (rice ton / ha) was significantly improved to 6.37 rice ton / ha and 6.43 rice ton / ha, respectively, compared to the control group (4.90 rice ton / ha) (p value = 0.025).

[0129] Analysis of plant growth indices and yields for the bioinoculant in the second-year pear cultivation experiment is ongoing, but it was more effective than in the first year. This is likely due to methane emissions measurement experiments, which revealed that increased methane production due to global warming likely stimulated the growth of methanotrophs, which use methane as a carbon source, thereby promoting rice growth.

[0130]

[0131] Effects of Methanotroph-Based Bioinoculants NCN8 and UCBe9 on Plant Growth

[0132] ItemsTreatments 1 SEM 2 p valueControlNCN8UCBe9Plant height (cm)84.00±3.6188.67±3.5185.33±1.151.110.223Straw weight (g)117.70±11.97160.85±5.15132.62±8.946.850.003*No. of tillers53.00±6.0857.97±7.5141.33±7.573.180.072No. of panicles per hill51.33±1.5374.00±4.5849.00±6.244.200.001*Weight of 1000 grains (g)8.07±0.757.51±1.218.79±0.450.330.194Ripened ratio (%)65.39±3.6265.23±1.0769.29±3.131.050.219Grain Yield (ton / ha)4.90±0.276.37±0.756.43±0.540.2980.025*

[0133] Data are expressed as mean ± standard deviation (SD). 1 Treatments: CON, 30ml of PBS buffer; NCN8, inoculation of NCN8 strain (1.5 x 10 9 cell / pot); UCBe9, inoculation of UCBe9 strain (1.5 x 10 9 cell / pot). 2 SEM, standard error of the mean. *p value <0.05. Effect of methanotroph-based bioinoculants on bacterial and archaeal communities.

[0134] A total of 7,108 bacterial amplified sequence variants (ASVs) were obtained from rice rhizosphere soil samples, and were analyzed and classified into approximately 800 genera, 300 families, 160 orders, 70 classes, and 32 phyla. Pseudomonas dorsi, Basilota, Chloroplexta, Actinomyceta, Myxococta, Acidobacteriota, Bacteroidesta, Thermodesulfobacteriota, Verrucomicrobiota, and Gemmatimonadota were the dominant phyla, accounting for more than 80% of all bacterial ASVs (Fig. 8A). In contrast, a total of 2,497 archaeal ASVs were detected and classified into 20 genera, 14 families, 12 orders, 7 classes, and 4 phyla in the archaeal community. The most dominant phyla were Euryarchaeota, Thermoplasmata, Nitrosphaerota, and Thermoproteota, accounting for nearly 70% of the total archaeal ASV (Fig. 8B). In all three experimental groups (control, NCN8-treated, and UCBe9-treated), the most dominant archaeal phylum was Euryarchaeota, accounting for 53.5%, 57.1%, and 49.1%, respectively. There was no significant change in the microbial community structure and relative abundance of the rhizosphere soil samples from the NCN8- and UCBe9-treated experimental groups (Figs. 8A and 8B). This suggests that the treatment with the methanotrophic bioinoculant did not alter the soil environment, but only had positive effects on reducing methane production and promoting rice growth. Both NCN8 and UCBe9 are composed of methanotrophs (Methylocystis sp.) and methylotrophs (Methylophilus sp.). The relative abundance of Methylocystis sp. in the control, NCN8-treated, and UCBe9-treated groups was 0.92%, 1.79%, and 2.62%, respectively. The relative abundance of the methanotroph Methylocystis sp. was 2- and 3-fold higher in NCN8 and UCB9 than in the control, respectively. However, the partner methylotroph Methylophilus sp. was observed at very low levels in all three experimental groups. In the rhizosphere of rice plants treated with the methanotroph-based bioinoculant, the methanotrophic isolates showed a strong ability to establish, proliferate, and persist.Colonization of these rice roots and rhizosphere soils with methanotrophic isolates resulted in a significant reduction in methane emissions.

[0135]

[0136] conclusion

[0137] The study results showed that applying a methanotroph-based bioinoculant to rice cultivation successfully reduced methane emissions by approximately 27%. Rice yields also increased by approximately 130%, and plant growth characteristics were significantly improved. Methanotroph-based bioinoculants have tremendous potential to mitigate methane emissions from rice farming and increase rice yields, contributing significantly to climate change responses and addressing food security challenges.

Claims

1. A composition for reducing methane emissions from a paddy field containing methanotrophic bacteria.

2. In claim 1, the methanotrophic bacteria is a composition for reducing methane emissions from a paddy field derived from the rhizosphere of rice.

3. A composition for reducing methane emissions in a paddy field according to claim 1, wherein the methanotrophic bacteria are bacteria belonging to the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylosaricna or Methylacidiphyllum.

4. A composition for reducing methane emissions in a paddy field further comprising methylotrophic bacteria according to claim 1.

5. In claim 4, the methylotrophic bacteria is a composition for reducing methane emissions from a paddy field derived from the root zone of rice.

6. A composition for reducing methane emissions in a paddy field according to claim 4, wherein the methylotrophic bacteria are bacteria of the genus Methylphyllus.

7. A method for reducing methane emissions from rice, comprising the step of treating the rhizosphere of rice with the composition of any one of claims 1 to 6.

8. A composition for promoting the growth of rice containing methanotrophic bacteria.

9. In claim 8, the methanotrophic bacteria is a composition for promoting the growth of rice derived from the rhizosphere of rice.

10. A composition for promoting the growth of rice according to claim 8, wherein the methanotrophic bacteria is a bacterium belonging to the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylosaricna or Methylacidiphyllum.

11. A composition for promoting the growth of rice, further comprising methylotrophic bacteria according to claim 8.

12. In claim 11, the methylotrophic bacteria is a composition for promoting the growth of rice derived from the root zone of rice.

13. A composition for promoting the growth of rice according to claim 11, wherein the methylotrophic bacteria is a bacterium of the genus Methylophilus.

14. A method for promoting growth of rice, comprising the step of treating the composition of any one of claims 8 to 13 to the root zone of a bone.

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