WO2025116669A1 - Composition for reducing methane emissions in rice paddies - Google Patents

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 fields

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

Rice, which is consumed as a staple food by more than half of the world’s population, is a critical element 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 expected that rice consumption will increase by about 6% by 2030. While rice production has contributed significantly to global food security, it is also a major source of methane, a greenhouse gas. Methane is a major contributor to climate change, with a global warming potential (GWP) that is about 84 times greater than that of _CO2 . It is estimated that about 100 g of methane is released into the atmosphere when producing 1 kg of rice. As the world’s population is expected to increase, the demand for rice production is expected to increase, leading to a significant increase in methane emissions.

Traditional methane reduction strategies in rice farming have focused primarily on water management, fertilizer management, and cultivation practices. More recently, attempts have been made to reduce methane emissions by optimizing plant carbon allocation, regulating root exudate composition, and changing root architecture, but this approach cannot avoid the controversy of genetically modified organisms (GMOs).

With the rapid development of microbiome technology, microbial inoculants are considered as promising tools in sustainable agricultural systems. Microbial inoculants improve nutrient availability to plants and alleviate abiotic/biotic stresses (e.g. drought, salinity, diseases) to enhance crop yields.

In this invention, we demonstrate that methanotrophs that utilize methane as their sole carbon source have the potential to reduce methane emissions in rice farming. In addition, some methanotrophs have the ability to fix atmospheric nitrogen, and thus may play an important ecological role in regulating methane and nitrogen metabolism in rice fields. The supply of nitrogen sources to rice by methanotrophs can reduce the use of nitrogen fertilizers, thereby improving the economic feasibility of agriculture and reducing environmental pollution. However, studies on the isolation and application of methanotrophs as bio-inoculants to alleviate methane emissions and supply nitrogen sources in rice farming have been limited.

The Haber-Bosch process used to manufacture nitrogen fertilizers is a typical example of environmental pollution, and reducing the use of nitrogen fertilizers is very beneficial for environmental conservation because nitrogen fertilizers are converted to nitrous oxide ( _N2O ), a strong greenhouse gas, in the field. Nitrous oxide has a global warming potential (GWP) that is about 350 times higher than _CO2 .

Methanotrophs can utilize methane as an energy and carbon source, which could be a promising solution to mitigate methane emissions from rice fields while potentially increasing rice yields. This invention suggests the potential use of methanotrophs as bio-inoculants for rice to reduce methane emissions while improving rice productivity.

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

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

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.

3. A composition for reducing methane emission in a paddy field, wherein the methanotrophic bacteria in the above 1 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 in the above 1.

5. In the above 4, the methylotrophic bacteria is a composition for reducing methane emissions from rice roots in a paddy field.

6. A composition for reducing methane emissions in a paddy field, wherein the methylotrophic bacteria in the above 4 are Methylophilus bacteria.

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.

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

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

10. A composition for promoting the growth of rice, wherein in the above 8, the methanotrophic bacteria are bacteria belonging to the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylosaricna or Methylacidiphyllum.

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

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

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

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

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

The present invention can improve the growth of rice.

The present invention can improve the yield of rice.

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.

Figure 2. Nitrogen fixation ability test 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.

Figure 3. SEM images of NCN8 and UCBe9 cells showing two morphologically different cell types.

Figure 4. A) Circular chromosome map including the complete genome of Methylocystis species and two plasmids, and comparison of the genomes 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.

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

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

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

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).

The present invention is described in detail below.

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

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.

Methanotrophic bacteria may be capable of growing and growing in the environment around rice roots.

For example, it may be capable of growing under anaerobic or partially aerobic conditions. This may be, for example, by having the ability to utilize oxygen-alternative electron acceptors under anaerobic conditions.

Methanotrophs may originate from the rhizosphere of rice.

Methanotrophic bacteria can be aerobic or anaerobic strains.

The methanotrophic bacteria can be, for example, strains of the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylosaricna or Methylacidiphyllum. For example, a strain of the genus Methylocystis can be Methylocystis fabus, Methylocystis rosea or Methylocystis econoides.

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

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

Methylotrophic bacteria may originate from the rhizosphere of rice.

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

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.

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

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

The method of the present invention comprises a step of treating the above-described composition for reducing methane emissions to the rhizosphere of rice.

The composition can be applied in various ways, for example, by applying it to a seedbed, applying it when transplanting rice seedlings, applying it by mixing it in irrigation water, applying it to the soil before tillage, applying it to the surface of a paddy field, or applying it by irrigating the soil around rice after transplanting.

In addition, the present invention relates to a composition for promoting the growth of rice, comprising methanotrophic bacteria.

Methanotrophic bacteria may be an example of this.

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

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

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.

The composition of the present invention is applied to rice fields and reproduces in the root zone of rice plants, thereby oxidizing methane produced by methanogenic bacteria in the soil and reducing methane emissions into the atmosphere. In addition, intermediate metabolites produced in the methane oxidation process promote rice growth and, depending on the nitrogen fixation ability, also promote rice growth.

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

The composition of the present invention may further contain conventional components included in fertilizers.

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

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

The composition can be applied in various ways, for example, by applying it to a seedbed, applying it when transplanting rice seedlings, applying it by mixing it in irrigation water, applying it to the soil before tillage, applying it to the surface of a paddy field, or applying it by irrigating the soil around rice after transplanting.

The present invention will be described more specifically with reference to the following examples.

Example

method

Enrichment and separation of methanotrophic consortia

Rice (Oryza sativa L. ssp. japonica var. Saeilmi) was uprooted from a paddy field in Sacheon-si, Korea, separated and washed to remove 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 and 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 mixed with fresh NMS-Cu or UMS-Cu medium and cultured again under the culture conditions mentioned above. This procedure was repeated six times for the enrichment of methanotrophic bacteria.

Then, the concentrated culture solution was filtered through a 0.2 μm pore size polycarbonate membrane (Sterlitech PCT027630), and the membrane filters with the concentrated culture microorganisms attached were transferred to a petri dish containing 30 mL of fresh NMS-Cu or UMS-Cu medium so that the polycarbonate membrane floated on the medium. Then, the dish was placed in a sealed chamber containing a mixture of methane and air (50:50) at 30°C. The membrane was 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. Then, the colonies were directly transferred to fresh liquid NMS-Cu or UMS-Cu medium and cultured in a serum bottle with shaking at 180 rpm at 30°C for 1 week. The isolated colonies were named NCN for colonies isolated using NMS-Cu medium, and UCB for colonies isolated using UMS-Cu medium.

Analysis of growth and morphological characteristics

Methanotrophic growth experiments were performed in 120-ml serum bottles containing 30 ml of NMS-Cu medium 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 for all growth experiments. Two methanotrophic isolates (NCN8 and UCB9) were cultured in a shaking incubator at 30°C and 180 rpm, and the growth rate was measured. The growth amount was observed by optical density (OD600) in an Ultrospec 10 cell density meter (Amersham Biosciences).

To investigate the ability of the above methanotrophic isolates to fix nitrogen from air, their ability to grow in a nitrogen-free medium was tested. A nitrogen-free medium was created by removing the nitrogen source _KNO3 from (NMS-Cu) in a nitrate mineral salt medium 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 a nitrogen-free medium without a nitrogen source, and centrifuged again to collect the cells, inoculate them into a nitrogen-free medium, and cultured according to the above culture method. The cell concentration (OD600) was analyzed every 12 h during 48 h of culture. In addition, the presence of major nitrogen fixation genes such as nifH and nifD was confirmed by PCR.

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

Genome feature analysis

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), 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, OrthoANIu algorithm, and EzAAI tool from EZBioCloud.

Rice pot experiment

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 (less than 2 mm in size). The soil was transferred to a Wagner pot (diameter 24 cm x height 30 cm) and filled to a bulk density of 1.2 g/cm ³ .

After culturing the isolates of NCN8 and UCBe9 on NMS-Cu and UMS-Cu media, respectively, they were washed with phosphate-buffered saline (PBS) and the final cell concentration was 5.1 x 10 ⁷ CFU/ml. In the first year, the roots of 3-week-old rice seedlings ( Oryza sativa L. ssp. japonica var., Saeil-mi) were soaked in UCBe9 or NCN8 suspensions at a concentration of 5.1 x 10 ⁷ CFU/ml for 5 h, and the roots of the control seedlings were soaked in PBS buffer for the same time. Next, the seedlings were transplanted into Wagner pots containing soil. Immediately after transplanting, 30 ml of NCN8 or UCBe9 suspensions were injected near the roots at a concentration of 1.5 x 10 ⁹ CFU/pot, and the same amount of isolates was injected in the same manner every 2 weeks until harvest. For the control group, 30 ml of PBS buffer was injected instead of the suspension of the isolated bacteria.

In the second year, the roots of 3-week-old rice seedlings ( Oryza sativa L. ssp. japonica var., Younghojin-mi) were soaked in low-concentration (7.7 x10 ⁷ CFU/ml) and high-concentration (7.7 x10 ⁸ CFU/ml) NCN8 culture suspensions for approximately 24 h, while the control seedlings were soaked in PBS buffer for the same period. The seedlings were then transplanted into Wagner pots containing soil. Approximately 100 mL of low-concentration and high-concentration NCN8 suspensions were injected near the roots immediately after transplanting. Low-concentration and high-concentration NCN8 were injected once per month until harvest.

A standard composition of chemical fertilizer consisting of urea (55 kg N/ha), molten superphosphate (45 kg P ₂ O ₅ /ha), and potassium chloride (40 kg K ₂ O /ha) was added to the planting soil one day before transplanting. An additional 22 kg N/ha was applied at the tillage stage about 2 weeks after transplanting, and 33 kg N/ha and 18 kg K ₂ O /ha were added at the panicle flowering stage about 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.

The harvested rice was naturally dried and then the grains and stems were separated. The rice growth indices, such as the number of ears per plant, the number of grains per ear, the ripening rate (%), and the weight of 1,000 grains, were measured according to the Korean research standards set by the Rural Development Administration.

CH ₄ Emission Measurement

Methane emissions were measured using the closed chamber method. Every Wednesday at 4:00 p.m., the pots were covered with a fan-equipped cylindrical transparent acrylic chamber (100 cm high × 24 cm diameter), and the gas inside the chamber was sampled using a 50-ml sealed syringe at 0 and 30 min. 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 80-100mesh).

Methane emission rates were calculated using the formula described below.

Methane emission rate (mg/ ^m2 /h) = △C/△tx (V/A) x ρ x (273/T)

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.

Microbiological analysis

After harvest, soil samples from each pot were taken to a depth of 10 cm using an auger aimed at 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.

Computational and statistical analysis

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

result

Isolation and characterization of NCN8 and UCBe9

Methanotrophs are known to exist in the roots and rhizosphere of rice plants. In this study, aerobic methanotrophs were isolated from the rice root system through a series of subcultures and repeated re-cultivation processes on agar plates. The colonies were then transferred to polycarbonate membranes to minimize contamination by heterotrophs that may be found on agar plates.

The growth of two promising isolates (NCN8 and UCBe9) obtained from polycarbonate membranes was compared at 30°C. They grew to OD600 values greater than 2 with specific growth rates of 0.1052 h ^-1 and 0.0967 h ^-1 within 36 h (Fig. 1) (Table 1).

Growth rates of two isolates, NCN8 and UCBe9

Temperature (°C) μ _max (h ^-1 ) Td (h) NCN8 30 0.1052±0.0041 6.6±0.25 UCBe9 30 0.0967±0.0058 7.2±0.44

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

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

PCR primers used for amplification of nitrogenase genes (nifH and nifD)

Primer name Product Sequence Reference nifH-F nifH TAYGGNAARGGNGGNATYGGNAARTC
(sequence number 1) Boulygina et al., (2002) nifH-R2 TCNGGNGARATGATGGC
(sequence number 2) nifD-f nifD GYGGYTGCGCCTAYGCCGG
(sequence number 3) Dedysh et al.,
(2004) nifD-r TCCCANGARTGCATCTGRCGGA
(sequence number 4)

SEM: Both NCN8 and UCBe9 appeared to be composed of two bacterial species, as two morphologically distinct cell types were detected by scanning electron microscopy (Figs. 3A and 3B). In NCN8, one type of cell appeared as a rod-shaped bacilli measuring approximately 2.4–2.9 x 0.8–1 micron in size. The second type appeared as a curved cocci with a rough surface measuring approximately 1.3–1.5 x 0.8–1 micron in size (Fig. 3A). Similarly, UCBe9 also showed two different cell types, one type appeared as a cocci measuring approximately 0.8 micron in diameter, and the second type appeared as a bacilli measuring approximately 1.5 x 0.45 micron in size (Fig. 3B). In addition, genome sequencing of the NCN8 culture revealed two genomic DNAs 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.

Genome analysis: NCN8 genome analysis 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 Methylocystis. 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 numbers were calculated using Prokka (Table 3). The genomes of NCN8, Methylocystis (Fig. 4A) and Methylophyllus (Fig. 4B), were visualized and compared with those of closely related species, as shown in Fig. 3.

Two circular chromosome genome features of NCN8

Traits Methylophilus sp. Methylocystis sp. Genome size 3.04 Mbp 4.22 Mbp Circular Yes Yes GC% 51.33 64.15 tRNA 45 52 rRNA 9 9 Proteins 2837 4066 16S 3 3 pmoAs - 2 Integrated plasmids - 2 Genbank Accession No CP173101 CP172975

SUBID BioProject BioSample Localid Accession Organism SUB14831851 PRJNA1180736 SAMN44527556 Methylocystis_NCN8 CP172975 Methylocystis sp. NCN8 SUB14831851 PRJNA1180736 SAMN44527556 pNCN8_1 CP172976 Methylocystis sp. NCN8 SUB14831851 PRJNA1180736 SAMN44527556 pNCN8_2 CP172977 Methylocystis sp. NCN8 SUB14831932 PRJNA1180737 SAMN44527557 Methylophilus_NCN8 CP173101 Methylophilus sp. NCN8

Genome-based comparisons of the Methylocystis and Methylophilus species present in NCN8 with those of closely related species were performed, and the average nucleotide identity (ANI), in silico DNA-DNA hybridization (DDH), and average amino-acid identity (AAI) were calculated. The ANI, AAI, and DDH values between the Methylocystis strains and their 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 propose that the Methylocystis strains in NCN8 represent a new species of the genus Methylocystis in the family Methylocystidae . Similarly, when the genome of Methylophilus sp. in NCN8 was compared with that of closely related Methylophilus sp., 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 only methanotrophs from NCN8, a consortium of methanotrophs and methylotrophs, and the growth rate of the isolated methanotrophs was seriously inhibited. In order to maximize methane reduction, it is ideal to use methanotrophs with high growth rates that can rapidly metabolize methane. Therefore, it may be best to utilize a consortium of methanotrophs and methylotrophs rather than pure methanotrophs for methane reduction in rice paddies. Methylotrophs can metabolize the excess methanol produced during methane oxidation, thereby reducing methanol toxicity and promoting the growth of methanotrophs. In addition, the exchange of essential nutrients between Methylocystis and Methylophilus can promote the overall growth performance of this consortium.

Methylocystis and Methylophilus genera of NCN8 Genome comparison with other closely related species. isDDH: in silico DNA-DNA hybridization, ANI: average nucleotide identity, AAI: average amino acid identity.

Rice isolate (NCN8) Closely related members isDDH (%) ANI (%) AAI (%) Methylocystis sp. Methylocystis parvus OBBP 35.5 [26.5-42.6] 83.23 85.04 Methylocystis iwaonis JCM 34278T 26.7 [25.3-28.3] 81.57 81.71 Methylocystis echinoides LMG 27198 26.3 [24.5-28.1] 80.94 80.07 Methylocystis_NCN8 (Rumen isolate) 26.2 [23.8-28.5] 80.65 80.55 Methylocystis rosea SV98 19.1 [17.8-21.6] 77.35 74.58 Methylosinus trichosporium OB3b 17.9 [16.0-21.5] 75.96 69.18 Methylophilus sp. Methylophilus sp. DW102 82.3 [76.7-85.9] 97.29 98.10 Methylophilus methylotrophus D22 31.2 [22.8-38.0] 79.64 85.92 Methylophilus sp. TWE2 30.6 [23.0-36.7] 79.64 85.68 Methylophilus medardicus MMS-M-51 24.6 [20.1-28.4] 76.90 83.73

In rice, methane emission is an ecological balance between two metabolic processes: methane production by methanogens and methane oxidation by methanotrophs. The continuous submergence of rice creates an anaerobic environment favorable to methanogens, which consume soil organic matter as a carbon source and release methane gas into the atmosphere. On the other hand, methanotrophs are aerobic bacteria that oxidize methane as a carbon source. Therefore, methane emissions from rice paddies can be reduced by significantly increasing the number of methanotrophs and oxidizing more methane. The simplest and most direct method is to directly inoculate methanotrophs-based bioinoculants into rice and rice paddies.

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

Effects of Methanotrophic Bioinoculation on Cumulative Methane Emissions and Methane Intensity

Items Treatments ¹ SEM ² p value Control NCN8 UCBe9 Methane emission
(kg CH ₄ /ha) 1,223.58±66.57 1,034.05±63.50 1,022.79±86.49 38.77 0.026* Methane intensity
(kg CH ₄ /ton rice) 250.36±18.49 163.63±18.37 160.39±26.16 15.97 0.003*

Data are expressed as mean ± standard deviation (SD). ¹ Treatments: CON, 30 ml 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 of all rice plants treated with LOW and HIGH NCN8 significantly decreased by more than 15% and 27%, respectively, compared to the untreated control (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 CH ₄ /ha, respectively (Table 7).

Most importantly, due to the effects of global warming, the average temperature in the second year was higher than in the first year, and there were more days when the temperature exceeded 40℃ (Fig. 5A and Fig. 6A). Due to these effects, the methane production in the control group tended to be higher in the second year, and it was confirmed that the reduction in methane production was greater in the second year when methanotrophic bioinoculants were treated (Fig. 5B and Fig. 6B). Therefore, as global warming worsens, the methane production reduction effect in rice paddies by methanotrophic bioinoculants will be more dramatically enhanced.

Effect of Methanotroph-Based Bioinoculant NCN8 on Cumulative Methane Emissions

Items Treatments ¹ SEM ² p value Control LOW HIGH Methane emission (kg CH ₄ /ha) 1,823±33 1,539±128 1,325±177 85.1 0.023*

In the first year, most of the measured growth indices were improved compared to the negative control when rice was inoculated with NCN8 and UCBe9 bioinoculants (Fig. 7A) (Table 8). Root length and weight, panicle length and weight, number of tillers, number of panicles per plant, and grain yield were significantly improved when NCN8 was inoculated. In particular, root weight was 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, grain yield (rice ton/ha) was significantly improved to 6.37 rice ton/ha and 6.43 rice ton/ha, respectively, when NCN8 and UCBe9 were inoculated compared to the control group (4.90 rice ton/ha) (p value = 0.025).

Analysis of plant growth indices and yields for the biological inoculant in the second-year pear cultivation experiment is in progress, but it was more effective than in the first year. The reason is that, as methane emissions are increased due to global warming, the growth of methanotrophs in the biological inoculant, which uses methane as a carbon source, is thought to have become more active, promoting rice growth.

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

Items Treatments ¹ SEM ² p value Control NCN8 UCBe9 Plant height (cm) 84.00±3.61 88.67±3.51 85.33±1.15 1.11 0.223 Straw weight (g) 117.70±11.97 160.85±5.15 132.62±8.94 6.85 0.003* No. of tillers 53.00±6.08 57.97±7.51 41.33±7.57 3.18 0.072 No. of panicles per hill 51.33±1.53 74.00±4.58 49.00±6.24 4.20 0.001* Weight of 1000 grains (g) 8.07±0.75 7.51±1.21 8.79±0.45 0.33 0.194 Ripened ratio (%) 65.39±3.62 65.23±1.07 69.29±3.13 1.05 0.219 Grain Yield (ton/ha) 4.90±0.27 6.37±0.75 6.43±0.54 0.298 0.025*

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

A total of 7,108 bacterial amplified sequence variants (ASVs) were obtained from rice rhizosphere soil samples, which were analyzed and classified into approximately 800 genera, 300 families, 160 orders, 70 classes, and 32 phyla. Pseudomonas dota, Basilota, Chloroplexta, Actinomyceta, Myxococcus, Acidobacteriota, Bacteroidesta, Thermodesulfobacteriota, Verrucomicrobiota, and Gemmatimonadota were the dominant phyla, accounting for more than 80% of the total bacterial ASVs (Fig. 8A). In contrast, a total of 2,497 archaeal ASVs were classified and detected in the archaeal community, which belonged to 20 genera, 14 families, 12 orders, 7 classes, and 4 phyla. The most dominant phylum was 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 in the experimental groups treated with NCN8 and UCBe9 (Figs. 8A and 8B). This suggests that the treatment with methanotrophic bioinoculants does not change the soil environment, but only has 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 abundances of Methylocystis sp. in the control, NCN8-treated, and UCBe9-treated groups were 0.92%, 1.79%, and 2.62%, respectively, indicating that the relative abundance of Methylocystis sp., a methanotroph, 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 inoculant, the methanotrophic isolates showed a strong ability to colonize, thrive, and persist. The colonization of rice roots and rhizosphere soils by these methanotrophic isolates resulted in a significant reduction in methane emissions.

conclusion

The results of the study showed that when methanotroph-based bioinoculants were used in rice cultivation, methane emissions could be successfully reduced by about 27% or more. Rice yields also increased by about 130% or more, and plant growth characteristics were also significantly improved. Methanotroph-based bioinoculants have enormous potential to mitigate methane emissions from rice farming and increase rice yields, which can greatly contribute to solving not only climate change responses but also food issues.

Claims (14)

A composition for reducing methane emissions from a paddy field comprising methanotrophic bacteria. In claim 1, the methanotrophic bacteria is a composition for reducing methane emissions from a paddy field derived from the rhizosphere of rice. 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. A composition for reducing methane emissions in a field, further comprising methylotrophic bacteria according to claim 1. In claim 4, the methylotrophic bacteria is a composition for reducing methane emissions in a paddy field derived from the rhizosphere of rice. A composition for reducing methane emissions in a paddy field according to claim 4, wherein the methylotrophic bacteria are bacteria of the genus Methylphyllus. A method for reducing methane emissions from rice, comprising the step of treating the rhizosphere of rice with a composition according to any one of claims 1 to 6. A composition for promoting the growth of rice, comprising methanotrophic bacteria. In claim 8, the methanotrophic bacteria is a composition for promoting the growth of rice derived from the rhizosphere of rice. A composition for promoting the growth of rice according to claim 8, wherein the methanotrophic bacteria are bacteria belonging to the genus Methylocystis, Methylomonas, Methylococcus, Methylosinus, Methylomicrobium, Methylobacter, Methylosaricna or Methylacidiphyllum. A composition for promoting rice growth, further comprising methylotrophic bacteria according to claim 8. In claim 11, the methylotrophic bacteria is a composition for promoting the growth of rice derived from the root zone of rice. A composition for promoting the growth of rice according to claim 11, wherein the methylotrophic bacteria is a bacterium of the genus Methylophilus. 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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