Short Communication
Analysis of Soil Microbial Features in a Rice Paddy Field with High Methane Emissions
2025 年 40 巻 4 号 論文ID: ME25044
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2025 年 40 巻 4 号 論文ID: ME25044
The soil microbial communities in two Japanese paddy fields were compared: site PA, which emits methane at the domestic average level, and site PX, which emits more than five times that amount. The transcription levels of methyl-coenzyme M reductase (mcrA) significantly increased during peak methane emissions at site PX, but not at site PA. The anaerobic methanotroph “Candidatus Methanoperedens” exhibited low activity and abundance exclusively at site PX. The genus Methanoregula was the most active methanogen at both sites; however, the dominant methanotrophs differed, with Methylocystis dominating at site PA and Methylomonas at site PX.
Methane is the second most abundant greenhouse gas emitted from natural sources and anthropogenic activities. In paddy fields, organic matter in soil is decomposed and metabolized into low-molecular-weight materials through microbial metabolism, and some may be used as substrates for methanogens (Alpana et al., 2017). Methane produced by methanogens is partially consumed by methane-oxidizing microorganisms, while the remainder is released into the atmosphere (Le Mer and Roger, 2001).
The amount of reducible ions in the soil significantly impacts methane emissions in paddy fields. When paddy field soil becomes flooded, substances are reduced in the order of thermodynamic favorability (Lovley, 1991). The genera Geobacter and Anaeromyxobacter are the main iron-reducing bacteria in paddy field soils (Hori et al., 2010; Masuda et al., 2024). In incubation experiments using a paddy field soil, Geobacter spp. used iron as an electron acceptor to oxidize acetic acid, thereby decreasing soil methane production (Hori et al., 2010).
A project investigating greenhouse gas emissions from agricultural fields in Japan (https://www.maff.go.jp/j/seisan/kankyo/tuti_chyosa.html) (Oura et al., unpublished) revealed that some paddy fields emitted methane at markedly higher levels annually than the national average. In the present study, we selected two paddy sites from this project to analyze soil microbial communities and identify candidate microbial groups linked to elevated methane emissions from rice fields. The composition and activity of soil microbes serve as an indicator of the environmental conditions present at each site.
Table S1 shows details of the two experimental sites (PA and PX). Site PA emits methane at the domestic average level, whereas site PX emits more than five times this amount. Rice straw-removed plots (N, no rice straw, removed after harvest) and rice straw-plowed plots (RS, 0.5 kg m–2) were run in triplicate at both sites. Soil samples were obtained at two time points: drained (D) and waterlogged (W). The soil sampling procedure is described in the supplemental material, Tx S1. Nucleic acids were extracted from soil samples according to a previously reported technique with some modifications, particularly to reduce DNA fragmentation. The copy numbers of the methyl-coenzyme M reductase (mcrA) gene and archaeal and bacterial 16S rRNA genes, as well as their transcripts, were quantified using a real-time PCR system (StepOne; Thermo Fisher Scientific). Nucleotide sequences were analyzed using a next-generation sequencer. The details of each procedure are shown in Tx S1.
The results of the quantitative PCR analysis showed that the copy numbers of the mcrA and 16S rRNA genes were lower during peak methane emissions (W) than before flooding (D) at site PA, but remained nearly unchanged at site PX (Fig. 1). Conversely, the transcription levels of the mcrA and archaeal 16S rRNA genes were significantly higher during peak methane emissions than before flooding at site PX, but remained unchanged or decreased at site PA. These results suggest that the soil at site PX maintained microbial populations under reducing conditions after flooding, which prevented a decrease in microbial abundance while significantly activating archaeal groups, including methanogens. The copy numbers and transcription levels of these genes typically fluctuate throughout the rice growing season (e.g., Watanabe et al., 2009; Itoh et al., 2013; Lee et al., 2014), and, thus, further studies are required to confirm the present results. Similar findings supporting this view were obtained in a subsequent survey conducted at site PX (Sakai et al., unpublished).
Comparison of copy numbers for genes and transcripts of mcrA, archaeal 16S rRNA, and bacterial 16S rRNA in PA and PX soil samples. N, rice straw removed; RS, rice straw plowed; D, drained sample; W, water-logged sample. Lowercase letters a, b, c, and d indicate significant differences in copy numbers within each gene or transcript (P<0.05).
An amplicon analysis of the soil at site PA revealed that most of the mcrA genes expressed in pre-flooded PA soil (D) were from “Candidatus Methanoperedens” (formally ANME-2d) (Fig. 2). This genus is closely related to the genus Methanosarcina, but is known to oxidize methane in an anaerobic environment using the mcrA gene in the reverse reaction of methane production (Evans et al., 2019). The abundance of “Ca. Methanoperedens” was negligible at site PX. Quantitative PCR yielded similar results (Fig. S1). “Ca. Methanoperedens” in paddy fields was previously examined in Italy (Vaksmaa et al., 2017) and China (Shen et al., 2021) and was also detected in the field soil samples of several countries, including Japan (e.g., Fernández-Baca et al., 2021; Sakoda et al., 2022). Therefore, “Ca. Methanoperedens” appears to be ubiquitous in paddy soil, and its absence may be one of the factors increasing methane emissions at site PX. Among methanogens, the group with the highest mcrA gene transcription level was common to both sites and belonged to the genus Methanoregula (Fig. 2).
Abundance and transcript counts of mcrA genes from each genus in soil samples from sites PA and PX, calculated using amplicon sequencing and real-time PCR data. N, rice straw removed; RS, rice straw plowed; D, drained sample; W, water-logged sample.
The amplicon analysis of 16S rRNA genes showed that the abundance and activity of the phyla Pseudomonadota (including most parts of the former genus Proteobacteria), Acidobacteriota, and Chloroflexota were high at both sites (Fig. S2A), which is consistent with previous findings (e.g., Lee et al., 2014; Vaksmaa et al., 2017). Aerobic methanotrophs were detected at high ratios at both sites, although the dominant group differed (Fig. 3A and S3). The genus Methylomonas, which was mainly detected at site PX, is a methanotroph that prefers higher concentrations of methane and higher pH (Ho et al., 2013; Kambara et al., 2022). This finding is consistent with the differences observed between the two sites. Archaeal 16S rRNA genes accounted for a small percentage of total 16S rRNA genes (Fig. S2B), with Thermoproteota (mostly comprising Nitrososphaeria and Bathyarchaeia) and Nanoarchaeota accounting for the majority, while Methanobacteriota, Thermoplasmatota, and Halobacteriota, to which the main methanogens belonged, accounted for less than 10% of archaeal 16S rRNA genes. The copy number and transcripts of 16S rRNA genes from Nanoarchaeota significantly increased during peak methane emissions (W) at site PX.
Heatmap showing the relative abundance of sequences from total 16S rRNA genes and transcripts in each soil sample from sites PA and PX (A), and a bar graph showing the genus level ratio of Geobacteraceae within total 16S rRNA genes sequences. (B). The heat map shows the 30 most abundant families. N, rice straw removed; RS, rice straw plowed; D, drained sample; W, water-logged sample.
Contrary to expectations, genes from the family Geobacteraceae, including Geobacter and its relatives, which are known as iron-reducing bacteria, were transcribed at higher levels under flooding (W) at site PX than at site PA (Fig. 3A), despite the lower content of extractable ions at site PX (Table S1). When the breakdown of Geobacteraceae sequences detected in the present study was examined, more than half were “unclassified” (Fig. 3B). The operational taxonomic units (OTU, 98%) of these “unclassified” sequences showed that the representative sequence of OTU2 was 98% identical to the sequence of Geobacter metallireducens GS-15, a representative strain of direct interspecies electron transfer (DIET) (Rotaru et al., 2014) (Fig. S4B). The sequence of OTU3 was 99% identical to the sequence of Oryzomonas japonica Red96, which exhibits the ability to fix N2 (Masuda et al., 2024). Therefore, some of the electron flow at site PX may be used by DIET and N2 fixation. DIET between the genera Geobacter and Methanothrix has been indicated in rice paddies in the United States (Holmes et al., 2017).
The plowing of rice straw into sites PA and PX enhanced methane emissions (Oura et al., unpublished), as previously reported (e.g., Yagi and Minami, 1990); however, the shift in the soil microbial organization was smaller than that observed among locations differing in soil type and climate (Fig. S5). Locations and soil types have been identified as important factors affecting the soil bacterial structure and activity (e.g., Bao et al., 2012).
Excessive methane production at site PX may have been due to a lack of microbially available iron in the soil. However, no other rice paddy fields emitted a similar amount of methane after the completion of iron reduction, making it difficult to attribute this solely to iron. While we detected several important microbial groups in the present study, their specific roles in methane metabolism warrant further investigation.
Sequence data were deposited in the DNA Data Bank under BioProject ID PRJDB15976.
The authors declare that there are no conflicts of interest.
Sakai, Y., Uezono, I., Shibuya, M., Oura, N., and Sudo, S. (2025) Analysis of Soil Microbial Features in a Rice Paddy Field with High Methane Emissions. Microbes Environ 40: ME25044.
https://doi.org/10.1264/jsme2.ME25044
We would like to thank R. Machida for providing experimental assistance. Nucleotide sequencing was supported by the Genome Breeding Support Office of the Institute of Crop Science, NARO (NICS) (Project ID: 22A20).
