Next Article in Journal
Microbial and Chemical Contamination in Springs of Northern and Central Lithuania
Previous Article in Journal
Ion Homeostasis, Osmotic Adjustment, and ROS Detoxification Underlie Pea Salinity Tolerance Induced by Pseudomonas putida RT12
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microbiology Research
Volume 16
Issue 11
10.3390/microbiolres16110228
microbiolres-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Efficacy of an Indigenously Isolated Rice Field Methanotroph as a Potential Bio-Inoculant for Promoting Rice Plant Growth

by
Shubha Manvi
1,2,†,
Kajal Pardhi
1,2,†,
Shirish Kadam
1,
Yash Kadam
1,
Yukta Patil
1,
Rahul A. Bahulikar
3,* and
Monali C. Rahalkar
1,*
1
C2-83,84, MACS Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, Maharashtra, India
2
Faculty of Science and Technology, Savitribai Phule Pune University, Ganeshkhind Road, Pune 411007, Maharashtra, India
3
Central Research Station, BAIF Development Research Foundation, Urulikanchan, Pune 412202, Maharashtra, India
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microbiol. Res. 2025, 16(11), 228; https://doi.org/10.3390/microbiolres16110228
Submission received: 15 September 2025 / Revised: 19 October 2025 / Accepted: 24 October 2025 / Published: 28 October 2025
Browse Figures
Versions Notes

Abstract

Methanotrophs offer promising avenues for sustainable agriculture and climate mitigation. This study evaluates the efficacy of indigenously isolated methanotrophs, particularly Methylomonas Kb3, as bioinoculants in rice cultivation. Kb3-treated plants exhibited early flowering, increased height, and a grain yield up to 17% higher than that of untreated controls. A mixed inoculation of Methylomonas and Methylomagnum resulted in a 15% increase in yield, indicating limited synergistic benefit. The root-dipping method during transplantation proved to be a practical and scalable inoculation technique for farmers. Genomic analysis revealed that Methylomonas Kb3 harbours genes associated with nitrogen fixation and resistance to heavy metals and antibiotics, potentially underpinning its agronomic performance. Beyond yield enhancement, the application of methanotrophs may contribute to reduced methane emissions in flooded paddy systems, offering dual benefits for both productivity and environmental sustainability. These findings warrant multilocation trials to validate efficacy across diverse agro-climatic zones and support the development of climate-smart biofertilizer strategies.

1. Introduction

Rice is a staple cereal consumed globally, with demand expected to rise significantly as the world population approaches 10 billion over the next 30 years. Asia accounts for nearly 90% of global rice production, and India ranks second after China, with approximately 43.7 million hectares under cultivation (Agricultural Statistics at a Glance, 2020). Traditional rice farming involves waterlogged conditions, which suppress weeds and pests but also create anoxic environments that promote methane production by anaerobic microbes. Methane, the second most potent greenhouse gas after CO2, traps 26 times more heat and has a relatively short atmospheric half-life (~12 years). Rice fields contribute 25–37 Tg of methane annually [1]—about 10% of global emissions—with India’s rice fields emitting ~3.9 Tg/year [2]. Peak emissions occur between August and September, coinciding with the peak rice cultivation period in India [2]. Methane emissions from rice fields are regulated by the balance between production and oxidation. Methanotrophs—methane-oxidising bacteria—act as natural mitigators by converting methane to CO2 [3] and are present in environments where methane and oxygen co-exist, such as lakes [4,5], wetlands [6,7,8], and rice fields [9]. These alpha and gamma proteobacteria inhabit the oxic-anoxic interface and rice roots [6], oxidizing up to 20% of the methane produced [10]. Their distribution is influenced by the heterogeneous oxygen and methane levels in the rhizosphere, where Type I methanotrophs dominate [11], though both Type I and II are present [11,12]. The rice rhizospheres are complex environments where methanogens, methanotrophs [4,13], nitrifiers, denitrifiers [7], nitrogen fixing bacteria [14,15,16] co-exist. Our recent experiments on rice plants showed that various species of indigenously isolated methanotrophs could enhance rice plant growth when applied as bioinoculants, which included strains of Methylobacter [17], Methylocucumis [18], Methylomonas [19], and Methylomagnum [20], showing positive effects [21]. Nitrogen is vital for plant growth, as it forms key components such as amino acids, chlorophyll, and enzymes. It enhances rice yield and quality by promoting tillering, increasing leaf area, promoting grain development, and enhancing protein synthesis. Each additional ton of rice requires 40–50 kg of nitrogen per hectare (http://www.knowledgebank.irri.org/training/fact-sheets/nutrient-management/item/nitrogen, accessed on 11 October 2025). However, conventional nitrogen fertilizers like urea and DAP (di-ammonium phosphate) often result in significant waste and environmental harm, making reduced fertilizer use a more sustainable strategy.
The novelty of the present study lies in the application of methanotrophs—previously identified for their plant growth-promoting potential and scalability—for enhancing rice yield under low nitrogen fertilizer input. Two strains, Methylomonas Kb3 and Methylomagnum ishizawai KRF4, were selected based on their success in prior pot experiments in 2021 and 2022 [21] and a further pot and a plot experiment in 2023 [22]. Both of these methanotrophs could be grown on a larger scale (20–40 L) under static and semi-sterile conditions and hence were shortlisted for the small-scale field experiment [23]. Building on the trials in the 2023 rice season [22], we chose a plant-dipping method for inoculation rather than conventional spraying. Dipping the plants in the methanotroph inoculum before transplantation was considered a more practical approach compared to spraying or direct field application, as the rice fields are often waterlogged, making spraying or direct field application ineffective.

2. Materials and Methods

2.1. Selection of a Rice Field for the Field Experiment

A rice farm in Malegaon village, near Lavasa (https://villageinfo.in/maharashtra/pune/mulshi/malegaon.html, accessed on 10 October 2025), was selected for the experiments after discussing farming practices with the farmer and obtaining his permission. The farmer agreed and allowed us to experiment on his rice field. All the experiments were performed in his presence. The field is located at 18.4489° N and 73.6095° E on the Lavasa road, within the interior of the village of Malegaon. This area has traditionally been used for rice farming, and the rice farmer practiced traditional rice farming by transplanting rice and using relatively low nitrogen fertilizer inputs, i.e., with low nitrogen inputs (50 kg N/ha), which were ideal for our experiments. An initial site visit was conducted in May 2024 to assess the field conditions. A follow-up visit took place on 20 June, approximately 15 days after sowing, when the rice plants were in the early nursery stage. No fertilizer was applied by the farmer during nursery preparation. The entire field experiment (Figure 1) was conducted from June to October 2024.
The methanotrophs Methylomonas Kb3 and Methylomagnum ishizawai KRF4 were initially grown in replicates in 1 L or 2 L sealed flasks with methane or biogas in the headspace. This inoculum was used for inoculation into a larger plastic tank with a 60 L capacity, using a modified NMS medium under stationary conditions at an ambient temperature of 28 °C [9] (Figure 2A,B). It is possible to grow ~20–40 L of methanotroph on a bench top using biogas or methane, as recently described [21,23]. On the day of transplantation, the inoculum was transported to the field. Approximately 2–3 L of each fully grown and fresh culture was transported to the field in plastic cans, which contained ~0.7 OD of the culture, which was equivalent to 3 × 108 cells/mL for Methylomagnum ishizawai strain KRF4 and 7 × 108 cells/mL for Methylomonas strain Kb3 (Figure 2C.) Methylomonas strain Kb3 is a small rod shaped methanotroph (Figure 2D) and Methylomagnum ishizawai strain KRF4 a large methanotroph (Figure 2E) were used for the field application, and a combination of the same, termed as mixed MOB (methane-oxidising bacteria). For the control treatment, plants were dipped in tap water without the addition of bacterial inoculum. Methylomonas Kb3, a pink-pigmented methanotroph, was isolated from an organic rice field [24] approximately 20 km from the experimental site. Its whole genome was subsequently sequenced and analysed, and is available as a draft assembly under the accession number PIZT01 [25]. Methylomagnum ishizawai KRF4 has been isolated from a rice field in Kerala, and the 16S rRNA gene sequence OR473174 indicates that it is closest to Methylomagnum ishizawai strain RS11D-PrT [20,26]. The experiment was continued until the end of the rice season, and the mature grains were harvested.

2.2. Treatment of Rice Plantlets with Selected Methanotroph Strains

The field experiment commenced in the transplantation stage on 3 July 2024. Experimental plots measuring 4 × 3 m were demarcated using wooden stakes and arranged in a randomised design. Approximately 25-day-old rice nursery plantlets were gently rinsed in clean water in a plastic tub and subsequently treated with methanotroph cultures by immersing them in a diluted inoculum (1:20 ratio) for one hour. A mixed consortium was prepared by combining equal volumes of Methylomonas Kb3 and Methylomagnum ishizawai KRF4 immediately prior to application. Four treatments were administered: Kb3, KRF4, Kb3 + KRF4 (1:1), and a control (tap water without bacteria), each using 1 L of inoculum diluted in 19 L of water. Following treatment, the plantlets were transplanted into the designated plots in rows, using a rope to maintain 25 cm spacing between hills. This configuration yielded approximately 16 hills per square meter, totaling 192–200 hills per treatment. Fertilizers were applied at minimal doses: urea at ~38 kg/acre and Suphala (15:15:15 NPK) at 25 kg/acre, one-week post-transplantation. The total nitrogen applied was approximately 50 kg/ha, which is below the standard recommendation of 100–125 kg N/ha as per ICAR guidelines (https://www.agrifarming.in/the-best-fertilizer-for-rice-crop-organic-npk-and-schedule-for-paddy, accessed on 10 October 2025).

2.3. Data Collection, Farm Visits, and Soil Sampling

The field was visited regularly, and data, as well as plant samples, were collected in the tillering, flowering-early grain filling, and grain maturation stages on 31 July 2024 (tillering stage), 19 September 2024 (flowering stage), and 28 October 2024 (grain maturation stage and harvest). The flowering stage and early grain formation stage were observed on 19 September 2024, and the plants were photographed using a mobile camera. At this stage, the plant height and panicle height were recorded, and the number of plants with grain formation and the number of plants with only flowering were noted in six random plants. The treated rice crops were harvested after 4 months of transplantation and assessed for their growth in terms of height, number of panicles, number of tillers, height of the panicle, weight of 1000 grains, and total weight of grains per hill. Rice plants from eight rice hills, each measuring approximately 100 cm by 50 cm (5000 cm2 = 0.5 square meters), were harvested for each treatment. The data were then extrapolated from grain yield in grams per square meter to quintals/ha (100 kg/ha = 0.1 tons/ha) by multiplying by the corresponding factor. One kilogram of soil was collected from the field in the tillering stage, following fertilizer application. The sample was air-dried, manually disaggregated to remove gravel, and sieved through a 100 µm mesh to obtain fine soil fractions. Subsequent analysis of organic carbon and NPK content was conducted at the Division of Soil Science, Mahatma Phule Krishi Vidyapeeth, Agricultural College, Pune, India.

2.4. Soil Sampling for Enrichment of Methanotrophs

Rhizosphere soil from the field was sampled in the tillering stage (31 July 2024) by uprooting three randomly selected plants from both the treated and control plots. Three plants from each treatment were uprooted, and the roots, along with the attached soil, were further used for isolation of methanotrophs. The serial endpoint dilution method was used for the enrichment and isolation of methanotrophs from the rhizosphere soil of the plants, as described before [20]. After 3–4 weeks, the grown dilutions were streaked on NMS agarose plates (all chemicals were purchased from Sisco Research Laboratories Pvt. Ltd., Taloja, Maharashtra, India). Colonies were purified, and the cultures were microscopically observed. Pure cultures were further subjected to pmoA PCR and sequencing analysis.

2.5. Data Analysis

MS Excel was used to store the data and also for the fundamental analysis, like averages, standard deviation, etc. PCA was carried out using PAST software (version 4.17) [27]. The difference between plant growth in terms of plant height, tiller numbers, panicle numbers, panicle height, grain yield in g per plant hill, and 1000 grain weight in g was noted.

2.6. Comparison of Methylomonas and Methylomagnum Genomes

The genome of Methylomonas Kb3 (accession: PIZT01 https://www.ncbi.nlm.nih.gov/nuccore/PIZT00000000.1) was compared with the complete genome of Methylomagnum ishizawai strain RS11-DPr (accession: NZ_AP019783.1; https://www.ncbi.nlm.nih.gov/nuccore/NZ_AP019783.1). (As the genome of Methylomagnum strain KRF4 is not yet available, the type strain RS11-DPr was used for comparative analysis, given its 99.5% similarity to KRF4 based on 16S rRNA gene sequences.) Genome-wide comparisons were conducted using the RAST annotation platform (https://rast.nmpdr.org/), where both genomes were uploaded and analyzed for functional gene content. Unique genes specific to each strain were identified and examined.

3. Results

3.1. Agri-Environmental Conditions

The village of Malegaon (18.4489° N and 73.6095° E), located near Lavasa, Mulshi, is a small village where rice farming is practised throughout the region. Detailed soil analysis classified the field soil as silt-clay loam, comprising 43.75% silt and 28.25% clay. The average organic carbon content was 0.4%, while the concentrations of nitrogen, phosphorus, and potassium were 275.96 kg/ha, 27.70 kg/ha, and 1437.18 kg/ha, respectively. These values indicate that the N was in the low–medium range, P in the medium range, and K was in the high range as per the chart for Indian soils (Table S1) (https://agritech.tnau.ac.in/agriculture/agri_soil_soilratingchart.html, accessed on 10 October 2025). The region has an average annual rainfall of ~2300 mm, with peaks in July and August. The highest temperature during the peak monsoon is 27 °C during the day (https://weather-and-climate.com/average-monthly-precipitation-Rainfall,lavasa-maharashtra-in,India, Lavasa, Maharashtra, India, accessed on 10 October 2025).

3.2. Overall Health of the Plants and Early Flowering Seen in Methylomonas Kb3-Treated Plants

Compared to the control plants (uninoculated), all methanotroph-treated plants remained consistently green and healthy throughout the growth stages, with no observable adverse effects. Notably, early flowering—approximately 10–12 days ahead of the control and other treatments—was observed in plants treated with Methylomonas Kb3. This was evidenced by grain formation and panicle drooping due to grain weight, whereas control plants exhibited upright panicles bearing only flowers. On 19 September 2024, 66% of the Kb3-treated plants (4 out of 6) showed grain formation. In contrast, plants inoculated with Methylomagnum ishizawai KRF4 and the control group displayed only flowering, with no grain development (Figure 3). Among the plants treated with a mixed consortium of Methylomonas Kb3 and Methylomagnum ishizawai KRF4, 50% (3 out of 6) exhibited grain formation. The remaining plants in this group showed either flowering or early grain development. These observations suggest that Methylomonas Kb3, alone or in combination, promotes earlier grain maturation, as indicated by drooping panicles (Figure 3).

3.3. Enhanced Growth Yield, Plant Height in Methylomonas Kb3-Treated Plants

Methylomonas strain Kb3 and the mixed methanotroph consortium (Kb3 + Methylomagnum ishizawai KRF4) outperformed other treatments in terms of plant height, panicle height, and tiller development (Table 1). Grain yield was highest in plants treated with Methylomonas Kb3, a 17% increase in total grain yield compared to the control, reaching 61.06 quintals/ha (6.106 tons/ha)—approximately 9 quintals/ha more than the control (52.13 quintals/ha; Table 1). The mixed consortium treatment yielded 59.99 quintals/ha, an increase of 8 quintals/ha over the control. In contrast, the Methylomagnum KRF4 treatment resulted in a slightly lower yield than the control (50.5 quintals/ha). Although the KRF4 treatment showed a marginally higher 1000-grain weight, the overall grain yield was likely reduced due to fewer panicles. The number of tillers, panicles, and panicle height were slightly higher in inoculated plants, but differences were not statistically significant. However, total plant height was significantly greater in Kb3- and MOB mix-treated plants compared to the control. Principal Component Analysis (PCA) was conducted using all growth parameters listed in Table 1. The first two principal components accounted for 90.6% of the total variance (Figure 4), clearly separating Kb3 and MOB mix treatments from the control and KRF4. Vectors associated with plant height, grain yield per hill, and estimated yield were closely aligned with Kb3 and MOB mix treatments, while control and KRF4 clustered together in the lower left quadrant (Figure 4). These results demonstrate the effectiveness of Kb3 and the mixed consortium in enhancing rice growth and yield.

3.4. Comparison of Genome Information

Comparative genomic analysis revealed key differences between the strains. Notably, Methylomagnum ishizawai lacked genes associated with the urea cycle, suggesting an inability to utilise urea, a readily available nitrogen source. Furthermore, the genome of Methylomagnum lacked genetic components related to heavy metal and antibiotic resistance, including multidrug efflux pumps and genes conferring resistance to arsenic, cobalt, cadmium, and copper homeostasis, which were present in Methylomonas Kb3 (Table S2).

3.5. Re-Isolation of Methanotrophs

Methanotrophs were observed to grow up to a 10−6 dilution in samples treated with Methylomonas Kb3 and the mixed methanotroph consortium (MOB), exhibiting characteristic pink pigmentation. Methylomonas Kb3 was successfully re-isolated from enrichment cultures prepared via endpoint dilution in liquid medium, followed by streaking on solid agarose plates. Colony and cell morphology were consistent with Methylomonas-like cells, and identity was further confirmed through pmoA gene amplification and sequencing, linking the isolate to rhizosphere-associated Methylomonas Kb3. In samples treated with Methylomagnum ishizawai KRF4, the strain was recovered alongside other Type II methanotrophs. In contrast, control soil samples showed lower methanotroph abundance, with growth detectable only up to a 10−4 dilution. Cultured methanotrophs from control samples included Type II genera such as Methylosinus and Methylocystis, identified by distinct colony and cell morphologies—Methylosinus forming cream-colored, half-moon or pear-shaped colonies, and Methylocystis forming white, coccoid-shaped colonies (Table S3).

4. Discussion

This study builds upon findings from our previous trials [13,14] and the demonstrated feasibility of large-scale methanotroph cultivation [8]. Conducted on a limited scale with the farmer’s consent and active participation, this field experiment represents one of the first applications of pure, indigenously isolated methanotroph cultures in rice cultivation. The selected field was ideal for experimentation, as the farmer had consistently applied only 50 kg N/ha annually—significantly lower than the standard recommendation of 100–150 kg N/ha—creating a low-nitrogen environment conducive to evaluating bioinoculant efficacy. Two methanotroph strains were selected: Methylomonas Kb3 and Methylomagnum ishizawai, based on their proven ability to promote rice plant growth [21] and their capacity for high-density cultivation [23]. Current estimates suggest that 15–25 L of methanotroph inoculum per hectare, diluted to approximately 500 L, would be sufficient for field application [28] and could be conveniently distributed to farmers in barrels or cans. Furthermore, recent findings indicate that methanotrophs can be cultivated using biogas [23], offering a practical and scalable approach for integrating this technology into agricultural systems, although further validation under field conditions is required.
In our pot experiments, Methylomonas strain Kb3 consistently demonstrated strong potential as a bioinoculant, promoting rice plant growth over the past four years [13,14]. While Methylobacter strain BLB1 and Methylocucumis oryzae showed superior growth-promoting effects in earlier trials, their slow growth rates and limited scalability hinder their practical application [15]. In contrast, Methylomonas Kb3 may have outperformed Methylomagnum ishizawai due to its genomic features, including genes conferring resistance to heavy metals such as arsenic, cadmium, and cobalt, as well as multidrug efflux pumps for antibiotic resistance. These traits likely enhance its competitiveness in the rhizosphere. Although not directly confirmed in this study, the growth-promoting effects of Methylomonas Kb3 may also be linked to its potential nitrogen fixation capabilities and its capacity to survive in harsh conditions, as depicted by the genome analysis. Methanotrophs are estimated to fix approximately 40 kg N/ha in rice fields and play a crucial role in atmospheric nitrogen fixation [29]. Methanotrophs also often possess nitrogen-fixation pathways [30] and are active participants in nitrogen fixation in rice roots. As per an estimate, methanotrophs are capable of fixing 1.2 to 1014 g N/year [31] globally. Stable isotope probing (SIP) experiments with 13CH4 and 15N2 have recently verified that methanotroph-mediated nitrogen fixation occurs in rice roots [32]. The nitrogen fixation in methane-consuming roots was found to be dominated by Methylomonas. The authors also noted that the microbial N2-fixing activity was stimulated by low nitrogen conditions. Advanced imaging techniques (e.g., NanoSIMS) have confirmed that individual methanotrophic cells in rice roots can simultaneously fix nitrogen and oxidise methane [32]. Methylomonas Kb3 possesses genes associated with the nitrogen fixation pathway and demonstrates the ability to grow in nitrogen-free environments [25]. Although nitrogen fixation was not directly confirmed in the present study, it may have contributed to the observed enhancement in plant growth. A similar approach had recently been adopted by Stringbio company (Bangalore, India), which developed a methanotroph-based biostimulant—CleanRise R—comprising Methylococcus capsulatus. In open-field trials conducted over three rice seasons, application of this biostimulant resulted in up to a 39% increase in grain yield under full-fertilizer conditions and approximately 34% yield improvement with 75% fertilizer input [17]. The proposed mechanisms underlying the biostimulant effect were found to include indole acetic acid production, enhanced photosynthesis, improved tillering, and panicle development, all of which contribute to increased yield [17]. Transcriptomic analysis from the same study provides further support for the hypothesis that methanotrophs can promote rice growth and mitigate methane emissions. Although the study focused on a single strain (Methylococcus capsulatus), similar biochemical pathways are conserved across methanotrophs, suggesting that comparable plant-microbe interactions may occur with other methanotrophs, e.g., Methylomonas Kb3. Methylomonas Kb3 possesses several genes for chemotaxis, aerotaxis, and hemerythrins, which are necessary for their movement towards oxygen in the plant root rhizospheres [33].
In the present study, the yield obtained with the treatment of Methylomonas Kb3 (61.06 quintals/ha) is equivalent to or even slightly better than the yield reported for the variety Indrayani from Maharashtra state in another study with the recommended full dose of fertilisers (100 kgN/ha), which was 59.50 quintals/ha [34]. The grain yields in our study (52.13 quintals/ha) with no methanotroph inoculum (control) were equivalent to the yields obtained using 75% of the recommended dose (53.26 quintals/ha) for the same variety.
One of the notable outcomes of this study was the early grain formation and maturation observed in rice plants inoculated with Methylomonas Kb3. This accelerated development suggests that rice can reach harvestable maturity sooner, offering potential agronomic benefits such as earlier harvesting and improved crop scheduling. In contrast, Methylomagnum ishizawai appeared less effective, likely due to genomic differences—specifically, the absence of genes associated with heavy metal and antibiotic resistance—which may have hindered its ability to establish a strong association with the plant.
Additionally, the method of inoculation played a crucial role. Dipping rice roots in the methanotroph inoculum during transplantation proved to be a simple and farmer-friendly technique. This approach was more effective than direct field application or foliar spraying, which are less reliable under flooded conditions typical of rice cultivation. Frequent rainfall during the transplantation period further limits the feasibility of spraying, making root dipping a practical and scalable method for field-level implementation.
The use of only ~50 kg N/ha (significantly below conventional levels) with improved yield suggests a shift toward low-input agriculture. This could lead to widespread adoption of integrated nutrient management strategies, combining minimal synthetic fertilizers with microbial enhancements. Methanotrophs naturally oxidise methane, a potent greenhouse gas emitted from flooded rice fields. Their dual role in enhancing yield and mitigating emissions aligns with global climate goals, positioning them as key agents in climate-smart agriculture. Enhanced plant height and panicle numbers indicate broader physiological benefits beyond yield. This could encourage holistic crop management approaches that prioritise plant vigour, resilience, and reproductive success. The success in a single field prompts the need for multilocation trials, which could lead to region-specific microbial formulations tailored to local soil and climate conditions. Extension services may evolve to include microbial literacy programs for farmers.

5. Conclusions

This study demonstrates the potential of indigenously isolated methanotrophs, particularly Methylomonas Kb3, as an effective bioinoculant for rice cultivation. Methylomonas Kb3-treated plants exhibited early flowering, increased plant height, and a grain yield up to 17% higher than that of the controls. The mixed treatment with Methylomonas and Methylomagnum showed a 15% yield increase, suggesting limited additional benefit. The root-dipping method during transplantation proved to be a practical and farmer-friendly inoculation technique. Genomic analysis revealed that Kb3 possesses traits for nitrogen fixation and resistance to heavy metals and antibiotics, likely contributing to its superior performance. These findings support the use of methanotrophs not only for enhancing rice productivity but also for mitigating methane emissions in flooded paddy systems. Methanotrophs may also contribute to methane mitigation, offering a dual advantage in terms of productivity and sustainability. These findings also support further multilocation trials to validate efficacy across diverse agro-climatic zones and inform future biofertilizer strategies for climate-smart agriculture.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microbiolres16110228/s1, Table S1: Analytical report of the soil collected from the experimental rice field in Malegaon, Lavasa, in 2024. Table S2: Functional genes which are present in Methylomonas Kb3 and absent in Methylomagnum ishizawai: Comparison was done using RAST annotation platform: https://rast.nmpdr.org/. Table S3: Re-isolation of methanotrophs from treated plants. Figure S1: Grain harvest stage: Kb3 inoculated plants can be seen, which were already lodged down (in the front). In the back, control plants can be seen.

Author Contributions

Conceptualization, M.C.R. and R.A.B.; methodology, R.A.B., S.M., K.P., S.K., Y.K., Y.P., and M.C.R.; validation, S.M., K.P., and S.K.; formal analysis, S.M., K.P., and S.K.; writing—original draft preparation, S.M., K.P., and M.C.R.; writing—review and editing, M.C.R. and S.M.; visualization, R.A.B. and M.C.R.; supervision, M.C.R.; project administration, M.C.R.; funding acquisition, M.C.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Anusandhan National Research Foundation (ANRF) (formerly SERB) through a POWER grant fellowship provided to MCR, with grant number SPF/2022/000045.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the sequence data mentioned are available in the NCBI database.

Acknowledgments

We sincerely thank Shivajirao Gund of Malegaon village, Lavasa Road, for generously permitting us to conduct field experiments on his farm. We are also grateful to his family and field workers for their kind cooperation and support throughout the study. Special thanks to Jyoti Mohite for her assistance in cultivating methanotroph cultures. K.P. acknowledges the University Grants Commission (UGC) for the award of a Junior Research Fellowship. The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
Methane-oxidizing bacteriaMOB

References

  1. Saunois, M.; Bousquet, P.; Poulter, B.; Peregon, A.; Ciais, P.; Canadell, J.G.; Dlugokencky, E.J.; Etiope, G.; Bastviken, D.; Houweling, S.; et al. The global methane budget 2000–2012. Earth Syst. Sci. Data 2016, 8, 697–751. [Google Scholar] [CrossRef]
  2. Ganesan, A.; Rigby, M.; Lunt, M.F.; Parker, R.J.; Boesch, H.; Goulding, N.; Umezawa, T.; Zahn, A.; Chatterjee, A.; Prinn, R.G.; et al. Atmospheric observations show accurate reporting and little growth in India’s methane emissions. Nat. Commun. 2017, 8, 836. [Google Scholar] [CrossRef]
  3. Hanson, R.S.; Hanson, T.E. Methanotrophic bacteria. Microbiol. Mol. Biol. Rev. 1996, 60, 439–471. [Google Scholar] [CrossRef] [PubMed]
  4. Auman, A.; Stolyar, S.; Lidstrom, M.E. Molecular characterization of methanotrophic isolates from freshwater lake sediment. Appl. Environ. Microbiol. 2000, 66, 5259. [Google Scholar] [CrossRef] [PubMed]
  5. Rahalkar, M. Aerobic Methanotrophic Bacterial Communities from Sediments of Lake Constance. Ph.D. Thesis, University of Konstanz, Konstanz, Germany, 2007. [Google Scholar]
  6. Lüke, C.; Krause, S.; Cavigiolo, S.; Greppi, D.; Lupotto, E.; Frenzel, P. Biogeography of wetland rice methanotrophs. Environ. Microbiol. 2010, 12, 862–872. [Google Scholar] [CrossRef]
  7. Yun, J.; Ma, A.; Li, Y.; Zhuang, G.; Wang, Y.; Zhang, H. Diversity of methanotrophs in Zoige wetland soils under both anaerobic and aerobic conditions. J. Environ. Sci. 2010, 22, 1232–1238. [Google Scholar] [CrossRef]
  8. Pardhi, K.; Manvi, S.; Bahulikar, R.A.; Patil, Y.; Kadam, Y.; Kadam, S.; Saraf, C.; Rahalkar, M.C. Cultivation of Diverse Type I and Type II Methanotrophs from Tropical Wetlands in India, Including Rare Taxa (Methylocucumis and Methylolobus). Methane 2025, 4, 17. [Google Scholar] [CrossRef]
  9. Lüke, C. Molecular Ecology and Biogeography of Methanotrophic Bacteria in Wetland Rice Fields. Ph.D. Thesis, Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, Germany, 2010. [Google Scholar]
  10. Conrad, R. The global methane cycle: Recent advances in understanding the microbial processes involved. Environ. Microbiol. Rep. 2009, 1, 285–292. [Google Scholar] [CrossRef]
  11. Wu, L.; Ma, K.; Lu, Y. Rice roots select for type I methanotrophs in rice field soil. Syst. Appl. Microbiol. 2009, 32, 421–428. [Google Scholar] [CrossRef] [PubMed]
  12. Gilbert, B.; Frenzel, P. Rice roots and CH4 oxidation: The activity of bacteria, their distribution and the microenvironment. Soil Biol. Biochem. 1998, 30, 1903–1916. [Google Scholar] [CrossRef]
  13. Fazli, P.; Man, H.C.; Shah, U.K.M.; Idris, A. Characteristics of methanogens and methanotrophs in rice fields: A review. Asia-Pac. J. Mol. Biol. Biotechnol. 2013, 21, 3–17. [Google Scholar]
  14. Hardoim, P.R.; Andreote, F.D.; Reinhold-Hurek, B.; Sessitsch, A.; van Overbeek, L.S.; van Elsas, J.D. Rice root-associated bacteria: Insights into community structures across 10 cultivars. FEMS Microbiol. Ecol. 2011, 77, 154–164. [Google Scholar] [CrossRef]
  15. Sessitsch, A.; Hardoim, P.; Doering, J.; Weilharter, A.; Krause, A.; Woyke, T.; Mitter, B.; Hauberg-Lotte, L.; Friedrich, F.; Rahalkar, M.; et al. Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Mol. Plant-Microbe Interact. 2012, 25, 28–36. [Google Scholar] [CrossRef] [PubMed]
  16. Engelhard, M.; Hurek, T.; Reinhold-Hurek, B. Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races. Environ. Microbiol. 2000, 2, 131–141. [Google Scholar] [CrossRef]
  17. Rahalkar, M.C.; Khatri, K.; Pandit, P.S.; Dhakephalkar, P.K. A putative novel Methylobacter member (KRF1) from the globally important Methylobacter clade 2: Cultivation and salient draft genome features. Antonie van Leeuwenhoek 2019, 112, 1399–1408. [Google Scholar] [CrossRef]
  18. Rahalkar, M.C.; Mohite, J.A.; Pardhi, K.; Manvi, S.S.; Kadam, Y.S.; Patil, Y.V. Insights into Methylocucumis oryzae, a Large-sized, Phylogenetically Unique Type Ia Methanotroph with Biotechnological Potential. Ind. J. Microbiol. 2024, 64, 1964–1969. [Google Scholar] [CrossRef]
  19. Pandit, P.S.; Rahalkar, M.; Dhakephalkar, P.; Ranade, D.R.; Pore, S.; Arora, P.; Kapse, N. Deciphering community structure of methanotrophs dwelling in rice rhizospheres of an Indian rice field using cultivation and cultivation independent approaches. Microb. Ecol. 2016, 71, 634–644. [Google Scholar] [CrossRef]
  20. Rahalkar, M.; Khatri, K.; Pandit, P.S.; Bahulikar, R.; Mohite, J.A. Cultivation of Important Methanotrophs From Indian Rice Fields. Front. Microbiol. 2021, 12, 669244. [Google Scholar] [CrossRef]
  21. Mohite, J.; Khatri, K.; Pardhi, K.; Manvi, S.S.; Jadhav, R.; Rathod, S.; Rahalkar, M.C. Exploring the potential of methanotrophs for plant growth promotion in rice agriculture. Methane 2023, 2, 361–371. [Google Scholar] [CrossRef]
  22. Kadam, S.; Saraf, C. Experimental trials for use of selected methanotrophs as bioinoculant in rice plant (Indrayani variety). Master’s Thesis, Swami Ramanand Teerth Marathwada University, Nanded, India, 2024. [Google Scholar]
  23. Mohite, J.; Pardhi, K.; Rahalkar, M.C. Single-Cell Protein Using an Indigenously Isolated Methanotroph Methylomagnum ishizawai, Using Biogas. Microbiol. Res. 2025, 16, 171. [Google Scholar] [CrossRef]
  24. Pandit, P.S.; Ranade, D.R.; Dhakephalkar, P.K.; Rahalkar, M.C. A pmoA-based study reveals dominance of yet uncultured Type I methanotrophs in rhizospheres of an organically fertilized rice field in India. 3 Biotech 2016, 6, 135. [Google Scholar] [CrossRef] [PubMed]
  25. Rahalkar, M.C.; Pandit, P. Genome-based insights into a putative novel Methylomonas species (strain Kb3), isolated from an Indian rice field. Gene Rep. 2018, 13, 9–13. [Google Scholar] [CrossRef]
  26. Khalifa, A.; Lee, C.G.; Ogiso, T.; Ueno, C.; Dianou, D.; Demachi, T.; Katayama, A.; Asakawa, S. Methylomagnum ishizawai gen. nov., sp. nov., a mesophilic type I methanotroph isolated from rice rhizosphere. Int. J. Syst. Evol. Microbiol. 2015, 65, 3527–3534. [Google Scholar] [CrossRef]
  27. Hammer, O.; Harper, D.A.T.; Ryan, P.D. PAST: Paleontological statistics software package for education and data analysis. Paleontol. Electron. 2001, 4, 9. [Google Scholar]
  28. Kumar, S.R.; David, E.M.; Pavithra, G.J.; Kumar, G.S.; Subbian, E. Methane-derived microbial biostimulant reduces greenhouse gas emissions and improves rice yield. Front. Plant Sci. 2024, 15, 1432460. [Google Scholar] [CrossRef]
  29. Cao, W.; Cai, Y.; Bao, Z.; Wang, S.; Yan, X.; Jia, Z. Methanotrophy Alleviates Nitrogen Constraint of Carbon Turnover by Rice Root-Associated Microbiomes. Front. Microbiol. 2022, 13, 885087. [Google Scholar] [CrossRef]
  30. Auman, A.J.; Speake, C.C.; Lidstrom, M.E. nifH Sequences and Nitrogen Fixation in Type I and Type II Methanotrophs. Appl. Environ. Microbiol. 2001, 67, 4009–4016. [Google Scholar] [CrossRef]
  31. Cai, Y.; Zheng, Y.; Bodelier, P.L.E.; Conrad, R.; Jia, Z. Conventional methanotrophs are responsible for atmospheric methane oxidation in paddy soils. Nat. Commun. 2016, 7, 11728. [Google Scholar] [CrossRef] [PubMed]
  32. Hara, S.; Wada, N.; Hsiao, S.S.; Zhang, M.; Bao, Z.; Iizuka, Y.; Lee, D.; Sato, S.; Tang, S.; Minamisawa, K. In Vivo Evidence of Single 13C and 15N Isotope–Labeled Methanotrophic Nitrogen-Fixing Bacterial Cells in Rice Roots. mBio 2022, 13, e0125522. [Google Scholar] [CrossRef] [PubMed]
  33. Rahalkar, M.; Bahulikar, R. Hemerythrins are widespread and conserved for methanotrophic guilds. Gene Rep. 2018, 11, 250–254. [Google Scholar] [CrossRef]
  34. Shende, S.M.; Hasure, R.R.; Gajbhiye, P.N.; Gedam, V.B.; Shinde, R.H. Response of dry seeding of kharif paddy (Oryza sativa L.) Varieties to different fertilizer levels with respect growth characters. Int. J. Chem. Stud. 2020, 8, 1272–1277. [Google Scholar] [CrossRef]
Microbiolres 16 00228 g001
Figure 1. Sequential stages of the field experiment conducted in a farmer’s field in Malegaon, located off Lavasa Road near Pune, Maharashtra.
Figure 1. Sequential stages of the field experiment conducted in a farmer’s field in Malegaon, located off Lavasa Road near Pune, Maharashtra.
Microbiolres 16 00228 g001
Microbiolres 16 00228 g002
Figure 2. (A). Growth of Methylomonas Kb3 in plastic tank. (B). Growth of Methylomagnum KRF4 in plastic tank. (C). Methanotropic bioinoculum taken to the field. (D). Phase contrast micrograph of Methylomonas Kb3. The bar represents 5 µm. (E). Phase contrast micrograph of Methylomagnum ishizawai. The bar represents 5 µm.
Figure 2. (A). Growth of Methylomonas Kb3 in plastic tank. (B). Growth of Methylomagnum KRF4 in plastic tank. (C). Methanotropic bioinoculum taken to the field. (D). Phase contrast micrograph of Methylomonas Kb3. The bar represents 5 µm. (E). Phase contrast micrograph of Methylomagnum ishizawai. The bar represents 5 µm.
Microbiolres 16 00228 g002
Microbiolres 16 00228 g003
Figure 3. Early flowering and grain formation in rice plants inoculated with Methylomonas Kb3, observed approximately 100 days after sowing (19 September 2024). Control plants, visible in the background, exhibited only flowering without grain development.
Figure 3. Early flowering and grain formation in rice plants inoculated with Methylomonas Kb3, observed approximately 100 days after sowing (19 September 2024). Control plants, visible in the background, exhibited only flowering without grain development.
Microbiolres 16 00228 g003
Microbiolres 16 00228 g004
Figure 4. Principal component analysis (PCA) based on the effect of inoculation of methanotrophs on plant growth parameters. The first two coordinates contribute to 90.6% of % variance.
Figure 4. Principal component analysis (PCA) based on the effect of inoculation of methanotrophs on plant growth parameters. The first two coordinates contribute to 90.6% of % variance.
Microbiolres 16 00228 g004
Table 1. Comparison of different methanotroph treatments and their effects on rice plant growth parameters, including plant height, tiller number, panicle development, and grain yield.
Table 1. Comparison of different methanotroph treatments and their effects on rice plant growth parameters, including plant height, tiller number, panicle development, and grain yield.
TreatmentTotal Plant Height (cm)No. of TillersNo. of PaniclesPanicle Height (cm)Weight of 1000 Grains gAverage Grain Weight per Hill gYield Quintal per haIncrease in Yield
Kb3 106.8 ± 4.619 ± 318 ± 322.8 ± 1.520.0138.161.0617%
KRF496.0 ± 617 ± 315 ± 123.3 ± 2.222.7131.650.5Little decrease (−3%)
Kb3 + KRF4 (MOB mix) 101.8 ± 6.418 ± 316 ± 324.1 ± 1.221.5937.559.9915%
Control 94.8 ± 6.419 ± 317 ± 323.3 ± 1.320.6832.652.13Control yield
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Manvi, S.; Pardhi, K.; Kadam, S.; Kadam, Y.; Patil, Y.; Bahulikar, R.A.; Rahalkar, M.C. Efficacy of an Indigenously Isolated Rice Field Methanotroph as a Potential Bio-Inoculant for Promoting Rice Plant Growth. Microbiol. Res. 2025, 16, 228. https://doi.org/10.3390/microbiolres16110228

AMA Style

Manvi S, Pardhi K, Kadam S, Kadam Y, Patil Y, Bahulikar RA, Rahalkar MC. Efficacy of an Indigenously Isolated Rice Field Methanotroph as a Potential Bio-Inoculant for Promoting Rice Plant Growth. Microbiology Research. 2025; 16(11):228. https://doi.org/10.3390/microbiolres16110228

Chicago/Turabian Style

Manvi, Shubha, Kajal Pardhi, Shirish Kadam, Yash Kadam, Yukta Patil, Rahul A. Bahulikar, and Monali C. Rahalkar. 2025. "Efficacy of an Indigenously Isolated Rice Field Methanotroph as a Potential Bio-Inoculant for Promoting Rice Plant Growth" Microbiology Research 16, no. 11: 228. https://doi.org/10.3390/microbiolres16110228

APA Style

Manvi, S., Pardhi, K., Kadam, S., Kadam, Y., Patil, Y., Bahulikar, R. A., & Rahalkar, M. C. (2025). Efficacy of an Indigenously Isolated Rice Field Methanotroph as a Potential Bio-Inoculant for Promoting Rice Plant Growth. Microbiology Research, 16(11), 228. https://doi.org/10.3390/microbiolres16110228

Article Metrics

Back to TopTop