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Communication

Cultivation of Diverse Type I and Type II Methanotrophs from Tropical Wetlands in India, Including Rare Taxa (Methylocucumis and Methylolobus)

by
Kajal Pardhi
1,2,
Shubha Manvi
1,2,
Rahul A. Bahulikar
3,
Yukta Patil
1,
Yash Kadam
1,
Shirish Kadam
1,
Chandani Saraf
1 and
Monali C. Rahalkar
1,2,*
1
C2-83,84, MACS Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, Maharashtra, India
2
Savitribai Phule Pune University, Ganeshkhind Road, Pune 411007, Maharashtra, India
3
BAIF Development Research Foundation, Central Research Station, Urulikanchan, Pune 412202, Maharashtra, India
*
Author to whom correspondence should be addressed.
Methane 2025, 4(3), 17; https://doi.org/10.3390/methane4030017
Submission received: 13 May 2025 / Revised: 25 June 2025 / Accepted: 10 July 2025 / Published: 16 July 2025
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Abstract

Wetlands are the most important natural sources of methane. Studies on the distribution and diversity of methanotrophs, especially in tropical wetlands, are limited. The studies on wetland methanotrophs help bridge the gap in the literature for understanding the community structure of methanotrophs in tropical wetlands. Our present study documents the methanotroph diversity from various wetland habitats across Western India. Samples from various sites, such as freshwater ponds, lake sediments, mangroves, etc., located in Western India, were collected and enriched for methanotroph isolation. An established protocol for the isolation of methanotrophs from Indian rice fields, involving serial dilution and long-term incubations, was slightly modified and used. Obtaining entirely pure cultures of methanotrophs is a labor-intensive and technically challenging process. Hence, for primary level characterization, ‘methanotroph monocultures’, which have a single methanotroph culture with minimal contamination, were established. Twenty monocultures and eight pure cultures of methanotrophs were obtained in this study. The pmoA gene has been used for the phylogenetic characterization of methanotrophs for the last 25 years. Monocultures were from seven genera: the Methylomonas, Methylocystis, Methylosinus, Methylocaldum, Methylocucumis, Methylomagnum, and Methylolobus genera. Eight pure cultures were obtained, which were strains of Methylomonas koyamae, Methylosinus sporium, and Methylolobus aquaticus. A maximum number of cultures belonged to the Type I genus Methylomonas and to the Type II genus Methylocystis. Thus, the cultivation-based community studies of methanotrophs from wetland habitats in India expanded the current knowledge about the methanotroph diversity in such regions. Additionally, the cultivation approach helped us obtain new methanotrophs from this previously unexplored habitat, which can be used for further biotechnological and environmental applications. The isolated monocultures can either be used as MMCs (mixed methanotroph consortia) for environmental applications or further purified and used as pure cultures.

1. Introduction

Wetlands are hotspots of high methanogenic activity, making these the most important natural sources of atmospheric methane (CH4), contributing to a relatively high percentage of global emissions [1]. Methane is produced in wetlands through the anaerobic microbial breakdown of organic matter. Methane emissions from wetlands account for 2 to 7% of net primary productivity [2]. Wetlands are essential to terrestrial and aquatic ecosystems and contribute significantly to greenhouse gas emissions by releasing methane. Wetlands are territories of marsh, fen, peat land, or water, either natural or manmade, permanent or temporary, with water that is static or flowing, fresh, brackish, or salty, and also includes portions of marine water whose depth at low tide does not exceed six meters [3]. As wetlands are the world’s most significant carbon sink and the primary natural source of methane, the type, area, distribution, timing, and amount of flooding of wetlands are critical factors to consider when calculating greenhouse gas emissions and carbon storage [3]. The high organic carbon concentration of the soil in wetlands makes them a significant contributor to the global carbon cycle despite making up only 5% of the planet’s land area [4]. Globally, ~20% of the methane emissions arise from wetlands. About 160 ± 40 Tg CH4 is contributed annually by natural wetlands, such as bogs, fens, flood plains, coastal zones of lakes, marshes, and swamps [5]. The majority of CH4 emissions to the atmosphere are released by tropical wetlands, which are followed by temperate and northern wetlands [5].
These methane-rich zones shelter diverse aerobic methanotrophs and anaerobic methane oxidizers, the only known biological filter for methane, thereby crucial in regulating the atmospheric methane flux. Aerobic methanotrophs consume methane, the second most significant greenhouse gas, as their only energy and carbon source [6]. Methanotrophs, or methane-oxidizing bacteria, are a special class of methylotrophic bacteria that derive their energy and carbon solely from methane. These are Gram-negative, obligately aerobic, and ubiquitous in various habitats such as freshwater, sediments, and soils [7]. Although our current work focuses on aerobic methanotrophs, anaerobic methane oxidation is an important process, which takes place via three major pathways: S-DAMO (sulfate-dependent anaerobic methane oxidation) [8,9,10], N-DAMO (nitrate/nitrite-dependent anaerobic methane oxidation) [11], and M-DAMO (metal-dependent Fe (III) Mn (IV) anaerobic methane oxidation) [12]. Additionally, there are reports that aerobic methanotrophs have been found in the anoxic environments of lakes, freshwater sediments, and wetlands [13]. Due to cultivation challenges associated with the culturing of anaerobic methane oxidizers, the present work was focused on understanding the cultivable diversity of aerobic and microaerophilic methanotrophs. The present study was undertaken to broaden the knowledge of methanotrophs from various wetland habitats in India, some of which are included under the biodiversity hotspots of India: The Western Ghats. The recently developed cultivation methodology, designed for the cultivation of methanotrophs from rice fields [14], was used in the current study, which included serial endpoint dilution in serum bottles/microtiter plates, followed by streaking on agarose-containing nitrate mineral salt media. This method resulted in the isolation of diverse methanotrophs from a single habitat, and serial dilution allowed the growth of slow-growing but abundant methanotrophs [14]. As methanotrophs are difficult to purify from the heterotrophs that co-exist with them, it might take years to obtain pure cultures in some cases [15]. Hence, in our studies, the methanotrophs were purified until morphologically, a single morphotype existed (3–4 times re-streaking), and we termed these cultures as ‘methanotroph monocultures’. All the monocultures had a minimal amount of contamination and resulted in a pure pmoA sequence, suggesting that a single methanotroph existed in the culture. A few of these were further purified, and pure cultures were obtained. Thus, by applying this method, our cultivation studies were completed in a time span of 1–2 years, starting from the sample collection. Methanotrophs have exciting applications, such as their use as single-cell proteins (for animal consumption), as methane mitigation agents, and for methane valorization [16]. Thus, the cultivation approach would enable us to acquire cultures, which can be explored and bio-prospected for various practical applications such as methane mitigation and valorization.

2. Results

The samples were collected from various sites in Maharashtra, Western India, representing various wetland habitats such as pond sediments, stone quarries, and lake sediments (freshwater), and a few samples were from mangroves (marshy sediments) (Figure 1). Enrichments that showed a decline in methane accompanied by visual growth in terms of turbidity, surface pellicle, or biofilm growth at the bottom were indicative of positive enrichment. The last positive serial dilution for each enrichment was noted. Methanotrophs grew in the collected samples in various abundance values, ranging from 10−2 to 10−12 (Table 1). It was seen that most of the pond types of freshwater and shallow habitats showed the presence of methanotrophs in relatively high numbers, reaching up to 10−12 cells/g of soil. However, in the case of the mangrove samples, the highest dilution reached was 10−3 or 10−4, indicating a low abundance of methanotrophs.
Serial dilution enrichment followed by isolation on agarose plates in the presence of methane and an air environment resulted in the cultivation of methanotrophs from various groups: Type Ia, Type Ib, and Type II methanotrophs. Our goal was to use a cultivation approach to document the biodiversity of methanotrophs and to expand the present knowledge of cultivable methanotrophs from Indian wetlands. In this study, 28 methanotrophs were cultured and were composed of 20 monocultures of methanotrophs (single dominant methanotrophs) and eight pure cultures from seven genera. A clear pmoA sequence was obtained for all of the 28 cultures, and their next neighbors were determined by NCBI blast analyses (Table 1). The pmoA gene sequencing helped assign the methanotrophs to their corresponding genera and species [17,18]. Amongst these, eight axenic cultures were identified as Methylomonas fluvii, Methylomonas sp., Methylolobus aquaticus, Methylosinus sporium, and Methylosinus trichosporium, respectively, after 16S rRNA gene and pmoA gene sequencing (Table 2).
Most of the cultures were found to be of the genera Methylocystis and Methylomonas, as seen after morphological and pmoA blast analyses. The Methylocystis genus represents Type II methanotrophs, which are usually small in size (~1 µm diameter) and coccoid in shape (Figure 2). Out of the 26 cultures, six cultures of methanotrophs, namely [strain name (pmoA accession number)] VLS12 (PQ821915), VLS6, VLW4 (PQ821917), MB5 (PQ821931), MG3 (PQ821909), and MMB (PQ821921) (Figure 2), isolated from freshwater, soil, mud, and mangroves (Table 1), belong to the Methylocystis genus and show ~97% nucleotide pmoA gene similarity with the pmoA sequence of Methylocystis hirsuta CSC1T (Figure 3) (Table 1). Nine cultures were from the Methylomonas genus (Table 1). They were seen as thick and short rods in microscopic analyses (Figure 2). Among these, five cultures, i.e., ASQA (PQ821920), MSA (PQ821928), TM3 (PQ821925), MgM2 (PQ821911) (Figure 2), and AL2 (PQ821908), showed 96.12, 95.81, 92.47, 91.88, and 87.79% pmoA gene similarity with Methylomonas koyamae Fw 12E-YT (Figure 3) (Table 1). The culture MgM2 showed 96.70% 16SrRNA gene similarity with Methylomonas aurea strain SURF-1T (Figure 4) (Table 2). Two cultures of Methylomonas, i.e., VUS3 (PQ821926) and TS2 (PQ821913), showed 95.19% pmoA and 99.41% 16SrRNA gene similarity with Methylomonas fluvii EbBT (Figure 3 and Figure 4) (Table 1 and Table 2). Two strains, AL2B (PQ821907) and PgA6 (PQ821927), showed 89.56% and 94.34 pmoA gene similarity with Methylomonas montana MW1T (Figure 3) (Table 1). Methylomonas species can be found in freshwater lakes and rivers, silt, wetland muds, activated sludge and wastewater, groundwater, and coal mine drainage water [19]. Three cultures belonged to the Type Ia methanotroph genus Methylocaldum. All the Methylocaldum cultures were cultivated from mangrove regions (Figure 1). These mangroves are in the saline and tropical regions of Konkan and Mumbai. The Methylocaldum genus has been detected earlier in India from cow dung, compost, and biogas slurry samples [20]. Three cultures isolated from the mangrove samples, i.e., MgM4 (PQ821912), MgN2 (PQ821930), and MgD2 (PQ821910) (Figure 2), show 99.18, 99.52, and 98.95% pmoA gene similarity with Methylocaldum gracile VKL-14LT (Figure 3) (Table 1). The members of the genus Methylocaldum are thermotolerant methanotrophs and are detected in diverse environments, including marine and aquatic habitats, upland soils, rice fields, and landfills [21]. Our previously published study isolated Methylocaldum gracile strain RS9 from biogas slurry [20]. Six cultures of the genus Methylosinus have been isolated in the study; four cultures belong to the species Methylosinus sporium, and two cultures belong to M. trichosporium. The cultures VLS4 (PQ821914), AS1B (PQ821919), and AW2A (PQ821929) (Figure 2) show 95.06, 99.31, and 95.05% pmoA gene similarity with M. sporium ATCC 35069T, and the culture PLW2 (PQ821916) shows 100% pmoA gene similarity with M. trichosporium OB3bT (Figure 3) (Table 1). The cultures VLS4 and AW1A show 98.96% and 98.89% 16SrRNA gene similarity with Methylosinus sporium NCIMB 11126T, and the cultures PLW2 and AW2B show 99.93% and 100% 16SrRNA gene similarity with Methylosinus trichosporium OB3bT (Figure 4) (Table 2). The cultures of the genera Methylomagnum and Methylocucumis, having a large size >5 µm, were also cultured in this study. The culture MSBM (PQ821923) (Figure 2) was isolated from a seaweed sample from a wetland patch on a hill near Mahatma Society, Pune (termed Mahatma Hill). This culture showed 99.53% pmoA gene similarity with Methylomagnum ishizawai RS11DT (Figure 4) (Table 1). Two cultures, MSBC (PQ821922) (Figure 2) and MWC (PQ821924), show 98.66% and 98.61% pmoA gene similarity with Methylocucumis oryzae Sn 10-6T (Figure 3) (Table 1). These cultures were obtained from seaweed and water samples from a wetland patch near Mahatma Society, Pune (Mahatma Hill). The culture isolated from freshwater samples from Pashan Lake, Pune, i.e., PLW4 (PQ821918) (Figure 2, Figure 3 and Figure 4), showed 97.25% pmoA and 99.35% gene similarity with Methylolobus aquaticus FWC3T (Table 1 and Table 2). PLW4 is the second strain of the newly described genus and species, Methylolobus aquaticus. Currently, only a single strain is available from this novel genus and species, the type strain FWC3T [22]. Thus, this study documented the cultivation of methanotrophs from two novel and newly described genera from India, Methylolobus and Methylocucumis, both described from India, and Methylocucumis has not been cultivated so far from any other country other than India [23].
Habitat-wise distribution of methanotrophs: In our study, we studied seven freshwater and three brackish habitats (mangroves). It was seen that the mangroves which lie beside the seashores with hot and humid habitats showed the presence of thermotolerant methanotrophs, mostly Methylocaldum sp., and Methylocystis sp. In contrast, all the freshwater samples showed strictly mesophilic methanotrophs, and no cultures of Methylocaldum were isolated from any of the freshwater wetland samples. All of the small ponds showed the presence of Methylomonas sp. Vetal Hill (also popularly known as ARAI Hill) and Mahatma Hill, both of which have stone quarries that are filled with water throughout the year, showed a little difference in the distribution of methanotrophs. Vetal Hill showed Methylosinus as the most dominant genus prevalent in the last positive dilutions, and Methylomonas in the lower dilutions. Mahatma Hill showed the presence of a newly described genus Methylocucumis, first isolated from a rice field in western India, and was also reported from Mahatma Hill in an earlier study [23,24,25,26].

3. Discussion

The main objective of this study was to expand the knowledge of wetland methanotrophs from India using a cultivation approach. The specialized cultivation protocol was developed earlier for rice field methanotrophs and was used to retrieve the maximum possible methanotroph members from the habitat [14], which can be further used for bio-prospecting. The method used in this study was a slight modification of the method that was developed by our group for the cultivation of methanotrophs from rice field habitats. Instead of two steps of serial dilutions, the first one in serum bottles followed by microtiter plates, we used a single serial dilution step followed by streaking on NMS agarose plates, and the repeated streaking of single colonies (3–5 streaking rounds). Though cultivation-independent analysis does provide a picture of the community structure, the cultures are not present, and hence applications such as using the methanotrophs as models in studying methane oxidation in wetlands, or using the methanotrophs in any other applications are not possible. A repository of methanotrophs has been developed as a result of ~10 years of cultivation studies in methanotrophs from Indian habitats, and hence, one of the objectives was also to expand this repository and characterize unique methanotrophs [14,20,22,23,24,25,26,27,28,29,30,31,32,33]. Methanotroph monocultures basically refer to methanotrophs that are purified, form a single type of colony, and have marginal levels of non-methanotrophs as contamination. Non-methanotrophs or heterotrophs grow on metabolites produced by the methanotrophs and either grow as satellite colonies, which are smaller and translucent, or grow with methanotrophs as mixed cultures, making the colony appear bigger [15]. Further purification of methanotrophs is achieved by picking up single colonies and re-streaking until a pure culture is established. After 3–5 re-streaking attempts, usually the methanotroph is ~90% pure, meaning that under a phase contrast microscope, a single morphotype persists [14,24]. Methanotrophs are usually >1 μm and appear darker in color under a phase contrast microscope. Type I methanotrophs are generally large (~1.5–5 μm), so it is easy to establish the purity of the culture by microscopic analysis. In case of Type II methanotrophs, Methylosinus sporium and Methylosinus trichosporium cells are large and have a characteristic shape (half-moon-shaped cells or rosette-forming pear-shaped cells). Methylocystis cells can be coccoid and are comparatively smaller. However, the non-methanotrophs associated with methanotrophs are much smaller (0.5–1 μm) and can be easily noticed using a phase contrast microscope. The monocultures described in this study showed >90% cells of the same morphology (large and dark cells) and, after streaking on nutrient agar (Himedia, Thane, India), showed some amount of growth and were, hence, termed as methanotroph monocultures. The methanotroph pure cultures showed 100% the same cells, and no growth was observed on nutrient agar plates. Though monocultures have a small amount of contamination, these can be directly used as ‘mixed methanotrophic consortium’ or MMCs, which can be used for direct applications [34]. Usually, compared to the pure cultures, MMCs have better growth, higher cell density, and methane-oxidizing activity; are more stable in open environments; and can be applied to open systems [34]. In our work, all the monocultures can be used as MMCs, a culture with a predominant methanotroph and other companion microorganisms. The co-existing heterotrophs usually can remove toxic metabolites produced by the methanotrophs and, in some cases, can supply key nutrients [34].
The pmoA gene encodes the β-subunit of the particulate methane monooxygenase (pMMO), which is highly conserved and has been used for a long time as a functional gene to probe methanotrophs [35,36]. This gene can provide phylogenetic information that is congruent with the 16S rRNA gene and hence widely used for taxonomy over the last 25–30 years. The pmoA gene is usually amplified using A189f primer and mb661r primer [37]. Several studies based on methanotroph diversity and community analysis have been based exclusively on the pmoA gene as the phylogenetic marker, for Lake Constance [27,38,39], Lake Washington [40,41], and from rice fields [28,42,43,44,45]. The functional pmoA gene is frequently used to probe the diversity and phylogeny of methane-oxidizing bacteria (MOB) in various environments [46]. The study proposed 10 and 17% dissimilarity of pmoA gene nucleotide cutoffs corresponding to 3 and 5% thresholds of the 16S rRNA gene [42] by assuming a 3.5 times higher substitution rate [47].
Although there is less information about the methanotrophs that are active in tropical wetlands, there is quite extensive information about the methanotroph community structure in rice fields from Italy, China, and India [28,36,42,44,45,48,49,50,51,52,53] using a culture-independent approach. Study of community structure using a cultivation approach provides direct access to the methanotrophs for future applications, and various methanotrophs from rice fields in India have been studied by a cultivation or a dual approach (combining culturing and culture-independent) [14,28,31] and from wetlands [22,24]. There are a large number of studies on northern wetland methanotrophs using cultivation-independent analysis [54,55,56], but studies on tropical wetlands are very few [57].
Varied types of wetlands were covered in this study, which included mangroves, hilltop stone quarries, lake sediments, and small ponds with aquatic plants. The geographical area covered spans Western India and mainly the Western Ghats, one of India’s two biodiversity hotspots. The Western Ghats, also known as the Sahyadris, are a mountain range that stretches 1600 km (990 mi) along the western coast of India [Western Ghats Biodiversity Hotspot—WorldAtlas]. They have a diverse flora and fauna, though the microbial diversity remains mostly undocumented. Samples from the Venna lake, located at Mahabaleshwar, the hill stone quarries in Pune, and the mangroves from Alibag and Mumbai all lie within the Western Ghats.
Most isolated cultures were observed to belong to the genera Methylocystis and Methylomonas. These two genera have been the major components of Sphagnum bog wetland methanotroph communities as seen in a study using a culture-independent approach [58]. Methylomonas species can be found in freshwater lakes and river silt, wetland muds, activated sludge, wastewater, groundwater, and coal mine drainage water [59]. One of the most ecologically significant methanotroph populations in terrestrial settings is the Methylocystis species. They live in various environments, including freshwater, rice paddies, peatlands, and landfills [59,60]. Groundwater and soil freshwater sediments are important ecosystems for Methylosinus species [59]. Among the unique methanotrophs, strains from the newly described genera—Methylocucumis and Methylolobus—were also isolated. Methylocucumis oryzae, the newly described genus isolated from a rice field, has been reported by our research group and has been exclusively isolated from the Western Ghat regions of India. This particular methanotroph is relatively large in size and oblong-shaped, isolated frequently from stone quarries in wetland patches of Pune city. Similarly, in this study, we could also isolate the strain Methylolobus aquaticus, a newly described genus and species, isolated from a wetland in the state of Maharashtra [22]. Thus, our technique helped isolate members from novel genera; e.g., Methylomagnum, a genus first described in rice fields [61,62], was recovered from a wetland sample. Three of the cultures belonged to Methylocaldum, all isolated from mangrove regions. These mangroves are basically in Konkan and Mumbai’s hot and moist regions. In India, Methylocaldum has been detected in cow dung, biogas slurries, and rice fields [20,31]. There are no major reports on the methanotroph diversity in mangroves, although some general studies are available [63], indicating the importance of aerobic methane oxidation in mangrove habitats [64].
Wetlands are often subjected to drying and exposure to sunlight and light-related damage. Therefore, many isolates belonging to the Type I methanotrophs were found to have colors like pink and red, mostly related to the carotenoid pigments. Beyond their ecological function, methanotrophs have earned more and more interest due to their biotechnological potential, which includes the synthesis of valuable substances like carotenoids, polyhydroxyalkanoates (PHAs), and single-cell proteins [16]. A broad class of pigmented isoprenoid chemicals, carotenoids, are used in the food, feed, and pharmaceutical industries for their antioxidant qualities. Carotenoids like β-carotene, canthaxanthin, and zeaxanthin have been reported to be produced by several methanotrophs, especially those from the genera Methylobacter, Methylomonas, and Methylococcus. However, strain screening is an essential step for finding high-yielding options for industrial applications because the potential for carotenoid synthesis differs greatly among strains [65]. Carotenoids are known to have a protective role in photoprotection [66]. Pink coloration was also seen in Methylocystis cultures, as found in Methylocystis rosea, which showed pink coloration [67]. Additionally, a few of the Methylocystis cultures isolated from Indian rice landraces also show pink coloration [31]. The isolated methanotrophs can be used for various mitigation and value-adding applications [16], including serving as models for research on methanotroph-based methane mitigation from wetland habitats. Methanotrophs are already being used for the production of single-cell proteins (e.g., Uni-protein-), carotenoids [65], and other valuable chemicals. Recently, two studies from India have focused on the use of methanotrophs in plant growth promotion in rice [68,69].

4. Materials and Methods

All the samples were collected using gloves in sterile plastic vials or bottles or sterile plastic bags and proceeded for the enrichment of methanotrophs and the oxidation of methane. Mud samples and water samples were collected in triplicate from the wetland site using sampling vials (50 mL capacity) from the littoral zones of lakes or ponds, or with about 2–15 cm of water layer on the top (Figure 1), Table 1.
Serial dilutions were set up using a modified Nitrate Mineral Salt (NMS) medium as described earlier [27] from 10−1 to 10−12 by adding 1 g of the sample to a 9 mL sterile (NMS) medium in 35 mL serum bottles with methane: air in headspace, were set up as described earlier [27]. Alternatively, a few samples, microtiter plates (48 wells), incubated in a desiccator with methane: air, were used for the serial dilution enrichments. Both serum bottles and microtiter plates can be used alternatively for enrichment purposes. All the enrichments were incubated at ambient temperatures (23–28 °C) with methane and air in the ratio of 20:80 as headspace gas. Gas Chromatography was performed by injecting the headspace gas into the Chemito 8610 Gas Chromatography machine equipped with a flame ionization detector (FID). All positive enrichments were streaked on modified NMS agarose medium plates and incubated in desiccators in a methane: air (20:80) environment. The last positive dilution was noted in each sample. After growth in a methane and an air environment, colonies were obtained. Single colonies were picked up and streaked on fresh plates until a single morphotype dominated. This was usually achieved after ~3 re-streaking procedures, confirmed by phase contrast microscopy with a camera (using Nikon 80i, Tokyo, Japan) of the cells under an oil immersion lens (100× magnification). Axenic or pure cultures were obtained by repeated streaking to eliminate heterotrophic contaminants, and the purity was confirmed by microscopy, as well as the absence of growth on nutrient agar plates (Himedia, Mumbai, India). Heterotrophic contaminants often accompany methanotrophs, and the cultures that remained non-axenic (having small numbers of heterotrophs) even after 3–5 purifications on agarose were termed ‘methanotroph monocultures’. Pure cultures of methanotrophs, which were devoid of any other heterotrophic bacteria, showed no contamination on microscopic observations or growth on nutrient agar plates. As methanotrophs can only utilize methane or methanol, they are unable to grow on nutrient agar or other complex media, a characteristic feature of methanotrophs [6,15].
DNA was extracted from the twenty methanotroph monocultures as well as from the eight pure cultures using 4–5 colonies, and the Gram-negative protocol of the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich Life Sciences, Bangalore, India) was used for the DNA extraction. The genes for particulate methane monooxygenase subunit (pmoA) were amplified using the A189f-mb661r primers [37]. In case of pure cultures, additionally, 16S rRNA genes were amplified using the universal primers: 27f [70] and 1492r [71]. All the amplifications were performed in a 96-well thermal cycler (Veriti, Applied Biosystems, Thermo Scientific, Mumbai, India) with a total volume of 25 μL using Takara® PCR Master Mix. The amplified PCR products were purified using a PCR purification kit (Alphagen Biotech Ltd., Pingtung City, Taiwan). The sequencing of the purified PCR products was performed at the Progene Life Sciences lab (GeneSpec, Kochi, Kerala, India). The sequences obtained using both primers of the 16S rRNA gene were aligned and assembled using SeqMan (DNASTAR, Lasergene software, version 7) and were subjected to a BLAST analysis (BLAST: Basic Local Alignment Search Tool, version 1.4.0). Sequences of all of the isolates were deposited in the National Center for Biotechnology Information (NCBI) database, and accession numbers were obtained (Table 1 and Table 2). For the gene pmoA, the nucleotides (~432 bases) were translated to amino acid sequences using SeqMan (DNASTAR, Lasergene software) and used for phylogenetic tree construction. The phylogenetic trees were constructed based on the amino acid sequence of the functional pmoA gene of monocultures and pure cultures. For the eight pure cultures, ~1380 bp of the universal 16S rRNA gene sequences were used. The ML phylogenetic trees were constructed using MEGA XI (version 11) with 1000 bootstraps [72]. For monocultures, each culture was classified based on its phylogenetic position in the tree into the respective genus. All the pure cultures were classified based on their phylogenetic positions using the 16S rRNA gene sequence and pmoA partial sequence, and assigned to the genus and possible species level, and any novelty was noted.

5. Conclusions

The current study reports the diversity, abundance, and community structure of methanotrophs from tropical wetlands spanning the strip of Western India, which falls under the Western Ghats and is categorized as one of the two biodiversity hotspots in the country. Cultures from seven major genera of methanotrophs were isolated in the study, amongst which Methylomonas and Methylocystis, the two prominent genera representing Type I and Type II methanotrophs, respectively, were seen to dominate the wetland community structure of various habitats. Methylocaldum, Methylosinus, Methylocucumis, Methylomagnum, and Methylolobus were a few other genera isolated in the study. The abundance of methanotrophs was relatively low in mangrove soils.

Author Contributions

Conceptualization, M.C.R. and R.A.B.; methodology, K.P., S.M., Y.P., Y.K., S.K. and C.S.; writing—original draft, K.P. and M.C.R.; writing—review and editing, M.C.R., R.A.B., K.P., M.C.R. and S.K. performed the sample collection. R.A.B. constructed the amino-acid phylogenetic tree. K.P. and M.C.R. wrote the draft manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

M.C.R. acknowledges ANRF (SPF/2022/000045) for providing the funds. K.P. acknowledges UGC for providing her with a junior research fellowship.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the sequence data are available in the NCBI database.

Acknowledgments

We thank Mahesh Shindikar, COEP, and Sagar Pandit, IISER, for collecting the mangrove samples from Mumbai. We would also like to thank Kaiwalya Bahulikar for assisting in the sample collection at Mahatma Hill.

Conflicts of Interest

The authors have no relevant financial or non-financial interests to disclose.

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Figure 1. Sampling sites of different wetland patches were visited for the study: (A) Mahatma Hill in winter. (B) Mahatma Hill in summer. (C) Venna Lake. (D) Vetal Hills. (E) Dive Agar Mangroves. (F) Pashan Lake.
Figure 1. Sampling sites of different wetland patches were visited for the study: (A) Mahatma Hill in winter. (B) Mahatma Hill in summer. (C) Venna Lake. (D) Vetal Hills. (E) Dive Agar Mangroves. (F) Pashan Lake.
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Figure 2. Morphology of the isolated cultures as seen under a phase-contrast microscope (Nikon 80i, Japan microscope with a camera) under 100× magnification with oil emulsion: (A) Methylocystis hirsuta culture VLS12; (B) Methylocystis hirsuta VLS6; (C) Methylosinus sporium strain VLS4; (D) Methylocystis hirsuta MG3; (E) Methylomonas fluvii strain TS2; (F) Methylomonas koyamae culture MgM2; (G) Methylocucumis oryzae MSBC; (H) Methylomangnum ishizawai culture MSBM; (I) Methylolobus aquaticus culture PLW4; (J) Methylocaldum gracile culture MgM4; (K) Methylocaldum gracile culture MgD2; (L) Methylocaldum gracile culture MgN2.
Figure 2. Morphology of the isolated cultures as seen under a phase-contrast microscope (Nikon 80i, Japan microscope with a camera) under 100× magnification with oil emulsion: (A) Methylocystis hirsuta culture VLS12; (B) Methylocystis hirsuta VLS6; (C) Methylosinus sporium strain VLS4; (D) Methylocystis hirsuta MG3; (E) Methylomonas fluvii strain TS2; (F) Methylomonas koyamae culture MgM2; (G) Methylocucumis oryzae MSBC; (H) Methylomangnum ishizawai culture MSBM; (I) Methylolobus aquaticus culture PLW4; (J) Methylocaldum gracile culture MgM4; (K) Methylocaldum gracile culture MgD2; (L) Methylocaldum gracile culture MgN2.
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Figure 3. Amino acid-based phylogenetic tree based on the pmoA gene of the methanotrophs from this study with their closest members (indicated in bold). The phylogenetic tree was constructed using the partial pmoA sequence (~412 bp) from the methanotrophs in comparison with the pmoA sequences of the type cultures using the MEGA XI software. It was inferred by the maximum likelihood method and the Tamura–Nei model. The bar showed a 5% divergence. The protein accession numbers are indicated before the strain name.
Figure 3. Amino acid-based phylogenetic tree based on the pmoA gene of the methanotrophs from this study with their closest members (indicated in bold). The phylogenetic tree was constructed using the partial pmoA sequence (~412 bp) from the methanotrophs in comparison with the pmoA sequences of the type cultures using the MEGA XI software. It was inferred by the maximum likelihood method and the Tamura–Nei model. The bar showed a 5% divergence. The protein accession numbers are indicated before the strain name.
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Figure 4. Maximum likelihood 1000 bootstrap tree of 16S rRNA-gene based (using ~1350 bases) phylogenetic tree of pure methanotrophic strains (shown in bold) with their closest members. The evolutionary history was inferred by using the maximum likelihood method and the Tamura–Nei model. Evolutionary analyses were conducted in MEGA XI. The bar represents 5% divergence.
Figure 4. Maximum likelihood 1000 bootstrap tree of 16S rRNA-gene based (using ~1350 bases) phylogenetic tree of pure methanotrophic strains (shown in bold) with their closest members. The evolutionary history was inferred by using the maximum likelihood method and the Tamura–Nei model. Evolutionary analyses were conducted in MEGA XI. The bar represents 5% divergence.
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Table 1. Tabular summary describing the pmoA identification of the representative sample isolated from the respective collection site with their names, dates, and dilution.
Table 1. Tabular summary describing the pmoA identification of the representative sample isolated from the respective collection site with their names, dates, and dilution.
Sampling DetailsDilution Used for Isolation of MethanotrophsRepresentative CulturesIdentification Using the pmoA Gene
Sampling Site and Geographical LocationSampling Date Strain NameGeneBank Accession NumberNearest Match (with Type Cultures)% Similarity (Nucleotide)% Similarity
(Protein)
Vetal Hill Pond a
(Fresh water sample)
18.525474° N
73.815292° E		15 January 202410−2AS1BPQ821919Methylosinus sporium strain ATCC 3506999.3198.61
15 January 202410−2ASQAPQ821920Methylomonas koyamae strain Fw12E-Y96.1299.31
15 January 202410−8AW2APQ821929Methylosinus sporium strain ATCC 3506995.0597.93
15 January 202410−8AW1APQ821929Methylosinus sporium strain ATCC 3506994.8497.99
15 January 202410−8AW2BPQ821919Methylosinus sporium strain ATCC 35069100100
Mahatma Hill Pond a
(Fresh water and mud sample)
18.4926° N
73.8013° E		21 April 202410−2MSBMPQ821923Methylomagnum ishizawai strain RS11D99.5399.29
18 December 202310−3TM3PQ821925Methylomonas koyamae strain Fw12E-Y92.4797.18
21 April 202410−5MMBPQ821921Methylocystis hirsuta strain CSC197.4698.62
21 April 202410−6MSAPQ821928Methylomonas koyamae strain Fw12E-Y95.81100
21 April 202410−7MWCPQ821924Methylocucumis oryzae strain Sn 10-698.61100
21 April 202410−8MSBCPQ821922Methylocucumis oryzae strain Sn 10-698.66100
ARI pond a
(Lotus root sample)
18.5173° N
73.8475° E		2 January 202410−2AL2PQ821908Methylomonas koyamae strain Fw12E-Y87.7995.16
2 January 202410−2AL2BPQ821907Methylomonas montana strain MW189.5697.90
Paragrass
BAIF pond a
(Seaweed sample)
18.491005° N
74.134544° E		9 January 202410−6PgA6PQ821927Methylomonas montana strain MW194.3498.03
Pashan Lake
(Fresh water sample)
18.533752° N
73.785717° E		8 July 202310−2PLW2PQ821916Methylosinus trichosporium strain OB3b 10099.31
8 July 202310−4PLW4PQ821918Methylolobus aquaticus strain FWC397.25100
Venna Lake
Sediments
(Fresh water and sediment sample)
17.934° N
73.665° E		28 December 202210−3VUS3PQ821926Methylomonas fluvii EbB95.19100
28 December 202210−4VLS4PQ821914Methylosinus sporium ATCC 3506995.0698.55
28 December 202210−4VLW4PQ821917Methylocystis hirsuta strain CSC197.3597.93
28 December 202210−5MB5PQ821931Methylocystis hirsuta strain CSC197.1298.66
28 December 202210−6VLS6PQ821915Methylocystis hirsuta CSC197.7098.56
28 December 202210−12VLS12PQ821915Methylocystis hirsuta CSC197.2598.60
Tamhini river
(Fresh water sediment sample)
18.134111° N
73.605728° E		16 June 202310−2TS2PQ821913Methylomonas fluvii strain EbB95.1998.53
Mumbai
Mangroves a
(Brackish water and soil sample)
19.0374° N, 72.981° E		21 September 202310−2MgM2PQ821911Methylomonas koyamae strain Fw12E-Y91.8897.14
21 September 202310−4MgM4PQ821912Methylocaldum gracile strain VKM-14L99.18100
Alibag mangroves
(Brackish water and soil sample)
18.64° N
72.88° E		23 March 202310−3MG3PQ821909Methylocystis hirsuta strain CSC197.5099.29
23 March 202310−3MgN2PQ821930Methylocaldum gracile strain VKM-14L99.52100
Diveagar mangroves
(Brackish water and soil sample)
18.1920° N
72.9789° E		29 December 202310−2MgD2PQ821910Methylocaldum gracile strain VKM-14L98.95100
Notes: The last positive enrichments are indicated in bold, superscript a indicates enrichment in microtiter plates, and all other enrichments were set up in serum bottles. All the pure culture strains are indicated in bold. The modified NMS medium [14] was used for all the enrichments: g/L: MgSO4 7H2O, 1; CaCl2 2H2O, 0.2; KNO3, 1; SL10 solution, 1 mL; Fe3NH4 citrate solution, 1 mL. After autoclaving, phosphate buffer (pH 6.8), 20 mL/L, and vitamin solution (1×), 10 mL/L, were added.
Table 2. Taxonomy of pure cultures identified using 16S rRNA gene sequencing with details on their sampling sites, dilution, and representative strain.
Table 2. Taxonomy of pure cultures identified using 16S rRNA gene sequencing with details on their sampling sites, dilution, and representative strain.
Sampling DetailsDilution Used for Isolation of MethanotrophsRepresentative StrainIdentification Using 16S rRNA Gene
Sampling Site
and
Geographical Location		Sampling Date Strain NameGene Accession NumberNearest Match with Type Strain% Similarity
Vetal Hill Pond
(Fresh water sample)
18.525474° N
73.815292° E		15 January 202310−8AW1APQ826297Methylosinus sporium strain NCIMB 1112698.89
15 January 202410−8AW2BPV637194Methylosinus trichosporium strain OB3b100
Venna Lake
(Sediment sample)
17.934° N
73.665° E		28 December 202210−3VUS3PQ826293Methylomonas fluvii strain EbB99.41
28 December 202210−4VLS4PQ826294Methylomonas sporium strain NCIMB 1112698.96
Pashan Lake
(Fresh water sample)
18.533752° N
73.785717° E		8 July 202310−2PLW2PV637193Methylosinus trichosporium strain OB3b99.93
8 July 202310−4PLW4PV804843Methylolobus aquaticus strain FWC399.43
Tamhini river
(Fresh water
sediment sample)
18.134111° N
73.605728° E		16 June 202310−2TS2PQ826293Methylomonas fluvii strain EbB99.41
Mumbai
Mangroves
(Soil sample)
19° N
72° E		21 September 202310−2MgM2PV630802Methylomonas aurea strain SURF-196.70
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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. https://doi.org/10.3390/methane4030017

AMA Style

Pardhi K, Manvi S, Bahulikar RA, Patil Y, Kadam Y, Kadam S, Saraf C, Rahalkar MC. Cultivation of Diverse Type I and Type II Methanotrophs from Tropical Wetlands in India, Including Rare Taxa (Methylocucumis and Methylolobus). Methane. 2025; 4(3):17. https://doi.org/10.3390/methane4030017

Chicago/Turabian Style

Pardhi, Kajal, Shubha Manvi, Rahul A. Bahulikar, Yukta Patil, Yash Kadam, Shirish Kadam, Chandani Saraf, and Monali C. Rahalkar. 2025. "Cultivation of Diverse Type I and Type II Methanotrophs from Tropical Wetlands in India, Including Rare Taxa (Methylocucumis and Methylolobus)" Methane 4, no. 3: 17. https://doi.org/10.3390/methane4030017

APA Style

Pardhi, K., Manvi, S., Bahulikar, R. A., Patil, Y., Kadam, Y., Kadam, S., Saraf, C., & Rahalkar, M. C. (2025). Cultivation of Diverse Type I and Type II Methanotrophs from Tropical Wetlands in India, Including Rare Taxa (Methylocucumis and Methylolobus). Methane, 4(3), 17. https://doi.org/10.3390/methane4030017

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