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In natural ecosystems such as forests topographical gradients, species composition variability and seasonality, are the potential drivers of methane (CH4) metabolism, and thus, of the population size and activities of methane oxidizing bacteria (MOB). To test the hypothesis that topography, tree species and seasonal variability influence MOB population and soil methane oxidation potential (MOP), we conducted two consecutive years of study selecting three sites in a dry deciduous tropical forest in Chandauli district of eastern Uttar Pradesh, India. The qPCR results showed a large variation in MOB population size (copies g-1 dws), ranging from 1.0 × 106 to 2.1 × 107 and 9.0 × 105 to 2.2 × 107 during 2016 and 2017 respectively. The distribution of MOB population revealed the trend: hilltop > middle > hillbase with its maxima in the winter and minima in the rainy season. Laboratory incubation study revealed a similar trend in soil MOP (ng CH4 g-1 h-1 dws), it varied from 10.6 to 20.6 and 10.5 to 20.7 during 2016 and 2017 respectively. The outer canopy soils showed lower MOB population and MOP compared to under canopy soils of tree species Butea monosperma, Madhuca indica and Tectona grandis during both years of study. The topography, season and tree species significantly influenced the MOB population size and MOP. Soil MOP showed a highly significant correlation (r = 0.89; p < 0.01) with MOB population, and a negative correlation was found with soil moisture (r = 0.76; p < 0.01). The results indicate that the dry deciduous tropical forest soils are potential sinks of atmospheric CH4 wherein, the MOB population characteristically responds to topographical changes, tree species and seasonal shifts driving collectively the overall MOP of forest soils. [ABSTRACT FROM AUTHOR]

The fate of the enormous amount of reactive nitrogen released to the environment by human activities in India is unknown. Here we show occurrence of seasonal stratification and generally low concentrations of dissolved inorganic combined nitrogen, and high molecular nitrogen (N2) to argon ratio, thus suggesting seasonal loss to N2 in anoxic hypolimnia of several dam-reservoirs. However, 15N-experiments yielded low rates of denitrification, anaerobic ammonium oxidation and dissimilatory nitrate reduction to ammonium--except in the presence of methane (CH4) that caused ~12-fold increase in denitrification. While nitrite-dependent anaerobic methanotrophs belonging to the NC10 phylum were present, previously considered aerobic methanotrophs were far more abundant (up to 13.9%) in anoxic hypolimnion. Methane accumulation in anoxic freshwater systems seems to facilitate rapid loss of reactive nitrogen, with generally low production of nitrous oxide (N2O), through widespread coupling between methanotrophy and denitrification, potentially mitigating eutrophication and emissions of CH4 and N2O to the atmosphere. [ABSTRACT FROM AUTHOR]

Biogas reactors run on various types of waste, with cattle dung and agricultural wastes being the primary sources in India. As biogas contains 50–60% methane, there is a possibility that the reactors harbour methanotrophs or methane-oxidizing bacteria. We set up serial endpoint dilution enrichments for the cultivation of methanotrophs using slurry from a small biogas reactor and cattle dung samples and obtained cultures of Methylocaldum gracile , a thermotolerant methanotroph. The study was expanded by sampling reactors of another small reactor of 20 L capacity and two 1000 L reactors. Dung samples were obtained from two Indian cattle breeds (Tharparkar and Gir). Pulverized rice straw used for feeding the biogas was also used for experiments. All the enrichment bottles were incubated at 39 °C, the reactors' in-situ temperature, and the rumen gut temperature. Our study isolated four pure cultures most related to Methylocaldum gracile VKM-14LT, two strains from cattle dung samples, and two from reactors. The study also resulted in the cultivation of four additional cultures of Methylocaldum gracile and Methylocaldum tepidum, which were non-axenic and identified by pmoA gene sequencing. Pure cultures Methylocaldum gracile RS-9 and CDP-2 were studied for optimum temperature and oxygen. Both the strains were thermotolerant and grew in the temperature range of 25–45 °C with the optimum between 37 and 45 °C. The cultures could grow with minimal oxygen (0.5%–1%) in the headspace, with growth up to 10% oxygen. To summarize, we report the cultivation and isolation of methanotrophs from biogas slurries and cattle dung samples. Methylocaldum was the dominant methanotroph cultured, probably due to its thermotolerant nature and the ability to grow under variable oxygen conditions. The present study also expands the existing knowledge about habitats known for the genus Methylocaldum. An analysis of the isolated cultures would help us design strategies for methane mitigation from ruminants. [Display omitted] • Methanotrophs were cultured from biogas slurries and cattle dung, and all of the cultures belonged to Methylocaldum. • The abundance values of methanotrophs were more in slurries from small biogas reactors than from large biogas reactors. • Cattle dung samples showed methanotrophs in low abundances. • Two strains (Methylocaldum gracile RS-9 and CDP-2) showed growth at low oxygen conditions and preferred 37-45°C. • One of the first studies documenting methanotrophs from biogas slurries and cattle dung samples. [ABSTRACT FROM AUTHOR]

To study the effect of crop phenology and cultivation practices on methanotrophic communities, two tropical rice fields located in the upper Gangetic plain of India with similar soil type and different cropping history were selected. A laboratory incubation experiment for the enumeration of methanotrophs and for the measurement of CH4 oxidation potential was conducted on a parallel basis. The methanotroph population size was found to be significantly higher in the Banaras Hindu University (BHU), Varanasi soil than the Indian Institute of Vegetable Research (IIVR), Varanasi soil. The population size increased with the age of the plant for both the sites. The CH4 oxidation potential was higher with the BHU soil compared to the IIVR soil. The CH4 oxidation rate increased significantly from tillering to flowering to grain-filling stages, and finally there was no significant difference between the grain-filling and the grain-maturation stages. A diagnostic microarray targeting the pmoA gene and a 16S rRNA denaturing gradient gel electrophoresis (DGGE)-based approach were applied to assess the diversity of the methanotrophic community for the two sites. A broad diversity of methanotrophs was detected at both sites, including type I and type II methanotrophs of the genera Methylobacter, Methylomonas, Methylosarcina, Methylosphaera, Methylomicrobium and Methylocystis. Type II methanotrophs were found in higher abundance as compared to type I methanotrophs at both the sites. DGGE analysis indicated that the methanotroph community in BHU soil was more or less stable, while little variation was found in IIVR soil during crop growth. [ABSTRACT FROM AUTHOR]

The article presents a study that investigates the impact of soil type and plant age on the population and activities of rhizospheric methanotrophs in tropical rice soils. The research was conducted by collecting different samples of soils from the rice fields of Gangetic plain in India and analyzing their soil moisture and ammonium content, methane oxidizing bacteria (MOB) or methanotrophs population and crop biomass. It reveals that soil types, cultivation practice, plant age and topography affect the methane oxidation and population size of MOB and rice rhizosphere has an essential role in the microbial ecology of methanotrophs.

Abstract: Soil samples were collected from Panchamarhi dry deciduous forest in Satpuda Biosphere Reserve, India to determine the effect of hill slopes and altitude on the population size of methanotrophic bacteria. Population size, in range of 4×105–3.6×107 g−1 dry soil, was negatively correlated with altitude and increased exponentially (, P<0.001) at steep slope (60°) while logarithmically (, P<0.001) at low slope (45°). Soil organic C, total N, and soil moisture increased while C/N ratio and temperature decreased down the hill slope. The results indicated that nutritional status of the soil across the slopes determines the methanotrophic bacterial population size. [Copyright &y& Elsevier]

The utilization of methanotrophs for reducing the global warming potential in a flooded paddy system is the progressive investigation in the recent. The field investigation was conducted in Tamil Nadu Rice Research Institute, Aduthurai. This study showed the effect of isolated methanotroph strain (MR 15 ) on the reduction of methane emission and improvement in growth parameters and yield of paddy. The treatments included the diverse dosages of methanotroph consortium (0, 25, 50, 75 & 100%). The total seasonal methane emission varied between 1.96 and 5.04 kg ha - 1 for the season of Kharif and 2.18 to 5.81 kg ha - 1 for the season of Rabi owing to the footprint of methanotroph. Irrespective of treatments, the mean seasonal emission was more prominent during Rabi compared to the Kharif season. The dosage of methanotroph consortium significantly influenced not only seasonal methane emission but also the grain yield percentage, which increased over 100% Recommended Dose of Fertilizers (RDF). Application of 100% methanotroph consortium (i.e.6.25 kg ha - 1 ) minimizes the methane emission by 60% with an increase in grain yield by 35% and lessens the usage of additional nutrients. Overall, this study showed a sign of atmospheric methane uptake with increase growth potential and yield of paddy cultivation using methanotroph consortium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel gammaproteobacterial methanotroph; strain FWC3 was isolated from a tropical freshwater wetland sample collected near a beach in Western India. Strain FWC3 forms flesh pink/peach colored colonies, is non-motile, and the cells are present as diplococci, triads, tetracocci and aggregates. Strain FWC3 grows only on methane and methanol. As the 16S rRNA gene of strain FWC3 showed low similarities with other Type I methanotrophs (less than 94.3%), it was further investigated for its novelty and characterisation by a polyphasic approach. ANI indices and DDH values deduced from the draft genome of strain FWC3 (SEYW00000000.1) with the other nearest type strains (Methylocaldum marinum S8 T and Methylococcus capsulatus Bath T ) were ~ 70% and ~ 15%, respectively. The low level similarities indicated that strain FWC3 can belong to a new genus and species. Additionally, strain FWC3 showed a unique fatty acid profile with the dominance of C16:1 ω7 and ω6c, C16:0 and C16:1 ω9c. During the characterisation of strain FWC3, a morphologically similar methanotroph, strain C50C1 was described (Ghashghavi et al. in mSphere 4:e00631-18, 2019) and named as 'Methylotetracoccus oryzae'. We found that strain FWC3 and strain C50C1 belonged to the same genus but could belong to different species based on the ANI indices and dDDH values (~ 94% and ~ 55%, respectively). However, strain C50C1 has not been deposited in two culture collections and not been validly described. Also, the 16S rRNA gene of strain C50C1 is neither available on the database nor can it be retrieved from the genome assembly. Based on the polyphasic characterisation and comparison to the other type strains of Methylococcaceae, we propose strain FWC3 (= JCM 33786 T , = KCTC 72733 T , = MCC 4198 T ) to be the type strain of a novel genus and species, for which the name Methylolobus aquaticus is proposed. Strain C50C1 (Ghashghavi et al. 2019) could represent another species ('Methylolobus oryzae').

Members of the genus Methylobacter (Mtb) have been identified to be the most dominant methanotrophs in aquatic as well as terrestrial habitats. Methylobacter shows four species with validly published names and these are grouped in two clades based on phylogenetic and genomic comparisons. Mtb luteus and Mtb marinus (synonym: Mtb whittenburyi) belong to clade 1 Methylobacter. Clade 2 Methylobacter comprises of two species: Mtb tundripaludum and Mtb psychrophilus (type strain, no longer available). We isolated a yellow pigmented, rod-shaped methanotroph, strain (KRF1), from a tropical rice field in India, which might represent a putative novel species within Methylobacter clade 2. The cells are long, thick and motile rods. The strain grows under variable oxygen concentration (5-80% air) and also in nitrogen free media (5-50% air). The morphological, chemotaxonomic and genomic features of KRF1 were investigated in details. The digital DNA-DNA hybridization values and average nucleotide identity (ANI) comparisons with the members of its closest species, Mtb tundripaludum, were in the range of 20-26% and ~ 73-81%, respectively. The fatty acid methyl esters profile of KRF1 was different from the profile of Mtb tundripaludum SV96 T . The major cell wall fatty acids of strain KRF1 are 16:1 ω7c/16:1 ω6c summed feature (55.4%) and 16:1 ω5c (28.6%). The draft genome of KRF1 is of 5.04 Mbp in size with a GC content of 49.3% and the whole genome shotgun sequencing project has the accession number RYFG00000000 (version: RYFG02000000). Due to the difficulties in the maintenance and cryopreservation of this culture, it could not be deposited in two international culture collections. We thereby propose KRF1 to be member of a Candidatus species, 'Candidatus Methylobacter oryzae' KRF1. The culture is maintained live in our laboratory and also in our institutional WDCM approved culture collection (MACS Collection of Microorganisms) as MCMB-1471.

The bacterial oxidation of sulfur and methane is central to the biogeochemical processes in sediments such as the tropical mangrove sediments. However, there is a lacuna of information on the seasonal interactions including the influence of monsoons which is a major driver of seasonal change, on sulfur-oxidizing bacteria (SOB) and methane-oxidizing bacteria (MOB), their activity and the environmental variables. To understand these interactions, the analysis was carried out on sediment samples that were sampled monthly for a year from Chorao mangrove, Goa, southwest coast of India. SOB (3.8×105CFU g−1) and MOB (0.90×105CFU g−1) had maximum average abundance in the surface sediments in the post-monsoon and monsoon season, respectively. The mean sulfur-oxidation activity (SOA) of 2.63 mM day−1 and methane-oxidation activity (MOA) of 110.94 mM day−1 were highest in surface sediments during the post-monsoon season. Generally, the activity of SOB and MOB in surface sediments of post-monsoon was 2.2 times(×) and 2.8× respectively higher than that in the monsoon season. Among the environmental parameters analyzed, protein and sulfide concentrations significantly (p < 0.001) influenced SOA and MOA, respectively. There was a significant difference in SOA (p < 0.003) and MOA (p < 0.036) in surface sediments between the monsoon and the post-monsoon season. During the monsoon season, when the system is a sink of terrestrial/anthropogenic material, the interrelationship of SOB with MOA (r = 0.617, p < 0.001) and SOB with SOA (r = 0.489, p < 0.05) aids in maintaining the homeostasis of the system. [ABSTRACT FROM AUTHOR]

Methane oxidation by methanotrophs is a very important environmental process in the mitigation of methane. Methylobacter (Mtb.) clade 2 members have been reported as dominant methane oxidisers in soils and sediments worldwide. We enriched and purified a methanotroph from a tropical rice field soil sample from India. The highly enriched culture showed the presence of motile, long and thick rods (3-5 µm × 0.9-1.2 µm) and minor presence of short, thin rods. The culture was purified on agarose medium and formed yellow colonies which showed the presence of only thick and long rods, henceforth termed as strain KRF1. Based on 16S rRNA gene sequence analysis, strain KRF1 shows close phylogenetic affiliation to Methylobacter tundripaludum SV96 T (98.6% similarity). Due to the taxonomic novelty, and being the first member of Mtb. related to Mtb. tundripaludum from the tropics, the draft genome was sequenced. From the blastx analysis of the contigs, it was clear that the culture still had contamination of another organism, a Methylophilus species. The data binned in two clear bins: Mtb. related contigs and Methylophilus-related contigs. The binned draft genome of KRF1 shows features including the typical pathways for methane metabolism, denitrification and the presence of molybdenum iron and vanadium-iron nitrogenase genes. KRF1 is phylogenetically distinct from the five strains of Mtb. tundripaludum including SV96 T , Lake Washington strains and OWC strains, showing ~ 26% DDH and ~ 81% ANIb values and a unique position in a phylogenomic tree. Subsequently, KRF1 has been completely purified from its methylotrophic partner and a pure culture has been established and maintained in a WDCM approved culture collection, the MACS Collection of Microorganisms (as MCM 1471). KRF1 is thus the first cultured member of a putative novel species of Methylobacter clade 2 isolated from the tropics.

Candidatus Methylocucumis (Mcu.) oryzae, strain Sn10-6, a Gram-negative, pale pink pigmented, motile, large Type I methanotroph (3-6 µm × 1.5-2 µm) was recently isolated from an Indian rice field. Due to the difficulties in preservation of the strain it could not be deposited and preserved in two recognised culture collections with public access located in two different countries. Hence, Sn10-6 was described as a member of a Candidatus genus and species, 'Ca. Mcu. oryzae'. Subsequent to the publication, the strain has been preserved in the Japan Collection of Microorganisms, Japan (JCM 32869 T ) in addition to its earlier preservation in the Korean collection for type cultures, KCTC, Korea and in National Centre for Microbial Resources, India. Addition of DMSO as a cryopreserving agent proved to be useful in the preservation of the strain. The deposition of the strain in two recognised culture collections in two different countries and additionally in the home country repository makes the strain eligible to be proposed as the type strain of the type species of a new genus and species with the name Methylocucumis oryzae strain Sn10-6 T . In this paper we formally propose the name Mcu. oryzae, with Sn10-6 T as the type strain, for the earlier described Ca. Mcu. oryzae. Other characteristics including the digital protologue of the culture (CA00040) remain the same as described earlier for Ca. Mcu. oryzae (Pandit et al. in Antonie Van Leeuwenhoek 111:2473-2484, 2018). Mcu. oryzae Sn10-6 T (= MCC 3492 = KCTC 15683 = JCM 32869) represents a new genus and species within the family Methylococcaceae of the class Gammaproteobacteria.

An elliptical to cucumber-shaped methanotroph with large cells was isolated from a rice rhizosphere in Western India. Strain Sn10-6 is one of the first methanotrophs to be isolated from Indian rice fields. Cells of Sn10-6 are Gram-negative, motile, large in size (3-6 µm × 1.5-2 µm) and contain intracellular cytoplasmic membrane stacks. Colonies of Sn10-6 and liquid cultures have a pale pink colour. Strain Sn10-6 was initially isolated under micro-oxic conditions but later adapted to grow under fully oxic conditions. The major fatty acids present were identified as C 16:1 ω6c, C 16:1 ω7c and C 16:0 and ubiquinone was found to be the major quinone. The 16S rRNA gene sequence of strain Sn10-6 displays high similarity to the genes of Methylovulum psychrotolerans Sph1 T (93.6%) followed by Methylosarcina fibrata AML-C10 T (93.5%) and about 90-93% similarity to the genes of known species of Type I methanotrophic genera from the family Methylococcaceae. The draft genome information indicated that the G + C content of strain Sn10-6 is 43.9 mol%. Phylogenetic trees using 16S rRNA gene and the particulate methane mono-oxygenase sequences unequivocally placed Sn10-6 close to the genus Methylovulum. Based on the 16S rRNA gene differences, morphological characters and draft genome information, strain Sn10-6 (=MCC 3492 =KCTC 15683) is described here as the type strain of a novel species within a new genus, 'Candidatus Methylocucumis oryzae'.