Diverse type I and type II methanotrophs cultivated from an Indian freshwater wetland habitat.

Wetlands are the main natural sources of methane emissions, which make up a significant portion of greenhouse gas emissions. Such wetland patches serve as rich habitats for aerobic methanotrophs. Limited knowledge of methanotrophs from tropical wetlands widens the scope of study from these habitats. In the present study, a freshwater wetland in a tropical region in India was sampled and serially diluted to obtain methanotrophs in culture. This was followed by the isolation of methanotrophs on agarose-containing plates, incubated under methane: air atmosphere. Methanotrophs are difficult to cultivate, and very few cultures of methanotrophs are available from tropical wetlands. Our current study reports the cultivation of a diverse community of methanotrophs from six genera, namely, Methylomonas, Methylococcus, Methylomagnum, Methylocucumis (type I methanotrophs) along with Methylocystis, Methylosinus (type II methanotrophs). A high abundance of methanotrophs (106-1010 methanotrophs/g fresh weight) was observed in the samples. A Methylococcus strain could represent a putative novel species that was also isolated. Cultures of Methylomagnum and Methylocucumis, two newly described type I methanotrophs exclusively found in rice fields, were obtained. A large number of Methylomonas koyamae strains were cultured. Our study is pioneering in the documentation of culturable methanotrophs from a typical tropical wetland patch. The isolated methanotrophs can act as models for studying methanotroph-based methane mitigation from wetland habitats and can be used for various mitigation and valorization applications.

Thermotolerant methanotrophs belonging to the Methylocaldum genus dominate the methanotroph communities in biogas slurry and cattle dung: A culture-based study from India.

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.

Cultivation of Important Methanotrophs From Indian Rice Fields.

Methanotrophs are aerobic to micro-aerophilic bacteria, which oxidize and utilize methane, the second most important greenhouse gas. The community structure of the methanotrophs in rice fields worldwide has been studied mainly using culture-independent methods. Very few studies have focused on culturing methanotrophs from rice fields. We developed a unique method for the cultivation of methanotrophs from rice field samples. Here, we used a modified dilute nitrate mineral salts (dNMS) medium, with two cycles of dilution till extinction series cultivation with prolonged incubation time, and used agarose in the solid medium. The cultivation approach resulted in the isolation of methanotrophs from seven genera from the three major groups: Type Ia (Methylomonas, Methylomicrobium, and Methylocucumis), Type Ib (Methylocaldum and Methylomagnum), and Type II (Methylocystis and Methylosinus). Growth was obtained till 10-6-10-8 dilutions in the first dilution series, indicating the culturing of dominant methanotrophs. Our study was supported by 16S rRNA gene-based next-generation sequencing (NGS) of three of the rice samples. Our analyses and comparison with the global scenario suggested that the cultured members represented the major detected taxa. Strain RS1, representing a putative novel species of Methylomicrobium, was cultured; and the draft genome sequence was obtained. Genome analysis indicated that RS1 represented a new putative Methylomicrobium species. Methylomicrobium has been detected globally in rice fields as a dominant genus, although no Methylomicrobium strains have been isolated from rice fields worldwide. Ours is one of the first extensive studies on cultured methanotrophs from Indian rice fields focusing on the tropical region, and a unique method was developed. A total of 29 strains were obtained, which could be used as models for studying methane mitigation from rice fields and for environmental and biotechnological applications.

Lethal Pneumonia Cases in Mojiang Miners (2012) and the Mineshaft Could Provide Important Clues to the Origin of SARS-CoV-2.

With the COVID-19 pandemic reaching its worst heights, people are interested in the origin of SARS-CoV-2. This study started with two important questions: first, were there any similar atypical pneumonia outbreaks, even on a smaller level, reported between SARS in 2004 and COVID-19 in 2019/20 in China. Second, examining the beta-coronavirus most closely related to date with SARS-CoV-2 at the genome sequence level, strain RaTG13 (CoV4991), which was sampled from a horseshoe bat in Yunnan province, we asked where exactly did it come from. It was found that RaTG13/CoV4991 was collected from Tongguan mineshaft in Mojiang, Yunnan, China, in 2013. Surprisingly, the same mineshaft was also associated with a severe pneumonia-like illness in miners in 2012 killing three of the six miners. A Master's thesis (in the Chinese language) was found on the cnki.net website which described in detail the severe illness in miners. The thesis concluded that a SARS-like CoV originating from Chinese horseshoe bats (Rhinolophus) was the predicted causative agent. The cases were remotely monitored by a prominent pulmonologist in China. Retrospective analysis of the pneumonia cases shows striking similarities with COVID-19. Bilateral pneumonia, vascular complications like pulmonary thromboembolism, and secondary infections are the main similarities. The treatment regimes were similar to the current treatments for COVID-19. We propose that the Mojiang mineshaft miners' illness could provide important clues to the origin of SARS-CoV-2. These cases should be studied by various academicians, researchers, and medical professionals as many important questions are raised in this context.

A novel Type I methanotroph Methylolobus aquaticus gen. nov. sp. nov. isolated from a tropical wetland.

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 S8T and Methylococcus capsulatus BathT) 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 33786T, = KCTC 72733T, = MCC 4198T) 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').

Description of 'Ca. Methylobacter oryzae' KRF1, a novel species from the environmentally important Methylobacter clade 2.

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 SV96T. 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.

A putative novel Methylobacter member (KRF1) from the globally important Methylobacter clade 2: cultivation and salient draft genome features.

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 SV96T (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 SV96T, 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.

Renaming of 'Candidatus Methylocucumis oryzae' as Methylocucumis oryzae gen. nov., sp. nov., a novel Type I methanotroph isolated from India.

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 32869T) 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 Sn10-6T. In this paper we formally propose the name Mcu. oryzae, with Sn10-6T 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 Sn10-6T (= MCC 3492 = KCTC 15683 = JCM 32869) represents a new genus and species within the family Methylococcaceae of the class Gammaproteobacteria.

Description of 'Candidatus Methylocucumis oryzae', a novel Type I methanotroph with large cells and pale pink colour, isolated from an Indian rice field.

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 C16:1ω6c, C16:1ω7c and C16:0 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 Sph1T (93.6%) followed by Methylosarcina fibrata AML-C10T (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'.

Cultivated methanotrophs associated with rhizospheres of traditional rice landraces from Western India belong to Methylocaldum and Methylocystis.

Aerobic methanotrophs associated with Indian rice plants have rarely been cultivated. In the present study, we cultured aerobic methanotrophic bacteria from the rhizosphere regions of rice plants. Rhizospheric soils from seven rice landraces traditionally grown and maintained by tribal people in Jawhar region belonging to part of the Western Ghats in India, were used. Seven methanotrophic cultures were isolated from the last positive dilution (10- 4). Methanotrophs were identified by analyzing the partial methane monooxygenase gene, pmoA gene and three of these belonged to the genus Methylocaldum (gammaproteobacterial, Type I methanotrophs) and four belonged to the genus Methylocystis (alphaproteobacterial, Type II methanotrophs). We present here the first report on the cultivation of methanotrophs from Indian traditional rice landraces originating from a biodiversity hotspot.

A pmoA-based study reveals dominance of yet uncultured Type I methanotrophs in rhizospheres of an organically fertilized rice field in India.

Rice fields are one of the important sources of anthropogenic methane. Methanotrophs can oxidize up to 30 % of the produced methane and thus have a pivotal environmental role in methane mitigation. India occupies the largest region under rice cultivation; however, most of the studies done on methanotrophic communities have focused on the Northern region. We studied methanotrophic community of a flooded, organically fertilized rice field using pmoA clone library approach. Organic rice fields impose a more serious threat as they produce more methane. pmoA gene is the main functional gene which is primarily used for taxonomical analysis of methanotrophs. Our results showed that the pmoA clone libraries from two growth stages of rice were dominated by pmoA sequences which were very distant from cultivated Type Ia methanotrophic genera (80-82 % nucleotide similarity) indicative of the presence of a putatively novel genus. We designated this group of clones as 'rice field clones' as this also includes many pmoA sequences originating from other rice fields. Thus, our current knowledge of methanotroph diversity from Indian rice fields has been expanded revealing that a substantial portion of methanotrophic diversity is unexplored.

Genome Characteristics of a Novel Type I Methanotroph (Sn10-6) Isolated from a Flooded Indian Rice Field.

Flooded rice fields are important sources of atmospheric methane. Aerobic methanotrophs living in the vicinity of rice roots oxidize methane and act as environmental filters. Here, we present genome characteristics of a gammaproteobacterial methanotroph, isolate Sn10-6, which was isolated from a rice rhizosphere of a flooded field in India. Sn10-6 has been identified as a member of a putative novel genus and species within the family Methylococcaceae (Type I methanotrophs). The draft genome of Sn10-6 showed pathways for the following: methane oxidation, formaldehyde assimilation (RuMP), nitrogen fixation, conversion of nitrite to nitrous oxide, and other interesting genes including the ones responsible for survival in the rhizosphere environment. The majority of genes found in this genome were most similar to Methylovulum miyakonese which is a forest isolate. This draft genome provided insight into the physiology, ecology, and phylogeny of this gammaproteobacterial methanotroph.

Deciphering Community Structure of Methanotrophs Dwelling in Rice Rhizospheres of an Indian Rice Field Using Cultivation and Cultivation-Independent Approaches.

Methanotrophs play a crucial role in filtering out methane from habitats, such as flooded rice fields. India has the largest area under rice cultivation in the world; however, to the best of our knowledge, methanotrophs have not been isolated and characterized from Indian rice fields. A cultivation strategy composing of a modified medium, longer incubation time, and serial dilutions in microtiter plates was used to cultivate methanotrophs from a rice rhizosphere sample from a flooded rice field in Western India. We compared the cultured members with the uncultured community as revealed by three culture-independent methods. A novel type Ia methanotroph (Sn10-6), at the rank of a genus, and a putative novel species of a type II methanotroph (Sn-Cys) were cultivated from the terminal positive dilution (10(-6)). From lower dilution (10(-4)), a strain of Methylomonas spp. was cultivated. All the three culture-independent analyses, i.e., pmoA clone library, terminal restriction fragment length polymorphism (T-RFLP), and metagenomics approach, revealed the dominance of type I methanotrophs. Only metagenomic analysis showed significant presence of type II methanotrophs, albeit in lower proportion (37 %). All the three isolates showed relevance to the methanotrophic community as depicted by uncultured methods; however, the cultivated members might not be the most dominant ones. In conclusion, a combined cultivation and cultivation-independent strategy yielded us a broader picture of the methanotrophic community from rice rhizospheres of a flooded rice field in India.

Draft genome of Elstera litoralis, a freshwater epilithic biofilm associated bacterium from littoral zone of Lake Constance.

Elstera litoralis, is a Rhodospirillaceae member which was isolated from the littoral zone of Lake Constance from a stone biofilm using diatom extracellular polymeric substances (EPS) as C source. We present here the draft genome of E. litoralis which has a genome size of 3.83 Mb and 61.2% G+C content. Genome analysis indicated utilization of multiple C substrates explaining its heterotrophic lifestyle as a bacterium present in natural biofilms. Further comparative genome analysis of Elstera with other members of Rhodospirillaceae would be helpful to understand the evolutionary relationships and divergence of hydrobacteria from terrabacteria.