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.

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.