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. (AU)

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.

Prevalence of Deltaproteobacterial sequences in nifH gene pools associated with the rhizosphere of native switchgrass from Tall Grass Prairie (Oklahoma, USA).

The potential nitrogen-fixing bacterial diversity in the rhizospheric soil of the native switchgrass (Panicum virgatum L.) from Tall Grass Prairies of Northern Oklahoma was studied using a partial region of nitrogenase structural gene-nifH. Eleven clone libraries constructed from nifH amplicons gave 407 good-quality sequences. More than 70% of sequences showed similarity of nifH with uncultured bacteria (< 98%). The dominance of sequences affiliated with Deltaproteobacterial nifH was observed, followed by Betaproteobacterial nifH sequences. The nifH gene library was dominated by the genera Geobacter, Rhizobacter, Paenibacillus, and Azoarcus. Sequences affiliated with rhizobia, such as Bradyrhizobium, Methylocystis, Ensifer, etc., were also in the rhizosphere in small numbers. From Deltaproteobacteria, five genera, namely Geobacter, Pelobacter, Geomonas, Desulfovibrio, and Anaeromyxobacter, contributed to 48% of the total sequences suggesting the dominance of group Deltaproteobacteria in the rhizosphere of native switchgrass. Considering the percent similarity of the nifH sequences with cultivated bacteria, this study demonstrated the presence of novel bacterial species in switchgrass rhizospheric soil from Tall Grass Prairie.

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.

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.

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.

Diversity of nitrogen-fixing bacteria associated with switchgrass in the native tallgrass prairie of northern Oklahoma.

Switchgrass (Panicum virgatum L.) is a perennial C4 grass native to North America that is being developed as a feedstock for cellulosic ethanol production. Industrial nitrogen fertilizers enhance switchgrass biomass production but add to production and environmental costs. A potential sustainable alternative source of nitrogen is biological nitrogen fixation. As a step in this direction, we studied the diversity of nitrogen-fixing bacteria (NFB) associated with native switchgrass plants from the tallgrass prairie of northern Oklahoma (United States), using a culture-independent approach. DNA sequences from the nitrogenase structural gene, nifH, revealed over 20 putative diazotrophs from the alpha-, beta-, delta-, and gammaproteobacteria and the firmicutes associated with roots and shoots of switchgrass. Alphaproteobacteria, especially rhizobia, predominated. Sequences derived from nifH RNA indicated expression of this gene in several bacteria of the alpha-, beta-, delta-, and gammaproteobacterial groups associated with roots. Prominent among these were Rhizobium and Methylobacterium species of the alphaproteobacteria, Burkholderia and Azoarcus species of the betaproteobacteria, and Desulfuromonas and Geobacter species of the deltaproteobacteria.

Elstera litoralis gen. nov., sp. nov., isolated from stone biofilms of Lake Constance, Germany.

An alphaproteobacterium, strain Dia-1(T), was isolated from algae-dominated biofilms on stones from the littoral zone of Lake Constance, Germany. This bacterium was isolated after initial enrichment in spent medium obtained after growth of a diatom culture. Numerous sugars and some organic acids and alcohols served as growth substrates. The bacterium grew slowly, was strictly aerobic but microaerophilic, and did not grow in cultures shaken under air. 16S rRNA gene sequence analysis indicated that strain Dia-1(T) was distantly related to representatives of the genera Azospirillum (90-91% sequence similarity), Skermanella (88-89%), Rhodocista (87-88%) and Dongia (88-89% sequence similarity). Based on this sequence comparison, on phenotypic characterization including substrate utilization patterns, and comparison of cellular fatty acids, quinones, polar lipids and polyamines, this isolate was found to be substantially different from the genera mentioned above. On the basis of these results, a novel genus and species is proposed for this strain. The name Elstera litoralis gen. nov., sp. nov. is suggested, with strain Dia-1(T) ( = DSM 19532(T) = LMG 24234(T)) as the type strain of the type species.

Bacteria associated with benthic diatoms from Lake Constance: phylogeny and influences on diatom growth and secretion of extracellular polymeric substances.

The composition of diatom-associated bacterial communities was studied with 14 different unialgal xenic diatom cultures isolated from freshwater epilithic biofilms of Lake Constance, Germany. A clear dominance of Alphaproteobacteria was observed, followed by Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. Pure cultures of the diatom Cymbella microcephala, which was found to be dominant in epilithic biofilms in Lake Constance, were cocultivated with six associated bacterial strains. All these bacterial strains were able to grow in C. microcephala cultures in the absence of organic cosubstrates. Diatom growth was generally enhanced in the presence of bacteria, and polysaccharide secretion was generally increased in the presence of Proteobacteria. The monomer composition of extracellular polysaccharides of C. microcephala changed in relation to the presence of different bacteria, but the dominant monomers were less affected. Our results indicate that these changes were caused by the diatom itself rather than by specific bacterial degradation. One Bacteroidetes strain strongly influenced carbohydrate secretion by the alga via extracellular soluble compounds. Biofilms were formed only in the presence of bacteria. Phylogenetic analysis and coculture studies indicate an adaptation of Proteobacteria and Bacteroidetes to the microenvironment created by the diatom biofilm.

THE COMPLEX EXTRACELLULAR POLYSACCHARIDES OF MAINLY CHAIN-FORMING FRESHWATER DIATOM SPECIES FROM EPILITHIC BIOFILMS(1).

Diatoms are dominant organisms in phototrophic biofilms in aquatic habitats. They produce copious amounts of extracellular polymeric substances (EPS), which mainly consist of carbohydrates and traces of proteins and glycoproteins. This study focuses on the characterization of EPS from a total of 14 diatoms belonging to the six genera Achnanthes, Cymbella, Fragilaria, Punctastriata, Staurosira, and Pseudostaurosira, all of which were isolated from epilithic biofilms of the littoral zone of Lake Constance. EPS from all isolates were extracted by a sequential extraction procedure resulting in five different fractions. The monosaccharide composition of each fraction was analyzed by HPLC equipped with a pulse amperiometric detector, yielding results similar to those obtained by probing the EPS structures with monomer-specific fluorophore-linked lectins. Significant differences in carbohydrate composition occurred in the different fractions of single isolates. Most of the diatom isolates in our study form chain-like colonies in which the cells are attached to each other by intercellular pads. Here we demonstrate that these pads can be dissolved in hot bicarbonate and that they show a heterogeneous composition of monosaccharides in contrast to other fractions, which mostly were dominated by one or two monosaccharides. Principal component analysis indicates a correlation between carbohydrate composition of EPS fractions and the phylogenetic relationship of the respective species, indicating that EPS analyses under defined culture conditions may support taxonomic analyses.

ISSR and AFLP analysis of the temporal and spatial population structure of the post-fire annual, Nicotiana attenuata, in SW Utah.

BACKGROUND: The native annual tobacco, Nicotiana attenuata, is found primarily in large ephemeral populations (typically for less than 3 growing seasons) after fires in sagebrush and pinyon-juniper ecosystems and in small persistent populations (for many growing seasons) in isolated washes typically along roadsides throughout the Great Basin Desert of the SW USA. This distribution pattern is due to its unusual germination behavior. Ephemeral populations are produced by the germination of dormant seeds from long-lived seed banks which are stimulated to germinate by a combination of unidentified positive cues found in wood smoke and the removal of inhibitors leached from the unburned litter of the dominant vegetation. Persistent populations may result where these inhibitors do not exist, as in washes or along disturbed roadsides. To determine if this germination behavior has influenced population structure, we conducted an AFLP (244 individuals), ISSR (175 individuals) and ISSR+ AFLP (175 individuals) analysis on plants originating from seed collected from populations growing in 11 wash and burns over 11 years from the SW USA. RESULTS: Genetic variance as measured by both ISSR and AFLP markers was low among sites and comparatively higher within populations. Cluster analysis of the Utah samples with samples collected from Arizona, California, and Oregon as out-groups also did not reveal patterns. AMOVA analysis of the combined AFLP and ISSR data sets yielded significantly low genetic differentiation among sites (Phict), moderate among populations within sites (Phisc) and higher genetic differentiation within populations (Phist). CONCLUSIONS: We conclude that the seed dormancy of this post-fire annual and its resulting age structure in conjunction with natural selection processes are responsible for significantly low among sites and comparatively high within-population genetic variation observed in this species.