Large freshwater phages with the potential to augment aerobic methane oxidation.

There is growing evidence that phages with unusually large genomes are common across various microbiomes, but little is known about their genetic inventories or potential ecosystem impacts. In the present study, we reconstructed large phage genomes from freshwater lakes known to contain bacteria that oxidize methane. Of manually curated genomes, 22 (18 are complete), ranging from 159 kilobase (kb) to 527 kb in length, were found to encode the pmoC gene, an enzymatically critical subunit of the particulate methane monooxygenase, the predominant methane oxidation catalyst in nature. The phage-associated PmoC sequences show high similarity to (>90%), and affiliate phylogenetically with, those of coexisting bacterial methanotrophs, including members of Methyloparacoccus, Methylocystis and Methylobacter spp. In addition, pmoC-phage abundance patterns correlate with those of the coexisting bacterial methanotrophs, supporting host-phage relationships. Future work is needed to determine whether phage-associated PmoC has similar functions to additional copies of PmoC encoded in bacterial genomes, thus contributing to growth on methane. Transcriptomics data from Lake Rotsee (Switzerland) showed that some phage-associated pmoC genes were highly expressed in situ and, of interest, that the most rapidly growing methanotroph was infected by three pmoC-phages. Thus, augmentation of bacterial methane oxidation by pmoC-phages during infection could modulate the efflux of this potent greenhouse gas into the environment.

A Novel Cold-adapted Methylovulum species, with a High C16:1ω5c Content, Isolated from an Arctic Thermal Spring in Spitsbergen.

A novel cold-adapted methane-oxidizing bacterium, termed TFB, was isolated from the thermoglacial Arctic karst spring, Trollosen, located in the South Spitsbergen National Park (Norway). The source water is cold and extremely low in phosphate and nitrate. The isolate belongs to the Methylovulum genus of gammaproteobacterial methanotrophs, with the closest phylogenetic affiliation with Methylovulum miyakonense and Methylovulum psychrotolerans (96.2 and 96.1% 16S rRNA gene sequence similarities, respectively). TFB is a strict aerobe that only grows in the presence of methane or methanol. It fixes atmospheric nitrogen and contains Type I intracellular membranes. The growth temperature range was 2-22°C, with an optimum at 13-18°C. The functional genes pmoA, mxaF, and nifH were identified by PCR, whereas mmoX and cbbL were not. C16:1ω5c was identified as the major fatty acid constituent, at an amount (>49%) not previously found in any methanotrophs, and is likely to play a major role in cold adaptation. Strain TFB may be regarded as a new psychrotolerant or psychrophilic species within the genus Methylovulum. The recovery of this cold-adapted bacterium from a neutral Arctic thermal spring increases our knowledge of the diversity and adaptation of extremophilic gammaproteobacterial methanotrophs in the candidate family "Methylomonadaceae".

Methylicorpusculum oleiharenae gen. nov., sp. nov., an aerobic methanotroph isolated from an oil sands tailings pond.

An aerobic methane oxidizing bacterium, designated XLMV4T, was isolated from the oxic surface layer of an oil sands tailings pond in Alberta, Canada. Strain XLMV4T is capable of growth on methane and methanol as energy sources. NH4Cl and sodium nitrate are nitrogen sources. Cells are Gram-negative, beige to yellow-pigmented, motile (via a single polar flagellum), short rods 2.0-3.3 µm in length and 1.0-1.6 µm in width. A thick capsule is produced. Surface glycoprotein or cup shape proteins typical of the genera Methylococcus, Methylothermus and Methylomicrobium were not observed. Major isoprenoid quinones are Q-8 and Q-7 at an approximate molar ratio of 71 : 22. Major polar lipids are phosphoglycerol and ornithine lipids. Major fatty acids are C16 : 1 ω8+C16 : 1 ω7 (34 %), C16 : 1 ω5 (16 %), and C18 : 1 ω7 (11 %). Optimum growth is observed at pH 8.0 and 25 °C. The DNA G+C content based on a draft genome sequence is 46.7 mol%. Phylogenetic analysis of 16S rRNA genes and a larger set of conserved genes place strain XLMV4T within the class Gammaproteobacteria and family Methylococcaceae, most closely related to members of the genera Methylomicrobium and Methylobacter (95.0-97.1 % 16S rRNA gene sequence identity). In silico genomic predictions of DNA-DNA hybridization values of strain XLMV4T to the nearest phylogenetic neighbours were all below 26 %. On the basis of the data presented, strain XLMV4T is considered to represent a new genus and species for which the name Methylicorpusculum oleiharenae is proposed. Strain XLMV4T (=DSMZ DSM 27269=ATCC TSD-186) is the type strain.

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.

Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification. This study was aimed at characterizing the biochemical properties of the malic enzyme (Mae) from the halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z. The His6-tagged Mae was obtained by heterologous expression in Escherichia coli BL21 (DE3) and purified by affinity metal chelating chromatography. As determined by gel filtration and non-denaturating gradient gel electrophoresis, the molecular mass of the native enzyme is 260 kDa. The homotetrameric Mae (65x4 kDa) catalyzed an irreversible NAD+-dependent reaction of L-malate decarboxylation into pyruvate with a specific activity of 32 ± 2 units mg-1 and Km value of 5.5 ± 0.8 mM for malate and 57 ± 5 µM for NAD+. The disruption of the mae gene by insertion mutagenesis resulted in a 20-fold increase in intracellular malate level in the mutant compared to the wild type strain. Based on both enzyme and mutant properties, we conclude that the malic enzyme is involved in the control of intracellular L-malate level in Mtm. alcaliphilum 20Z. Genomic analysis has revealed that Maes present in methanotrophs fall into two different clades in the amino acid-based phylogenetic tree, but no correlation of the division with taxonomic affiliations of the host bacteria was observed.

Methylotetracoccus oryzae Strain C50C1 Is a Novel Type Ib Gammaproteobacterial Methanotroph Adapted to Freshwater Environments.

Methane-oxidizing microorganisms perform an important role in reducing emissions of the greenhouse gas methane to the atmosphere. To date, known bacterial methanotrophs belong to the Proteobacteria, Verrucomicrobia, and NC10 phyla. Within the Proteobacteria phylum, they can be divided into type Ia, type Ib, and type II methanotrophs. Type Ia and type II are well represented by isolates. Contrastingly, the vast majority of type Ib methanotrophs have not been able to be cultivated so far. Here, we compared the distributions of type Ib lineages in different environments. Whereas the cultivated type Ib methanotrophs (Methylococcus and Methylocaldum) are found in landfill and upland soils, lineages that are not represented by isolates are mostly dominant in freshwater environments, such as paddy fields and lake sediments. Thus, we observed a clear niche differentiation within type Ib methanotrophs. Our subsequent isolation attempts resulted in obtaining a pure culture of a novel type Ib methanotroph, tentatively named "Methylotetracoccus oryzae" C50C1. Strain C50C1 was further characterized to be an obligate methanotroph, containing C16:1ω9c as the major membrane phospholipid fatty acid, which has not been found in other methanotrophs. Genome analysis of strain C50C1 showed the presence of two pmoCAB operon copies and XoxF5-type methanol dehydrogenase in addition to MxaFI. The genome also contained genes involved in nitrogen and sulfur cycling, but it remains to be demonstrated if and how these help this type Ib methanotroph to adapt to fluctuating environmental conditions in freshwater ecosystems.IMPORTANCE Most of the methane produced on our planet gets naturally oxidized by a group of methanotrophic microorganisms before it reaches the atmosphere. These microorganisms are able to oxidize methane, both aerobically and anaerobically, and use it as their sole energy source. Although methanotrophs have been studied for more than a century, there are still many unknown and uncultivated groups prevalent in various ecosystems. This study focused on the diversity and adaptation of aerobic methane-oxidizing bacteria in different environments by comparing their phenotypic and genotypic properties. We used lab-scale microcosms to create a countergradient of oxygen and methane for preenrichment, followed by classical isolation techniques to obtain methane-oxidizing bacteria from a freshwater environment. This resulted in the discovery and isolation of a novel methanotroph with interesting physiological and genomic properties that could possibly make this bacterium able to cope with fluctuating environmental conditions.

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.

Key Physiology of a Nitrite-Dependent Methane-Oxidizing Enrichment Culture.

Nitrite-dependent methane-oxidizing bacteria couple the reduction of nitrite to the oxidation of methane via a unique oxygen-producing pathway. This process is carried out by members of the genus Methylomirabilis that belong to the NC10 phylum. Contrary to other known anaerobic methane oxidizers, they do not employ the reverse methanogenesis pathway for methane activation but instead a canonical particulate methane monooxygenase similar to those used by aerobic methanotrophs. Methylomirabilis-like bacteria are detected in many natural and manmade ecosystems, but their physiology is not well understood. Here, using continuous cultivation techniques, batch activity assays, and state-of-the-art membrane-inlet mass spectrometry, we determined growth rate, doubling time, and methane and nitrite affinities of the nitrite-dependent methane-oxidizing bacterium "Candidatus Methylomirabilis lanthanidiphila." Our results provide insight into understanding the interactions of these microorganisms with methanotrophs and other nitrite-reducing microorganisms, such as anaerobic ammonium-oxidizing bacteria. Furthermore, our data can be used in modeling studies as well as wastewater treatment plant design.IMPORTANCE Methane is an important greenhouse gas with a radiative forcing 28 times that of carbon dioxide over a 100-year time scale. The emission of methane to the atmosphere is controlled by aerobic and anaerobic methanotrophs, which are microorganisms that are able to oxidize methane to conserve energy. While aerobic methanotrophs have been studied for over a century, knowledge on the physiological characteristics of anaerobic methanotrophs is scarce. Here, we describe kinetic properties of "Candidatus Methylomirabilis lanthanidiphila," a nitrite-dependent methane-oxidizing microorganism, which is ecologically important and can be applied in wastewater treatment.

Methylococcaceae are the dominant active aerobic methanotrophs in a Chinese tidal marsh.

Although coastal marshes are net carbon sinks, they are net methane sources. Aerobic methanotrophs in coastal marsh soils are important methane consumers, but their activity and populations are poorly characterized. DNA stable-isotope probing followed by sequencing was used to determine how active methanotrophic populations differed in the main habitats of a Chinese coastal marsh. These habitats included mudflat, native plant-dominated, and alien plant-dominated habitats. Methylococcaceae was the most active methanotroph family across four habitats. Abundant methylotroph sequences, including methanotrophs and non-methane-oxidizing methylotrophs (Methylotenera and Methylophaga), constituted 50-70% of the 16S rRNA genes detected in the labeled native plant-dominated and mudflat soils. Methylotrophs were less abundant (~ 20%) in labeled alien plant-dominated soil, suggesting less methane assimilation into the target community or a different extent of carbon cross-feeding. Canonical correspondence analysis indicated a significant correlation between the active bacterial communities and soil properties (salinity, organic carbon, total nitrogen, pH, and available phosphorus). Importantly, these results highlight how changing vegetation or soil features in coastal marshes may change their resident active methanotrophic populations, which will further influence methane cycling.

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.

Upland soil cluster γ dominates the methanotroph communities in the karst Heshang Cave.

Microorganisms are thought to play a critical role in methane (CH4) consumption in karst caves and yet the presence and diversity of methane-oxidizing bacteria (MOB) remain a mystery. In Heshang Cave, CH4 concentration decreases from 1.9 ppm at the entrance to 0.65 ppm inside the cave. To explore the presence and diversity of MOB in this cave, weathered rocks and sediment samples were collected from the cave and subjected to molecular analysis. The abundances of MOB were 107-108 copies g-1 dry sample via quantification of the pmoA gene, which are comparable to or even higher than those reported in other terrestrial environments, and account for up to 20% of the total microbial communities. Phylogenetically, MOB communities were dominated by the 'high-affinity' upland soil cluster γ (USCγ), although the predominance of Type Ia MOB was also detected in the permanently waterlogged stream sediment. The estimated CH4 oxidation potential varied dramatically among samples in the range of 0.6-80 CH4 m-3 d-1. Collectively, this study provides compelling evidence that the high-affinity MOB capable of oxidizing CH4 at the atmospheric level are present in Heshang Cave, which may play an important role in the CH4 consumption, and supports karst caves as important atmospheric CH4 sinks.

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'.

[Effects of Gypsum on CH4 Emission and Functional Microbial Communities in Paddy Soil].

In this study, the effects of gypsum (FGD) on CH4 emission and functional microbial community in paddy soil were identified under five treatments, including FGDG0(0 t·hm-2), FGDG1(2 t·hm-2), FGDG2(4 t·hm-2), FGDG3(8 t·hm-2), and FGDG4(16 t·hm-2). The methane flux was determined using static chamber and chromatography. Bacterial community structure and its effect on soil bacterial community structure, and the abundance of methanogenic and methanotrophs were measured via high-throughput sequencing and quantitative PCR. The results showed that after treatment with desulfurated gypsum, pH of the soil increased significantly (P<0.05). Redox potential, organic carbon, and available potassium content increased, with no significant difference (P>0.05). The average emission flux of CH4 reduced with the increase of desulfurated gypsum content, following the following trend:FGDG1 > FGDG2 > FGDG3 > FGDG4. They decreased by 31.56%, 57.30%, 83.60%, and 90.66%, respectively, compared with the control. Compared with the control, FGDG1 and FGDG2 increased the richness and variety of soil bacteria. However, when the application amount exceeds 4 t·hm-2, the richness and variety of soil bacteria decrease. Compared with the control, the relative abundance of sulfate-reducing bacteria in paddy soil increased significantly by 6.98%-13.56%. The abundance of the methane-oxidizing bacteria pmoA gene increased by 0.3%-6.2%. The abundance of the methanogen gene, mrcA decreased significantly by 2.4%-15.8%, while the abundance ratio (pmoA/mcrA)increased with the increase of the amount of desulfurated gypsum. Correlation analysis showed that the average emission of CH4 was markedly negatively correlated with the relative abundance of the sulfate-reducing bacteria and pmoA/mcrA percentage in soil, and significantly positively correlated with methanogenic gene, mcrA. In summary, desulfurated gypsum can improve the diversity of bacterial communities and reduce the emission of CH4 in the paddy soils.

The characteristics and comparative analysis of methanotrophs reveal genomic insights into Methylomicrobium sp. enriched from marine sediments.

Methanotrophic bacteria are widespread and use methane as a sole carbon and energy source. They also play a crucial role in marine ecosystems by preventing the escape of methane into the atmosphere from diverse methane sources, such as methane seeps and hydrothermal vents. Despite their importance for methane carbon cycling, relatively few marine methanotrophic bacteria have been isolated and studied at the genomic level. Herein, we report the genome of a marine methanotrophic member of the genus Methylomicrobium, metagenome-assembled genome (MAG) wino1, which was obtained through enrichment using methane as the sole carbon source. Phylogenetic analysis based on 16S rRNA sequences and comparison of pmoA genes supported the close relationship of MAG-wino1 to the genus Methylomicrobium and it possessed a genome of 5.06 Mb encoding many specialized methanotrophic genes. A comparison of MAG-wino1 with the genomes of other strains (Methylomicrobium alcaliphilum 20ZT and Methylomicrobium buryatense 5G) showed that genes (e.g. ectABC, ask, and mscLS) involved in the accumulation of compatible solutes required for survival in marine environments might be conserved. Methane utilization genes, including methanol dehydrogenase, and key enzymes related to ribulose monophosphate (RuMP) metabolism were identified. The wino1 genome harbored nitrogen fixation, urease, urea and nitrate transporter genes involved in the exploitation of nitrogen sources. Poly-ß-hydroxybutyrate degradation and glycogen synthesis-related genes may facilitate survival under nutrient-limiting conditions. Additionally, genome analysis revealed three dominant taxa in the enrichment culture, methanotroph Methylomicrobium sp., methylotroph Methyloceanibacter sp., and non-methylotroph Labrenzia sp., which provided insights into microbial associations in marine sediments.

Activity and diversity of methane-oxidizing bacteria along a Norwegian sub-Arctic glacier forefield.

Methane (CH4) is one of the most abundant greenhouse gases in the atmosphere and identification of its sources and sinks is crucial for the reliability of climate model outputs. Although CH4 production and consumption rates have been reported from a broad spectrum of environments, data obtained from glacier forefields are restricted to a few locations. We report the activities of methanotrophic communities and their diversity along a chronosequence in front of a sub-Arctic glacier using high-throughput sequencing and gas flux measurements. CH4 oxidation rates were measured in the field throughout the growing season during three sampling times at eight different sampling points in combination with laboratory incubation experiments. The overall results showed that the methanotrophic community had similar trends of increased CH4 consumption and increased abundance as a function of soil development and time of year. Sequencing results revealed that the methanotrophic community was dominated by a few OTUs and that a short-term increase in CH4 concentration, as performed in the field measurements, altered slightly the relative abundance of the OTUs.

Molecular and stable isotopic evidence for the occurrence of nitrite-dependent anaerobic methane-oxidizing bacteria in the mangrove sediment of Zhangjiang Estuary, China.

Nitrite-dependent anaerobic methane oxidation (n-damo), which is mediated by "Candidatus Methylomirabilis oxyfera-like" bacteria, is unique in linking the carbon and nitrogen cycles. However, the niche and activity of n-damo bacteria in the mangrove ecosystem have not been confirmed. Here, we report the occurrence of the n-damo process in the mangrove wetland of the Zhangjiang Estuary, China. The widespread occurrence of n-damo bacteria in mangrove wetland was confirmed using real-time quantitative polymerase chain reaction (qPCR) assay, which showed that the abundance of Methylomirabilis oxyfera-like bacterial 16S rRNA and pmoA genes ranged from 2.43 × 106 to 2.09 × 107 and 2.07 × 106 to 3.38 × 107copies per gram of dry soil in the examined sediment cores. The highest amount of targeting genes was all detected in the upper layer (0-20 cm). Phylogenetic analyses of n-damo bacterial 16S rRNA and pmoA genes illustrated the depth-specific distribution and high diversity of n-damo bacteria in the mangrove wetland. Stable isotope experiments further confirmed the occurrence of n-damo in the examined mangrove sediments, and the potential n-damo rates ranged from 25.93 to 704.08 nmol CO2 per gram of dry soil per day at different depths of the sediment cores, with the n-damo being more active in the upper layer of the mangrove sediments. These results illustrate the existence of active M. oxyfera-like bacteria and indicate that the n-damo process is a previously overlooked microbial methane sink in the mangrove wetlands.

Aerobic and anaerobic methanotrophic communities in urban landscape wetland.

Both aerobic methane-oxidizing bacteria (MOB) and nitrite-dependent anaerobic methane oxidation (n-damo) organisms can be important methane sinks in a wetland. However, the influences of the vegetation type on aerobic MOB and n-damo communities in wetland, especially in constructed wetland, remain poorly understood. The present study investigated the influences of the vegetation type on both aerobic MOB and n-damo organisms in a constructed urban landscape wetland. Sediments were collected from eight sites vegetated with different plant species. The abundance (1.19-3.27 × 107 pmoA gene copies per gram dry sediment), richness (Chao1 estimator = 16.3-81.5), diversity (Shannon index = 2.10-3.15), and structure of the sediment aerobic MOB community were found to vary considerably with sampling site. In contrast, n-damo community abundance (8.74 × 105-4.80 × 106 NC10 16S rRNA gene copies per gram dry sediment) changed slightly with the sampling site. The richness (Chao1 estimator = 1-11), diversity (Shannon index = 0-0.78), and structure of the NC10 16S rRNA gene-based n-damo community illustrated slight site-related changes, while the spatial changes of the pmoA gene-based n-damo community richness (Chao1 estimator = 1-8), diversity (Shannon index = 0-0.99), and structure were considerable. The vegetation type could have a profound impact on the wetland aerobic MOB community and had a stronger influence on the pmoA-based n-damo community than on the NC10 16S-based one in urban wetland. Moreover, the aerobic MOB community had greater abundance and higher richness and diversity than the n-damo community. Methylocystis (type II MOB) predominated in urban wetland, while no known type I MOB species was detected. In addition, the ratio of total organic carbon to total nitrogen (C/N) might be a determinant of sediment n-damo community diversity and aerobic MOB richness.

Diversity and potential activity of methanotrophs in high methane-emitting permafrost thaw ponds.

Lakes and ponds derived from thawing permafrost are strong emitters of carbon dioxide and methane to the atmosphere, but little is known about the methane oxidation processes in these waters. Here we investigated the distribution and potential activity of aerobic methanotrophic bacteria in thaw ponds in two types of eroding permafrost landscapes in subarctic Québec: peatlands and mineral soils. We hypothesized that methanotrophic community composition and potential activity differ regionally as a function of the landscape type and permafrost degradation stage, and locally as a function of depth-dependent oxygen conditions. Our analysis of pmoA transcripts by Illumina amplicon sequencing and quantitative PCR showed that the communities were composed of diverse and potentially active lineages. Type I methanotrophs, particularly Methylobacter, dominated all communities, however there was a clear taxonomic separation between the two landscape types, consistent with environmental control of community structure. In contrast, methanotrophic potential activity, measured by pmoA transcript concentrations, did not vary with landscape type, but correlated with conductivity, phosphorus and total suspended solids. Methanotrophic potential activity was also detected in low-oxygen bottom waters, where it was inversely correlated with methane concentrations, suggesting methane depletion by methanotrophs. Methanotrophs were present and potentially active throughout the water column regardless of oxygen concentration, and may therefore be resilient to future mixing and oxygenation regimes in the warming subarctic.

Characterization of the first rice paddy cluster I isolate, Methyloterricola oryzae gen. nov., sp. nov. and amended description of Methylomagnum ishizawai.

Three gammaproteobacterial methanotrophic strains (73aT, 175 and 114) were isolated from stems of rice plants. All strains are Gram-negative, motile and grow on methane or methanol as sole carbon sources. They oxidize methane using the particulate methane monooxygenase. Strains 114 and 175 possess additionally a soluble methane monooxygenase. All strains contain significant amounts of the cellular fatty acids C16 : 0, C16 : 1ω6c and C16 : 1ω7c, typical for type Ib methanotrophs. Characteristic for strains 114 and 175 are high amounts of C14 : 0 and C16 : 1ω6c , while strain 73aT contains high quantities of C16 : 1ω5c. 16S rRNA gene sequence analyses showed that strains 114 and 175 are most closely related to Methylomagnum ishizawai (≥99.6 % sequence identity). Strain 73aT is representing a new genus within the family Methylococcaceae, most closely related to Methylococcus capsulatus (94.3 % sequence identity). Phylogenetic analysis of the PmoA sequence indicates that strain 73aT represents rice paddy cluster I (RPCI), which has almost exclusively been detected in rice ecosystems. The G+C content of strain 73aT is 61.0 mol%, while strains 114 and 175 have a G+C content of 63.3 mol%. Strain 73aT (=LMG 29185T, =VKM B-2986T) represents the type strain of a novel species and genus, for which the name Methyloterricola oryzae gen. nov., sp. nov. is proposed and a description is provided. Strains 175 (=LMG 28717, VKM B-2989) and 114 are members of the species Methylomagnum ishizawai. This genus was so far only represented by one isolate, so an amended description of the species is given.