Methylocystis silviterrae sp.nov., a high-affinity methanotrophic bacterium isolated from the boreal forest soil.

A novel species is proposed for a high-affinity methanotrophic representative of the genus Methylocystis. Strain FST was isolated from a weakly acidic (pH 5.3) mixed forest soil of the southern Moscow area. Cells of FST are aerobic, Gram-negative, non-motile, curved coccoids or short rods that contain an intracytoplasmic membrane system typical of type-II methanotrophs. Only methane and methanol are used as carbon sources. FST grew at a temperature range of 4-37 °C (optimum 25-30 °C) and a pH range of 4.5 to 7.5 (optimum pH 6.0-6.5). The major fatty acids were C18  :  1ω8c, C18  :  1ω7c and C18  :  0; the major quinone as Q-8. FST displays 16S rRNA gene sequences similarity to other taxonomically recognized members of the genus Methylocystis, with Methylocystis hirsuta CSC1T (99.6 % similarity) and Methylocystis rosea SV97T (99.3 % similarity) as its closest relatives. The genome comprises 3.85 Mbp and has a DNA G+C content of 62.6 mol%. Genomic analyses and DNA-DNA relatedness with genome-sequenced members of the genus Methylocystis demonstrated that FST could be separated from its closest relatives. FST possesses two particulate methane monooxygenases (pMMO): low-affinity pMMO1 and high-affinity pMMO2. In laboratory experiments, it was demonstrated that FST might oxidize methane at atmospheric concentration. The genome contained various genes for nitrogen fixation, polyhydroxybutyrate synthesis, antibiotic resistance and detoxification of arsenic, cyanide and mercury. On the basis of genotypic, phenotypic and chemotaxonomic characteristics, it is proposed that the isolate represents a novel species, Methylocystis silviterrae sp. nov. The type strain is FST (=KCTC 82935T=VKM B-3535T).

Chthonobacter rhizosphaerae sp. nov., a bacterium isolated from rhizosphere soil of Citrus sinenesis.

A Gram-staining negative, facultative anaerobic, motile and short rod-shaped bacterium, designated strain yh7-1T, was isolated from rhizosphere soil of Citrus sinenesis collected from the garden of Citrus sinenesis in Ailao Mountain, south-west China. Cells grew at 15-45 °C, pH 5.0-9.0 and were able to tolerate up to 1% (w/v) NaCl on R2A medium. The respiratory lipoquinone was Q-10 and the major cellular fatty acids contained summed feature 8 (C18:1 ω7c or C18:1 ω6c) and C18:0. Polar lipids in the cellular membrane were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and one unidentified aminophospholipid. The genomic DNA G+C content was 69.9 mol%. On basis of 16S rRNA gene sequence analysis, strain yh7-1T showed the highest similarities with Chthonobacter albigriseus KCTC 42450T (97.6%), Mongoliimonas terrestris KCTC 42635T (97.0%) and lower than 97.0% to other species. Phylogenetic trees based on 16S rRNA gene sequences indicated that strain yh7-1T clustered with C. albigriseus KCTC 42450T. The ANI values ranged between 78.1 and 82.7% for C. albigriseus KCTC 42450T, M. terrestris KCTC 42635T and strain yh7-1T, which were lower than the prokaryotic species delineation threshold of 95.0-96.0%. The digital DNA-DNA hybridization values between C. albigriseus KCTC 42450T, M. terrestris KCTC 42635T and strain yh7-1T indicated that the new isolate represents a novel genomic species. According to the phenotypic and genotypic characteristics, strain yh7-1T should belong to the genus Chthonobacter, for which the name Chthonobacter rhizosphaerae sp. nov. (type strain yh7-1T = CGMCC 1.17236T = CCTCC AB 2019258T = KCTC 82185T) is proposed.

Shifts in methanogenic archaea communities and methane dynamics along a subtropical estuarine land use gradient.

In coastal aquatic ecosystems, prokaryotic communities play an important role in regulating the cycling of nutrients and greenhouse gases. In the coastal zone, estuaries are complex and delicately balanced systems containing a multitude of specific ecological niches for resident microbes. Anthropogenic influences (i.e. urban, industrial and agricultural land uses) along the estuarine continuum can invoke physical and biochemical changes that impact these niches. In this study, we investigate the relative abundance of methanogenic archaea and other prokaryotic communities, distributed along a land use gradient in the subtropical Burnett River Estuary, situated within the Great Barrier Reef catchment, Australia. Microbiological assemblages were compared to physicochemical, nutrient and greenhouse gas distributions in both pore and surface water. Pore water samples from within the most urbanised site showed a high relative abundance of methanogenic Euryarchaeota (7.8% of all detected prokaryotes), which coincided with elevated methane concentrations in the water column, ranging from 0.51 to 0.68 µM at the urban and sewage treatment plant (STP) sites, respectively. These sites also featured elevated dissolved organic carbon (DOC) concentrations (0.66 to 1.16 mM), potentially fuelling methanogenesis. At the upstream freshwater site, both methane and DOC concentrations were considerably higher (2.68 µM and 1.8 mM respectively) than at the estuarine sites (0.02 to 0.66 µM and 0.39 to 1.16 mM respectively) and corresponded to the highest relative abundance of methanotrophic bacteria. The proportion of sulfate reducing bacteria in the prokaryotic community was elevated within the urban and STP sites (relative abundances of 8.0%- 10.5%), consistent with electron acceptors with higher redox potentials (e.g. O2, NO3-) being scarce. Overall, this study showed that ecological niches in anthropogenically altered environments appear to give an advantage to specialized prokaryotes invoking a potential change in the thermodynamic landscape of the ecosystem and in turn facilitating the generation of methane-a potent greenhouse gas.

Atmospheric Methane Oxidizers Are Dominated by Upland Soil Cluster Alpha in 20 Forest Soils of China.

Upland soil clusters alpha and gamma (USCα and USCγ) are considered a major biological sink of atmospheric methane and are often detected in forest and grassland soils. These clusters are phylogenetically classified using the particulate methane monooxygenase gene pmoA because of the difficulty of cultivation. Recent studies have established a direct link of pmoA genes to 16S rRNA genes based on their isolated strain or draft genomes. However, whether the results of pmoA-based assays could be largely represented by 16S rRNA gene sequencing in upland soils remains unclear. In this study, we collected 20 forest soils across China and compared methane-oxidizing bacterial (MOB) communities by high-throughput sequencing of 16S rRNA and pmoA genes using different primer sets. The results showed that 16S rRNA gene sequencing and the semi-nested polymerase chain reaction (PCR) of the pmoA gene (A189/A682r nested with a mixture of mb661 and A650) consistently revealed the dominance of USCα (accounting for more than 50% of the total MOB) in 12 forest soils. A189f/A682r successfully amplified pmoA genes (mainly RA14 of USCα) in only three forest soils. A189f/mb661 could amplify USCα (mainly JR1) in several forest soils but showed a strong preferential amplification of Methylocystis and many other type I MOB groups. A189f/A650 almost exclusively amplified USCα (mainly JR1) and largely discriminated against Methylocystis and most of the other MOB groups. The semi-nested PCR approach weakened the bias of A189f/mb661 and A189f/A650 for JR1 and balanced the coverage of all USCα members. The canonical correspondence analysis indicated that soil NH4+-N and pH were the main environmental factors affecting the MOB community of Chinese forest soils. The RA14 of the USCα group prefers to live in soils with low pH, low temperature, low elevation, high precipitation, and rich in nitrogen. JR1's preferences for temperature and elevation were opposite to RA14. Our study suggests that combining the deep sequencing of 16S rRNA and pmoA genes to characterize MOB in forest soils is the best choice.

Hansschlegelia quercus sp. nov., a novel methylotrophic bacterium isolated from oak buds.

Novel aerobic, restricted facultatively methylotrophic bacteria were isolated from buds of English oak (Quercus robur L.; strain DubT) and northern red oak (Quercus rubra L.; strain KrD). The isolates were Gram-negative, asporogenous, motile short rods that multiplied by binary fisson. They utilized methanol, methylamine and a few polycarbon compounds as carbon and energy sources. Optimal growth occurred at 25 °C and pH 7.5. The dominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and phoshatidylglycerol. The major cellular fatty acids of cells were C18 : 1 ω7c, 11-methyl C18 : 1 ω7c and C16 : 0. The major ubiquinone was Q-10. Analysis of 16S rRNA gene sequences showed that the strains were closely related to the members of the genus Hansschlegelia: Hansschlegelia zhihuaiae S113T(97.5-98.0 %), Hansschlegelia plantiphila S1T (97.4-97.6 %) and Hansschlegelia beijingensis PG04T(97.0-97.2 %). The 16S rRNA gene sequence similarity between strains DubT and KrD was 99.7 %, and the DNA-DNA hybridization (DDH) result between the strains was 85 %. The ANI and the DDH values between strain DubT and H. zhihuaiae S113T were 80.1 and 21.5  %, respectively. Genome sequencing of the strain DubT revealed a genome size of 3.57 Mbp and a G+C content of 67.0 mol%. Based on the results of the phenotypic, chemotaxonomic and genotypic analyses, it is proposed that the isolates be assigned to the genus Hansschlegelia as Hansschlegelia quercus sp. nov. with the type strain DubT (=VKM B-3284T=CCUG 73648T=JCM 33463T).

Methylopila carotae sp. nov., a facultative methylotroph, isolated from a root of Daucus carota L.

An aerobic facultatively methylotrophic bacterium, designated strain Das4.1T, was isolated from a root of Daucus carota L. The cells of this strain were observed to be Gram-stain negative, asporogenous, non-motile short rods multiplying by binary fission. Strain Das4.1T can utilise methanol, methylamine and a variety of polycarbon compounds as carbon and energy sources. C1-compounds were found to be assimilated via the isocitrate lyase-negative variant of the serine pathway. On medium with 0.5% methanol, growth of strain Das4.1T was observed at pH 5.5-9.0 (optimum, pH 6.0-7.0) and 18-37 °C (optimum, 24-29 °C) and in the presence of 0-2% (w/v) NaCl (optimum, 0.05%). Cells are catalase and oxidase positive and synthesise indole from L-tryptophan. The major fatty acids of methanol-grown cells were identified as C18:1ω7c, C18:0 and 11-methyl-C18:1ω7c. The predominant phospholipids were found to be phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major respiratory quinone was identified as Q-10. The DNA G + C content of strain Das4.1T was determined to be 67.3 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Das4.1T belongs to the genus Methylopila and shows high sequence similarity to Methylopila oligotropha 2395AT (98.4%) and Methylopila capsulata IM1T (98.0%). However, the DNA-DNA relatedness of strain Das4.1T with M. oligotropha 2395AT was only 22 ± 3%. Based on genotypic, chemotaxonomic and physiological characterisation, the isolate can be classified as a novel species of the genus Methylopila, for which the name Methylopila carotae sp. nov. is proposed. The type strain is Das4.1T (= VKM B-3244T = CCUG 72399T).

Genome sequence of Methylocystis hirsuta CSC1, a polyhydroxyalkanoate producing methanotroph.

Polyhydroxyalkanoates (PHAs) are biodegradable plastics that can be produced by some methanotrophic organisms such as those of the genus Methylocystis. This allows the conversion of a detrimental greenhouse gas into an environmentally friendly high added-value bioproduct. This study presents the genome sequence of Methylocystis hirsuta CSC1 (a high yield PHB producer). The genome comprises 4,213,043 bp in 4 contigs, with the largest contig being 3,776,027 bp long. Two of the other contigs are likely to correspond to large size plasmids. A total of 4,664 coding sequences were annotated, revealing a PHA production cluster, two distinct particulate methane monooxygenases with active catalytic sites, as well as a nitrogen fixation operon and a partial denitrification pathway.

Anaerobic carbon monoxide metabolism by Pleomorphomonas carboxyditropha sp. nov., a new mesophilic hydrogenogenic carboxydotroph.

Carbon monoxide (CO)-metabolism and phenotypic and phylogenetic characterization of a novel anaerobic, mesophilic and hydrogenogenic carboxydotroph are reported. Strain SVCO-16 was isolated from anaerobic sludge and grows autotrophically and mixotrophically with CO. The genes cooS and cooF, coding for a CO dehydrogenase complex, and genes similar to hycE2, encoding a CO-induced hydrogenase, were present in its genome. The isolate produces H2 and CO2 from CO, and acetate and formate from organic substrates. Based on the 16S rRNA sequence, it is an Alphaproteobacterium most closely related to the genus Pleomorphomonas (98.9%-99.2% sequence identity). Comparison with other previously characterized Pleomorphomonas showed that P. diazotrophica and P. oryzae do not metabolize CO, and P. diazotrophica does not grow anaerobically with organic substrates. Average nucleotide identity values between strain SVCO-16 and P. diazotrophica, P. oryzae or P. koreensis were 86.66 ± 0.21%. These values are below the boundary to define species (95%-96%). Digital DNA-DNA hybridization estimates between strain SVCO-16 and reference strains were also below the 70% threshold for species delineation: 29.1%-34.5%. Based on the differences in CO metabolism, genome analyses and cellular fatty acid composition, the isolate should be classified into the genus Pleomorphomonas as a representative of a novel species, Pleomorphomonas carboxyditropha. The type strain of Pleomorphomonas carboxyditropha is SVCO-16T (strain deposit numbers, DSM 106132T and TSD-119T).

Terasakiella salincola sp. nov., a marine alphaproteobacterium isolated from seawater, and emended description of the genus Terasakiella.

A Gram-reaction-negative, S-shaped, motile, poly-ß-hydroxybutyrate-accumulating, facultatively anaerobic, beige-pigmented bacterium, designated strain KMU-80T, was isolated from seawater collected from the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Methylocystaceae, of the class Alphaproteobacteria, and that it possessed the greatest sequence similarity (96.7 %) to Terasakiella pusilla NBRC 13613T. The DNA G+C content of KMU-80T was 48.3 mol%, and ubiquinone 10 was the sole respiratory quinone. The predominant cellular fatty acids consisted of C18 : 1ω7c (60.2 %), C16 : 0 (13.4 %) and C16 : 1ω7c and/or C16 : 1ω6c (11.1 %). Strain KMU-80T had phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminolipid, an unidentified phospholipid and four unidentified lipids as polar lipids. Based on its distinct phylogenetic position and the combination of genotypic and phenotypic characteristics, this strain is considered to represent a novel species of the genus Terasakiella, for which the name Terasakiella salincola sp. nov. is proposed. The type strain of T. salincola sp. nov. is KMU-80T (= KCCM 90274T = NBRC 112846T). An amended description of the genus Terasakiella is also provided.

Chthonobacter albigriseus gen. nov., sp. nov., isolated from grass-field soil.

A novel aerobic, Gram-stain-negative and non-motile bacterial strain, designated ED7T, was isolated from grass-field soil in Cheonan, Korea. Strain ED7T utilized methanol and methylamine, but not formate, as carbon and energy sources. The strain was able to grow at 20-42 °C (optimum 30-35 °C), at pH 7.0-8.5 (optimum pH 7.5-8.0), and in the absence of NaCl. According to the similarities of the 16S rRNA gene sequences, strain ED7T was most closely related to the genera Labrenzia (≤93.3 % 16S rRNA gene sequence similarity), Pleomorphomonas (≤93.1 %) and Prosthecomicrobium (≤93.1 %). A phylogenetic analysis based on the 16S rRNA gene sequence of strain ED7T revealed that it was affiliated with the family Methylocystaceae, being most closely related to the genus Pleomorphomonas. In contrast to Pleomorphomonas koreensis and Pleomorphomonas oryzae, strain ED7T did not contain the nifH gene. The DNA G+C content of the genomic DNA of strain ED7T was 71.8 mol%. The predominant fatty acids of strain ED7T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 2 (C14 : 0 3-OH, and/or C16 : 1 iso I), 11-methyl C18 : 1ω7c and C16 : 0 3-OH. The major isoprenoid quinone was ubiquinone 10 (Q-10). The major polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unknown aminophospholipid. Based on phenotypic, chemotaxonomic and genotypic characteristic data, strain ED7T could be differentiated from other genera, suggesting that strain ED7T represents a novel species of a new genus in the family Methylocystaceae, for which the name Chthonobacter albigriseus gen. nov., sp. nov. is proposed. The type strain of the type species is ED7T (=JCM 30603T=KCTC 42450T).

Oharaeibacter diazotrophicus gen. nov., sp. nov., a diazotrophic and facultatively methylotrophic bacterium, isolated from rice rhizosphere.

A novel facultatively methanol-utilizing bacterial strain, SM30T, was isolated from rice rhizosphere. Strain SM30T was Gram-stain-negative, aerobic, motile, short rods, and grew optimally at pH 7 and at 28 °C. It could tolerate 0 to 2 % (w/v) NaCl. Based on 16S rRNA gene sequence comparisons, strain SM30T was most closely related to Pleomorphomonas oryzae DSM 16300T, with a low similarity of 94.17 %. One of the lanthanide metals, lanthanum, could enhance its growth slightly on methanol. Phylogenetic trees, based on the mxaF, xoxF and cpn60 genes of SM30T showed its distinct phylogenetic position with respect to species with validly published names. Polymerase chain reaction (PCR) amplification of the nifH and growth on nitrogen-free medium indicated that strain SM30T is a diazotroph. The major cellular fatty acids were summed feature 8 (containing 18 : 1ω7c and 18 : 1ω6c) and cyclo 19 : 0ω8c. The major quinone was ubiquinone 10. The DNA G+C content was 74.6 mol%. Based on the genotypic and phenotypic characteristics, strain SM30T represents a novel genus and species, for which the name Oharaeibacter diazotrophicus gen. nov., sp. nov. is proposed with the type strain SM30T (=NBRC 111955T=DSM 102969T).

Salinity Affects the Composition of the Aerobic Methanotroph Community in Alkaline Lake Sediments from the Tibetan Plateau.

Lakes are widely distributed on the Tibetan Plateau, which plays an important role in natural methane emission. Aerobic methanotrophs in lake sediments reduce the amount of methane released into the atmosphere. However, no study to date has analyzed the methanotroph community composition and their driving factors in sediments of these high-altitude lakes (>4000 m). To provide new insights on this aspect, the abundance and composition in the sediments of six high-altitude alkaline lakes (including both freshwater and saline lakes) on the Tibetan Plateau were studied. The quantitative PCR, terminal restriction fragment length polymorphism, and 454-pyrosequencing methods were used to target the pmoA genes. The pmoA gene copies ranged 104-106 per gram fresh sediment. Type I methanotrophs predominated in Tibetan lake sediments, with Methylobacter and uncultivated type Ib methanotrophs being dominant in freshwater lakes and Methylomicrobium in saline lakes. Combining the pmoA-pyrosequencing data from Tibetan lakes with other published pmoA-sequencing data from lake sediments of other regions, a significant salinity and alkalinity effect (P = 0.001) was detected, especially salinity, which explained ∼25% of methanotroph community variability. The main effect was Methylomicrobium being dominant (up to 100%) in saline lakes only. In freshwater lakes, however, methanotroph composition was relatively diverse, including Methylobacter, Methylocystis, and uncultured type Ib clusters. This study provides the first methanotroph data for high-altitude lake sediments (>4000 m) and shows that salinity is a driving factor for the community composition of aerobic methanotrophs.

Identification of active aerobic methanotrophs in plateau wetlands using DNA stable isotope probing.

Sedge-dominated wetlands on the Qinghai-Tibetan Plateau are methane emission centers. Methanotrophs at these sites play a role in reducing methane emissions, but relatively little is known about the composition of active methanotrophs in these wetlands. Here, we used DNA stable isotope probing to identify the key active aerobic methanotrophs in three sedge-dominated wetlands on the plateau. We found that Methylocystis species were active in two peatlands, Hongyuan and Dangxiong. Methylobacter species were found to be active only in Dangxiong peat. Hongyuan peat had the highest methane oxidation rate, and cross-feeding of carbon from methanotrophs to methylotrophic Hyphomicrobium species was observed. Owing to a low methane oxidation rate during the incubation, the labeling of methanotrophs in Maduo wetland samples was not detected. Our results indicate that there are large differences in the activity of methanotrophs in the wetlands of this region.

Chenggangzhangella methanolivorans gen. nov., sp. nov., a member of the family Methylocystaceae, transfer of Methylopila helvetica Doronina et al. 2000 to Albibacter helveticus comb. nov. and emended description of the genus Albibacter.

A Gram-stain-negative, rod-shaped, non-motile and aerobic bacterial strain, designated CHL1T, was isolated from a sludge sample collected from a sewage treatment tank of an agricultural chemical factory. The strain grew at salinities of 0.5-5 % (w/v) NaCl (optimum 2.5 %). Growth occurred at pH 6.0-8.0 (optimum pH 7.0) and 5-40 °C (optimum 28-30 °C). The genomic DNA G+C content was determined to be 70.4 mol%. Q-10 was detected as the respiratory quinone. The major fatty acids (>10 %) were C18 : 1ω7c and/or C18 : 1ω6c and C16 : 0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified phospholipids and two unidentified aminophospholipids. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CHL1T formed a distinct clade with Albibacter methylovorans DSM 22840T and Methylopila helvetica DM9T within the family Methylocystaceae. On the basis of phenotypic, chemotaxonomic and phylogenetic characteristics, the strain merits recognition as a representative of a novel species of a new genus within the family Methylocystaceae, for which the name Chenggangzhangella methanolivorans gen. nov., sp. nov. is proposed. The type strain of the type species is CHL1T (=KCTC 42661T=CCTCC AB 2015175T). In addition, the species Methylopila helveticaDoronina et al. (2000) is proposed to be transferred to the genus Albibacter as Albibacterhelveticus comb. nov. (type strain DM9T=CIP 106788=VKM B-2189) on the basis of the phylogenetic analysis. An emended description of the genus Albibacter is also provided.

Terasakiella brassicae sp. nov., isolated from the wastewater of a pickle-processing factory, and emended descriptions of Terasakiella pusilla and the genus Terasakiella.

A Gram-stain-negative, motile, polyhydroxybutyrate-accumulating, aerobic, S-shaped bacterium, designated B3T, was isolated from the wastewater of a pickle-processing factory. 16S rRNA gene sequence similarity analysis showed that it was most closely related to the type strain, Terasakiella pusilla (96.6% similarity). Strain B3T was able to grow at 4-40 °C (optimum 32-37 °C), pH 5.5-9.0 (optimum 6.5-7.5) and with 0.5-8% (w/v) NaCl present (optimum 1-2%, w/v). Chemotaxonomic analysis showed that the respiratory quinone was ubiquinone Q-10, the major fatty acids included C16:0, C18:1ω7c and C16:1ω7c and/or iso-C15:2-OH. The major polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, aminophospholipid and three uncharacterized phospholipids. The genomic DNA G+C content of strain B3T was 42.3 mol%. The DNA-DNA relatedness value between B3T and T. pusilla DSM 9263T was 23.9%. On the basis of the phenotypic, chemotaxonomic and genotypic characteristics of strain B3T, it represents a novel species of the genus Terasakiella, for which the name Terasakiella brassicae sp. nov. is proposed. The type strain is B3T (=KCTC 42652T=CGMCC 1.15254T). Emended descriptions of T. pusilla and the genus Terasakiella are also presented.

Conversion of methane-derived carbon and microbial community in enrichment cultures in response to O2 availability.

Methanotrophs not only play an important role in mitigating CH4 emissions from the environment, but also provide a large quantity of CH4-derived carbon to their habitats. In this study, the distribution of CH4-derived carbon and microbial community was investigated in a consortium enriched at three O2 tensions, i.e., the initial O2 concentrations of 2.5 % (LO-2), 5 % (LO-1), and 21 % (v/v) (HO). The results showed that compared with the O2-limiting environments (2.5 and 5 %), more CH4-derived carbon was converted into CO2 and biomass under the O2 sufficient condition (21 %). Besides biomass and CO2, a high conversion efficiency of CH4-derived carbon to dissolved organic carbon was detected in the cultures, especially in LO-2. Quantitative PCR and Miseq sequencing both showed that the abundance of methanotroph increased with the increasing O2 concentrations. Type II methanotroph Methylocystis dominated in the enrichment cultures, accounting for 54.8, 48.1, and 36.9 % of the total bacterial 16S rRNA gene sequencing reads in HO, LO-1, and LO-2, respectively. Methylotrophs, mainly including Methylophilus, Methylovorus, Hyphomicrobium, and Methylobacillus, were also abundant in the cultures. Compared with the O2 sufficient condition (21 %), higher microbial biodiversity (i.e., higher Simpson and lower Shannon indexes) was detected in LO-2 enriched at the initial O2 concentration of 2.5 %. These findings indicated that compared with the O2 sufficient condition, more CH4-derived carbon was exuded into the environments and promoted the growth of non-methanotrophic microbes in O2-limiting environments.

Isolation of methanotrophic bacteria from termite gut.

The guts of termites feature suitable conditions for methane oxidizing bacteria (MOB) with their permanent production of CH4 and constant supply of O2 via tracheae. In this study, we have isolated MOB from the gut contents of the termites Incisitermes marginipennis, Mastotermes darwiniensis, and Neotermes castaneus for the first time. The existence of MOB was indicated by detecting pmoA, the gene for the particulate methane monooxygenase, in the DNA of gut contents. Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction supported those findings. The MOB cell titer was determined to be 10(2)-10(3) per gut. Analyses of the 16S rDNA from isolates indicated close similarity to the genus Methylocystis. After various physiological tests and fingerprinting methods, no exact match to a known species was obtained, indicating the isolation of new MOB species. However, MALDI-TOF MS analyses revealed a close relationship to Methylocystis bryophila and Methylocystis parvus.

Effect of CH4/O2 ratio on fatty acid profile and polyhydroxybutyrate content in a heterotrophic-methanotrophic consortium.

Understanding the role of heterotrophic-methanotrophic (H-Meth) communities is important for improvement of methane (CH4) oxidation capacities (MOC) particularly in conjunction with bio-product development in industrial bio-filters. Initially, a H-Meth consortium was established and enriched from marine sediments and characterized by next generation sequencing of the 16s rDNA gene. The enriched consortium was subjected to 10-50% CH4 (i.e., 0.20-1.6 CH4/O2 ratios) to study the effects on MOCs, biomass growth, fatty acid profiles and biopolymer (e.g. polyhydroxybutyrate; PHB) content. Methylocystis, Methylophaga and Pseudoxanthomonas dominated the H-Meth consortium. Culture enrichment of the H-Meth consortium resulted in 15-20-folds higher MOC compared to seed sediments. Increasing CH4 concentration (and decreased O2 levels) yielded higher MOCs, but did not improve total fatty acid contents. PHB contents varied between 2.5% and 8.5% independently of CH4/O2 ratios. The results suggest that H-Meth consortia could potentially be used in industrial bio-filters for production of biopolymer/biofuel precursors from CH4.

Microform-related community patterns of methane-cycling microbes in boreal Sphagnum bogs are site specific.

Vegetation and water table are important regulators of methane emission in peatlands. Microform variation encompasses these factors in small-scale topographic gradients of dry hummocks, intermediate lawns and wet hollows. We examined methane production and oxidization among microforms in four boreal bogs that showed more variation of vegetation within a bog with microform than between the bogs. Potential methane production was low and differed among bogs but not consistently with microform. Methane oxidation followed water table position with microform, showing higher rates closer to surface in lawns and hollows than in hummocks. Methanogen community, analysed by mcrA terminal restriction fragment length polymorphism and dominated by Methanoregulaceae or 'Methanoflorentaceae', varied strongly with bog. The extent of microform-related variation of methanogens depended on the bog. Methanotrophs identified as Methylocystis spp. in pmoA denaturing gradient gel electrophoresis similarly showed effect of bog, and microform patterns were stronger within individual bogs. Our results suggest that methane-cycling microbes in boreal Sphagnum bogs with seemingly uniform environmental conditions may show strong site-dependent variation. The bog-intrinsic factor may be related to carbon availability but contrary to expectations appears to be unrelated to current surface vegetation, calling attention to the origin of carbon substrates for microbes in bogs.

Methylopila henanense sp. nov., a novel methylotrophic bacterium isolated from tribenuron methyl-contaminated wheat soil.

A bacterial strain, designated LYBFD3-16A2(T), was isolated from tribenuron methyl contaminated wheat soil. Cells were observed to be Gram-negative short rods with a single flagellum. The strain was found to utilize methanol, glucose, maltose and mannitol as carbon and energy sources, and utilized glutamate, leucine, phenylalanine as organic nitrogen sources. Strain LYBFD3-16A2(T) was found to be aerobic, to form urease, produce hydrogen sulfide and reduce nitrate to nitrite. The indole test in tryptone broth was observed to be positive. The major cellular fatty acids were identified as C18:1ω7c (81.3 %), 11-methylC18:1ω7c (7.9 %), C18:0 (3.0 %) and C16:0 (3.0 %). The major phospholipids were identified as phosphatidylcholine, phosphatidylethanolamine, phosphatidyglycerol and diphosphatidylglycerol. The main ubiquinone was identified as Q-10. The DNA G+C content was determined to be between 70.2 and 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated the affiliation of strain LYBFD3-16A2 to members of the genus Methylopila. The DNA-DNA hybridization values of the novel strain with the type strains of the most closely related species Methylopila musalis MUSA(T) and Methylopila jiangsuensis JZL-4(T) were 35.4 % and 31.4 % respectively. The genotypic and phenotypic characterization, along with chemotaxonomic properties of strain LYBFD3-16A2(T), showed that the strain represents a novel species of the genus Methylopila for which the name Methylopila henanense sp. nov. is proposed. The type strain is LYBFD3-16A2(T) (=CGMCC1.10703(T) = LMG 25959(T)).