Methylomonas defluvii sp. nov., a type I methane-oxidizing bacterium from a secondary sedimentation tank of a wastewater treatment plant.

An aerobic methanotroph was isolated from a secondary sedimentation tank of a wastewater treatment plant and designated strain OY6T. Cells of OY6T were Gram-stain-negative, pink-pigmented, motile rods and contained an intracytoplasmic membrane structure typical of type I methanotrophs. OY6T could grow at a pH range of 4.5-7.5 (optimum pH 6.5) and at temperatures ranging from 20 °C to 37 °C (optimum 30 °C). The major cellular fatty acids were C14 : 0, C16 : 1ω7c/C16 : 1ω6c and C16 : 1ω5c; the predominant respiratory quinone was MQ-8. The genome size was 5.41 Mbp with a DNA G+C content of 51.7 mol%. OY6T represents a member of the family Methylococcaceae of the class Gammaproteobacteria and displayed 95.74-99.64 % 16S rRNA gene sequence similarity to the type strains of species of the genus Methylomonas. Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) confirmed that OY6T should be classified as representing a novel species. The most closely related type strain was Methylomonas fluvii EbBT, with 16S rRNA gene sequence similarity, ANI by blast (ANIb), ANI by MUMmer (ANIm) and dDDH values of 99.64, 90.46, 91.92 and 44.5 %, respectively. OY6T possessed genes encoding both the particulate methane monooxygenase enzyme and the soluble methane monooxygenase enzyme. It grew only on methane or methanol as carbon sources. On the basis of phenotypic, genetic and phylogenetic data, strain OY6T represents a novel species within the genus Methylomonas for which the name Methylomonas defluvii sp. nov. is proposed, with strain OY6T (=GDMCC 1.4114T=KCTC 8159T=LMG 33371T) as the type strain.

A novel bioprospecting strategy via 13C-based high-throughput probing of active methylotrophs inhabiting oil reservoir surface soil.

Methane-oxidizing bacteria (MOB) have long been considered as a microbial indicator for oil and gas prospecting. However, due to the phylogenetically narrow breath of ecophysiologically distinct MOB, classic culture-dependent approaches could not discriminate MOB population at fine resolution, and accurately reflect the abundance of active MOB in the soil above oil and gas reservoirs. Here, we presented a novel microbial anomaly detection (MAD) strategy to quantitatively identify specific indicator methylotrophs in the surface soils for bioprospecting oil and gas reservoirs by using a combination of 13C-DNA stable isotope probing (SIP), high-throughput sequencing (HTS), quantitative PCR (qPCR) and geostatistical analysis. The Chunguang oilfield of the Junggar Basin was selected as a model system in western China, and type I methanotrophic Methylobacter was most active in the topsoil above the productive oil wells, while type II methanotrophic Methylosinus predominated in the dry well soils, exhibiting clear differences between non- and oil reservoir soils. Similar results were observed by quantification of Methylobacter pmoA genes as a specific bioindicator for the prediction of unknown reservoirs by grid sampling. A microbial anomaly distribution map based on geostatistical analysis further showed that the anomalous zones were highly consistent with petroleum, geological and seismic data, and validated by subsequent drilling. Over seven years, a total of 24 wells have been designed and drilled into the targeted anomaly, and the success rate via the MAD prospecting strategy was 83 %. Our results suggested that molecular techniques are powerful tools for oil and gas prospecting. This study indicates that the exploration efficiency could be significantly improved by integrating multi-disciplinary information in geophysics and geomicrobiology while reducing the drilling risk to a greater extent.

Unlocking synergies: Harnessing the potential of biological methane sequestration through metabolic coupling between Methylomicrobium alcaliphilum 20Z and Chlorella sp. HS2.

A halotolerant consortium between microalgae and methanotrophic bacteria could effectively remediate in situ CH4 and CO2, particularly using saline wastewater sources. Herein, Methylomicrobium alcaliphilum 20Z was demonstrated to form a mutualistic association with Chlorella sp. HS2 at a salinity level above 3.0%. Co-culture significantly enhanced the growth of both microbes, independent of initial inoculum ratios. Additionally, increased methane provision in enclosed serum bottles led to saturated methane removal. Subsequent analyses suggested nearly an order of magnitude increase in the amount of carbon sequestered in biomass in methane-fed co-cultures, conditions that also maintained a suitable cultural pH suitable for methanotrophic growth. Collectively, these results suggest a robust metabolic coupling between the two microbes and the influence of the factors other than gaseous exchange on the assembled consortium. Therefore, multi-faceted investigations are needed to harness the significant methane removal potential of the identified halotolerant consortium under conditions relevant to real-world operation scenarios.

Targeting methanotrophs and isolation of a novel psychrophilic Methylobacter species from a terrestrial Arctic alkaline methane seep in Lagoon Pingo, Central Spitsbergen (78° N).

The microbial diversity associated with terrestrial groundwater seepage through permafrost soils is tightly coupled to the geochemistry of these fluids. Terrestrial alkaline methane seeps from Lagoon Pingo, Central Spitsbergen (78°N) in Norway, with methane-saturated and oxygen-limited groundwater discharge providing a potential habitat for methanotrophy. Here, we report on the microbial community's comparative analyses and distribution patterns at two sites close to Lagoon Pingo's methane emission source. To target methane-oxidizing bacteria from this system, we analysed the microbial community pattern of replicate samples from two sections near the main methane seepage source. DNA extraction, metabarcoding and subsequent sequencing of 16S rRNA genes revealed microbial communities where the major prokaryotic phyla were Pseudomonadota (42-47%), Gemmatimonadota (4-14%) and Actinobacteriota (7-11%). Among the Pseudomonadota, members of the genus Methylobacter were present at relative abundances between 1.6 and 4.7%. Enrichment targeting the methane oxidising bacteria was set up using methane seep sediments as inoculum and methane as the sole carbon and energy source, and this resulted in the isolation of a novel psychrophilic methane oxidizer, LS7-T4AT. The optimum growth temperature for the isolate was 13 °C and the pH optimum was 8.0. The morphology of cells was short rods, and TEM analysis revealed intracytoplasmic membranes arranged in stacks, a distinctive feature for Type I methanotrophs in the family Methylomonadaceae of the class Gammaproteobacteria. The strain belongs to the genus Methylobacter based on high 16S rRNA gene similarity to the psychrophilic species of Methylobacter psychrophilus Z-0021T (98.95%), the psychrophilic strain Methylobacter sp. strain S3L5C (99.00%), and the Arctic mesophilic species of Methylobacter tundripaludum SV96T (99.06%). The genome size of LS7-T4AT was 4,338,157 bp with a G + C content of 47.93%. The average nucleotide identities (ANIb) of strain LS7-T4AT to 10 isolated strains of genus Methylobacter were between 75.54 and 85.51%, lower than the species threshold of 95%. The strain LS7-T4AT represents a novel Arctic species, distinct from other members of the genus Methylobacter, for which the name Methylobacter svalbardensis sp. nov. is proposed. The type of strain is LS7-T4AT (DSMZ:114308, JCM:39463).

Ridge with no-tillage facilitates microbial N2 fixation associated with methane oxidation in rice soil.

Aerobic methane-oxidizing bacteria (MOB) play a crucial role in mitigating the greenhouse gas methane emission, particularly prevalent in flooded wetlands. The implementation of ridge with no-tillage practices within a rice-rape rotation system proves effective in overcoming the restrictive redox conditions associated with waterlogging. This approach enhances capillary water availability from furrows, especially during periods of low rainfall, thereby supporting plant growth on the ridges. However, the microbe-mediated accumulation of soil organic carbon and nitrogen remains insufficiently understood under this agricultural practice, particularly concerning methane oxidation, which holds ecological and agricultural significance in the rice fields. In this study, the ridge and ditch soils from a 28-year-old ridge with no-tillage rice field experiment were utilized for incubation with 13C-CH4 and 15NN2 to estimate the methane-oxidizing and N2-fixing potentials. Our findings reveal a significantly higher net production of fresh soil organic carbon in the ridge compared to the ditch soil during methane oxidation, with values of 626 and 543 µg 13C g-1 dry weight soil, respectively. Additionally, the fixed 15N exhibited a twofold increase in the ridge soil (14.1 µg 15N g-1 dry weight soil) compared to the ditch soil. Interestingly, the result of DNA-based stable isotope probing indicated no significant differences in active MOB and N2 fixers between ridge and ditch soils. Both Methylocystis-like type II and Methylosarcina/Methylomonas-like type I MOB catalyzed methane into organic biomass carbon pools. Soil N2-fixing activity was associated with the 15N-labeling of methane oxidizers and non-MOB, such as methanol oxidizers (Hyphomicrobium) and conventional N2 fixers (Burkholderia). Methane oxidation also fostered microbial interactions, as evidenced by co-occurrence patterns. These results underscore the dual role of microbial methane oxidation - not only as a recognized sink for the potent greenhouse gas methane but also as a source of soil organic carbon and bioavailable nitrogen. This emphasizes the pivotal role of microbial methane metabolism in contributing to soil carbon and nitrogen accumulation in ridge with no-tillage systems.

Seasonal dynamics of the microbial methane filter in the water column of a eutrophic coastal basin.

In coastal waters, methane-oxidizing bacteria (MOB) can form a methane biofilter and mitigate methane emissions. The metabolism of these MOBs is versatile, and the resilience to changing oxygen concentrations is potentially high. It is still unclear how seasonal changes in oxygen availability and water column chemistry affect the functioning of the methane biofilter and MOB community composition. Here, we determined water column methane and oxygen depth profiles, the methanotrophic community structure, methane oxidation potential, and water-air methane fluxes of a eutrophic marine basin during summer stratification and in the mixed water in spring and autumn. In spring, the MOB diversity and relative abundance were low. Yet, MOB formed a methane biofilter with up to 9% relative abundance and vertical niche partitioning during summer stratification. The vertical distribution and potential methane oxidation of MOB did not follow the upward shift of the oxycline during summer, and water-air fluxes remained below 0.6 mmol m-2 d-1. Together, this suggests active methane removal by MOB in the anoxic water. Surprisingly, with a weaker stratification, and therefore potentially increased oxygen supply, methane oxidation rates decreased, and water-air methane fluxes increased. Thus, despite the potential resilience of the MOB community, seasonal water column dynamics significantly influence methane removal.

Soil bacterial community composition in rice-turtle coculture systems with different planting years.

The rice-turtle coculture system is the most special rice-fish integrated farming system. In this study, we selected four paddy fields, including a rice monoculture paddy and three rice-turtle paddies with different planting years, to investigate the soil bacterial community composition with Illumina MiSeq sequencing technology. The results indicated that the contents of soil available nitrogen (AN), soil available phosphorus (AP) and soil organic matter (OM) in 9th year of rice-turtle paddy (RT9) were increased by 5.40%, 51.11% and 23.33% compared with rice monoculture paddy (CK), respectively. Significant differences of Acidobacteria, Desulfobacteria, Crenarchaeota were observed among the different rice farming systems. The relative abundance of Methylomonadaceae, Methylococcaceae and Methylophilaceae in RT9 was significantly higher than that in other treatments. RT9 had significantly lower relative abundance of Acidobacteria, but significantly higher relative abundance of Proteobacteria than other treatments. Redundancy analysis showed that soil AN and AP contents were the major factors influencing the abundance of the dominant microbes, wherein Methylomonadaceae, Methylococcaceae and Methylophilaceae were positively correlated with OM. The findings revealed the rice-turtle coculture system in the 9th year had higher soil nutrients and soil bacterial diversity, but there was also a risk of increasing methane emissions.

Evidence for Nitrous Oxide Emissions by Nitrite-Dependent Anaerobic Methane Oxidizing Bacteria.

Nitrite-dependent anaerobic methane oxidizing (n-DAMO) bacteria generally convert nitrite to dinitrogen and bypass the nitrous oxide (N2O) formation step. However, N2O is often detected in n-DAMO bacteria dominated cultures and it remains an open question as to the microbial origin of N2O in these enrichments. Using a stable nitrite consuming microbial community enriched for n-DAMO bacteria, we demonstrated that N2O production was coupled to methane oxidation and the higher initial nitrite concentrations led to increased quantities of N2O being formed. Moreover, continuous exposure of the enrichment culture to about 5 mg of N L-1 nitrite resulted in constant N2O being produced (12.5% of nitrite was reduced to N2O). Metatranscriptomic analyses revealed that nitrite reductase (nirS) and nitric oxide reductase (norZ) transcripts from n-DAMO bacteria increased in response to nitrite exposure. No other bacteria significantly expressed nor genes under these conditions, suggesting n-DAMO bacteria are responsible for N2O being produced. In a 35-day bioreactor experiment, N2O produced by the n-DAMO bacteria accumulated when nitrite was in excess; this was found to be up to 3.2% of the nitrogen that resulted from nitrite removal. Together, these results suggested that excess nitrite is an important driver of N2O production by n-DAMO bacteria. To this end, proper monitoring and control of nitrite levels in wastewater treatment plants would be effective strategies for mitigating N2O emissions to the atmosphere.

Versatile methanotrophs form an active methane biofilter in the oxycline of a seasonally stratified coastal basin.

The potential and drivers of microbial methane removal in the water column of seasonally stratified coastal ecosystems and the importance of the methanotrophic community composition for ecosystem functioning are not well explored. Here, we combined depth profiles of oxygen and methane with 16S rRNA gene amplicon sequencing, metagenomics and methane oxidation rates at discrete depths in a stratified coastal marine system (Lake Grevelingen, The Netherlands). Three amplicon sequence variants (ASVs) belonging to different genera of aerobic Methylomonadaceae and the corresponding three methanotrophic metagenome-assembled genomes (MOB-MAGs) were retrieved by 16S rRNA sequencing and metagenomic analysis, respectively. The abundances of the different methanotrophic ASVs and MOB-MAGs peaked at different depths along the methane oxygen counter-gradient and the MOB-MAGs show a quite diverse genomic potential regarding oxygen metabolism, partial denitrification and sulphur metabolism. Moreover, potential aerobic methane oxidation rates indicated high methanotrophic activity throughout the methane oxygen counter-gradient, even at depths with low in situ methane or oxygen concentration. This suggests that niche-partitioning with high genomic versatility of the present Methylomonadaceae might contribute to the functional resilience of the methanotrophic community and ultimately the efficiency of methane removal in the stratified water column of a marine basin.

Methanol bioconversion in Methylotuvimicrobium buryatense 5GB1C through self-cycling fermentation.

Methanol is an abundant and low-cost next-generation carbon source. While many species of methanotrophic bacteria can convert methanol into valuable bioproducts in bioreactors, Methylotuvimicrobium buryatense 5GB1C stands out as one of the most promising strains for industrialization. It has a short doubling time compared to most methanotrophs, remarkable resilience against contamination, and a suite of tools enabling genetic engineering. When approaching industrial applications, growing M. buryatense 5GB1C on methanol using common batch reactor operation has important limitations; for example methanol toxicity leads to mediocre biomass productivity. Advanced bioreactor operation strategies, such as fed-batch and self-cycling fermentation, have the potential to greatly improve the industrial prospects of methanotrophs growing on methanol. Herein, implementation of fed-batch operation led to a 26-fold increase in biomass density, while two different self-cycling fermentation (SCF) strategies led to 3-fold and 10-fold increases in volumetric biomass productivity. Interestingly, while synchronization is a typical trait of microbial populations undergoing SCF, M. buryatense 5GB1C cultures growing under this mode of operation led to stable, reproducible cycles but no significant synchronization.

Characterisation of 'Candidatus Methylobacter titanis' sp. nov., a putative novel species of Methylobacter clade 2 and their distribution in sediments of freshwater lakes in maritime Antarctica.

Global warming has a strong impact on the polar regions, in particular, the Antarctic Peninsula and nearby islands. Methane (CH4) is a major factor in climate change and mitigation of CH4 emissions can be accomplished through microbial oxidation by methanotrophic bacteria. Understanding this biological process is crucial given the shortage of research carried out in this geographical area. The aim of this study was to characterise psychrophilic enrichment cultures of aerobic methanotrophs obtained from lake sediments of the Fildes Peninsula (King George Island, South Shetland Islands) and revealing the distribution of the genus Methylobacter in different lake sediments of the peninsula. Four stable methanotrophic enrichment cultures were obtained and analysed by metagenome-assembled genomes (MAGs). The phylogeny of methanotroph MAGs recovered from these enrichment cultures based on the 16S rRNA gene showed that K-2018 MAG008 and D1-2020 MAG004Ts clustered within the Methylobacter clade 2, with high similarity to Methylobacter tundripaludum SV96T (97.88 and 98.56% respectively). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with M. tundripaludum were < 95% (84.8 and 85.0%, respectively) and < 70% (30.2 and 30.3%, respectively), suggesting that they represent a putative novel species for which the name 'Ca. Methylobacter titanis' is proposed. This is the first species of clade 2 of the genus Methylobacter obtained from Antarctica. The bacterial diversity assessed by 16S rRNA gene sequencing of 21 samples of different lakes (water column and sediments) revealed 54 ASVs associated with methanotrophs and the genus Methylobacter as the most abundant. These results suggest that aerobic methanotrophs belonging to the Methylobacter clade 2 would be the main responsible for CH4 oxidation in these sediments.

Methanotrophs dominate methanogens and act as a methane sink in a subterranean karst cave.

Karst caves are potential sinks of atmospheric methane due to microbial consumption. However, knowledge gaps on methanogens (methane producing microorganisms) and their interaction with methane-oxidizing bacteria (MOB) hinder our further understanding about methane dynamics in karst caves. Here we reported methanogenic community composition and their interaction with MOBs in the Heshang Cave to comprehensively understand methane cycling in subsurface biosphere. MOBs in karst cave were dominated by high-affinity MOB, upland soil cluster (USC), with USCγ pmoA gene abundance within the range of 1.34 × 104 to 1.8 × 107 copies·g-1 DW. In contrast, methanogens were dominated by Methanoregula and cluster ZC-I. The mcrA numbers were 7.21 × 103 to 8.31 × 104 copies·g-1 DW, 1-3 orders of magnitude lower than those of MOB. The inter-domain network analysis indicated that MOBs and methanogens cooperated more in the interior of the cave. Despite of the higher number of methanogenic nodes in the network, MOB dominated the keystone taxa, suggesting a leading functional role of MOB. MOB in caves showed a comparable with or higher potential methane oxidizing rate (PMOR, 0.63 ng CH4·g-1 DW·h-1 in sediment versus 11.02 ng CH4·g-1 DW·h-1 in weathered rock) than those in soils, whereas methane produced by methanogens was undetected. Collectively, high absolute abundances of MOB, high PMORs, the dominance of methanotrophic keystone taxa in the inter-domain network confirmed the superiority of MOBs over methanogens in the oligotrophic karst cave, mounting new evidence on caves as an important methane sink in terms of the interaction between methanogens and MOBs.

Type-specific quantification of particulate methane monooxygenase gene of methane-oxidizing bacteria at the oxic-anoxic interface of a surface paddy soil by digital PCR.

Aerobic methane-oxidizing bacteria (MOB) play an important role in mitigating methane emissions from paddy fields. In this study, we developed a differential quantification method for the copy number of pmoA genes of type Ia, Ib, and IIa MOB in paddy field soil using chip-based digital PCR. Three probes specific to the pmoA of type Ia, Ib, and IIa MOB worked well in digital PCR quantification when genomic DNA of MOB isolates and PCR-amplified DNA fragments of pmoA were examined as templates. When pmoA genes in the surface soil layer of a flooded paddy were quantified by digital PCR, the copy numbers of type Ia, Ib, and IIa MOB were 105 -106 , 105 -106 , and 107 copies g-1 dry soil, respectively, with the highest values in the top 0-2-mm soil layer. Especially, the copy numbers of type Ia and Ib MOB increased by 240% and 380% at the top layer after soil flooding, suggesting that the soil circumstances at the oxic-anoxic interfaces were more preferential for growth of type I MOB than type II MOB. Thus, type I MOB likely play an important role in the methane consumption at the surface paddy soil.

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.

Community structure and network interaction of aerobic methane-oxidizing bacteria in Chongqing's central urban area in the Three Gorges Reservoir, China.

A reservoir is an important source of methane (CH4), which is consumed by aerobic methane-oxidizing bacteria (MOB), representing the main CH4 sink in water. The central urban area of Chongqing in the Three Gorges Reservoir (TGR) area was selected as the study area in 2021. High-throughput sequencing was used to analyze the community structure and abundance of MOBs. The results showed that Methylocystis (Type II) was the dominant MOB in water, whereas Methylococcus (Type I) and Methylocystis co-dominated the sediments. High water temperature in the study area largely accounted for the predominance of Type II MOBs in the two habitats. Moreover, the influence of environmental factors on MOB community and its interspecific relationship were significantly regulated by the operation of the TGR. In the low-water-level period, NO2--N and CO2 concentration significantly correlated with Methylocystis, whereas in the high-water-level period, the higher discharge and velocity weakened the influence of all environmental factors on Methylocystis. In addition, the scouring of sediments by increasing discharge in the high-water-level period caused a significant decrease in dissolved CH4 concentration. The decrease in substrate increased interspecific competition within the MOB community, especially between different types of MOBs, in the high-water-level period.

Characterization of methanotrophic community and activity in landfill cover soils under dimethyl sulfide stress.

Landfill cover soil is the environmental interface between landfills and the atmosphere and plays an important role in mitigating CH4 emission from landfills. Here, stable isotope probing microcosms with CH4 or CH4 and dimethyl sulfide (DMS) were carried out to characterize activity and community structure of methanotrophs in landfill cover soils under DMS stress. The CH4 oxidation activity in the landfill cover soils was not obviously influenced at the DMS concentration of 0.05%, while it was inhibited at the DMS concentrations of 0.1% and 0.2%. DMS-S was mainly oxidized to sulfate (SO42-) in the landfill cover soils. In the landfill cover soils, DMS could inhibit the expression of bacteria and decrease the abundances of pmoA and mmoX genes, while it could prompt the expression of pmoA and mmoX genes. γ-Proteobacteria methanotrophs including Methylocaldum, Methylobacter, Crenothrix and unclassified Methylococcaceae and α-Proteobacteria methanotrophs Methylocystis dominated in assimilating CH4 in the landfill cover soils. Of them, Methylobacter and Crenothrix had strong tolerance to DMS or DMS could promote the growth and activity of Methylobacter and Crenothrix, while Methylocaldum had weak tolerance to DMS and showed an inhibitory effect. Metagenomic analyses showed that methanotrophs had the genes of methanethiol oxidation and could metabolize CH4 and methanethiol simultaneously in the landfill cover soils. These findings suggested that methanotrophs might metabolize sulfur compounds in the landfill cover soils, which may provide the potential application in engineering for co-removal of CH4 and sulfur compounds.

Activity and abundance of methanotrophic bacteria in a northern mountainous gradient of wetlands.

Methane uptake and diversity of methanotrophic bacteria was investigated across six hydrologically connected wetlands in a mountainous forest landscape upstream of lake Langtjern, southern Norway. From floodplain through shrubs, forest and sedges to a Sphagnum covered site, growing season CH4 production was insufficiently consumed to balance release into the atmosphere. Emission increased by soil moisture ranging 0.6-6.8 mg CH4 m-2  h-1 . Top soils of all sites consumed CH4 including at the lowest 78 ppmv CH4 supplied, thus potentially oxidizing 17-51 nmol CH4 g-1 dw h-1 , with highest Vmax 440 nmol g-1 dw h-1 under Sphagnum and lowest Km 559 nM under hummocked Carex. Nine genera and several less understood type I and type II methanotrophs were detected by the key functional gene pmoA involved in methane oxidation. Microarray signal intensities from all sites revealed Methylococcus, the affiliated Lake Washington cluster, Methylocaldum, a Japanese rice cluster, Methylosinus, Methylocystis and the affiliated Peat264 cluster. Notably enriched by site was a floodplain Methylomonas and a Methylocapsa-affiliated watershed cluster in the Sphagnum site. The climate sensitive water table was shown to be a strong controlling factor highlighting its link with the CH4 cycle in elevated wetlands.

Coupling methanotrophic denitrification to anammox in a moving bed biofilm reactor for nitrogen removal under hypoxic conditions.

Simultaneous removal of ammonium and nitrate was achieved in a methane-fed moving bed biofilm reactor (MBBR). In the reactor, methanotrophic microorganisms oxidized methane under hypoxic conditions likely to methanol, hence providing an electron donor to denitrifiers to reduce nitrate to nitrite that then allowed anaerobic ammonium oxidizing bacteria (Anammox) to remove excess ammonium as N2. The ammonium and nitrate removal rates reached 72.09 ± 5.81 mgNH4+-N/L/d and 62.61 ± 4.17 mgNO3--N/L/d when the MBBR was operated in continuous mode. Nitrate removal by the methane-fed mixed consortia was confirmed in a batch test revealing a CH4/NO3- molar removal ratio of 1.15. The functional populations were unveiled by FISH analysis and 16S rRNA gene sequencing, which showed that the biofilm was dominated by Anammox bacteria (Candidatus Kuenenia) and diverse taxa associated with the capacity for denitrification: aerobic methanotrophs (Methylobacter, Methylomonas, and unclassified Methylococcaceae), methylotrophic denitrifiers (Opitutaceae and Methylophilaceae), and other heterotrophic denitrifiers (Ignavibacteriaceae, Anaerolineaceae, Comamonadaceae, Rhodocyclaceae and Thauera). Neither DAMO archaea nor DAMO bacteria were found in the sequencing analysis, indicating that more unknown community members possess the metabolic capacity of methanotrophic denitrification.

pH dependence of arsenic speciation in paddy soils: The role of distinct methanotrophs.

Arsenic (As) is a priority environmental pollutant in paddy field. The coupling of arsenate (As(V)) reduction with anaerobic methane (CH4) oxidation was recently demonstrated in paddy soils and has been suggested to serve as a critical driver for As transformation and mobilization. However, whether As(V)-dependent CH4 oxidation is driven by distinct methanotrophs under different pH conditions remains unclear. Here, we investigated the response of As(V)-dependent CH4 oxidation to pH shifts (pH 5.5-8.0) by employing isotopically labelled CH4. Furthermore, the underlying mechanisms were also investigated in well-controlled anoxic soil suspension incubations. Our results showed that As(V)-dependent CH4 oxidation is highly sensitive to pH changes (1.6-6.8 times variation of arsenite formation). A short-term (0-10 d) pH shift from near-neutral pH to acidic conditions (i.e., pH 5.5, -85% arsenite formation) had an inhibitory effect on As(V)-dependent CH4 oxidation. However, prolonged acidic conditions (i.e., >15 d) had no significant influence on As(V)-dependent CH4 oxidation. The microbial analyses indicated that As reduction in paddies can be driven by anaerobic CH4 oxidation archaea (ANME) and methanotrophs. And, methanotrophs may serve as a critical driver for As(V)-dependent CH4 oxidation. Moreover, type I methanotrophs Methylobacter were more active in oxidizing CH4 than type II methanotrophs Methylocystis when the pH ≥ 6.5. However, Methylocystis had a higher tolerance to soil acidification than Methylobacter. This study illustrates that As(V)-dependent CH4 oxidation could be dominated by distinct methanotrophs along with pH shifts, which eventually enhances As release in paddy soils.

Engineering type I methanotrophic bacteria as novel platform for sustainable production of 3-hydroxybutyrate and biodegradable polyhydroxybutyrate from methane and xylose.

Methylotuvimicrobium alcaliphilum20Z recombinant strain co-utilizing methane and xylose from anthropogenic activities and lignocellulose biomassis a promising cell factory platform. In this study, the production of (R)-3-hydroxybutyrate and poly (3-hydroxybutyrate) inM. alcaliphilum20Z was demonstrated. The production of (R)-3-hydroxybutyrate was optimized by introducing additional thioesterase, and a tunable genetic module. The final recombinant strain produced the highest titer of 334.52 ± 2 mg/L (R)-3-hydroxybutyrate (yield of 1,853 ± 429 mg/g dry cell weight). The poly (3-hydroxybutyrate) yielded 1.29 ± 0.08% (w/w) from methane and xylose in one-stage cultivation. Moreover, the study demonstrated the importance of pathway reversibility as an effective design strategy for balancing the driving force and intermediate accumulation. This is the first demonstration of the production ofbiodegradablepoly (3-hydroxybutyrate) from methane in type I methanotrophs, which is a key step toward sustainable biomanufacturing and carbon-neutral society.