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ABSTRACT: Large amounts of gas hydrate are distributed in the northern slope of the South China Sea, which is a potential threat of methane leakage. Aerobic methane oxidation by methanotrophs, significant methane biotransformation that occurs in sediment surface and water column, can effectively reduce atmospheric emission of hydrate-decomposed methane. To identify active aerobic methanotrophs and their methane oxidation potential in sediments from the Shenhu Area in the South China Sea, multi-day enrichment incubations were conducted in this study. The results show that the methane oxidation rates in the studied sediments were 2.03–2.36 μmol/gdw/d, which were higher than those obtained by sediment incubations from other areas in marine ecosystems. Thus the authors suspect that the methane oxidation potential of methanotrophs was relatively higher in sediments from the Shenhu Area. After the incubations family Methylococcaea (type I methanotrophs) mainly consisted of genus Methylobacter and Methylococcaea_Other were predominant with an increased proportion of 70.3%, whereas Methylocaldum decreased simultaneously in the incubated sediments. Collectively, this study may help to gain a better understanding of the methane biotransformation in the Shenhu Area.©2022 China Geology Editorial Office.

Rivers are hotspots for methane (CH 4 ) emissions, and aerobic methane oxidation is a crucial process in controlling emissions. The spatio-temporal heterogeneity of river environment can greatly affect the methane oxidation process. However, currently, few studies have focused on the spatio-temporal changes in activity of methane oxidation and the associated microbiome in riverine ecosystems, which hinders a comprehensive understanding the role of this process in reducing emissions of CH 4 . Here, we investigated the variations in methane oxidation activity and community of methanotrophs in sediment of a mountain river across different reaches and seasons. The potential methane oxidation rate ranged from 24.11 to 493.03 nmol CH 4 g -1 (sediment) d -1 , which was significantly greater in sediment obtained during the winter than in that obtained during the summer. Moreover, the rate in middle reaches was significantly greater than that in upper and lower reaches in summer. The abundance of pmoA gene of methanotrophs ranged from 2.45×10⁶ to 2.98×10⁷ copies g -1 (sediment), which was also significantly greater in winter than in summer and showed significant variations among reaches. Additionally, methanotrophic diversity and community composition exhibited significant variations across both reaches and seasons, and the relative abundance of Methylococcus and Methylocystis was closely associated with methane oxidation activity. Sediment NH 4 + content, pH and temperature were potentially crucial factors affecting the activity or methanotrophic community. In conclusion, it is necessary to consider both temporal and spatial scales to improve our understanding of the significance and driving mechanisms of methane oxidation in controlling CH 4 emissions from rivers., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024. Published by Elsevier Ltd.)

Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the #916;mbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. [ABSTRACT FROM AUTHOR]

Methanobactins (MBs) are small (,1,300-Da) posttranslationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some being reduced after binding, e.g., Cu2+ reduced to Cu1. Other metal ions, however, are bound but not reduced, e.g., K1. The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H2 18O, we show that when MBs from Methylocystis sp. strain SB2 (MB-SB2) and Methylosinus trichosporium OB3+ (MB-OB3) were incubated in the presence of either Au31, Cu2, or Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed in the presence of either MB-SB2 or MB-OB3b alone, gold alone, copper alone, or silver alone or when K1 or Mo2+ was incubated with MB-SB2. In contrast to MB-OB3b, MB-SB2 binds Fe31 with an N2S2 coordination and will also reduce Fe31 to Fe2+. Iron reduction was also found to be coupled to the oxidation of 2H2O and the generation of O2. MB-SB2 will also couple Hg2+, Ni2+, and Co2+ reduction to the oxidation of 2H2O and the generation of O2, but MB-OB3+ will not, ostensibly as MB-OB3+ binds but does not reduce these metal ions. To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3+ was monitored under anoxic conditions. The results demonstrate that O2 generation from metal ion reduction by MB-OB3+ can support methane oxidation. [ABSTRACT FROM AUTHOR]

Aerobic methane (CH4) oxidation plays a significant role in marine CH4 consumption. Temperature changes resulting from, for example, global warming, have been suggested to be able to influence methanotrophic communities and their CH4 oxidation capacity. However, exact knowledge regarding temperature controls on marine aerobic methane oxidation is still missing. In this study, CH4 consumption and the methanotrophic community structure were investigated by incubating sediments from shallow (Bohai Bay) and deep marine environments (East China Sea) at 4, 15, and 28 °C for up to 250 days. The results show that the abundance of the methanotrophic population, dominated by the family Methylococcaceae (type I methanotrophs), was significantly elevated after all incubations and that aerobic methane oxidation for both areas had a strong temperature sensitivity. A positive correlation between the CH4 oxidation rate and temperature was witnessed in the Bohai Bay incubations, whereas for the East China Sea incubations, the optimum temperature was 15 °C. The systematic variations of pmoA OTUs between the Bohai Bay and East China Sea incubations indicated that the exact behaviors of CH4 oxidation rates with temperature are related to the different methanotrophic community structures in shallow and deep seas. These results are of great significance for quantitatively evaluating the biodegradability of CH4 in different marine environments.

ABSTRACT 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.

Methane oxidation coupled to selenate reduction has been suggested as a promising technology to bio-remediate selenium contaminated environments. However, the effect of dissolved oxygen (DO) on this process remained unclear. Here, we investigate the feasibility of selenate removal at two distinct DO concentrations. A membrane biofilm reactor (MBfR) was initially fed with ∼5 mg Se/L and then lowered to ∼1 mg Se/L of selenate, under anoxic condition containing ∼0.2 mg/L of influent DO. Selenate removal reached approximately 90% without selenite accumulation after one-month operation. Then 6–7 mg/L of DO was introduced and showed no apparent effect on selenate reduction in the subsequent operation. Electron microscopy suggested elevated oxygen exposure did not affect microbial shapes. 16S rDNA sequencing showed the aerobic methanotroph Methylocystis increased, while possible selenate reducers, Ignavibacterium and Bradyrhizobium , maintained stable after oxygen boost. Gene analysis indicated that nitrate/nitrite reductases positively correlated with selenate removal flux and were not remarkably affected by oxygen addition. Reversely, enzymes related with aerobic methane oxidation were obviously improved. This study provides a potential technology for selenate removal from oxygenated environments in a methane-based MBfR. Image 1 • Influent 6–7 mg/L DO had no inhibition on selenate reduction in a CH 4 -based MBfR. • Aerobic methanotroph Methylocystis remarkably increased after DO elevating. • Likely selenate reducers Ignavibacterium and Bradyrhizobium weren't affected by DO. [ABSTRACT FROM AUTHOR]

Mangrove forests are one of the important ecosystems in tropical coasts because of their high primary production, which they sustain by sequestering a substantial amount of CO2 into plant biomass. These forests often experience various levels of inundation and play an important role in CH4 emissions, but the taxonomy of methanotrophs in these systems remains poorly understood. In this study, DNA-based stable isotope probing showed significant niche differentiation in active aerobic methanotrophs in response to niche differentiation in upstream and downstream mangrove soils of the Tamsui estuary in northwestern Taiwan, in which salinity levels differ between winter and summer. Methylobacter and Methylomicrobium-like Type I methanotrophs dominated methane-oxidizing communities in the field conditions and were significantly 13C-labeled in both upstream and downstream sites, while Methylobacter were well adapted to high salinity and low temperature. The Type II methanotroph Methylocystis comprised only 10–15% of all the methane oxidizers in the upstream site but less than 5% at the downstream site under field conditions. 13C-DNA levels in Methylocystis were significantly lower than those in Type I methanotrophs, while phylogenetic analysis further revealed the presence of novel methane oxidizers that are phylogenetically distantly related to Type Ia in fresh and incubated soils at a downstream site. These results suggest that Type I methanotrophs display niche differentiation associated with environmental differences between upstream and downstream mangrove soils.

Methanotrophy of arable soils is affected by N fertilization, but the knowledge about the effect of oxygen level is poorly understood; soil aeration can fluctuate and zones of low oxygen are widespread in soil. We monitored CH4 oxidation in three mineral soils (Eutric Cambisol, Haplic Podzol, Mollic Gleysol) under laboratory conditions by varying the O2 level (from 20 to 2% O2), with or without NH4+ (100 mg N kg−1). In controls (without NH4+), CH4 was oxidized completely in the O2 range from oxia (20% O2) to high hypoxia (5% O2), while the process was inhibited under microoxia (2% O2). Ammonium application decreased CH4 consumption in all soils. This negative effect was stronger at 20% and 2% O2 than under hypoxia. The highest CH4 oxidation rates and the shortest initial (lag) phases in both control and NH4+-amended soils were observed under high (5% O2) and low (10% O2) hypoxia. [ABSTRACT FROM AUTHOR]

The O2:CH4 ratio significantly effects nitrogen removal in mixed cultures where aerobic methane oxidation is coupled with denitrification (AME-D). The goal of this study was to investigate nitrogen removal of the AME-D process at four different O2:CH4 ratios [0, 0.05, 0.25, and 1 (v/v)]. In batch tests, the highest denitrifying activity was observed when the O2:CH4 ratio was 0.25. At this ratio, the methanotrophs produced sufficient carbon sources for denitrifiers and the oxygen level did not inhibit nitrite removal. The results indicated that the synergy between methanotrophs and denitrifiers was significantly improved, thereby achieving a greater capacity of nitrogen removal. Based on thermodynamic and chemical analyses, methanol, butyrate, and formaldehyde could be the main trophic links of AME-D process in our study. Our research provides valuable information for improving the practical application of the AME-D systems.

The O2:CH4 ratio significantly effects nitrogen removal in mixed cultures where aerobic methane oxidation is coupled with denitrification (AME-D). The goal of this study was to investigate nitrogen removal of the AME-D process at four different O2:CH4 ratios [0, 0.05, 0.25, and 1 (v/v)]. In batch tests, the highest denitrifying activity was observed when the O2:CH4 ratio was 0.25. At this ratio, the methanotrophs produced sufficient carbon sources for denitrifiers and the oxygen level did not inhibit nitrite removal. The results indicated that the synergy between methanotrophs and denitrifiers was significantly improved, thereby achieving a greater capacity of nitrogen removal. Based on thermodynamic and chemical analyses, methanol, butyrate, and formaldehyde could be the main trophic links of AME-D process in our study. Our research provides valuable information for improving the practical application of the AME-D systems. [ABSTRACT FROM AUTHOR]

Hydrothermal vents are an important contributor to marine biogeochemistry, producing large volumes of reduced fluids, gasses, and metals and housing unique, productive microbial and animal communities fueled by chemosynthesis. Methane is a common constituent of hydrothermal vent fluid and is frequently consumed at vent sites by methanotrophic bacteria that serve to control escape of this greenhouse gas into the atmosphere. Despite their ecological and geochemical importance, little is known about the ecophysiology of uncultured hydrothermal vent-associated methanotrophic bacteria. Using metagenomic binning techniques, we recovered and analyzed a near-complete genome from a novel gammaproteobacterial methanotroph (B42) associated with a white smoker chimney in the Southern Lau basin. B42 was the dominant methanotroph in the community, at ~80x coverage, with only four others detected in the metagenome, all on low coverage contigs (7x - 12x). Phylogenetic placement of B42 showed it is a member of the Methylothermaceae, a family currently represented by only one sequenced genome. Metabolic inferences based on the presence of known pathways in the genome showed that B42 possesses a branched respiratory chain with A- and B-family heme copper oxidases, cytochrome bd oxidase and a partial denitrification pathway. These genes could allow B42 to respire over a wide range of oxygen concentrations within the highly dynamic vent environment. Phylogenies of the denitrification genes revealed they are the result of separate horizontal gene transfer from other proteobacteria and suggest that denitrification is a selective advantage in conditions where extremely low oxygen concentrations require all oxygen to be used for methane activation.

We examined the potential for CH4 oxidation to be coupled with oxygen derived from the dissimilatory reduction of perchlorate, chlorate or via chlorite (ClO2-) dismutation. Although dissimilatory reduction of ClO4- and ClO3- could be inferred from the accumulation of chloride ions either in spent media or in soil slurries prepared from exposed freshwater lake sediment, neither of these oxyanions evoked methane oxidation when added to either anaerobic mixed cultures or soil enriched in methanotrophs. In contrast, ClO2- amendment elicited such activity. Methane (0.2 kPa) was completely removed within several days from the headspace of cell suspensions of Dechloromonas agitata CKB incubated with either Methylococcus capsulatus Bath or Methylomicrobium album BG8 in the presence of 5 mM ClO2-. We also observed complete removal of 0.2 kPa CH4 in bottles containing soil enriched in methanotrophs when co-incubated with D. agitata CKB and 10 mM ClO2-. However, to be effective these experiments required physical separation of soil from D. agitata CKB to allow for the partitioning of O2 liberated from chlorite dismutation into the shared headspace. Although a link between ClO2- and CH4 consumption was established in soils and cultures, no upstream connection with either ClO4- or ClO3- was discerned. This result suggests that the release of O2 during enzymatic perchlorate reduction was negligible, and that the oxygen produced was unavailable to the aerobic methanotrophs.

Aerobic methane (CH4) oxidation plays a significant role in marine CH4 consumption. Temperature changes resulting from, for example, global warming, have been suggested to be able to influence methanotrophic communities and their CH4 oxidation capacity. However, exact knowledge regarding temperature controls on marine aerobic methane oxidation is still missing. In this study, CH4 consumption and the methanotrophic community structure were investigated by incubating sediments from shallow (Bohai Bay) and deep marine environments (East China Sea) at 4, 15, and 28 °C for up to 250 days. The results show that the abundance of the methanotrophic population, dominated by the family Methylococcaceae (type I methanotrophs), was significantly elevated after all incubations and that aerobic methane oxidation for both areas had a strong temperature sensitivity. A positive correlation between the CH4 oxidation rate and temperature was witnessed in the Bohai Bay incubations, whereas for the East China Sea incubations, the optimum temperature was 15 °C. The systematic variations of pmoA OTUs between the Bohai Bay and East China Sea incubations indicated that the exact behaviors of CH4 oxidation rates with temperature are related to the different methanotrophic community structures in shallow and deep seas. These results are of great significance for quantitatively evaluating the biodegradability of CH4 in different marine environments. [ABSTRACT FROM AUTHOR]

Mangrove forests are one of the important ecosystems in tropical coasts because of their high primary production, which they sustain by sequestering a substantial amount of CO2 into plant biomass. These forests often experience various levels of inundation and play an important role in CH4 emissions, but the taxonomy of methanotrophs in these systems remains poorly understood. In this study, DNA-based stable isotope probing showed significant niche differentiation in active aerobic methanotrophs in response to niche differentiation in upstream and downstream mangrove soils of the Tamsui estuary in northwestern Taiwan, in which salinity levels differ between winter and summer. Methylobacter and Methylomicrobium-like Type I methanotrophs dominated methane-oxidizing communities in the field conditions and were significantly 13C-labeled in both upstream and downstream sites, while Methylobacter were well adapted to high salinity and low temperature. The Type II methanotroph Methylocystis comprised only 10–15% of all the methane oxidizers in the upstream site but less than 5% at the downstream site under field conditions. 13C-DNA levels in Methylocystis were significantly lower than those in Type I methanotrophs, while phylogenetic analysis further revealed the presence of novel methane oxidizers that are phylogenetically distantly related to Type Ia in fresh and incubated soils at a downstream site. These results suggest that Type I methanotrophs display niche differentiation associated with environmental differences between upstream and downstream mangrove soils. [ABSTRACT FROM AUTHOR]

Chromate contamination can pose a high risk to both the environment and public health. Previous studies have shown that CH 4 -based membrane biofilm reactor (MBfR) is a promising method for chromate removal. In this study, we developed a multispecies biofilm model to study chromate reduction and its interaction with nitrate reduction in a CH 4 -based MBfR. The model-simulated results were consistent with the experimental data reported in the literature. The model showed that the presence of nitrate in the influent promoted the growth of heterotrophs, while suppressing methanotrophs and chromate reducers. Moreover, it indicated that a biofilm thickness of 150 μm and an influent dissolved oxygen concentration of 0.5 mg O 2 /L could improve the reactor performance by increasing the chromate removal efficiency under the simulated conditions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2019 Zhejiang University.)

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 C 16:1 ω9c 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., (Copyright © 2019 Ghashghavi et al.)

The O 2 :CH 4 ratio significantly effects nitrogen removal in mixed cultures where aerobic methane oxidation is coupled with denitrification (AME-D). The goal of this study was to investigate nitrogen removal of the AME-D process at four different O 2 :CH 4 ratios [0, 0.05, 0.25, and 1 (v/v)]. In batch tests, the highest denitrifying activity was observed when the O 2 :CH 4 ratio was 0.25. At this ratio, the methanotrophs produced sufficient carbon sources for denitrifiers and the oxygen level did not inhibit nitrite removal. The results indicated that the synergy between methanotrophs and denitrifiers was significantly improved, thereby achieving a greater capacity of nitrogen removal. Based on thermodynamic and chemical analyses, methanol, butyrate, and formaldehyde could be the main trophic links of AME-D process in our study. Our research provides valuable information for improving the practical application of the AME-D systems.