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Methane-oxidizing bacteria (MOB) is a group of planktonic microorganisms that use methane as their primary source of cellular energy. For tropical lakes in monsoon Asia, there is currently a knowledge gap on MOB community diversity and the factors influencing their abundance. Herewith, we present a preliminary assessment of the MOB communities in three maar lakes in tropical monsoon Asia using Catalyzed Reporter Deposition, Fluorescence In-Situ Hybridization (CARD-FISH), 16S rRNA amplicon sequencing, and pmoA gene sequencing. Correlation analysis between MOB abundances and lakes' physicochemical parameters following seasonal monsoon events were performed to explain observed spatial and temporal patterns in MOB diversity. The CARD-FISH analyses detected the three MOB types (I, II, and NC10) which aligned with the results from 16S rRNA amplicons and pmoA gene sequencing. Among community members based on 16S rRNA genes, Proteobacterial Type I MOB (e.g., Methylococcaceae and Methylomonadaceae), Proteobacterial Type II (Methylocystaceae), Verrucomicrobial (Methylacidiphilaceae), Methylomirabilota/NC10 (Methylomirabilaceae), and archaeal ANME-1a were found to be the dominant methane-oxidizers in three maar lakes. Analysis of microbial diversity and distribution revealed that the community compositions in Lake Yambo vary with the seasons and are more distinct during the stratified period. Temperature, DO, and pH were significantly and inversely linked with type I MOB and Methylomirabilota during stratification. Only MOB type I was influenced by monsoon changes. This research sought to establish a baseline for the diversity and ecology of planktonic MOB in tropical monsoon Asia to better comprehend their contribution to the CH4 cycle in tropical freshwater ecosystems. [ABSTRACT FROM AUTHOR]

Methane-oxidizing bacteria (MOB) is a group of planktonic microorganisms that use methane as their primary source of cellular energy. For tropical lakes in monsoon Asia, there is currently a knowledge gap on MOB community diversity and the factors influencing their abundance. Herewith, we present a preliminary assessment of the MOB communities in three maar lakes in tropical monsoon Asia using Catalyzed Reporter Deposition, Fluorescence In-Situ Hybridization (CARD-FISH), 16S rRNA amplicon sequencing, and pmoA gene sequencing. Correlation analysis between MOB abundances and lakes’ physicochemical parameters following seasonal monsoon events were performed to explain observed spatial and temporal patterns in MOB diversity. The CARD-FISH analyses detected the three MOB types (I, II, and NC10) which aligned with the results from 16S rRNA amplicons and pmoA gene sequencing. Among community members based on 16S rRNA genes, Proteobacterial Type I MOB (e.g., Methylococcaceae and Methylomonadaceae), Proteobacterial Type II (Methylocystaceae), Verrucomicrobial (Methylacidiphilaceae), Methylomirabilota/NC10 (Methylomirabilaceae), and archaeal ANME-1a were found to be the dominant methane-oxidizers in three maar lakes. Analysis of microbial diversity and distribution revealed that the community compositions in Lake Yambo vary with the seasons and are more distinct during the stratified period. Temperature, DO, and pH were significantly and inversely linked with type I MOB and Methylomirabilota during stratification. Only MOB type I was influenced by monsoon changes. This research sought to establish a baseline for the diversity and ecology of planktonic MOB in tropical monsoon Asia to better comprehend their contribution to the CH4 cycle in tropical freshwater ecosystems.

Abstract It is estimated that up to half of global methane (CH4) emissions are derived from microbial processes in aquatic ecosystems. However, it is not fully understood which factors explain the spatial and temporal variability of these emissions. For example, light has previously been shown to both inhibit and stimulate aerobic methane‐oxidizing bacteria (i.e., methanotrophs) in the water column. These contrasting results indicate that the mechanisms that light has on CH4 oxidation are not yet clearly known, even less so for benthic aerobic methanotrophs. Here, we tested whether light reaching the seafloor can inhibit methanotrophic activity on the sediment surface. We sampled and distributed over 40 intact sediment cores from two coastal sites (illuminated 10 m, and a dark site at 33 m water depth) into 0, 50, and 100 PAR light treatments. After 10 days, we found no difference between treatments for each site in pore‐water CH4 concentrations, relative abundance of aerobic methanotrophs, or the number of RNA transcripts related to methane oxidation. Our results suggest that light attenuation in coastal waters does not significantly affect aerobic methanotrophs in coastal sediments.

Biochar as an agricultural soil amendment plays vital roles in mediating methane (CH4) and nitrous oxide (N2O) emissions in soils. The link between different types of biochar, bulk soil, and rhizosphere microbial communities in relation to CH4 and N2O emissions is being investigated in this study. The rice pot experiment was conducted using biochar at two temperatures (300°C and 500°C) in combination with three biochar levels (0, 2, 10% w/w). Soil properties and the abundance of genes associated with CH4 and N2O emissions from both rhizosphere and bulk soils were investigated. The study also aimed to examine the structure of microbial communities (pmoA, nosZ) in rhizosphere and bulk soils whereas CH4 and N2O emissions were monitored while growing rice. Results showed that biochar at 300°C and 10% incorporation significantly increased the CH4 emissions by up to 59% rise compared to the control group. Random Forest analysis revealed that the ratio of mcrA/pmoA along with the abundance of mcrA from both rhizosphere and bulk soils, the abundance of AOA, TN, DOC, and the community composition of pmoA-harboring microorganisms from both bulk and rhizosphere soils were important predictors of CH4 emissions. Therefore, the ratio of mcrA/pmoA in rhizosphere soil and the abundance of AOA in bulk soil were the main factors influencing CH4 emissions. Variation Partitioning Analysis (VPA) results indicated that the effects of these factors on bulk soil were 9% of CH4 emissions variations in different treatments, which contributed more than rhizosphere soils' factors. Moreover, random forest analysis results indicated that the abundance of AOB in bulk soil was the most important predictor influencing N2O emissions. The VPA result revealed that the factors in rhizosphere soil could explain more than 28% of the variations in N2O emissions. Our study highlights that rhizosphere soil has a more significant effect than bulk soil on N2O production. Our findings further the understanding of the link between bulk and rhizosphere attributes, and their impact on CH4 and N2O emissions in paddy soils. In summary, we recommend the application of biochar at 500°C and 2% incorporation rate for agricultural production in the area. [ABSTRACT FROM AUTHOR]

Biochar as an agricultural soil amendment plays vital roles in mediating methane (CH4) and nitrous oxide (N2O) emissions in soils. The link between different types of biochar, bulk soil, and rhizosphere microbial communities in relation to CH4 and N2O emissions is being investigated in this study. The rice pot experiment was conducted using biochar at two temperatures (300°C and 500°C) in combination with three biochar levels (0, 2, 10% w/w). Soil properties and the abundance of genes associated with CH4 and N2O emissions from both rhizosphere and bulk soils were investigated. The study also aimed to examine the structure of microbial communities (pmoA, nosZ) in rhizosphere and bulk soils whereas CH4 and N2O emissions were monitored while growing rice. Results showed that biochar at 300°C and 10% incorporation significantly increased the CH4 emissions by up to 59% rise compared to the control group. Random Forest analysis revealed that the ratio of mcrA/pmoA along with the abundance of mcrA from both rhizosphere and bulk soils, the abundance of AOA, TN, DOC, and the community composition of pmoA-harboring microorganisms from both bulk and rhizosphere soils were important predictors of CH4 emissions. Therefore, the ratio of mcrA/pmoA in rhizosphere soil and the abundance of AOA in bulk soil were the main factors influencing CH4 emissions. Variation Partitioning Analysis (VPA) results indicated that the effects of these factors on bulk soil were 9% of CH4 emissions variations in different treatments, which contributed more than rhizosphere soils’ factors. Moreover, random forest analysis results indicated that the abundance of AOB in bulk soil was the most important predictor influencing N2O emissions. The VPA result revealed that the factors in rhizosphere soil could explain more than 28% of the variations in N2O emissions. Our study highlights that rhizosphere soil has a more significant effect than bulk soil on N2O production. Our findings further the understanding of the link between bulk and rhizosphere attributes, and their impact on CH4 and N2O emissions in paddy soils. In summary, we recommend the application of biochar at 500°C and 2% incorporation rate for agricultural production in the area.

A viral shunt can occur when phages going through a lytic cycle, including lysogenic phages triggered by inducing agents (e.g. mitomycin C), results in host lysis and the release of cell constituents and virions. The impact of a viral shunt on the carbon, including methane cycle in soil systems is poorly understood. Here, we determined the effects of mitomycin C on the aerobic methanotrophs in a landfill cover soil. To an extent, our results support a mitomycin C-induced viral shunt, as indicated by the significantly higher viral-like particle (VLP) counts relative to bacteria, elevated nutrient concentrations (ammonium, succinate), and initially impaired microbial activities (methane uptake and microbial respiration) after mitomycin C addition. The trend in microbial activities at <2 days largely corresponded to the expression of the pmoA and 16S rRNA genes. Thereafter (>11 days), the active bacterial community composition significantly diverged in the mitomycin C-supplemented incubations, suggesting the differential impact of mitomycin C on the bacterial community. Collectively, we provide insight on the effects of mitomycin C, and potentially a viral shunt, on the bacteria in the soil environment. [ABSTRACT FROM AUTHOR]

Cold seeps are oasis for the microbes in the deep-sea ecosystems, and various cold seeps are located along the northern slope of the South China Sea (SCS). However, by far most microbial ecological studies were limited to specific cold seep in the SCS, and lack of comparison between different regions. Here, the surface sediments (0–4 cm) from the Site F/Haima cold seeps and the Xisha trough in the SCS were used to elucidate the biogeography of microbial communities, with particular interest in the typical functional groups involved in the anaerobic oxidation of methane (AOM) process. Distinct microbial clusters corresponding to the three sampling regions were formed, and significantly higher gene abundance of functional groups were present in the cold seeps than the trough. This biogeographical distribution could be explained by the geochemical characteristics of sediments, such as total nitrogen (TN), total phosphorus (TP), nitrate (NO3−), total sulfur (TS) and carbon to nitrogen ratios (C/N). Phylogenetic analysis demonstrated that mcrA and pmoA genotypes were closely affiliated with those from wetland and mangroves, where denitrifying anaerobic methane oxidation (DAMO) process frequently occurred; and highly diversified dsrB genotypes were revealed as well. In addition, significantly higher relative abundance of NC10 group was found in the Xisha trough, suggesting that nitrite-dependent DAMO (N-DAMO) process was more important in the hydrate-bearing trough, although its potential ecological contribution to AOM deserves further investigation. Our study also further demonstrated the necessity of combining functional genes and 16S rRNA gene to obtain a comprehensive picture of the population shifts of natural microbial communities among different oceanic regions.

Oil palm (OP) plantations are gradually replacing tropical rainforest in Malaysia, one of the largest palm oil producers globally. Conversion of lands to OP plantations has been associated with compositional shifts of the microbial community, with consequences on the greenhouse gas (GHG) emissions. While the impact of the change in land use has recently been investigated for microorganisms involved in N2O emission, the response of the aerobic methanotrophs to OP agriculture remains to be determined. Here, we monitored the bacterial community composition, focusing on the aerobic methanotrophs, in OP agricultural soils since 2012, 2006, and 1993, as well as in a tropical rainforest, in 2019 and 2020. High-affinity methane uptake was confirmed, showing significantly lower rates in the OP plantations than in the tropical rainforest, but values increased with continuous OP agriculture. The bacterial, including the methanotrophic community composition, was modified with ongoing OP agriculture. The methanotrophic community composition was predominantly composed of unclassified methanotrophs, with the canonical (Methylocystis) and putative methanotrophs thought to catalyze high-affinity methane oxidation present at higher relative abundance in the oldest OP plantation. Results suggest that the methanotrophic community was relatively more stable within each site, exhibiting less temporal variations than the total bacterial community. Uncharacteristically, a 16S rRNA gene-based co-occurrence network analysis revealed a more complex and connected community in the OP agricultural soil, which may influence the resilience of the bacterial community to disturbances. Overall, we provide a first insight into the ecology and role of the aerobic methanotrophs as a methane sink in OP agricultural soils. [ABSTRACT FROM AUTHOR]

ABSTRACT Methanotrophs, which help regulate atmospheric levels of methane, are active in diverse natural and man-made environments. This range of habitats and the feast-famine cycles seen by many environmental methanotrophs suggest that methanotrophs dynamically mediate rates of methane oxidation. Global methane budgets require ways to account for this variability in time and space. Functional gene biomarker transcripts are increasingly studied to inform the dynamics of diverse biogeochemical cycles. Previously, per-cell transcript levels of the methane oxidation biomarker pmoA were found to vary quantitatively with respect to methane oxidation rates in the model aerobic methanotroph Methylosinus trichosporium OB3b. In the present study, these trends were explored for two additional aerobic methanotroph pure cultures grown in membrane bioreactors, Methylocystis parvus OBBP and Methylomicrobium album BG8. At steady-state conditions, per-cell pmoA mRNA transcript levels strongly correlated with per-cell methane oxidation across the three methanotrophs across many orders of magnitude of activity (R2 = 0.91). The inclusion of both type I and type II aerobic methanotrophs suggests a universal trend between in situ activity level and pmoA RNA biomarker levels which can aid in improving estimates of both subsurface and atmospheric methane. Additionally, genome-wide expression data (obtained by transcriptome sequencing [RNA-seq]) were used to explore transcriptomic responses of steady-state M. album BG8 cultures to short-term CH4 and O2 limitation. These limitations induced regulation of genes involved in central carbon metabolism (including carbon storage), cell motility, and stress response. IMPORTANCE Methanotrophs are naturally occurring microorganisms capable of oxidizing methane, having an impact on global net methane emissions. Additionally, they have also gained interest for their biotechnological applications in single-cell protein production, biofuels, and bioplastics. Having better ways of measuring methanotroph activity and understanding how methanotrophs respond to changing conditions is imperative for both optimization in controlled-growth applications and understanding in situ methane oxidation rates. In this study, we explored the applicability of methane oxidation biomarkers as a universal indicator of methanotrophic activity and explored methanotroph transcriptomic response to short-term changes in substrate availability. Our results contribute to better understanding the activity of aerobic methanotrophs, their core metabolic pathways, and their stress responses.

Although the study of aerobic methane-oxidizing bacteria (MOB, methanotrophs) has been carried out for more than a hundred years, there are many uncultivated methanotrophic lineages whose metabolism is largely unknown. Here, we reconstructed a nearly complete genome of a Beijerinckiaceae methanotroph from the enrichment of paddy soil by using nitrogen-free M2 medium. The methanotroph labeled as MO3_YZ.1 had a size of 3.83 Mb, GC content of 65.6%, and 3442 gene-coding regions. Based on phylogeny of pmoA gene and genome and the genomic average nucleotide identity, we confirmed its affiliation to the MO3 lineage and a close relationship to Methylocapsa. MO3_YZ.1 contained mxaF- and xoxF-type methanol dehydrogenase. MO3_YZ.1 used the serine cycle to assimilate carbon and regenerated glyoxylate through the glyoxylate shunt as it contained isocitrate lyase and complete tricarboxylic acid cycle-coding genes. The ethylmalonyl-CoA pathway and Calvin–Benson–Bassham cycle were incomplete in MO3_YZ.1. Three acetate utilization enzyme-coding genes were identified, suggesting its potential ability to utilize acetate. The presence of genes for N2 fixation, sulfur transformation, and poly-β-hydroxybutyrate synthesis enable its survival in heterogeneous habitats with fluctuating supplies of carbon, nitrogen, and sulfur. [ABSTRACT FROM AUTHOR]

Maintaining rice yield, soil function, and fertility are essential components of long-term compost fertilization. However, paddy fields are major sources of anthropogenic methane emissions. The aim of the study is to evaluate the changes in soil chemical properties and their concurrent impact on the abundance of methanogenesis (mcrA) and methane oxidation (pmoA) related genes among compost (Com), NPK+Compost (NPKCom), and unfertilized (NF) fallow paddy fields under long-term compost fertilization. Results showed that compost and NPK+Compost fertilization altered the soil chemical properties of paddy fields with a significant increase in the functional gene abundance potentially associated with Methanobacteriaceae for mcrA (1.23 × 106 to 3.84 × 106 copy number g−1 dry soil) and methane oxidizing bacteria such as Methylomonas and Methylobacter for pmoA (1.65 × 106 to 4.3 × 106 copy number g−1 dry soil). Ordination plots visualized these changes, where treatments clustered distinctly indicating that Com and NPKCom treatments were characterized by paddy soils with elevated OM, TN, K and P content and higher abundances of methanogenesis and methane oxidation related genes. The study showed that long-term compost fertilization resulted in paddy fields with high nutrient content and high gene abundance, attributed to methanogens and methane oxidizing bacteria that responded well with compost fertilization. These results indicated the potential of these fallow paddy fields for methane emission and methane oxidation and that they are 'primed', potentially influencing subsequent paddy field responses to long-term compost application. [ABSTRACT FROM AUTHOR]

Ammonium-induced stimulatory, inhibitory, and/or neutral effects on soil methane oxidation have been attributable to the ammonium concentration and mineral forms, confounded by other edaphic properties (e.g., pH, salinity), as well as the site-specific composition of the methanotrophic community. We hypothesize that this inconsistency may stem from the discrepancy in the cation adsorption capacity of the soil. We postulate that the effects of ammonium on the methanotrophic activity in soil are more accurately portrayed by relating methane uptake rates to the soluble ammonium (bioavailable), rather than the exchangeable (total) ammonium. To reduce adsorption (exchangeable) sites for ammonium in a paddy soil, two successive pre-incubation steps were introduced resulting in a 1000-fold soil dilution (soil enrichment), to be compared to a soil slurry (tenfold dilution) incubation. Ammonium was supplemented as NH4Cl at 0.5–4.75gL−1 after pre-incubation. While NH4Cl significantly stimulated the methanotrophic activity at all concentrations in the soil slurry incubation, methane uptake showed a dose-dependent effect in the soil enrichment. The trend in methane uptake could be explained by the soluble ammonium concentration, which was proportionate to the supplemented ammonium in the soil enrichment. In the soil slurry incubation, a fraction (36–63%) of the supplemented ammonium was determined to be adsorbed to the soil. Accordingly, Methylosarcina was found to predominate the methanotrophic community after the incubation, suggesting the relevance of this methanotroph at elevated ammonium levels (< 3.25gL−1 NH4Cl). Collectively, our results showed that the soluble, rather than the exchangeable ammonium concentration, is relevant when determining the effects of ammonium on methane oxidation, but this does not exclude other (a)biotic factors concurrently influencing methanotrophic activity. [ABSTRACT FROM AUTHOR]

Experiments were carried out to elucidate linkage between methane consumption and mineralization of phosphorous (P) from different P sources. The treatments were (i) no CH4 + no P amendment (absolute control), (ii) with CH4 + no P amendment (control), (iii) with CH4 + inorganic P as Ca3(PO4)2, and (iv) with CH4 + organic P as sodium phytate. P sources were added at 25 lg P·(g soil)-1. Soils were incubated to undergo three repeated CH4 feeding cycles, referred to as feeding cycle I, feeding cycle II, and feeding cycle III. CH4 consumption rate k (lg CH4 consumed·(g soil)-1·day-1) was 0.297 6 0.028 in no P amendment control, 0.457 60.016 in Ca3(PO4)2, and 0.627 6 0.013 in sodium phytate. Rate k was stimulated by 2 to 6 times over CH4 feeding cycles and followed the trend of sodium phytate > Ca3(PO4)2 > no P amendment control. CH4 consumption stimulated P solubilization from Ca3(PO4)2 by a factor of 2.86. Acid phosphatase (lg paranitrophenol released· (g soil)-1·h-1) was higher in sodium phytate than the no P amendment control. Abundance of 16S rRNA and pmoA genes increased with CH4 consumption rates. The results of the study suggested that CH4 consumption drives mineralization of unavailable inorganic and organic P sources in the soil ecosystem. [ABSTRACT FROM AUTHOR]

In stratified lakes, methane-oxidizing bacteria (MOB) are strongly mitigating methane fluxes to the atmosphere by consuming methane entering the water column from the sediments. MOB communities in lakes are diverse and vertically structured, but their spatio-temporal dynamics along the water column as well as physico-chemical parameters and interactions with other bacterial species that drive the community assembly have so far not been explored in depth. Here, we present a detailed investigation of the MOB and bacterial community composition and a large set of physico-chemical parameters in a shallow, seasonally stratified, and sub-alpine lake. Four highly resolved vertical profiles were sampled in three different years and during various stages of development of the stratified water column. Non-randomly assembled MOB communities were detected in all compartments. We could identify methane and oxygen gradients and physico-chemical parameters like pH, light, available copper and iron, and total dissolved nitrogen as important drivers of the MOB community structure. In addition, MOB were well-integrated into a bacterial-environmental network. Partial redundancy analysis of the relevance network of physico-chemical variables and bacteria explained up to 84% of the MOB abundances. Spatio-temporal MOB community changes were 51% congruent with shifts in the total bacterial community and 22% of variance in MOB abundances could be explained exclusively by the bacterial community composition. Our results show that microbial interactions may play an important role in structuring the MOB community along the depth gradient of stratified lakes.

Despite the recognition of streams and rivers as sources of methane (CH4) to the atmosphere, the role of CH4 oxidation (MOX) in these ecosystems remains poorly understood to date. Here, we measured the kinetics of MOX in stream sediments of 14 sites to resolve the ecophysiology of CH4 oxidizing bacteria (MOB) communities. The streams cover a gradient of land cover and associated physicochemical parameter and differed in stream- and porewater CH4 concentrations. Michealis–Menten kinetic parameter of MOX, maximum reaction velocity (Vmax), and CH4 concentration at half Vmax (KS) increased with CH4 supply. KS values in the micromolar range matched the CH4 concentrations measured in shallow stream sediments and indicate that MOX is mostly driven by low-affinity MOB. 16S rRNA gene sequencing identified MOB classified as Methylococcaceae and particularly Crenothrix. Their relative abundance correlated with pmoA gene counts and MOX rates, underscoring their pivotal role as CH4 oxidizers in stream sediments. Building on the concept of enterotypes, we identify two distinct groups of co-occurring MOB. While there was no taxonomic difference among the members of each cluster, one cluster contained abundant and common MOB, whereas the other cluster contained rare operational taxonomic units (OTUs) specific to a subset of streams. These integrated analyses of changes in MOB community structure, gene abundance, and the corresponding ecosystem process contribute to a better understanding of the distal controls on MOX in streams.

Although the study of aerobic methane-oxidizing bacteria (MOB, methanotrophs) has been carried out for more than a hundred years, there are many uncultivated methanotrophic lineages whose metabolism is largely unknown. Here, we reconstructed a nearly complete genome of a Beijerinckiaceae methanotroph from the enrichment of paddy soil by using nitrogen-free M2 medium. The methanotroph labeled as MO3_YZ.1 had a size of 3.83 Mb, GC content of 65.6%, and 3442 gene-coding regions. Based on phylogeny of pmoA gene and genome and the genomic average nucleotide identity, we confirmed its affiliation to the MO3 lineage and a close relationship to Methylocapsa. MO3_YZ.1 contained mxaF- and xoxF-type methanol dehydrogenase. MO3_YZ.1 used the serine cycle to assimilate carbon and regenerated glyoxylate through the glyoxylate shunt as it contained isocitrate lyase and complete tricarboxylic acid cycle-coding genes. The ethylmalonyl-CoA pathway and Calvin–Benson–Bassham cycle were incomplete in MO3_YZ.1. Three acetate utilization enzyme-coding genes were identified, suggesting its potential ability to utilize acetate. The presence of genes for N2 fixation, sulfur transformation, and poly-β-hydroxybutyrate synthesis enable its survival in heterogeneous habitats with fluctuating supplies of carbon, nitrogen, and sulfur.

Methanotrophic microorganisms are characterized by their ability to oxidize methane. Globally they have a significant impact on methane emissions by attenuating net methane fluxes to the atmosphere in natural and engineered systems, though the populations are dynamic in their activity level in soils and waters. Methanotrophs oxidize methane using methane monooxygenase (MMO) enzymes, and selected subunit genes of the most common MMOs, specifically pmoA and mmoX, are used as biomarkers for the presence and abundance of populations of bacterial methanotrophs. The relative expression of these biomarker genes is dependent on copper-to-biomass ratios. Empirically derived quantitative relationships between methane oxidation biomarker transcript amounts and methanotrophic activity could facilitate determination of methane oxidation rates. In this study, pure cultures of a model type II methanotroph, Methylosinus trichosporium OB3b, were grown in hollow-fiber membrane bioreactors (HFMBR) under different steady-state methane oxidation conditions. Methanotroph biomass (DNA based) and methane oxidation biomarker mRNA transcript amounts were determined using quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR), respectively. Under both copper-present and copperlimited conditions, per-cell pmoA mRNA transcript levels positively correlated with measured per-cell methane oxidation rates across 3 orders of magnitude. These correlations, if maintained across different methanotrophs, could prove valuable for inferring in situ oxidation rates of methanotrophs and understanding the dynamics of their impact on net methane emissions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In stratified lakes, methane-oxidizing bacteria (MOB) are strongly mitigating methane fluxes to the atmosphere by consuming methane entering the water column from the sediments. MOB communities in lakes are diverse and vertically structured, but their spatio-temporal dynamics along the water column as well as physico-chemical parameters and interactions with other bacterial species that drive the community assembly have so far not been explored in depth. Here, we present a detailed investigation of the MOB and bacterial community composition and a large set of physico-chemical parameters in a shallow, seasonally stratified, and sub-alpine lake. Four highly resolved vertical profiles were sampled in three different years and during various stages of development of the stratified water column. Non-randomly assembled MOB communities were detected in all compartments. We could identify methane and oxygen gradients and physico-chemical parameters like pH, light, available copper and iron, and total dissolved nitrogen as important drivers of the MOB community structure. In addition, MOB were well-integrated into a bacterial-environmental network. Partial redundancy analysis of the relevance network of physico-chemical variables and bacteria explained up to 84% of the MOB abundances. Spatio-temporal MOB community changes were 51% congruent with shifts in the total bacterial community and 22% of variance in MOB abundances could be explained exclusively by the bacterial community composition. Our results show that microbial interactions may play an important role in structuring the MOB community along the depth gradient of stratified lakes. [ABSTRACT FROM AUTHOR]

Microorganisms may reciprocally select for specific interacting partners, forming a network with interdependent relationships. The methanotrophic interaction network, comprising methanotrophs and non-methanotrophs, is thought to modulate methane oxidation and give rise to emergent properties beneficial for the methanotrophs. Therefore, microbial interaction may become relevant for community functioning under stress. However, empirical validation of the role and stressor-induced response of the interaction network remains scarce. Here, we determined the response of a complex methane-driven interaction network to a stepwise increase in NH4Cl-induced stress (0.5–4.75 g L−1, in 0.25–0.5 g L−1 increments) using enrichment of a naturally occurring complex community derived from a paddy soil in laboratory-scale incubations. Although ammonium and intermediates of ammonium oxidation are known to inhibit methane oxidation, methanotrophic activity was unexpectedly detected even in incubations with high ammonium levels, albeit rates were significantly reduced. Sequencing analysis of the 16S rRNA and pmoA genes consistently revealed divergent communities in the reference and stressed incubations. The 16S rRNA-based co-occurrence network analysis revealed that NH4Cl-induced stress intensification resulted in a less complex and modular network, likely driven by less stable interaction. Interestingly, the non-methanotrophs formed the key nodes, and appear to be relevant members of the community. Overall, stressor intensification unravels the interaction network, with adverse consequences for community functioning. [ABSTRACT FROM AUTHOR]

Combination products (CPs) are nothing but medical products that do not adequately apt solely into a regulatory category, but reasonably comprise of any combination of drug, device and biological product. The use of combination products and delivery systems are to deal with a chronic condition or to relieve an acute condition or to treat the various lives threaten & complex diseases. This article assesses the regulation of combination products in USA, India, China, Japan and Europe and likens the regulatory regime in pharmaceutical environments. Though many of these types of products have been available for years, recent FDA guidelines have spelled out viewpoint on both a development and commercialization perspective. These elucidations have led to augment regulatory expectations resulting in new and increased challenges. To make sure compliance with these regulations and beat challenges, it is essential to remain familiar with the dynamic changing regulations. This piece of writing spotlights on key aspects to understand the concept of combination products, its classification, review period and most importantly facts to consider when any organization is gearing up for a regulatory submission to regulatory agency and its prospective for PLCM of the combination product. [ABSTRACT FROM AUTHOR]

Methane-oxidizing bacteria (MOB) possess the metabolic potential to assimilate the highly potent greenhouse gas, CH 4 , and can also synthesize valuable products. Depending on their distinct and fastidious metabolic pathways, MOB are mainly divided into Type I and Type II; the latter are known as producers of polyhydroxyalkanoate (PHA). Despite the metabolic potential of MOB to synthesize PHA, the ecophysiology of MOB, especially under high CH 4 flux conditions, is yet to be understood. Therefore, in this study, a rice paddy soil receiving a high CH 4 flux from underground was used as an inoculum to enrich MOB using fed-batch operation, then the enriched Type II MOB were characterized. The transitions in the microbial community composition and CH 4 oxidation rates were monitored by 16S rRNA gene amplicon sequencing and degree of CH 4 consumption. With increasing incubation time, the initially dominant Methylomonas sp., affiliated with Type I MOB, was gradually replaced with Methylocystis sp., Type II MOB, resulting in a maximum CH 4 oxidation rate of 1.40 g-CH 4 /g-biomass/day. The quantification of functional genes encoding methane monooxygenase, pmoA and PHA synthase, phaC , by quantitative PCR revealed concomitant increases in accordance with the Type II MOB enrichment. These increases in the functional genes underscore the significance of Type II MOB to mitigate greenhouse gas emission and produce PHA. • Semi-batch incubation enriched Type II MOB inoculated from a high CH 4 emission paddy soil. • The enriched biomass possessed a maximum methane oxidizing rate of 1.40 g-CH 4 /g-biomass/day. • Microbial transitions from Type I Methylomonas to Type II Methylocystis were observed. • The enrichment procedure increased functional gene abundances of phaC and pmoA. [ABSTRACT FROM AUTHOR]

Anaerobic wastewater treatment offers several advantages; however, the effluent of anaerobic digesters still contains high levels of ammonium and dissolved methane that need to be removed before these effluents can be discharged to surface waters. The simultaneous anaerobic removal of methane and ammonium by denitrifying (N-damo) methanotrophs in combination with anaerobic ammonium-oxidizing (anammox) bacteria could be a potential solution to this challenge. After a molecular survey of a wastewater plant treating brewery effluent, indicating the presence of both N-damo and anammox bacteria, we started an anaerobic bioreactor with a continuous supply of methane, ammonium, and nitrite to enrich these anaerobic microorganisms. After 14 months of operation, a stable enrichment culture containing two types of 'Candidatus Methylomirabilis oxyfera' bacteria and two strains of 'Ca. Brocadia'-like anammox bacteria was achieved. In this community, anammox bacteria converted 80% of the nitrite with ammonium, while 'Ca. Methylomirabilis' contributed to 20% of the nitrite consumption. The analysis of metagenomic 16S rRNA reads and fluorescence in situ hybridization (FISH) correlated well and showed that, after 14 months, 'Ca. Methylomirabilis' and anammox bacteria constituted approximately 30 and 20% of the total microbial community. In addition, a substantial part (10%) of the community consisted of Phycisphaera-related planctomycetes. Assembly and binning of the metagenomic sequences resulted in high-quality draft genome of two 'Ca. Methylomirabilis' species containing the marker genes pmoCAB, xoxF, and nirS and putative NO dismutase genes. The anammox draft genomes most closely related to 'Ca. Brocadia fulgida' included the marker genes hzsABC, hao, and hdh. Whole-reactor and batch anaerobic activity measurements with methane, ammonium, nitrite, and nitrate revealed an average anaerobic methane oxidation rate of 0.12 mmol h−1 L−1 and ammonium oxidation rate of 0.5 mmol h−1 L−1. Together, this study describes the enrichment and draft genomes of anaerobic methanotrophs from a brewery wastewater treatment plant, where these organisms together with anammox bacteria can contribute significantly to the removal of methane and ammonium in a more sustainable way. Key points: • An enrichment culture containing both N-damo and anammox bacteria was obtained. • Simultaneous consumption of ammonia, nitrite, and methane under anoxic conditions. • In-depth metagenomic biodiversity analysis of inoculum and enrichment culture. [ABSTRACT FROM AUTHOR]

The effect of grazing on the abundance, composition, and methane (CH4) uptake of methanotrophs in grasslands has been well documented in the past few decades, but the dominant communities of active methanotrophs responsible for CH4 oxidation activity in grazed soils are still poorly understood. In this study, we characterized the metabolically active, aerobic methanotrophs in grasslands with three different levels of grazing (light, medium, and heavy) by combining DNA-stable isotope probing (SIP) and quantitative PCR (qPCR) for methane monooxygenase (pmoA) gene– and 16S rRNA gene–based amplicon sequencing. The CH4 oxidation potential was as low as 0.51 μmol g dry weight−1 day−1 in the ungrazed control, while it decreased as grazing intensity increased in grazed fields, ranging from 2.25 μmol g dry weight−1 day−1 in light grazed fields to 1.59 in heavily grazed fields. Increased CH4 oxidation activity was paralleled by twofold increases in abundance of pmoA genes and relative abundance of methanotroph-affiliated 16S rRNA genes in the total microbial community in grazed soils. SIP and sequencing revealed that the genera Methylobacter and Methylosarcina (type I; Gammaproteobacteria) and Methylocystis (type II; Alphaproteobacteria) were active methanotrophs responsible for CH4 oxidation in grazed soils. Light and intermediate grazing stimulated the growth and activity of methanotrophs, while heavy grazing decreased the abundance and diversity of the active methanotrophs in the typical steppe. Redundancy and correlation analysis further indicated that the variation of bulk density and soil C and N induced by grazing determined the abundance, diversity of active methanotrophs, and methane oxidation activity in the long-term grazed grassland soil. [ABSTRACT FROM AUTHOR]

Despite the recognition of streams and rivers as sources of methane (CH4) to the atmosphere, the role of CH4 oxidation (MOX) in these ecosystems remains poorly understood to date. Here, we measured the kinetics of MOX in stream sediments of 14 sites to resolve the ecophysiology of CH4 oxidizing bacteria (MOB) communities. The streams cover a gradient of land cover and associated physicochemical parameter and differed in stream- and porewater CH4 concentrations. Michealis–Menten kinetic parameter of MOX, maximum reaction velocity (V max ), and CH4 concentration at half V max (K S ) increased with CH4 supply. K S values in the micromolar range matched the CH4 concentrations measured in shallow stream sediments and indicate that MOX is mostly driven by low-affinity MOB. 16S rRNA gene sequencing identified MOB classified as Methylococcaceae and particularly Crenothrix. Their relative abundance correlated with pmoA gene counts and MOX rates, underscoring their pivotal role as CH4 oxidizers in stream sediments. Building on the concept of enterotypes, we identify two distinct groups of co-occurring MOB. While there was no taxonomic difference among the members of each cluster, one cluster contained abundant and common MOB, whereas the other cluster contained rare operational taxonomic units (OTUs) specific to a subset of streams. These integrated analyses of changes in MOB community structure, gene abundance, and the corresponding ecosystem process contribute to a better understanding of the distal controls on MOX in streams. [ABSTRACT FROM AUTHOR]

The pmoA gene number is considered as a soil microbiological parameter indicating abundance and potential activity of methanotrophic bacteria. The efficiency of this parameter for prediction and modeling of the real soil methane consumption rates remains an open issue. In the current study rate of methane oxidation by soil at its ambient concentration and the number of the pmoA genes determined by quantitative polymerase chain reaction were compared. Soil samples were collected in May, July, and September 2018 in the middle taiga subzone forest ecosystems near Khanty-Mansiysk, Russia. Soil methane oxidation rate varied from 0.01 to 8 ng СН4/g dry weight per hour. The pmoA gene numbers per g of dry weight varied from 107 to 109. The correlation between these two parameters became smaller as the scale changed from the soil profile level to ecosystem and interseasonal levels. Methane oxidation rate increased significantly above the threshold methanotroph abundance of 2 × 108pmoA genes/g. Within the intervals below and above this threshold value, no significant changes in methane oxidation rate occurred while methanotroph abundance increased. Thus, quantification of methanotroph abundance alone is insufficient for assessment of methanotrophic activity in upland soils, and the effect of other factors should be considered. [ABSTRACT FROM AUTHOR]

Understanding soil microbial populations influencing biogeochemical cycles with potential implications for greenhouse gas (GHG) fluxes emissions is crucial. Methanotrophic, nitrifying and N 2 O-reducing microorganisms are major drivers of CH 4 and N 2 O fluxes in soils. The metabolism of these organisms relies on enzymes that require as cofactors metal ions scarcely available in the soil, such as copper (Cu) and zinc (Zn). Despite the importance of these ions, how their concentrations relate to the abundance of these microbes at the global scale has not been addressed yet. Here, we used data from a global survey carried out in 47 drylands from 12 countries to evaluate the role of soil Cu and Zn concentrations, and their relationship with aridity, as drivers of the abundance of methanotrophs, archaeal and bacterial nitrifiers, and N 2 O reducers. To do so, we performed qPCR analyses of the marker genes pmoA , archaeal and bacterial amoA and nosZI. We did not find an association between the abundance of methanotrophs and Cu or Zn availability. However, our results highlight the importance of Cu influencing the abundance of nitrifying bacteria and N 2 O reducers, two main actors involved in the N 2 O cycle. Our findings indicate that dryland soils can be prone to reduce the N 2 O coming from nitrification to innocuous N 2 , but reductions in soil Cu availability (e.g., by increased aridity conditions due to climate change) could shift this trend. • The role of Cu and Zn on the abundance of microbes associated to GHG was evaluated. • Dryland soils may be prone to reduce N 2 O to N 2 through bacterial metabolism. • A greater Cu content promotes a lower potential N 2 O: N 2 emission ratio in drylands. • Increases in aridity may enhance N releasing to the atmosphere in the form of N 2 O. • Methanotrophs abundance was not associated to Cu or Zn in drylands. [ABSTRACT FROM AUTHOR]

Oxygenic denitrification represents a new route in reductive nitrogen turnover which differs from canonical denitrification in how nitric oxide (NO) is transformed into dinitrogen gas. Instead of NO reduction via N2O to N2, NO is proposed to be directly disproportionated into N2 and O2 in oxygenic denitrification, catalyzed by the putative NO dismutase (Nod). Although a high diversity of nod genes has been recovered from various environments, still little is known about the niche partitioning and ecophysiology of oxygenic denitrifiers. One constraint is that nod as a functional marker for oxygenic denitrifiers is not well established. To address this issue, we compared the diversity and phylogeny of nod, 16S rRNA and pmoA gene sequences of four NC10 enrichments that are capable of methane-driven oxygenic denitrification and one environmental sample. The phylogenies of nod, 16S rRNA and pmoA genes of these cultures were generally congruent. The diversity of NC10 bacteria inferred from different genes was also similar in each sample. A new set of NC10-specific nod primers was developed and used in qPCR. The abundance of NC10 bacteria inferred from nod genes was constantly lower than via 16S rRNA genes, but the difference was within one order of magnitude. These results suggest that nod is a suitable molecular marker for studying the diversity and phylogeny of methane-driven oxygenic denitrifiers, the further investigation of which may be of value to develop enhanced strategies for sustainable nitrogen or methane removal.

Oxygenic denitrification represents a new route in reductive nitrogen turnover which differs from canonical denitrification in how nitric oxide (NO) is transformed into dinitrogen gas. Instead of NO reduction via N2O to N2, NO is proposed to be directly disproportionated into N2 and O2 in oxygenic denitrification, catalyzed by the putative NO dismutase (Nod). Although a high diversity of nod genes has been recovered from various environments, still little is known about the niche partitioning and ecophysiology of oxygenic denitrifiers. One constraint is that nod as a functional marker for oxygenic denitrifiers is not well established. To address this issue, we compared the diversity and phylogeny of nod , 16S rRNA and pmoA gene sequences of four NC10 enrichments that are capable of methane-driven oxygenic denitrification and one environmental sample. The phylogenies of nod , 16S rRNA and pmoA genes of these cultures were generally congruent. The diversity of NC10 bacteria inferred from different genes was also similar in each sample. A new set of NC10-specific nod primers was developed and used in qPCR. The abundance of NC10 bacteria inferred from nod genes was constantly lower than via 16S rRNA genes, but the difference was within one order of magnitude. These results suggest that nod is a suitable molecular marker for studying the diversity and phylogeny of methane-driven oxygenic denitrifiers, the further investigation of which may be of value to develop enhanced strategies for sustainable nitrogen or methane removal. [ABSTRACT FROM AUTHOR]

• MOR of mudflats with rice planting and soil microbial communities was determined. • Long-term rice cultivation in mudflats promoted the MOR. • High MOR was related to the synergistic growth of MPA and MCB. • MOA coupled to sulfate and nitrite reductions contributed to the methane oxidation. This study aimed to determine the methane oxidation rates (MOR), pmoA gene abundance and diversity, and microbial community composition using Illumina high-throughput sequencing. Mudflats located within Yancheng City, divided into different plots with 0-, 11-, and 20-year successive rice planting histories, were selected and sampled. The study found that the relative MOR (normalized with the 16S rRNA gene) increased dramatically after 11-year cultivation and remained stable in 20-year treatment, indicating that long-term rice cultivation in mudflats promoted MOR. The sequencing data analysis revealed that high MOR was related to the synergistic growth of methane-producing archaea (MPA) and aerobic and facultative methane-consuming bacteria (MCB) mainly belonging to Proteobacteria. Redundancy and correlation analyses showed that Methylophilaceae and Methylococcaceae affiliated within β- and γ-Proteobacterial methanotrophs were closely related to the relative MOR. Methane-oxidizing archaea (MOA) coupled to sulfate and nitrite reductions contributed more to the high and stable MOR compared with Proteobacterial MCB. Chloroflexi and Geobacter were the potential hydrogen donors for hydrogenotrophic MPA. The results showed that long-term rice cultivation in mudflats promoted the relative MOR. The unknown MOA coupled to sulfate and nitrite reductions, besides the necessary hydrogenotrophic MPA and their hydrogen donors (Chloroflexi and Geobacter) collectively contributed to methane cycling. [ABSTRACT FROM AUTHOR]

Abstract Colonies of mammals and sea birds are known hotspots for biogeochemical cycles with potentially large element stocks and fast turnover in the soil. Although penguin and seal colonies are sites with potentially extremely fast biogeochemical turnover, these sites as potential sources of GHG have largely been neglected. This study aims to quantify magnitudes and environmental drivers of GHG fluxes effected by the activity of Antarctic fur seal (Arctocephalus gazella) and King penguin (Aptenodytes patagonicus) in South Georgia, sub-Antarctica. In-situ CO 2 and CH 4 gas flux measurements were combined with laboratory incubations of intact soil cores collected from sites along two transects away from a seal and a penguin colony. Variations in laboratory incubations were consistent with the in-situ fluxes measured in February 2017, and showed high ecosystem respiration rates at colonies (mean 44.3 μg CO 2 cm−3 h−1 for seal colony, and 52.9–159 μg CO 2 cm−3 h−1 for penguin colony) and a marked decrease away from these hotspots. Moderate methane production rates were found within the colonies (mean 0.1 ng CH 4 cm−3 h−1 for seal colony, 44–145.5 ng CH 4 cm−3 h−1 for penguin colony), while relatively high consumption rates (mean −1.8 ng CH 4 cm−3 h−1) occurred outside the colonies. Incubations also included N 2 O production rates, which were highly variable within the colonies (1.1–293 ng N 2 O cm−3 h−1 for seal colony, 0.8–594.7 ng N 2 O cm−3 h−1 for penguin colony) and decreased markedly to near zero away from the colonies. Bacterial and pmoA communities and drivers of GHG turnover and microbe community along both transects varied according to the sea animal activity intensity gradient. This is the first study at these latitudes to quantify the overall capacity of in-situ soil methane uptake at hot spots linked to sea animal colonies versus ambient conditions. All colony sites in this study were net sources of N 2 O, while sites beyond colonies, which dominate the ice-free areas in South Georgia, were sinks of methane. Highlights • High CH 4 uptake rates are observed in bare soil areas but decreased towarsds seal and penguin colonies. • Colonies were hotspots for CO 2 , CH 4 and N 2 O emissions due to large excreta deposits. • Spatial trends in gas rates were correlated to microbial community distribution. • Dominate methanotrophs differed between sites. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • High macro and microscopic robustness of CH 4 -treating bioreactors to CH 4 -shock load. • Both reactors (continuous and feast-famine feeding) were CH 4 mass transfer limited. • pmoA gene responded differently based on the previous CH 4 feeding regime. • More moderate response of pmoA gene to CH 4 -shock load in the feast-famine reactor. Abstract A rapid recovery of methanotrophic activity from process fluctuations such as surges in CH 4 inlet load is of utmost importance in bioreactors to guarantee a robust abatement of this potent greenhouse gas. However, the response of methanotrophs in CH 4 abatement engineered ecosystems facing recurrent operational upsets has not been systematically investigated. The genetic and process performance response of methanotrophs to a gradual 2.8-fold increase in CH 4 inlet load in continuous and feast-famine bioreactors was characterized by measuring their pmoA gene transcripts and the CH 4 degradation capacities and CO 2 production rates, respectively. Both bioreactors exhibited a high robustness in terms of CH 4 removal towards CH 4 inlet load increase. Higher CH 4 elimination and CO 2 production during shock load suggested the occurrence of CH 4 mass transfer limitations. Consistently, a rapid regulation of the pmoA gene (less than one hour after CH 4 -shock load), characterized by several transient events of increased expression, was observed in both bioreactors. However, the pmoA gene expression in the feast-famine bioreactor exhibited a less intense response to the increase in CH 4 load compared to the continuous system, which suggested a possible adaptation of the pmoA transcriptional response of methanotrophs to the previous history of the culture. [ABSTRACT FROM AUTHOR]

Abstract Aerobic oxidation of methane at (circum-)atmospheric concentrations (<40 ppm v) has long been assumed to be catalyzed by the as-yet-uncultured high-affinity methanotrophs in well-aerated, non-wetland (upland) soils, the only known biological methane sink globally. Although the low-affinity canonical methanotrophs with cultured representatives have been detected along with the high-affinity ones, their role as a methane sink in upland soils remains enigmatic. Here, we show that canonical methanotrophs can contribute to (circum-)atmospheric methane uptake in agricultural soils. We performed a stable-isotope 13C CH 4 labelling incubation in the presence and absence of bio-based residues that were added to the soil to track the flow of methane. Residue amendment transiently stimulated methane uptake rate (<50 days). Soil methane uptake was sustained throughout the incubation (130 days), concomitant to the enrichment of 13C CO 2. The 13C-enriched phospholipid fatty acids (PLFAs) were distinct in both soils, irrespective of amendments, and were unambiguously assigned almost exclusively to canonical alphaproteobacterial methanotrophs with cultured representatives. 16S rRNA and pmoA gene sequence analyses revealed that the as-yet-uncultured high-affinity methanotrophs were virtually absent in these soils. The stable-isotope labelling approach allowed to attribute soil methane uptake to canonical methanotrophs, whereas these were not expected to consume (circum-)atmospheric methane. Our findings thus revealed an overlooked reservoir of high-affinity methane-oxidizers represented by the canonical methanotrophs in agriculture-impacted upland soils. Given that upland agricultural soils have been thought to marginally or do not contribute to atmospheric methane consumption due to the vulnerability of the high-affinity methanotrophs, our findings suggest a thorough revisiting of the contribution of agricultural soils, and the role of agricultural management to mitigation of climate change. Highlights • Stable isotope (13C-CH 4) probing was used to detect high-affinity methanotrophs. • Soil methane uptake rate was stimulated by residue amendments. • The active methanotophs were determined based on their PLFA profiles. • 13C-enriched PLFA belonged to canonical alphaproteobacterial methanotrophs. • High-affinity methane oxidation was catalyzed by canonical methanotrophs. [ABSTRACT FROM AUTHOR]

Abstract The ability of methanotrophs to rapidly respond to intentional or accidental stress conditions caused by operational failures or process fluctuations is of utmost importance to guarantee the robustness of CH 4 abatement biotechnologies. In this study, the performance of a continuous and two feast-famine (5:5 days feast-famine cycles) stirred tank reactors treating diluted CH 4 emissions (4–5% v/v) was comparatively assessed for 149 days. The robustness of the three bioreactors towards a 5 days CH 4 deprivation episode was thoroughly evaluated at a molecular level (pmoA gene expression level) and correlated to macroscopic process performance. The bioreactors recovered their steady-state abatement performance (in terms of CH 4 elimination capacity and CO 2 production rate) within 1.5–2 h following CH 4 supply resumption concomitantly with a maximum in pmoA gene expression, regardless of the previous operational mode. However, while methanotrophs from the continuous unit maintained higher basal levels of pmoA expression as a strategy for a rapid CH 4 metabolism initiation, the strategy of the feast-famine adapted-methanotrophs consisted on a more accurate regulation of their pmoA transcripts levels along with a higher and/or more rapid induction of the pmoA gene by CH 4 availability. Graphical abstract Image 1 Highlights • High robustness of CH 4 -treatment biotechnologies towards inlet load fluctuations. • Recovery of CH 4 abatement performance within 1.5–2 h after CH 4 resumption. • Different recovery strategies (pmoA -based) of methanotrophs against CH 4 starvation. [ABSTRACT FROM AUTHOR]

Abstract There is considerable evidence that environmental properties are important for microbial niche partitioning in general. However, little is known about the environmental factors explaining this for soil methane-oxidising bacteria (or methanotrophs), which play an essential role in ecosystem functioning and climate regulation through mitigation of net CH 4 emissions worldwide. This knowledge gap limits the inclusion of taxon-based information to improve predictions of climate change-simulation models. In this study, 697 soil samples were collected across Scotland and 62 climo-edaphic properties were analysed. Combined with a set of hybrid geostatistical modelling approaches, the aim of this study was to investigate the biogeographical distribution (pmoA gene relative abundance) of key methanotrophic operational taxonomic units named Terminal-Restriction Fragments (T-RFs) and of methanotrophic community structure. The main objectives were to: 1) identify major environmental drivers influencing the distribution and composition of methanotrophs; and 2) perform spatial modelling and mapping of soil methanotrophic community assemblage and distribution of those dominant T-RFs. Herein, it was hypothesised that the assemblage of methanotrophic community and distribution of key populations across various landscapes could be predicted using a range of climo-edaphic factors optimised for spatial, climate and terrain attributes. The findings presented here suggest that the distribution of methanotrophs is strongly linked to land use and some edaphic properties, predominantly soil moisture/rainfall, nutrients and metal ions. The hybrid geostatistical approach allowed for spatial prediction of methanotrophic T-RFs and community, and demonstrated a clear niche partitioning between dominant T-RFs. Overall, these results provide novel evidence that the distribution of methanotrophs could be explained and mapped in terms of niche partitioning and predicted at the regional scale. The findings of the present study have significance for the sustainable management of ecosystems and improvement of simulation models for better prediction of ecosystem functions under predicted global changes. Highlights • Methanotroph population investigated at a large, geographical scale across Scotland. • New modelling approach identified ecosystem drivers of methanotroph distribution. • Niche-partitioning showed methanotroph populations driven by ecosystem properties. • Incorporation of microbial data into simulation models and management policies. [ABSTRACT FROM AUTHOR]

We investigated the response of aerobic methane oxidation and the associated methanotrophs to salt-stress in a NaCl gradient ranging from 0 M (un-amended reference) to 0.6 M NaCl (seawater salinity) using a rice paddy soil as a model system. Salt-stress significantly inhibited methanotrophic activity at > 0.3 M NaCl; at 0.6 M NaCl amendment, methanotrophic activity fully ceased. MiSeq sequencing of the pmoA gene and group-specific qPCR analyses revealed that type Ia methanotroph ( Methylobacter ) appeared to be favored under salinity up to 0.3 M NaCl, increasing in numerical abundance, while the type Ib was adversely affected. This suggests niche differentiation within members of the gammaproteobacterial methanotrophs. Overall, rice paddy soil methanotrophs showed remarkable resistance to salt-stress. [ABSTRACT FROM AUTHOR]

Paddy soil accounts for 10% of global atmospheric methane (CH 4 ) emissions. Many types of fertilizers may enhance CH 4 emissions, especially organic fertilizer. The aim of this study was to explore the effects of different fertilizers on CH 4 and methanogen patterns in paddy soil. This experiment involved four treatments: chemical fertilizer (CT), organic fertilizer (OT), mixed with chemical and organic fertilizer (MT), and no fertilizer (ctrl). The three fertilization treatments were applied with total nitrogen at the same rate of 300 kg N ha −1 . Paddy CH 4 , soil physicochemical variables and methanogen communities were quantitatively analyzed. Rhizosphere soil mcrA and pmoA gene copy numbers were determined by qPCR. Methanogenic 16S rRNA genes were identified by MiSeq sequencing. The results indicated CH 4 emissions were significantly higher in OT (145.31 kg ha −1 ) than MT (84.62 kg ha −1 ), CT (77.88 kg ha −1 ) or ctrl (32.19 kg ha −1 ). Soil organic acids were also increased by organic fertilization. CH 4 effluxes were significantly and negatively related to mcrA and pmoA gene copy numbers, and positively related to mcrA / pmoA . Above all, hydrogenotrophic Methanocella and acetoclastic Methanosaeta were the predominant methanogenic communities; these communities were strictly associated with soil potassium, oxalate, acetate, and succinate. Application of organic fertilizer promoted the dominant acetoclastic methanogens, but suppressed the dominant hydrogenotrophic methanogens. The transformation in methanogenic community structure and enhanced availability of C substrates may explain the increased CH 4 production in OT compared to other treatments. Compared to OT, MT may partially mitigate CH 4 emissions while guaranteeing a high rice yield. On this basis, we recommend the local fertilization pattern should change from 300 N kg ha −1 of organic manure to the same level of mixed fertilization. Moreover, we suggest multiple combinations of mixed fertilization merit more investigation in the future. [ABSTRACT FROM AUTHOR]

A broad methanotrophic community consisting of 16 different operational taxonomic units (OTUs) was detected by particulate methane monooxygenase A (pmoA) gene analyses of reactor sludge samples obtained from an industrial biogas plant. Using a cloning-sequencing approach, 75% of the OTUs were affiliated to the group of type I methanotrophs (γ-Proteobacteria) and 25% to type II methanotrophs (α-Proteobacteria) with a distinct predominance of the genus Methylobacter. By database matching, half of the total OTUs may constitute entirely novel species. For evaluation of process conditions that support growth of methanotrophic bacteria, qPCR analyses of pmoA gene copy numbers were performed during a sampling period of 70 days at varying reactor feeding scenarios. During the investigation period, methanotrophic cell counts estimated by qPCR fluctuated between 3.4 × 10 4 and 2 × 10 5 cells/mL with no distinct correlation to the organic loading rate, the amount of CH 4 , O 2 and NH 4 -N. Methanotrophic activity was proofed even at low O 2 levels (1%) by using stable carbon isotope labelling experiments of CH 4 in batch experiments inoculated with reactor sludge. Supplementation of 13 C labelled CH 4 in the headspace of the reaction vials unambiguously confirmed the formation of 13 C labelled CO 2 . Thus, industrial biogas reactors can be considered as a further methanotrophic habitat that exhibits a unique methanotrophic community which is specifically adapted to high CH 4 and low O 2 concentrations. To the best of our knowledge, our study is the first accurate detection and quantification of methanotrophic bacteria in industrial biogas reactors. [ABSTRACT FROM AUTHOR]

Sitting at ∼5,000 m water depth on the Congo-Angola margin and ∼760 km offshore of the West African coast, the recent lobe complex of the Congo deep-sea fan receives large amounts of fluvial sediments (3–5% organic carbon). This organic-rich sedimentation area harbors habitats with chemosynthetic communities similar to those of cold seeps. In this study, we investigated relative abundance, diversity and distribution of aerobic methane-oxidizing bacteria (MOB) communities at the oxic–anoxic interface of sedimentary habitats by using fluorescence in situ hybridization and comparative sequence analysis of particulate mono-oxygenase (pmoA) genes. Our findings revealed that sedimentary habitats of the recent lobe complex hosted type I and type II MOB cells and comparisons of pmoA community compositions showed variations among the different organic-rich habitats. Furthermore, the pmoA lineages were taxonomically more diverse compared to methane seep environments and were related to those found at cold seeps. Surprisingly, MOB phylogenetic lineages typical of terrestrial environments were observed at such water depth. In contrast, MOB cells or pmoA sequences were not detected at the previous lobe complex that is disconnected from the Congo River inputs.

Methanotrophic bacteria in soil serve as the only known biological sink of atmospheric methane, and impact net methane emission from soils. Most members of methanotrophs use particulate methane monooxygenase enzyme, encoded by the pmoA gene, to oxidize methane. Here, Illumina sequencing (iTAG) of pmoA gene was carried out to examine methanotrophic bacterial diversity in soils under different land use. Additionally, the effect of soil-type on pmoA amplicon sequence abundance was analyzed. Two sites in Ohio, each comprising of long-term no-tillage plots, plow-tillage plots, grassland, and forest were sampled. The tillage plots also included continuous-corn and corn-soybean crop rotations. By referencing a curated database of the pmoA gene, we were able to identify eight genera of methanotrophs with varying relative abundances in the samples. Additionally, four genera were differentially abundant across samples (alpha = 0.005) and included Methylocystis , Methylocococcus , Methylosoma , and USCa . About 30% of processed sequences were unclassified. Nonmetric multidimensional scaling (NMDS) of unweighted UniFrac and Bray-Curtis metrics showed clear community distinction based on sites. Soil type had the most significant effect (p < 0.005) on community (dis)similarity, despite the lack of clear diversity patterns with respect to land use. Although community composition of samples from agricultural fields appeared to cluster closer to each other as compared to forested areas and grass areas, nominal variable modeling showed soil type (which were specific to the sites sampled) as the strongest driver of methanotrophic community structure. This study was the first to employ high-throughput paired-end iTAG sequencing of a functional gene to understand microbial community dynamics. Our findings also indicate that functional gene sequencing on advanced sequencing platforms may be used to infer community dynamics of microbes belonging to unique functional guilds. This study also highlights the need to develop functional genes' reference databases that are compatible with the current iTAG sequence analyses software. [ABSTRACT FROM AUTHOR]

Soddy-podzolic soils are widely distributed in European Russia, but their role as a sink for atmospheric methane is poorly documented and there is no information on the methanotroph diversity. We analysed the potential CH 4 -oxidation rates in soil samples and showed that the rate was significantly higher in forest soil than in arable soil, 1.21 and 0.40 ng CH 4 g soil −1 day −1 , respectively. PCR-DGGE and clone library analysis indicated the distinct methanotrophic communities in these soils. The pmoA sequences associated with uncultured soil methanotrophs, referred to as NUSC, dominated forest soil, while in agricultural soil, type I ( Methylobacter, Methylocaldum ) and type II ( Methylocystis, Methylosinus ) methanotrophs were dominant. A newly developed primer set was applied in qPCR analysis and revealed that the copy number of pmoA genes of NUSC methanotrophs in forest soil was (9.2 ± 0.87) × 10 4 g soil −1 , whereas the transcript number was (1.33 ± 0.31) × 10 6 g soil −1 . We concluded that differences between the CH 4 oxidation rates between forest and agriculture soils were driven by the structure of the methane-oxidizing community and that a novel group of methanotrophs may be an active participant in this process. [ABSTRACT FROM AUTHOR]

Glacier forefield soils can provide a substantial sink for atmospheric CH4, facilitated by aerobic methane-oxidizing bacteria (MOB). However, MOB activity, abundance, and community structure may be affected by soil age, MOB location in different forefield landforms, and temporal fluctuations in soil physical parameters. We assessed the spatial and temporal variability of atmospheric-CH4 oxidation in an Alpine glacier forefield during the snow-free season of 2013. We quantified CH4 flux in soils of increasing age and in different landforms (sandhill, terrace, and floodplain forms) by using soil gas profile and static flux chamber methods. To determine MOB abundance and community structure, we employed pmoA gene-based quantitative PCR and targeted amplicon sequencing. Uptake of CH4 increased in magnitude and decreased in variability with increasing soil age. Sandhill soils exhibited CH4 uptake rates ranging from -3.7 to -0.03 mg CH4 m-2 day-1. Floodplain and terrace soils exhibited lower uptake rates and even intermittent CH4 emissions. Linear mixed-effects models indicated that soil age and landform were the dominating factors shaping CH4 flux, followed by cumulative rainfall (weighted sum ≤4 days prior to sampling). Of 31 MOB operational taxonomic units retrieved, ~30% were potentially novel, and ~50% were affiliated with upland soil clusters gamma and alpha. The MOB community structures in floodplain and terrace soils were nearly identical but differed significantly from the highly variable sandhill soil communities. We concluded that soil age and landform modulate the soil CH4 sink strength in glacier forefields and that recent rainfall affects its shortterm variability. This should be taken into account when including this environment in future CH4 inventories. [ABSTRACT FROM AUTHOR]

Sitting at ~5,000 m water depth on the Congo-Angola margin and 760 km offshore of the West African coast, the recent lobe complex of the Congo deep-sea fan receives large amounts of fluvial sediments (3-5% organic carbon). This organic-rich sedimentation area harbors habitats with chemosynthetic communities similar to those of cold seeps. In this study, we investigated relative abundance, diversity and distribution of aerobic methane-oxidizing bacteria (MOB) communities at the oxic-anoxic interface of sedimentary habitats by using fluorescence in situ hybridization and comparative sequence analysis of particulate mono-oxygenase (pmoA) genes. Our findings revealed that sedimentary habitats of the recent lobe complex hosted type I and type II MOB cells and comparisons of pmoA community compositions showed variations among the different organic-rich habitats. Furthermore, the pmoA lineages were taxonomically more diverse compared to methane seep environments and were related to those found at cold seeps. Surprisingly, MOB phylogenetic lineages typical of terrestrial environments were observed at such water depth. In contrast, MOB cells or pmoA sequences were not detected at the previous lobe complex that is disconnected from the Congo River inputs. [ABSTRACT FROM AUTHOR]

Proteobacteria capable of converting the greenhouse gas methane to biomass, energy, and carbon dioxide represent a small but important sink in global methane inventories. Currently, 23 genera of methane oxidizing (methanotrophic) proteobacteria have been described, although many are represented by only a single validly described species. Here we describe a new methanotrophic isolate that shares phenotypic characteristics and phylogenetic relatedness with the marine methanotroph Methylomarinum vadi. However, the new isolate derives from a terrestrial saline mud pot at the northern terminus of the Eastern Pacific Rise (EPR). This new cultivar expands our knowledge of the ecology of Methylomarinum, ultimately towards a fuller understanding of the role of this genus in global methane cycling.