Diverse type I and type II methanotrophs cultivated from an Indian freshwater wetland habitat.

Wetlands are the main natural sources of methane emissions, which make up a significant portion of greenhouse gas emissions. Such wetland patches serve as rich habitats for aerobic methanotrophs. Limited knowledge of methanotrophs from tropical wetlands widens the scope of study from these habitats. In the present study, a freshwater wetland in a tropical region in India was sampled and serially diluted to obtain methanotrophs in culture. This was followed by the isolation of methanotrophs on agarose-containing plates, incubated under methane: air atmosphere. Methanotrophs are difficult to cultivate, and very few cultures of methanotrophs are available from tropical wetlands. Our current study reports the cultivation of a diverse community of methanotrophs from six genera, namely, Methylomonas, Methylococcus, Methylomagnum, Methylocucumis (type I methanotrophs) along with Methylocystis, Methylosinus (type II methanotrophs). A high abundance of methanotrophs (106-1010 methanotrophs/g fresh weight) was observed in the samples. A Methylococcus strain could represent a putative novel species that was also isolated. Cultures of Methylomagnum and Methylocucumis, two newly described type I methanotrophs exclusively found in rice fields, were obtained. A large number of Methylomonas koyamae strains were cultured. Our study is pioneering in the documentation of culturable methanotrophs from a typical tropical wetland patch. The isolated methanotrophs can act as models for studying methanotroph-based methane mitigation from wetland habitats and can be used for various mitigation and valorization applications.

Linking Transcriptional Dynamics of Peat Microbiomes to Methane Fluxes during a Summer Drought in Two Rewetted Fens.

Rewetted peatlands are reestablished hot spots for CH4 emissions, which are subject to increased drought events in the course of climate change. However, the dynamics of soil methane-cycling microbiomes in rewetted peatlands during summer drought are still poorly characterized. Using a quantitative metatranscriptomic approach, we investigated the changes in the transcript abundances of methanogen and methanotroph rRNA, as well as mcrA and pmoA mRNA before, during, and after the 2018 summer drought in a coastal and a percolation fen in northern Germany. Drought changed the community structure of methane-cycling microbiomes and decreased the CH4 fluxes as well as the rRNA and mRNA transcript abundances of methanogens and methanotrophs, but they showed no recovery or increase after the drought ended. The rRNA transcript abundance of methanogens was not correlated with CH4 fluxes in both fens. In the percolation fen, however, the mcrA transcript abundance showed a positive and significant correlation with CH4 fluxes. Importantly, when integrating pmoA abundance, a stronger correlation was observed between CH4 fluxes and mcrA/pmoA, suggesting that relationships between methanogens and methanotrophs are the key determinant of CH4 turnover. Our study provides a comprehensive understanding of the methane-cycling microbiome feedbacks to drought events in rewetted peatlands.

Vallisneria natans decreased CH4 fluxes in wetlands: Interactions among plant physiological status, nutrients and epiphytic bacterial community.

Submerged macrophytes provide niches for epiphytic microbes (including aerobic methanotrophs) growth. However, little is known about the impacts of submerged macrophytes growth status and nutrients loadings on methanotroph community and methane release in wetlands. In the present study, methane fluxes, bacterial and methanotroph community in epiphytic biofilm, and environmental parameters were investigated during Vallisneria natans senescence in wetlands under low (VnL) and high (VnH) nutrients for seven weeks. Relative conductivity and concentration of H2O2, total chlorophyll and malondialdehyde were higher in leaves of V. natans in VnH than VnL at the same sampling time. Nutrients loading increased methane fluxes in treatments with or without (Control) macrophytes, while healthy V. natans plants reduced the methane flux and nutrients concentration in water columns. CH4 fluxes were positively correlated to temperature and COD (p < 0.05). Methane oxidation rates were 3.04-31.68 µmol methane mg-1 fresh weight of V. natans leaves - epiphytic biofilm within 1 h. Proteobacteria, Cyanobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, Actinobacteria and Acidobacteria were dominant phylum in all epiphytic biofilms. The mean abundances of pmoA/16S rRNA were higher in VnL than VnH. According to Illumina sequencing results of pmoA gene, γ-proteobacteria and α-proteobacteria were the dominant methanotroph class in epiphytic biofilm from VnH and VnL, respectively. Among seven detected methanotrophic genera, Methylomonas was significantly higher in VnH than VnL. Network analysis revealed that there were much closer relationships between the environmental parameters and epiphytic bacterial community in VnH than in VnL. COD and MDA were negatively correlated with Methyloglobulus, Methylosarcina, Methylobacter and Methylocystis, but positively correlated with Methylomonas and Methylosinus. This study highlights that methanotrophs in epiphytic biofilm play important roles in methane-oxidizing, which can be affected by plant physiological status and environmental parameters.

Soil edaphic factors and climate seasonality explain the turnover of methanotrophic communities in riparian wetlands.

Aerobic CH4-oxidizing bacteria (methanotrophs) represent a biological model system for the removal of atmospheric CH4, which is sensitive to the dynamics of water tables. However, little attention has been given to the turnover of methanotrophic communities across wet and dry periods in riparian wetlands. Here, by sequencing the pmoA gene, we investigated the turnover of soil methanotrophic communities across wet and dry periods in typical riparian wetlands that experience intensive agricultural practices. The results demonstrated that the methanotrophic abundance and diversity were significantly higher in the wet period than in the dry period, probably owing to the climatic seasonal succession and associated variation in soil edaphic factors. The co-occurrence patterns of the interspecies association analysis demonstrated that the key ecological clusters (i.e., Mod#1, Mod#2, Mod#4, Mod#5) showed contrasting correlations with soil edaphic properties between wet and dry periods. The linear regression slope of the relationships between the relative abundance of Mod#1 and the carbon to nitrogen ratio was higher in the wet period than in the dry period, whereas the linear regression slope of the relationships between the relative abundance of Mod#2 and soil nitrogen content (i.e., dissolved organic nitrogen, nitrate, and total nitrogen) was higher in the dry period than in the wet period. Moreover, Stegen's null model combined with phylogenetic group-based assembly analysis demonstrated that the methanotrophic community exhibited a higher proportion of drift (55.0%) and a lower contribution of dispersal limitation (24.5%) in the wet period than in the dry period (43.8% and 35.7%, respectively). Overall, these findings demonstrate that the turnover of methanotrophic communities across wet and dry periods were soil edaphic factors and climate dependent.

Effects of abrupt and gradual increase of atmospheric CO2 concentration on methanotrophs in paddy fields.

The simulation of abrupt atmospheric CO2 increase is a common way to examine the response of soil methanotrophs to future climate change. However, atmosphere is undergoing a gradual CO2 increase, and it is unknown whether the previously reported response of methanotrophs to abrupt CO2 increase can well represent their response to the gradual increase. To improve the understanding of the effect of elevated CO2 (eCO2) on methanotrophs in paddy ecosystems, the methane oxidation potential and communities of methanotrophs were examined via open top chambers under the three following CO2 treatments: an ambient CO2 concentration (AC); an abrupt CO2 increase by 200 ppm above AC (AI); a gradual CO2 increase by 40 ppm each year until 200 ppm above AC (GI). Relative to AC treatment, AI and GI treatments significantly (p < 0.05) increased the methane oxidation rate by 43.8% and 36.7%, respectively, during rice growth period. Furthermore, the abundance of pmoA genes was significantly (p < 0.05) increased by 62.4% and 32.5%, respectively, under AI and GI treatments. However, there were no significant variations in oxidation rate or gene abundance between the two eCO2 treatments. In addition, no obvious change of overall community composition of methanotrophs was observed among treatments, while the proportions of Methylosarcina and Methylocystis significantly (p < 0.05) changed. Taken together, our results indicate similar response of methanotrophs to abrupt and gradual CO2 increase, although the magnitude of response under gradual increase was smaller and the abrupt increase may somewhat overestimate the response.

Aerobic Methanotrophy and Co-occurrence Networks of a Tropical Rainforest and Oil Palm Plantations in Malaysia.

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.

Methanotrophic Community Detected by DNA-SIP at Bertioga's Mangrove Area, Southeast Brazil.

Methanotrophic bacteria can use methane as sole carbon and energy source. Its importance in the environment is related to the mitigation of methane emissions from soil and water to the atmosphere. Brazilian mangroves are highly productive, have potential to methane production, and it is inferred that methanotrophic community is of great importance for this ecosystem. The scope of this study was to investigate the functional and taxonomic diversity of methanotrophic bacteria present in the anthropogenic impacted sediments from Bertioga´s mangrove (SP, Brazil). Sediment sample was cultivated with methane and the microbiota actively involved in methane oxidation was identified by DNA-based stable isotope probing (DNA-SIP) using methane as a labeled substrate. After 4 days (96 h) of incubation and consumption of 0.7 mmol of methane, the most active microorganisms were related to methanotrophs Methylomonas and Methylobacter as well as to methylotrophic Methylotenera, indicating a possible association of these bacterial groups within a methane-derived food chain in the Bertioga mangrove. The abundance of genera Methylomonas, able to couple methane oxidation to nitrate reduction, may indicate that under low dissolved oxygen tensions, some aerobic methanotrophs could shift to intraerobic methane oxidation to avoid oxygen starvation.

Do methanotrophs drive phosphorus mineralization in soil ecosystem?

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 µg 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 (µg CH4 consumed·(g soil)-1·day-1) was 0.297 ± 0.028 in no P amendment control, 0.457 ± 0.016 in Ca3(PO4)2, and 0.627 ± 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 (µg 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.

Methane Monooxygenase Gene Transcripts as Quantitative Biomarkers of Methanotrophic Activity in Methylosinus trichosporium OB3b.

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 copper-limited 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.IMPORTANCE Methanotrophs are naturally occurring microorganisms capable of oxidizing methane and have an impact on global net methane emissions. The genes pmoA and mmoX are used as biomarkers for bacterial methanotrophs. Quantitative relationships between transcript amounts of these genes and methane oxidation rates could facilitate estimation of methanotrophic activity. In this study, a strong correlation was observed between per-cell pmoA transcript levels and per-cell methane oxidation rates for pure cultures of the aerobic methanotroph M. trichosporium OB3b grown in bioreactors. If similar relationships exist across different methanotrophs, they could prove valuable for inferring in situ oxidation rates of methanotrophs and better understanding their impact on net methane emissions.

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

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

Population dynamics of methanogens and methanotrophs along the salinity gradient in Pearl River Estuary: implications for methane metabolism.

Methane, a major greenhouse gas, plays an important role in global carbon cycling and climate change. Methanogenesis is identified as an important process for methane formation in estuarine sediments. However, the metabolism of methane in the water columns of estuaries is not well understood. The goal of this research was to examine the dynamics in abundance and community composition of methanogens and methanotrophs, and to examine whether and how they take part in methane metabolism in the water columns from the lower Pearl River (freshwater) to the coastal South China Sea (seawater). Quantitative PCR (qPCR) and high-throughput sequencing results showed that the abundance of methanogens decreased with increasing salinity, suggesting that growth of these methanogens in the Pearl River Estuary may be influenced by high salinity. Also, the methane concentration in surface waters was lower than that in near-bottom waters at most sites, suggesting sediment methanogens are a likely source of methane. In the estuarine mixing zone, significantly high methane concentrations existed with the presence of salt-tolerant methanogens (e.g., Methanomicrobiaceae, Methanocella, Methanosaeta and Methanobacterium) and methanotrophs (e.g., Methylocystis and Methylococcaceae), which were found in brackish habitats. Furthermore, a number of methanotrophic OTUs (from pmoA gene sequence data) had specific positive correlations with methanogenic OTUs (from mcrA gene sequence data), and some of these methanogenic OTUs were correlated with concentrations of particulate organic carbon (POC). The results indicate that methanotrophs and methanogens may be intimately linked in methane metabolism attached with particles in estuarine waters.

Diversity, enrichment, and genomic potential of anaerobic methane- and ammonium-oxidizing microorganisms from a brewery wastewater treatment plant.

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.

Community Structure of Active Aerobic Methanotrophs in Red Mangrove (Kandelia obovata) Soils Under Different Frequency of Tides.

Methanotrophs are important microbial communities in coastal ecosystems. They reduce CH4 emission in situ, which is influenced by soil conditions. This study aimed to understand the differences in active aerobic methanotrophic communities in mangrove forest soils experiencing different inundation frequency, i.e., in soils from tidal mangroves, distributed at lower elevations, and from dwarf mangroves, distributed at higher elevations. Labeling of pmoA gene of active methanotrophs using DNA-based stable isotope probing (DNA-SIP) revealed that methanotrophic activity was higher in the dwarf mangrove soils than in the tidal mangrove soils, possibly because of the more aerobic soil conditions. Methanotrophs affiliated with the cluster deep-sea-5 belonging to type Ib methanotrophs were the most dominant methanotrophs in the fresh mangrove soils, whereas type II methanotrophs also appeared in the fresh dwarf mangrove soils. Furthermore, Methylobacter and Methylosarcina were the most important active methanotrophs in the dwarf mangrove soils, whereas Methylomonas and Methylosarcina were more active in the tidal mangrove soils. High-throughput sequencing of the 16S ribosomal RNA (rRNA) gene also confirmed similar differences in methanotrophic communities at the different locations. However, several unclassified methanotrophic bacteria were found by 16S rRNA MiSeq sequencing in both fresh and incubated mangrove soils, implying that methanotrophic communities in mangrove forests may significantly differ from the methanotrophic communities documented in previous studies. Overall, this study showed the feasibility of 13CH4 DNA-SIP to study the active methanotrophic communities in mangrove forest soils and revealed differences in the methanotrophic community structure between coastal mangrove forests experiencing different tide frequencies.

High Temporal and Spatial Variability of Atmospheric-Methane Oxidation in Alpine Glacier Forefield Soils.

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 short-term variability. This should be taken into account when including this environment in future CH4 inventories.IMPORTANCE Oxidation of methane (CH4) in well-drained, "upland" soils is an important mechanism for the removal of this potent greenhouse gas from the atmosphere. It is largely mediated by aerobic, methane-oxidizing bacteria (MOB). Whereas there is abundant information on atmospheric-CH4 oxidation in mature upland soils, little is known about this important function in young, developing soils, such as those found in glacier forefields, where new sediments are continuously exposed to the atmosphere as a result of glacial retreat. In this field-based study, we investigated the spatial and temporal variability of atmospheric-CH4 oxidation and associated MOB communities in Alpine glacier forefield soils, aiming at better understanding the factors that shape the sink for atmospheric CH4 in this young soil ecosystem. This study contributes to the knowledge on the dynamics of atmospheric-CH4 oxidation in developing upland soils and represents a further step toward the inclusion of Alpine glacier forefield soils in global CH4 inventories.

Oxidative mitigation of aquatic methane emissions in large Amazonian rivers.

The flux of methane (CH4 ) from inland waters to the atmosphere has a profound impact on global atmospheric greenhouse gas (GHG) levels, and yet, strikingly little is known about the dynamics controlling sources and sinks of CH4 in the aquatic setting. Here, we examine the cycling and flux of CH4 in six large rivers in the Amazon basin, including the Amazon River. Based on stable isotopic mass balances of CH4 , inputs and outputs to the water column were estimated. We determined that ecosystem methane oxidation (MOX) reduced the diffusive flux of CH4 by approximately 28-96% and varied depending on hydrologic regime and general geochemical characteristics of tributaries of the Amazon River. For example, the relative amount of MOX was maximal during high water in black and white water rivers and minimal in clear water rivers during low water. The abundance of genetic markers for methane-oxidizing bacteria (pmoA) was positively correlated with enhanced signals of oxidation, providing independent support for the detected MOX patterns. The results indicate that MOX in large Amazonian rivers can consume from 0.45 to 2.07 Tg CH4 yr(-1) , representing up to 7% of the estimated global soil sink. Nevertheless, climate change and changes in hydrology, for example, due to construction of dams, can alter this balance, influencing CH4 emissions to atmosphere.

Evaluation and update of cutoff values for methanotrophic pmoA gene sequences.

The functional pmoA gene is frequently used to probe the diversity and phylogeny of methane-oxidizing bacteria (MOB) in various environments. Here, we compared the similarities between the pmoA gene and the corresponding 16S rRNA gene sequences of 77 described species covering gamma- and alphaproteobacterial methanotrophs (type I and type II MOB, respectively) as well as methanotrophs from the phylum Verrucomicrobia. We updated and established the weighted mean pmoA gene cutoff values on the nucleotide level at 86, 82, and 71 % corresponding to the 97, 95, and 90 % similarity of the 16S rRNA gene. Based on these cutoffs, the functional gene fragments can be entirely processed at the nucleotide level throughout software platforms such as Mothur or QIIME which provide a user-friendly and command-based alternative to amino acid-based pipelines. Type II methanotrophs are less divergent than type I both with regard to ribosomal and functional gene sequence similarity and GC content. We suggest that this agrees with the theory of different life strategies proposed for type I and type II MOB.

Co-occurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in subtropical acidic forest soils.

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two new processes of recent discoveries linking the microbial nitrogen and carbon cycles. In this study, 16S ribosomal RNA (rRNA) gene of anammox bacteria and pmoA gene of n-damo bacteria were used to investigate their distribution and diversity in natural acidic and re-vegetated forest soils. The 16S rRNA gene sequences retrieved featured at least three species in two genera known anammox bacteria, namely Candidatus Brocadia anammoxidans, Candidatus Brocadia fulgida, and Candidatus Kuenenia stuttgartiensis while the pmoA gene amplified was affiliated with two species of known n-damo bacteria Candidatus Methylomirabilis oxyfera and a newly established Candidatus Methylomirabilis sp. According to the results, the diversity of anammox bacteria in natural forests was lower than in re-vegetated forests, but no significant difference was observed in n-damo community between them. Quantitative real-time PCR showed that both anammox and n-damo bacteria were more abundant in the lower layer (10-20 cm) than the surface layer (0-5 cm). The abundance of anammox bacteria varied from 2.21 × 10(5) to 3.90 × 10(6) gene copies per gram dry soil, and n-damo bacteria quantities were between 1.69 × 10(5) and 5.07 × 10(6) gene copies per gram dry soil in the two different layers. Both anammox and n-damo bacteria are reported for the first time to co-occur in acidic forest soil in this study, providing a more comprehensive information on more defined microbial processes contributing to C and N cycles in the ecosystems.

Current advances in molecular methods for detection of nitrite-dependent anaerobic methane oxidizing bacteria in natural environments.

Nitrite-dependent anaerobic methane oxidation (n-damo) process uniquely links microbial nitrogen and carbon cycles. Research on n-damo bacteria progresses quickly with experimental evidences through enrichment cultures. Polymerase chain reaction (PCR)-based methods for detecting them in various natural ecosystems and engineered systems play a very important role in the discovery of their distribution, abundance, and biodiversity in the ecosystems. Important characteristics of n-damo enrichments were obtained and their key significance in microbial nitrogen and carbon cycles was investigated. The molecular methods currently used in detecting n-damo bacteria were comprehensively reviewed and discussed for their strengths and limitations in applications with a wide range of samples. The pmoA gene-based PCR primers for n-damo bacterial detection were evaluated and, in particular, several incorrectly stated PCR primer nucleotide sequences in the published papers were also pointed out to allow correct applications of the PCR primers in current and future investigations. Furthermore, this review also offers the future perspectives of n-damo bacteria based on current information and methods available for a better acquisition of new knowledge about this group of bacteria.

Unexpected stimulation of soil methane uptake as emergent property of agricultural soils following bio-based residue application.

Intensification of agriculture to meet the global food, feed, and bioenergy demand entail increasing re-investment of carbon compounds (residues) into agro-systems to prevent decline of soil quality and fertility. However, agricultural intensification decreases soil methane uptake, reducing, and even causing the loss of the methane sink function. In contrast to wetland agricultural soils (rice paddies), the methanotrophic potential in well-aerated agricultural soils have received little attention, presumably due to the anticipated low or negligible methane uptake capacity in these soils. Consequently, a detailed study verifying or refuting this assumption is still lacking. Exemplifying a typical agricultural practice, we determined the impact of bio-based residue application on soil methane flux, and determined the methanotrophic potential, including a qualitative (diagnostic microarray) and quantitative (group-specific qPCR assays) analysis of the methanotrophic community after residue amendments over 2 months. Unexpectedly, after amendments with specific residues, we detected a significant transient stimulation of methane uptake confirmed by both the methane flux measurements and methane oxidation assay. This stimulation was apparently a result of induced cell-specific activity, rather than growth of the methanotroph population. Although transient, the heightened methane uptake offsets up to 16% of total gaseous CO2 emitted during the incubation. The methanotrophic community, predominantly comprised of Methylosinus may facilitate methane oxidation in the agricultural soils. While agricultural soils are generally regarded as a net methane source or a relatively weak methane sink, our results show that methane oxidation rate can be stimulated, leading to higher soil methane uptake. Hence, even if agriculture exerts an adverse impact on soil methane uptake, implementing carefully designed management strategies (e.g. repeated application of specific residues) may compensate for the loss of the methane sink function following land-use change.

The methane-oxidizing microbial communities of three maar lakes in tropical monsoon Asia.

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.