pmoA Gene Sequences Research Articles

Seasonal variations of n-damo bacterial community in the subtropical Mai Po mangrove wetland of Hong Kong

Mangrove wetland is one of the most important ecosystems along the coast and often suffers from global climate change as well as the anthropogenic activities. Nitrite-dependent anaerobic methane oxidization (n-damo) bacteria play an important role in linking the microbial geochemical cycles of carbon and nitrogen in the wetlands, but the pattern of their diversity and abundance to a changing mangrove wetland is poorly understood. In the present work, we investigated the seasonal variations of n-damo bacteria in vegetated and non-vegetated sediments of a subtropical mangrove wetland by amplifying 16 S rDNA and pmoA gene sequences. Results indicated that n-damo bacterial communities in the surface sediments maintained freshwater lineage characteristics as the majority after the long rainy season, while the abundance of marine lineages dominated in all the subsurface sediments. Shannon-Wiener and Chao1 indices based on 16 S rDNA gene sequences decreased largely in reed sediments by the end of rainy season, but increased in both surface and subsurface sediments of mangrove and mudflat zones, especially in the non-vegetated surface sediments. The majority of n-damo bacterial pmoA gene sequences shifted from Cluster SCS-2 to Cluster SCS-1 by the end of the dry season. Results suggested that Clusters SCS-1 and SCS-3 would be more likely detected in relatively pristine marine sediments, while Cluster SCS-2 tended to be abundant in coastal habitats with a strong terrestrial/anthropogenic influence. Vegetation had either direct or indirect influence on the decrease of alpha diversity of n-damo pmoA gene when the abundance showed an increase by the end of the wet season. The abundances of n-damo bacteria increased by two orders of magnitude to 5.1–9.4 × 107 copies/g dry sediment after the rainy season. Results suggested that NH4+ and NH4+/NO2− were significant factors governing the diversity of n-damo community in mangrove wetland sediments. The findings of this present work provided an in-depth understanding on the variations of n-damo bacterial community to seasonal changes in the subtropical mangrove wetland.

Distribution pattern of N-damo bacteria along an anthropogenic nitrogen input gradient from the coastal mangrove wetland to the South China sea sediments

Microbial nitrite-dependent anaerobic methane oxidation (n-damo) process is important for mitigating methane emission and anthropogenic nitrogen inputs in the marine environment. However, the distribution pattern of n-damo bacteria along an anthropogenic N-input gradient from the coastal wetland to the pristine South China Sea is poorly understood. This study investigated the diversity and abundance of n-damo bacteria in samples collected along a N-input gradient from Mai Po (MP) mangrove wetland sediments of the Pearl River Estuary (PRE) to the deep ocean sediments of the South China Sea (SCS). Retrieved 16S rDNA sequences showed a shift of n-damo community composition of complex structures with both freshwater and marine n-damo lineages in MP intertidal sediments to marine dominated characteristic in SCS sediments. The observed variation of Shannon and Chao1 indexes of n-damo bacteria shared a similar trend of a decrease at first followed by an increase along the targeting gradient with previously investigated methanogens, anaerobic methanotrophic archaea, ammonia-oxidizing archaea and ammonia-oxidizing bacteria, but had a reverse pattern with anammox bacteria. The community structure of pmoA gene sequences contained freshwater lineages only in SCS continental shelf sediments closer to the PRE, and turned to group with other marine samples in deeper and pristine sediments. Results suggested that n-damo bacteria might be a major contributor to anaerobic denitrification in the SCS sediments because their abundances were much higher than previously studied anammox bacteria in the same sample set. The distribution pattern of n-damo bacterial diversity, richness and abundance along the anthropogenic N-input gradient implies that they could be used as a bio-indicator for monitoring the anthropogenic/terrestrial inputs in marine environments.

Diversity and Effect of Increasing Temperature on the Activity of Methanotrophs in Sediments of Fildes Peninsula Freshwater Lakes, King George Island, Antarctica.

Global warming has a strong impact on polar regions. Particularly, the Antarctic Peninsula and nearby islands have experienced a marked warming trend in the past 50 years. Therefore, higher methane (CH4) emissions from this area could be expected in the future. Since mitigation of these emissions can be carried out by microbial oxidation, understanding this biological process is crucial since to our knowledge, no related studies have been performed in this area before. In this work, the aerobic CH4 oxidation potential of five freshwater lake sediments of Fildes Peninsula (King George Island, South Shetland Islands) was determined with values from 0.07 to 10 μmol CH4 gdw–1 day–1 and revealed up to 100-fold increase in temperature gradients (5, 10, 15, and 20°C). The structure and diversity of the bacterial community in the sediments were analyzed by next-generation sequencing (Illumina MiSeq) of 16S rRNA and pmoA genes. A total of 4,836 ASVs were identified being Proteobacteria, Actinobacteriota, Acidobacteriota, and Bacteroidota the most abundant phyla. The analysis of the pmoA gene identified 200 ASVs of methanotrophs, being Methylobacter Clade 2 (Type I, family Methylococcaceae) the main responsible of the aerobic CH4 oxidation. Moreover, both approaches revealed the presence of methanotrophs of the classes Gammaproteobacteria (families Methylococcaceae and Crenotrichaceae), Alphaproteobacteria (family Methylocystaceae), Verrucomicrobia (family Methylacidiphilaceae), and the candidate phylum of anaerobic methanotrophs Methylomirabilota. In addition, bacterial phospholipid fatty acids (PLFA) biomarkers were studied as a proxy for aerobic methane-oxidizing bacteria and confirmed these results. Methanotrophic bacterial diversity was significantly correlated with pH. In conclusion, our findings suggest that aerobic methanotrophs could mitigate in situ CH4 emissions in a future scenario with higher temperatures in this climate-sensitive area. This study provides new insights into the diversity of methanotrophs, as well as the influence of temperature on the CH4 oxidation potential in sediments of freshwater lakes in polar regions of the southern hemisphere.

Unique high Arctic methane metabolizing community revealed through in situ 13CH4-DNA-SIP enrichment in concert with genome binning

Greenhouse gas (GHG) emissions from Arctic permafrost soils create a positive feedback loop of climate warming and further GHG emissions. Active methane uptake in these soils can reduce the impact of GHG on future Arctic warming potential. Aerobic methane oxidizers are thought to be responsible for this apparent methane sink, though Arctic representatives of these organisms have resisted culturing efforts. Here, we first used in situ gas flux measurements and qPCR to identify relative methane sink hotspots at a high Arctic cytosol site, we then labeled the active microbiome in situ using DNA Stable Isotope Probing (SIP) with heavy 13CH4 (at 100 ppm and 1000 ppm). This was followed by amplicon and metagenome sequencing to identify active organisms involved in CH4 metabolism in these high Arctic cryosols. Sequencing of 13C-labeled pmoA genes demonstrated that type II methanotrophs (Methylocapsa) were overall the dominant active methane oxidizers in these mineral cryosols, while type I methanotrophs (Methylomarinovum) were only detected in the 100 ppm SIP treatment. From the SIP-13C-labeled DNA, we retrieved nine high to intermediate quality metagenome-assembled genomes (MAGs) belonging to the Proteobacteria, Gemmatimonadetes, and Chloroflexi, with three of these MAGs containing genes associated with methanotrophy. A novel Chloroflexi MAG contained a mmoX gene along with other methane oxidation pathway genes, identifying it as a potential uncultured methane oxidizer. This MAG also contained genes for copper import, synthesis of biopolymers, mercury detoxification, and ammonia uptake, indicating that this bacterium is strongly adapted to conditions in active layer permafrost and providing new insights into methane biogeochemical cycling. In addition, Betaproteobacterial MAGs were also identified as potential cross-feeders with methanotrophs in these Arctic cryosols. Overall, in situ SIP labeling combined with metagenomics and genome binning demonstrated to be a useful tool for discovering and characterizing novel organisms related to specific microbial functions or biogeochemical cycles of interest. Our findings reveal a unique and active Arctic cryosol microbial community potentially involved in CH4 cycling.

Effect of elevated atmospheric CO2 concentration on the activity, abundance and community composition of aerobic methanotrophs in paddy soils

The elevated atmospheric CO2 concentration (eCO2) has a great impact on soil microbial communities and their functions; however, how the activity and communities of methanotrophs respond to eCO2 in paddy ecosystem is poorly known. Here, we examined the effects of a 4-year exposure to eCO2 (EC; ambient + 200 ppm) in open top chambers on the potential activity, abundance and community composition of methanotrophs in paddy fields in Nanjing City, China. The potential activity was measured in a slurry incubation under aerobic condition, and the community composition and abundance of methanotrophs were analyzed through Illumina Miseq sequencing and quantitative PCR of pmoA genes. The results demonstrated that EC significantly increased (p < 0.05) the methane oxidation potential and pmoA gene abundance of methanotrophs by 52.9%–56.7% and 83.5%–213.6%, respectively, during late rice growth stages as compared with ambient condition (CK). Further, the community composition of methanotrophs in soil changed significantly (p < 0.05), with the dominant methanotrophs shifting from type II under CK to type I under EC. The variation of soil dissolved organic carbon and inorganic nitrogen levels caused by eCO2 had an important impact on the methane oxidation potential and methanotrophic community structure. Taken together, our results indicate that eCO2 can have an important impact on the activity, abundance and community composition of methanotrophs, adding new insights into the effects of rising CO2 on methane cycling in paddy ecosystems.

USCγ Dominated Community Composition and Cooccurrence Network of Methanotrophs and Bacteria in Subterranean Karst Caves.

ABSTRACTKarst caves have recently been demonstrated to act as a sink for atmospheric methane, due in part to consumption by microbes residing in caves that can oxidize methane at atmospheric levels. However, our knowledge about the responsible atmospheric methane-oxidizing bacteria (atmMOB) in this vast habitat remains limited to date. To address this issue, weathered rock samples from three karst caves were collected in Guilin City and subjected to high-throughput sequencing of pmoA and 16S rRNA genes. The results showed that members of the high-affinity upland soil cluster (USC), especially upland soil cluster gamma (USCγ), with absolute abundances of 104 to 109 copies · g−1 dry sample, dominated the atmMOB communities, while Proteobacteria and Actinobacteria dominated the overall bacterial communities. Moreover, USCγ was a keystone taxon in cooccurrence networks of both the atmMOB and the total bacterial community, whereas keystone taxa in the bacterial network also included Gaiella and Aciditerrimonas. Positive links overwhelmingly dominated the cooccurrence networks of both atmMOB and the total bacterial community, indicating a consistent response to environmental disturbances. Our study shed new insights on the diversity and abundances underlining atmMOB and total bacterial communities and on microbial interactions in subterranean karst caves, which increased our understanding about USC and supported karst caves as a methane sink.IMPORTANCE Karst caves have recently been demonstrated to be a potential atmospheric methane sink, presumably due to consumption by methane-oxidizing bacteria. However, the sparse knowledge about the diversity, distribution, and community interactions of methanotrophs requires us to seek further understanding of the ecological significance of methane oxidation in these ecosystems. Our pmoA high-throughput results from weathered rock samples from three karst caves in Guilin City confirm the wide occurrence of atmospheric methane-oxidizing bacteria in this habitat, especially those affiliated with the upland soil cluster, with a gene copy number of 104 to 109 copies per gram dry sample. Methanotrophs and the total bacterial communities had more positive than negative interactions with each other as indicated by the cooccurrence network, suggesting their consistent response to environmental disturbance. Our results solidly support caves as an atmospheric methane sink, and they contribute to a comprehensive understanding of the diversity, distribution, and interactions of microbial communities in subsurface karst caves.

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.

Multifunctional Periphytic Biofilms: Polyethylene Degradation and Cd2+ and Pb2+ Bioremediation under High Methane Scenario.

Priority pollutants such as polyethylene (PE) microplastic, lead (Pb2+), and cadmium (Cd2+) have attracted the interest of environmentalists due to their ubiquitous nature and toxicity to all forms of life. In this study, periphytic biofilms (epiphyton and epixylon) were used to bioremediate heavy metals (HMs) and to biodegrade PE under high (120,000 ppm) methane (CH4) doses. Both periphytic biofilms were actively involved in methane oxidation, HMs accumulation and PE degradation. Epiphyton and epixylon both completely removed Pb2+ and Cd2+ at concentrations of 2 mg L−1 and 50 mg L−1, respectively, but only partially removed these HMs at a relatively higher concentration (100 mg L−1). Treatment containing 12% 13CH4 proved to be most effective for biodegradation of PE. A synergistic effect of HMs and PE drastically changed microbial biota and methanotrophic communities. High-throughput 16S rRNA gene sequencing revealed that Cyanobacteria was the most abundant class, followed by Gammaproteobacteria and Alphaproteobacteria in all high-methane-dose treatments. DNA stable-isotope probing was used to label 13C in a methanotrophic community. A biomarker for methane-oxidizing bacteria, pmoA gene sequence of a 13C-labeled fraction, revealed that Methylobacter was most abundant in all high-methane-dose treatments compared to near atmospheric methane (NAM) treatment, followed by Methylococcus. Methylomonas, Methylocystis, Methylosinus, and Methylocella were also found to be increased by high doses of methane compared to NAM treatment. Overall, Cd+2 had a more determinantal effect on methanotrophic activity than Pb2+. Epiphyton proved to be more effective than epixylon in HMs removal and PE biodegradation. The findings proved that both epiphyton and epixylon can be used to bioremediate HMs and biodegrade PE as an efficient ecofriendly technique under high methane concentrations.

Enhanced Adsorptive Bioremediation of Heavy Metals (Cd2+, Cr6+, Pb2+) by Methane-Oxidizing Epipelon.

Cadmium (Cd), chromium (Cr) and lead (Pb) are heavy metals that have been classified as priority pollutants in aqueous environment while methane-oxidizing bacteria as a biofilter arguably consume up to 90% of the produced methane in the same aqueous environment before it escapes into the atmosphere. However, the underlying kinetics and active methane oxidizers are poorly understood for the hotspot of epipelon that provides a unique micro-ecosystem containing diversified guild of microorganisms including methane oxidizers for potential bioremediation of heavy metals. In the present study, the Pb2+, Cd2+and Cr6+ bioremediation potential of epipelon biofilm was assessed under both high (120,000 ppm) and near-atmospheric (6 ppm) methane concentrations. Epipelon biofilm demonstrated a high methane oxidation activity following microcosm incubation amended with a high concentration of methane, accompanied by the complete removal of 50 mg L−1 Pb2+ and 50 mg L−1 Cd2+ (14 days) and partial (20%) removal of 50 mg L−1 Cr6+ after 20 days. High methane dose stimulated a faster (144 h earlier) heavy metal removal rate compared to near-atmospheric methane concentrations. DNA-based stable isotope probing (DNA-SIP) following 13CH4 microcosm incubation revealed the growth and activity of different phylotypes of methanotrophs during the methane oxidation and heavy metal removal process. High throughput sequencing of 13C-labelled particulate methane monooxygenase gene pmoA and 16S rRNA genes revealed that the prevalent active methane oxidizers were type I affiliated methanotrophs, i.e., Methylobacter. Type II methanotrophs including Methylosinus and Methylocystis were also labeled only under high methane concentrations. These results suggest that epipelon biofilm can serve as an important micro-environment to alleviate both methane emission and the heavy metal contamination in aqueous ecosystems with constant high methane fluxes.

A Novel Moderately Thermophilic Type Ib Methanotroph Isolated from an Alkaline Thermal Spring in the Ethiopian Rift Valley.

Aerobic moderately thermophilic and thermophilic methane-oxidizing bacteria make a substantial contribution in the control of global warming through biological reduction of methane emissions and have a unique capability of utilizing methane as their sole carbon and energy source. Here, we report a novel moderately thermophilic Methylococcus-like Type Ib methanotroph recovered from an alkaline thermal spring (55.4 °C and pH 8.82) in the Ethiopian Rift Valley. The isolate, designated LS7-MC, most probably represents a novel species of a new genus in the family Methylococcaceae of the class Gammaproteobacteria. The 16S rRNA gene phylogeny indicated that strain LS7-MC is distantly related to the closest described relative, Methylococcus capsulatus (92.7% sequence identity). Growth was observed at temperatures of 30–60 °C (optimal, 51–55 °C), and the cells possessed Type I intracellular membrane (ICM). The comparison of the pmoA gene sequences showed that the strain was most closely related to M. capsulatus (87.8%). Soluble methane monooxygenase (sMMO) was not detected, signifying the biological oxidation process from methane to methanol by the particulate methane monooxygenase (pMMO). The other functional genes mxaF, cbbL and nifH were detected by PCR. To our knowledge, the new strain is the first isolated moderately thermophilic methanotroph from an alkaline thermal spring of the family Methylococcaceae. Furthermore, LS7-MC represents a previously unrecognized biological methane sink in thermal habitats, expanding our knowledge of its ecological role in methane cycling and aerobic methanotrophy.

Characteristics of aerobic methane-oxidising bacterial community at the sea-floor surface of the Nankai Trough

Methane hydrate is one of the new energy sources, but methane leakage could cause environmental issues such as marine ecosystem changes and global warming. The methane-oxidising bacterial (MOB) community could reflect the methane concentration, thus it may be an indicator of methane leakage. We obtained two sea-floor surface samples from a methane seep area and 12 samples from other general sea-floor areas of the Nankai Trough for the detection and phylogenetic analysis of the particulate methane monooxygenase (pmoA) gene. Using quantitative polymerase chain reaction analysis, the methane seep samples were found to have 106 copies of the pmoA gene per gram of sediment, whereas the general sea-floor area samples of the Nankai Trough contained 103–104 copies of the gene per gram of sediment. Phylogenetic analysis of the pmoA gene sequences showed that the sequences detected in the general and methane seep area samples differed significantly. Specifically, the pmof1–pmor primer pair could detect pmoA genes for the methane seep area, whereas pmoA gene from the general seafloor samples could be detected only using the A189–mb661 primer pair. The results of this study may facilitate the detection of unintended leakage of methane at methane hydrate production sites by monitoring MOB communities using pmoA-targeted phylogenetic analysis and quantification.

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.

Nitric Oxide Dismutase (nod) Genes as a Functional Marker for the Diversity and Phylogeny of Methane-Driven Oxygenic Denitrifiers

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.

Upland Soil Cluster Gamma dominates methanotrophic communities in upland grassland soils

Aerobic methanotrophs in upland soils consume atmospheric methane, serving as a critical counterbalance to global warming; however, the biogeographic distribution patterns of their abundance and community composition are poorly understood, especial at a large scale. In this study, soils were sampled from 30 grasslands across >2000 km on the Qinghai-Tibetan Plateau to determine the distribution patterns of methanotrophs and their driving factors at a regional scale. Methanotroph abundance and community composition were analyzed using quantitative PCR and Illumina Miseq sequencing of pmoA genes, respectively. The pmoA gene copies ranged from 8.2 × 105 to 1.1 × 108 per gram dry soil. Among the 30 grassland soil samples, Upland Soil Cluster Gamma (USCγ) dominated the methanotroph communities in 26 samples. Jasper Ridge Cluster (JR3) was the most dominant methanotrophic cluster in two samples; while Methylocystis, cluster FWs, and Methylobacter were abundant in other two wet soil samples. Interestingly, reanalyzing the pmoA genes sequencing data from existing publications suggested that USCγ was also the main methanotrophic cluster in grassland soils in other regions, especially when their mean annual precipitation was <500 mm. Canonical Analysis of Principal Coordinates including all soil samples indicated that the methanotrophic community composition was significantly correlated with local environmental factors, among which mean annual precipitation and pH showed the strongest correlations. Variance partitioning analysis showed that environmental factors and spatial distance were significant factors affecting the community structure of methanotrophs, and environmental properties were more important factors. Collectively, these findings indicate that atmospheric methane may be mainly oxidized by USCγ in upland soils. They also highlight the key role of water availability and pH in determining the abundance and community profiles of grassland soil methanotrophs.

Molecular Existence and Diversity of Nitrite-Dependent Anaerobic Methane Oxidizing (n-Damo) Bacteria in the Lakes of Badain of the Gobi Desert

Nitrite-dependent anaerobic methane oxidation (n-damo) process, mediated by Candidatus Methylomirabilis oxyfera of the candidate phylum NC10, was discovered recently which plays an important role in coupling the global nitrogen and carbon cycles. However, the distribution and diversity of this new anaerobic methane-oxidizing microorganism have not been investigated in desert lakes yet. The present study successfully retrieved n-damo bacterial 16S rRNA and pmoA gene sequences using PCR technique from lakes in Badain Jaran Desert of China. Phylogenetic analyses showed that n-damo bacteria widely occurred in brine and freshwater lakes on the desert with high diversity, including both sediment and water samples. The results of quantitative PCR indicated that the abundance of the 16S rRNA gene in lake sediments varied from 1.12 ± 0.68 × 105 to 1.64 ± 0.70 × 105 copies g−1 (dry weight), while that in water samples per milliliter was generally one order of magnitude lower than sediments. Correlation analyses suggested that n-damo bacterial abundance and diversity strongly depended on salinity. In lake sediments, the distribution, abundance, and diversity of n-damo bacteria were significantly associated with depth due to the concentration gradient of the NOx- and ammonium. This study provided new insights into both the n-damo community patterns and its interaction with ambient environmental factors in the desert lake ecosystem.

Composition, diversity and abundance of Candidatus M. oxyfera-like bacteria in response to the estuary salinity gradient

“Candidatus Methylomirabilis oxyfera” (M. oxyfera) bacteria has attracted much attention recently since it plays an important role in performing methane oxidation. Most studies on M. oxyfera-like bacteria have been carried out in freshwater ecosystems, however it remains unclear about its distribution along the salinity gradient of estuarine and coastal environments. In this study, we used molecular analyses to reveal the community, diversity and abundance of M. oxyfera-like bacteria in the Yangtze estuarine and coastal sediments. Clone library analyses confirmed the wide distributions of M. oxyfera-like bacterial 16S rRNA and pmoA genes in the study area. The M. oxyfera-like bacterial 16S rRNA gene sequences were grouped into eleven separate OTUs, which were assigned to groups A and B of M. oxyfera-like bacteria. Most sequences (89.4–97.6% of total sequences) of M. oxyfera-like bacterial 16S rRNA gene were affiliated with group B, while only few clone sequences (2.4–10.6% of total sequences) were affiliated with group A. The retrieved M. oxyfera-like bacterial pmoA gene sequences were grouped into five distinct clusters. Bray–Curtis dissimilarity of M. oxyfera-like bacterial 16S rRNA and pmoA genes differed greatly among the low, middle and high salinity sites. M. oxyfera-like bacterial 16S rRNA and pmoA abundances were mainly recovered from the oligohaline sites rather than from the saline sites. In addition, the large differences in 16S rRNA and pmoA gene sequences between estuarine and land freshwater environments suggested that salinity could be able to affect the community distribution of M. oxyfera-like bacteria. Also, sediment nitrate/ammonium played an important role in affecting the M. oxyfera-like bacteria. Overall, these results indicate that salinity has a great influence on M. oxyfera-like bacteria community in estuarine environments.

Stable isotope probing of active methane oxidizers in rice field soils from cold regions

DNA-based stable isotope probing (DNA-SIP) was employed to establish direct link between methane oxidation activity and the taxonomic identity of active methanotrophs in three rice field soils from Jian-San-Jiang (one baijiang origin soil, JB and one meadow origin soil, JM) and Qing-An (meadow origin soil, QA) districts in Northeastern China. Following microcosm incubation under 1% v/v13CH4 condition, soil organic 13C atom percent significantly increased from background 1.08 to 1.21% in average, indicating the biomass synthesis supported by methanotrophy. Real-time PCR analysis of methanotroph-specific biomarker pmoA genes of the buoyant density for DNA gradient, following the ultracentrifugation of the total DNA extracted from SIP microcosms, indicated an enrichment of methanotroph genomes in 13C-labeled DNA. It suggested propagation of microbial methane oxidizers in soils. High-throughput sequencing of 16S rRNA and pmoA genes from 13C-labeled DNA further revealed a diverse guild of both type I and II methanotrophs in all three soils. Specifically, Methylobacter-affiliated type I methanotrophs dominated the methanotrophic activity in JB and JM soils, whereas Methylocystis-affiliated type II methanotrophs dominated QA soil. This implied the physiological diversification of soil methanotrophs that might be due to constant environmental fluctuations in paddies.

Unexpected role of canonical aerobic methanotrophs in upland agricultural soils

Aerobic oxidation of methane at (circum-)atmospheric concentrations (<40 ppmv) 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 13CCH4 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 13CCO2. 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.

Phylogenetically distinct methanotrophs modulate methane oxidation in rice paddies across Taiwan

Rice paddies are considerable sources of methane because of the highly reduced soil oxidation–reduction conditions during rice cultivation. In this ecosystem, active methane-oxidizing bacteria are important, because they consume methane while reducing the overall emissions from rice paddy soils. However, the biogeographic distribution of active methanotrophs in paddy soils across Taiwan remains poorly understood. We used DNA-based stable isotope probing (DNA-SIP) to show that phylogenetically distinct type I and type II methanotrophs dominated methane oxidation in geographically different paddy soils across Taiwan. High-throughput sequencing of soil 16S rRNA and pmoA genes under field conditions revealed that a type II methanotroph, Methylocystis, was predominant in rice paddy soils. In addition, an uncultured novel type I methanotroph cluster (Rice Paddy Clusters) was detected with the closest relatedness to Methylocaldum 16S rRNA genes in all rice field soils tested. SIP microcosm incubation, however, suggested that the type I methanotrophs Methylosarcina and Methylobacter were significantly stimulated during the consumption of high-concentration methane in five paddy soils with pH > 6.07, whereas the Methylosinus-like microorganisms of type II methanotrophs dominated aerobic methane-oxidizing communities in acidic soil with pH = 5.17. Furthermore, high-throughput sequencing of 13C-pmoA genes indicated the presence of novel methanotrophs that are phylogenetically distantly related to the type I methanotrophs Methylosarcina in four out of five non-acidic paddy soils studied, and the high proportions in the 13C-DNA suggested that these uncultured methanotrophs play an important role in methane oxidation. These results provide strong evidence for the environmental selection of phylogenetically distinct methanotrophs under field conditions. Moreover, community shifts in active methanotrophs likely occurred in response to environmental variations with fluctuating methane concentrations.

A New Primer to Amplify pmoA Gene From NC10 Bacteria in the Sediments of Dongchang Lake and Dongping Lake.

Nitrite-dependent anaerobic methane oxidation (n-damo) is catalyzed by the NC10 phylum bacterium "Candidatus Methylomirabilis oxyfera" (M. oxyfera). Generally, the pmoA gene is applied as a functional marker to test and identify NC10-like bacteria. However, it is difficult to detect the NC10 bacteria from sediments of freshwater lake (Dongchang Lake and Dongping Lake) with the previous pmoA gene primer sets. In this work, a new primer cmo208 was designed and used to amplify pmoA gene of NC10-like bacteria. A newly nested PCR approach was performed using the new primer cmo208 and the previous primers cmo182, cmo682, and cmo568 to detect the NC10 bacteria. The obtained pmoA gene sequences exhibited 85-92% nucleotide identity and 95-97% amino acid sequence identity to pmoA gene of M. oxyfera. The obtained diversity of pmoA gene sequences coincided well with the diversity of 16S rRNA sequences. These results indicated that the newly designed pmoA primer cmo208 could give one more option to detect NC10 bacteria from different environmental samples.