Evolutionary ecology of denitrifying methanotrophic NC10 bacteria in the deep-sea biosphere.

The NC10 phylum links anaerobic methane oxidation to nitrite denitrification through a unique O2-producing intra-aerobic methanotrophic pathway. Although numerous amplicon-based studies revealed the distribution of this phylum, comprehensive genomic insights and niche characterization in deep-sea environments were still largely unknown. In this study, we extensively surveyed the NC10 bacteria across diverse deep-sea environments, including waters, sediments, cold seeps, biofilms, rocky substrates, and subseafloor aquifers. We then reconstructed and analysed 38 metagenome-assembled genomes (MAGs), and revealed the extensive distribution of NC10 bacteria and their intense selective pressure in these harsh environments. Isotopic analyses combined with gene expression profiling confirmed that active nitrite-dependent anaerobic methane oxidation (n-DAMO) occurs within deep-sea sediments. In addition, the identification of the Wood-Ljungdahl (WL) and 3-hydroxypropionate/4-hydroxybutyrat (3HB/4HP) pathways in these MAGs suggests their capability for carbon fixation as chemoautotrophs in these deep-sea environments. Indeed, we found that for their survival in the oligotrophic deep-sea biosphere, NC10 bacteria encode two branches of the WL pathway, utilizing acetyl-CoA from the carbonyl branch for citric acid cycle-based energy production and methane from the methyl branch for n-DAMO. The observed low ratios of non-synonymous substitutions to synonymous substitutions (pN/pS) in n-DAMO-related genes across these habitats suggest a pronounced purifying selection that is critical for the survival of NC10 bacteria in oligotrophic deep-sea environments. These findings not only advance our understanding of the evolutionary adaptations of NC10 bacteria but also underscore the intricate coupling between the carbon and nitrogen cycles within deep-sea ecosystems, driven by this bacterial phylum.

Role of n-DAMO in Mitigating Methane Emissions from Intertidal Wetlands Is Regulated by Saltmarsh Vegetations.

Coastal wetlands are hotspots for methane (CH4) production, reducing their potential for global warming mitigation. Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) plays a crucial role in bridging carbon and nitrogen cycles, contributing significantly to CH4 consumption. However, the role of n-DAMO in reducing CH4 emissions in coastal wetlands is poorly understood. Here, the ecological functions of the n-DAMO process in different saltmarsh vegetation habitats as well as bare mudflats were quantified, and the underlying microbial mechanisms were explored. Results showed that n-DAMO rates were significantly higher in vegetated habitats (Scirpus mariqueter and Spartina alterniflora) than those in bare mudflats (P < 0.05), leading to an enhanced contribution to CH4 consumption. Compared with other habitats, the contribution of n-DAMO to the total anaerobic CH4 oxidation was significantly lower in the Phragmites australis wetland (15.0%), where the anaerobic CH4 oxidation was primarily driven by ferric iron (Fe3+). Genetic and statistical analyses suggested that the different roles of n-DAMO in various saltmarsh wetlands may be related to divergent n-DAMO microbial communities as well as environmental parameters such as sediment pH and total organic carbon. This study provides an important scientific basis for a more accurate estimation of the role of coastal wetlands in mitigating climate change.

Enhancement of electron transfer via magnetite in nitrite-dependent anaerobic methane oxidation system.

Nitrite-dependent anaerobic methane oxidation (n-DAMO) is a novel denitrification process that simultaneously further removes and utilizes methane from anaerobic effluent from wastewater treatment plants. However, the metabolic activity of n-DAMO bacteria is relative low for practical application. In this study, conductive magnetite was added into lab-scale sequencing batch reactor inoculated with n-DAMO bacteria to study the influence on n-DAMO process. With magnetite amendment, the nitrogen removal rate could reach 34.9 mg N·L-1d-1, nearly 2.5 times more than that of control group. Magnetite significantly facilitated the interspecies electron transfer and built electrically connected community with high capacitance. Enzymatic activities of electron transport chain were significantly elevated. Functional gene expression and enzyme activities associated with nitrogen and methane metabolism had been highly up-regulated. These results not only propose a useful strategy in n-DAMO application but also provide insights into the stimulating mechanism of magnetite in n-DAMO process.

Impact of operating conditions on N2O accumulation in Nitrate-DAMO system: Kinetics and microbiological analysis.

Nitrate-dependent anaerobic methane oxidation (Nitrate-DAMO) is a novel and sustainable process that removes both nitrogen and methane. Previously, the metabolic pathway of Nitrate-DAMO has been intensively studied with some results. However, the production and consumption of nitrous oxide (N2O) in the Nitrate-DAMO system were widely disregarded. In this study, a Nitrate-DAMO system was used to investigate the effect of operational parameters (C/N ratio, pH, and temperature) on N2O accumulation, and the optimal operating conditions were determined (C/N = 3, pH = 6.5, and temperature = 20 °C). In this study, an enzyme kinetic model was used to fit the nitrate nitrogen degradation and the nitrous oxide production and elimination under different operating conditions. The thermodynamic model of N2O production and elimination in the system also has been constructed. Multiple linear regression analysis found that pH was the most important factor influencing N2O accumulation. The Metagenomics sequencing results showed that alkaline pH promoted the abundance of Nor genes and denitrifying bacteria, which were significantly and positively correlated with N2O emissions. And alkaline pH also promoted the production of Mdo genes related to the N2O-driven AOM reaction, indicating that part of the N2O was consumed by denitrifying bacteria and the other part was consumed by the N2O-driven AOM reaction. These findings reveal the mechanism of N2O production and consumption in DAMO systems and provide a theoretical basis for reducing N2O production and greenhouse gas emissions in actual operation.

Microbial Stratification Affects Conversions of Nitrogen and Methane in Biofilms Coupling Anammox and n-DAMO Processes.

A methane-based membrane biofilm reactor (MBfR) has a suitable configuration to incorporate anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) processes because of its high gas-transfer efficiency and efficient biomass retention. In this study, the spatial distribution of microorganisms along with the biofilm depth in methane-based MBfRs was experimentally revealed, showing the dominance of anammox bacteria, n-DAMO bacteria, and n-DAMO archaea in the outer layer, middle layer, and inner layer of biofilms, respectively. The long-term and short-term experimental investigations in conjunction with mathematical modeling collectively revealed that microorganisms living in the outer layer of biofilms tend to use substrates from wastewater, while microorganisms inhabiting the inner layer of biofilms tend to use substrates originating from biofilm substratum. Specifically, anammox bacteria dominating the biofilm surface preferentially removed the nitrite provided from wastewater, while n-DAMO bacteria mostly utilized the nitrite generated from n-DAMO archaea as these two methane-related populations spatially clustered together inside the biofilm. Likewise, the methane supplied from the membrane was mostly consumed by n-DAMO archaea, while the dissolved methane in wastewater would be primarily utilized by n-DAMO bacteria. This study offers novel insights into the impacts of microbial stratification in biofilm systems, not only expanding the fundamental understanding of biofilms and microbial interactions therein but also providing a rationale for the potential applications of methane-based MBfRs in sewage treatment.

Heterotrophic denitrification: An overlooked factor that contributes to nitrogen removal in n-DAMO mixed culture.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) has been recognized as a sustainable process for simultaneous removal of nitrogen and methane. The metabolisms of denitrifying anaerobic methanotrophs, including Candidatus Methanoperedens and Candidatus Methylomirabilis, have been well studied. However, potential roles of heterotrophs co-existing with these anaerobic methanotrophs are generally overlooked. In this study, we pulse-fed methane and nitrate into an anaerobic laboratory sequencing batch bioreactor and enriched a mixed culture with stable nitrate removal rate (NRR) of ∼28 mg NO3--N L-1 d-1. Microbial community analysis indicates abundant heterotrophs, e.g., Arenimonas (5.3%-18.9%) and Fimbriimonadales ATM1 (6.4%), were enriched together with denitrifying anaerobic methanotrophs Ca. Methanoperedens (10.8%-13.2%) and Ca. Methylomirabilis (27.4%-34.3%). The results of metagenomics and batch tests suggested that the denitrifying anaerobic methanotrophs were capable of generating methane-derived intermediates (i.e., formate and acetate), which were employed by non-methanotrophic heterotrophs for denitrification and biomass growth. These findings offer new insights into the roles of heterotrophs in n-DAMO mixed culture, which may help to optimize n-DAMO process for nitrogen removal from wastewater.

Reactivated biofilm coupling n-DAMO with anammox achieved high-rate nitrogen removal in membrane aerated moving bed biofilm reactor.

As a promising technology, the combination of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) with Anammox offers a solution to achieve effective and sustainable wastewater treatment. However, this sustainable process faces challenges to accumulate sufficient biomass for reaching practical nitrogen removal performance. This study developed an innovative membrane aerated moving bed biofilm reactor (MAMBBR), which supported sufficient methane supply and excellent biofilm attachment, for cultivating biofilms coupling n-DAMO with Anammox. Biofilms were developed rapidly on the polyurethane foam with the supply of ammonium and nitrate, achieving the bioreactor performance of 275 g N m-3 d-1 within 102 days. After the preservation at -20 °C for 8 months, the biofilm was successfully reactivated and achieved 315 g N m-3 d-1 after 188 days. After reactivation, MAMBBR was applied to treat synthetic sidestream wastewater. Up to 99.9% of total nitrogen was removed with the bioreactor performance of 4.0 kg N m-3 d-1. Microbial community analysis and mass balance calculation demonstrated that n-DAMO microorganisms and Anammox bacteria collectively contributed to nitrogen removal in MAMBBR. The MAMBBR developed in this study provides an ideal system of integrating n-DAMO with Anammox for sustainable wastewater treatment.

SSThe coexistence and diversity of Candidatus methylomirabilis oxyfera-like and anammox bacteria in sediments of an urban eutrophic lake.

Tangxun Lake is the largest urban lake in China, which is polluted by multiple wastewaters, and now is severely eutrophic. We detected diversity, abundance, and the coexistence of Candidatus Methylomirabilis oxyfera-like and anammox bacteria in different horizontal and vertical directions of the lake sediments through qPCR and clone library. Phylogenetic tree analysis showed that the Ca. Methylomirabilis oxyfera-like and anammox bacteria exhibited high diversity, and they belonged to group B-E and Ca. Brocadia genus, respectively. These two bacteria displayed higher diversity in polluted area than in other areas. Furthermore, they had great spatial variation of abundance both horizontally and vertically. The abundance of anammox bacteria was significantly higher than that of Ca. Methylomirabilis oxyfera-like bacteria. The stronger the human interference were, the higher abundances these two bacteria exhibited horizontally, whereas both their abundances and the ratio of anammox to Ca. Methylomirabilis oxyfera-like bacteria decreased with the increasing depth. Redundancy analysis indicated that nitrate was the most influential environmental factor to the abundance of these two bacteria. Ammonia, nitrite, total nitrogen, and organic matters were in positive correlation with the abundance of these two bacteria. Nitrate was slightly negatively correlated with the abundance of Ca. Methylomirabilis oxyfera-like bacteria, while it was positively correlated with that of anammox bacteria. Our results provided an insight into the effects of environmental factors such as ammonia, nitrite, and nitrate on the diversity and abundances of these two bacteria and theoretical basis for restoration of water.

Nitrite-dependent anaerobic methane oxidation bacteria and potential in permafrost region of Daxing'an Mountains.

Nitrite-dependent anaerobic methane oxidation (n-damo) acts as a crucial link between biogeochemical carbon and nitrogen cycles. Nevertheless, very few studies have characterized n-damo microorganisms in high-latitude permafrost regions. Therefore, this study investigated the vertical distribution and diversity of n-damo bacterial communities in soil from three forest types in the permafrost regions of the Daxing'an Mountains. A total of 11 and 8 operational taxonomic units (OTUs) of n-damo 16S rRNA and pmoA genes were observed, respectively. Remarkable spatial variations in n-damo bacteria community richness, diversity, and structure were observed at different soil depths. Moreover, the abundances of n-damo bacteria (16S rRNA and pmoA genes) varied between 1.55 × 104 to 1.47 × 105 and 1.31 × 103 to 3.11 × 104 copies g-1 dry soil (ds), as demonstrated by quantitative PCR analyses. 13CH4 stable isotope tracer assays indicated that the potential n-damo rates varied from 0 to 1.26 nmol g-1 day-1, with the middle layers (20-40 cm and 40-60 cm) exhibiting significantly higher values than the upper (0-20 cm) and deeper layers (80-100 cm) in all three forest types. Redundancy analyses (RDA) indicated that total organic carbon (TOC), nitrate (NO3--N), and nitrite (NO2--N) were key modulators of the distribution of n-damo bacterial communities. This study thus demonstrated the widespread occurrence of n-damo bacteria in cold and high-latitude regions of forest ecosystems and provided important insights into the global distribution of these bacteria. KEY POINTS: • This study detected n-damo bacteria in soil samples obtained from the permafrost region of three forest types in the Daxing'an Mountains. • The community composition of n-damo bacteria was mainly affected by soil depth and not forest type. • The abundances of n-damo bacteria first increased and then decreased at higher soil depths.

Nitrate/nitrite dependent anaerobic methane oxidation coupling with anammox in membrane biotrickling filter for nitrogen removal.

Combining nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) and anaerobic ammonium oxidation (Anammox) is a promising sustainable wastewater treatment technology, which simultaneously achieve nitrogen removal and methane emission mitigation. However, the practical application of n-DAMO has been greatly limited by its extremely slow growth-rate and low reaction rate. This work proposes an innovative Membrane BioTrickling Filter (MBTF), which consist of hollow fiber membrane for effective methane supplementation and polyurethane sponge as support media for the attachment and growth of biofilm coupling n-DAMO with Anammox. When steady state with a hydraulic retention time (HRT) of 6.00 h was reached, above 99.9% of nitrogen was removed from synthetic sidestream wastewater at a rate of 3.99 g N L-1 d-1. This system presented robust capacity to withstand unstable partial nitritation effluent, achieving complete nitrogen removal at a varied nitrite to ammonium ratio in the range of 1.10-1.40. It is confirmed that n-DAMO and Anammox microorganisms jointly dominated the microbial community by pyrosequencing technology. The complete nitrogen removal potential at high-rate and efficient biomass retention (12.4 g VSS L-1) of MBTF offers promising alternative for sustainable wastewater treatment by the combination of n-DAMO and Anammox.

Denitrifying anaerobic methane oxidation (DAMO) cultures: Factors affecting their enrichment, performance and integration with anammox bacteria.

The recently discovered process, denitrifying anaerobic methane oxidation (DAMO), links the carbon and nitrogen biogeochemical cycles via coupling the anaerobic oxidation of methane to denitrification. The DAMO process, in this respect, has the potential to mitigate the greenhouse effect through the assimilation of dissolved methane. Denitrification via methane oxidation rather than organic matter, provides a new perspective to performing this once thought to be well established process. The two main species responsible for this process are "Candidatus Methylomirabilis oxyfera (M. oxyfera), and "Candidatus Methanoperedens nitroreducens" (M. nitroreducens). M. oxyfera is responsible of reducing nitrite while M. nitroreducens reduces nitrate to nitrite. These two microorganisms, despite their different pathways, were found to exist together in nature through a syntrophic relationship. Their co-existence with anaerobic ammonium oxidation (Anammox) bacteria was also revealed in the last decade. Anammox bacteria are chemolithoautotrophs, converting ammonium and nitrite to N2 and nitrate. They are responsible for the release of more than 50% of oceanic N2, hence play an important role in the global nitrogen cycle. Factors leading to the enrichment of DAMO cultures and their cultivation with Anammox cultures are of significance for improved nitrogen removal systems with decreased greenhouse effect, and even for further full-scale applications. This study, therefore, aims to present an updated review of the DAMO process, by focusing on the factors that might have a significant role in enrichment of DAMO microorganisms and their co-existence with Anammox bacteria. Factors such as temperature, pH, inoculum and feed type, trace metals and reactor configuration are among the ones discussed in detail. Factors, which have not been investigated, are also elucidated to provide a better understanding of the process and set research goals that will aid in the development of DAMO-centered wastewater treatment alternatives.

Enhancing mainstream nitrogen removal by employing nitrate/nitrite-dependent anaerobic methane oxidation processes.

Due to serious eutrophication in water bodies, nitrogen removal has become a critical stage for wastewater treatment plants (WWTPs) over past decades. Conventional biological nitrogen removal processes are based on nitrification and denitrification (N/DN), and are suffering from several major drawbacks, including substantial aeration consumption, high fugitive greenhouse gas emissions, a requirement for external carbon sources, excessive sludge production and low energy recovery efficiency, and thus unable to satisfy the escalating public needs. Recently, the discovery of anaerobic ammonium oxidation (anammox) bacteria has promoted an update of conventional N/DN-based processes to autotrophic nitrogen removal. However, the application of anammox to treat domestic wastewater has been hindered mainly by unsatisfactory effluent quality with nitrogen removal efficiency below 80%. The discovery of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) during the last decade has provided new opportunities to remove this barrier and to achieve a robust system with high-level nitrogen removal from municipal wastewater, by utilizing methane as an alternative carbon source. In the present review, opportunities and challenges for nitrate/nitrite-dependent anaerobic methane oxidation are discussed. Particularly, the prospective technologies driven by the cooperation of anammox and n-DAMO microorganisms are put forward based on previous experimental and modeling studies. Finally, a novel WWTP system acting as an energy exporter is delineated.

Molecular and stable isotopic evidence for the occurrence of nitrite-dependent anaerobic methane-oxidizing bacteria in the mangrove sediment of Zhangjiang Estuary, China.

Nitrite-dependent anaerobic methane oxidation (n-damo), which is mediated by "Candidatus Methylomirabilis oxyfera-like" bacteria, is unique in linking the carbon and nitrogen cycles. However, the niche and activity of n-damo bacteria in the mangrove ecosystem have not been confirmed. Here, we report the occurrence of the n-damo process in the mangrove wetland of the Zhangjiang Estuary, China. The widespread occurrence of n-damo bacteria in mangrove wetland was confirmed using real-time quantitative polymerase chain reaction (qPCR) assay, which showed that the abundance of Methylomirabilis oxyfera-like bacterial 16S rRNA and pmoA genes ranged from 2.43 × 106 to 2.09 × 107 and 2.07 × 106 to 3.38 × 107copies per gram of dry soil in the examined sediment cores. The highest amount of targeting genes was all detected in the upper layer (0-20 cm). Phylogenetic analyses of n-damo bacterial 16S rRNA and pmoA genes illustrated the depth-specific distribution and high diversity of n-damo bacteria in the mangrove wetland. Stable isotope experiments further confirmed the occurrence of n-damo in the examined mangrove sediments, and the potential n-damo rates ranged from 25.93 to 704.08 nmol CO2 per gram of dry soil per day at different depths of the sediment cores, with the n-damo being more active in the upper layer of the mangrove sediments. These results illustrate the existence of active M. oxyfera-like bacteria and indicate that the n-damo process is a previously overlooked microbial methane sink in the mangrove wetlands.

Faunal Burrows Alter the Diversity, Abundance, and Structure of AOA, AOB, Anammox and n-Damo Communities in Coastal Mangrove Sediments.

In the present work, the diversity, community structures, and abundances of aerobic ammonia-oxidizing archaea (AOA) and bacteria (AOB), anaerobic ammonium-oxidizing (anammox) bacteria, and denitrifying anaerobic methane oxidization (n-damo) bacteria were unraveled in the bioturbated areas of the coastal Mai Po mangrove sediments. Results indicated that the bioturbation by burrowing in mangrove sediments was associated with higher concentration of NH4+ but lower concentrations of both NO2- and NO3-, and increase in diversity and richness of both AOA and AOB, but relatively lower diversity and richness of n-damo bacteria. The phylotypes of anammox bacterial community were significantly increased while their phylogenetic lineages observed in the less bioturbated areas were also maintained. Infauna also showed a great impact on the composition of n-damo bacterial phylotypes and burrowing activity altered the n-damo community structure profoundly in the sampled areas. The communities of n-damo bacteria in the surrounding bulk sediments showed similar structures to marine n-damo communities, but those on the burrow wall and in the ambient surface layer had a freshwater pattern, which was different from previous findings in Mai Po wetland. On the other hand, the abundances of AOA, AOB, and n-damo bacteria were greatly stimulated on burrow walls while the abundance of anammox bacteria remained unchanged. Infaunal burrows and mangrove roots affected the relative abundance of AOA and AOB. The benthic infauna stimulated the abundances of AOA, AOB, anammox, and n-damo bacteria. Furthermore, NH4+ and NO2- were important environmental factors changing the structure of each group. The communities of anammox and n-damo bacteria in bioturbated areas showed a competitive relationship.

Spatial and temporal distribution of nitrite-dependent anaerobic methane-oxidizing bacteria in an intertidal zone of the East China Sea.

Nitrite-dependent anaerobic methane oxidation (N-DAMO), which couples anaerobic methane oxidation and nitrite reduction, is a recently discovered bioprocess coupling microbial nitrogen and carbon cycles. The discovery of this microbial process challenges the traditional knowledge of global methane sinks and nitrogen losses. In this study, the abundance and activity of N-DAMO bacteria were investigated and their contributions to methane sink and nitrogen loss were estimated in different seasons and different partitions of an intertidal zone of the East China Sea. The results showed that N-DAMO bacteria were extensively and continuously present in the intertidal zone, with the number of cells ranging from 5.5 × 104 to 2.8 × 105 copy g-1 soil and the potential activity ranging from 0.52 to 5.7 nmol CO2 g-1 soil day-1, contributing 5.0-36.6% of nitrite- and sulfate-dependent anaerobic methane oxidation in the intertidal zone. The N-DAMO activity and its contribution to the methane consumption were highest in the spring and in the low intertidal zone. These findings showed that the N-DAMO process is an important methane and nitrogen sink in the intertidal zone and varies with the seasons and the partitions of the intertidal zone.

Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake.

Anaerobic methane oxidation coupled to denitrification, also known as "nitrate/nitrite-dependent anaerobic methane oxidation" (n-damo), was discovered in 2006. Since then, only a few studies have identified this process and the associated microorganisms in natural environments. In aquatic sediments, the close proximity of oxygen- and nitrate-consumption zones can mask n-damo as aerobic methane oxidation. We therefore investigated the vertical distribution and the abundance of denitrifying methanotrophs related to Candidatus Methylomirabilis oxyfera with cultivation-independent molecular techniques in the sediments of Lake Constance. Additionally, the vertical distribution of methane oxidation and nitrate consumption zones was inferred from high-resolution microsensor profiles in undisturbed sediment cores. M. oxyfera-like bacteria were virtually absent at shallow-water sites (littoral sediment) and were very abundant at deep-water sites (profundal sediment). In profundal sediment, the vertical distribution of M. oxyfera-like bacteria showed a distinct peak in anoxic layers that coincided with the zone of methane oxidation and nitrate consumption, a strong indication for n-damo carried out by M. oxyfera-like bacteria. Both potential n-damo rates calculated from cell densities (660-4,890 µmol CH4⋅m(-2)⋅d(-1)) and actual rates calculated from microsensor profiles (31-437 µmol CH4⋅m(-2)⋅d(-1)) were sufficiently high to prevent methane release from profundal sediment solely by this process. Additionally, when nitrate was added to sediment cores exposed to anoxic conditions, the n-damo zone reestablished well below the sediment surface, completely preventing methane release from the sediment. We conclude that the previously overlooked n-damo process can be the major methane sink in stable freshwater environments if nitrate is available in anoxic zones.

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.

Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments.

In the current study, we investigated nitrite-dependent anaerobic methane oxidation (N-DAMO) as a potential methane sink in the Hangzhou Bay and the adjacent Zhoushan sea area. The potential activity of the N-DAMO process was primarily observed in Hangzhou Bay by means of (13)C-labeling experiments, whereas very low or no potential N-DAMO activity could be detected in the Zhoushan sea area. The measured potential N-DAMO rates ranged from 0.2 to 1.3 nmol (13)CO2 g(-1) (dry sediment) day(-1), and the N-DAMO potentially contributed 2.0-9.4 % to the total microbial methane oxidation in the examined sediments. This indicated that the N-DAMO process may be an alternative pathway in the coastal methane cycle. Phylogenetic analyses confirmed the presence of Candidatus Methylomirabilis oxyfera-like bacteria in all the examined sediments, while the group A members (the dominant bacteria responsible for N-DAMO) were found mainly in Hangzhou Bay. Quantitative PCR showed that the 16S rRNA gene abundance of Candidatus M. oxyfera-like bacteria varied from 5.4 × 10(6) to 5.0 × 10(7) copies g(-1) (dry sediment), with a higher abundance observed in Hangzhou Bay. In addition, the overlying water NO3 (-) concentration and salinity were identified as the most important factors influencing the abundance and potential activity of Candidatus M. oxyfera-like bacteria in the examined sediments. This study showed the evidence of N-DAMO in coastal environments and indicated the importance of N-DAMO as a potential methane sink in coastal environments.

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.

Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria.

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3) copies g(-1) dry sed. for the environmental samples compared to approximately 10(5) copies g(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems.