Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N2 g(-1) (dry weight) day(-1) and the potential n-damo rates varied from 0.2 to 2.1 nmol CO2 g(-1) (dry weight) day(-1) in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10(5) to 2.0 × 10(6) copies g(-1) (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10(5) to 6.1 × 10(6) copies g(-1) (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with "Candidatus Brocadia" and "Candidatus Kuenenia" and n-damo bacteria related to "Candidatus Methylomirabilis oxyfera" were present in the soil cores. It is estimated that a total loss of 50.7 g N m(-2) per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH4 m(-2) per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.

Evidence for the Cooccurrence of Nitrite-Dependent Anaerobic Ammonium and Methane Oxidation Processes in a Flooded Paddy Field

The intertidal zone is an open ecosystem rich in organic matter and plays an important role in global biogeochemical cycles. It was previously considered that methane was mainly removed by sulfate-dependent anaerobic methane oxidation (sulfate-AOM) process in marine ecosystems while other anaerobic methane oxidation processes were ignored. Recent researches have demonstrated that denitrifying anaerobic methane oxidation (DAMO), consisting of nitrite-dependent anaerobic methane oxidation (nitrite-AOM) and nitrate-dependent anaerobic methane oxidation (nitrate-AOM), can also oxidize methane. In this work, the community structure, quantity and potential methane oxidizing rate of DAMO archaea and bacteria in the intertidal zone were studied by high-throughput sequencing, qPCR and stable isotope tracing method. The results showed that nitrate-AOM and nitrite-AOM were both active in the intertidal zone and showed approximate methane oxidation rates. The copy number of 16S rRNA gene of DAMO archaea and DAMO bacteria were 104 ∼ 105 copies g-1 (dry sediment), whereas NC10 bacteria were slightly higher. The contribution rate of DAMO process to total anaerobic methane removal in the intertidal zone reached 65.6% ∼ 100%, which indicates that DAMO process is an important methane sink in intertidal ecosystem. Laboratory incubations also indicated that DAMO archaea were more sensitive to oxygen and preferred a more anoxic environment. These results help us draw a more complete picture of methane and nitrogen cycles in natural habitats.

Denitrifying Anaerobic Methane Oxidation: A Previously Overlooked Methane Sink in Intertidal Zone

Paddy fields are important ecosystems, as rice is the primary food source for about half of the world’s population. Paddy fields are impacted by nitrogen fertilization and are a major anthropogenic source of methane. Microbial diversity and methane metabolism were investigated in the upper 60cm of a paddy soil by qPCR, 16S rRNA gene amplicon sequencing and anoxic 13C-CH4 turnover with a suite of electron acceptors. The bacterial community consisted mainly of Acidobacteria, Chloroflexi, Proteobacteria, Planctomycetes and Actinobacteria. Among archaea, Euryarchaeota and Bathyarchaeota dominated over Thaumarchaeota in the upper 30cm of the soil. Bathyarchaeota constituted up to 45% of the total archaeal reads in the top 5cm. In the methanogenic community, Methanosaeta were generally more abundant than the versatile Methanosarcina. The measured maximum methane production rate was 444 nmol gdwh-1, and the maximum rates of nitrate-, nitrite- and iron-dependent anaerobic oxidation of methane (AOM) were 57 nmol, 55 nmol and 56 nmol gdwh-1, respectively, at different depths. qPCR revealed a higher abundance of ‘Candidatus Methanoperedens nitroreducens’ than methanotrophic NC10 phylum bacteria at all depths, except at 60cm. These results demonstrate that there is substantial potential for anaerobic oxidation of methane in fertilized paddy fields, with ‘Candidatus Methanoperedens nitroreducens’ archaea as a potential important contributor.

/pdf/stratification-of-diversity-and-activity-of-methanogenic-and-5fwnh1z947.pdf

Stratification of Diversity and Activity of Methanogenic and Methanotrophic Microorganisms in a Nitrogen-Fertilized Italian Paddy Soil.

The distribution and importance of anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) have been identified in aquatic ecosystems; their role in agricultural upland soils however has not yet been well investigated. In this study, we examined spatio-temporal distributions of anammox and n-damo bacteria in soil profiles (300 cm depth) from an agricultural upland. Monitoring nitrogen (N) conversion activity using isotope-tracing techniques over the course of one year showed denitrification (99.0% N-loss in the winter and 85.0% N-loss in the summer) predominated over anammox (1.0% N-loss in the winter and 14.4% N-loss in the summer) and n-damo (0.6% N-loss in the winter) in surface soils (0-20 cm). While below 20 cm depth, N-loss was dominated by anammox (79.4 ± 14.3% in the winter and 65.4 ± 12.5% in the summer) and n-damo was not detected. Phylogenetic analysis showed that Candidatus Brocadia anammoxidans dominated the anammox community in the surface soil and Candidatus Brocadia fulgida dominated below 20 cm depth. Dissimilatory nitrate reduction to ammonium (DNRA), another nitrite reduction process, was found to play a limited role (4.9 ± 3.5%) in the surface soil compared with denitrification; below 80 cm DNRA rates were much higher than rates of anammox and denitrification. Ammonium oxidation was the main source of NO2- above 80 cm (70.9 ± 23.3%), the key influencing factor on anammox rates, and nitrate reduction (100%) was the main NO2- source below 80 cm. Considering the anammox, n-damo and denitrification rates as a whole in the sampled soil profile, denitrification is still the main N-loss process in upland soils.

Microbial pathways for nitrogen loss in an upland soil.

The nitrogen and methane cycles are important biogeochemical processes. Recently, ‘Candidatus Methanoperedens nitroreducens,’ archaea that catalyze nitrate-dependent anaerobic oxidation of methane (AOM), were enriched, and their genomes were analyzed. Diagnostic molecular tools for the sensitive detection of ‘Candidatus M. nitroreducens’ are not yet available. Here, we report the design of two novel mcrA primer combinations that specifically target the alpha sub-unit of the methyl-coenzyme M reductase (mcrA) gene of ‘Candidatus M. nitroreducens’. The first primer pair produces a fragment of 186-bp that can be used to quantify ‘Candidatus M. nitroreducens’ cells, whereas the second primer pair yields an 1191-bp amplicon that is with sufficient length and well suited for more detailed phylogenetic analyses. Six different environmental samples were evaluated with the new qPCR primer pair, and the abundances were compared with those determined using primers for the 16S rRNA gene. The qPCR results indicated that the number of copies of the ‘Candidatus M. nitroreducens’ mcrA gene was highest in rice field soil, with 5.6 ± 0.8 × 106 copies g−1 wet weight, whereas Indonesian river sediment had only 4.6 ± 2.7 × 102 copies g−1 wet weight. In addition to freshwater environments, sequences were also detected in marine sediment of the North Sea, which contained approximately 2.5 ± 0.7 × 104 copies g−1 wet weight. Phylogenetic analysis revealed that the amplified 1191-bp mcrA gene sequences from the different environments all clustered together with available genome sequences of mcrA from known ‘Candidatus M. nitroreducens’ archaea. Taken together, these results demonstrate the validity and utility of the new primers for the quantitative and sensitive detection of the mcrA gene sequences of these important nitrate-dependent AOM archaea. Furthermore, the newly obtained mcrA sequences will contribute to greater phylogenetic resolution of ‘Candidatus M. nitroreducens’ sequences, which have been only poorly captured by general methanogenic mcrA primers.

/pdf/mcra-primers-for-the-detection-and-quantification-of-the-1h2jsf1o5d.pdf

McrA primers for the detection and quantification of the anaerobic archaeal methanotroph ‘Candidatus Methanoperedens nitroreducens’

The discovery of nitrite-dependent anaerobic methane oxidation (n-damo) mediated by 'Candidatus Methylomirabilis oxyfera' with nitrite and methane as substrates has connected biogeochemical carbon and nitrogen cycles in a new way. The paddy fields often carry substantial methane and nitrate, thus may be a favorable habitat for n-damo bacteria. In this paper, the vertical-temporal molecular fingerprints of M. oxyfera-like bacteria, including abundance and community composition, were investigated in a paddy soil core in Jiangyin, near the Yangtze River. Through qPCR investigation, high abundance of M. oxyfera-like bacteria up to 1.0 × 10(8) copies (g d.w.s.)(-1) in summer and 8.5 × 10(7) copies (g d.w.s.)(-1) in winter was observed in the ecotone of soil and groundwater in the paddy soil core, which was the highest in natural environments to our knowledge. In the ecotone, the ratio of M. oxyfera-like bacteria to total bacteria reached peak values of 2.80% in summer and 4.41% in winter. Phylogenetic analysis showed n-damo bacteria in the paddy soil were closely related to M. oxyfera and had high diversity in the soil/groundwater ecotone. All of the results indicated the soil/groundwater ecotone of the Jiangyin paddy field was a favorable environment for the growth of n-damo bacteria.

methane-oxidizing bacteria in a paddy field profile

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methane-oxidizing bacteria in a paddy field profile