Project description:Paddy fields are a significant source of methane and contribute up to 20% of total methane emissions from wetland ecosystems. These inundated, anoxic soils featuring abundant nitrogen compounds and methane are an ideal niche for nitrate-dependent anaerobic methanotrophs. After 2 years of enrichment with a continuous supply of methane and nitrate as the sole electron donor and acceptor, a stable enrichment dominated by 'Candidatus Methanoperedens nitroreducens' archaea and 'Candidatus Methylomirabilis oxyfera' NC10 phylum bacteria was achieved. In this community, the methanotrophic archaea supplied the NC10 phylum bacteria with the necessary nitrite through nitrate reduction coupled to methane oxidation. The results of qPCR quantification of 16S ribosomal RNA (rRNA) gene copies, analysis of metagenomic 16S rRNA reads, and fluorescence in situ hybridization (FISH) correlated well and showed that after 2 years, 'Candidatus Methanoperedens nitroreducens' had the highest abundance of (2.2 ± 0.4 × 108) 16S rRNA copies per milliliter and constituted approximately 22% of the total microbial community. Phylogenetic analysis showed that the 16S rRNA genes of the dominant microorganisms clustered with previously described 'Candidatus Methanoperedens nitroreducens ANME2D' (96% identity) and 'Candidatus Methylomirabilis oxyfera' (99% identity) strains. The pooled metagenomic sequences resulted in a high-quality draft genome assembly of 'Candidatus Methanoperedens nitroreducens Vercelli' that contained all key functional genes for the reverse methanogenesis pathway and nitrate reduction. The diagnostic mcrA gene was 96% similar to 'Candidatus Methanoperedens nitroreducens ANME2D' (WP_048089615.1) at the protein level. The 'Candidatus Methylomirabilis oxyfera' draft genome contained the marker genes pmoCAB, mdh, and nirS and putative NO dismutase genes. Whole-reactor anaerobic activity measurements with methane and nitrate revealed an average methane oxidation rate of 0.012 mmol/h/L, with cell-specific methane oxidation rates up to 0.57 fmol/cell/day for 'Candidatus Methanoperedens nitroreducens'. In summary, this study describes the first enrichment and draft genome of methanotrophic archaea from paddy field soil, where these organisms can contribute significantly to the mitigation of methane emissions.

Project description:Biological nitrogen (N) fixation, an important pathway of N, inputs from the atmosphere to Earth's ecosystems, is well demonstrated to decline under N input. However, it remains unclear why N fixers sustain N fixation in many forests under high atmospheric N deposition. To address this knowledge gap, we analyzed the response of the diazotroph community to low N loads (short-term and low N addition; 3-year N addition at the rates of 25-50 kg N ha-1 year-1) vs high loads (chronic and high N addition; 9-year N addition at the rate of 150 kg N ha-1 year-1) in forest soils using high-throughput sequencing. Rates of N fixation decreased under low and high N loads (by 13%-27% and 10%-12%, respectively). Richness and alpha diversity (ACE and Chao1) of the soil diazotroph community decreased under low but not high N loads. Approximately 67.1%-74.4% of the nifH gene sequences at the OTU level overlapped between the control and low N loads, but only 52.0%-53.6% of those overlapped between the control and high N loads, indicating a larger shift of diazotroph community composition under high N loads. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ concentrations under high N loads did not have negative impacts on the structure and function of the diazotroph community. These findings indicate that diazotrophs sustain N fixation under high N deposition via adjustment of their community composition in forest soils.ImportanceThis study examined the changes in soil diazotroph community under different loads of simulated N deposition and analyzed its relationship with N fixation rates in in five forests using high-throughput sequencing. The magnitudes of N fixation rates reduced by low N loads were higher than those by high N loads. Low N loads decreased richness and diversity of diazotroph community, whereas diazotroph community structure remained stable under high N loads. Compared with low N loads, high N loads resulted in a less similarity and overlap of nifH gene sequences among the treatments and a larger adjustment of diazotroph community. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ under high N loads did not have negative impacts on diazotroph community structure and N fixation. Based on these findings, it is urgently needed to incorporate the loads of N deposition and the composition of diazotroph community into terrestrial N-cycling models for accurate understanding of N inputs in forest ecosystems.

Project description:Nitrogen gas (N2) fixation driven by diazotrophs is a crucial process for supplying nitrogen to paddy soil ecosystems. The genus Geomonas has been considered to be an important potential diazotroph in paddy soils, but direct experimental evidence of the nitrogen-fixing ability of Geomonas in pure culture is still lacking. Hence, we aimed to demonstrate this nitrogen-fixing capability and shed light on how this process was regulated in response to ammonium (NH4 +) in Geomonas. In this study, we determined that a key nitrogenase gene (nifH) was present in 50 isolates from paddy soils. Members of Geomonas contained the minimum nitrogen fixation gene cluster (nifBHDKEN) based on genomic analysis, implying Geomonas species had the potential to fix nitrogen. Acetylene reduction assay (ARA), 15N2 isotope labeling, and total nitrogen accumulation assays validated that Geomonas was, indeed, able to fix nitrogen in pure culture. Under nitrogen-fixing conditions, the cell morphology of Geomonas changed from short rod-shaped (with NH4 +) to long rod-shaped and flagella became longer and thicker. The expression of genes correlated to nitrogen fixation in the Geomonas transcriptome was quantified in response to NH4 +. Expression of genes associated with nitrogenase, flavin-based electron bifurcation complexes (such as the FixAB system), NH4 + uptake, and transformation (e.g., glutamine and glutamate synthetases) were significantly upregulated under nitrogen-fixing conditions, suggesting these mechanisms might be involved in N2 fixation in Geomonas. These results were verified by RT-qPCR. Taken together, our results demonstrate that Geomonas species possess the ability to fix N2 and expand our understanding on the ecological significance and potential applications of Geomonas in paddy soil ecosystems. IMPORTANCE The ability of Geomonas species to fix nitrogen gas (N2) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria. We demonstrated N2-dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas.

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Ferrihydrite-mediated methanotrophic nitrogen fixation in paddy soil under hypoxia

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