Methane-oxidizing microorganisms perform an important role in reducing emissions of the greenhouse gas methane to the atmosphere. To date, known bacterial methanotrophs belong to the Proteobacteria, Verrucomicrobia, and NC10 phyla. Within the Proteobacteria phylum, they can be divided into type Ia, type Ib, and type II methanotrophs. Type Ia and type II are well represented by isolates. Contrastingly, the vast majority of type Ib methanotrophs have not been able to be cultivated so far. Here, we compared the distributions of type Ib lineages in different environments. Whereas the cultivated type Ib methanotrophs (Methylococcus and Methylocaldum) are found in landfill and upland soils, lineages that are not represented by isolates are mostly dominant in freshwater environments, such as paddy fields and lake sediments. Thus, we observed a clear niche differentiation within type Ib methanotrophs. Our subsequent isolation attempts resulted in obtaining a pure culture of a novel type Ib methanotroph, tentatively named "Methylotetracoccus oryzae" C50C1. Strain C50C1 was further characterized to be an obligate methanotroph, containing C_16:1ω9c as the major membrane phospholipid fatty acid, which has not been found in other methanotrophs. Genome analysis of strain C50C1 showed the presence of two pmoCAB operon copies and XoxF5-type methanol dehydrogenase in addition to MxaFI. The genome also contained genes involved in nitrogen and sulfur cycling, but it remains to be demonstrated if and how these help this type Ib methanotroph to adapt to fluctuating environmental conditions in freshwater ecosystems.

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.

The biological nitrogen cycle is driven by a plethora of reactions transforming nitrogen compounds between various redox states. Here, we investigated the metagenomic potential for nitrogen cycle of the in situ microbial community in an oligotrophic, brackish environment of the Bothnian Sea sediment. Total DNA from three sediment depths was isolated and sequenced. The characterization of the total community was performed based on 16S rRNA gene inventory using SILVA database as reference. The diversity of diagnostic functional genes coding for nitrate reductases (napA;narG), nitrite:nitrate oxidoreductase (nxrA), nitrite reductases (nirK;nirS;nrfA), nitric oxide reductase (nor), nitrous oxide reductase (nosZ), hydrazine synthase (hzsA), ammonia monooxygenase (amoA), hydroxylamine oxidoreductase (hao), and nitrogenase (nifH) was analyzed by blastx against curated reference databases. In addition, Polymerase chain reaction (PCR)-based amplification was performed on the hzsA gene of anammox bacteria. Our results reveal high genomic potential for full denitrification to N₂, but minor importance of anaerobic ammonium oxidation and dissimilatory nitrite reduction to ammonium. Genomic potential for aerobic ammonia oxidation was dominated by Thaumarchaeota. A higher diversity of anammox bacteria was detected in metagenomes than with PCR-based technique. The results reveal the importance of various N-cycle driving processes and highlight the advantage of metagenomics in detection of novel microbial key players.

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.

Oxygen minimum zones (OMZ) are areas in the global ocean where oxygen concentrations drop to below one percent. Low oxygen concentrations allow alternative respiration with nitrate and nitrite as electron acceptor to become prevalent in these areas, making them main contributors to oceanic nitrogen loss. The contribution of anammox and denitrification to nitrogen loss seems to vary in different OMZs. In the Arabian Sea, both processes were reported. Here, we performed a metagenomics study of the upper and core zone of the Arabian Sea OMZ, to provide a comprehensive overview of the genetic potential for nitrogen and methane cycling. We propose that aerobic ammonium oxidation is carried out by a diverse community of Thaumarchaeota in the upper zone of the OMZ, whereas a low diversity of Scalindua-like anammox bacteria contribute significantly to nitrogen loss in the core zone. Aerobic nitrite oxidation in the OMZ seems to be performed by Nitrospina spp. and a novel lineage of nitrite oxidizing organisms that is present in roughly equal abundance as Nitrospina. Dissimilatory nitrate reduction to ammonia (DNRA) can be carried out by yet unknown microorganisms harbouring a divergent nrfA gene. The metagenomes do not provide conclusive evidence for active methane cycling; however, a low abundance of novel alkane monooxygenase diversity was detected. Taken together, our approach confirmed the genomic potential for an active nitrogen cycle in the Arabian Sea and allowed detection of hitherto overlooked lineages of carbon and nitrogen cycle bacteria.