Enrichment of anaerobic nitrate-dependent methanotrophic ‘Candidatus Methanoperedens nitroreducens’ archaea from an Italian paddy field soil

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-017-8416-0) contains supplementary material, which is available to authorized users.

Lateral gene transfer drives metabolic flexibility in the anaerobic methane oxidising archaeal familyMethanoperedenaceae

“…As previously reported, the MAGs M.Nitro, BLZ2, and IPS-1 encode respiratory nitrate reductases that are part of the CISM superfamily, allowing them to independently mediate AOM coupled to nitrate reduction (15, 24, 65). Based on phylogenetic analysis ( Figure 3 ), genes encoding cytoplasmic nitrite oxidoreductases (NxrA) were identified in the IPS-1, BLZ2, and M.Nitro MAGs, and a nitrate reductase closely related to NarG proteins was identified in the BLZ2 MAG.…”

ANME-2d anaerobic methanotrophic archaea differ from other ANME archaea in lipid composition and carbon source

“…strain BLZ2. Analysis of the microbial community in the Italian paddy field ANME-2d enrichment has been described by Vaksmaa et al, 2017. For this bioreactor a similar proportion (22%) of ANME-2d archaea, in this case Ca .…”

“…Under these conditions nitrite was not detectable with a colorimetric test with a lower detection limit of 2 mg/L (MQuant test stripes, Merck, Darmstadt, Germany). Growth conditions and operation of the bioreactor containing ANME-2d archaea enriched from an Italian paddy field soil are described by Vaksmaa et al ., 2017 (Vaksmaa, Jetten, et al , 2017). Sampled material from both reactors was centrifuged (10000 x g , 20 min, 4°C) and pellets were kept at −80°C until subsequent freeze-drying and following lipid and isotope analysis.…”

“…Instead, ANME-2d archaea are the main players in nitrate-dependent AOM. Microorganisms conducting nitrate AOM have been enriched from anoxic freshwater sediments, digester sludge and rice paddies (Raghoebarsing et al , 2006; Hu et al , 2009; Arshad et al , 2015; Vaksmaa, Guerrero-Cruz, et al , 2017). Denitrifying AOM can either be conducted by a consortium of nitrate-reducing ANMEs, Ca .…”

Key Physiology of a Nitrite-Dependent Methane-Oxidizing Enrichment Culture

“…When nitrate and nitrite were supplied together to anoxic sediments collected from agricultural ditch canals, both nitrate-and nitrite-dependent methane-oxidizing microorganisms were enriched (6,36). Enrichments solely fed with nitrate resulted in similar cocultures where nitrate-AOM supports nitrite-AOM by producing nitrite (10,37,38). On the other hand, when ammonium, methane, and nitrite were supplied as substrates, cocultures of Methylomirabilis species and anaerobic ammonium-oxidizing (anammox) bacteria were achieved (14,(39)(40)(41).…”