New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches

Abstract

The significance of ANME-2d in methane sink in the environment has been overlooked, and there was no any study evaluating the distribution of ANME-2d in the environment. New primers were thus needed to be designed for following research. In this paper, a pair of primers (DP397F and DP569R) was designed to quantify ANME-2d. The specificity and amplification efficiency of this primer pair were acceptable. PCR amplification of another pair of primers (DP142F and DP779R) generated a single, bright targeted band from the enrichment sample, but yielded faint, multiple bands from the environmental samples. Nested PCR was conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the copies of the 16s ribosomal RNA gene of ANME-2d in these samples ranged from 3.72 × 10(4) to 2.30 × 10(5) copies μg(-1) DNA, indicating that the percentage of ANME-2d was greatest in a polluted river sample and least in a rice paddy sample. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples; the latter finding suggests that ANME-2d may spread in environments.

Diversity of NC10 Bacteria and ANME-2d Archaea in Sediments of Fault Zones at Lake Baikal

“…no. FN429785); 4% of sequences (OTU74) formed a cluster with denitrifying anaerobic methane oxidation archaea [19]; the remaining sequences (approximately 2% of all analyzed ANME-2d sequences, OTU101) grouped with sequences of archaea from sediments of Honghu Lake (HM244209).…”

“…In recent years, there has been an active search for microbial agents that carry out AOM in freshwater ecosystems. Recent studies have shown that in oxygen-free soil layers and bottom sediments, anaerobic methanotrophic bacteria, and/or archaea, which are used as electron acceptors of nitrite or nitrate ions, consume methane [19][20][21][22][23][24]. Methane oxidation under anaerobic conditions, which is associated with nitrite reduction, was first demonstrated in the enrichment cultures derived from freshwater sediments [23].…”

“…Analyses of various environmental samples using primers on the pmoA and nod genes have indicated M. oxyfera various anoxic environments including lake sediments, wastewater treatment systems [27][28][29][30][31]. Members of the ANME-2d subcluster named M. nitroreducens perform AOM using nitrate as the terminal electron acceptor [19,22]. The resulting nitrite is reduced to gaseous nitrogen due to syntrophic interaction with ANAMMOX-bacteria (in the presence of ammonium) or with M. oxyfera (phylum NC10) [32].…”

Nitrate‐ and nitrite‐dependent anaerobic oxidation of methane

“…A). Few aspects about the physiology of ‘ M. nitroreducens ’ are known, and its environmental detection has been limited to molecular methods based on 16S rRNA and mcrA genes (Ding et al ., ). The overview of environmental distribution based on those biomarkers is summarized in Table .…”

Reverse Methanogenesis and Respiration in Methanotrophic Archaea

“…As ANME-2d archaea were only recently recognized, their environmental preferences remain insufficiently studied. So far they have been found in freshwater canals [31], soils and rice paddy fields [34–36], lakes and rivers [35], and wastewater treatment plants [33]. In situ AOM activity of ANME-2d was determined recently for the first time [36].…”