Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation

Since the discovery that anaerobic methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane coupled to sulfate reduction in marine sediments, different primers and probes specifically targeting the 16S rRNA gene of these archaea have been developed. Microbial investigation of the different ANME subtypes (ANME-1; ANME-2a, b, and c; and ANME-3) was mainly done in sediments where specific subtypes of ANME were highly enriched and methanogenic cell numbers were low. In different sediments with higher archaeal diversity and abundance, it is important that primers and probes targeting different ANME subtypes are very specific and do not detect other ANME subtypes or methanogens that are also present. In this study, primers and probes that were regularly used in AOM studies were tested in silico on coverage and specificity. Most of the previously developed primers and probes were not specific for the ANME subtypes, thereby not reflecting the actual ANME population in complex samples. Selected primers that showed good coverage and high specificity for the subclades ANME-1, ANME-2a/b, and ANME-2c were thoroughly validated using quantitative polymerase chain reaction (qPCR). From these qPCR tests, only certain combinations seemed suitable for selective amplification. After optimization of these primer sets, we obtained valid primer combinations for the selective detection and quantification of ANME-1, ANME-2a/b, and ANME-2c in samples where different ANME subtypes and possibly methanogens could be present. As a result of this work, we propose a standard workflow to facilitate selection of suitable primers for qPCR experiments on novel environmental samples.This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009). Research of PHATand AJMS is supported by the SIAM Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO).info:eu-repo/semantics/publishedVersio

Anaerobic oxidation of methane associated with sulfate reduction in a natural freshwater gas source

The occurrence of anaerobic oxidation of methane (AOM) and trace methane oxidation (TMO) was investigated in a freshwater natural gas source. Sediment samples were taken and analyzed for potential electron acceptors coupled to AOM. Long-term incubations with 13C-labeled CH4 (13CH4) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, 13C-labeled CO2 (13CO2) simultaneously increased with methane formation, which is typical for TMO. In the presence of nitrate, neither methane formation nor methane oxidation occurred. Net AOM was measured only with sulfate as electron acceptor. Here, sulfide production occurred simultaneously with 13CO2 production and no methanogenesis occurred, excluding TMO as a possible source for 13CO2 production from 13CH4. Archaeal 16S rRNA gene analysis showed the highest presence of ANME-2a/b (ANaerobic MEthane oxidizing archaea) and AAA (AOM Associated Archaea) sequences in the incubations with methane and sulfate as compared with only methane addition. Higher abundance of ANME-2a/b in incubations with methane and sulfate as compared with only sulfate addition was shown by qPCR analysis. Bacterial 16S rRNA gene analysis showed the presence of sulfate-reducing bacteria belonging to SEEP-SRB1. This is the first report that explicitly shows that AOM is associated with sulfate reduction in an enrichment culture of ANME-2a/b and AAA methanotrophs and SEEP-SRB1 sulfate reducers from a low-saline environment.We thank Douwe Bartstra (Vereniging tot Behoud van de Gasbronnen in Noord-Holland, The Netherlands), Carla Frijters (Paques BV, The Netherlands) and Teun Veuskens (Laboratory of Microbiology, WUR, The Netherlands) for sampling; Martin Meirink (Hoogheemraadschap Hollands Noorderkwartier, The Netherlands) for physicochemical data; Freek van Sambeek for providing Figure 1; Lennart Kleinjans (Laboratory of Microbiology, WUR, The Netherlands) for help with pyrosequencing analysis, Irene Sánchez-Andrea (Laboratory of Microbiology, WUR, The Netherlands) for proof-reading and Katharina Ettwig (Department of Microbiology, Radboud University Nijmegen, The Netherlands) for providing M. oxyfera DNA. We want to thank all anonymous reviewers for valuable contributions. This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)

A methanotrophic archaeon couples anaerobic oxidation of methane to Fe(III) reduction

Microbially mediated anaerobic oxidation of methane (AOM) is a key process in the regulation of methane emissions to the atmosphere. Iron can serve as an electron acceptor for AOM, and it has been suggested that Fe(III)-dependent AOM potentially comprises a major global methane sink. Although it has been proposed that anaerobic methanotrophic (ANME) archaea can facilitate this process, their active metabolic pathways have not been confirmed. Here we report the enrichment and characterisation of a novel archaeon in a laboratory-scale bioreactor fed with Fe(III) oxide (ferrihydrite) and methane. Long-term performance data, in conjunction with the C- and Fe-labelling batch experiments, demonstrated that AOM was coupled to Fe(III) reduction to Fe(II) in this bioreactor. Metagenomic analysis showed that this archaeon belongs to a novel genus within family Candidatus Methanoperedenaceae, and possesses genes encoding the "reverse methanogenesis" pathway, as well as multi-heme c-type cytochromes which are hypothesised to facilitate dissimilatory Fe(III) reduction. Metatranscriptomic analysis revealed upregulation of these genes, supporting that this archaeon can independently mediate AOM using Fe(III) as the terminal electron acceptor. We propose the name Candidatus "Methanoperedens ferrireducens" for this microorganism. The potential role of "M. ferrireducens" in linking the carbon and iron cycles in environments rich in methane and iron should be investigated in future research

Catabolic Pathways and Enzymes Involved in Anaerobic Methane Oxidation

Microbes use two distinct catabolic pathways for life with the fuel methane: aerobic methane oxidation carried out by bacteria and anaerobic methane oxidation carried out by archaea. The archaea capable of anaerobic oxidation of methane, anaerobic methanotrophs (ANME), are phylogenetically related to methanogens. While the carbon metabolism in ANME follows the pathway of reverse methanogenesis, the mode of electron transfer from methane oxidation to the terminal oxidant is remarkably versatile. This chapter discusses the catabolic pathways of methane oxidation coupled to the reduction of nitrate, sulfate, and metal oxides. Methane oxidation with sulfate and metal oxides are hypothesized to involve direct interspecies electron transfer and extracellular electron transfer. Cultivation of ANME, their mechanisms of energy conservation, and details about the electron transfer pathways to the ultimate oxidants are rather new and quickly developing research fields, which may reveal novel metabolisms and redox reactions. The second section focuses on the carbon catabolism from methane to CO2 and the biochemistry in ANME with its unique enzymes containing Fe, Ni, Co, Mo, and W that are compared with their homologues found in methanogens

Anaerobic Methane Oxidizers

The anaerobic oxidation of methane (AOM) with sulfate as the final electron acceptor according to the net reaction CH4 + SO42- -> HCO3- -> HS- + H2O is the major sink of methane in the ocean floor and hence a significant process in the marine methane budget and the global carbon cycle. Since its discovery, much has been learned about the distribution of the AOM process, its activity in different settings, and connections to other metabolic reactions in the seafloor. AOM is performed by consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). Since all known ANME and most of their partner bacteria have so far resisted isolation, the physiology of both organisms has been largely inferred from culture-independent approaches on natural enrichments or enrichment cultures. All known ANME are related to methanogenic Euryarchaeota, and as such they reverse the methanogenesis pathway to activate and completely oxidize methane. The reducing equivalents are shuttled to the partner bacteria, which use them for sulfate reduction. Recently, evidence has been found for ANME that can use nitrate or iron as electron acceptors. The exact mechanisms for the required exchange of reducing equivalents in AOM and their genetic codes are yet poorly understood, but recently discovered accumulations of cytochromes and nanowire connections in the intercellular space of the consortia suggest direct electron transfer between both partners

Anaerobic Methane Oxidizers

The anaerobic oxidation of methane (AOM) with sulfate as the final electron acceptor according to (CH4 + SO4 2− → HCO3 − + HS− + H2O) is the major sink of methane in the oceans and hence a significant process in the global carbon cycle and methane budget. Anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) are assumed to act as a syntrophic consortium where the archaeal partner activates and metabolizes methane, leading to an intermediate that is scavenged as electron donor by the sulfate-reducing partner. All known anaerobic methanotrophs are related to the methanogenic Euryarchaeota. Recently, much has been learned about the distribution, activity, and physiology of the ANME, however, not a single member of these groups has been obtained in culture and the biochemical functioning of AOM remains unknown