Archaea catalyze iron-dependent anaerobic oxidation of methane

Abstract

Anaerobic oxidation of methane (AOM) is crucial for controlling the emission of this potent greenhouse gas to the atmosphere. Nitrite-, nitrate-, and sulfate-dependent methane oxidation is well-documented, but AOM coupled to the reduction of oxidized metals has so far been demonstrated only in environmental samples. Here, using a freshwater enrichment culture, we show that archaea of the order Methanosarcinales, related to "Candidatus Methanoperedens nitroreducens," couple the reduction of environmentally relevant forms of Fe³⁺ and Mn⁴⁺ to the oxidation of methane. We obtained an enrichment culture of these archaea under anaerobic, nitrate-reducing conditions with a continuous supply of methane. Via batch incubations using [¹³C]methane, we demonstrated that soluble ferric iron (Fe³⁺, as Fe-citrate) and nanoparticulate forms of Fe³⁺ and Mn⁴⁺ supported methane-oxidizing activity. CO₂ and ferrous iron (Fe²⁺) were produced in stoichiometric amounts. Our study connects the previous finding of iron-dependent AOM to microorganisms detected in numerous habitats worldwide. Consequently, it enables a better understanding of the interaction between the biogeochemical cycles of iron and methane.

Keywords: anaerobic oxidation of methane; archaea; iron reduction; manganese reduction; multiheme proteins.

Fig. 1.

Fluorescence in situ hybridization of biomass from the enrichment culture of AAA and M. oxyfera-like bacteria. AAA were hybridized with Cy3-labeled AAA-specific probe S-*-AAA-FW-641 (red) (33); M. oxyfera-like bacteria were hybridized with Fluos-labeled NC10 bacteria-specific probe S-*-DBACT-1027-a-A-18 (yellow) and a Cy5-labeled mixture of EUB I to III and V (blue; overlay appears green). Other bacteria only hybridizing with EUB I to III and V appear blue. (Scale bar, 5 µm.)

Fig. 2.

(A) Stoichiometric coupling of [¹⁵N]nitrate reduction (open squares) to ³⁰N₂ (open triangles) and ¹³CO₂ (filled circles) production in the presence of ¹³CH₄ in batch incubations. Data of CO₂ and N₂ are the average of four replicate incubations measured twice each; nitrate values represent two replicates. Each incubation contained 77.0 ± 3.6 μg protein. Error bars indicate SD. (B) Transient formation of nitrite (open diamonds) and accumulation of ammonium (open circles) after repeated pulse feeding (arrows) of nitrate (open squares) in the continuous culture reactor with constant methane supply. Note the different scale for nitrite.

Fig. 3.

Metal-dependent oxidation of ¹³C-labeled CH₄ to ¹³CO₂ by an enrichment culture of AAA. (A) Fe³⁺-citrate addition at the start of the experiment and after 150 h (arrow) stimulates methane oxidation. Control incubations of the enrichment culture without any addition, sulfate or citrate only, as well as an archaea-free enrichment culture of M. oxyfera (34) incubated with Fe³⁺-citrate were not active. Data are the average of two incubations containing 1 ± 0.1 mg protein each. (B) Stoichiometric coupling of CH₄ oxidation to Fe²⁺ production. Methane oxidation was deduced from ¹³CO₂ production; data points represent the average of four incubations of 77 ± 3.6 µg protein each. (C) Stimulation of ¹³CO₂ production from ¹³CH₄ by the addition of nanoparticulate suspensions of ferrihydrite (Fe³⁺) and birnessite (Mn⁴⁺). Protein content in these experiments was 77.0 ± 3.6 µg per vial. Error bars indicate SD.

Fig. S1.

(A) Phylogenetic position of AOM-associated archaea (AAA) (red) among other archaeal methanotrophs (blue) and methanogens (gray) based on sequences of 16S rRNA gene sequences obtained from GenBank. (B) Detailed phylogenetic tree of 16S rRNA genes of the AAA group within Methanosarcinales. Sequences from enrichment cultures are highlighted in bold letters; sequences from sites or cultures with demonstrated AOM activity are shown in blue; those from particularly metal-rich or iron-reducing environments are shown in orange. The evolutionary analysis was conducted with the maximum-likelihood algorithm implemented in MEGA6 (39). There were a total of 949 positions in the final dataset. Bootstrap support >90 is shown at the branches. (Scale bar, number of substitutions per site.)