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

Abstract

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 ¹³C- and ⁵⁷Fe-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.

Fig. 1

Bioreactor performance data, and mass- and electron balance tests results. a Simultaneous methane consumption and dissolved Fe(II) production. Blue and red arrows represent bioreactor flushing with methane and ferrihydrite addition, respectively. The sharp drop in dissolved Fe(II) following each ferrihydrite addition was determined to be due to abiotic reactions between dissolved Fe(II) and ferrihydrite. b The profiles of Fe(III), Fe(II) and methane during the batch test started on Day 601 as an example. c Average ratios of Fe(III) consumption and Fe(II) production rates, respectively, to methane consumption rate determined in the three batch tests. Error bars represent standard deviations of results using three subsamples from the bioreactor

Fig. 2

AOM coupled to Fe(III) reduction by the enrichment culture in one of the duplicate isotopic labelling tests (grey area in Supplementary Figure 2a). a–d Conversion of ¹³CH₄ to ¹³CO₂ (a), ⁵⁷Fe(III) to ⁵⁷Fe(II) (b), CH₄ to CO₂ (c), and Fe(III) to Fe(II) (d)

Fig. 3

Phylogenetic placement of the dominant “M. ferrireducens” population. a, b Genome tree (a) and 16 S rRNA gene tree (b) showing the phylogenetic placement of the “M. ferrireducens” genome inferred by maximum-likelihood. In both trees, the Methanoperedenaceae genomes and 16 S rRNA gene sequences are highlighted in red with “M. ferrireducens” indicated with an asterisk. Black and white dots indicate >90% and >70% bootstrap values, respectively. The scale bars in a and b represent amino acids and nucleotide changes, respectively

Fig. 4

Metabolic construction of the putative pathway for AOM coupled to Fe(III) reduction in “M. ferrireducens“. Electrons from methane are generated through the “reverse methanogenesis” pathway and transferred to the menaquinone pool (MK/MKH₂) via the Fpo and Hdr complexes, which oxidise F₄₂₀H₂ and CoM-SH+CoB-SH, respectively. CoM-SH and CoB-SH can be oxidised by the HdrDE complex [53] or the cytoplasmic HdrABC–FrhB complex that also reoxidise ferredoxin and reduce two F₄₂₀ [21]. Reducing equivalents are transferred via the menaquinone:cytochrome c oxidoreductases to MHCs located outside the cytoplasm to reduce the Fe(III) oxides. Abbreviations for enzymes and co-factors: H₄MPT, tetrahydromethanopterin; MFR, methanofuran; Fwd, formyl-methanofuran dehydrogenase; Ftr, formylmethanofuran/H₄MPT formyltransferase; Mch, methenyl-H₄MPT cyclohydrolase; Mtd, F₄₂₀-dependent methylene H₄MPT dehydrogenase; Mer, F₄₂₀-dependent methylene-H₄MPT reductase; Mtr, Na⁺-translocating methyl-H₄MPT:coenzyme M methyltransferase; Mcr, methyl-coenzyme M reductase; Fpo, F₄₂₀H₂ dehydrogenase; MK, menaquinone; CoB-SH, coenzyme B; CoM-SH, coenzyme M; Fd, ferredoxin; Hdr, heterodisulfide reductase; FrhB, F₄₂₀-reducing hydrogenase subunit B; Cytb, b-type cytochrome; NrfD, polysulfide reductase subunit D; FeS, ferredoxin iron sulfur protein. The MHCs are denoted in as blue and the number of hemes are indicated as red diamonds. Normalised gene expression is indicated as TPM. More information on the metabolism in ANME archaea can be found in the previous reviews [61, 62]