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Expanding methanogens with genetic potential for extracellular electron transfer capabilities in anaerobic wastewater treatment ecosystems

Nature Water (2025)Cite this article

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Abstract

Methanogens have key roles in wastewater treatment, coupling water quality control and bioenergy recovery. However, our understanding of direct interspecies electron transfer via extracellular electron transfer (EET)—a newly discovered methanogenic pathway for CO2 reduction—remains limited because pure-culture cultivation is difficult and few strains are confirmed. Here we show that a survey of 378 methanogen genomes reveals key methanogenesis-related genes and widespread EET-associated structures, including proton-pumping Fpo complexes, conductive flagellin, conductive sheaths and multihaem c-type cytochromes. We identify 84 strains with genomic potential for EET, greatly expanding the candidate pool. Analysis of over 500 anaerobic digestion samples, including those from wastewater treatment systems, revealed that putative EET-capable methanogen genera are widespread, environmentally correlative and central to syntrophic networks. These findings deepen our understanding of methanogenic diversity in advancing wastewater treatment and sustainability while also broadening insights into methanogenesis across diverse aquatic ecosystems.

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Fig. 1: Overview of the screening workflow to identify putative EET-capable methanogens.
Fig. 2: Identification of CO2 reduction pathway genes and substrate utilization in methanogen strains.
Fig. 3: Identification and characterization of membrane-associated and membrane-integral Fpo proteins in methanogens.
Fig. 4: Distribution of conductive structures in methanogens.
Fig. 5: Global distribution and environmental context of putative EET-capable methanogens.
Fig. 6: Role of putative EET-capable methanogens in the global syntrophic network.

Data availability

The datasets generated and/or analysed during this study are available within this article and its Supplementary Information. No custom software or unpublished code was developed in this study.

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Acknowledgements

This study was partially supported by the Taighde Éireann–Research Ireland (formerly Science Foundation Ireland) and the Sustainable Energy Authority of Ireland under the SFI Frontiers for the Future Awards Programme (22/FFP-A/10346) (G.W.) and National Natural Science Foundation of China (52400107) (Q.Y. and B.L.). Q.Y. thanks the EU Marie Skłodowska Curie Actions Postdoctoral fellowship (101103499), C.L. and Y.D. thank the scholarship from the China Scholarship Council (202206510027), and G.W. is grateful for the support from the Galway University Foundation.

Author information

Authors and Affiliations

  1. Civil Engineering, School of Engineering, College of Science and Engineering, University of Galway, Galway, Ireland

    Qidong Yin & Guangxue Wu

  2. School of Civil Engineering, Sun Yat-Sen University, Zhuhai, China

    Qidong Yin

  3. College of Environmental Science and Engineering, Beijing Forestry University, Beijing, China

    Chuanqi Liu & Yan Dang

  4. School of Ecology, Environment and Resource, Guangdong University of Technology, Guangzhou, China

    Bo Li

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  1. Qidong Yin
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  2. Chuanqi Liu
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Contributions

Conceptualization: Q.Y. and G.W. Investigation: Q.Y., B.L. and C.L. Writing—original draft: Q.Y. and G.W. Writing—review and editing: Q.Y., Y.D., B.L., C.L. and G.W. Supervision: Y.D. and G.W. Funding acquisition: Q.Y. and G.W.

Corresponding author

Correspondence to Guangxue Wu.

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Nature Water thanks Joonyeob Lee and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sensitivity analysis of the number and taxonomic composition of putative EET-capable methanogens identified at different aromatic amino acid content thresholds in flagellin proteins.

Bars represent the counts of methanogen genera and species identified under thresholds ranging from 4.9% to 10%. Colours indicate different genera.

Extended Data Fig. 2 Metatranscriptomic results of a microbial electrolysis cell system.

a. Schematic representation of methane production comparing MEC (poised cathode, −0.5 V vs. SHE) and non-energized (NE) control conditions. Statistical significance was assessed using a two-sided Student’s t-test. p for CH4 (%):3.94 × 10−2⁰; p for CH4 (mL): 9.01 × 10−2⁰. b. Taxonomic composition of dominant methanogenic species detected in biocathode and bulk sludge samples across treatments. c. The expression levels (Transcripts Per Kilobase Million, TPM) of key fpo genes in Methanothrix and Methanosarcina taxa under MEC and NE conditions. d. The expression levels of CO2 reduction genes in Methanothrix and Methanosarcina taxa. e. The expression levels of flaB gene attributed to Methanosarcina and Methanospirillum. f. The expression levels of Mspa (Mthe_1070) gene attributed to Methanothrix. ***: p < 0.001.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Texts 1–9 and Tables 1–6.

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Source Data for Supplementary Figures

Source data for Supplementary Figs. 1, 2, 4, 5a, 5b and 6.

Source data

Source Data Table 1.

Source data for all data presented in graphs within the figures.

Source Data Table 2.

Source data for all data presented in graphs within the extended data figures.

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Yin, Q., Liu, C., Li, B. et al. Expanding methanogens with genetic potential for extracellular electron transfer capabilities in anaerobic wastewater treatment ecosystems. Nat Water (2025). https://doi.org/10.1038/s44221-025-00524-6

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