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Photoholes within cyanobacterial mats can account for the origin of anammox bacteria and ancient nitrogen loss

Nature Ecology & Evolution (2026)Cite this article

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Abstract

Anaerobic ammonium oxidation (anammox) bacteria contribute to nearly half of global nitrogen loss. However, the driving force responsible for the origin of anammox bacteria remains poorly understood. Here we show that anammox bacteria can oxidize ammonium to N2 for growth using photoholes—the positive charge carriers generated from photosensitizers—potentially supporting their origin. Such photoholes could have been generated in sunlit benthic environments by cyanobacterial mats and semiconducting minerals under the intense solar radiation of the Late Archaean (3.0–2.5 billion years ago). Moreover, cyanobacterial mats absorbed harmful short-wavelength light for anammox bacteria, while allowing longer-wavelength infrared light to penetrate. Light-driven enrichment of nitrite-reductase-deficient anammox bacteria in long-term-cultured cyanobacterial mats, DNA stable-isotope probing and evolutionary analysis collectively suggest that the ancestral anammox bacteria tended to be photoelectrotrophic instead of nitrite-dependent. Our discovery provides a paradigm shift in our understanding of the origin of ammonium oxidation and may explain the nitrogen loss on early Earth.

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Fig. 1: Light-driven enrichment of anammox bacteria in the cyanobacterial mats.
Fig. 2: Evidence for photohole-driven ammonium oxidation by anammox bacteria.
Fig. 3: Impact of light with different wavelengths on anammox bacteria.
Fig. 4: Timetree inferred under a Bayesian node-dating approach, showing the divergence time of anammox bacteria.
Fig. 5: Evolution of the metabolic traits of anammox bacteria.
Fig. 6: Proposed schematic model illustrating biophotoelectrochemical ammonium oxidation by anammox bacteria in sunlit benthic cyanobacterial mats.

Data availability

All raw metagenomes and metatranscriptomes are deposited in the NCBI Sequence Read Archive under BioProject accession numbers PRJNA1144341 and PRJNA1260665. All information on the publicly available MAGs used in this study is provided in Supplementary Datasets 2 and 3. The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

Analysis scripts are publicly available at https://github.com/zhengru-pku/Metatranscriptomic_analyze.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (W2412119 (S.L.), 52270016 (S.L.), 52200029 (K.Z.) and 523B2095 (L.K.)) and Peking University-BHP Carbon and Climate Wei-Ming PhD Scholars (WM202510 (L.K.)). We are grateful for the support of the High-Performance Computing Platform of Peking University.

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  1. These authors contributed equally: Lingrui Kong, Ru Zheng.

Authors and Affiliations

  1. College of Environmental Sciences and Engineering, Peking University, Beijing, P. R. China

    Lingrui Kong, Ru Zheng, Jinnan Feng, Yiming Feng, Baiyizhuo Chen, Yimin Mao, Jiangwei Wang, Kuo Zhang, Ansheng Cheng & Sitong Liu

  2. Key Laboratory of Water and Sediment Sciences, Ministry of Education of China, Beijing, P. R. China

    Lingrui Kong, Ru Zheng, Jinnan Feng, Yiming Feng, Baiyizhuo Chen, Yimin Mao, Jiangwei Wang, Kuo Zhang, Ansheng Cheng & Sitong Liu

  3. Eco-environment and Resource Efficiency Research Laboratory, Peking University Shenzhen Graduate School, Shenzhen, P. R. China

    Kuo Zhang & Sitong Liu

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Contributions

S.L., L.K. and R.Z. designed the study. L.K., R.Z. and J.F. conducted the research with the help of Y.F., B.C., Y.M., J.W. and A.C. K.Z., L.K., R.Z. and S.L. wrote the paper. Y.F. and B.C. performed the software analysis. Y.M., J.W. and A.C. investigated the related information. All the authors contributed to the interpretation of the findings.

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Correspondence to Sitong Liu.

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Nature Ecology & Evolution thanks Tianhua Liao, Eva Stüeken 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 EPR spectroscopy of the cyanobacterial mats.

(a) The EPR signal was detected in the presence of 5 mM spin probe TEMPO in cyanobacterial mats under dark conditions. (b) The TEMPO signal was detected with the addition of electron scavenger (5 mM AgNO3) in cyanobacterial mats under light conditions. All EPR spectra were recorded at room temperature (25 °C) and recorded every 5 minutes with the following instrumental parameters: center magnetic field, 3505.00 G; modulation frequency, 100.00 kHz; modulation amplitude, 1.000 G; microwave power, 0.6325 mW; sweep width, 100.0 G; and sweep time, 30 s. Magnified views of the selected region were shown to highlight subtle variations in signal intensity.

Source data

Extended Data Fig. 2 Expression of genes in enriched AMX1 at different days during the reactor operation.

Gene expression levels, quantified as Log2(TPM + 1), are shown for metatranscriptomic samples collected at key time points: under dark conditions on day 60 and under light conditions on days 200 and 450. Rows denote genes categorized by function, including light sensing and adaptation, nitrogen metabolism, electron transfer, and cyanate assimilation. The gene encoding hydrazine synthase subunit C (hzsC) was not detected in the AMX1 genome, likely due to the incompleteness of the recovered MAG. For each condition, TPM values represent the mean ± standard deviation derived from three biological replicates.

Source data

Extended Data Fig. 3 Morphological and structural characterization of hematite and hematite-anammox biohybrid system.

(a) Scanning electron microscopy (SEM) image showing the morphology of the hematite-anammox biohybrid system. Micrographs are representative results from 10 independent images. (b) Energy dispersive X-ray spectroscopy (EDS) mapping highlighting the distribution of the Fe element. (c) EDS mapping showing the distribution of the C element within the biohybrid system. (d) EDS mapping illustrating the distribution of the O element. (e) X-ray photoelectron spectroscopy (XPS) spectrum of hematite, revealing characteristic binding energies of Fe, O, and C. (f) X-ray diffraction (XRD) pattern of hematite, with key diffraction peaks corresponding to hematite (indexed in blue).

Source data

Extended Data Fig. 4 Formation of iron mineral on the surface of cyanobacterial mats.

SEM-EDS analysis of cyanobacterial mats collected from the photobioreactor on day 450. The SEM image shows cyanobacterial filaments partially covered by granular deposits identified as iron oxides. Elemental mapping of Fe, O, and C reveals strong co-localization of Fe and O, with negligible carbon signal, indicating the formation of iron mineral coatings on the cyanobacterial surface. Micrographs are representative results from 10 independent images.

Extended Data Fig. 5 The gene expression profile of anammox bacteria during the batch assays of biophotoelectrochemical ammonium oxidation.

Gene expression profiles (in terms of TPM) of three recovered MAGs of anammox bacteria (AMX1–3) under different experimental conditions: hematite-anammox biohybrid under illumination with ammonium (R0), anammox consortia without hematite under illumination (R1), and dark anammox consortia supplied with ammonium and nitrite (R2). TPM values represent means of three biological replicates. Red and blue indicate up- and down-regulated genes, respectively. Statistical significance was assessed using an unpaired two-sided Student’s t-test (*P < 0.05; **P < 0.01; **P < 0.001).

Source data

Extended Data Fig. 6 The variation of specific anammox activity (SAA) over time in the batch assays under different light conditions.

(a) Blue light groups. (b) Infrared light groups. (c) White light groups. Batch assays were conducted under a light intensity of 8000 lux, with each illuminated group paired with a corresponding dark control. Specific anammox activity (SAA) was calculated based on the substrate consumption rate to evaluate the metabolic activity of anammox bacteria over time. Data represent mean ± s.d. of three biological replicates. Statistical significance was determined using a two-tailed Student’s t-test (*P < 0.05; **P < 0.01; **P < 0.001).

Source data

Extended Data Fig. 7 Co-occurrence network of the microbial community.

The network was constructed at the genus level based on Spearman’s correlation coefficients across all metagenomic samples collected during the reactor operation. Node size corresponds to the relative abundance of each genus. A threshold of R > 0.7 and P < 0.05 was used to filter for genera most strongly correlated with anammox bacteria (Ca. Brocadia) and cyanobacteria (Leptolyngbya).

Extended Data Fig. 8 Phylogenetic analysis of genes encoding bacterioferritin and hydrazine synthase from anammox bacteria.

(a) Phylogenetic tree of genes encoding bacterioferritin (bfr). (b) Phylogenetic tree of genes encoding hydrazine synthase subunit A (hzsA). Trees show the classification of these sequences from different anammox bacterial lineages. The phylogenetic tree is constructed based on the alignments of gene sequences from all anammox reference MAGs used in Supplementary Dataset 3. All conserved protein phylogenomic and phylogenetic alignments are based on MAFFT, and the trees that were built by the IQ-Tree method with model LG + C60 + F + G using SH approximate likelihood ratio test implemented with 1000 bootstrap replicates with bootstrap higher than 0.8 are shown with grey squares on tree branches.

Supplementary information

Supplementary Information

Supplementary Texts 1–7, Figs. 1–7 and Tables 1–3.

Reporting Summary

Supplementary Datasets 1–4

This Excel file contains Supplementary Datasets 1–4. The title and description are provided within the file itself.

Source data

Source Data Figs. 1–3 and Extended Data Figs. 1–3, 5 and 6

Source data for all figures and Extended Data figures.

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Kong, L., Zheng, R., Feng, J. et al. Photoholes within cyanobacterial mats can account for the origin of anammox bacteria and ancient nitrogen loss. Nat Ecol Evol (2026). https://doi.org/10.1038/s41559-026-02976-9

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