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Nitrogen fixation and diazotroph diversity in groundwater systems

The ISME Journal volume 17, pages 2023–2034 (2023)Cite this article

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Abstract

Biological nitrogen fixation (BNF), the conversion of N2 into bioavailable nitrogen (N), is the main process for replenishing N loss in the biosphere. However, BNF in groundwater systems remains poorly understood. In this study, we examined the activity, abundance, and community composition of diazotrophs in groundwater in the Hetao Plain of Inner Mongolia using 15N tracing methods, reverse transcription qPCR (RT-qPCR), and metagenomic/metatranscriptomic analyses. 15N2 tracing incubation of near in situ groundwater (9.5–585.4 nmol N L−1 h−1) and N2-fixer enrichment and isolates (13.2–1728.4 nmol N g−1 h−1, as directly verified by single-cell resonance Raman spectroscopy), suggested that BNF is a non-negligible source of N in groundwater in this region. The expression of nifH genes ranged from 3.4 × 103 to 1.2 × 106 copies L−1 and was tightly correlated with dissolved oxygen (DO), Fe(II), and NH4+. Diazotrophs in groundwater were chiefly aerobes or facultative anaerobes, dominated by Stutzerimonas, Pseudomonas, Paraburkholderia, Klebsiella, Rhodopseudomonas, Azoarcus, and additional uncultured populations. Active diazotrophs, which prefer reducing conditions, were more metabolically diverse and potentially associated with nitrification, sulfur/arsenic mobilization, Fe(II) transport, and CH4 oxidation. Our results highlight the importance of diazotrophs in subsurface geochemical cycles.

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Fig. 1: Distribution of nifH gene abundances and their response to environment variables.
Fig. 2: Diversity and community composition of native and active diazotrophs in groundwater samples.
Fig. 3: Relationship between geochemical parameters and diazotrophic communities.
Fig. 4: Metabolic potential and genomic bins related to N2 fixation and N/S/Fe/CH4/As cycles in groundwater.
Fig. 5: N2 fixation rates in field groundwater and N2-fixer enrichments and isolates.

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Data availability

The raw data have been submitted to the NCBI database under BioProject numbers PRJNA937386, PRJNA996345, PRJNA996367, PRJNA882225, and PRJNA884812.

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Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (Grant No 42177068, 91851115, 91851209, and 91851101).

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Authors and Affiliations

  1. State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan, 430074, PR China

    Xiaohan Liu, Ping Li, Helin Wang, Yanhong Wang & Zhou Jiang

  2. Hubei Key Laboratory of Yangtze Catchment Environmental Aquatic Science, School of Environmental Studies, China University of Geosciences, Wuhan, 430074, PR China

    Xiaohan Liu, Ping Li, Helin Wang, Yanhong Wang & Zhou Jiang

  3. State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, PR China

    Li-Li Han & Shuh-Ji Kao

  4. Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, PR China

    Kai Yang & Li Cui

Authors
  1. Xiaohan Liu
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  2. Ping Li
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  3. Helin Wang
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  8. Li Cui
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Contributions

Conceptualization: PL, XL, and HW. Sample collection and methodology: HW, XL, L-LH, PL, YW, and ZJ. Analysis and writing-original draft preparation: XL and HW. Writing-review and editing and funding acquisition: PL, LC, and S-JK. 15N analysis: L-LH and S-JK. Single-cell resonance Raman-15N2 SIP analysis: KY and LC.

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Correspondence to Ping Li.

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Liu, X., Li, P., Wang, H. et al. Nitrogen fixation and diazotroph diversity in groundwater systems. ISME J 17, 2023–2034 (2023). https://doi.org/10.1038/s41396-023-01513-x

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