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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The raw data have been submitted to the NCBI database under BioProject numbers PRJNA937386, PRJNA996345, PRJNA996367, PRJNA882225, and PRJNA884812.
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This research was financially supported by the National Natural Science Foundation of China (Grant No 42177068, 91851115, 91851209, and 91851101).
The authors declare no competing interests.
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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