Nitrogen fixation and diazotroph diversity in groundwater systems

Biological nitrogen fixation (BNF), the conversion of N 2 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 15 N tracing methods, reverse transcription qPCR (RT-qPCR), and metagenomic/metatranscriptomic analyses. 15 N 2 tracing incubation of near in situ groundwater (9.5–585.4 nmol N L −1 h −1 ) and N 2 -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 × 10 3 to 1.2 × 10 6 copies L −1 and was tightly correlated with dissolved oxygen (DO), Fe(II), and NH 4 + . 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 CH 4 oxidation. Our results highlight the importance of diazotrophs in subsurface geochemical cycles.