Nitrogen fixation and diazotroph diversity in groundwater systems

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.

Fig. 1. Distribution of nifH gene abundances and their response to environment variables.

Comparison of (A) DNA-based and (B) RNA-based nifH/16S rRNA genes (%) between the reducing area and oxidizing area and linear correlations between nifH/16S rRNA genes and DO, ORP, Fe(II), and NH₄⁺ concentrations. The symbol * indicates significant differences between the reducing area and oxidizing area at p < 0.05. Solid lines represent linear regression modules. The shaded area represents the 95% confidence interval.

Fig. 2. Diversity and community composition of native and active diazotrophs in groundwater samples.

A Comparison of the diversity index between oxidizing and reducing areas based on DNA and RNA levels (* and ** represent p < 0.05 and 0.01, respectively). B Nonmetric multidimensional scaling (NMDS) based on the Bray‒Curtis distance of diazotrophic communities. The relative abundances at the (C) class and (D) genus levels are based on the DNA/RNA level nifH gene amplicon sequencing.

Fig. 3. Relationship between geochemical parameters and diazotrophic communities.

A DNA level. B RNA level. The symbol * indicates Pearson correlation with significance (*, **, and *** represent p < 0.05, 0.01, and 0.001, respectively).

Fig. 4. Metabolic potential and genomic bins related to N₂ fixation and N/S/Fe/CH₄/As cycles in groundwater.

A Pearson correlation analysis between nitrogenase abundances and geochemical parameters/other essential functional genes of N, S, Fe, CH₄, and As cycling identified by the KEGG Orthologs database based on metatranscriptome. B The co-expression network of N₂ fixation and other biogeochemical processes were analyzed by the KEGG module database (Pearson correlations r > |0.6| and p < 0.05). C Taxonomic distribution and metabolic potential of the bacterial medium-quality metagenomic bins (completeness > 50% and contamination < 10%). For the taxonomy of the bins, “c” stands for class level, and “g” stands for genus level.

Fig. 5. N₂ fixation rates in field groundwater and N₂-fixer enrichments and isolates.

The verified function of N₂ fixation in the (A) field groundwater and (B) N₂-fixer enrichments and isolates (detected by the ¹⁵N₂ tracing incubation). The activities of N₂-fixer isolates were also analyzed using the (C) acetylene reduction method and (D) single-cell resonance Raman spectroscopy. ¹⁵N₂-induced shifts in the resonance Raman band of Cyt c are a sensitive and robust indicator of N₂ fixation. Single-cell Raman spectra from four isolates (D) indicate that the 1129 cm⁻¹ band (C-N stretch) shifted markedly to 1114 cm⁻¹ under ¹⁵N₂ incubation conditions, implying that it is the substitution of light ¹⁴N with heavier ¹⁵N in the C-N bond resulting in a decrease in the vibrational frequency of the C-N stretch. The pink shading indicates the ¹⁵N₂-induced shifts in the resonance Raman spectra.