The nrfA-type microbial communities are widespread in hot springs of the Tibet-Yunnan geothermal zone

Abstract

The microorganisms are main drivers of biogeochemical processes in geothermal ecosystems. The dissimilatory nitrate-to-ammonium reduction pathway (DNRA) could act as an alternative source of ammonium and provide an important nitrogen supply for the maintenance of geothermal ecosystems. Investigating the distribution of DNRA-functional bacteria is of great significance to understanding the source of biological nitrogen production in geothermal environments. In this study, we characterized the community distribution of microorganisms harboring nrfA genes in the sediments of hot springs from the Tibet-Yunnan geothermal zone, with the use of Illumina MiSeq high-throughput sequencing of nrfA genes and R language software for statistical analysis. In the present study, the nrfA genes were successfully amplified from the hot springs with a temperature of 38°C-80°C. The nrfA-based phylogenetic analysis showed that the DNRA pathway is widespread within the geothermal ecosystems, with microorganisms harboring nrfA genes predominantly belonging to phyla Chloroflexi, Proteobacteria, Deinococcus-Thermus (top 10), etc. Genus-level analysis revealed Thermoflexus (Chloroflexi) as the dominant taxon in the DGQ, while Geothrix (Acidobacteria) showed peak abundance in weakly acidic sites. The DNRA-functional community structure and nrfA gene abundance also showed a sample variability, even among samples from the same region, there were differences in dominant populations and overall nrfA gene abundance between them. Statistical analysis results indicate that the distribution of nrfA type microorganisms was mainly influenced by physicochemical factors, including pH, SO₄ ^2-, and NO₂ ^- concentrations. These findings deepen our understanding of the nitrogen cycle in extreme environments and provide valuable perspectives on the role of nitrogen metabolism in both contemporary and ancient geothermal systems.

Keywords: DNRA; Tibetan-Yunnan geothermal zone; hot springs; microbial community; nrfA gene.

Figure 1

Map depicting the locations of the studied area within the Tibet-Yunnan geothermal zone. TC, Tengchong, Yunnan; DGJ, Dagejia, Tibet; and DGQ, Duoguoqu, Tibet.

Figure 2

Distribution of nrfA gene across 53 sediment samples in relation to spring water pH and temperature. Orange indicates environments where amplicons were detected while blue represents environments with no amplicons detected.

Figure 3

DNRA community abundance based on nrfA functional gene analysis in sediment samples from the study area.

Figure 4

Box plots depicting the alpha diversity analysis of DNRA communities. Significance was determined by ANOVA and Kruskal Wallis test, followed by Bartlett’s test, Dunn test and Bonferroni correction. Statistical significance is marked as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001, and ns for no significant difference.

Figure 5

Principal coordinates analysis (PCoA) ordination of DNRA communities from positive sample sites. Each dot represents an independent biological sample. Proximity of points represents similarity in community structure, with horizontal and vertical axes represent different dimensions of the variable.

Figure 6

Phylogenetic analysis of nrfA gene sequences from positive hot spring samples (a), heatmap showing DNRA bacterial composition at the genus level for positive hot spring samples across the study area (b), color gradient indicates relative abundance (%) of genera across samples.

Figure 7

Pearson correlations between relative abundance of functional DNRA microbial populations. Statistical significance is marked as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001, and no * for no significant difference.

Figure 8

Principal component analysis (PCA) of positive sample sites across the three sampling regions (TC, DGJ, and DGQ), based on physicochemical parameters (pH, temperature, ion concentrations, etc.).

Figure 9

Mantel test of physicochemical factors at the phylum and genus level (a), RDA analysis of DNRA microbial communities in relation to physicochemical factors (b), the arrow length represents the explanatory strength of environmental factors on DNRA community variation, with longer arrows indicating stronger influences. The angle between arrows and axes reflects their correlation with the principal components (smaller angles indicate higher correlation), while the perpendicular distance from sample points to arrows quantifies the impact of each factor on specific samples (closer distances imply stronger effects).

Figure 10

Pearson correlations between measured environmental variables and relative abundance of functional microbial populations in DNRA, Statistical significance is marked as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001, and no * for no significant difference.

Figure 11

RDA analysis illustrating the influence of spatial factors and DNRA microbial community structure (A), Variance Partitioning Analysis (VPA) quantifying the relative contributions of physicochemical (X1) and spatial (X2) factors on DNRA microbial community structure (B).