Temperature-Dependent Regulation of Denitrification Intermediates in High-Temperature Ecosystems

Incomplete denitrification generates diverse nitrogen
intermediates
(e.g., NO2–, N2O), which may
reshape nitrogen redox dynamics and modulate ecosystem functions.
While prevalent in high-temperature ecosystems, the mechanisms regulating
temperature-dependent denitrification remain unclear. To address this
gap, we used hot springs with different temperatures (ranging from
37 to 75 °C) as representative environments and conducted enrichment
cultures with both in situ hot spring water and defined media with
varying acetate/NO3– ratios. Denitrification
product profiles shifted systematically with temperature: NO2– dominated above 60 °C, NO2– and N2O at 55 °C, and N2 below 45 °C. Multiomics analyses revealed temperature-driven
succession of dominant denitrifiers, with microorganisms affiliated
with Thermus (>60 °C), Tepidimonas (55 °C), and Thauera/Uliginosibacterium (<45 °C). Denitrification core and accessory gene expressions
showed temperature-specific patterns associated with intermediate
formation. High expression of upstream core genes (e.g., narG) and limited expression of accessory genes in Thermus spp. promote incomplete denitrification at high temperatures, while
low-temperature communities upregulated complementary pathways (e.g., nosRD with nosZ in Thauera spp.) for complete denitrification. Division of metabolic labor
appears to be more important in denitrifying communities at high temperatures,
where dominant denitrifiers perform incomplete denitrification and
downstream intermediates are reduced by minor species (e.g., N2O reduction). These findings improve understanding of denitrification
in thermally dynamic environments and reveal mechanisms underlying
its temperature-dependent regulation.