Temperature and substrate jointly shape significant anammox contributions to nitrogen removal in hot springs

Hot springs are extreme environments that serve as critical natural laboratories for understanding microbial biogeochemistry. However, the relative importance of anammox versus denitrification remains poorly resolved, particularly regarding their quantitative partitioning and potential rates in natural terrestrial geothermal systems. This study integrated geochemical analyses, ¹⁵N isotope labelling, and quantitative PCR to investigate reactive nitrogen (Nr) removal across a wide thermal gradient (60.2-91.5 °C) in the Tengchong hot springs. While undetectable in nitrogen-poor sites, anammox contributes substantially (∼50 %) to Nr removal in two specific nitrogen-rich springs, revealing a significant and previously underestimated thermal niche for this process up to 72.1 °C. Temperature and total nitrogen jointly regulated pathway partitioning and Nr removal: below 75 °C°C, denitrification and anammox co-occur in nitrogen-rich niches, with their competitive yet complementary interactions boosting overall Nr removal efficiency, albeit with a potential for elevated nitrous oxide (N₂O) yield under acidic conditions. In contrast, above 75 °C°C, Nr removal proceeded mainly through a markedly constrained denitrification pathway, resulting in lower efficiency but minimal N₂O production. Total nitrogen content positively influenced both pathways, partially alleviating thermal inhibition. These findings highlight a fundamental thermal mechanistic coupling between the rate and environmental impact of microbial Nr removal and suggest that coordinated control of temperature and substrate supply could steer Nr removal toward lower greenhouse gas production in both natural and engineered systems.