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 (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.不完全反硝化作用（denitrification）会产生多种氮中间体（nitrogen intermediates，如NO2–、N2O），可重塑氮氧化还原动态（redox dynamics）并调控生态系统功能（ecosystem functions）。尽管该过程在高温生态系统中普遍存在，但调控温度依赖性反硝化作用的机制仍不明晰。为填补这一研究空白，本研究以温度范围为37~75 ℃的温泉作为代表性生境，分别采用原位（in situ）温泉水与乙酸盐/硝酸盐（acetate/NO3–）比例可变的限定培养基开展富集培养（enrichment cultures）。反硝化产物谱（denitrification product profiles）随温度发生系统性变化：60 ℃以上以NO2–为主，55 ℃时同时存在NO2–与N2O，45 ℃以下则以N2为主。多组学分析（multiomics analyses）揭示了温度驱动的优势反硝化微生物演替：栖热菌属（Thermus）在>60 ℃环境中占优，温单胞菌属（Tepidimonas）在55 ℃环境中占优，携带narG基因的陶厄氏菌属（Thauera）/粘杆菌属（Uliginosibacterium）则在低温环境中占优；栖热菌属（Thermus spp.）的辅助基因表达受限，这会在高温下促进不完全反硝化过程，而低温群落则上调了完整反硝化所需的互补代谢通路（如陶厄氏菌属（Thauera spp.）中的nosRD与nosZ基因）。高温反硝化群落中代谢劳动分工似乎更为关键：优势反硝化微生物仅完成不完全反硝化，下游中间体则由稀有类群完成还原（如N2O还原）。本研究结果加深了我们对热动态环境（thermally dynamic environments）中反硝化作用的理解，并揭示了其温度依赖性调控的潜在机制。