Microbial denitrification is a main source of nitrous oxide (N₂O) emissions which have strong greenhouse effect and destroy stratospheric ozone. Though the importance of sulfide driven chemoautotrophic denitrification has been recognized, its contribution to N₂O emissions in nature remains elusive. We built up long-term sulfide-added microcosms with sediments from two freshwater lakes. Chemistry analysis confirmed sulfide could drive nitrate respiration in long term. N₂O accumulated to over 1.5% of nitrate load in both microcosms after long-term sulfide addition, which was up to 12.9 times higher than N₂O accumulation without sulfide addition. Metagenomes were extracted and sequenced during microcosm incubations. 16 S rRNA genes of Thiobacillus and Defluviimonas were gradually enriched. The nitric oxide reductase with c-type cytochromes as electron donors (cNorB) increased in abundance, while the nitric oxide reductase receiving electrons from quinols (qNorB) decreased in abundance. cnorB genes similar to Thiobacillus were enriched in both microcosms. In parallel, enrichment was observed for enzymes involved in sulfur oxidation, which supplied electrons to nitrate respiration, and enzymes involved in Calvin Cycle, which sustained autotrophic cell growth, implying the coupling relationship between carbon, nitrogen and sulfur cycling processes. Our results suggested sulfur pollution considerably increased N₂O emissions in natural environments.

微生物反硝化作用是一氧化二氮（N ₂ O）排放的主要来源，其具有强烈的温室效应并破坏平流层臭氧。尽管硫化物驱动的化学自养反硝化作用的重要性已经得到认可，但其对自然界中N ₂ O 排放的贡献仍然难以捉摸。我们用两个淡水湖的沉积物建立了长期添加硫化物的微观世界。化学分析证实硫化物可以长期驱动硝酸盐呼吸。长期添加硫化物后，N ₂ O 在两个微观世界中积累到硝酸盐负荷的 1.5% 以上，比不添加硫化物的N ₂ O 积累量高出 12.9 倍。宏基因组在微观孵化过程中被提取和测序。16 个 S rRNA 基因Thiobacillus和Defluviimonas逐渐富集。以 c 型细胞色素为电子供体的一氧化氮还原酶 (cNorB) 丰度增加，而接受羟基喹啉电子的一氧化氮还原酶 (qNorB) 丰度减少。与硫杆菌相似的cnorB基因在两个微观世界中都富集。同时，观察到参与硫氧化的酶（为硝酸盐呼吸提供电子）和参与卡尔文循环的酶（维持自养细胞生长）富集，这意味着碳、氮和硫循环过程之间的耦合关系。我们的结果表明硫污染大大增加了自然环境中的N ₂ O 排放。