Producing stable nitrite is a necessity for anaerobic ammonium oxidation (anammox) but remains a huge challenge. Here, we describe the design and operation of a hydrogenotrophic denitratation system that stably reduced >90% nitrate to nitrite under self-alkaline conditions of pH up to 10.80. Manually lowering the pH to a range of 9.00–10.00 dramatically decreased the nitrate-to-nitrite transformation ratio to <20%, showing a significant role of high pH in denitratation. Metagenomics combined with metatranscriptomics indicated that six microorganisms, including a Thauera member, dominated the community and encoded the various genes responsible for hydrogen oxidation and the complete denitrification process. During denitratation at high pH, transcription of periplasmic genes napA, nirS, and nirK, whose products perform nitrate and nitrite reduction, decreased sharply compared to that under neutral conditions, while narG, encoding a membrane-associated nitrate reductase, remained transcriptionally active, as were genes involved in intracellular proton homeostasis. Together with no reduction in only nitrite-amended samples, these results disproved the electron competition between reductions of nitrate and nitrite but highlighted a lack of protons outside cells constraining biological nitrite reduction. Overall, our study presents a stably efficient strategy for nitrite production and provides a major advance in the understanding of denitratation.

中文翻译：

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基于自碱化的 pH 依赖性氢营养反硝化

生产稳定的亚硝酸盐是厌氧氨氧化（anammox）的必要条件，但仍然是一个巨大的挑战。在这里，我们描述了氢营养型反硝化系统的设计和操作，该系统在 pH 高达 10.80 的自碱性条件下稳定地将 >90% 的硝酸盐还原为亚硝酸盐。手动将 pH 值降低到 9.00–10.00 的范围内，硝酸盐到亚硝酸盐的转化率显着降低至 <20%，表明高 pH 值在反硝化中的重要作用。宏基因组学与宏转录组学相结合表明，包括Thauera成员在内的六种微生物在群落中占主导地位，并编码了负责氢氧化和完全反硝化过程的各种基因。在高 pH 值脱硝过程中，周质基因napA的转录，nirS和nirK的产物执行硝酸盐和亚硝酸盐还原，与中性条件下相比急剧下降，而编码膜相关硝酸盐还原酶的narG保持转录活性，参与细胞内质子稳态的基因也是如此。再加上只有亚硝酸盐修饰的样品没有还原，这些结果反驳了硝酸盐和亚硝酸盐还原之间的电子竞争，但强调细胞外缺乏质子限制生物亚硝酸盐还原。总的来说，我们的研究提出了一种稳定有效的亚硝酸盐生产策略，并在理解反硝化方面取得了重大进展。