Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series ¹³C and ²H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO₂ fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO₂ followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

厌氧氨氧化 (anammox) 细菌介导生物地球化学氮循环中的关键步骤，并已在全球范围内用于高效去除废水中的氮。然而，除了它们的核心能量代谢之外，人们对驱动厌氧氨氧化细菌合成代谢的代谢网络以及基于基因组预测之外的不同碳和能量底物的使用知之甚少。^{在这里，我们使用时间序列13} C 和² H 同位素示踪、代谢组学和同位素非平稳代谢通量分析，实验性地解决了 anammox 细菌Candidatus 'Kuenenia stuttgartiensis'的中心碳代谢。我们的研究结果证实了用于 CO _{2的预测代谢途径}K. stuttgartiensis的固定、中枢代谢和氨基酸生物合成，并揭示了体内代谢通量不支持基因组预测的几个实例。这包括使用不完全三羧酸循环的氧化分支进行 α-酮戊二酸生物合成，尽管基因组没有注释的柠檬酸合酶。我们还证明K. stuttgartiensis能够通过 Wood-Ljungdahl 途径直接同化细胞外甲酸盐，而不是将其完全氧化为 CO ₂然后再同化。相比之下，我们的数据表明K. stuttgartiensis不能原位使用乙酸盐作为碳源或能源，并且乙酸盐氧化是通过生物反应器侧群中低丰度微生物的代谢活动发生的。总之，这些发现为在系统层面了解厌氧氨氧化细菌的碳代谢奠定了基础，并将为未来的研究提供信息，这些研究旨在阐明在自然和工程生态系统中控制其功能和生态位分化的因素。