Autotrophic and mixotrophic metabolic network fluxes suggest versatile lifestyle for the anammox bacterium Candidatus Kuenenia stuttgartiensis

Abstract

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 mixotrophy beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus Kuenenia stuttgartiensis using time-series ¹³C isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). Our findings confirm predicted metabolic pathways used for CO ₂ 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 an incomplete oxidative tricarboxylic acid cycle, despite the genome not encoding a known citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate formate via the Wood-Ljungdahl pathway instead of oxidizing it completetly to CO ₂ followed by reassimilation. In contrast, our data suggests that acetate is fully oxidized to CO ₂ via reversal of the Wood-Ljungdahl pathway and partial TCA cycle activity, followed by reassimilation of the produced CO ₂. Together, these findings highlight the versatility of central carbon metabolism in anammox bacteria and will enable the construction of accurate metabolic models that predict their function in natural and engineered ecosystems.