Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

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 and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). 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 an oxidative tricarboxylic acid cycle, despite the genome not encoding a known 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 suggests 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 bioreactors 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.