Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo13C and2H 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 use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacteriumCandidatus'Kuenenia stuttgartiensis' using time-series(13)C and(2)H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO(2)fixation, central metabolism, and amino acid biosynthesis inK. 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 thatK. stuttgartiensisis able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO(2)followed by reassimilation. In contrast, our data suggest thatK. stuttgartiensisis 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.

Automated summary

This study experimentally resolved the central carbon metabolism of the anammox bacterium 'Kuenenia stuttgartiensis', confirming predicted metabolic pathways and revealing instances where genomic predictions did not align with in vivo metabolic fluxes. The findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and inform future studies on factors governing their function and niche differentiation in ecosystems.