The enrichment culture KS is one of the few existing autotrophic, nitrate-reducing, Fe(II)-oxidizing cultures that can be continuously transferred without an organic carbon source. We used a combination of catalyzed amplification reporter deposition fluorescence in situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (NanoSIMS) to analyze community dynamics, singlecell activities, and interactions among the two most abundant microbial community members (i.e., Gallionellaceae sp. and Bradyrhizobium spp.) under autotrophic and heterotrophic growth conditions. CARD-FISH cell counts showed the dominance of the Fe(II) oxidizer Gallionellaceae sp. under autotrophic conditions as well as of Bradyrhizobium spp. under heterotrophic conditions. We used NanoSIMS to monitor the fate of ¹³C-labeled bicarbonate and acetate as well as ¹⁵N-labeled ammonium at the single-cell level for both taxa. Under autotrophic conditions, only the Gallionellaceae sp. was actively incorporating ¹³C-labeled bicarbonate and ¹⁵N-labeled ammonium. Interestingly, both Bradyrhizobium spp. and Gallionellaceae sp. became enriched in [¹³C]acetate and [¹⁵N]ammonium under heterotrophic conditions. Our experiments demonstrated that Gallionellaceae sp. was capable of assimilating [¹³C]acetate while Bradyrhizobium spp. were not able to fix CO₂, although a metagenomics survey of culture KS recently revealed that Gallionellaceae sp. lacks genes for acetate uptake and that the Bradyrhizobium sp. carries the genetic potential to fix CO₂. The study furthermore extends our understanding of the microbial reactions that interlink the nitrogen and Fe cycles in the environment.