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 hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (NanoSIMS) to analyze community dynamics, single-cell activities, and interactions among the two most abundant microbial community members (i.e., sp. and spp.) under autotrophic and heterotrophic growth conditions. CARD-FISH cell counts showed the dominance of the Fe(II) oxidizer sp. under autotrophic conditions as well as of spp. under heterotrophic conditions. We used NanoSIMS to monitor the fate of C-labeled bicarbonate and acetate as well as N-labeled ammonium at the single-cell level for both taxa. Under autotrophic conditions, only the sp. was actively incorporating C-labeled bicarbonate and N-labeled ammonium. Interestingly, both spp. and sp. became enriched in Cacetate and Nammonium under heterotrophic conditions. Our experiments demonstrated that sp. was capable of assimilating Cacetate while spp. were not able to fix CO , although a metagenomics survey of culture KS recently revealed that sp. lacks genes for acetate uptake and that the sp. carries the genetic potential to fix CO The study furthermore extends our understanding of the microbial reactions that interlink the nitrogen and Fe cycles in the environment. Microbial mechanisms by which Fe(II) is oxidized with nitrate as the terminal electron acceptor are generally referred to as "nitrate-dependent Fe(II) oxidation" (NDFO). NDFO has been demonstrated in laboratory cultures (such as the one studied in this work) and in a variety of marine and freshwater sediments. Recently, the importance of NDFO for the transport of sediment-derived Fe in aquatic ecosystems has been emphasized in a series of studies discussing the impact of NDFO for sedimentary nutrient cycling and redox dynamics in marine and freshwater environments. In this article, we report results from an isotope labeling study performed with the autotrophic, nitrate-reducing, Fe(II)-oxidizing enrichment culture KS, which was first described by Straub et al. (1) about 20 years ago. Our current study builds on the recently published metagenome of culture KS (2).