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The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to ∼3.4 μM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 μM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 × 107 cells ml−1). Incubation experiments with 15N-nitrite revealed nitrogen loss occurring in the chemocline through denitrification (∼3 nM N h−1). At the same depth, incubations experiments with 15N2- and 13CDIC-labelled bicarbonate, indicated substantial N2 fixation (31.7–42.1 pM h−1) and inorganic carbon assimilation (40–85 nM h−1). Catalysed reporter deposition fluorescencein situhybridization (CARD-FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacteriumChlorobium clathratiforme. Phylogenetic analyses of thenifHgenes expressed as mRNA revealed a high diversity of N2 fixers, with the highest expression levels right at the chemocline. The majority of N2 fixers were related toChlorobium tepidum/C. phaeobacteroides.By using HalogenIn SituHybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), we could for the first time directly linkChlorobiumto N2 fixation in the environment. Moreover, our results show that N2 fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.