Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland)

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 M nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 M). 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 ¹³C_DIC-labelled bicarbonate, indicated substantial N ₂ fixation (31.7-42.1 pM h^-1) and inorganic carbon assimilation (40-85 nM h^-1). Catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N ₂ fixers, with the highest expression levels right at the chemocline. The majority of N₂ fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), we could for the first time directly link Chlorobium to 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.