The consequences of detrimental alterations caused to the natural nitrogen (N) cycle are manifold. To tackle problems, such as eutrophication of coastal marine and lacustrine environments, or increasing emissions of greenhouse gas nitrous oxide (N2O), requires a clear understanding of the microbial N cycle. A promising tool to study N transformations is the measurement of the stable isotope composition of N compounds. The overall goal of this project was to improve the understanding of N transformation pathways and associated isotope effects, using the meromictic northern and the monomictic southern basins of Lake Lugano as natural model systems. Toward this goal, we collected samples from the water column of both basins for dissolved inorganic nitrogen (DIN) analyses (including N2:Ar, N2O), molecular microbiological phylogenetic analyses, 15N-labeling experiments (water column and sediments), and stable N and O isotope (and N2O isotopomer) measurements.

First, we identified the main processes responsible for fixed N elimination in the Lake Lugano north basin. The stable redox transition zone (RTZ) in the mid-water column provides environmental conditions that are favorable for both, anaerobic ammonium oxidation (anammox), as well as sulfur-driven denitrification. Previous marine studies suggested that sulfide (H2S) inhibits the anammox reaction. In con- trast to this we demonstrated that anammox bacteria coexist with sulfide-dependent denitrifiers in the water column of the Lake Lugano north basin. The maximum potential rates of both processed were comparatively low, but consistent with nu- trient fluxes calculated from concentration gradients. Furthermore, we showed that organotrophic denitrification is a negligible nitrate-reducing pathway in the Lake Lugano north basin.

Based on these findings, we next interpreted the N and O isotope signatures in the Lake Lugano north basin. Anammox and sulfide-dependent denitrification left clear N (in NO–3 and NH+4 ) and O (in NO–3) isotope patterns in the water column. However, the associated isotope effects were low compared to previous reports on iso- tope fractionation by organotrophic denitrification and aerobic ammonium oxidation. We attribute this apparent under-expression to two possible explanations: 1) The biogeochemical conditions (i.e., substrate limitation, low cell specific N transformation rates) that are particularly conducive in the Lake Lugano RTZ to an N isotope effect under-expression at the cellular-level, or 2) a low process-specific isotope fractionation at the enzyme-level. Moreover, an 18O to 15N enrichment ratio of ∼0.66 associated with NO–3 reduction suggested that the periplasmic dissimilatory nitrate reductase Nap was more important than the membrane-bound dissimilatory Nar.