Nitrogen transformation pathways, rates, and isotopic signatures in Lake Lugano

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 contrast 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 nutrient 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 NO3- and NH4+) and O (in NO3-) isotope patterns in the water column. However, the associated isotope effects were low compared to previous reports on isotope 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 ~0.89 associated with NO3- reduction suggested that the periplasmic dissimilatory nitrate reductase Nap was more important than the membrane-bound dissimilatory Nar.
While in the meromictic north basin, most fixed N elimination took place within the water column RTZ, seasonal mixing and re-oxygenation of the water column in the south basin suggests N2 production within the sediments. We showed that denitrification was the major benthic NO3- reduction pathway in the southern basin. Benthic anammox and dissimilatory nitrate reduction to ammonium (DNRA) rates remained close to the detection limit. A comparison between benthic N2 production rates and water column N2 fluxes revealed that during anoxic bottom water conditions, ~40% of total N2 production was associated with benthic and ~60% with pelagic processes. This quantitative partitioning was confirmed by N isotope analysis of water column NO3-. The N isotope enrichment factor associated with total NO3- reduction was ~14‰. This translates into a sedimentary N2 contribution of 36-51%, if canonical assumptions for N isotope fractionation associated with water column (15εwater = 20-25‰) and sedimentary (15εsed = 1.5-3‰) denitrification are made.
Finally, we compared the N2O production and consumption pathways in the northern and southern basin and found contrasting N2O dynamics. Maximum N2O concentrations in the south basin (>900 nmol L-1) greatly exceeded maximum concentrations in the north basin (<13 nmol L-1). 15N site preference (SP) values >32‰ in the south basin indicated nitrification via hydroxylamine (NH2OH) oxidation as the prime N2O source, whereas in the north basin N2O production was attributed to nitrifier denitrification. In the north basin, N2O was completely reduced within the RTZ. This chemolithotrophic N2O reduction occurred with an 18O to 15N enrichment ratio of ~2.5, which is consistent with previous reports for organotrophic N2O reduction.
In conclusion, our study highlights the importance of chemolithotrophic processes in aquatic ecosystems. Moreover, the expression of N isotope fractionation can be variable in nature and depends on various factors such as the pathways of NO3- dissimilation (organotrophic vs. chemolithotrophic), the main catalyzing enzymes, the pathways of NH4+ oxidation (nitrification vs. anammox), and the controlling environmental conditions (e.g., substrate limitation, cell specific N transformation rates). Hence, this study suggests to refrain from universal, canonical assumptions of N isotope fractionation in N budget calculations. Additional stable isotope measurements such as O isotopes in NO3-, or the 15N site preference in N2O are powerful tools to identify and quantify microbial N transformation pathways occurring simultaneously or in close vicinity. For a successful interpretation of such data, however, a mechanistic understanding of the processes leading to certain characteristic isotopic signatures in the environment is needed.