Community N and O isotope fractionation by sulfide-dependent denitrification and anammox in a stratified lacustrine water column

We investigated the community nitrogen (N) and oxygen (O) isotope effects of fixed N loss in the northern basin of Lake Lugano, where sulfide-dependent denitrification and anammox are the main drivers of suboxic N2 production. A decrease in nitrate (NO3−) concentration toward the redox transition zone (RTZ) at mid-water depth was paralleled by an increase in δ15N and δ18O from approximately 5‰ to >20‰ and from 0‰ to >10‰, respectively. Ammonium (NH4+) concentrations were highest in the near-bottom water and decreased toward the RTZ concomitant with an increase in δ15N-NH4+ from ∼7‰ to >15‰. A diffusion-reaction model yielded N and O isotope enrichment factors that are significantly smaller than isotope effects reported previously for microbial NO3− reduction and NH4+ oxidation (15εNO3 ≈ 10‰, 18εNO3 ≈ 7‰, and 15εNH4 ≈ 10−12‰). For the Lake Lugano north basin, we constrain the apparent under-expression of the N isotope effects to: (1) environmental conditions (e.g., substrate limitation, low cell specific N transformation rates), or (2) low process-specific (chemolithotrophic denitrification and anammox) isotope fractionation. Our results have confirmed the robust nature of the co-linearity between N and O isotope enrichment during microbial denitrification beyond its organotrophic mode. However, the ratio of 18O to 15N enrichment (18εNO3:15εNO3) associated with NO3− reduction in the RTZ was ∼0.89, which is lower than observed in marine environments and in most culture experiments. We propose that chemolithotrophic NO3− reduction in the Lake Lugano north basin was partly catalyzed by the periplasmic dissimilatory nitrate reductase (Nap) (rather than the membrane-bound dissimilatory Nar), which is known to express comparably low 18εNO3:15εNO3 ratios in the ambient NO3− pool. However, NO2− re-oxidation, e.g., during anammox or microaerobic nitrification, could have contributed to the lowered 18O to 15N enrichment ratios. Although we do not yet understand the exact controls on the observed N (and O) isotope fractionation in the Lake Lugano north basin, our study implies that caution is advised when assuming canonical (i.e., high) N isotope effects for NO3− reduction and NH4+ oxidation in natural environments. In Lake Lugano, the community N (and O) isotope effects by sulfide-dependent denitrification and anammox in a natural ecosystem appear to be significantly lower than for organotrophic denitrification and aerobic ammonium oxidation.