Nitrogen and oxygen isotope effects during enzymatic nitrate reduction in vitro and by natural lake water consortia

Nitrate (NO ₃ ^- ) isotope ratios are useful indicators for nitrogen (N)-transformation processes if the associated isotope effects and their environmental controls are well-constrained. The NO ₃ ^- isotope effects in natural environments may depend on the type of dissimilatory nitrate reductases involved and the degree of isotopic overprinting via NO ₃ ^- regeneration. We measured the coupled N and oxygen (O) isotope effects of NO ₃ ^- reduction in anoxic incubation experiments with laboratory cultures ( Pseudomonas sp. and Escherichia coli ) harboring different nitrate reductase enzymes (Nar and/or Nap) as well as with natural freshwater consortia from Lake Lugano (Switzerland) and Lake La Cruz (Spain). For comparison, isotope effects were also evaluated through coupled N and O isotope measurements in the redox transition zone of Lake Lugano North Basin. Incubation-based Rayleigh model N isotope effects (ϵ _N ) were variable, ranging from 9 to 30‰. In comparison, in situ ϵ _N values (5 to 14‰) estimated by the closed system model were lower, likely due to substrate limitation in the water column. Experiments with Pseudomonas sp. and E. coli cultures possessing Nar yielded N isotope effects of similar magnitudes and, consistent with previous data, robust Δδ ¹⁸ O:Δδ ¹⁵ N enrichment ratios of ~0.9 - 1.0. Nitrate reduction by cultures possessing solely Nap led to lower Δδ ¹⁸ O:Δδ ¹⁵ N of ~0.7. In anoxic incubations of lake water, where the effect of nitrification could be excluded, Δδ ¹⁸ O:Δδ ¹⁵ N values between 0.6 and 1.0 suggest "community activity" of both Nar and Nap. Interestingly, stimulation of lithotrophic nitrate reduction in incubations with amended NO ₃ ^- + sulfide resulted in a Δδ ¹⁸ O:Δδ ¹⁵ N slope of 0.90 ± 0.03 (standard error, SE), indicating Nar as the more dominant nitrate-reducing enzyme. On the contrary, stimulation of organotrophic nitrate reduction in NO ₃ ^- + acetate amended incubations resulted in a significantly lower slope of 0.72 ± 0.03 SE, suggesting a greater contribution by Nap. In contrast to the nitrate-reduction incubation experiments, the in situ Δδ ¹⁸ O:Δδ ¹⁵ N value of 1.36 ± 0.14 SE observed in the Lake Lugano water column was unusually high for a freshwater environment, likely reflecting the superimposed effect of NO ₃ ^- production by nitrification. Our study thus underscores that both variations in activity of Nar versus Nap during nitrate reduction, as well as isotopic overprinting by nitrate regeneration, may impact ecosystem NO ₃ ^- isotope dynamics in natural denitrifying environments.