Nitrogen isotope effects can be used to diagnose N transformations in wastewater anammox systems

Abstract

Anaerobic ammonium oxidation (anammox) plays an important role in aquatic systems as a sink of bioavailable nitrogen (N), and in engineered processes by removing ammonium from wastewater. The isotope effects anammox imparts in the N isotope signatures (¹⁵N/¹⁴N) of ammonium, nitrite, and nitrate can be used to estimate its role in environmental settings, to describe physiological and ecological variations in the anammox process, and possibly to optimize anammox-based wastewater treatment. We measured the stable N-isotope composition of ammonium, nitrite, and nitrate in wastewater cultivations of anammox bacteria. We find that the N isotope enrichment factor ¹⁵ε for the reduction of nitrite to N₂ is consistent across all experimental conditions (13.5‰ ± 3.7‰), suggesting it reflects the composition of the anammox bacteria community. Values of ¹⁵ε for the oxidation of nitrite to nitrate (inverse isotope effect, - 16 to - 43‰) and for the reduction of ammonium to N₂ (normal isotope effect, 19-32‰) are more variable, and likely controlled by experimental conditions. We argue that the variations in the isotope effects can be tied to the metabolism and physiology of anammox bacteria, and that the broad range of isotope effects observed for anammox introduces complications for analyzing N-isotope mass balances in natural systems.

Figure 1

The variation in concentration (a) and N isotopic composition (b) of substrates (NH₄⁺ and NO₂^–) and product (NO₃^–) over the course of a typical anammox incubation experiment (exemplary experiment with enrichment culture, 28 January 2019). Note that the nitrate δ¹⁵N is not corrected for the fraction of nitrate that was already present at the beginning of the incubation (see text).

Figure 2

The N isotope effect associated with the oxidation of ammonium, ¹⁵ε(NH₄⁺), for all experiments. Colored diamonds represent average values for different anammox incubations measured in this study, with 1-standard-deviation error bars. Small black circles represent the results of individual experiments. For comparison, the results of Brunner and coworkers (black square) and Kobayashi and coworkers (brown circles) are also shown^,.

Figure 3

The N isotope effect associated with the consumption of nitrite, ¹⁵ε(NO₂^–), reflecting the composite effect of conversion both to nitrate and N₂, for all experiments. Colored diamonds represent average values, surrounded by 1-standard-deviation error bars. Small black circles represent the results of individual experiments. For comparison, the results of Brunner and coworkers (black square) and Kobayashi and coworkers (brown circles) are also shown.

Figure 4

The N isotope effect associated with the oxidation of nitrite to nitrate, ¹⁵ε(NO₂^––NO₃^–), for all experiments. Colored diamonds represent average values, surrounded by 1-standard-deviation error bars. Small black circles represent the results of individual experiments. For comparison, the results of Brunner and coworkers (black square) and Kobayashi and coworkers. (brown circles) are also shown.

Figure 5

The N isotope effect associated with the reduction of nitrite to N₂, ¹⁵ε(NO₂^––N₂), for all experiments. Colored diamonds represent average values, surrounded by 1-standard-deviation error bars. Small black circles represent the results of individual experiments. For comparison, the results of Brunner and coworkers (black square) and Kobayashi and coworkers (brown circles) are also shown.

Figure 6

¹⁵ε(NH₄⁺) for each individual experiment, compared to the concentration of ammonium at the start of that experiment. Mainstream experiments are plotted in yellow circles, sidestream experiments in blue squares, and enrichment experiments in grey diamonds.