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

Variation in the N isotope effect imparted by ammonium oxidation

While the ammonium isotope effect, ¹⁵ε(NH₄⁺), varies by over 13‰ across all experiments, it exhibits a narrower range for a specific experimental setting with distinct cultivation conditions (mainstream, enrichment, sidestream), it exhibits a broader range across all experiments (Fig. 2). There are a number of possible physiological and experimental conditions that differ among experiments, including reaction rate, temperature, and initial concentration of substrates. As shown in Fig. 6, there is a systematic decrease in ¹⁵ε(NH₄⁺) at decreasing initial ammonium concentration, while at the highest ammonium concentrations, the value of ¹⁵ε(NH₄⁺) appears to plateau at a value near 32‰, close to the maximum value (32.7 ± 0.7‰) observed by Kobayashi and coworkers in chemostat experiments with enriched cultures¹⁹. Typically, the isotope effect imparted into a substrate pool by a kinetic process is set at the first irreversible step; any isotope effects that occur before this point can be expressed, while any that occur after it are concealed^37,38. Substrate supply limitations can decrease the reversibility of a given step, and thereby let it modulate the net isotope effect of a multi-step process. This behavior has been proposed to play important roles in controlling the isotopic signatures of microbial sulfate^39,40 and nitrate^41,42 reduction.

In the case of anammox bacteria, this pattern suggests that the relative kinetics of ammonium uptake and oxidation control the observed value of ¹⁵ε(NH₄⁺). The typical path of an ammonium molecule through an anammox cell requires crossing several cell membranes to the eventual site of reaction, inside the anammoxosome^43,44. When ammonium concentrations are relatively high and ammonium oxidation is not uptake-limited, ¹⁵ε(NH₄⁺) is set at the hydrazine synthase (Hzs) enzyme, at which ammonium binds and is subsequently oxidized to hydrazine⁴³. The maximum observed value of ¹⁵ε(NH₄⁺) would then be the expression of all isotope effects up to, and including, this bond-breaking step. But if, at relatively low ammonium concentrations in the external medium, the rate of ammonium oxidation is limited by its uptake, then active transport or passive diffusion of ammonium will become the first irreversible step and thereby set the observed value of ¹⁵ε(NH₄⁺). Indeed, for assimilatory uptake by a marine bacterium, ¹⁵ε(NH₄⁺) has been shown to depend on the external ammonium concentration, varying from 3.8 to 26.5‰ across an ammonium concentration range of 0.3 to 316 mg-N/L, as the first irreversible step changes from active transport at low NH₄⁺ concentrations to diffusion at intermediate concentrations and the enzymatic reaction associated with assimilation at high concentrations¹⁸.

In the process of NH₄⁺ uptake by anammox bacteria, a number of steps could imaginably impact the isotope effect imparted on the ammonium pool. Prior to oxidation within the anammoxosome, ammonium must cross three membranes to reach the Hzs enzyme⁴⁴. This is distinct from many other respiratory processes in the N cycle, including aerobic ammonia oxidation, where ammonia is thought to be oxidized in the periplasm^45,46. Metagenomic characterization of anammox bacteria in the mainstream system used in these experiments reveals the presence of genes for amt ammonium transporters in these species²³, while past studies of Ca. Kuenenia stuttgartiensis shows that anammox bacteria feature a number of genes homologous to those that express AmtB ammonium transporters in other bacteria^47,48. These transporters could function to transport ammonium across two membranes, first into the cytoplasm and then into the anammoxosome⁴⁹. In addition, passive, diffusive influx of ammonium into the cell could play a role, especially at relatively high external ammonium concentrations. Therefore, it is easily imaginable that under different physiological conditions, the observed value of ¹⁵ε(NH₄⁺) could reflect (1) free motion of ammonium into the anammoxosome and the full expression of the isotope effect associated with NH₄⁺ oxidation, (2) irreversibility in either of two active transport steps, or (3) irreversibility of diffusive transport of ammonium into the periplasm.

We do not know the N isotope effects for ammonium diffusion into the cell and ammonium transport to the anammoxosome. But in analogy to considerations made for the diffusion and active transport of nitrate and active transport into the cells of denitrifying bacteria⁴¹, we conclude that it is reasonable to expect the N isotope effects for both passive ammonium diffusion and active transport to be much smaller that the enzyme-level N isotope effect associated with the actual ammonium oxidation. Therefore, when external ammonium is relatively low, and NH₄⁺ transport becomes the rate-limiting step in anaerobic ammonium oxidation, the overall N isotope effect will approach that associated with NH₄⁺ uptake or transport, and will likely be lower than under NH₄⁺-replete conditions, where the full expression of the isotope effect associated with NH₄⁺ oxidation may be expressed. This also supports the observation (Fig. 6) of decreasing ¹⁵ε(NH₄⁺) under decreasing ammonium availability.

Importantly, in a different microbial setting, e.g., in an oceanic environment, which has anammox bacteria with different (e.g., higher) affinities for ammonium uptake and oxidation, we predict that the same endmember values of ¹⁵ε(NH₄⁺) that are seen in these experiments will be observed, but with the relationship between them unfolding at different (e.g., lower) values of ammonium concentration, cell densities, and in turn different cell-specific anammox rates. It is also important to note that the cell-specific anammox rate, not the bulk reaction rate, is the essential parameter for understanding the balance between the different processes at work. Unfortunately, because of the biofilm-dwelling nature of the anammox communities in this study, it is challenging to estimate accurately the number of anammox cells present, and so we were not able to determine the cell-specific anammox rate in our experimental setup. Yet, even at substrate concentrations that are much higher than those typically found in the natural environment, NH₄⁺ uptake can be limiting if the bacterial cell density is high, as is the case in this study.

For understanding the role that the balance between ammonium uptake and oxidation may play in controlling ¹⁵ε(NH₄⁺), it is useful to compare anammox bacteria to aerobic ammonia oxidizing bacteria (AOB) and archaea (AOA). It is notable that the range of ¹⁵ε(NH₄⁺) is similar for anammox bacteria and aerobic ammonia oxidizers; the AOB and AOA express values of ¹⁵ε(NH₄⁺) between 14 and 42‰^14–17. AOB and AOA perform catabolic ammonia oxidation using the ammonia monooxygenase enzyme. In AOB, this enzyme is located in the periplasm^45,46, not in an internal cell structure like the anammoxosome, and so it is unlikely that active transport controls observed isotope effects. Instead, it has been proposed that variations in ¹⁵ε(NH₄⁺) for the AOB are related to sequence variations in ammonia monooxygenase¹⁶. But recent results from Kobayashi and coworkers suggest that ¹⁵ε(NH₄⁺) for anammox bacteria is species-independent; for the three different species tested under similar experimental conditions, the N isotope effects were consistent¹⁹. In our experiments, variations in the ¹⁵ε(NH₄⁺) values were observed in mainstream and enrichment experiments, where the anammox bacteria population is expected to be similar, which also argues against species dependence. Indeed, the Hzs enzyme seems well conserved across anammox clades⁵⁰, and, therefore, the ammonium N isotope effect variation observed here cannot be attributed to sequence variations, and is more likely due to the changing experimental conditions (NH₄⁺ concentrations), as discussed above.

Irrespective of the explanations for the observed N-isotope effect variability for both ammonium oxidation modes, the overlap in the ranges of values for ¹⁵ε(NH₄⁺) for anammox and aerobic ammonia oxidation suggests that in a system that might be either aerobic or anaerobic, the mechanism of ammonium oxidation cannot necessarily be identified based on the ammonium N isotope signature. That is, an enrichment in ¹⁵ N associated with ammonium consumption cannot be attributed to ammonia oxidizing bacteria or anammox bacteria based on this measurement alone. Further work to compare the responses of both aerobic ammonia oxidizers and anammox bacteria to changing concentrations and cell-specific reaction rates would be helpful for identifying the overall environmental controls on ¹⁵ε(NH₄⁺) under both oxic and anoxic conditions.

¹⁵ε(NO₂
^–) reflects a mixture of processes

The parameter ¹⁵ε(NO₂^–) reflects the weighted sum of the isotope effects for the consumption of nitrite by reduction to N₂ and by oxidation to nitrate, and so its value depends on these two processes, as well as upon the stoichiometric ratio between them. For considering the physiology of anammox and its role in a biogeochemical N cycling network, it is of limited use, but in a system where the δ¹⁵N of nitrite can be readily measured it is valuable to know how to interpret it. It exhibits relatively little variation across the experimental conditions described here, and is also consistent with the result reported by Brunner and coworkers for Ca. K. stuttgartiensis enrichment cultures (Fig. 3)¹⁰, but our results differ from ¹⁵ε(NO₂^–) values for other anammox species by Kobayashi and coworkers¹⁹. The principal cause of the constancy of ¹⁵ε(NO₂^–) in these experiments is likely the stability of ¹⁵ε(NO₂^––N₂) across all experiments, and is discussed in the sections that follow.

The N isotope effect associated with the reduction of nitrite by anammox, ¹⁵ε(NO₂
^––N₂), reflects the microbial community

The N isotope effect associated with the reduction of nitrite to N₂ in anammox, ¹⁵ε(NO₂^––N₂), is consistent across all three experimental settings (Fig. 5), which is notable when compared to the broad range in ¹⁵ε(NO₂^––N₂) observed in previous pure culture experiments with members of the genera Ca. Kuenenia, Ca. Scalindua, Ca. Jettenia, and Ca. Brocadia^10,19, as well as in anammox incubation experiments²⁰. This consistancy is also striking in light of the variation of ¹⁵ε(NH₄⁺) observed in this study, and suggests that variations in substrate concentrations, reaction rates, or other physiological conditions are not strong controls on ¹⁵ε(NO₂^––N₂). Instead, the identity of the anammox bacteria, and in turn its biochemical processing of nitrite, appears to exert control over this isotope effect. In the mainstream system used for these experiments, it has been shown that species in the genus Ca. Brocadia are the principal members of the anammox community present, but that Ca. Kuenenia and Ca. Jettenia are also represented (Table S1)²³. Indeed, using the metagenomic characterization of the mainstream system reported by Niederdorfer and coworkers²³, as well as the observed values of ¹⁵ε(NO₂^––N₂) from previous studies^10,19, we calculate an expected value of ¹⁵ε(NO₂^––N₂) for the mainstream system of 7.5‰ ± 5.5‰ (1 s.d.). This result is close to, but distinct from, the observed value, and leads to the conclusion that ¹⁵ε(NO₂^––N₂) in these systems is the result of a stable mixture of different anammox species, but that the contribution of different species to anammox activity under a specific set of experimental conditions may not directly reflect their cellular abundance. Likewise, although we do not yet know the microbial community composition of the material used in the sidestream experiment, we predict, based on its consistent value for ¹⁵ε(NO₂^––N₂), that it is similar to that seen in the mainstream and enrichment settings, and we speculate that this microbial community has been stable over the course of the ~ 5 years between 2014 and 2019.

The distinct values of ¹⁵ε(NO₂^––N₂) observed for different species can be connected to key variations in the anammox metabolism. Although the canonical anammox mechanism includes the reduction of nitrite to NO by a nitrite reductase enzyme⁵¹, genomes of anammox bacteria of the Genus Ca. Brocadia^52,53, including 5 of 6 metagenome-assembled genomes for bacteria in the mainstream system used in this study²³, typically lack any canonical nitrite reductase in their genomes. Instead, it has been proposed that Ca. Brocadia do not produce NO and instead have hydroxylamine as the intermediate between nitrite and hydrazine⁵². This hypothesis is supported further by the nature of Hzs, which has two catalytic centers, one of which reduces NO to hydroxylamine, while the second conproportionates hydroxylamine and ammonia to generate hydrazine⁵⁴; it is possible that Ca. Brocadia can bypass NO entirely and deliver hydroxylamine directly to Hzs.

In contrast, Ca. Kuenenia, Ca. Scalindua, and Ca. Jettenia all include a canonical nitrite reductase in their genomes. Indeed, Kobayashi and coworkers¹⁹ observed that the offset in ¹⁵ε(NO₂^––N₂) between measured values for Ca. Kuenenia and Ca. Scalindua, which have the iron-bearing nitrite reductase NirS, and Ca. Jettenia, which has the copper-bearing nitrite reductase NirK, corresponds to that observed for NirK and NirS in bacterial denitrifiers⁵⁵. This interpretation is complicated by the observation that the genes for these canonical nitrite reductases are often not expressed^49,56 or translated⁵⁷ under environmental conditions. Nevertheless, the differences in N isotopic discrimination of nitrite among anammox clades appear to correspond to fundamental differences in the conversion of nitrite, but the molecular mechanisms of these steps remain poorly understood.

Inverse isotope effect imparted in ¹⁵ε(NO₂
^––NO₃
^–) by nitrite oxidation

A pronounced inverse isotope effect, in which nitrate becomes enriched in ¹⁵ N relative to nitrite from which it is produced, was observed in all experimental settings. Such an inverse isotope effect appears to be a signature feature of microbial nitrite oxidation to nitrate, both under oxic and anoxic (i.e., anammox) conditions^58,59 In culture-based experiments with nitrite oxidizing bacteria (NOB), ¹⁵ε(NO₂^––NO₃^–) has been found to vary between − 7.8‰ and − 23.6‰^59,60, while anammox bacteria have been shown to express ¹⁵ε(NO₂^––NO₃^–) values in pure or highly-enriched cultures between − 30 and − 45‰^10,19, with values as low as − 78‰ in a wastewater incubation experiment²⁰. In our experiments, we found N isotope effects that cover nearly this whole range (Fig. 4). In both anammox and the NOB, nitrite oxidation is thought to be performed by the enzyme nitrate:nitrite oxidoreductase (Nxr)^25,43,59, which is also closely related to bacterial membrane-bound and periplasmic nitrate reductases^61,62. The structural details of the Nxr enzyme family are not yet well explored, especially in light of its diverse metabolic roles⁶³, and so it remains unclear what metabolic or microbial processes are responsible for the observed and reported variation in ¹⁵ε(NO₂^––NO₃^–). At least for anammox bacteria, the inverse kinetic N isotope effect associated with the enzymatic oxidation of nitrite to nitrate may be superposed in part by a relatively large equilibrium N isotope effect between nitrite and nitrate¹⁰, perhaps promoted by the reversibility of the enzymatic nitrite oxidation reaction⁶⁴. It is notable that the most negative end of the observed range for ¹⁵ε(NO₂^––NO₃^–) in this study approaches the theoretical limit for the isotope effect set by the N isotope equilibrium between nitrite and nitrate, which at 20 °C is – 54.6‰⁵⁹, and which the NOB have not been observed to approach. This suggests that under the metabolic conditions of anammox, the Nxr enzyme is more likely to catalyze reversible reactions, and so the corresponding N isotope effect is closer to the equilibrium limit, than in aerobic nitrite oxidation. However, the great range observed in ¹⁵ε(NO₂^––NO₃^–) for anammox makes it difficult to predict a priori how much fractionation anammox will impart on a nitrate pool.

On the other hand, observations in this study and elsewhere^10,19 (Fig. 4) of values of ¹⁵ε(NO₂^––NO₃^–) falling near − 30‰ for nitrate generated by anammox match a prediction from water column measurements of N isotope ratios in nitrate and nitrite in the Peru oxygen deficient zone (ODZ)⁶⁵. The large and variable magnitude of this inverse isotope effect means that even though only ~ 25% of the nitrite oxidized by anammox is converted to nitrate, it can have an outsize effect on nitrate and nitrite pools that can be mistaken for either nitrite oxidation by NOB or nitrite generation by denitrification.

Implications for N isotope measurements in natural and engineered environments

Taken together, the results measured in this study suggest both potential and pitfalls for the application of N isotope measurements to disentangle the systematics of microbial N cycling processes. By its interaction with the nitrate, nitrite, ammonium, and N₂ pools, anammox already complicates analysis of the N cycle in any setting where it acts; not only does it impact the stable isotope pools of these molecules, but also its effects on each of these pools can vary greatly depending on physiological or metabolic variables or the identity of the dominant anammox bacteria species. In the context of a wastewater treatment process, measurements of δ¹⁵N alone may not be able to directly diagnose what processes are occurring, but when coupled to rate and stoichiometry measurements, may provide insights into the efficiency or limitations of those processes.

The removal of ¹⁵ N-depleted N by anammox may partly explain the heavy N isotope values (> 15‰) for nitrate in ODZs that have been formerly attributed to denitrification alone. It remains uncertain, however, what the exact expression of the range of N isotope effects reported here is under natural and variable substrate concentrations. First, as shown here for ammonium, concentration levels will have an effect on the relative kinetics of uptake and oxidation, and in turn on the cell-specific N isotope effect. Second, ammonium concentrations are generally at or below detection in the interior of OMZs, indicating that ammonium supplied by degradation of organic matter may be quantitatively oxidized to N₂ by anammox. Under these conditions, the N isotope effect associated with the conversion of ammonium to N₂ will be suppressed. Previously published estimates of the overall N isotope effect of dissolved inorganic N (DIN) elimination to N₂ in ODZs based on comparing nitrate δ¹⁵N values to observed water-column nitrate deficits has inherently included any potential non-fractionating loss of ammonium, and has thus implicitly represented a community N loss isotope effect that depends on the balance between anammox and canonical denitrification. The overall expression on DIN lost by the combined processes of denitrification and anammox in sediments, however, may be completely different. In contrast to nitrate and nitrite⁶⁶, ammonium is usually not limiting in sediments and its fractional loss to overlying waters allows the N isotope effect of ammonium oxidation to N₂ by benthic anammox to be expressed.

In both natural and engineered systems where anammox is known to be occurring, measurements of ¹⁵ε(NH₄⁺) may be able to diagnose substrate limitations or other physiological limitations. And in the case where ammonium is observed to be consumed by an unknown pathway, it can be expected that isotope effects will fall into a similar range for both aerobic and anaerobic ammonium oxidation; on the other hand, ¹⁵ε(NH₄⁺) is of little use for distinguishing ammonium consumption by AOB and anammox. We have also found that despite the great possibility for variation in ¹⁵ε(NO₂^––N₂) amongst different anammox species, values of ¹⁵ε(NO₂^––N₂) remain relatively stable in a given system that has a stable microbial community, and so this parameter has some potential to be used in monitoring such microbial community stability. Further work is needed to explore how metabolic variation amongst anammox species is related to variations in this parameter, but it appears that subtle changes in the mix of anammox species present, which have no observed effect on anammox rates or stoichiometry can lead to major changes in ¹⁵ε(NO₂^––N₂). Finally, this study expands the range of ¹⁵ε(NO₂^––NO₃^–) for anammox bacteria. On one hand, this result lends further support to the observation that through a strong manifestation of the inverse isotope effect associated with nitrite oxidation, anammox can produce nitrate strongly enriched in ¹⁵ N, thereby complicating N mass balances based on tracking the nitrate pool. But we also find that anammox can have ¹⁵ε(NO₂^––NO₃^–) values much closer to 0‰, falling in the same range as for NOB bacteria, so the contribution of anammox to the δ¹⁵N composition of the nitrate pool can in fact vary greatly. Finally, we find that there are no systematic relationships among ¹⁵ε(NH₄⁺), ¹⁵ε(NO₂^––N₂), ¹⁵ε(NO₂^––NO₃^–), which is consistent with the conclusion that each of these parameters is controlled at a distinct point in the anammox metabolism.

Source: Ecology - nature.com