Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities

AOA kinetic properties

In this study we investigated the kinetic properties of 12 AOA strains, including representatives from all four described AOA phylogenetic lineages: Nitrosopumilales (Group I.1a), ‘Ca. Nitrosotaleales’ (Group I.1a-associated), Nitrososphaerales (Group I.1b), and ‘Ca. Nitrosocaldales’ (thermophilic AOA clade) [58, 59] (Fig. 1). These AOA isolates and enrichments were obtained from a variety of habitats (marine, soil, sediment, hot spring) and have optimal growth pH and temperatures ranging from 5.3–7.8 to 25–72 °C, respectively (Table S2). The substrate-dependent oxygen consumption rates for all AOA tested followed Michaelis–Menten kinetics. Below, the kinetic properties of these AOA are put into a broader context with comparisons to previously characterized AOM. It is important to note that the whole cell kinetic properties, such as substrate competitiveness, detailed here were generated from instantaneous activity measurements in the absence of growth. It is unknown how the substrate competitiveness of nitrifiers may or may not differ from their competitiveness when cellular processes such as growth, division, stress, and repair are involved.

Nitrosopumilales (Group I.1a)

From this lineage, three mesophilic marine (N. piranensis D3C, N. adriaticus NF5, and N. maritimus SCM1) [3, 60], two agricultural soil (N. koreense MY1 and ‘Ca. N. chungbukensis’ MY2) [61, 62] and one thermal spring isolate (‘Ca. N. uzonensis’ N4) [40] were kinetically characterized (Fig. S1). These AOA all displayed a high substrate affinity for NH₃, ranging from ~2.2 to 24.8 nM. Thus, all characterized Nitrosopumilales, and not just marine isolates, are adapted to oligotrophic conditions. All possess substrate affinities several orders of magnitude higher (lower K_m(app)) than any characterized AOB, with the exception of the recently characterized acidophilic gammaproteobacterial AOB ‘Ca. Nitrosacidococcus tergens’ [55] (Fig. 2a). This finding appears to support the widely reported hypothesis that regardless of the environment, AOA in general are adapted to lower substrate concentrations than AOB [22, 29, 30]. However, as described later, this trend does not apply to all AOA.

As the substrate oxidation kinetics of the marine AOA strain, N. maritimus SCM1, originally characterized by Martens-Habbena et al. [29] have recently been disputed [63], they were revisited in this study (Fig. S2). With the same strain of N. maritimus used in Hink et al. [63] (directly obtained by the authors), we were able to reproduce (Figs. S1 and S2) the original kinetic properties of N. maritimus SCM1 reported in Martens-Habbena et al. [29] ruling out strain domestication during lab propagation as cause for the observed discrepancy. Therefore, the reported differences in the literature possibly reflect the measurements of two distinct cellular properties, K_m(app) [29] and K_s [63], representing the half saturation of activity and growth, respectively. In addition, differences in pre-measurement cultivation and growth conditions could also contribute to these unexpected differences [63, 64]. More details are provided in the Supplementary Results and Discussion.

‘Ca. Nitrosotaleales’ (Group I.1a-associated)

The only isolated AOA strains in this lineage ‘Ca. Nitrosotalea devanaterra’ Nd1 and ‘Ca. Nitrosotalea sinensis’ Nd2, are highly adapted for survival in acidic environments and grow optimally at pH 5.3 [25, 65]. Both display a relatively low affinity for total ammonium (K_m(app) = 3.41–11.23 μM), but their affinity for NH₃ is among the highest calculated of any AOA characterized (K_m(app) = ~0.6–2.8 nM) (Fig. 2a,c, and Fig. S3). This seemingly drastic difference in substrate affinity for total ammonium versus NH₃ is due to the combination of the high acid dissociation constant of ammonium (pKa = 9.25) and the kinetic properties of these strains being carried out at pH 5.3. The very limited availability of NH₃ under acidic conditions has led to the hypothesis that these acidophilic AOA should be highly adapted to very low NH₃ concentrations and possess a high substrate affinity (low K_m(app)) for NH₃ [66, 67]. Our data corroborate this hypothesis.

Nitrososphaerales (Group I.1b)

The AOA strains ‘Ca. N. nevadensis’ GerE (culture information provided in Supplementary Results and Discussion), ‘Ca. N. oleophilus’ MY3 [68] and ‘Ca. N. franklandus’ C13 [69] were kinetically characterized, and contextualized with the previously published kinetic characterization of Nitrososphaera viennensis EN76 and ‘Ca. Nitrososphaera gargensis’ [5]. Together, the Nitrososphaerales AOA possess a wide range of affinities for NH₃ (K_m(app) = ~0.14–31.5 µM) (Fig. 2a and Fig. S4). Although this range of NH₃ affinities spans more than two orders of magnitude, none of the Nitrososphaerales AOA possess an affinity for NH₃ as high as any Nitrosopumilales or ‘Ca. Nitrosotaleales’ AOA (Fig. 2a).

The moderately thermophilic enrichment culture ‘Ca. N. nevadensis’ GerE displayed a higher substrate affinity (lower K_m(app)) for NH₃ (0.17 ± 0.03 µM) than the other characterized AOA strains within the genus Nitrososphaera (Fig. 2a). In contrast, ‘Ca. N. oleophilus’ MY3 and ‘Ca. N. franklandus’ C13, which belong to the genus Nitrosocosmicus, had the lowest affinity (highest K_m(app)) for NH₃ (12.37 ± 6.78 μM and 16.32 ± 14.11 μM, respectively) of any AOA characterized to date. In fact, their substrate affinity is comparable to several characterized AOB (Fig. 2a). In this context it is interesting to note that several Nitrosocosmicus species have been shown to tolerate very high ammonium concentrations [68,69,70], a trait usually associated with AOB [24, 54]. The low substrate affinity observed in Nitrosocosmicus AOA correlates with the absence of a putative Amt-type high affinity ammonium transporter in the genome of any sequenced Nitrosocosmicus species to date [68, 69, 71].

‘Ca. Nitrosocaldales’ (Thermophilic AOA lineage)

The thermophilic AOA enrichment cultures, ‘Ca. Nitrosocaldus yellowstonensis’ HL72 [72] and ‘Ca. N. tenchongensis’ DRC1 (culture information provided in Supplementary Results and Discussion), possess affinities for NH₃ (K_m(app) = ~1.36 ± 0.53 μM and ~0.83 ± 0.01 μM; respectively comparable to AOA within the genus Nitrososphaera (Fig. 2a). Notably, the substrate oxidation rate of these two AOA quickly dropped with increasing substrate concentrations after V_max was reached (Fig. S5). This trend was not observed with any other AOA tested here and may reflect an increased susceptibly to NH₃ stress at high temperatures, as the free NH₃ concentration increases with increasing temperatures [33]. It should be noted that both of these AOA cultures are enrichment cultures, as no member of the ‘Ca. Nitrosocaldales’ has been isolated to date.

Together, these results highlight that the substrate affinity for NH₃ among AOA species is much more variable than previously hypothesized, spanning several orders of magnitude and in some cases overlapping with the substrate affinity values of characterized non-oligotrophic AOB. In addition, the substrate affinity of AOA is related, to a certain degree, to their phylogenetic placement within each of the four AOA phylogenetic lineages mentioned above (Fig. 2). Although the substrate affinity ranges of these AOA lineages overlap, the link between AOA phylogeny and kinetic properties provides deeper insights into the physiological and evolutionary differences among AOA species. As a limited number of AOA have been isolated and characterized to date, the continued isolation and characterization of AOA from underrepresented phylogenetic lineages and new habitats is needed. While substrate affinity is certainly one of multiple factors that contribute to niche differentiation between AOM in general, it may also present a previously under acknowledged factor in AOA niche differentiation.

Maximum substrate oxidation rates (V
_max)

The normalized maximum substrate oxidation rate of all the AOA characterized to date only span about one order of magnitude from 4.27 to 54.68 μmol N mg protein⁻¹ h⁻¹. These normalized AOA V_max values are in the same range as the recorded V_max for the comammox N. inopinata (~12 μmol N mg protein⁻¹ h⁻¹) and the marine AOB strain Nitrosococcus oceani ATCC 19707 (~38 μmol N mg protein⁻¹ h⁻¹) but are lower than the normalized V_max of the AOB Nitrosomonas europaea ATCC 19718 (average of 84.2 μmol N mg protein⁻¹ h⁻¹; Fig. 2e). The high V_max value for N. europaea is the only real outlier among the AOM characterized to date and it remains to be determined whether other AOB related to N. europaea also possess such a high V_max or if members of the Nitrosomonadales possess a broad range of V_max values. Similarly, as additional comammox strains become available as pure cultures their kinetic characterization will be vital in understanding the variability of these ecologically important parameters within this guild.

Specific substrate affinity (a°)

Although the K_m(app) and V_max of AOM can be compared by themselves and provide useful information on cellular properties, the ability of an AOM to scavenge (and compete for) substrate from a dilute solution is most appropriately represented by the a°, which takes into account both the cellular K_m(app) and V_max [28]. In previous studies, the a° of AOM has been calculated using the K_m(app) value for total ammonium (NH₃ + NH₄⁺) and not the K_m(app) value for NH₃ [5, 29]. Calculating the a° based on the K_m(app) value for total ammonium allows for the a° of AOM to be compared with the a° of microorganisms that do not use NH₃ as a sole energy generating substrate, such as ammonium assimilating heterotrophic bacteria or diatoms [29]. While this is useful when evaluating competition for total ammonium in mixed communities or environmental settings, an a° calculated using the K_m(app) value for NH₃ may be more useful when directly comparing the interspecies competitiveness of AOM for the following reasons: (i) our data support the hypothesis that the substrate for all AOM is NH₃ and not NH₄⁺ (see below) and (ii) the K_m(app) value for total ammonium is more dependent on the environmental factors it was measured at (e.g., pH, temperature, salinity) than the K_m(app) for NH₃.

All characterized AOA (with the exception of representatives of the genus Nitrosocosmicus) and the comammox bacterium N. inopinata possess much higher a° for total ammonium or NH₃ (~10–3000×) than the AOB, N. oceani or N. europaea (Fig. 2b–d), indicating that they are highly competitive in environments limited in either total ammonium or only NH₃. However, due to the low number of published normalized V_max values for AOB, a° could only be calculated for these two AOB representatives. Thus, extrapolations to the a° of all AOB species, based solely on these observations should be approached with caution.

The low variation in experimentally measured V_max values (Fig. 2e) across all measured AOM in combination with the high variation in K_m(app) values leads to a strong relationship between cellular a° and the reciprocal of K_m(app) (Fig. 3) according to Eq. 2 (see Materials and Methods). AOM adapted to oligotrophic (low substrate) conditions should possess both a high substrate affinity (low K_m(app)) and a high a^o [28]. Therefore, the AOM best suited for environments limited in total ammonium are the AOA belonging to the Nitrosopumilales and the comammox isolate N. inopinata, (top right corner of Fig. 3a). Overall, when looking at solely NH₃ or total ammonium, the separation of species in these plots remains similar, with the exception that the acidophilic AOA belonging to the ‘Ca. Nitrosotaleales’ are predicted to be best suited for life in environments limited in NH₃ (Fig. 3b). The adaptation correlates well with the fact the AOA ‘Ca. Nitrosotalea devanaterra’ Nd1 and ‘Ca. Nitrosotalea sinensis’ Nd2 were isolated from acidic soils with a pH of 4.5 and 4.7, respectively [25, 65], where the NH₃:NH₄⁺ equilibrium is heavily shifted toward NH₄⁺.

In either case, when looking at NH₃ or total ammonium, the AOA belonging to the genus Nitrosocosmicus (‘Ca. N. oleophilus’ MY3 and ‘Ca. N. franklandus’ C13) and AOB populate the lower left section of these plots, indicating that they are not strong substrate competitors in NH₃ or total ammonium limited environments (Fig. 3). Here, the V_max of all the AOM reported spans ~10×, whereas the difference in K_m(app) spans about five orders of magnitude. If the cellular kinetic property of V_max really is so similar across all AOB, AOA, and comammox species (Fig. 2e) compared to the large differences in K_m(app) values, then substrate competitiveness can be predicted from an AOMs K_m(app) for either NH₃ or total ammonium (Fig. 2a–c). This may prove especially helpful when characterizing enrichment cultures, where normalizing ammonia-oxidizing activity to cellular protein in order to obtain a comparable V_max value is not possible. However, there is also a need for more kinetically characterized AOB and comammox species to confirm this hypothesis. In addition, when comparing AOM, differences in the V_max cellular property will play a larger role, the closer the K_m(app) values of the AOM strains are. This is important to consider when comparing AOM from similar habitats and likely adapted to similar substrate concentrations.

The effect of environmental and cellular factors on AOA kinetic properties

The concentration of NH₃ present in a particular growth medium or environment can vary by orders of magnitude, based solely on the pH, temperature, or salinity of the system [73]. This is notable because at a given total ammonium concentration, the concentration of NH₃ is ~10 times higher at 70 °C versus 30 °C and ~1000 times lower at pH 5.3 versus pH 8.4 (representative of maximum ranges tested). While it should be recognized that in our dataset no AOM were included that have a pH optimum between 5.3 and 7.0, the effect of pH and temperature on the ammonia oxidation kinetics of AOM must be considered in order to understand their ecophysiological niches. However, there was no correlation between the kinetic properties of AOM (K_m(app), V_max, and a°) measured in this study and their optimal growth temperature or pH. This lack of correlation between AOM species kinetic properties and growth conditions does not imply that the cellular kinetic properties of an individual AOM species will remain the same over a range of pH and temperature conditions. Therefore, we investigated the effect of pH and temperature variation on the substrate-dependent kinetic properties of the AOA strain ‘Ca. N. oleophilus’ MY3, and the effect of pH on the comammox strain N. inopinata. Here, the AOA ‘Ca. N. oleophilus’ MY3 was selected based on the fact that it is a non-marine, mesophilic, pure culture, that does not require external hydrogen peroxide scavengers for growth. These traits are shared with the previously characterized AOB, N. europaea [35], and the comammox organism, N. inopinata (this study) and thus facilitate comparison.

The effect of temperature

The effects of short-term temperature changes on the substrate-dependent kinetic properties of ‘Ca. N. oleophilus’ MY3 were determined. Temperature shifts of 5 °C above and below the optimal growth temperature (30 °C) had no effect on the K_m(app) for total ammonium. However, the K_m(app) for NH₃, V_max, and a° of ‘Ca. N. oleophilus’ MY3 all increased with increasing temperatures (Fig. S6). Therefore, as temperature increased, ‘Ca. N. oleophilus’ MY3 displayed a lower substrate affinity (higher K_m(app) for NH₃) but would be able to turnover substrate with a higher V_max and better compete for substrate with a higher a°. Increasing AOA K_m(app) values for NH₃ with increasing temperatures have also been observed across studies with N. viennensis EN76 (Fig. S2), and this is discussed in more detail in the Supplementary Results and Discussion. In addition, similar observations have previously been made for AOB strains belonging to the genus Nitrosomonas [33, 34]. The increase in V_max and a° can be explained in terms of the Van’t Hoff rule (reaction velocity increases with temperature) [74], or in terms of a temperature sensitivity coefficient (Q₁₀; change in reaction velocity over 10 °C) [75]. Here, the maximal reaction velocity of ‘Ca. N. oleophilus’ MY3, displays a relative Q₁₀ of 2.17 between 25 and 35 °C, which is in line with more general microbial respiration measurements [75, 76].

The increase in K_m(app) for NH₃ (lower NH₃ affinity) with increasing temperature is less straightforward to interpret. As this is a whole cell measurement, the observed differences may result from either broad cellular changes or from changes in individual enzymes involved in the ammonia oxidation pathway specifically. At the cellular level, changes in the proteinaceous surface layer (S-layer) or lipid cell membrane could affect substrate movement/transport and enzyme complex stability. It has been suggested that the negatively charged AOA S-layer proteins act as a substrate reservoir, trapping NH₄⁺ and consequently increasing the NH₃ concentration in the AOA pseudo-periplasmic space [77]. It is interesting to note that sequenced representatives from the genus ‘Ca. Nitrosocosmicus’ lack the main S-layer protein (slp1) found in all Nitrosopumilales, Nitrososphaerales, and ‘Ca. Nitrosotaleales’ sequenced isolates [71], although it remains to be demonstrated whether ‘Ca. Nitrosocosmicus’ members actually lack a S-layer or form S-layers composed of other proteins. In addition, it has been demonstrated that elevated temperatures significantly alter the lipid composition in the AOA cell membrane [78, 79]. However, it is unclear how differences in the cell membrane or S-layer composition between AOA species may affect the observed kinetic properties. In this context it is important to note that on the single enzyme level, previous studies have shown the same trend of decreasing substrate affinity and increasing maximal reaction velocity with increasing temperatures, due to altered protein structures and an increased enzyme-substrate dissociation constant [80, 81].

Notably, differing optimum growth and activity conditions were previously determined for the marine AOB strain Nitrosomonas cryotolerans [34]. These observations raise interesting, albeit unanswered, questions about why the growth and activity temperature optima are or can be uncoupled in AOM, and what this means for AOM niche differentiation and their competitiveness in-situ. Moving forward, investigations into the growth and cellular kinetic properties of AOM across a range of environmental factor gradients will be essential in understanding competition between AOM in engineered and environmental systems.

The effect of pH

The effects of short-term pH changes on the substrate-dependent kinetics of ‘Ca. N. oleophilus’ MY3 and N. inopinata were determined. The V_max of both ‘Ca. N. oleophilus MY3’ and N. inopinata were stable at 37.3 ± 6.6 μmol N mg protein⁻¹ h⁻¹ and 11.2 ± 2.5 μmol N mg protein⁻¹ h⁻¹, respectively, in medium with a pH between ~6.5 and ~8.5 (Table S3). The K_m(app) for total ammonium of ‘Ca. N. oleophilus MY3’ and N. inopinata decreased by more than an order of magnitude (~11×) across this pH range, while the K_m(app) for NH₃ remained more stable, increasing only 3–4 times (Fig. 4). This stability of the K_m(app) for NH₃ compared with the larger change in the K_m(app) for total ammonium across this pH range suggests that the actual substrate used by AOA and comammox is indeed the undissociated form (NH₃) rather than the ammonium ion (NH₄⁺), as previously demonstrated for AOB [34, 35, 54, 82]. As these kinetic measurements were performed with whole cells, the change in K_m(app) for NH₃ across this pH range may be due to cellular effects of the differing pH values unrelated to the direct ammonia oxidation pathway. The changes in Km_(app) for NH₃ and K_m(app) for total ammonium demonstrated here for ‘Ca. N. oleophilus’ MY3 and N. inopinata are similar to what has been observed for AOB. That AOA and AOB utilize the NH₃ as a substrate, aligns with the fact that both are competitively inhibited by the non-polar acetylene compound [83, 84].

Interestingly, the only exception to this rule to date is the gammaproteobacterial marine AOB Nitrosococcus oceani. The reported K_m(app) for total ammonium of N. oceani remained more stable (~2.3×) than the K_m(app) for NH₃ (78×) when the pH was shifted from 6.3 to 8.6 [85]. With this exception in mind, our results support the hypothesis that AOA, AOB, and comammox utilize NH₃ as their substrate. As only a few AOM have been characterized in this manner, the characterization of a more diverse set of AOM species is needed in order to make broader conclusions.

It is important to note that the substrate affinities reported here represent whole cell affinities and not the substrate affinity of ammonia monooxygenase (AMO) enzymes. Therefore, further experimental investigation with purified AMO and ammonia/ammonium transporter proteins is warranted. Although NH₃ can freely diffuse passively into AOM, this does not mean that the cellular affinity reported here is necessarily unrelated to the transporter-mediated movement of NH₃/NH₄⁺ into AOM cells. For example, AOB have previously been shown to accumulate very high (1 M) intracellular NH₄⁺ concentrations [86]. This high intracellular NH₄⁺ concentration may provide a concentrated substrate reservoir, indirectly increasing the concentration of NH₃ around the AMO enzyme complex. In addition, the negatively charged S-layer of N. maritimus has been shown to act as a substrate reservoir for the positively charged NH₄⁺. This total ammonium concentration in the pseudo-periplasmic space of AOA, may also act to indirectly increase NH₃ concentrations around the AMO enzyme complex [77]. However, the impact of total ammonium capture and transport on the ammonia oxidation kinetic properties of AOB, AOA, and comammox are not yet well understood. It is unknown if such a concentration mechanism would be more important for an AOB with a low substrate affinity (e.g., N. europaea) or for an AOA living in extremely substrate-limited environments (e.g., N. maritimus).

The effect of cell morphology

All AOM share the primary enzyme involved in ammonia oxidation, AMO, which is located in the cytoplasmic membrane with its substrate-binding site most likely facing the outside of the cell [77]. Therefore, a higher cellular SA/V ratio likely contributes to an increase in a°, as it increases the space available for AMO and the chance to bind NH₃ at very low concentrations. This assumption is based on the hypothesis that an increased abundance of uptake enzymes (e.g., permeases) leads to a higher a° [28, 56]. In fact, the SA/V ratio of AOM (Table S4) correlates to the log of their observed K_m(app) for NH₃ (R² = 0.88), K_m(app) for total ammonium (R² = 0.70), a° for NH₃ (R² = 0.78), and a° for total ammonium (R² = 0.72; Fig. 5). This type of casual semi-log relationship between cell size and nutrient affinities has previously been observed between microorganisms and their affinity for phosphate [87] as well as theoretically modeled for nutrient uptake and utilization in general [88]. Therefore, the SA/V ratio of newly cultured AOM might be a useful general indicator for these cellular kinetic properties. Consequently, AOM with a high SA/V ratio will likely outcompete other AOM in many natural aquatic and terrestrial environments, such as the pelagic marine water column that has a very low standing total ammonium pool. Consistently, these oligotrophic environments have already been postulated to select for organisms with a high SA/V ratio, enhancing their nutrient uptake capabilities [89, 90].

The correlation between the SA/V ratio and cellular kinetic properties of AOM sheds some light on the unusual kinetic properties of the AOA belonging to the genus Nitrosocosmicus. Both ‘Ca. N. oleophilus’ MY3 and ‘Ca. N. franklandus’ C13 possess a very low SA/V ratio compared to other AOA isolates and they both possess several characteristics normally associated with AOB—high substrate tolerances [68,69,70], low affinities for NH₃, and a low a° for NH₃—that are not consistent with the long-held convention that all AOA are much stronger competitors for NH₃ than AOB in substrate-limited environments. Therefore, the individual cell morphology of AOM may have a direct relationship with their cellular kinetic properties. Although this is only a correlation-based observation, it highlights that further investigation into these characteristics is warranted.

In addition to cellular morphology, the size of cell aggregates can affect the kinetic properties of AOM [48]. Cell aggregates have a lower SA/V ratio than individual cells, which can decrease diffusion rates and create microscale substrate/oxygen gradients within aggregates [91]. In order to ensure that the large differences in substrate affinity among AOA are not caused by differences in cell aggregation, the aggregate size of ‘Ca. N. uzonensis’ N4, ‘Ca. N. oleophilus’ MY3, and N. piranensis D3C cultures were inspected before and after MR experiments (Fig. S7).

These three AOA were chosen to highlight the aggregate sizes observed in cultures that displayed the lowest (‘Ca. N. oleophilus’ MY3) and among the highest (‘Ca. N. uzonensis’ N4 and N. piranensis D3C) substrate affinities. No aggregation pattern was observed that would explain the multiple orders of magnitude differences in substrate affinity between these AOA. In fact, of the three AOA investigated, the only strain to form large cell aggregates either before or after MR experiments was N. piranensis D3C, which has one of the highest measured substrate affinities (lowest K_m(app) for NH₃). In contrast, the cell aggregate size of ‘Ca. N. oleophilus’ MY3 and ‘Ca. N. uzonensis’ N4 were unaffected by the MR experiment and remained relatively small (Fig. S7). As ‘Ca. N. oleophilus’ MY3 has one of the lowest substrate affinities (highest K_m(app) for NH₃) and formed only small cell aggregates, the low substrate affinity of ‘Ca. N. oleophilus’ MY3 was not an artefact caused by cell aggregation.

Taken together, both environmental (pH and temperature) and AOM cellular (SA/V ratio) factors affect or are related to the observable cellular kinetic properties of individual AOM species. These factors need to be considered when investigating AOM competition or niche differentiation in-situ, as they are often in flux in environmental settings. This can be especially true considering cell morphology, which is often dependent on growth conditions [92]. However, the plasticity of the cellular kinetic properties within individual AOM species does not explain the larger trends observed here across AOA lineages or between AOM (Figs. 2, S1, S3–5, S8).

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