Nitrification is an important process in the nitrogen cycle, and it was widely believed as a two-step process for more than one century with ammonia oxidized to nitrite by ammonia-oxidizing bacteria (AOBs) or ammonia-oxidizing archaea (AOAs) (Könneke et al., 2005), and subsequently nitrite oxidization to nitrate by nitrite-oxidizing bacteria (NOBs). Complete ammonia oxidizers (comammoxs) were initially proposed according to optimal pathway length analysis (Costa et al., 2006), functionally enriched and finally isolated in recent efforts (Daims et al., 2015; van Kessel et al., 2015; Kits et al., 2017; Sakoula et al., 2021). The identification of comammox raises questions on their diversity and distribution in the environment. Metagenome and marker gene sequencing data analyses have demonstrated that newly discovered comammox populations are widely distributed in natural and engineered systems (Palomo et al., 2016; Pinto et al., 2016; Wang et al., 2017; Annavajhala et al., 2018; Orellana et al., 2018; Xia et al., 2018; Yang et al., 2020), which are highly overlapped with the habitat of AOAs, AOBs, and NOBs (Hatzenpichler, 2012; Prosser and Nicol, 2012; Stahl and de la Torre, 2012; Daims et al., 2016; Lawson and Lucker, 2018). These results indicate comammox is a common nitrifier in the environment, raising further questions about their niche differentiation and interactions in the environment (Daims et al., 2016; Santoro, 2016).

Niches of nitrifiers are highly overlapped as they could share similar habitats and substrates in the environment (Martens-Habbena et al., 2009). Cultivation studies of niche differentiation could reveal mechanisms about the origin and maintenance of species biodiversity, community assembly (HilleRisLambers et al., 2012), coexistence (File et al., 2012), species physiology (Hatzenpichler et al., 2008; Hatzenpichler, 2012), and habitat preferences (Bauer et al., 2018). For example, the high ammonia affinity of comammox indicated their prevalence at oligotrophic environments (Kits et al., 2017) and the cooperation possibility between anammox and AOAs determined by their ammonia affinity according to a biofilm model (Straka et al., 2019). As ammonia and nitrite are key substrates of nitrification (Falkowski et al., 2008; Canfield et al., 2010; Stein, 2015), of particular interest is niche differentiation among nitrifiers in response to ammonium concentrations, which may provide new insights into the biodiversity, ecophysiology, and evolution of metabolically distinct nitrifiers in the environment.

To identify patterns of niche differentiation and interactions of nitrifiers driven by ammonium, synthetic microbial ecology theories and approaches may provide a new strategy. Synthetic microbial communities are designed and built by microorganisms with known genome information, physiology, and metabolic characteristics in a well-defined medium (Johns et al., 2016). Natural environmental communities are generally complex and subjected to the influence of a multitude of environmental factors, making them difficult, if not impossible, to identify drivers of niche differentiation. While a major drawback of laboratory studies on pure cultures is the inherited inability to study population interactions and underlying mechanisms, which is critical to the manifestation of niche differentiation. To bridge such a gap, synthetic microbial ecology has recently been developed (Dolinsek et al., 2016; Lindemann et al., 2016; Zomorrodi and Segre, 2016; Lawson et al., 2019), and synthetic microbial communities have been used as a powerful tool to simplify natural microbial communities with reduced complexity and a controlled environment (Shou et al., 2007; Momeni et al., 2011; De Roy et al., 2014; Grosskopf and Soyer, 2014), which is promising to address niche differentiation of nitrifiers and their interactions in the environment.

In this study, we aimed to study niche differentiation among four types of nitrifiers and their underlying mechanisms in response to ammonium concentrations using synthetic nitrifying communities (SNCs). We used a bottom-up approach to construct SNCs with different combinations of Nitrospira inopinata (comammox), Nitrososphaera gargensis (AOA), Nitrosomonas communis (AOB), and Nitrospira moscoviensis (NOB) and examined their responses to comparable or beyond environmentally ammonium concentrations (0.2 to 20 mM). Our results showed that the niche differentiation of nitrifiers was largely driven by their ammonia affinity, specific growth rates, and ammonium/nitrite tolerance. This study advances our understanding of niche differentiation, coexistence, and interactions driven by ammonium among metabolically distinct nitrifiers in the environment.

Understanding the niche differentiation, interaction, and coexistence of nitrifiers and their associated mechanisms is a central issue in microbial ecology. Our current knowledge of nitrifiers is generally based on limited monocultures and environmental studies, and their coexistence, niche differentiation, and interactions in the environment are extremely difficult or impossible to address. As various differences in physiology of monocultures exist, it is well inferred that substrate (ammonium and nitrite), temperature, pH, and H₂O₂ detoxification are considered the main drivers of niche differentiation among metabolically distinct nitrifiers (Prosser and Nicol, 2012; Hu and He, 2017; Kits et al., 2017). The physiology and diversity of cultivated AOAs indicated that they had a wider range of temperature and pH adaption than most AOBs (Lehtovirta-Morley et al., 2011; Zhang et al., 2017; Prosser et al., 2020; Picone et al., 2021); α-keto acids (e.g., pyruvate) which were reported to enhance the growth of some AOAs (Tourna et al., 2011; Qin et al., 2014) were further confirmed as H₂O₂ scavengers (Kim et al., 2016). Recently, several studies further indicated that nitrifiers were metabolically versatile beyond the N cycle and involved in hydrogen and sulfide oxidation (Lehtovirta-Morley et al., 2011; Tourna et al., 2011; Stahl and de la Torre, 2012; Daims et al., 2016). Among such many drivers, we gave priority to answering niche differentiation to ammonium as it is the energy source for nitrifiers. Thus, in this study, we applied synthetic microbial ecology theories and approaches to construct SNCs under unified medium and temperature and tested niche differentiation with comammox and other three types of metabolically distinct nitrifiers under a gradient of environmentally comparable ammonium concentrations to reflect the performance of nitrifiers in the environment. We found that those nitrifiers could coexist at low ammonium concentrations, and their niches differentiated under different ammonium concentrations with possible mechanisms, including ammonia/nitrite affinity, specific growth rate, and ammonium/nitrite tolerance. In addition, the results allowed us to explore possible interactions among those nitrifiers. Specifically, N. inopinata was the most competitive population at low ammonium concentrations, and N. communis was dominant at high ammonium concentrations. While N. gargensis and N. moscoviensis were less competitive than N. inopinata and N. communis for ammonium and N. inopinata for nitrite, respectively, at our tested ammonium ranges, the growth of N. inopinata, N. gargensis, and N. moscoviensis were inhibited at high ammonium concentrations.

First, ammonia/nitrite affinity of nitrifiers is the most important theoretical basis of niche differentiation at low ammonium environments (Hatzenpichler, 2012; Prosser and Nicol, 2012; Kits et al., 2017). For ammonia affinity, how AOAs and AOBs establish their niches and function in the environment has been discussed for decades, and comammox has recently joined in this debate. Previous laboratory studies showed that N. inopinata had the highest ammonia affinity, followed by non-marine AOAs and AOBs (Kits et al., 2017), which was reflected in the apparent half-saturation constant value [K_m(app)] of N. inopinata, N. gargensis, and Nitrosomonas AOBs at 0.65, 5.6, and ~1,000 μM total ammonium, respectively (Martens-Habbena et al., 2009; Kits et al., 2017). Comammox populations have been considered as oligotrophs and could be prevalent at low ammonium environments, which is evidenced by many studies with microbial enrichments or environmental samples from oligotrophic environments, such as drinking water systems, rapid gravity sand filter, and groundwater-fed rapid sand filter (Palomo et al., 2016; Kits et al., 2017; Pjevac et al., 2017; Fowler et al., 2018; Xia et al., 2018). Also, AOAs were reported to have an advantage over AOBs in oligotrophic environments like open ocean (Könneke et al., 2005; Prosser and Nicol, 2008; Santoro et al., 2008; Walker et al., 2010), while eutrophic environments (e.g., fertilized agricultural soils) favored AOBs (Jia and Conrad, 2009; Prosser and Nicol, 2012; Hink et al., 2018). In this study, we found that the dominant species in N4 was N. inopinata, followed by N. gargensis and N. communis at low ammonium concentrations. N. gargensis exhibited the lowest K_m(app), followed by N. inopinata and N. communis (282 and 2,553 μM total ammonium, respectively). Such relatively high numerical values of K_m(app) observed may be due to higher ammonium concentrations used in this study than in previous physiological studies, and the contrast of K_m(app) between N. inopinata and N. gargensis could also result from the low activity and growth of N. gargensis in our experiments, thus affecting the degree of model fitting. These results generally support the predominance of comammox at low ammonium concentrations due to its high ammonia affinity, which is consistent with previous studies that comammox preferred oligotrophic lifestyle (Palomo et al., 2016; Pinto et al., 2016; Kits et al., 2017; Fowler et al., 2018; Xia et al., 2018). For nitrite affinity, known NOBs are affiliated with seven genera (Daims et al., 2016), of which Nitrospira NOBs are considered as K strategists and Nitrobacter NOBs as r-strategists (Nowka et al., 2015), and coincidently, all known comammoxs belong to Nitrospira. Nevertheless, the K_m(app) of N. inopinata to nitrite was 372 μM, close to that of Nitrobacter NOB (Kits et al., 2017), which was higher than Nitrospira NOB. In this study, we found that the K_m(app) of N. moscoviensis was 228 μM nitrite, which was lower than that of N. inopinata, indicating a niche of N. inopinata and N. moscoviensis in nitrite oxidation; thus, N. inopinata may prefer higher nitrite concentrations than N. moscoviensis.

Second, the ammonium/nitrite inhibition of nitrifiers may play important roles in their niche differentiation (Prosser and Nicol, 2012) as the growth and activity of nitrifiers were inhibited by different concentrations of FA and FNA (Anthonisen et al., 1976; Liu et al., 2019). A previous study showed that FA inhibition concentrations of AOB and NOB were 10–150 mg NH₃-N/L and 0.1–1.0 mg NH₃-N/L, respectively (Anthonisen et al., 1976). Also, the oxidation rate of Nitrosomonas and Nitrobacter decreased when FNA concentrations reached 0.10 (Vadivelu et al., 2006) and 0.011 mg HNO₂-N/L, respectively, and was completely inhibited at the FNA concentration of 0.40 mg and 0.023 mg HNO₂-N/L, respectively (Vadivelu et al., 2007), which are consistent with the results of this study, showing that high ammonium concentrations inhibited the growth of N. inopinata and N. moscoviensis, but no obvious inhibition was observed for N. communis. In addition, we found 2 mM ammonium would inhibit both ammonium oxidation and growth of AOA, which agrees with a previous study that shows an inhibition concentration of 3.08 mM ammonium (Hatzenpichler et al., 2008). Therefore, our results indicated that the ammonium/nitrite tolerance was different among these nitrifiers, which could explain the dominance of AOBs at high ammonium concentrations.

Third, the specific growth rate of nitrifiers under different ammonium concentrations may provide new insights into our understanding of niche differentiation. Generally, the growth rate of nitrifiers against substrate concentrations is described by the Monod equation (Prosser and Nicol, 2012). Previous studies showed that AOBs had higher specific growth rates than AOAs (Prosser and Nicol, 2012; Terada et al., 2013), and comammox nitrifiers were predicted to have a lower specific growth rate than canonical ammonia oxidizers (Costa et al., 2006). Kits et al. (2017) reported that the maximum specific ammonia oxidation activity of N. inopinata and N. gargensis was 0.032 h⁻¹ (37°C) and 0.028 h⁻¹ (46°C), respectively, and the specific ammonia oxidation activity of N. gargensis was 0.014 h⁻¹ at 37°C. In this study, we found that N. communis had the highest maximum specific growth rate, followed by N. inopinata, N. moscoviensis, and N. gargensis from N1, N2A, and N2B, respectively, which was different from the prediction of comammox (Costa et al., 2006). However, the maximum specific growth rates of nitrifiers were different in N4, with the highest maximum specific growth rate for N. inopinata, followed by N. communis, N. gargensis, and N. moscoviensis, indicating complex interactions among metabolically distinct nitrifiers. Such interactions and underlying mechanisms need to be further explored in future.

Based on the aforementioned results and current knowledge, we developed a conceptual model to understand kinetics-driven niche differentiation among those nitrifiers. It may be generally assumed that comammox had the highest ammonia affinity, followed by non-marine AOAs and AOBs, and NOBs had higher nitrite affinity than comammox when ammonium was used as the substrate. AOBs had the highest maximum specific growth rate or activity, followed by comammoxs, NOBs, and AOAs. The growth or activity of nitrifiers was inhibited by ammonium for AOAs, comammoxs, and AOBs sequentially, and NOBs appeared to tolerate higher nitrite than comammoxs. It is also noted that nitrite produced by N. inopinata (comammox) may be converted in the periplasm (Daims et al., 2015), and nitrite may leak out of comammox cells for NOB growth, as observed in this study, which may relieve its ammonium/nitrite inhibition and entail NOB abundance, which were higher in N4 at high ammonium concentrations (Figure 5B). Also, we used the aforementioned kinetics parameters (e.g., ammonia affinity, maximum specific growth rate, and ammonium oxidation rate) to construct the niche differentiation model of nitrifiers (Figure 6A), predicting that comammox might be dominant at low and moderate ammonium concentrations, and AOBs at high ammonium concentrations, which was different from our hypothesis. However, the chosen temperature was not optimal for AOAs, and the tested ammonium concentrations appeared not to be sufficiently low, preventing us from observing the predominance of AOAs at the lowest ammonium concentration tested in this study (Figure 6B). Our results showed that NOBs would utilize nitrite leaked by comammox when ammonium was high (e.g., 2 mM). The niche differentiation of comammoxs and NOBs for nitrite oxidation with ammonium as substrate was predicted (Figure 6C), and NOBs would also take leaked nitrite from comammox for growth, indicating their complex interactions. These results indicated that ammonia affinity, substrate inhibition, and specific growth rates could drive niche differentiation and potential interactions among metabolically distinct nitrifiers in response to ammonium concentrations in the environment.

In summary, we constructed SNCs with four types of nitrifiers to understand niche differentiation in response to ammonium concentrations and found that niche differentiation was driven by ammonia affinity, specific growth rate, and ammonium/nitrite tolerance under a gradient of ammonium concentrations among those nitrifiers. In the N4 community, the dominant species was shifted from comammoxs to AOBs as ammonium concentrations increased. Comammox predominated at low ammonium concentrations due to its high ammonia affinity and high maximum specific growth rates. AOB predominated at high ammonium concentrations due to its high maximum specific growth rate, high ammonium oxidation rate and no obvious inhibition by ammonium. AOA had constantly low growth rates due to its weak competitiveness with comammox (0.2 to 1 mM) and AOB (2 mM), and inhibition by high ammonium concentrations (≥10 mM). Niche differentiation of NOBs could be compromised by comammox at 0.2 to 2 mM ammonium and inhibited by high ammonium/nitrite concentrations. Although the mechanism of nitrifier interactions and trade-offs between ammonium concentration and abundance detection sensitivity needs to be further explored, this study advances our understanding of niche differentiation of nitrifiers in response to ammonium concentrations in the environment, and it has important implications for exploring interactions among metabolically distinct nitrifiers using the established SNCs and multi-omics technologies.

The original data presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

XYa, BW, and ZH conceptualized the research. XYa performed experiments and analyzed data with contributions from XYu, XG, and TD. XYa, QH, and ZH led the writing with contributions from all the other authors. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (31770539, 91951207) and by the Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) (SML2020SP004, 311021006).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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