Substrate competition contributes to the niche differentiation between ammonia-oxidizing bacteria and archaea in ammonium-rich alkaline soils

Background The niche differentiation (AOA) has been believed to primarily stem from for ammonia, such that the latter group dominates in ammonium-rich neutral-alkaline soils. However, recent surveys indicate that direct competition between them might be of importance in driving this phenomenon. As such, diverse AOA lineages might grow in ammonium-rich alkaline arable soils if AOB growth was suppressed. To test this hypothesis, a microcosm study using three alkaline arable soils differing substantially in biogeochemical properties was established in which high ammonium concentration (200 μg N g -1 dry soil) was maintained by routinely replenishing urea, and activities of AOB were selectively inhibited by 1-octyne or 3, 4-dimethylpyrazole phosphate (DMPP). The dynamics of AOA and AOB population were monitored throughout the incubation. Results Our results suggested that amendment with either 1-octyne or DMPP retarded the growth of AOB to varying extents, but AOA proliferation accelerated in both treatments; this fitted well with the magnitude to which AOB growth was inhibited. Unexpectedly, nearly identical AOA OTUs were enriched by high-ammonium amendment across the soils tested, and they were taxonomically related to Nitrososphaera sp. JG1, N. viennensis EN76, and N. garnensis . These phylotypes have been previously demonstrated to be sensitive to high ammonia concentrations adopted here. Besides, the changed OTUs in the same lineage of AOA showed a similar response pattern to a given treatment across soils. Conclusions Our results demonstrate that some AOA phylotypes occupy a wider niche than previously believed, and it is the competitiveness for ammonia rather than disparities in substrate affinity and tolerance threshold for ammonia that drives niche differentiation between these AOA and AOB in ammonium-rich alkaline soils. These results further expand our understanding of microbial nitrogen cycling in the terrestrial environment. AOB, we demonstrate that taxonomically constrained AOA, which had been believed to be sensitive to high ammonia concentration, prevail in copiotrophic alkaline soil conditions. These findings strongly support the notion that it is direct competition for the substrate that plays a key role in driving niche differentiation between these AOA phylotypes and AOB in ammonium-rich alkaline soils. Furthermore, our results revealed the coherence of ecophysiology within the same phylogenetic lineage of Nitrososphaerales.

the biogeochemistry of nitrogen cycling in aquatic and terrestrial environments [1,2], since ammonia oxidization, the key component and rate-limiting step of nitrogen cycling, had been thought to be primarily conducted by certain lineages of Beta-and Gamma-proteobacteria for more than a century [3]. In many ecosystems, AOA are numerically more abundant than AOB; this leads to the speculation that AOA assume a functionally-significant role in nitrification and, consequently, regulation of the ecosystem N-cycling [1,2].
In soils, AOB typically prevail in ammonium-rich conditions while AOA dominate environments with low NH 4 + concentrations [4][5][6]. These niche-associated differences, linking to AOA and AOB numerical and functional dominance, have been frequently invoked to explain the variation in nitrification activity among soil ecosystems [4][5][6], and demonstrated to have a profound mechanistic basis, as cultivated AOA and AOB show substantial disparities in physiological traits [4,7,8]. Importantly, most cultivated AOA have a higher affinity for ammonia/ammonium (i.e. K m value) than AOB [4][5][6]8], and this has been proposed as a key mechanism by which AOA dominate nitrification activity in oligotrophic environments including acidic soils where the availability of substrate, ammonia, is exponentially decreased due to the ionization [5,9]. On the other hand, their higher sensitivity to ammonia/ammonium has been believed to restrict growth under high ammonium/ammonia concentrations [4,5,10], such that AOB dominate nitrification in ammonium-rich neutral-alkaline soils [10][11][12].
While these physiological disparities between AOA and AOB provide compelling explanations for their varying activities in different environments, these prevailing paradigms have recently been questioned. Emerging physiological analyses failed to support that there are meaningful differences in ammonia affinity between several typical AOA and AOB strains [13,14]. And, isolates of the Nitrosocosmicus lineage that grow in ammonia concentration as high as 1.49 mM, similar to that of copiotrophic AOB, have been documented [15][16][17]. Therefore, it is likely that some, at least a few, AOA phylotypes potentially adapt to growth in ammonia-rich neutral-alkaline soils. This notion is supported by studies reporting that multiple phylotypes of Nitrosopumilales and Nitrososphaerales grew moderately in soils that were continually amended with a high amount of ammonium [10,18], and further strengthened by recent researches reporting that AOA growth was accelerated under ammonium-rich conditions when the growth of AOB was selectively repressed by inhibitors, 1-octyne or DMPP [19,20]. These studies point out that niche differentiation between AOA and AOB might stem not only from their differences in ammonia oxidization dynamics but also from direct inter-group competition for the substrate [19]. As such, it is reasonable to hypothesize that release from competition with AOB may support the growth of AOA in ammonium-rich alkaline soils; and if present, this phenomenon may not be soil specific or restricted to a narrow range of AOA lineages.
Therefore, the main objective of this study was to explore the possibility that diverse AOA lineages could thrive in high ammonium alkaline arable soils (7.7 < pH < 8.4). To achieve this, a microcosm experiment using three contrasting soils was established, in which a high concentration of NH 4 + (i.e. 200 µg g − 1 N dry weight soil) was maintained by routinely amending with urea and the growth of AOB was selectively inhibited by 1-octyne [21]. In comparison, DMPP was further selected to retard the growth of AOB based on earlier findings [20,22]. Taking advantage of multiple molecular ecological tools, we focused on testing the hypothesis that diverse AOA lineages, in particular those of Nitrosocosmicus lineage, would prevail in ammonium-rich alkaline arable soils if the growth of AOB was suppressed.

Results
The changes in ammonium, nitrate, and pH The dynamics of soil ammonium and nitrate concentration, as well as pH, were monitored over the incubation. Around 30-50% of added urea was hydrolyzed to ammonium within the 2 hours stabilization period in urea-amended treatments (Fig. 1), and complete hydrolysis was achieved within 12 hours for all three soils tested (data not shown). The NH 4 + concentrations of no amendment control (CK) approached the analytical detection limit throughout the whole incubation period for all soils, and a detectable buildup of NO 3 concentration in this treatment was only observed in soil from Shihezi (one-way ANOVA; P < 0.001; Fig. 1E). The NH 4 + concentrations in the C 2 H 2 treatments exhibited minor temporal variation, but no significant accumulation of NO 3 was observed at the end of incubation across all soils (Tukey HSD test; P > 0.05). In contrast, NH 4 + in the urea-only treatment (Urea) was rapidly nitrified into NO 3  and replenished urea completely converted within 72 h after day 12, leading to a decline of 0.6, 1.0 and 0.5 units in pH for the Luoyang, Shihezi, and Tianshui soils, respectively, relative to their value of CK at the end of incubation (Fig. S1). These findings indicated the occurrence of strong autotrophic nitrification activity for all soils tested. Amendment with DMPP strongly retarded oxidation of NH 4 + to NO 3 - (Fig. 1). Its effect was higher in soils from Shihezi (Fig. 1B) and Tianshui (Fig. 1C), resulting in relatively consistent nitrification rates and stable NH 4 + concentrations during incubation, such that a decline of smaller magnitude in pH was observed in this treatment of Luoyang (0.31, P < 0.001) and Shihezi soils (0.22, P < 0.001), but a minor increase (0.07 relative to CK) was observed in the Tianshui soil at day 27 (P < 0.02) (Fig. S1). In comparison, 1-Octyne treatment was less effective at inhibiting nitrification, particularly in the Luoyang soil ( Fig. 1D). In these treatments with 1-octyne, the pH dropped by 0.51, 0.95 and 0.27 units for Luoyang, Shihezi, and Tianshui soils, respectively (Fig. S1).
AOA and AOB amoA gene abundances Across all soils tested, AOA growth was inhibited in the C 2 H 2 -treated microcosms (i.e. C 2 H 2 treatment), but strongly promoted in both DMPP and 1-Octyne treatments ( Fig. 2A-C). The highest AOA growth rates were generally found in the DMPP-treated soils; the exception was the Tianshui soil in which similar AOA growth was observed in DMPP and 1-Octyne treatments after day 15 (Tukey post hoc test, P > 0.05), while the growth of AOA was moderately stimulated in the Urea treatment in Luoyang and Tianshui soils (one-way ANOVA, P < 0.001).
For all soils, the Urea treatment led to the fastest increase in AOB amoA gene numbers, but the addition of C 2 H 2 and DMPP with urea completely suppressed AOB growth in soils ( Fig. 2D-F). In comparison, the inhibitory effect of 1-Ocytne treatment was moderate, especially in the Shihezi soil ( Fig. 2E). The varying degrees of AOA and AOB growth led to different ratios of AOA to AOB among treatments, showing a trend of increase following the order of Urea, 1-Octyne, and DMPP across soils tested at the end of incubation, and this index was significantly higher in DMPP treated soils than in the rest treatments (Fig. S2).

Temporal changes in community structures of AOA and AOB
The temporal shift in AOA and AOB community structures was explored by terminal restriction fragment length polymorphism (T-RFLP) in combination with principal component analysis (PCA) ordination (Fig. S3, S4). The major two principal axes accounted for > 80% of the overall separation among samples for all soils (Fig. S4). Permutational analysis of variance (PERMANOVA) tests revealed significant effects of sampling time and treatment on both guilds across soils (Table S1). The

Phylogenetic composition of AOA and AOB
The phylogenetic composition of AOA and AOB ammonia oxidizers were analyzed by high-throughput sequencing of amoA genes from samples collected on day 27. Rarefaction curves indicated that the sequencing depth was sufficient to capture the vast majority of the total diversity present (Fig. S5). For AOB taxa, only two OTUs clustered within the Nitrosomonas family (Shihezi soil specific), the remaining being Nitrosospira (Fig. S6). The dominant lineages of AOB were similar among soils and primarily composed of taxa from cluster 3. Cluster 3a.1 accounted for > 60% of reads across soils, followed by cluster 3a.2 in Luoyang (25% relative abundance) and Tianshui soils (22%), and cluster 3b (18%) in soil from Shihezi.

Treatment effects on AOA and AOB amoA genotypes
The effects of treatment on compositional changes in AOA and AOB amoA OTUs were analyzed by non-metric multidimensional scaling (NMDS) (Fig. 4) and further visualized with heatmaps ( Fig. 5, S7).
A similar resolution for β-diversity analysis of treatment effect was observed between the NMDS ordination and the PCA analysis of T-RFLP data (Table S1; Fig. 4), and heatmaps revealed strong differences in compositions of amoA OTUs among the soils tested.
For AOA, the relative abundances of 28, 37, and 18 OTUs significantly differed among treatments for the Luoyang, Shihezi, and Tianshui soils, respectively (Kruskal-Wallis rank test, P < 0.05; Fig. S8A), but none of them were in the ε-2.2, γ-2.2.3, or Nitrosocosmicus lineages (Fig. 3, 4). Among these, seven OTUs were shared by all soils, each of which showed a similar response pattern to given treatment lineages, which are related to Nitrososphaera viennensis EN76 and Nitrososphaera sp. JG1, as well as Nitrosophaera gargensis strains with high bootstrap confidence (Fig. 3). However, their close relatives in α-3.2.3 lineage declined in both Urea and C 2 H 2 treatments compared with other treatments, implying diversification of physiology within phylogenetically associated lineages. In contrast, changed OTUs in β-1, δ-1, γ-1.1, and γ-2.1 lineages were generally reduced by 1-Octyne and DMPP treatments; interestingly, those of γ lineages mostly showed a trend of increase in C 2 H 2 treatment (Fig. 5).
For AOB, the relative abundances of 17 OTUs for Luoyang, 18 OTUs for Shihezi, and 19 OTUs for Tianshui were significantly changed by treatments (Kruskal-Wallis rank test, P < 0.05; Fig. S8B). All of them were assigned to Nitrosospira clusters 2 or 3 ( Fig. 4; S7). Seven of these treatment-responsive OTUs were shared across all soils, with six associated with Nitrosospira cluster 3a.1 and one to cluster 3a.2 ( Fig. S6B; S7). The relative abundances of OTUs in cluster 3b were generally reduced by either Octyne treatment across the soils, while OTUs 54 and 80 were inhibited by 1-octyne (Fig. S7).

Discussion
Underlying ammonia oxidization dynamics in cultivated strains of AOA and AOB, alongside biogeochemical data from field and microcosm studies, has established an understanding that AOA are favored under oligotrophic conditions whereas AOB thrive in moderate acid-alkaline soils with elevated concentrations of NH 4 + [4][5][6]. Extended from this, it has been proposed that the relative differences in abundance and functional role of AOB vs. AOA in various ecosystems are primarily driven by their disparities in metabolic traits including substrate affinity and tolerance threshold for ammonia, as well as preference for a supply rate of ammonia [4-6, 19, 23]. Here, by continually supplying urea and selectively inhibiting AOB, we demonstrate that taxonomically constrained AOA, which had been believed to be sensitive to high ammonia concentration, prevail in copiotrophic alkaline soil conditions. These findings strongly support the notion that it is direct competition for the substrate that plays a key role in driving niche differentiation between these AOA phylotypes and AOB in ammonium-rich alkaline soils. Furthermore, our results revealed the coherence of ecophysiology within the same phylogenetic lineage of Nitrososphaerales.
Ammonia rather than ammonium serves as the direct substrate for autotrophic ammonia oxidizers; differences in substrate affinity and tolerance threshold for ammonia have been proposed as overarching mechanisms driving niche differentiation between AOA and AOB [4][5][6]. Indeed, the majority of cultivated AOA own a much higher substrate affinity for ammonia than their bacterial counterpart, which has been postulated to provide them with advantages under oligotrophic conditions [5,6]. This appeared to be evident in the untreated Shihezi soil where no AOB growth was observed but AOA populations were sustained by scavenging ammonia released during organic matter mineralization [24]. However, it would be of little relevance in treatments such as 1-Octyne, as the soil NH 4 + was maintained at a high concentration in the first 15 days, during which AOA and AOB both grew rapidly and simultaneously. In fact, the NH 4 + -N concentrations fluctuated between 20 and 200 µg g -1 soil during this period, in particular for Shihezi and Tianshui soils in which the lowest NH 4 + -N concentrations were found to be as high as 117 µg g -1 soil. According to the ionization equilibrium  [19,20]. Therefore, our results provide strong evidence that low AOA growth in ammonium-rich alkaline arable soils extends from their poor competitiveness with AOB rather than disparities in physiological characteristics such as substrate affinity and tolerance threshold for ammonia, as well as preference for a supply rate of ammonia.
Besides, when directly assessed in environmental studies including those using stable isotopic probing analysis, phylotypes within these lineages grew to a moderate extent in microcosms that were regularly spiked with urea at a concentration of 100 µg NH 4 + -N g -1 soil [12,22,25,26]. Thus, it appears that AOA affiliating to Nitrososphaerales-α-3.2.1 and α-3.2.4 lineages adapt to growth in ammonium-rich alkaline soils. Notwithstanding, their rapid growth under such high ammonium concentrations adopted here (i.e. 200 µg NH 4 + -N g -1 soil) was still unexpected, as the reported inhibitory concentrations of ammonia for these taxa are much lower than the theoretical values in 1-Octyne, especially for DMPP treatments (the highest value of 356 µM ammonia for EN76 vs. lowest concentration of 2 375 µM in DMPP treatments) [27,28]. Thus, it is likely that these phylotypes occupy a much broader niche than previously described, and they are highly susceptible to the competition from their bacterial counterpart.
On the other hand, the promotion of growth of Nitrososphaerales-α-3.

and α-3.2.4 lineages in 1-
Octyne treatment might be associated with mixotrophic activities, since their representative strains are equipped with genes encoding transporters for organics [29,32,33], and their growth could be enhanced by the addition of low-molecular-weight organic compound [27,34]. To exclude this possibility, DMPP, a nitrification inhibitor with distinctive heterocyclic chemical structure, was additionally adopted to inhibit the growth of AOB based on earlier findings [20,22]. Our results suggested that the amendment of soils with 1-octyne or DMPP led to the enrichment of the same AOA phylotypes both within and across each soil tested, thus proving that the acceleration of AOA growth in 1-Octyne and DMPP treatments was indeed attributed to their chemolithotrophy. However, unexpectedly, 1-octyne was far less effective at reducing the growth of AOB than previously reported [19,21]. Some AOB OTUs increased in the 1-Octyne treatment, particularly those related to Nitrosospira multiformis whose growth had been previously demonstrated to be abolished by a lower concentration of 1-octyne [21]. It is less likely that this stems from restricted diffusion or leakage of 1octyne in the experimental systems, given that low concentrations of C 2 H 2 completely abolished the growth of both AOA and AOB. Regardless of mechanisms, the lower inhibition efficiency of 1-octyne did not compromise our conclusion. Rather, the inhibition gradient created by Urea, 1-Octyne and DMPP treatments provided a unique opportunity to disentangle the effect of interaction between AOA and AOB. Hereby, we were able to show that the growth of AOA was closely coupled with the magnitude to which the growth of AOB was inhibited, and DMPP is a more effective and specific inhibitor against AOB than 1-octyne in the soils examined (see supplemental discussion).
Besides, the experimental design adopted here allowed us to explore the potential ecological coherence of major phylogenetic lineages. Indeed, we found that, in addition to those enriched by the high-ammonium amendment, the majority of changed OTUs within the same lineage showed a similar response pattern to a given treatment across soils. For instance, our results confirmed that the neutral-alkalinophilic Nitrososphaerales-δ lineage dominated the AOA assemblage in all soils tested [30], but most of OTUs therein were insensitive to amendment with urea or inhibitors across soils.
Currently, no cultured representative has been identified in this lineage, therefore its ecological function in the soil is still unknown [18]. In contrast, changed OTUs in β-1, γ-1.1, and γ-2.1 lineages were generally reduced by 1-Octyne and DMPP treatments. In particular, those of γ-1.1 and γ-2.1 lineages were exceptionally enriched in C 2 H 2 treatment of all soils tested, implying a mixotrophic lifestyle of these lineages. Similarly, one earlier report documented that the addition of 10 Pa C 2 H 2 failed to inhibit the growth of AOA in an alkaline arable soil [35]. Intriguingly, despite being closely related to α-3.2.4, OTUs of Nitrososphaeralesα-3.2.3 lineage were inhibited by Urea but not by DMPP and 1-Octyne treatment, this concurred with the finding that phylogenetically closely related AOA could show contrasting metabolic traits [36]. Overall, our results confirm that AOA are functionally heterogeneous [36], but the coherence of response to treatments indicates similar ecophysiology between phylotypes in the same AOA lineages [30].
Finally, our results reaffirmed that AOB of Nitrosospira cluster 3a dominated the nitrification activity in the ammonium-rich alkaline soils (see supplemental discussion) [10-12, 35, 37], whereas the growth of AOA in Urea and 1-Octyne treatments implies that they assume a considerable role in contributing to overall nitrification in these soils as well. Although our experimental design did not allow for an absolute partitioning of their relative contribution, the nearly linear buildup of NO 3 and occurrence of only AOA growth in DMPP treatment across the soils enabled estimates to be calculated. Assuming that each AOA strain contains two amoA gene copies [32] and all AOA growth is due to autotrophic nitrification activity, the specific AOA nitrification rates estimated ranged from 1.06 to 2.48 fmol cell -1 Consequently, it is estimated that AOA accounted for 2%, 9% and 10% nitrification activities in Urea treatments of the Shihezi, Tianshui, and Luoyang soils, respectively. These matched the estimates reported in ref. [12] (1.51%-23.4%) and were marginally lower than measurements in ref.

Conclusions
Our results revealed that release from the competition by bacterial ammonia oxidizers allowed AOA to thrive under ammonium-rich alkaline arable soils. Phylotypes that were particularly supported by high ammonium were from a narrow taxonomical group closely related to Nitrososphaera sp. JG1, N. viennensis EN76, and N. garnensis; these isolated strains have been previously demonstrated to be sensitive to high ammonia concentration adopted here. Moreover, 1-Octyne and DMPP treatment of urea-enriched soils supported identical AOA lineages, highlighting that growth acceleration of these AOA under high ammonia conditions was attributed to chemolithotrophy. Together, these findings strongly support our hypothesis that it is direct competition for the substrate that drives the niche differentiation between these AOA phylotypes and AOB in ammonium-rich alkaline soils, thereby providing an alternative explanation for this phenomenon. Overall, this research strongly demonstrates that AOA plays an important role in nitrification in ammonium-rich alkaline arable soils, further expanding our understanding of microbial nitrification and N cycling.

Material And Methods Soil sampling and determination of soil properties
Three alkaline arable soils (i.e. fluvo-aquic soil, grey desert soil, and a loessial soil), all previously characterized as being AOB-dominated with regard to nitrification activity [12,40,41] were used in this study. The fluvo-aquic soil was collected from Henan province, the grey desert soil from Xinjiang Uyghur autonomous region, and the loessial soil from the Zhongliang long-term fertilization experiment station, Gansu province (see Table 1 for georeferencing). These sites, spanning a wide geographic range, differed greatly in meteorological and physio-chemical properties, as well as management practices (Table 1). Bulk soil (0-20 cm) was collected at each site, sieved through a 2 mm mesh, and stored at 4 °C. The detailed sampling design and determination of basic soil properties were presented in supplemental materials. Table 1 The meteorological parameters of sampling sites and basic physicochemical properties of soils Microcosm experiment The 27-day microcosm incubation experiment with five treatments for each of the three soil types was established. These treatments included no fertilizer control ('CK'), the addition of urea ('Urea'), the addition of urea with 10 Pa C 2 H 2 ('C 2 H 2 '), the addition of urea with 1-octyne (C aq =4 µM; '1-Octyne'), and addition of urea with DMPP (0.15% of added urea N; 'DMPP'). The C 2 H 2 treatment was used to inhibit all autotrophic nitrification activity, while 1-octyne and DMPP are considered to have specificity to inhibition of AOB growth [20,21]. All soils were pre-incubated at 25 Table S1 and raw data analysis procedures have been reported previously [41].

Statistical analysis
The phylogenetic analysis of AOA and AOB was conducted in MEGA 6.0 [42] and visualized using iTOL (http://itol.embl.de) [43]. Statistical analysis was conducted in R. 3.6.1 [44]. One-way ANOVA was used to assess the temporal changes in pH, NH 4 + , and NO 3 concentrations, as well as the abundances of AOA and AOB among soil types. Variation in treatment means was assessed using Tukey's post hoc test. Differences in relative abundances of OTUs among treatments were assessed with the Kruskal-Wallis rank test at α = 0.05. Variation in the presence of amoA OTUs (AOA and AOB) in soils was conducted in the 'vegan' package [45]. PCA was used to explore the temporal shift in the occurrence of AOA and AOB genes across treatments and soils. PERMANOVA was used to test the effect size and significance of treatments, and these were visually interpreted using NMDS.  OTUs that were significantly different among treatments within soil were labeled with the red asterisk.  The composition of AOA OUTs that were significantly different among treatments within soils. The unrooted phylogenetic tree was shown on the left of the heatmap. The cluster analysis result of samples based on the Bray-Curtis dissimilarity matrix was shown above the heatmap. For clarity, only OTUs with relative abundance higher than 0.1% across all samples were shown.

Supplementary Files
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