Diversity and characterization of the ammonia-oxidizing bacteria responsible for nitrification in tea field soils

Aoyagi, Luciano N.; Wang, Yong; Ohbayashi, Tsubasa; Hirono, Yuhei; Hayatsu, Masahito; Tago, Kanako

doi:10.1038/s41598-025-18596-5

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Published: 26 September 2025

Diversity and characterization of the ammonia-oxidizing bacteria responsible for nitrification in tea field soils

Luciano N. Aoyagi^1,2,
Yong Wang¹^nAff4,
Tsubasa Ohbayashi¹,
Yuhei Hirono³,
Masahito Hayatsu¹ &
…
Kanako Tago^1,2

Scientific Reports volume 15, Article number: 33221 (2025) Cite this article

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Abstract

Ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and comammox Nitrospira have been considered to coexist in acidic soils, oxidizing ammonia within their respective niches. However, their relative contributions to soil nitrification, as well as their properties and ecological roles in acidic soils, remain poorly understood. This study focused on AOB in acidic tea field soils, investigating their ecology and physiology through metagenomic analysis and the genomic and physiological characterization of AOB isolates. β-AOB were significantly more abundant than γ-AOB and AOA in soil layers with the highest potential for nitrification activity, indicating that β-AOB play a key role in acidic tea soil. Diversity analysis of this dominant group identified Nitrosospira as the major genus present in tea fields, and four pure strains representing some of the main operational taxonomic units in this environment were isolated. Physiological and genetic characterization of these isolates revealed some distinct traits compared to other species of the genus and closely related taxa, suggesting adaptations that may contribute to niche differentiation and survival in acidic soils. These findings provide new insights into the ecological role of β-AOB in acidic soils and may inform strategies to manage soil nitrification and reduce nitrogen loss in agricultural ecosystems.

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Introduction

Nitrification, the oxidation of ammonia to nitrate via nitrite mediated by microorganisms, is a crucial step in the nitrogen cycle and plant nutrition. However, it also leads to fertilizer loss through nitrate leaching and contributes to greenhouse gas (GHG) emissions, particularly nitrous oxide, into the environment¹. Ammonia oxidation, the first and rate-limiting step of nitrification, is carried out by ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (β-AOB and γ-AOB), and the recently discovered complete ammonia-oxidizing bacteria (comammox Nitrospira)². The diversity, population, and distribution of these ammonia oxidizers are influenced by various environmental factors, with pH being particularly significant. The highest nitrification rates are typically observed in acidic soils (pH < 5.0), which account for approximately 30% of the total land area³.

The discovery of AOA and their high abundance in acidic soils led to the assumption that AOA play an important role in nitrification. However, recent studies have expanded the understanding of AOA and AOB abundance and distribution, showing that AOB, despite being less abundant in some cases, are significantly correlated with nitrification in acidic forest soils⁴, terrace paddy soils⁵, tea plantations⁶, and upland soils⁷. These findings suggest that β-AOB may play a more substantial role in nitrification than previously thought. Conversely, recent reports indicate that comammox bacteria may contribute more significantly to nitrification in acidic soil⁸. Therefore, the relative contributions of AOA, β-AOB, and comammox to nitrification in acid soils remain unclear.

Tea cultivation requires large amounts of nitrogen fertilizer to maintain quality and yield. For instance, in Japan and China, the recommended application rate is 450–540 kg N ha⁻¹ yr⁻¹⁹. Tea field soils become strongly acidified owing to protons released during ammonium uptake by tea roots and the nitrification process. Many tea plantations in China¹⁰ and Japan¹¹ have soil pH values below 4.5, while in India, typically below 5.0¹². Due to heavy fertilization and extreme acidity, tea field soils exhibit high nitrification activity and N₂O production¹³, prompting research into nitrification and ammonia-oxidizing microorganisms in tea field soils^6,14,15.

The TAO100 strain, the first soil-originated γ-AOB capable of surviving in extremely acidic conditions (pH 2), was isolated from tea field soil in Japan¹⁴. Subsequently, comammox Nitrospira, presumably adapted to acidic environments, was enriched from the same tea field. Characterization revealed that these two nitrifiers may occupy distinct niches due to physiological differences¹⁵. Although there have been a few publications on the isolation and characterization of pure-cultured β-AOB from soil^16,17, information on isolates from acidic environments remains limited^14,18. Studies on AOB in acidic soils have focused on metagenomics to determine abundance and diversity^19,20, genomics^21,22 or isolation and physiological characterization²³. However, few studies have integrated these approaches to evaluate their contribution to soil nitrification^14,24 and explore their relationship with other nitrifiers.

This study aimed to analyze the diversity and abundance of nitrifying bacteria and to characterize AOB isolates from an acidic tea field to determine their role in nitrification in this environment. We assessed the distribution and diversity of nitrifiers based on the ammonia monooxygenase subunit A (amoA) gene and examined the correlation between their abundance and soil properties. Physiological analysis of the β-AOB isolates enabled a comparison of their characteristics with the other nitrifiers from the same tea field. Finally, genomic characterization was conducted to investigate potential adaptations for inhabiting acidic tea fields.

Results

Distribution, abundance, and nitrification activity of β-AOB in tea fields

The properties of three different tea fields, each with six soil layers, were analyzed (Fig. 1). All soil samples had an acidic pH (2.83–5.50) (Supplementary Table S1). The potential for nitrification activity (PNA) was significantly higher (P < 0.05) in the surface layers (organic layer and 0–2 cm) of the tea soil (Fig. 1a). The pH of the organic, 0–2 cm, and 2–4 cm layers was significantly less acidic than that of the 4–10 cm soil (Fig. 1b). The surface layers also had significantly higher nitrate concentrations, total C (%) and total N (%) than the other layers (Fig. 1c, e, and f). Ammonia concentration (Fig. 1d) did not show significant differences across soil layers.

The abundance of the β-AOB amoA gene ranged from 8.18 × 10³–5.81 × 10⁸ copies g⁻¹ dry soil, with the highest values found in the surface layers (Fig. 2), which also had the highest PNA (Fig. 1a). The γ-AOB amoA gene was less abundant than β-AOB amoA and AOA amoA, ranging from 1.30 × 10³–1.32 × 10⁷ copies g⁻¹ dry soil. AOA amoA was also less abundant than β-AOB amoA (Fig. 2a–c), with values ranging from 2.69 × 10³–1.47 × 10⁷ copies g⁻¹ dry soil. β-AOB amoA dominated over AOA amoA in the organic (Fig. 2) and 0–2 cm (Fig. 2b, c) layers, where the AOA/β-AOB ratio was the lowest among all layers (Supplementary Fig. S1). In fact, β-AOB was the only group that showed significant differences in amoA genes distribution across soil layers (Fig. 2).

Pearson’s correlation coefficient (r) between ammonia-oxidizers abundance and soil physicochemical properties is shown in Table 1. Ammonia-oxidizers abundance was positively correlated with soil pH, nitrate concentration, total C, and total N, but not with ammonium concentration. PNA was positively correlated with both AOB groups, more strongly with β-AOB (r = 0.81, P < 0.01) than with γ-AOB (r = 0.55, P < 0.01), but not with AOA (Table 1). This suggests that AOB, particularly β-AOB, may play a more significant role in nitrification in this environment (Table 1). For this reason, the focus of this study was to isolate and characterize β-AOB from the surface soils of tea fields.

Table 1 Pearson’s correlation (r) between AmoA gene abundance and soil physicochemical properties.

Full size table

Isolation and taxonomic characterization of β-AOB from tea field soils

At three tested pH levels (pH 5, 6, and 7), several AOB enrichment cultures were obtained from β-AOB medium at pH 7.0. These cultures were tested for purity using fluorescence in situ hybridization (FISH) with fluorescent probes specific for the 16S rRNA gene of β-AOB²⁵ (Supplementary Fig. S2). The final cultures of four isolates, designated TNE100, TNL100, TNL200, and TNL300, were confirmed to contain only a single cell type. TNE100 has a short rod shape with a length of 1.0 μm, whereas TNL100 and TNL300 consisted of spherical cells, each 1.0 μm in diameter. TNL200 has elongated, compartmentalized cells, 0.8 μm in length (Supplementary Fig. S3). Cell aggregation in TNL100, TNL200, and TNL300 was occasionally observed under stress conditions (below pH 6.0 and during ammonia depletion), while it was rare in TNE100 strain (Supplementary Fig. S4).

Diversity of β-AOB in tea field soils based on AmoA amplicon analysis

The diversity of β-AOB in tea fields was analyzed by sequencing amoA amplicons (Supplementary Fig. S5). A total of 206,406 β-AOB amoA sequences were clustered into 20 operational taxonomic units (OTUs). The AOB community in tea soils is dominated by the genus Nitrosospira, which accounted for 99.99% of all sequences and compose 19 out of the 20 OTUs, both in the organic and in the 0–2 cm layers (Supplementary Fig. S6).

A phylogenetic tree constructed based on β-AOB-amoA sequences showed that TNL100 and TNL300 clustered in OTU2, while TNL200 clustered in OTU5 (Supplementary Fig. S6). OTU2 and OTU5 accounted for 19.16% and 6.74% of all sequenced amoA genes, respectively (Supplementary Fig. S5), suggesting that the Nitrosospira isolates belong to one of the major AOB groups in the tea field. TNE100 grouped within OTU15, which was composed of Nitrosomonas spp. (Supplementary Fig. S6), accounting for 0.01% of all amoA sequences. While less abundant than Nitrosospira spp. in the tea field, its successful isolation indicates that it can survive in this environment and warrants its inclusion in subsequent analyses.

Effects of pH and ammonium concentration on growth

The effect of pH on the growth of the four strains is shown in Fig. 3. All strains were neutrophilic and exhibited poor growth at pH 6. TNE100 grew within the pH range 6.5–8.5, with optimal growth at pH 8.0. Below pH 6.5, growth was significantly slow (Fig. 3a). At the optimal pH (8.0), the specific growth rate (µ) was 0.48 ± 0.0017 d⁻¹ (Fig. 3b).

The TNL100, TNL200, and TNL300 isolates grew between pH 6.0 and 8.0 (Fig. 3c, e, and g, respectively), with optimal growth at pH 7.5 (Fig. 3d, f, and h, respectively). At their optimal pH, the µ values were similar for TNL100 (0.31 ± 0.01 d⁻¹) and TNL300 (0.34 ± 0.01 d⁻¹), whereas TNL200 exhibited slower growth (0.20 ± 0.02 d⁻¹) (Fig. 3d, h, and f, respectively).

The effect of ammonia concentration on growth at pH 7.5 was also examined (Fig. 4). Strains TNE100, TNL100, and TNL300 tolerated concentrations up to 100 mM ammonium (Fig. 4a, c, and g), whereas TNL200 tolerated only up to 20 mM, with slower growth above this concentration (Fig. 4e, f). TNE100 exhibited the fastest growth between 20 and 80 mM (Fig. 4b), while TNL100 and TNL300 grew rapidly between 40 and 80 mM (Fig. 4d, h).

Ammonia oxidation kinetics properties

The ammonia oxidation kinetics of the four isolates were investigated by analyzing ammonium-dependent oxygen uptake. The half-saturation constant (K_m) values for ammonium oxidation by TNE100, TNL100, TNL200, and TNL300 were 0.98, 0.85, 0.39, and 0.99 mM (NH₄⁺ + NH₃), respectively (Supplementary Fig. S7).

Genome phylogeny

The genome-based phylogenetic analysis revealed that TNE100 strain clustered with other species of the Nitrosomonas genus, including Nitrosomonas eutropha C91 (Fig. 5a). Average nucleotide identity (ANI) comparison of TNL100 and TNL300 with other Nitrosospira species showed high similarity to Nitrosospira multiformis ATCC25196 (ANI = 90.35%) and N. multiformis Nl12 (ANI = 93.93%) at the genome level. A similar cluster was observed in the amoA-based phylogenetic tree, where TNE100 strain grouped in the cluster 7 with Nitrosomonas europaea and N. eutropha C91, while TNL100 and TNL300 were grouped in cluster 3a, composed exclusively of N. multiformis species (Fig. 5b). The TNL200 strain clustered alongside Nitrosospira sp. 56 − 18 and Nitrosospira sp. Nsp65 (Fig. 5b), and its comparison with Nitrosospira sp. 56 − 18 showed 97.07% identity (Supplementary Table S2). Although TNL100 and TNL300 differed slightly in the number of coding sequences (CDS) and the structure of a few genes, their ANI was nearly 100%, indicating that they belong to the same species. Overall, the four isolates showed both similarities and some differences compared to closely related species (Supplementary Tables S2–S4).

Gene inventory of β-AOB strains

To analyze the metabolic potential of the four isolates and compare them with other AOB genomes, a heatmap was generated based on their protein sequences, representing the metabolic pathway completeness of 46 β-AOB species, as determined by the KEGG Decoder (Supplementary Fig. S8).

Analysis of the AMO gene cluster (amoCABED) showed that, similar to other β-AOB^22,26,27, the four AOB isolates possess a complete amoCABED cluster along with an ammonia transporter gene (Table 2, Supplementary Fig. S9). TNE100 has a similar amoCAB organization to N. eutropha C91 (Table S4). Meanwhile, TNL100, TNL200, and TNL300 have three copies of amoCAB, two of which are complete with the amoDE operon (Supplementary Fig. S9a).

Table 2 Genome characteristics of AOB isolates from acidic tea soil.

Full size table

Hydroxylamine oxidoreductase (HAO) catalyzes the second step of nitrification and contributes to N₂O production by AOB²⁴. Due to their importance in the nitrogen cycle and GHG production, HAO clusters, encoded by the haoAB-cycAB genes, were analyzed. TNE100 has one complete haoAB-cycAB and one additional copy lacking cycB (Supplementary Fig. S9b, Table S5), similar to N. eutropha C91²⁷. TNL100, TNL200, and TNL300 contain three complete haoAB-cycAB copies, as seen in other Nitrosospira spp^22,27. (Supplementary Table S6).

While most members of the N. multiformis cluster possess the nitric oxide reduction pathway, it appears to be absent in TNL100, TNL200, and TNL300. In contrast, TNE100 and most Nitrosomonas strains in the cluster have a complete pathway (Table 2). All four β-AOB isolates contain the genes necessary for N₂O production through hydroxylamine oxidation, but only TNE100 possesses nitrifier denitrification pathways (Supplementary Tables S4–S7, Fig. S10). The absence of the norCBQD cluster may affect the ability of Nitrosospira isolates to prevent the accumulation of nitrite and NO, as well as their capacity to produce N₂O²⁷. However, like TNE100, they may avoid the accumulation of these intermediates via cytochrome c’-beta and sNOR (Supplementary Fig. S10).

Genome analysis revealed that all three Nitrosospira strains have a complete urease operon with three structural genes (ureABC) and four accessory genes (ureD, ureEFG) required for activation^28,29, and a urea transporter gene (Table 2, Supplementary Table S8). Urea availability and urease activity were examined in these isolates using culture-based methods, confirming that all three strains hydrolyze and utilize urea. While the TNE100 strain, despite lacking the ureABCDEFG cluster, exhibited weak urease activity (Supplementary Fig. S11).

Genes related to exopolysaccharide (EPS), lipopolysaccharide (LPS), biofilm biosynthesis, and transport were identified in the genomes of all four isolates (Supplementary Tables S8–S11). A comparison of N. multiformis putative conserved gene clusters for EPS (Nmul_A0238–A0262), LPS and capsule (Nmul_A2530–A2515), and polysaccharide capsule biosynthesis (Nmul_A0264–A0292)²¹ in Nitrosospira spp. provided insights into genome similarities. The EPS and LPS clusters were well conserved in the three Nitrosospira spp. (Fig. 6a, b, Supplementary Table S8). Among the three isolates, the polysaccharide capsule cluster was the least complete and contained the highest number of unique genes (Fig. 6c, Supplementary Table S10).

Discussion

In this study, β-AOB amoA genes were significantly more abundant in the surface layers of the tea field (organic layer and 0–2 cm) (Fig. 2), which are more exposed to N inputs from fertilizers^30,31 and exhibited the highest PNA (Fig. 1a). The soil pH in these layers was also slightly but significantly higher than in deeper layers. The correlation analysis revealed that PNA was significantly associated (P < 0.05) only with β-AOB and γ-AOB amoA abundance (Table 1), demonstrating that, unlike in other acidic soils^32,33,34, AOB were more abundant than AOA in the acidic tea field in Japan and likely served as the primary nitrifiers. AOA were more abundant than AOB in unfertilized environments^30,35,36, while inorganic N inputs decrease AOA amoA and increase AOB amoA gene abundance⁶. Our results indicate that this pattern also applies to tea field soil in Japan, which typically receives high annual N fertilizer inputs³⁰. Additionally, the high polyphenol concentrations in tea may slow organic matter decomposition, reducing mineralization rates³⁷, particularly in the topsoil, where pruned leaves accumulate. Both AOA and AOB populations decrease under polyphenol addition; however, AOA appear more sensitive, as shown by a greater reduction in amoA gene copies and a declining AOA/AOB ratio over time³⁸. This suggests that AOA are likely more impacted, which is consistent with their dominance in soils where NH₃ is mineralized from organic N sources, while AOB tend to thrive in environments rich in inorganic N, such as tea field surface layers.

Factors such as pH¹⁹ and soil depth³⁹ also influence the distribution and abundance of AOA and AOB. In this study, pH was negatively correlated with AOA abundance (r = −0.61, P < 0.01), and the AOA/AOB ratio was lower in surface layers (Fig. 2), which had lower pH values (Fig. 1b). AOB predominated in the topsoil, whereas increasing soil depth resulted in higher AOA/AOB ratios (Fig. 2, Supplementary Fig. S1), consistent with previous findings^39,40,41. The difference in nitrifier distribution with depth may be attributed to greater nutrient availability^40,42, higher oxygen levels^43,44, and lower moisture content^44,45 in the topsoil. AOA may be better adapted to the oligotrophic conditions found in subsoil, as they exhibit a higher affinity for NH₃ and oxygen than AOB⁴⁴. Moreover, N fertilizer inputs enhance AOB abundance, which was positively correlated with PNA in surface layers^{40,41,42,43,44,45} (Table 1). While AOA were more abundant in deeper layers (Supplementary Fig. S1), their increase did not correlate with PNA (Table 1), unlike AOB, aligning with previous reports^40,42,45. The significant correlation between β-AOB and γ-AOB abundance and PNA highlights their crucial role in nutrient-rich soil layers, supporting the hypothesis that N fertilizers favor AOB over AOA⁴³. In tea fields, the intensive application of N fertilizers may be sufficient to shift the community composition in favor of AOB. These findings suggest that AOB are the primary nitrifiers in tea field soil and likely play a major role in nitrification processes in other acidic soils as well. Future studies should investigate the potential contribution of comammox bacteria to nitrification in tea field soils.

The isolation process from the tea soil layers with the highest PNA resulted in three Nitrosospira spp. isolates and one Nitrosomonas sp. isolate (Supplementary Fig. S3). Nitrosospira strains have previously been isolated from acidic environments^23,46, including tea fields⁴⁷. Nitrosospira are known to dominate acidic soils, whereas Nitrosomonas are more common in neutral soils³⁴. The β-AOB community diversity analysis, based on amoA sequencing, revealed that Nitrosospira overwhelmingly dominated the soil (99.99% of all sequences) (Supplementary Fig. S5), similar to findings in other acidic soils^{4,5,19,48,49,50,51,52,53}, while Nitrosomonas sp. represented only a small fraction of the community.

Phylogenetic analysis based on genomes and amoA sequences (Fig. 5) placed TNE100 in cluster 7, which includes Nitrosomonas spp. capable of tolerating high ammonia concentrations^54,55. Meanwhile, TNL100 and TNL300 grouped within cluster 3a, a group that dominates acidic soils¹⁹. Although TNL100 and TNL300 were closely related to the well-known N. multiformis ATCC25196, neither strain exhibited ANI > 95% with other species within the cluster (Supplementary Fig. S8), indicating that both represent new species of Nitrosospira. Since most genomes in cluster 3 are draft genomes, only N. multiformis ATCC25196, TNL100, and TNL300 have been fully sequenced and analyzed.

Our analyses revealed that, despite being isolated from the same tea field, the four β-AOB strains exhibited differences in physiological characteristics. TNE100 did not grow at the lowest pH tested (6.0) (Fig. 3b) but was able to grow within a pH range (pH 6.5–8.5), similar to some Nitrosomonas strains isolated from acidic soils⁵⁶. However, its growth rate was slower than that of previously reported Nitrosomonas spp^57,58. (Fig. 3b). Among Nitrosospira spp., TNL200 exhibited the slowest growth across all tested pH values (Fig. 3f). The TNL100, TNL200, and TNL300 strains grew in the same pH range (pH 6.0–8.0) as Nitrosospira sp. isolated from acid soils⁵⁶. Their growth rates (0.008–0.014 h⁻¹) (Fig. 3) were consistent with those of soil-derived Nitrosospira (0.005–0.013 h⁻¹)³². Although all four β-AOB strains were isolated from acidic soil (pH 3.2–5.5), they exhibited optimal growth at neutral to alkaline pH (Fig. 3), with slower growth observed at pH 6.5 or lower. Similar findings have been reported for Nitrosomonas and Nitrosospira isolated from acidic soils, all of which were acid-sensitive^23,56. However, despite their preference for higher pH (> 6.5), Nitrosospira spp. can still show metabolic activity under acidic conditions²³ and have been shown to proliferate and nitrify at pH as low as ~ 3.0⁵⁹, particularly after adapting to pH fluctuations⁴⁶. The presence of β-AOB amoA in acidic tea fields¹⁴ and the known activity of β-AOB in low pH environments, facilitated by protective cell aggregates^60,61, further support this observation. Furthermore, in this study, only β-AOB abundance was significantly correlated with PNA in the tea field (Table 1), indicating that they are actively contributing to nitrification.

The physiological analysis revealed that the growth rates of TNE100, TNL100, and TNL300 were highest at 40–80 mM ammonium (Fig. 4), corresponding to an estimated free NH₃ concentration of 1413.25 µM at 80 mM. In contrast, TNL200 showed the highest growth at 10–20 mM, with 176.66 µM NH₃ at 10 mM. The optimal NH₃ concentration for all four strains was higher than that for comammox Nitrospira (1.94 µM) and γ-AOB TAO100 (28.42 µM), previously isolated from a Kanaya tea field^14,15. A similar pattern was observed for maximum NH₃ tolerance, with TNE100, TNL100, and TNL300 tolerating up to 1766.57 µM NH₃, while TNL200 tolerated up to 353.31 µM NH₃, both values exceeding those reported for TAO100 (227.34 µM)¹⁴ and comammox Nitrospira (46.50 µM)¹⁵. The high NH₃ tolerance of these four AOB isolates may contribute to their dominance over AOA in this nutrient-rich environment, as AOA are more sensitive to NH₃ inhibition³².

The NH₃ affinity was also higher in these isolates, with K_m values (expressed as free ammonia) estimated at 17.31, 15.02, 6.89, and 17.49 µM for TNE100, TNL100, TNL200, and TNL300, respectively, compared to TAO100 (33.3 µM)¹⁴. The K_m for TNE100 is lower than that observed for N. europaea (23–58 µM NH₃) from soil⁶², while the values for the Nitrosospira strains are lower than those reported for other AOB from soil^63,64. This higher substrate affinity (lower K_m) observed in β-AOB isolates from tea fields is likely crucial for their survival in acidic conditions and may represent an adaptation to such environments, where NH₃ is limited as a result of ionization to ammonium. Furthermore, competition with tea plants and other nitrifiers can further reduce NH₃ availability between fertilizer applications^30,31.

These results suggest that substrate affinity and tolerance are key factors in niche differentiation among nitrifiers in tea fields. The higher NH₃ tolerance of the four β-AOB strains, compared to other nitrifiers from tea fields such as comammox Nitrospira and γ-AOB TAO100, may reflect their adaptation to higher nutrient content and pH in the surface layer from which they were isolated. In contrast, TAO100 and comammox likely occupy niches with moderate NH₃ levels, such as intermediate soil layers, while AOA, being more sensitive to substrate inhibition, may inhabit deeper layers where NH₃ availability is further reduced due to acidity and gradient effects. The distribution of nitrifiers in our study supports these ecological distinctions, with β-AOB and γ-AOB dominating the surface layers (Fig. 2b, c), while AOA are more abundant in the more acidic deeper layers (Fig. 2a).

Lastly, genomic analysis of the four isolates revealed the presence of genes associated with N₂O emission in this agricultural soil, as well as genes conferring protection from acidic conditions, enhancing nutrient acquisition, and other vital survival traits. Notably, the TNL100 and TNL300 strains harbor four copies of the amoC and two complete amoCABED clusters, double that of N. multiformis (Supplementary Fig. S9, Table S3). Experimental evidence indicates that the amoC gene is involved in responses to starvation and stress⁶⁵, potentially giving both strains an advantage in recovering after nutrient limitation between fertilizer applications in the tea field. The potential role of extra amoC/E/D in AOB adaptation to acidic soils warrants further investigation. All three Nitrosospira isolates possess an ammonia transporter gene, unlike TNE100, which lacks amtB similarly to N. eutropha C91 (Table 2). Likewise, TAO100 also lacks an ammonia transporter¹⁴, suggesting that these AOB may rely on passive diffusion for NH₃ uptake.

Gene annotation revealed that TNE100 contains all the necessary components for N₂O production (Supplementary Fig. S10) via both hydroxylamine oxidation (hao and cytS) and nitrifier denitrification (nirK, cytL, cytochrome c₅₅₂, and nor) pathways^66,67,68, a trait shared with N. eutropha C91 (Table 2). Except for norCBQD and cytL, these genes are also present in the TNL100, TNL200, and TNL300 strains (Table 2), similar to N. multiformis²¹. Although Nitrosospira spp. lack norB, the presence of cytochrome c’-beta and sNOR enables them to reduce NO to N₂O^67,68,69. The norSY-norSenC gene is absent in TNL200, where cytochrome c’-beta likely prevents NO accumulation⁶⁷ (Supplementary Fig. S10). Although N₂O production under oxygen-limited conditions has been described in N. europaea^66,67, its functional validation in Nitrosospira remains pending. Moreover, N₂O production by Nitrosospira spp. has been observed⁵³ even in the absence of norB homologs or a complete denitrification pathway^27,70. These ammonia-oxidizers could contribute significantly to N₂O emission in tea fields, which receive high N fertilizer inputs and emit elevated levels of this GHG^13,71.

The genomes of TNL100, TNL200, and TNL300 contain a complete ureABCDEFG cluster as well as urea transporter genes, and we confirmed that these strains exhibit urease activity (Supplementary Fig. S11). In contrast, although TNE100 lacks the ureABCDEFG cluster (Supplementary Fig. S8), it showed weak urease activity (Supplementary Fig. S11), a pattern also observed in N. eutropha C91²⁸. Urease activity has been proposed as a mechanism that enables nitrifying bacteria to survive in acidic environments, as ureolysis locally increases pH and provides NH₃^28,54,60. The identification of the ureABCDEFG cluster in AOB isolates suggests that ureolysis may play an important role in colonizing tea fields and other acidic environments.

Additionally, acid-sensitive isolates may survive by migrating to microsites that offer protection from low pH conditions^72,73. All four isolates in this study possess most of the genes encoding the bacterial flagellum, supporting their potential for motility (Supplementary Fig. S12, Table S14). This motility, combined with adhesion mediated by EPS, is associated with cellular aggregation⁷⁴ and enables acid-sensitive AOB to survive in acidic conditions^60,61. A search for genes related to EPS and LPS biosynthesis, based on sequences from N. multiformis ATCC25196²¹, identified putative genes in the genomes of Nitrosospira isolates, revealing genetic differences among them (Fig. 6). Despite these differences, all three Nitrosospira spp. form aggregates (Supplementary Fig. S4), suggesting that the genes present are sufficient for biosynthesis. Aggregation was rarely observed in TNE100, which harbors only the EPS synthesis gene (ExoD, TNE_09840), a gene that is upregulated in Nitrosomonas mobilis Ms1 cells during aggregation⁷⁴. These findings provide insight into the composition of EPS and LPS gene clusters in AOB and may serve as references for future comparisons (Supplementary Tables S8–S10).

Although this study identified key traits of AOB isolates that enable their survival in acidic soils, an important limitation is the temporal and spatial variation of conditions in the tea field. Since the study was conducted at a specific time point, the results may not reflect seasonal fluctuations in soil conditions, such as those occurring when nitrogen fertilizer is applied, which could impact the dynamics of the nitrifier population. This limitation underscores the need for further research to understand how variable agricultural conditions over time can influence the abundance and activity of nitrifiers. In future work, we hope to broaden our investigation by incorporating temporal and spatial sampling strategies, along with more comprehensive molecular analyses, including 16S rRNA gene amplicon sequencing or shotgun metagenomics. These efforts would enable a more robust assessment of the nitrifying communities’ dynamics, potentially including contributions from comammox and other microbial groups, such as denitrifiers, heterotrophic nitrifying bacteria, anammox bacteria, and fungi, under changing environmental conditions.

In summary, the results of genomic and physiological analyses indicate that neutrophilic AOB isolates possess key characteristics that enable them to survive in acidic soils, including high tolerance and affinity for ammonia, motility to avoid stress and seek nutrients in microsites, urease activity, and acid-resistant aggregate/biofilm formation.

This study highlights the importance of integrating data on nitrifier abundance and diversity from metagenomic analyses, soil properties, and comparative physiological and genetic studies of pure AOB isolates to better understand their distribution and the survival strategies required to inhabit acidic tea fields. Our findings reveal that the physiological differences observed among the four β-AOB isolates, γ-AOB, and comammox Nitrospira from the same tea field, such as tolerance and affinity to NH₃, likely lead to niche differentiation and potential coexistence, further emphasizing the complexity of nitrification in this environment. This research also demonstrates the major role of β-AOB in the nitrification process in strongly acidic agricultural soils amended with N fertilizers, especially β-AOB Nitrosomonas spp. and Nitrosospira spp. Furthermore, these findings provide valuable insights into the relationships among these nitrifiers and soil characteristics, enhancing our understanding of the dynamics of the nitrification process in such unique environments.

Methods

Soil samples and sampling site

Soil samples were collected from three experimental fields at the Kanaya Tea Research Station, Institute of Fruit Tree and Tea Science, NARO–NIFTS in Shimada, Shizuoka, Japan (34°48’28.0"N 138°08’00.0"E). The soil type at the sampling sites is classified as yellow soil, corresponding to Acrisols, Alisols, or Cambisols (World Reference Base for Soil Resources). Soil samples were collected in June 2015 using a core sampler, from the surface layer, including tea plant residues (O horizon), down to a depth of 10 cm (one sample every 2 cm). Triplicate samples were collected from three locations: site-1 (“naka7”), with the tea cultivar “Fushun”; site-2 (“naka49”), with “Yabukita”; and site-3 (“minami9”), with “Benifuki” or “Benifuji.” Nitrification activity was measured within two days of soil sampling. Samples were immediately stored at −80 °C or−4 °C until further molecular and biochemical analyses.

Isolation and cultivation of ammonia-oxidizing bacteria from tea field soils

The most probable number (MPN) method was used to isolate AOB, following a dilution-to-extinction approach. Briefly, soil was suspended in 100 mL of Schmidt’s AOB medium⁷⁵, adjusted to pH 5, 6, and 7, and supplemented with ammonium sulfate at concentrations of 10, 50, and 100 mM at each pH. The suspensions were incubated at 25 °C with shaking (110 rpm) until nitrite production indicated AOB activity. Cultures were serially diluted 10-fold and re-incubated, and this process was repeated until no heterotrophic growth was detected¹⁴. AOB growth in MPN test tubes, incubated for several weeks, was monitored by nitrite production, and the most diluted test tube in which AOB growth was detected was further diluted and inoculated into fresh medium. A detailed description of the method can be found in¹⁴. The purity of the isolated AOB cultures was assessed using Fluorescence In Situ Hybridization (FISH).

Measurement of ammonia, nitrite, and nitrate concentrations

Absorbance wavelengths were measured using a microplate reader (BioTek, Winooski, VT, USA). Ammonia was measured at 635 nm using the indophenol reaction⁷⁶, nitrite at 520 nm using the diazotization reaction⁷⁷, and nitrate at 410 nm using the salicylic acid nitration method⁷⁸.

Abundance of ammonia oxidizers in the tea field

Soil DNA was extracted from each of the three subsamples (0.4 g) using the Fast DNA Spin Kit for Soil (Qbiogene, Inc., Irvine, CA, USA) according to the manufacturer’s protocol. The extracted DNA was analyzed by quantitative PCR with SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan) and 200 nM of primers. The primers and PCR temperature profiles for AOA amoA and AOB amoA genes were used as described previously^{79,80,81,82,83} (Supplementary Table S12). The primers and PCR conditions for γ-AOB amoA were the same as previously reported¹⁴.

Electron microscopy

Isolated AOB cells were harvested from batch cultures by centrifugation, washed, and resuspended in fresh media diluted tenfold with sterilized distilled water. The preparations were flash-frozen in liquid propane at −175 °C, then freeze-substituted with 2% osmium tetroxide in acetone and 2% distilled water. Cells were dehydrated using anhydrous acetone and 100% ethanol, then embedded in resin (Quetol-651; Nisshin EM Co., Tokyo, Japan). Thin sections were stained with uranyl acetate and lead stain (Sigma-Aldrich Co., Tokyo, Japan) and examined under a JEM 1400Plus electron microscope (JEOL, Tokyo, Japan) operated at 80 kV.

FISH

AOB isolate cells were applied to well glass slides and air-dried for in situ hybridization, following a protocol described previously⁸⁴. The Cy3-labeled probe Nso1225 (5′-CGCCATTGTATTACGTGTGA-3′)²⁵, specific for β-proteobacteria 16S rRNA was used (Supplementary Table S12). Total bacterial cells were stained with SYTOX Green nucleic acid stain (Invitrogen, Eugene, OR, USA). After double staining, samples were observed under a Nikon ECLIPSE Ni-U fluorescence microscope with a Plan Fluor 100/1.3 oil lens (Nikon, Co. Ltd., Tokyo, Japan).

Genome sequencing, assembly, and gene annotation

Genomic DNA was extracted from the cells of Nitrosomonas sp. TNE100 and Nitrosospira spp. TNL100, TNL200, and TNL300 and collected by centrifugation (10,000 × g, 10 °C, 10 min) using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO, USA). The extracted DNA was purified using the Genomic DNA Clean & Concentrator™ −25 Kit (Zymo Research). All procedures were performed according to the manufacturers’ instructions. Genomic DNA sequencing of TNE100, TNL100, and TNL200 was performed using an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) and single-molecule real-time sequencing (PacBio, Menlo Park, CA, USA)⁸⁵. MiSeq reads shorter than 127 bases or with an average quality score below 30 were excluded from the original sequence files using Sickle (ver. 1.33). PacBio long reads were filtered using Filtlong (ver. 0.2.1), discarding reads shorter than 1 kbp and removing the lowest-quality reads until only 500 Mbp remained. Sequencing errors in the PacBio long reads were corrected with FMLRC2 (ver. 0.1.5)⁸⁶ using an FM-index of short reads constructed using RopeBWT2. Finally, the corrected PacBio long reads and filtered MiSeq short reads were used for hybrid assembly using Unicycler (ver. 0.4.8)⁸⁷. The DNA from TNL300 was sequenced using the PacBio HiFi sequencing system. The acquired PacBio HiFi reads were filtered by Filtlong (ver. 0.2.1), discarding reads shorter than 1 kbp and removing the lowest 5% of reads. Then assembly was done using Trycycler (ver. 0.5.3)⁸⁸ with the default pipeline. The quality of the assembled genomes was evaluated with CheckM (ver. 1.0.18)⁸⁹.

Automatic annotation was performed using DFAST (ver. 1.2.18)⁹⁰ and Bakta (ver. 1.6.1)⁹¹. Manual annotation was conducted by comparing the results of these two automatic annotations. Protein domain information predicted by InterProScan (5.62-94.0)⁹² was referenced when necessary.

To determine the completeness of metabolic pathways, functional annotation of protein sequences was performed using eggNOG-mapper (ver. emapper-2.1.9)⁹³. Heatmaps were generated with KEGG Decoder (ver. 1.3)^94,95,96,97 and Interactive Tree Of Life (ver. 6)⁹⁸. OrthoVenn2⁹⁹ was used to compare orthologous gene clusters among AOB genomes.

Phylogenetic analysis

Sequences from 46 AOB genomes were obtained from the National Center for Biotechnology Information (NCBI) (Supplementary Table S13). Phylogenetic analysis was performed using JolyTree¹⁰⁰. DNA sequences encoding the amoA gene from 83 AOB were aligned using the Multiple Alignment using Fast Fourier Transform method¹⁰¹. A molecular phylogenetic tree of amoA sequences was generated using the maximum likelihood method with gap-including and ambiguous sites removed. Bootstrap analysis (1,000 replicates) was conducted in MEGA software (ver. 10.1.8)^102,103. The Tamura–Nei model of nucleotide substitution with gamma-distributed and invariant sites (G + I) was selected¹⁰⁴.

Identity analysis

ANI values were calculated to compare the genomes of Nitrosomonas sp. TNE100, Nitrosospira sp. TNL100, TNL200, and TNL300 with other Nitrosomonas and Nitrosospira¹⁰⁵. The identity of DNA and protein sequences for amoA, HAO subunit A (haoA), EPS cluster genes, LPS cluster genes, and 16S rRNA were calculated using the Basic Local Alignment Search Tool.

Growth experiments

β-AOB seed cultures were inoculated at a concentration of 1% (v/v) in Erlenmeyer flasks containing 40 mL of medium for AOB¹⁰⁶ buffered with 50 mM HEPES. Cultures were incubated in a rotary shaker at 25 °C and 110 rpm. To determine the effect of pH on AOB growth, media were buffered with 20 mM MES to pH 5.0, 5.5, 6.0, 6.5, and 7.0 or with 20 mM HEPES to pH 7.0, 7.5, and 8.0. Fresh AOB medium (20 mM HEPES, pH 7.5) with final concentrations of 5, 10, 20, 40, 80, and 100 mM of NH₄⁺ and pH 7.5 was used to investigate the effect of substrate on AOB growth. The pH of cultures in all experiments was adjusted with a sterile 10% sodium carbonate solution whenever necessary. All experiments were performed in triplicates. Nitrite production was measured to estimate strain growth. Samples (1 mL) of culture medium were regularly collected during incubation to quantify nitrite concentration. The µ value was calculated at the optimum pH of each strain from the slope of the log-transformed nitrite production data plotted against time during the exponential growth of AOB^35,107. The µ value was calculated for days 1–4 for Nitrosomonas sp. TNE100, days 2–8 for Nitrosospira sp. TNL100, days 3–9 for Nitrosospira sp. TNL200, and days 1–5 for Nitrosospira sp. TNL300. The generation time (G) was calculated using the expression G = ln2/µ⁵⁸. The four isolated strains were inoculated in medium containing urea instead of ammonium sulfate, and ammonia and nitrite levels were periodically measured to assess urease activity.

Kinetics of ammonia oxidation

The K_m and the maximum reaction velocity (V_max) values for ammonia oxidation were determined by estimating the oxygen consumption of a suspension of AOB cells in a stirred vessel equipped with a Clark-type oxygen electrode (Rank Brothers, Bootisham, CAM, England) at 25 °C. AOB cells were collected from the culture medium by centrifugation (8,000 × g, 10 °C, 10 min), washed, and suspended in 20 mM HEPES buffer (pH 7.5). AOB cells (2.0 × 10⁹ cells mL⁻¹) were added to a vessel containing 1 mL of buffer solution with 20 mM HEPES buffer (pH 7.5) and various concentrations of ammonium sulfate (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6, 2.0, 2.4, 3.0, 4.0, 6.0 mM NH₄⁺). To adjust the concentration of cells used in the experiment, cell counts were performed under a phase contrast microscope model (Nikon ECLIPSE Ni-U, Ph2 40/0.65 lens, Nikon, Co. Ltd., Tokyo, Japan) using a Neubauer improved cell counting chamber (EKDS F9479, depth = 0.02 mm). The K_m values were calculated using SigmaPlot (ver. 12.5) (Systat Software, San Jose, CA, USA), based on the oxygen consumption rate obtained from the slope of a 5-min linear regression interval. All experiments were performed in triplicate.

Statistical analysis

The significance of differences among mean values of amoA gene abundance, tea soil layers, and soil properties was analyzed using analysis of variance and Tukey’s Honestly Significant Difference test with the “agricolae” package (ver. 1.3-5)¹⁰⁸ in R Statistical Software (ver. 4.1.1)¹⁰⁹ and RStudio (ver. 2021.09.0)¹¹⁰. The r value was calculated using Microsoft Excel for Windows (Regression and CORREL function) to assess correlations between tea soil properties and amoA gene abundance in ammonia-oxidizing microbes.

Data availability

Genome and plasmid sequences files of strains TNE100, TNL100, TNL200, and TNL300 have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers AP040997 - AP040999, AP041000 - AP041002, AP041003 - AP041004, and AP041005 - AP041006, respectively.

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Acknowledgements

We thank Udagawa, M. and Nakamori, T. (NARO) for their technical assistance and Fujitani, H. (Chuo University) for support in fluorescence in situ hybridization methodology.

Funding

This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) through the Moonshot Research Program [JPNP18016], the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry [28004 A] by BRAIN, and MEXT KAKENHI [19H01156, 16K14874, and 26310315].

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Yong Wang
Present address: TanBIO Inc, Tsukuba, Ibaraki, Japan

Authors and Affiliations

Institute for Agro-Environmental Sciences (NIAES), National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan
Luciano N. Aoyagi, Yong Wang, Tsubasa Ohbayashi, Masahito Hayatsu & Kanako Tago
School of Veterinary Medicine, Kitasato University, Sagamihara, Kanagawa, Japan
Luciano N. Aoyagi & Kanako Tago
Institute of Fruit Tree and Tea Science, NARO, Shimada, Shizuoka, Japan
Yuhei Hirono

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Luciano N. Aoyagi
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Yong Wang
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Tsubasa Ohbayashi
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Yuhei Hirono
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Masahito Hayatsu
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Kanako Tago
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Contributions

Resources: M.H., K.T., and Y.H. Data curation: Y.W. and M.H. Formal Analysis and Investigation: L.N.A, Y.W., T.O., K.T., and M.H. Writing—original draft: L.N.A. Writing—review and editing: all authors. Conceptualization, project administration and Supervision: K.T. and M.H. All authors read, reviewed, and approved the final manuscript.

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Correspondence to Kanako Tago.

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Aoyagi, L.N., Wang, Y., Ohbayashi, T. et al. Diversity and characterization of the ammonia-oxidizing bacteria responsible for nitrification in tea field soils. Sci Rep 15, 33221 (2025). https://doi.org/10.1038/s41598-025-18596-5

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Received: 10 June 2025
Accepted: 02 September 2025
Published: 26 September 2025
DOI: https://doi.org/10.1038/s41598-025-18596-5