Physiological and genomic characterization of a new 'Candidatus Nitrotoga' isolate.

Oxidation of nitrite to nitrate is an important process in the global nitrogen cycle. Recent molecular biology-based studies have revealed that the widespread nitrite-oxidizing bacteria (NOB) belonging to the genus 'Candidatus Nitrotoga' may be highly important for the environment. However, the insufficient availability of pure Nitrotoga cultures has limited our understanding of their physiological and genomic characteristics. Here, we isolated the 'Ca. Nitrotoga' sp. strain AM1P, from a previously enriched Nitrotoga culture, using an improved isolation strategy. Although 'Ca. Nitrotoga' have been recognized as cold-adapted NOB, the strain AM1P had a slightly higher optimum growth temperature at 23°C. Strain AM1P showed a pH optimum of 8.3 and was not inhibited even at high nitrite concentrations (20 mM). We obtained the complete genome of the strain and compared the genome profile to five previously sequenced 'Ca. Nitrotoga' strains. Comparative genomics suggested that lactate dehydrogenase may be only encoded in the strain AM1P and closely related genomes. While the growth yield of AM1P did not change, we observed faster growth in the presence of lactate in comparison to purely chemolithoautotrophic growth. The characterization of the new strain AM1P sheds light on the physiological adaptation of this environmentally important, but understudied genus 'Ca. Nitrotoga'.

Enrichment and Physiological Characterization of a Cold-Adapted Nitrite-Oxidizing Nitrotoga sp. from an Eelgrass Sediment.

Nitrite-oxidizing bacteria (NOB) are responsible for the second step of nitrification in natural and engineered ecosystems. The recently discovered genus Nitrotoga belongs to the Betaproteobacteria and potentially has high environmental importance. Although environmental clones affiliated with Nitrotoga are widely distributed, the limited number of cultivated Nitrotoga spp. results in a poor understanding of their ecophysiological features. In this study, we successfully enriched the nonmarine cold-adapted Nitrotoga sp. strain AM1 from coastal sand in an eelgrass zone and investigated its physiological characteristics. Multistep-enrichment approaches led to an increase in the abundance of AM1 to approximately 80% of the total bacterial population. AM1 was the only detectable NOB in the bacterial community. The 16S rRNA gene sequence of AM1 was 99.6% identical to that of "Candidatus Nitrotoga arctica," which was enriched from permafrost-affected soil. The highest nitrogen oxidation rate of AM1 was observed at 16°C. The half-saturation constant (Km ) and the generation time were determined to be 25 µM NO2- and 54 h, respectively. The nitrite oxidation rate of AM1 was stimulated at concentrations of <30 mM NH4Cl but completely inhibited at 50 mM NH4Cl. AM1 can grow well under specific environmental conditions, such as low temperature and in the presence of a relatively high concentration of free ammonia. These results help improve our comprehension of the functional importance of NitrotogaIMPORTANCE Nitrite-oxidizing bacteria (NOB) are key players in the second step of nitrification, which is an important process of the nitrogen cycle. Recent studies have suggested that the organisms of the novel NOB genus Nitrotoga were widely distributed and played a functional role in natural and engineered ecosystems. However, only a few Nitrotoga enrichments have been obtained, and little is known about their ecology and physiology. In this study, we successfully enriched a Nitrotoga sp. from sand in a shallow coastal marine ecosystem and undertook a physiological characterization. The laboratory experiments showed that the Nitrotoga enrichment culture could adapt not only to low temperature but also to relatively high concentrations of free ammonia. The determination of as-yet-unknown unique characteristics of Nitrotoga contributes to the improvement of our insights into the microbiology of nitrification.

Detection and Diversity of the Nitrite Oxidoreductase Alpha Subunit (nxrA) Gene of Nitrospina in Marine Sediments.

Nitrite-oxidizing bacteria (NOB) are chemolithoautotrophs that catalyze the oxidation of nitrite to nitrate, which is the second step of aerobic nitrification. In marine ecosystems, Nitrospina is assumed to be a major contributor to nitrification. To date, two strains of Nitrospina have been isolated from marine environments. Despite their ecological relevance, their ecophysiology and environmental distribution are understudied owing to fastidious cultivation techniques and the lack of a sufficient functional gene marker. To estimate the abundance, diversity, and distribution of Nitrospina in various marine sediments, we used nxrA, which encodes the alpha subunit of nitrite oxidoreductase, as a functional and phylogenetic marker. We observed that Nitrospina diversity in polar sediments was significantly lower than that of non-polar samples. Moreover, nxrA-like sequences revealed an unexpected diversity of Nitrospina, with approximately 41,000 different sequences based on a 95% similarity cutoff from six marine sediments. We detected nxrA gene copy numbers of up to 3.57 × 104 per gram of marine sediment sample. The results of this study provide insight into the distribution and diversity of Nitrospina, which is fundamentally important for understanding their contribution to the nitrogen cycle in marine sediments.

Isolation of sublineage I†Nitrospira by a novel cultivation strategy.

Nitrification is an important process in the biogeochemical nitrogen cycle and is widely exploited in biological wastewater treatment. Recently, Nitrospira has been recognized as the numerically dominant nitrite-oxidizing bacterial genus and is primarily responsible for the second step of aerobic nitrification. Nevertheless, the physiological properties of Nitrospira remain poorly understood because the organisms are difficult to isolate and culture. Here, we report a novel cultivation strategy for obtaining members of the Nitrospira sublineage I in pure culture. The method combines: (i) selective enrichment of Nitrospira using a continuous feeding reactor and (ii) purification followed by sub-cultivation via a cell sorting system by focusing on the unique characteristics of Nitrospira forming spherical micro-colonies. This strategy is potentially applicable to other uncultured or unisolated Nitrospira and could accelerate the physiological and biochemical understandings of this important group of organisms.

Variation in growth rates between cultures hinders the cultivation of ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria, key players in the nitrogen cycle, have been the focus of extensive research. Numerous novel species have been isolated and their growth dynamics were studied. Despite these efforts, controlling their growth to obtain diverse physiological findings remains a challenge. These bacteria often fail to grow, even under optimal conditions. This unpredictable growth pattern could be viewed as a survival strategy. Understanding this heterogeneous behavior could enhance our ability to culture these bacteria. In this study, the variation in the growth rate was quantified for the ammonia-oxidizing bacterium Nitrosomonas mobilis Ms1. Our findings revealed significant growth rate variation under low inoculum conditions. Interestingly, higher cell densities resulted in more stable cultures. A comparative analysis of three Nitrosomonas species showed a correlation between growth rate variation and culture failure. The greater the variation in growth rate, the higher the likelihood of culture failure.

Isolation of a Moderately Acidophilic Nitrobacter from a Nitrifying Community Supplied with Urea.

Nitrite-oxidizing bacteria (NOB), which perform the second step of aerobic nitrification, play an important role in soil. In the present study, we report a novel isolate from agricultural soil affiliated with the genus Nitrobacter and its physiological characteristics. We sampled the surface soil of a vegetable field and obtained mixed culture A31 using the most probable number (MPN) method with inorganic medium containing 0.75| |mM urea (pH 5.5). The dilution-extinction procedure on culture A31 led to the isolation of a strain that was designated as Nitrobacter sp. A67. The nxrB1 gene sequence of Nitrobacter sp. A67 (302 bp) was classified into Cluster 5, and the highest sequence identity was 96.10% with Nitrobacter sp. BS5/19. The NO2- oxidation activity of Nitrobacter sp. A67 was investigated at various pH. The optimum pH for NO2- oxidation was 5.8-6.4. This result indicates that Nitrobacter sp. A67 is a moderately acidophilic nitrite-oxidizing bacterium.

Characterisation of bacteria representing a novel Nitrosomonas clade: Physiology, genomics and distribution of missing ammonia oxidizer.

Members of the genus Nitrosomonas are major ammonia oxidizers that catalyse the first step of nitrification in various ecosystems. To date, six subgenus-level clades have been identified. We have previously isolated novel ammonia oxidizers from an additional clade (unclassified cluster 1) of the genus Nitrosomonas. In this study, we report unique physiological and genomic properties of the strain PY1, compared with representative ammonia-oxidising bacteria (AOB). The apparent half-saturation constant for total ammonia nitrogen and maximum velocity of strain PY1 were 57.9 ± 4.8 µM NH3 + NH4 + and 18.5 ± 1.8 µmol N (mg protein)-1 h-1 , respectively. Phylogenetic analysis based on genomic information revealed that strain PY1 belongs to a novel clade of the Nitrosomonas genus. Although PY1 contained genes to withstand oxidative stress, cell growth of PY1 required catalase to scavenge hydrogen peroxide. Environmental distribution analysis revealed that the novel clade containing PY1-like sequences is predominant in oligotrophic freshwater. Taken together, the strain PY1 had a longer generation time, higher yield and required reactive oxygen species (ROS) scavengers to oxidize ammonia, compared with known AOB. These findings expand our knowledge of the ecophysiology and genomic diversity of ammonia-oxidising Nitrosomonas.

Effects of Co-existing Heterotrophs on Physiology of and Nitrogen Metabolism in Autotrophic Nitrite-oxidizing Candidatus Nitrotoga.

Interactions between autotrophic nitrifiers and heterotrophs have attracted considerable attention in microbial ecology. However, the mechanisms by which heterotrophs affect the physiological activity of and nitrogen metabolism in autotrophic nitrite oxidizers remain unclear. We herein focused on nitrite-oxidizing Candidatus Nitrotoga and compared an axenic culture including only Ca. Nitrotoga with a co-culture of both Ca. Nitrotoga and Acidovorax in physiological experiments and transcriptomics. In the co-culture with Acidovorax, nitrite consumption by Ca. Nitrotoga was promoted, and some genes relevant to nitrogen metabolism in Ca. Nitrotoga were highly expressed. These results provide insights into the mechanisms by which co-existing heterotrophs affect autotrophic nitrifiers.

Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort.

Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip "fluorescent nucleic acid probe in droplets for bacterial sorting" (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.

Transcriptome Analysis of the Ammonia-Oxidizing Bacterium Nitrosomonas mobilis Ms1 Reveals Division of Labor between Aggregates and Free-living Cells.

Bacteria change their metabolic states to increase survival by forming aggregates. Ammonia-oxidizing bacteria also form aggregates in response to environmental stresses. Nitrosomonas mobilis, an ammonia-oxidizing bacterium with high stress tolerance, often forms aggregates mainly in wastewater treatment systems. Despite the high frequency of aggregate formation by N. mobilis, its relationship with survival currently remains unclear. In the present study, aggregates were formed in the late stage of culture with the accumulation of nitrite as a growth inhibitor. To clarify the significance of aggregate formation in N. mobilis Ms1, a transcriptome analysis was performed. Comparisons of the early and late stages of culture revealed that the expression of stress response genes (chaperones and proteases) increased in the early stage. Aggregate formation may lead to stress avoidance because stress response genes were not up-regulated in the late stage of culture during which aggregates formed. Furthermore, comparisons of free-living cells with aggregates in the early stage of culture showed differences in gene expression related to biosynthesis (ATP synthase and ribosomal proteins) and motility and adhesion (flagella, pilus, and chemotaxis). Biosynthesis genes for growth were up-regulated in free-living cells, while motility and adhesion genes for adaptation were up-regulated in aggregates. These results indicate that N. mobilis Ms1 cells adapt to an unfavorable environment and grow through the division of labor between aggregates and free-living cells.

Enrichment of Comammox and Nitrite-Oxidizing Nitrospira From Acidic Soils.

In agricultural soils fertilized with a high amount of ammonium nitrogen, the pH decreases because of the oxidation of ammonia by nitrifiers. Molecular-based analyses have revealed that members of the genus Nitrospira dominate over other nitrifiers in some acidic soils. However, terrestrial Nitrospira are rarely cultivated and little is known about their ecophysiology. In addition, recent studies discovered a single microbe with the potential to oxidize both ammonia and nitrite (complete ammonia oxidizer; comammox) within Nitrospira, which had been previously recognized as a nitrite oxidizer. Despite their broad distribution, there are no enrichment samples of comammox from terrestrial or acidic environments. Here, we report the selective enrichment of both comammox and nitrite-oxidizing Nitrospira from the acidic soil of a heavily fertilized tea field. Long-term enrichment was performed with two individual continuous-feeding bioreactors capable of controlling ammonia or nitrite concentration and pH. We found that excessive ammonium supply was a key factor to enhance the growth of comammox Nitrospira under acidic conditions. Additionally, a low concentration of nitrite was fed to prevent the accumulation of free nitrous acid and inhibition of cell growth under low pH, resulting in the selective enrichment of nitrite-oxidizing Nitrospira. Based on 16S rRNA gene analysis, Nitrospira accounting for only 1.2% in an initial soil increased to approximately 80% of the total microorganisms in both ammonia- and nitrite-fed bioreactors. Furthermore, amoA amplicon sequencing revealed that two phylotypes belonging to comammox clade A were enriched in an ammonia-fed bioreactor. One group was closely related to previously cultivated strains, and the other was classified into a different cluster consisting of only uncultivated representatives. These two groups coexisted in the bioreactor controlled at pH 6.0, but the latter became dominant after the pH decreased to 5.5. Additionally, a physiological experiment revealed that the enrichment sample oxidizes ammonia at pH <4, which is in accordance with the strongly acidic tea field soil; this value is lower than the active pH range of isolated acid-adapted nitrifiers. In conclusion, we successfully enriched multiple phylotypes of comammox and nitrite-oxidizing Nitrospira and revealed that the pH and concentrations of protonated N-compounds were potential niche determinants.

MazF Endoribonucleolytic Toxin Conserved in Nitrospira Specifically Cleaves the AACU, AACG, and AAUU Motifs.

MazF is an endoribonucleolytic toxin that cleaves intracellular RNAs in sequence-specific manners. It is liberated in bacterial cells in response to environmental changes and is suggested to contribute to bacterial survival by inducing translational regulation. Thus, determining the cleavage specificity provides insights into the physiological functions of MazF orthologues. Nitrospira, detected in a wide range of environments, is thought to have evolved the ability to cope with their surroundings. To investigate the molecular mechanism of its environmental adaption, a MazF module from Nitrospira strain ND1, which was isolated from the activated sludge of a wastewater treatment plant, is examined in this study. By combining a massive parallel sequencing method and fluorometric assay, we detected that this functional RNA-cleaving toxin specifically recognizes the AACU, AACG, and AAUU motifs. Additionally, statistical analysis suggested that this enzyme regulates various specific functions in order to resist environmental stresses.

Successful enrichment of low-abundant comammox Nitrospira from nitrifying granules under ammonia-limited conditions.

In artificial engineered systems, nitrification is a key reaction that accounts for the removal of biological nitrogen. Recently, a single microbe capable of oxidizing ammonia to nitrate, known as a complete ammonia oxidizer (comammox), has been discovered. Although the abundance and diversity of comammox Nitrospira in engineered systems have been identified through molecular-based approaches, the enrichment and isolation of comammox Nitrospira remains a challenge. Therefore, the aim of this study was to enrich comammox Nitrospira from nitrifying granules, which were used to increase the efficiency of biological nitrogen removal in wastewater treatment plants. We sought to accomplish this through the use of a fixed-bed continuous feeding bioreactor. Fluorescence in situ hybridization, 16S rRNA gene amplicon sequencing and qPCR of functional genes were utilized to monitor the growth of nitrifiers including comammox Nitrospira. Cloning of comammox amoA genes identified amoA phylogeny of enriched comammox Nitrospira. This work is an example demonstrating that continuous supply of low ammonium concentrations alongside biomass carriers is effective in cultivating comammox Nitrospira from engineered systems.

Genomic and Physiological Characteristics of a Novel Nitrite-Oxidizing Nitrospira Strain Isolated From a Drinking Water Treatment Plant.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, which is an important process of the biogeochemical nitrogen cycle and is exploited extensively as a biological nitrogen removal process. Members of the genus Nitrospira are often identified as the dominant NOB in a diverse range of natural and artificial environments. Additionally, a number of studies examining the distribution, abundance, and characterization of complete ammonia oxidation (comammox) Nitrospira support the ecological importance of the genus Nitrospira. However, niche differentiation between nitrite-oxidizing Nitrospira and comammox Nitrospira remains unknown due to a lack of pure cultures. In this study, we report the isolation, physiology, and genome of a novel nitrite-oxidizing Nitrospira strain isolated from a fixed-bed column at a drinking water treatment plant. Continuous feeding of ammonia led to the enrichment of Nitrospira-like cells, as well as members of ammonia-oxidizing genus Nitrosomonas. Subsequently, a microcolony sorting technique was used to isolate a novel nitrite-oxidizing Nitrospira strain. Sequences of strains showing the growth of microcolonies in microtiter plates were checked. Consequently, the most abundant operational taxonomic unit (OTU) exhibited high sequence similarity with Nitrospira japonica (98%) at the 16S rRNA gene level. The two other Nitrospira OTUs shared over 99% sequence similarities with N. japonica and Nitrospira sp. strain GC86. Only one strain identified as Nitrospira was successfully subcultivated and designated as Nitrospira sp. strain KM1 with high sequence similarity with N. japonica (98%). The half saturation constant for nitrite and the maximum nitrite oxidation rate of strain KM1 were orders of magnitude lower than the published data of other known Nitrospira strains; moreover, strain KM1 was more sensitive to free ammonia compared with previously isolated Nitrospira strains. Therefore, the new Nitrospira strain appears to be better adapted to oligotrophic environments compared with other known non-marine nitrite oxidizers. The complete genome of strain KM1 was 4,509,223 bp in length and contained 4,318 predicted coding sequences. Average nucleotide identities between strain KM1 and known cultured Nitrospira genome sequences are 76.7-78.4%, suggesting at least species-level novelty of the strain in the Nitrospira lineage II. These findings broaden knowledge of the ecophysiological diversity of nitrite-oxidizing Nitrospira.

Nitrogen and Oxygen Isotope Signatures of Nitrogen Compounds during Anammox in the Laboratory and a Wastewater Treatment Plant.

Isotopic fractionation factors against 15N and 18O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ18O of water to investigate the effects of water δ18O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect (15εNXR of -77.8 to -65.9‰ and 15ΔNXR of -31.3 to -30.4‰) and possibly O isotopic effect (18εNXR of -20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ18O vs δ15N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ18O vs δ15N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.

Draft Genome Sequence of Acidovorax sp. Strain NB1, Isolated from a Nitrite-Oxidizing Enrichment Culture.

Here, we report the draft genome sequence of Acidovorax sp. strain NB1, isolated from an enrichment culture of nitrite-oxidizing bacteria (NOB). Genes involved in denitrification were found in the draft genome of NB1. The closest strain to NB1 based on genomic relatedness is Acidovorax sp. strain GW101-3H11, with 91.5% average nucleotide identity.

Seabird-affected taluses are denitrification hotspots and potential N2O emitters in the High Arctic.

In High Arctic tundra ecosystems, seabird colonies create nitrogen cycling hotspots because of bird-derived labile organic matter. However, knowledge about the nitrogen cycle in such ornithocoprophilous tundra is limited. Here, we determined denitrification potentials and in-situ nitrous oxide (N2O) emissions of surface soils on plant-covered taluses under piscivorous seabird cliffs at two sites (BL and ST) near Ny-Ålesund, Svalbard, in the European High Arctic. Talus soils at both locations had very high denitrification potentials at 10 °C (2.62-4.88 mg N kg-1 dry soil h-1), near the mean daily maximum air temperature in July in Ny-Ålesund, with positive temperature responses at 20 °C (Q10 values, 1.6-2.3). The talus soils contained abundant denitrification genes, suggesting that they are denitrification hotspots. However, high in-situ N2O emissions, indicating the presence of both active aerobic nitrification and anaerobic denitrification, were observed only at BL (max. 16.6 µg N m-2 h-1). Rapid nitrogen turnover at BL was supported by lower carbon-to-nitrogen ratios, higher nitrate content, and higher δ15N values in the soils at BL compared with those at ST. These are attributed to the 30-fold larger seabird density at BL than at ST, providing the larger organic matter input.

A rapid collection of yet unknown ammonia oxidizers in pure culture from activated sludge.

Nitrification is an important reaction in the biological nitrogen removal process in wastewater treatment plants (WWTPs). As ammonia-oxidizing microbes are slow-growing and sensitive to environmental factors such as free ammonia, pure strains are hard to obtain, preventing our understanding of their physiological characteristics. To conquer this hurdle, we report a high-throughput isolation technique based on scattering signatures, which exploits the tendency of many ammonia-oxidizing bacteria (AOB) to form microcolonies in activated sludge. The AOB microcolonies were directly sorted from the activated sludge without long incubation and enrichment bias, and were sequentially inoculated into 96-well microtiter plates containing growth medium. Phylogenetic analysis of the pure strains isolated in this study revealed a deeply branching and unrecognized lineage and diversity within the genus Nitrosomonas, beyond our expectation.

Genomic Analysis of Two Phylogenetically Distinct Nitrospira Species Reveals Their Genomic Plasticity and Functional Diversity.

The genus Nitrospira represents a dominant group of nitrite-oxidizing bacteria in natural and engineered ecosystems. This genus is phylogenetically divided into six lineages, for which vast phylogenetic and functional diversity has been revealed by recent molecular ecophysiological analyses. However, the genetic basis underlying these phenotypic differences remains largely unknown because of the lack of genome sequences representing their diversity. To gain a more comprehensive understanding of Nitrospira, we performed genomic comparisons between two Nitrospira strains (ND1 and NJ1 belonging to lineages I and II, respectively) previously isolated from activated sludge. In addition, the genomes of these strains were systematically compared with previously reported six Nitrospira genomes to reveal their similarity and presence/absence of several functional genes/operons. Comparisons of Nitrospira genomes indicated that their genomic diversity reflects phenotypic differences and versatile nitrogen metabolisms. Although most genes involved in key metabolic pathways were conserved between strains ND1 and NJ1, assimilatory nitrite reduction pathways of the two Nitrospira strains were different. In addition, the genomes of both strains contain a phylogenetically different urease locus and we confirmed their ureolytic activity. During gene annotation of strain NJ1, we found a gene cluster encoding a quorum-sensing system. From the enriched supernatant of strain NJ1, we successfully identified seven types of acyl-homoserine lactones with a range of C10-C14. In addition, the genome of strain NJ1 lacks genes relevant to flagella and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated genes) systems, whereas most nitrifying bacteria including strain ND1 possess these genomic elements. These findings enhance our understanding of genomic plasticity and functional diversity among members of the genus Nitrospira.

Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation.

Nitrite oxidation is an aerobic process of the nitrogen cycle in natural ecosystems, and is performed by nitrite-oxidizing bacteria (NOB). Also, nitrite oxidation is a rate-limiting step of nitrogen removal in wastewater treatment plants (WWTPs). Although Nitrospira is known as dominant NOB in WWTPs, information on their physiological properties and kinetic parameters is limited. Here, we report the kinetic parameters and inhibition of nitrite oxidation by free ammonia in pure cultures of Nitrospira sp. strain ND1 and Nitrospira japonica strain NJ1, which were previously isolated from activated sludge in a WWTP. The maximum nitrite uptake rate ( [Formula: see text] ) and the half-saturation constant for nitrite uptake ( [Formula: see text] ) of strains ND1 and NJ1 were 45 ± 7 and 31 ± 5 (µmol NO2-/mg protein/h), and 6 ± 1 and 10 ± 2 (µM NO2-), respectively. The [Formula: see text] and [Formula: see text] of two strains indicated that they adapt to low-nitrite-concentration environments like activated sludge. The half-saturation constants for oxygen uptake ( [Formula: see text] ) of the two strains were 4.0±2.5 and 2.6±1.1 (µM O2), respectively. The [Formula: see text] values of the two strains were lower than those of other NOB, suggesting that Nitrospira in activated sludge could oxidize nitrite in the hypoxic environments often found in the interiors of biofilms and flocs. The inhibition thresholds of the two strains by free ammonia were 0.85 and 4.3 (mg-NH3 l-1), respectively. Comparing the physiological properties of the two strains, we suggest that tolerance for free ammonia determines competition and partitioning into ecological niches among Nitrospira populations.