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

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e9, 2017 www.elsevier.com/locate/jbiosc

Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation Norisuke Ushiki,1 Masaru Jinno,1 Hirotsugu Fujitani,1 Toshikazu Suenaga,2 Akihiko Terada,2 and Satoshi Tsuneda1, * Department of Life Science and Medical Bioscience, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan1 and Department of Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan2 Received 28 November 2016; accepted 28 December 2016 Available online xxx

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 (Vmax NO2 ) and the half-saturation constant for nitrite L uptake (Km NO2 ) of strains ND1 and NJ1 were 45 ± 7 and 31 ± 5 (mmol NOL 2 /mg protein/h), and 6 ± 1 and 10 ± 2 (mM NO2 ), respectively. The Vmax NO2 and Km NO2 of two strains indicated that they adapt to low-nitrite-concentration environments like activated sludge. The half-saturation constants for oxygen uptake (Km O2 ) of the two strains were 4.0 ± 2.5 and 2.6 ± 1.1 (mM O2), respectively. The Km O2 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 lL1), 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. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Nitrospira; Affinity for nitrite; Affinity for oxygen; Growth parameters; Free ammonia; Inhibitory constant; Activated sludge]

Nitrification, in which ammonia is transformed to nitrate via nitrite, is an aerobic step in the global nitrogen cycle. Nitrification consists of two stages, ammonia oxidation and nitrite oxidation, which are performed by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB). Recently, it was reported that complete ammonia oxidation (COMAMMOX) was performed by a single Nitrospira bacterium, which was known previously as NOB that catalyzed only nitrite oxidation (1,2). Nitrification is also a key rate-limiting step in biological nitrogen removal from activated sludge in wastewater treatment plants (WWTPs), and contributes to prevention of eutrophication of coastal ecosystems (3). It is widely accepted that nitrification in activated sludge is stable in normal conditions, and that nitrite as an intermediate of nitrification is instantly converted into nitrate by close cooperation between AOB and NOB (4). However, nitrite occasionally accumulates when this cooperation collapses because of different growth rates of AOB and NOB (5). Nitrite accumulation may also be caused by inhibition of nitrite-oxidizing activity in NOB; temperature, pH, dissolved oxygen, light, free ammonia and free nitrous acid are known to inhibit nitrite

* Corresponding author. Tel./fax: þ81 3 5369 7325. E-mail address: [email protected] (S. Tsuneda).

oxidation (6). Thus, understanding of the microbial communities and physiological properties of NOB is required for stabilization of nitrite oxidation in activated sludge. Over the last two decades and based on cultivation approaches, the genus Nitrobacter within the class Alphaproteobacteria has been considered as the dominant NOB in activated sludge. Since isolation and cultivation of Nitrobacter are relatively uncomplicated, the metabolism and biochemistry of NOB were investigated using pure cultures of Nitrobacter as a model bacterium (7e11). However, it was recently reported that previously unrecognized NOB belonging to genera outside the Alphaproteobacteria were found in activated sludge. Candidatus Nitrospira defluvii, belonging to the phylum Nitrospirae, was successfully enriched from activated sludge by cultivation at low nitrite concentration and selective repression with ampicillin, and appeared to prefer low-nitrite-concentration environments (12). Candidatus Nitrotoga arctica, belonging to the class Betaproteobacteria, was enriched from permafrost-affected soils in Siberia (13), and the genus Nitrotoga was recognized as cold-adapted NOB in activated sludge (14,15). Nitrolancea hollandica, belonging to the phylum Chloroflexi, was isolated from a nitrifying bioreactor with a high loading of ammonium bicarbonate and was characterized as a thermotolerant, neutrophilic NOB with high nitrite-tolerance (16,17). Thus, the huge phylogenetic diversity of

1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.12.016

Please cite this article in press as: Ushiki, N., et al., Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.016

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nitrite oxidizers indicates that the insights obtained to date into the physiology of NOB are the tip of an iceberg. Molecular approaches have revealed that Nitrospira, not Nitrobacter, are the dominant NOB in ubiquitous activated sludge (15,18e21). Since Nitrospira are known as notoriously recalcitrant bacteria and the availability of pure strains is limited, detailed physiological characteristics of Nitrospira are still uncertain. Affinities for substrates (nitrite and oxygen) and inhibition by environmental factors (pH, temperature and free ammonia) of enriched Nitrospira cells in nitrifying bioreactors have been investigated using a microsensor system (20,22,23). Based on the physiological characteristics determined from enrichment samples, it was revealed that the affinity for nitrite and maximum nitrite uptake rates were major factors in the ecological niche differentiation between Nitrobacter and Nitrospira (20). It was suggested that their differentiation could be explained by K and r strategies: Nitrobacter (r strategists) have relatively low affinity for nitrite and a high maximum nitrite uptake rate, whereas Nitrospira (K strategists) have relatively high affinity for nitrite and a low maximum nitrite uptake rate (20,24). Moreover, it was recently reported that competition and partitioning between ecological niches among phylogenetically different populations of Nitrospira in WWTPs were caused by different physiological properties such as affinities for substrates, formate utilization and relationships with AOB (25e27). However, these insights were obtained from mixed cultures containing other species, not from pure Nitrospira cultures. The kinetic and growth parameters of NOB were determined using pure cultures of Nitrobacter and Nitrospira, and major factors determining ecological niche differentiation among NOB communities were also demonstrated (28). However, the nitrite affinities and maximum uptake rates determined by a few strains are not enough to explain competition and partitioning into ecological niches among Nitrospira communities in activated sludge, and more information is required from the available pure cultures. Here, using two pure Nitrospira cultures of Nitrospira sp. strain ND1 and Nitrospira japonica strain NJ1, we investigated kinetics and growth parameters, and evaluated inhibition by free ammonia, which is well known as an inhibitor of nitrite oxidation (29). In previous studies (30e32), we selectively enriched phylogenetically distinct Nitrospira lineages (I and II) using a continuous feeding bioreactor, and successfully isolated strains ND1 and NJ1 using a cell sorting system. Although both strains were selectively enriched by controlling nitrite concentration in the continuous feeding bioreactor, it was not clear whether nitrite concentration or other factors determined their competition and ecological niche partitioning in activated sludge. Thus, different properties of the two strains as representative strains within two Nitrospira lineages, were expected to help elucidate the competition and ecological niche partitioning between two Nitrospira lineages found in activated sludge. MATERIALS AND METHODS Nitrospira strains and culture conditions In this study, we investigated pure cultures of Nitrospira sp. strain ND1 and N. japonica strain NJ1, which were previously isolated from activated sludge in a wastewater treatment plant by our research group (30,31). The two Nitrospira strains were cultured in mineral medium containing nitrite in the same manner as in previous reports (30,31). The medium consisted of NaNO2 (49.3 mg l1), KH2PO4 (38.2 mg l1), MgSO4$7H2O (61.1 mg l1), CaCl2$2H2O (10.0 mg l1), FeSO4$7H2O (5.0 mg l1), NaHCO3 (200.0 mg l1), MnSO4$5H2O (54.2 mg l1), H3BO3 (49.4 mg l1), ZnSO4$7H2O (43.1 mg l1), Na2Mo4O4 (27.6 mg l1) and CuSO4$5H2O (25.0 mg l1). Other culture conditions were darkness, pH 7.8e8.0, 29 C, and shaking at 100 rpm. Determination of kinetic parameters for nitrite oxidation To investigate kinetic parameters of nitrite oxidation, nitrite affinity and the maximum nitrite uptake rate, nitrite concentration was measured over time. Fig. S1 shows a flow chart of the sampling method. As in a previous report (28), we used pure cultures in early stationary phase, which is between 12 and 48 h after nitrite consumption. Since the

cell number in Nitrospira cultures is low, 40-ml aliquots of the pure cultures were enriched using a membrane filter with a pore size of 0.22 mm (Merck Milipore, Billerrica, MA, USA), and the cells were resuspended in 4 ml mineral medium. The initial nitrite concentration of the samples was adjusted to 3 mg-N l1 (214 mM NO 2 ). Incubation conditions were: shaking at 150 rpm, 25 C, pH 8.0. Every 5e20 min during incubations, 50 ml aliquots of incubated samples were collected and treated at 95 C for 10 min to inactivate the nitrite-oxidizing activity. Occasionally, we checked the remaining nitrite concentration in the samples using Griess reagent (33). The initial interval of sampling was 20 min; once the remaining nitrite concentration in the incubated samples was below approximately 1.5 mg-N l1, the sampling interval was changed to 5 min. When the remaining nitrite concentration in the incubated samples reached 0 mg-N l1, the nitrite concentrations of all the samples collected were measured using Griess reagent and a microplate spectrophotometer (PowerScan HT, BioTek, Winooski, VT, USA). The nitrite uptake rate of pure Nitrospira cultures was calculated based on the measured nitrite concentrations. Subsequently, the nitrite affinities and the maximum nitrite uptake rates of Nitrospira cultures were calculated by fitting the data to a MichaeliseMenten kinetic equation (Eq. 1): VNO2 ¼ Vmax

NO2 $½S




Km

NO2

þ ½S




(1)

Here, VNO2 is the nitrite uptake rate (mmol NO2/mg protein/h), Vmax NO2 is the maximum nitrite uptake rate (mmol NO 2 /mg protein/h), Km NO2 is the halfsaturation constant for nitrite uptake (mM NO 2 ), and S is the nitrite concentration (mM NO 2 ). The data fitting was performed using non-linear least square method with solver in Excel. In these experiments, we did not use purified nitrite-oxidizing enzymes (nitrite oxidoreductases) from Nitrospira. Since the kinetic parameters were determined in short-term (2e3 h) experiments, we can ignore growth of the Nitrospira cells and regard them as enzymes. Thus, we prefer to use the terms Vmax and Km. Oxygen affinity of pure Nitrospira cultures To investigate the oxygen affinity of strains ND1 and NJ1, the dissolved oxygen concentration was measured over time. The strains were harvested in early stationary phase. After they were concentrated using a membrane filter with a pore size of 0.22 mm (Merck Milipore), the initial nitrite concentration and pH were adjusted to approximately 21 mg-N l1 (1.5 mM NO 2 ) and 8.0, respectively. Each suspension was transferred into a 2-ml glass chamber, followed by commencement of an oxygen consumption test at  29 C with complete mixing by a magnetic stirrer. The oxygen consumption of each strain was monitored using a microrespiration system (Unisense AS, Denmark). After measurements, the dissolved oxygen concentration data were subjected to smoothing to remove noise using Sigma plot 13.0 software (Systat Software GmbH, Erkrath, Germany). The oxygen affinity and the maximum oxygen uptake rate of strains ND1 and NJ1 were calculated by fitting the data to the following a MichaeliseMenten kinetic equation (Eq. 2): VO2 ¼ Vmax

O2 $½S




Km

O2

þ ½S




(2)

Here, VO2 is the oxygen uptake rate (mmol O2/h), Vmax O2 is the maximum oxygen uptake rate (mmol O2/h), Km O2 is the half-saturation constant for oxygen (mM O2), and S is the dissolved oxygen concentration (mM O2). The data fitting was performed using non-linear least square method with solver in Excel. Determination of growth parameters Subcultured pure Nitrospira cultures (50 ml) were added to an autoclaved 5-l glass bottle containing 950-ml mineral medium. The initial nitrite concentration was adjusted to 1 mM, and other culture conditions were 29 C, pH 8.0, shaking at 100 rpm and darkness. Sampling was performed once a day during several days of incubation. During sampling, since both strains ND1 and NJ1 formed large flocs, 20 ml aliquots were taken from incubated samples and sonicated at 25% amplitude for 20 s to disrupt the flocs using an ultrasonic processor (Vibra-cell VCX500, Sonics & Materials, Inc., CT, USA). After sonication treatment, 1 ml aliquots were taken from the sonicated samples for chemical analysis and DNA extraction, and were stored at 20 C. After the incubation finished, total DNA was extracted from 1 ml aliquots of each sampling point using a DNA extraction kit (NucleoSpin Tissue, Takara Bio, Otsu, Japan), and stored at 20 C. The cell growth yield of Nitrospira pure cultures was calculated based on cells produced per millimole of nitrite oxidized. Measurement of nitrite concentration Nitrite concentration in samples taken from nitrite-oxidizing activity experiments was measured using ion chromatography (IC 2010, Tosoh, Tokyo, Japan). Before measuring nitrite concentrations, samples were sterilized using a membrane filter with a pore size of 0.22 mm (Advantec, Tokyo, Japan). Extraction and quantification of protein The incubated samples in each experiment were concentrated by centrifuge (15,000 rpm, 15 min), and lysed by incubation with 0.15 M NaOH at 90 C for 30 min following a published protocol (28). Protein quantification was performed using the Qubit protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Calculation of Nitrospira cell numbers To calculate cell numbers of the two Nitrospira strains, qPCR was performed targeting the nxrB gene in the DNA extracted from each sample. The nxrB gene is known as a functional and phylogenetic marker gene for Nitrospira. Based on genomic information for both strains (unpublished data), the genome of strain ND1 contains one copy of the nxrB gene, and the strain NJ1 genome contains three copies of the nxrB gene. Thus, the cell numbers of strains

Please cite this article in press as: Ushiki, N., et al., Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.016

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ND1 and NJ1 were calculated by dividing the copy numbers of the nxrB gene in the extracted DNA by 1 and 3, respectively. qPCR targeting the nxrB genes was performed using primers 169f (50 -TACATGTGGTGGAACA-30 ) and 638r (50 CGGTTCTGGTCRATCA-30 ) (34). To confirm the detection of nxrB, we checked that the nxrB gene was amplified from DNA of both strains by PCR using the primer pair 169f638r with KOD SYBR qPCR Mix (Toyobo, Osaka, Japan). The following thermal profile was used in this nxrB gene amplification: an initial denaturation step was conducted at 98 C for 2 min, followed by 30 cycles of denaturation at 98 C for 10 s, annealing at 52 C for 10 s, and elongation at 68 C for 40 s. The amplification of the nxrB gene from the DNA of both strains was confirmed by electrophoresis using 1.5% agarose gels. The PCR products were purified using the Wizard SV Gel and PCR Clean-up System (Promega KK, Tokyo, Japan), and concentrations of the purified PCR products were measured using the Qubit dsDNA assay kit (Thermo Fisher Scientific). Based on the concentration and the length of the amplified nxrB genes, copy numbers of the purified nxrB genes were calculated, and the PCR products were diluted to create standard samples (102e108 nxrB gene copies/ml). The qPCR targeting the nxrB genes was performed using KOD SYBR qPCR Mix (Toyobo) and an Applied Biosystems Step One system (Thermo Fisher Scientific). The following thermal profile was used in the qPCR targeting nxrB: an initial denaturation step was conducted at 98 C for 2 min, followed by 40 cycles of denaturation at 98 C for 10 s, annealing at 52 C for 10 s, and elongation at 68 C for 40 s, with a melting curve analysis. The R2 values of the qPCRs targeting the nxrB genes were >0.99.

ammonia (0 mg-NH3 l1) was defined as 1, and the relative nitrite-oxidizing activity was calculated by dividing the amount of oxidized nitrite for each sample by the amount of oxidized nitrite in the absence of free ammonia. The concentration of free ammonia in each sample was calculated from the total ammonium concentration, temperature and pH, according to a previous report (29).

Inhibition of nitrite-oxidizing activity with free ammonia Nitriteoxidizing activity tests were performed to investigate inhibition of nitrite oxidation in pure Nitrospira cultures by different concentrations of free ammonia. Samples for the activity tests consisted of Nitrospira cultures (8 ml) and mineral medium (2 ml) with nitrite (20 mg-N l1) and NH4Cl (0e100 mg-N l1) in a 50-ml glass test-tube. The pH was adjusted to 8.0 and the temperature was 29 C. All samples were incubated in the dark with shaking at 100 rpm for 3 days. The nitrite concentration in each sample was measured on days 0 and 3, and the nitriteoxidizing activity of each sample was compared in the presence of different concentrations of free ammonia. The amount of nitrite oxidation without free

3

Inhibitory constants for free ammonia in nitrite oxidation kinetics The inhibitory constants (Ki) for free ammonia in nitrite oxidation kinetics were determined for strains ND1 and NJ1. The apparent kinetic parameters, a maximum nitrite 0 0 uptake rate Vmax_NO and a half-saturation constant Km NO2 , were determined 2 similarly to the above-mentioned experiments for determining kinetic parameters of nitrite oxidation but in the presence of different concentration of free ammonia. The inhibitory constants (Ki) for free ammonia in nitrite oxidation kinetics were calculated by fitting the apparent kinetic parameters to the inhibitory constant equations: 0 Vmax

NO2

¼ Vmax


 NO2

 1 þ ½ I  Ki


V

(3)

and 0 Km

NO2

¼ Km

NO2 $

 1 þ ½ I  Ki


K

(4)

0 is the apparent maximum nitrite uptake rate (mmol NO Here, Vmax_NO 2 /mg protein/h), 2 0 Vmax NO2 is the maximum nitrite uptake rate (mmol NO 2 /mg protein/h), Km_NO2 is the apparent half-saturation constant for nitrite uptake (mM NO 2 ), Km NO2 is the halfsaturation constant for nitrite uptake (mM NO 2 ), Ki_V is the inhibitory constant for Vmax NO2 (mg-NH3 l1), Ki_K is the inhibitory constant for Km NO2 (mg-NH3 l1), and I is 1 the free ammonia concentration (mg-NH3 l ). The data fitting was performed using non-linear least square method with solver in Excel.

FIG. 1. Nitrite oxidation kinetics of Nitrospira sp. strain ND1 and Nitrospira japonica strain NJ1. (A) Nitrite uptake of Nitrospira sp. strain ND1. (B) Nitrite uptake of Nitrospira japonica strain NJ1. (C) Nitrite uptake of Nitrospira sp. strain ND1 fitted a MichaeliseMenten plot. Circle plots indicate measured data. Solid line indicates fitted data. (D) Nitrite uptake of Nitrospira japonica strain NJ1 fitted a MichaeliseMenten plot. Square plots indicate measured data. Solid line indicates fitted data.

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RESULTS AND DISCUSSION Determination and comparison of kinetic parameters for nitrite oxidation of strains ND1 and NJ1 Nitrite concentration changes in a test-tube containing concentrated strain ND1 or NJ1 were measured using Griess reagent (Fig. 1A and B). Both concentrated strains consumed the nitrite initially added (200 mM NO 2 ) during incubation for 100e130 min. Using Eq. 1, the maximum nitrite uptake rate (Vmax NO2 ) and the half-saturation constant for nitrite (Km NO2 ) of each strain were calculated from the amounts of nitrite consumed (Fig. 1C and D). The Vmax NO2 and Km NO2 of strain ND1 were 45  7 (mmol NO 2 /mg protein/h) and 6  1 (mM NO 2 ), respectively (Table 1). The Vmax NO2 and Km NO2 of strain NJ1 were 31  5 (mmol NO 2 /mg protein/h) and 10  2 (mM NO 2 ), respectively. Comparing kinetic parameters for nitrite oxidation between strains ND1 and NJ1, their half-saturation constants for nitrite (Km NO2 ) were not significantly different, but their maximum nitrite uptake rates (Vmax NO2 ) were significantly different (Table 1). The kinetic parameters in nitrite oxidation reflect the ecological niche differentiation between Nitrospira lineages I (strain ND1) and II (strain NJ1) in our nitrifying bioreactor (32), from which we previously isolated ND1 and NJ1. In our bioreactor, a coexisting population of lineages I and II occurred at low nitrite concentration (0e71 mM NO 2 ), whereas the population of lineage I increased and became dominant at high nitrite concentration (143e429 mM NO 2) (32). The higher Vmax NO2 of strain ND1 belonging to lineage I could account for the dominant population of lineage I at high nitrite concentration. That there was no significant different in Km NO2 between strains ND1 and NJ1 would reflect the coexisting population of lineages I and II at low nitrite concentration (0e71 mM NO 2 ). Thus, the Vmax NO2 of strains ND1 and NJ1 would be a major factor providing the ecological niche differentiation between lineages I and II in our nitrifying bioreactor. Moreover, comparing kinetic parameters in nitrite oxidation among all Nitrospira cultures, the Km NO2 values of strains ND1 and NJ1 were very similar to those of N. defluvii and Nitrospira moscoviensis (Table 1) (28). The Vmax NO2 of Nitrospira lineage I (strains ND1 and N. defluvii) tends to be higher than that of Nitrospira lineage II (strains NJ1, N. moscoviensis and Nitrospira lenta) (Table 1) (28). Considering these consistent tendencies, ecological niche differentiation between lineages I and II based on differences in Vmax NO2 would not be expected to be exclusive to our bioreactor. In a previous report, kinetic parameters for nitrite oxidation were determined by stoichiometrically converting oxygen consumption measured using a microsensor system in NOB cultures into their nitrite consumption (28). In contrast, in our method,

nitrite uptake in pure Nitrospira cultures was directly determined using Griess regent (Figs. 1 and S1). The kinetic values obtained using our method were consistently similar to those for other Nitrospira cultures determined in previous study (Table 1). Our method was simple and enabled parallel determinations of nitrite oxidation kinetics with different concentrations of an inhibitor at the same time. Therefore, the measurement of nitrite concentrations using Griess reagent is expected to become a general procedure for the determination of kinetic parameters of pure NOB cultures.

Growth of pure Nitrospira cultures Strains ND1 and NJ1 in batch cultures consumed approximately 1 mM nitrite after 9 days of incubation (Fig. 2A and B). Unexpectedly, the cell number of strain ND1 in batch cultures increased slowly in the initial incubation (lag phase), but increased dramatically on days 8e9 (Fig. 2A and C). Based on the increase of the cell number on days 8e9, the maximum generation time of strain ND1 was 14 h (Table 1). The average generation time of strain ND1 between days 0 and 9 was 74 h (Table 1). In contrast, the cell number of strain NJ1 in batch cultures increased exponentially (Fig. 2B and C), and the maximum generation time of strain NJ1 was 19 h (Table 1). Since the cell number of strain NJ1 increased exponentially, the growth of strain NJ1 was approximated by an exponential equation (Fig. 2C). According to this equation, the average generation time of strain NJ1 was 39 h (Table 1). Besides, the growth yields of strains ND1 and NJ1 in batch cultures were calculated from the cell numbers and nitrite uptake. The growth yields of strains ND1 and NJ1 were 4.05 (109 cell/mmol NO 2 ) and 2.33 (109 cell/mmol NO 2 ), respectively (Table 1). Strain NJ1 increased its cell number exponentially in batch culture with nitrite, whereas strain ND1 grew unstably with a lag phase in batch culture (Fig. 2). Comparing the growth in mineral medium containing nitrite among Nitrospira strains, the maximum generation time (14 h) of strain ND1 was shorter than that of other Nitrospira strains, although the average generation time of strain ND1 including the lag phase (74 h) was long (Table 1). Similarly, it was reported that N. lenta grew in a batch culture with a lag phase and was inhibited by nitrite and nitrate (28,35). In batch culture, the cell growth of strain ND1 could be inhibited by nitrite or nitrate, which could cause a lag phase. However, nitrite consumption in the batch culture of strain ND1 was confirmed in the initial incubation (Fig. 2A), suggesting that its nitrite oxidizing activity was not likely inhibited by nitrite. Indeed, it was reported that nitrite oxidation of the two strains was not inhibited by 0.71e2.14 mM nitrite in our previous reports (30,31). Therefore, since inhibition of nitrite oxidation by nitrite

TABLE 1. Kinetic and growth parameters of pure NOB cultures. Organism (reference) Nitrobacter vulgaris (28) Nitrobacter hamburgensis (28) Nitrobacter winogradskyi (28) Nitrospira defluviia (28) Nitrospira sp. strain ND1a Nitrospira japonica strain NJ1b Nitrospira moscoviensisb (28) Nitrospira lentab (28) Nitrotoga arcticac (28) Nitrolancea hollandica (16)

Km NO2  SD (mM NO 2)

Vmax NO2  SD (mmol NO 2 /mg protein/h)

Generation time (h)

Growth yield (Log cell/mmol NO 2)

49  11 544  55 309  92 93 61 10  2 93 27  11 58  28 1000

164  9 64  1 78  5 48  2 45  7 31  5 18  1 20  2 26  3 NA

13 43 26 37 14 (74d) 19 (39d) 32 37 44 36

10.32 9.95 10.26 9.93 9.61 9.37 10.55 10.4 10.14 NA

NA indicates not available data. SD indicates standard deviation of three biological replicates. a Nitrospira lineage I. b Nitrospira lineage II. c Enriched culture. d Average generation time in hour.

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FIG. 2. Growth of two Nitrospira pure cultures in mineral medium with nitrite as batch cultures. (A) Growth of Nitrospira sp. strain ND1. Filled circle plots indicate consumption of nitrite. Open circle plots with dashed line indicate cell number of strain ND1. The time points at which maximum generation time were calculated are marked by arrows. (B) Growth of Nitrospira japonica strain NJ1. Filled square plots indicate consumption of nitrite. Open square plots with dashed line indicate cell number of strain NJ1. The time points at which the maximum generation time were calculated are marked by arrows. (C) Non-constant cell growth of Nitrospira sp. strain ND1 and exponential cell growth of Nitrospira japonica strain NJ1. Circle plots with dashed line indicate cell number of strain ND1. Square plots with dashed line indicate cell number of strain NJ1. Solid line indicates approximately exponential growth of strain NJ1. Error bars indicate standard deviation of three technical replicates.

was ignorable, the above-mentioned values of Vmax strains appeared correct.

NO2

of two

Oxygen affinity in nitrite oxidation of strains ND1 and NJ1 Dissolved oxygen concentration changes were measured in a test-tube containing concentrated strain ND1 or NJ1 using a microsensor system (Fig. S2A and B). From Eq. 2, a half-saturation constant for oxygen (Km O2 ) was calculated for each strain (Fig. S2C and D). The Km O2 values of strains ND1 and NJ1 were 4.0  2.5 and 2.6  1.1 (mM O2), respectively (Table 2). The Km O2 in nitrite oxidation were determined for the first time using pure Nitrospira cultures. There was no significant difference in the Km O2 between strains ND1 and NJ1 (Table 2). Since the isolation of Nitrospira is a hurdle, previous research used Nitrospiraenriched cultures instead of pure Nitrospira cultures to determine the Km O2 of Nitrospira (23). The Km O2 of the Nitrospira-enriched culture was 16.9  4.4 mM O2, which was much higher than the values we determined for strains ND1 and NJ1 (Table 2). It is likely that the difference in the Km O2 between pure cultures and the enriched culture was caused by uptake of dissolved oxygen by other microorganisms coexisting in the nitrifying bioreactor in the enriched culture. Park et al. (26) reported that dissolved oxygen

was an important factor in determining the composition of Nitrospira communities in a nitrifying bioreactor. According to their report, a population of Nitrospira lineages I and II coexisted at high dissolved oxygen concentration (266 mM O2) in the nitrifying bioreactor, whereas the population of lineage I was dominant at low dissolved oxygen concentration (3.75e7.5 mM O2) (26). However, in the present study, there was no significant difference in the Km O2 between strains ND1 (lineage I) and NJ1 (lineage II) (Table 2). Thus, the dissolved oxygen concentration might not be an important factor in determining the ecological niche differentiation between lineages I and II. The previously reported dominant lineage II population in a nitrifying bioreactor was phylogenetically distinct from strain NJ1 (26); thus, lineage II appears to contain physiologically diverse Nitrospira populations with different affinities for oxygen. Hanaki et al. (36) reported that dissolved oxygen concentration strongly affected nitrite accumulation in activated sludge, which was due to the difference of oxygen affinity between AOB and NOB. Indeed, the Km O2 of Nitrosomonas europaea, an AOB type strain, was 3.0e14.9 mM O2, whereas that of Nitrobacter winogradskyi, a NOB type strain, was 22.1e165.8 mM O2, suggesting the oxygen affinity of AOB was higher than that of NOB (37). However, here, the

Please cite this article in press as: Ushiki, N., et al., Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.016

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J. BIOSCI. BIOENG., TABLE 2. Oxygen affinity and inhibition by free ammonia of NOB.

Organism (reference) Nitrospira sp. strain ND1a Nitrospira japonica strain NJ1a Nitrospirab (23) Nitrobacterb (23) Nitrobacter winogradskyib (37) Nitrobacter winogradskyia (46)

Km

O2

 SD (mM O2)

Inhibition concentration of FA (mg-NH3/l)

Ki for Vmax NO2  SD (mg-NH3/l)

Ki for Km NO2  SD (mg-NH3/l)

0.85 4.3 0.04e0.08 50 NA 14.8

31  2.0 42  6.1 NA NA NA NA

8.5  0.9 16  2.3 NA NA NA NA

4.0  2.5 2.6  1.1 16.9  4.4 13.4  2.5 22.1e165.8 NA

NA indicates not available data. SD indicates standard deviation of three bilogical replicates. a Pure culture. b No pure culture.

Km O2 values of strains ND1 and NJ1 were 4.0  2.5 mM O2 and 2.6  1.1 mM O2, respectively (Table 2), which were not significantly different from that of N. europaea. Interestingly, Harada et al. (38) reported that nitrite was not accumulated in thick biofilms at the bottom in which the dissolved oxygen concentration was extremely low. Nitrospira is detected ubiquitously in biofilms and flocs in activated sludge (21). Therefore, nitrification is most likely performed by AOB and Nitrospira in low oxygen environments such as inside biofilms and flocs. According to previous studies, Vmax NO2 and Km NO2 are important factors for the ecological niche differentiation of Nitrobacter and Nitrospira (20,22,28). In this study, the difference in oxygen affinity between Nitrobacter and Nitrospira also appeared to be an important factor in the ecological niche differentiation among NOB (Table 2). We predict that the difference in oxygen affinity between Nitrobacter and Nitrospira is caused by the difference in the terminal oxidase in their electron transport chains. Based on previous genomic analyses of NOB, Nitrospira genomes contain a gene encoding a cytochrome bd oxidase, while Nitrobacter genomes contain a gene encoding a cytochrome c oxidase (39e42). It was reported that cytochrome bd oxidase was activated at low oxygen concentrations for the growth of Bacteroides fragilis as a strictly anaerobic bacterium (43). Thus, Nitrospira bacteria appear to possess high oxygen affinity because of the presence of cytochrome bd oxidase as the terminal oxidase in their electron transport systems.

FIG. 3. Inhibition of nitrite oxidation of two Nitrospira pure cultures in mineral medium including free ammonia. Circle plots indicate relative nitrite-oxidizing activity of strain ND1 with different free ammonia concentrations. Square plots indicate relative nitriteoxidizing activity of strain NJ1 with different free ammonia concentrations. Error bars indicate standard deviation of three technical replicates.

Inhibition of nitrite oxidation of strains ND1 and NJ1 by free ammonia The inhibition of nitrite oxidation of strains ND1 and NJ1 at different concentrations of free ammonia was evaluated by nitrite-oxidizing activity tests. The nitrite-oxidizing activity of strain ND1 was initially inhibited by a low concentration of free ammonia (0.85 mg-NH3 l1) (Fig. 3). The nitrite-oxidizing activity of strain NJ1 was not inhibited by low concentrations (0.85e2.1 mg-NH3 l1) of free ammonia; the threshold of inhibition was 4.3 mg-NH3 l1 (Fig. 3). Thus, strain NJ1 was more tolerant of free ammonia than strain ND1. Besides, the inhibitory constants (Ki) for free ammonia in nitrite oxidation were calculated from inhibitory constant equations Eqs. 3 and 4. Determination of Ki for both strains was performed similarly to the above-mentioned kinetic parameter experiments. The 0 0 apparent kinetic parameters Vmax NO2 and Km NO2 of both strains changed in mineral medium containing different concentrations of NH4Cl (Fig. 4). Based on Eqs. 3 and 4, Ki_V for Vmax and Ki_K for 0 0 Km were calculated from the values of Vmax NO2 and Km NO2 , respectively. The Ki_V and Ki_K of strain ND1 were 31  2.0 and 8.5  0.9 mg-NH3 l1, respectively, and the Ki_V and Ki_K of strain NJ1 were 42  6.1 and 16  2.3 mg-NH3 l1, respectively (Table 2). Thus, comparison of the inhibitory constants confirmed that strain NJ1 was more tolerant of free ammonia than strain ND1. It was previously known that free ammonia inhibits the nitrification process (29), and free ammonia leads to nitrite accumulation by inhibition of the nitrite-oxidizing activity of NOB (6,44,45). Recently, Blackburne et al. (23) reported that the inhibition thresholds of Nitrospira and Nitrobacter by free ammonia were 0.04e0.08 and 50 mg-NH3 l1, respectively (Table 2), based on enrichment samples in nitrifying bioreactors and measurements using a microsensor system. Based on pure culture, Sayavedra-Soto et al. (46) reported that Nitrobacter winogradskyi was inhibited by 35 mM NH4Cl (NH3: 14.8 mg-NH3 l1). The inhibition thresholds of free ammonia for strains ND1 and NJ1 in the present study were 0.85 and 4.3 mg-NH3 l1, respectively (Fig. 3 and Table 2). Comparing inhibition of pure NOB strains by free ammonia, Nitrospira appears more sensitive to free ammonia than Nitrobacter. Moreover, the inhibition threshold of strain ND1 by free ammonia was significantly lower than that of strain NJ1. Therefore, differences in tolerance to free ammonia appear to be an important factor in ecological niche differentiation of strains. Indeed, in previous study, it was reported that within a biofilm the density of lineage I in the vicinity of AOB appeared to be higher than that of lineage II (25). The vicinity of AOB within the biofilm appeared to contain high nitrite and low ammonia concentrations as a consequence of ammonia oxidation. Although Maixner et al. (25) assumed nitrite concentration was one important factor in the niche differentiation among different Nitrospira populations, it is likely that the free ammonia inhibition recognized in this study is another major factor. Although affinities for nitrite and oxygen, formate utilization, and relationships with AOB have been considered as major factors in Nitrospira ecological niche determination in previous studies

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FIG. 4. Inhibition of nitrite oxidizing activity of strain ND1 (A) and strain NJ1 (B) with different free ammonia concentrations. Circle plots indicate nitrite uptake without free ammonia and ammonium. Diamond plots indicate nitrite uptake with 10 mg total ammonium per liter. Square plots indicate nitrite uptake with 50 mg total ammonium per liter. Triangle plots indicate nitrite uptake with 100 mg total ammonium per liter.

(25e27), tolerance of free ammonia has not been considered to date. Recently, it was reported that Nitrospira possess activity for urea and cyanate degradation (42,47), and COMAMMOX-Nitrospira bacteria that oxidize ammonia were discovered (1,2). Since then, the difference in tolerance to free ammonia of Nitrospira that oxidize nitrite only, Nitrospira that produce ammonia and COMAMMOX-Nitrospira that oxidize ammonia, has provided an interesting insight into the microbial ecology of Nitrospira. Here, we successfully calculated Ki for free ammonia in two pure Nitrospira cultures (Table 2); remarkably, free ammonia inhibited the Vmax NO2 and Km NO2 of the cultures. Although the mechanism of inhibition of nitrite oxidation by the free ammonia is not known, the free ammonia is most likely toxic to Nitrospira cells. It was reported that nitrite oxidation by NOB was inhibited by several

factors, including free ammonia, temperature, pH, organic substances, salinity and antibiotics (6,23,48,49). Our method of calculation of Ki values is expected to be applied to the determination of Ki values for other parameters and inhibitors. In conclusions, we investigated kinetic and growth parameters, and inhibition by free ammonia, of strains ND1 and NJ1, to reveal the competition and partitioning into ecological niches of two Nitrospira lineages found in activated sludge. Comparing several physiological properties between strains ND1 and NJ1, such as affinities for nitrite and oxygen, maximum nitrite uptake rates, growth parameters, and inhibition by free ammonia, a significant difference in the tolerance of the two strains to free ammonia appeared to be an important factor for their competition and partitioning. Although environmental factors of Nitrospira ecological

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niches, such as affinities for nitrite and oxygen, formate utilization and relationships with AOB, have been considered in previous studies, tolerance of free ammonia was unrecognized to date. Free ammonia is generally found in activated sludge, and appears an important factor in the cooperation of NOB with AOB in nitrification in biological nitrogen removal processes. Thus, the tolerance of Nitrospira should be an emphasized physiological property alongside its kinetic and growth parameters. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2016.12.016.

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ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) (16K18609) (to H.F.) and by the Large Research Projects program of the Institute for Fermentation, Osaka (to S.T.). References 1. Daims, H., Lebedeva, E. V., Pjevac, P., Han, P., Herbold, C., Albertsen, M., Jehmlich, N., Palatinszky, M., Vierheilig, J., Bulaev, A., and other 6 authors: Complete nitrification by Nitrospira bacteria, Nature, 528, 504e509 (2015). 2. van Kessel, M. A., Speth, D. R., Albertsen, M., Nielsen, P. H., Op den Camp, H. J., Kartal, B., Jetten, M. S., and Lücker, S.: Complete nitrification by a single microorganism, Nature, 528, 555e559 (2015). 3. Okabe, S., Aoi, Y., Satoh, H., and Suwa, Y.: Nitrification in wastewater treatment, pp. 405e433, in: Ward, B. B., Arp, D. J., and Klotz, M. G. (Eds.), Nitrification. American Society for Microbiology, Washington, DC (2011). 4. Randall, C. W. and Buth, D.: Nitrite build-up in activated sludge resulting from temperature effects, J. Water Pollut. Control Fed., 56, 1039e1044 (1984). 5. Smith, R. V., Doyle, R. M., Burns, L. C., and Stevens, R. J.: A model for nitrite accumulation in soils, Soil Biol. Biochem., 29, 1241e1247 (1997). 6. Philips, S., Laanbroek, H. J., and Verstraete, W.: Origin, causes and effects of increased nitrite concentrations in aquatic environments, Rev. Environ. Sci. Biotechnol., 1, 115e141 (2002). 7. Alleman, J. E.: Elevated nitrite occurrence in biological wastewater treatment systems, Water Sci. Technol., 17, 409e419 (1984). 8. Bock, E. H., Koops, P., Harms, H., and Ahlers, B.: The biochemistry of nitrifying organism, pp. 171e200, in: Shively, J. M. and Barton, L. L. (Eds.), Variation in autotrophic life. Academic Press, San Diego, CA (1991). 9. Hooper, A. B. and DiSpirito, A. A.: In bacteria which grow on simple reductants, generation of a proton gradient involves extracytoplasmic oxidation of substrate, Microbiol. Rev., 49, 140e157 (1985). 10. Wood, P. M.: Nitrification as a bacterial energy source, pp. 39e62, in: Prosser, J. I. (Ed.), Nitrification. IRL, Oxford, UK (1986). 11. Yamanaka, T. and Fukumori, Y.: The nitrite oxidizing system of Nitrobacter winogradskyi, FEMS Microbiol. Rev., 4, 259e270 (1988). 12. Spieck, E., Hartwig, C., McCormack, I., Maixner, F., Wagner, M., Lipski, A., and Daims, H.: Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge, Environ. Microbiol., 8, 405e415 (2006). 13. Alawi, M., Lipski, A., Sanders, T., Pfeiffer, E. M., and Spieck, E.: Cultivation of a novel cold-adapted nitrite oxidizing betaproteobacterium from the Siberian Arctic, ISME J., 1, 256e264 (2007). 14. Alawi, M., Off, S., Kaya, M., and Spieck, E.: Temperature influences the population structure of nitrite-oxidizing bacteria in activated sludge, Environ. Microbiol. Rep., 1, 184e190 (2009). 15. Lücker, S., Schwarz, J., Gruber-Dorninger, C., Spieck, E., Wagner, M., and Daims, H.: Nitrotoga-like bacteria are previously unrecognized key nitrite oxidizers in full-scale wastewater treatment plants, ISME J., 9, 708e720 (2015). 16. Sorokin, D. Y., Lücker, S., Vejmelkova, D., Kostrikina, N. A., Kleerebezem, R., Rijpstra, W. I., Damste, J. S., Le Paslier, D., Muyzer, G., Wagner, M., van Loosdrecht, M. C., and Daims, H.: Nitrification expanded: discovery, physiology and genomics of a nitrite-oxidizing bacterium from the phylum Chloroflexi, ISME J., 6, 2245e2256 (2012). 17. Sorokin, D. Y., Vejmelkova, D., Lücker, S., Streshinskaya, G. M., Rijpstra, W. I., Sinninghe Damste, J. S., Kleerbezem, R., van Loosdrecht, M., Muyzer, G., and Daims, H.: Nitrolancea hollandica gen. nov., sp. nov., a chemolithoautotrophic nitrite-oxidizing bacterium isolated from a bioreactor belonging to the phylum Chloroflexi, Int. J. Syst. Evol. Microbiol., 64, 1859e1865 (2014). 18. Juretschko, S., Timmermann, G., Schmid, M., Schleifer, K. H., PommereningRoser, A., Koops, H. P., and Wagner, M.: Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge:

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