Low-temperature-resistant nitrosomonas bacteria and application thereof

Abstract

The present application relates to the technical field of microorganism, and provide a kind of low temperature resistant nitrosomonas and its application.The nitrosomonas is nitrosomonas (Nitrosomonas europaea.) H1, and the preservation number is CCTCC NO:M 2024693.The nitrosomonas H1 of the present application has the characteristics of low temperature resistance, grows well at 10 DEG C, and has good ammonia oxidation capacity, is suitable for the treatment of northern winter low temperature wastewater, and does not need to be heated or take other measures to maintain the stability of biochemical system.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a low-temperature-resistant nitrosomonas and its application. BACKGROUND

[0002] Ammonia nitrogen in water body is a common water pollution, and its main sources include agricultural discharge, industrial wastewater and municipal sewage, etc. In the water environment, ammonia nitrogen mainly exists in the form of ammonium ion (NH4 + ) and non-ionic ammonia (NH3). High concentration of ammonia nitrogen not only promotes eutrophication of water body and triggers water bloom, but also destroys the balance of water ecosystem, causing serious negative impact on human health and environment. Nitrification and denitrification are the key ways to treat ammonia nitrogen pollution in water body, and this process is considered as an efficient, sustainable and economic solution. Specifically, in the nitrification process, ammonia nitrogen is first converted into nitrite (NO2 - ), and then further converted into nitrate (NO3 - ); and in the denitrification process, nitrate is reduced to nitrogen (N2) or nitrous oxide (N2O).

[0003] In this conversion process, ammonia-oxidizing bacteria (AOB) plays a key role in converting ammonia nitrogen in water body into nitrite nitrogen, is the rate-limiting step of the entire nitrification process, and plays an important role in global nitrogen cycle. This type of bacteria has a wide application in wastewater treatment. However, ammonia-oxidizing bacteria are sensitive to environmental changes, and usually need to establish a close symbiotic relationship with other microorganisms, which makes their isolation and cultivation difficult and time-consuming.

[0004] Nitrosomonas is a major genus of ammonia-oxidizing bacteria, which maintains its growth by oxidizing ammonia nitrogen in water, and synthesizes the required substances by taking carbonate as carbon source and ammonia nitrogen as nitrogen source, and belongs to typical autotrophic bacteria. The patent with publication number CN112625940A reports a high-temperature-resistant nitrosomonas application, which exhibits good ammonia oxidation activity under high-temperature conditions, and has important practical significance for stable denitrification in high-temperature environment. However, there are few reports on nitrosomonas under low-temperature environment, therefore, developing a nitrosomonas which can survive and maintain good ammonia oxidation activity under low-temperature conditions has extremely important value for wastewater treatment in northern region in winter. SUMMARY

[0005] Therefore, the present application provides a low-temperature-resistant nitrosomonas H1, which has good ammonia oxidation performance, can quickly start in a short time, and has low-temperature resistance, and has good treatment effect when applied to wastewater treatment in northern region in winter.

[0006] The technical scheme of the present application is implemented as follows: in the first aspect, the present application provides a low-temperature-resistant Nitrosomonas europaea H1, with a preservation number of CCTCC M 2024693.

[0007] On the basis of the above technical scheme, preferably, the 16S rDNA nucleotide sequence of the Nitrosomonas europaea H1 is shown in SEQ ID NO: 1.

[0008] On the basis of the above technical scheme, preferably, the temperature tolerance of the Nitrosomonas europaea H1 is 10-40℃, the salinity is 0.2%-2%, and the pH value is 4-12.

[0009] In the second aspect, the present application provides a sewage treatment bacterial agent, comprising the above-mentioned Nitrosomonas europaea H1.

[0010] In the third aspect, the present application provides an application of the low-temperature-resistant Nitrosomonas europaea in winter low-temperature wastewater treatment, with a wastewater temperature of ≥10℃.

[0011] On the basis of the above technical scheme, preferably, the method comprises the following steps:

[0012] S1, culturing the Nitrosomonas europaea H1 at 10℃ for 3-5 rounds until the data is stable, then centrifuging (10000g, 6min) the Nitrosomonas europaea H1 cultured to the logarithmic growth phase, removing the supernatant, washing with sterilized normal saline for 3 times, and obtaining a mother liquor of the Nitrosomonas europaea H1;

[0013] The components of the autotrophic culture medium are as follows: ammonium chloride (NH4Cl) 0.4-0.6g / L, magnesium sulfate (MgSO4) 0.5-0.7g / L, potassium hydrogen phosphate (K2HPO4) 0.1-0.2g / L, sodium bicarbonate (NaHCO3) 0.5-1.5g / L, iron trichloride hexahydrate (FeCl3·6H2O) 0.34-0.70g / 10mL, EDTA 0.12-0.56g / 10mL (added to 1L of the culture medium after sterilization), sodium chloride (NaCl) 0.2-2g / L, trace element stock solution 1mL, distilled water 1000mL, and pH 6-9.

[0014] The components of the trace element stock solution are as follows: copper sulfate pentahydrate (CuSO4·5H2O) 0.1g / L, zinc sulfate heptahydrate (ZnSO4·7H2O) 0.5g / L, cobalt chloride hexahydrate (CoCl2·6H2O) 0.5g / L, manganese sulfate tetrahydrate (MnSO4·4H2O) 0.2g / L, sodium molybdate dihydrate Na2MoO4·2H2O 0.1g / L, and boric acid (H3BO3) 0.1 / L.

[0015] The autotrophic culture medium is sterilized in a high-pressure sterilization pot at 103.4 kpa and 121 DEG C for 20 min, and is cooled to room temperature;

[0016] S2, the mother liquor of the obtained Nitrosomonas H1 is added into the cooled autotrophic culture medium for fermentation culture, to obtain liquid bacterial agent of the Nitrosomonas H1; then the obtained liquid bacterial agent of the Nitrosomonas H1 is added into the low-temperature wastewater.

[0017] The low-temperature-resistant Nitrosomonas and the application thereof have the following beneficial effects relative to the prior art:

[0018] (1) The low-temperature-resistant Nitrosomonas (Nitrosomonas europaea.) H1 has the low-temperature-resistant property, grows well at 10 DEG C, has good ammonia oxidation capacity, and is suitable for the treatment of low-temperature wastewater in the northern winter, without the need of heating or taking other measures to maintain the stability of the biochemical system. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0020] Figure 1 It is a morphological feature of Nitrosomonas H1 under scanning electron microscope;

[0021] Figure 2 It is a denitrification performance graph of Nitrosomonas H1 under different salinities;

[0022] Figure 3 It is a denitrification performance graph of Nitrosomonas H1 under different temperatures;

[0023] Figure 4 It is a denitrification performance graph of Nitrosomonas H1 under different pH values;

[0024] Figure 5 It is a denitrification performance graph of Nitrosomonas H1 under different rotating speeds;

[0025] Figure 6 It is a denitrification performance graph of Nitrosomonas H1 under different salinities at low temperature.

[0026] Figure 7 It is a denitrification performance graph of Nitrosomonas H1 under different pH values at low temperature.

[0027] Figure 8 Figure A is a denitrification performance diagram of the Nitrosomonas europaea H1 at different rotating speeds at low temperature.

[0028] Figure 9 Figure B is an application effect of the Nitrosomonas europaea H1 in low-temperature wastewater.

[0029] Figures 2-9 In the figures, A is a denitrification performance diagram of ammonia nitrogen, and B is a denitrification performance diagram of nitrous nitrogen. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0031] The present application provides a low-temperature-resistant Nitrosomonas europaea, which is Nitrosomonas europaea H1, deposited in the China Center for Type Culture Collection (CCTCC) in Wuhan, China, on April 15, 2024, with the deposit number CCTCC NO: M 2024693, and identified as active on April 22, 2024.

[0032] The present application also provides a sewage treatment bacterial agent containing Nitrosomonas europaea H1, and application of the low-temperature-resistant Nitrosomonas europaea H1 in winter low-temperature wastewater treatment.

[0033] The present application is further explained and described by the following examples, but does not constitute a limitation on the present application. It should be understood that these examples are only used to illustrate the present application and do not limit the scope of the present application.

[0034] Example 1: Isolation and identification of Nitrosomonas europaea H1

[0035] The present application provides a low-temperature-resistant Nitrosomonas europaea H1, which was isolated from a natural foul-smelling water body in 2022.

[0036] 1.1 Isolation method

[0037] The isolation method of Nitrosomonas H1 is as follows: 2 mL of natural foul-smelling wastewater is inoculated into 100 mL of sterilized autotrophic nitrification medium, which is placed in a constant temperature incubator (10°C, 150 rpm) for 3-5 days, 5 mL of the enriched bacterial liquid is transferred into the autotrophic nitrification medium containing 100 mg / L of ammonia nitrogen, and is cultured in the constant temperature incubator, and the ammonia nitrogen and nitrous nitrogen are detected in turn, the ammonia oxidation performance is tested, and the growth curve is drawn, and the next round of culture is carried out by subculturing at a ratio of 1% in the logarithmic growth phase. After 3-5 rounds of repeated enrichment culture, the enrichment culture is diluted, and then coated on the solid medium. When the colonies can be observed by the naked eye, a single colony is selected and inoculated into the sterilized autotrophic medium for 3-5 days, gradient dilution (10 -1 ~10 -8 ) is carried out, and 10 -8 dilutions are selected for large-scale culture, and the plate coating is confirmed to be free of impurities, so that a pure culture of the low-temperature resistant Nitrosomonas H1 is obtained.

[0038] The autotrophic nitrification medium for strain enrichment and isolation comprises the following components: ammonium chloride (NH4Cl) 0.4 g / L, magnesium sulfate (MgSO4) 0.05 g / L, potassium hydrogen phosphate (K2HPO4) 0.01 g / L, sodium bicarbonate (NaHCO3) 0.5 g / L, ferric chloride hexahydrate (FeCl3·6H2O) 0.052 g / 10 mL (added separately into 1 L of medium after sterilization), sodium chloride (NaCl) 2 g / L, trace elements 1 mL, distilled water 1000 mL, and pH value 7-8.

[0039] The formula of the trace element stock solution is: copper sulfate pentahydrate (CuSO4·5H2O) 0.1 g / L, zinc sulfate heptahydrate (ZnSO4·7H2O) 0.5 g / L, cobalt chloride hexahydrate (CoCl2·6H2O) 0.5 g / L, manganese sulfate tetrahydrate (MnSO4·4H2O) 0.2 g / L, sodium molybdate dihydrate Na2MoO4·2H2O 0.1 g / L, and boric acid (H3BO3) 0.1 / L.

[0040] The solid medium is obtained by adding 1.5% of agar powder by mass to the enrichment medium.

[0041] 1.2 Strain identification

[0042] 1.2.1 Morphological and physiological and biochemical characteristics of bacteria

[0043] Morphological characteristics of bacteria: the colony morphological characteristics of the strain H1 are transparent light yellow, needle tip size, and the cells are rod-shaped, 1-1.5 μm long and about 0.5 μm wide. The cell suspension is light yellow during the enrichment culture process (see Figure 1 ).

[0044] Physiological and biochemical characteristics: Strain H1 is a gram-negative bacterium with good ammonia oxidation capacity.

[0045] 1.2.2 16S rDNA identification of strain H1

[0046] The 16S rRNA was amplified by using bacterial genome as DNA template, 27F (5'- AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 25 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25 cycles; 72℃ final extension for 5 min. After amplification, the PCR product was subjected to 1% agarose gel electrophoresis to detect the integrity of the PCR product. Finally, the PCR product was sent to the sequencing department of Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing.

[0047] The effective length of the 16S rDNA gene amplified was about 1443 bp nucleotide sequence, and the 16S rDNA sequence was shown as SEQ ID NO: 1. The sequence was input into GenBank, and the Blast software was used for comparison and analysis with the database sequence. The results showed that the similarity with the 16S rDNA sequence of Nitrosomonas europaea. was high, which was 99.93%. Based on the phylogenetic analysis results of 16S rDNA gene and physiological and biochemical characteristics, it was identified as a new strain of Nitrosomonas europaea., named Nitrosomonas europaea. H1 (hereinafter referred to as "strain H1"). The strain was preserved in China Center for Type Culture Collection, and the preservation number was CCTCC NO: M 2024693.

[0048] Example 2 Preparation of strain H1

[0049] The preparation method of strain H1 includes the following steps:

[0050] S1, culturing Nitrosomonas europaea. H1 at 0.2% salinity and 10℃ for 3-5 rounds until the data is stable. Then, centrifuging Nitrosomonas europaea. H1 in the logarithmic growth phase, removing the supernatant, and washing with sterilized physiological saline for three times to obtain the mother liquor of Nitrosomonas europaea. H1;

[0051] S2, configuring autotrophic nitrification medium. The medium was sterilized in a high-pressure sterilization pot at 103.4 kpa and 121℃ for 20 min, and then cooled to room temperature;

[0052] S3, the mother liquor of the Nitrosomonas H1 obtained in step S1 is added into the culture medium cooled to room temperature in step S2 for fermentation culture, to obtain a liquid bacterial solution of the Nitrosomonas H1, i.e. a bacterial suspension.

[0053] Example 3 Strain H1 Test

[0054] 3.1 Denitrification effect of strain H1 at different salinities

[0055] Under the conditions of an ammonia nitrogen concentration of 100 mg / L, a temperature of 30°C, and a pH of 8, the bacterial suspension of strain H1 was inoculated into 100 mL of autotrophic nitrification medium in a 250 mL triangular flask at an inoculation ratio of 1%, and was cultured at different salinities (0.2%, 0.5%, 1%, 1.5%, and 2%). During the culture process, the shaking speed was set to 150 rpm. The ammonia nitrogen and nitrite nitrogen were alternately detected during the culture process, the denitrification performance was tested, and the growth curve was plotted. The results are shown in Figure 2 .

[0056] As shown in Figure 2 , the strain can grow and exhibit autotrophic nitrification activity at a salinity of 0.2%-2%, and the ammonia nitrogen removal rate can reach 57.78%-100% in 72 h. The data show that strain H1 can remove ammonia nitrogen in 72 h at a salinity of less than 1%.

[0057] 3.2 Denitrification effect of strain H1 at different temperatures

[0058] Under the conditions of an ammonia nitrogen concentration of 100 mg / L, a salinity of 0.2%, and a pH of 8, the bacterial suspension of strain H1 was inoculated into 100 mL of autotrophic nitrification medium in a 250 mL triangular flask at an inoculation ratio of 1%, and was cultured at different temperatures (10°C, 20°C, 30°C, 40°C, and 50°C). During the culture process, the shaking speed was set to 150 rpm. The ammonia nitrogen and nitrite nitrogen were alternately detected during the culture process, the denitrification performance was tested, and the growth curve was plotted. The results are shown in Figure 3 .

[0059] As shown in Figure 3 , the strain can grow at a temperature of 10-40°C, and the strain growth and ammonia nitrogen conversion efficiency are higher as the temperature increases. The ammonia nitrogen degradation rate all reaches more than 99% in 72 h, and the strain stops growing at 50°C.

[0060] 3.3 Denitrification effect of strain H1 at different pHs

[0061] Under conditions of 100 mg / L ammonia nitrogen concentration, 30℃ culture temperature, and 0.2% salt concentration, a bacterial suspension of strain H1 was inoculated at a 1% inoculation ratio into 250 ml Erlenmeyer flasks containing 100 mL of autotrophic nitrification medium. The flasks were cultured at different pH values ​​(4, 6, 8, 10, and 12) with a rotation speed of 150 rpm. Ammonia nitrogen and nitrite nitrogen were alternately measured during the culture process to test the nitrogen removal performance, and growth curves were plotted. The results are shown below. Figure 4 .

[0062] Figure 4 As shown, this strain exhibits good growth adaptability within a pH range of 4-12. Specifically, within the pH range of 6-10, the growth and ammonia nitrogen conversion efficiency of the strain increase with increasing pH. Within 72 hours, strain H1 achieved an ammonia nitrogen degradation rate exceeding 99%, after which the strain's activity decreases with further increases in pH. Notably, at pH=4 and pH=12, the growth and metabolism of the strain are inhibited to some extent, but it still maintains a certain ammonia nitrogen conversion capacity. Compared to alkaline conditions, the strain demonstrates good acid tolerance.

[0063] 3.4 Denitrification effect of strain H1 at different rotation speeds

[0064] Under conditions of 100 mg / L ammonia nitrogen, sodium acetate as carbon source, pH 8.0, culture temperature 30℃, and salt concentration 0.2%, a bacterial suspension of strain H1 was inoculated at a 1% inoculation ratio into 250 mL Erlenmeyer flasks containing 100 mL of autotrophic nitrification medium. The flasks were then cultured at different rotation speeds (0 rpm, 50 rpm, 100 rpm, 150 rpm, and 200 rpm). Ammonia nitrogen and nitrite nitrogen were alternately measured during the culture process to test the nitrogen removal performance, and growth curves were plotted. The results are shown below. Figure 5 .

[0065] like Figure 5 As shown, within the shaking speed range of 0-200 rpm, the removal efficiency of ammonia nitrogen by strain H1 significantly increased with increasing shaking speed. When the shaking speed exceeded 50 rpm, the ammonia nitrogen degradation rate of strain H1 remained at approximately 100% for 72 hours. Further increasing the speed to above 100 rpm, strain H1 completely degraded ammonia nitrogen with an initial concentration of 100 mg / L in just 48 hours. These results indicate that optimizing the shaking speed has a significant impact on improving the ammonia nitrogen removal efficiency of strain H1.

[0066] Example 4: Tolerance of strain to salinity, rotation speed, and pH at 10°C.

[0067] 4.1 Denitrification effect of strain H1 at low temperature and different salinities

[0068] Under the conditions of ammonia nitrogen concentration of 100 mg / L, temperature of 10℃, pH of 8, the bacterial suspension of strain H1 was inoculated into 250 mL triangular flask containing 100 mL autotrophic nitrification medium at 1% inoculation ratio, and cultured under different mass fraction of salinity (0.2%, 0.5%, 1%, 1.5%, 2%). The rotation speed of the shaker was set to 150 rpm during the culture process. The ammonia nitrogen and nitrite nitrogen were detected every 24 h to test the denitrification performance, and the growth curve was drawn. The results are shown in Figure 6 .

[0069] Figure 6 As shown in the figure, under the synergistic effect of low temperature and high salinity, the denitrification performance of strain H1 decreased. Strain H1 could completely metabolize 100 mg / L of ammonia nitrogen within 72 h under the conditions of 10℃ and 0.2% salinity. With the increase of salinity, the performance decreased significantly. Strain H1 could still metabolize ammonia nitrogen under the conditions of 2% salinity, but the conversion rate was less than 10% within 120 h.

[0070] 4.2 Denitrification effect of strain H1 under low temperature and different pH

[0071] Under the conditions of ammonia nitrogen concentration of 100 mg / L, culture temperature of 10℃, and salinity of 0.2%, the bacterial suspension of strain H1 was inoculated into 250 mL triangular flask containing 100 mL autotrophic nitrification medium at 1% inoculation ratio, and cultured under different pH (4, 6, 8, 10, 12). The rotation speed was set to 150 rpm during the culture process. The ammonia nitrogen and nitrite nitrogen were detected every 24 h to test the denitrification performance, and the growth curve was drawn. The results are shown in Figure 7 .

[0072] Figure 7 As shown in the figure, under the low temperature and alkaline environment, strain H1 was significantly inhibited, and stopped growing at pH = 12. However, it still had certain denitrification efficiency under acidic conditions. Strain H1 could tolerate the minimum pH = 4 under low temperature environment, and could completely metabolize 100 mg / L of ammonia nitrogen within 120 h at pH = 6.

[0073] 4.3 Denitrification effect of strain H1 under low temperature and different rotation speeds

[0074] Under the conditions of ammonia nitrogen concentration of 100 mg / L, sodium acetate as carbon source, pH of 8.0, culture temperature of 10℃, and salinity of 0.2%, the bacterial suspension of strain H1 was inoculated into 250 mL triangular flask containing 100 mL of the above-mentioned medium at 1% inoculation ratio, and cultured under different rotation speeds (0 rpm, 50 rpm, 100 rpm, 150 rpm, 200 rpm). The ammonia nitrogen and nitrite nitrogen were detected every 24 h to test the denitrification performance, and the growth curve was drawn. The results are shown in Figure 8 .

[0075] As shown in the figure, under the conditions of 10℃ and 0.2% salinity, strain H1 could completely metabolize 100 mg / L of ammonia nitrogen within 72 h. With the increase of rotation speed, the performance decreased significantly. Strain H1 could still metabolize ammonia nitrogen under the conditions of 200 rpm, but the conversion rate was less than 10% within 120 h. Figure 8As shown, at 0 rpm, the growth of the strain was significantly inhibited, and it was in the stationary phase within 120h, and then the denitrification ability was enhanced with the increase of the rpm, and the optimal rpm was still 150rpm, and at 100rpm and 200rpm, the strain H1 had nearly similar denitrification rates.

[0076] Example 5 Application effect of the strain H1 in low-temperature wastewater

[0077] 30L sewage was collected from a waste transfer station in Xunying District, Yichang City, Hubei Province, and the initial water quality parameters of the sewage were as follows: ammonia nitrogen 2236mg / L, nitrite nitrogen 30mg / L, and salinity 2%. The sewage was divided into 6 plastic cups with a volume of 8L, 3 of which were added with 1% strain H1 bacterial suspension (OD600≈0.01), and the other 3 were used as a control group (without adding bacterial agents); all the barrels were placed in a 10℃ incubator for cultivation, and at the same time, a microporous sand core air head was used for aeration, and the aeration amount was 180mL / min; during the test, the changes of ammonia nitrogen and nitrite nitrogen in the system were detected regularly to analyze the denitrification ability of the strain, and the results are shown in Figure 9 .

[0078] Figure 9 As shown, after 9 days, the ammonia nitrogen removal rate of the inoculated group reached 100%, while the control group had almost no ammonia nitrogen removal, and the denitrification efficiency of the strain in the sewage was very significant; thus, it is proved that the strain H1 has good low-temperature ammonia oxidation ability.

[0079] The above only describes the preferred embodiments of the present application and should not be used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0080]

[0081]

Claims

1. A low temperature resistant Nitrosomonas bacterium, characterized in that: The said Nitrosomonas is Nitrosomonas eutropha (ATCC 19778) Nitrosomonas europaea. ) H1, with the preservation number of CCTCC NO: M 2024693; The Nitrosomonas europaea H1 has a tolerance temperature of 10-40℃, a salinity of 0.2%-2%, and a pH value of 4-12.

2. The psychrophilic Nitrosomonas bacterium of claim 1, wherein: The 16S rDNA nucleotide sequence of the Nitrosomonas europaea H1 is shown as SEQ ID NO:

1.

3. A sewage treatment bacterial agent, characterized by: The Nitrosomonas europaea H1 according to any one of claims 1-2.

4. The use of a psychrotolerant Nitrosomonas bacteria according to any one of claims 1 to 2 for the treatment of wastewater at low winter temperatures, characterized in that: The wastewater temperature is ≥10℃.

5. The use of a psychrotolerant Nitrosomonas bacteria according to claim 4 for the treatment of wastewater at low winter temperatures, characterized in that: The method comprises the following steps: S1, culturing the Nitrosomonas europaea H1 at 10℃ to the logarithmic growth phase, centrifuging, removing the supernatant, and obtaining the mother liquor of the Nitrosomonas europaea H1 after washing; S2, fermenting and culturing the mother liquor of the Nitrosomonas europaea H1 obtained in step S1, and then adding it into the low-temperature wastewater to degrade ammonia nitrogen.

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