Nitrosomonas sp. And application of nitrosomonas sp. In ammonia nitrogen degradation under reverse conditions

Abstract

The invention relates to the technical field of microorganisms and environmental protection, in particular to a strain of European nitrosomonas and application thereof in ammonia nitrogen degradation under a reverse condition. The European nitrosomonas sp. SZG-AOB-004 is classified and named as Nitrosomonas sp. SZG-AOB-004, the preservation number of the European nitrosomonas sp. SZG-AOB-004 is CCTCC (China Center For Type Culture Collection) NO: M20251507, and the European nitrosomonas sp. SZG-AOB-004 not only has a higher nitration rate than the same European nitrosomonas sp. SZG-AOB-004, but also can keep a higher nitration rate under the inverse conditions of high alkalinity, high salinity or containing benzene series, toxic metal and the like, and has a better application prospect in degrading ammonia nitrogen in sewage.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms and environmental protection, and particularly relates to a strain of Nitrosomonas europaea and application of the strain in degradation of ammonia nitrogen under adverse conditions. BACKGROUND

[0002] Ammonia-oxidizing bacteria (AOB) is the core microorganism of nitrogen cycle, which can oxidize ammonia nitrogen (NH3 / NH4 + ) into nitrite (NO2 - ). Since the metabolic mode of ammonia-oxidizing bacteria is chemoautotrophic, ammonia nitrogen is used as energy, the growth is slow, the average generation time is more than 10h, and the pure culture process is easily contaminated by heterotrophic bacteria. It is difficult to obtain pure culture by using traditional separation method. Winogradsky first isolated a strain of AOB named Nitrosomonas europaea in 1904, which has been widely studied as a model strain in the following more than 100 years.

[0003] The most common AOB is Nitrosomonas europaea, and there are still limited reports on pure culture strains of the AOB. The strain has low resistance to toxic and harmful substances such as high alkalinity, high salinity, benzene series and toxic metals, and cannot efficiently remove ammonia nitrogen in these adverse environments. SUMMARY

[0004] Therefore, the present application provides a strain of Nitrosomonas europaea and application thereof, so as to solve the problem that the existing Nitrosomonas europaea cannot efficiently remove ammonia nitrogen under the conditions of high alkalinity, high salinity, benzene series, toxic metals and other toxic and harmful substances.

[0005] The technical scheme of the present application is as follows:

[0006] In a first aspect, the present application provides an ammonia-oxidizing bacteria, which is Nitrosomonas europaea SZG-AOB-004, the classification name of which is Nitrosomonas europaea SZG-AOB-004, the preservation number of which is CCTCC NO: M20251507, and which was preserved in China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on July 2, 2025; and the 16S rRNA sequence (SEQ ID NO. 1) thereof is as follows:

[0007] AAGTCGAACGGCAGCGGGGGCTTCGGCCTGCCGGCGAGTGGCGAACGGG

[0008] TGAGTAATGCATCGGAACGTGTCCTTAAGTGGGGAATAACGCATCGAAAGA

[0009] TGTGCTAATACCGCATATCTCTGAGGAGAAAAGCAGGGGATCGTAAGACCT

[0010] TGCGCTAAAGGAGCGGCCGATGTCTGATTAGCTAGTTGGTGGGGTAAAGG

[0011] CTTACCAAGGCAACGATCAGTAGTTGGTCTGAGAGGACGGCCAACCACAC

[0012] TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAAT

[0013] TTTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGAGTGAAGAA

[0014] GGCCTTCGGGTTGTAAAGCTCTTTTAGTCGGAAAGAAAGGATTGCAACGA

[0015] ATAATTGTGATTTATGACGGTACCGACAGAAAAAGCACCGGCTAACTACGT

[0016] GCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGG

[0017] GCGTAAAGGGTGCGCAGGCGGTTTTGCAAGTCAGATGTGAAAGCCCCGGG

[0018] CTTAACCTGGGAATTGCGTTTGAAACTACAAGGCTAGAGTGCAGCAGAGG

[0019] GGAGTGGAATTCCATGTGTAGCAGTGAAATGCGTAGAGATGTGGAAGAAC

[0020] ACCGATGGCGAAGGCAGCTCCCTGGGTTGACACTGACGCTCATGCACGAA

[0021] AGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAAC

[0022] GATGTCAACTGGTTGTCGGATCTAATTAAGGATTTGGTAACGTAGCTAACG

[0023] CGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGG

[0024] AATTGACGGGGACCCGCACAAGCGGTGGATTATGTGGATTAATTCGATGCA

[0025] ACGCGAAAAACCTTACCTACCCTTGACATGCTTGGAATCTAATGGAGACAT

[0026] AAGAGTGCCCGAAAGGGAGCCAAGACACAGGTGCTGCATGGCTGTCGTC

[0027] AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT

[0028] GTCACTAATTGCTATCATTTTTAATGAGCACTTTAGTGAGACTGCCGGTGAC

[0029] AAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGT

[0030] AGGGCTTCACACGTAATACAATGGCGTGTACAGAGGGTTGCCAACCCGCG

[0031] AGGGGGAGCCAATCTCAGAAAGCACGTCGTAGTCCGGATCGGAGTCTGCA

[0032] ACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGC

[0033] GGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGT

[0034] GGTTTTCACCAGAAGCAGGTAGCTTAACCGCAAGGAGGGCGCTTGCCACGGTGGGGGTCATGACTGGGGTGAAGTCGAAAGG.

[0035] In a second aspect, there is provided the use of the ammonia-oxidizing bacteria as described above in degrading ammonia nitrogen in wastewater.

[0036] Preferably, the ammonia nitrogen content in the wastewater is less than or equal to 35 mg / L.

[0037] Preferably, the alkalinity in the wastewater is less than or equal to 30000 ppm.

[0038] Preferably, the salinity in the wastewater is less than or equal to 30000 ppm.

[0039] Preferably, the content of benzene series in the wastewater is less than or equal to 20 mg / L.

[0040] Preferably, the benzene series comprises at least one of phenol and aniline.

[0041] Preferably, the wastewater contains heavy metal ions Cd 2+ and Hg 2+ .

[0042] Preferably, the content of Cd 2+ is less than or equal to 0.3 mg / L, and the content of Hg 2+ is less than or equal to 0.03 mg / L.

[0043] In a third aspect, there is provided a microbial agent for degrading ammonia nitrogen in wastewater, preferably comprising the ammonia-oxidizing bacteria as described above.

[0044] The European Nitrosomonas of the present application has the following beneficial effects over the prior art:

[0045] The European Nitrosomonas SZG-AOB-004 screened in the present application has a nitrification rate of up to 600 mg NH4 + -N / L / h after fermentation, which is higher than the nitrification rate of similar microbial agent products. Meanwhile, the strain can not only normally reproduce but also exert good nitrification capacity in the presence of high alkalinity, high salinity, and benzene series, toxic metals, and other adverse environments, helping factories or sewage plants to better and faster establish a nitrification system, degrade ammonia nitrogen in the incoming water until the discharge standard is met, and protect the environment. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and all other embodiments obtained by those of ordinary skill in the art without creative effort based on these accompanying drawings also belong to the protection scope of the present application.

[0047] Figure 1 Figure for alkalinity resistance of European Nitrosomonas in the present application;

[0048] Figure 2 Figure for salinity resistance of European Nitrosomonas in the present application;

[0049] Figure 3 Figure for aniline resistance of European Nitrosomonas in the present application;

[0050] Figure 4 Figure for phenol resistance of European Nitrosomonas in the present application;

[0051] Figure 5 Figure for toxic metal resistance of European Nitrosomonas in the present application;

[0052] Figure 6 Figure for ammonia nitrogen degradation change of European Nitrosomonas in the present application in electroplating wastewater;

[0053] Figure 7 Figure for ammonia nitrogen degradation change of European Nitrosomonas in the present application in industrial wastewater. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments only constitute some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0055] The formula of AOB culture medium used in the present application is as follows:

[0056] Ingredients (1L): potassium dihydrogen phosphate 54mg, potassium chloride 74mg, magnesium sulfate heptahydrate 49mg, calcium chloride dihydrate 147mg, sodium chloride 584mg, calcium carbonate 5g; the solid culture medium also contains 10g agar powder.

[0057] Each component is weighed according to the above formula, 10% NaHCO3 is used to adjust pH to 8.0, 90mL per bottle, and sterilized at 121℃ for 20 minutes.

[0058] Example 1: Screening of AOB strain

[0059] 1. Enrichment of AOB strains

[0060] (1) Water samples from each sewage plant, pharmaceutical factory, chemical plant, etc. are divided into pharmaceutical, petrochemical, chemical, resin, coal wastewater, electroplating / battery, food, municipal domestic wastewater according to the type;

[0061] (2) The medium is improved according to the characteristics of each water sample, and adverse conditions are added, as shown in Table 1.

[0062] Table 1 AOB experimental grouping

[0063]

[0064] (3) Take 10 mL of the bottom sludge sample of the above water sample, add 90 mL of enrichment liquid medium, 1 mL of ammonium sulfate, and cultivate at 30°C, 150 rpm, in the dark;

[0065] (4) Adjust the pH to 8 using sodium bicarbonate every morning and evening.

[0066] 2. Transfer of AOB strains

[0067] The transfer amount is 10 vol%, centrifuged at 8000 rpm for 10 min, and transferred to a new culture medium.

[0068] 3. Screening and purification of AOB strains

[0069] The samples with good enrichment effect are respectively transferred to 10 -2 , 10 -3 and 10 -4The agar plates were coated, 2 plates for each dilution, and then cultured in a 30°C constant temperature incubator. The culture time of the agar plates was 4-5 weeks; single colonies on the agar plates were selected and streaked on agar plates for 4-5 weeks of moist culture at 30°C; single colonies on the streaked plates were picked and transferred to liquid culture medium for culture for 3 weeks, and the sub-salt content was detected every week; the strain culture liquid with nitrification reaction was streaked on LB to verify whether it was mixed with heterotrophic bacteria. If it contains heterotrophic bacteria, it needs to be further streaked and separated until a pure strain is obtained. The purified AOB strain was subjected to 16S rRNA sequencing to confirm its species. After repeated screening and purification, a pure strain of AOB was finally screened, identified by 16S rRNA sequencing, and the sequence is shown as SEQ ID NO. 1, which is Nitrosomonas europaea, named Nitrosomonas europaea SZG-AOB-004, and its classification is Nitrosomonas europaea SZG-AOB-004, which was preserved in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on July 2, 2025, and the preservation number is CCTCC NO: M 20251507.

[0070] 4. Culture of AOB strain

[0071] An AOB enrichment culture medium was prepared, and AOB bacterial liquid was added to the culture medium, with a total inoculation amount of 10 vol%. 1 mL of 100 g / L ammonium sulfate solution was added as feed each time, and the ammonia nitrogen consumption in the shake flask was detected qualitatively every day. When the ammonia nitrogen was consumed, 1 mL of feed solution was added again, and 100 g / L NaHCO3 solution was used to adjust the pH to 8.5-9.0. After 150 h of feed culture, the nitrification rate was detected according to the industry standard “HG / T 5748-2020 Performance Evaluation Method for Nitrifying Bacteria for Water Treatment”.

[0072] Example 2: Evaluation of stress resistance of different AOB strains

[0073] 1. Strain: Four AOB strains were used in this example, all of which were Nitrosomonas europaea, but the actual stress resistance was different. Among them, A1 strain was a model strain Nitrosomonas europaea ATCC 25978 purchased from a preservation agency, B1 was a commercially available nitrifying bacteria product Nitrosomonas europaea B1, C1 was a previously screened Nitrosomonas europaea SZG-AOB-002, preservation number CCTCC NO: M 20232716, and the other was the Nitrosomonas europaea SZG-AOB-004 of the application (referred to as AA-1).

[0074] 2. Evaluation method

[0075] The above four AOB strains were all cultured to the logarithmic growth phase, the bacterial cells were collected by centrifugation, and resuspended in PBS to the same biomass (OD 600 = 0.5 ± 0.05). The culture medium used in the experiment was the national standard nitrification rate detection medium, and the performance comparison index was the nitrification rate.

[0076] 3. Stress resistance experiment

[0077] Strain culture: The four strains A1, B1, C1 and AA-1 used in the experiment were cultured according to the AOB strain culture method in Example 1, and the total culture time was 150 h.

[0078] Different experimental gradients were designed according to the content of various stress components in actual wastewater in the industry, as follows:

[0079] Alkalinity: 5000, 10000, 15000, 20000, 25000, 30000 ppm

[0080] Salinity: 5000, 10000, 15000, 20000, 25000, 30000 ppm

[0081] Aniline: 0, 2, 5, 10, 20 mg / L

[0082] Phenol: 0, 5, 10, 15, 20 mg / L

[0083] Heavy metal: Cd 2+ (0.1 mg / L), Hg 2+ (0.01 mg / L), Cd 2+ (0.2 mg / L), Hg 2+ (0.02 mg / L), Cd 2+ (0.3 mg / L), Hg 2+ (0.03 mg / L).

[0084] After the culture was completed, the nitrification rate of the four strains was detected according to the industry standard “HG / T 5748-2020 Performance Evaluation Method for Nitrifying Bacteria for Water Treatment” by adding the above stress conditions to the speed culture medium. The results of the alkalinity resistance experiment are shown in Tables 2 and Figure 1

[0085] Table 2 Nitrification rate data of alkalinity resistance experiment

[0086]

[0087] It can be seen that when the alkalinity is 25000 ppm, the strain AA-1 still has 550 mg NH4 + ​- 0.5 N / L / h nitrification rate, while the rest of the strains had only 100 or less nitrification rate. It can be seen that strain AA-1 was superior to the rest of the strains in terms of resistance to salinity. + - 0 N / L / h nitrification rate, while the rest of the strains had 0 nitrification rate. It can be seen that strain AA-1 was superior to the rest of the strains in terms of resistance to salinity.

[0088] The results of the salinity resistance experiment are shown in Table 3 and Figure 2

[0089] Table 3 Nitrification rate data for salinity resistance experiment

[0090]

[0091] At salinity of 30000 ppm, strain AA-1 still had 486 mg NH4 + - 0.5 N / L / h nitrification rate, while the rest of the strains had only 100 or less nitrification rate. It can be seen that strain AA-1 was superior to the rest of the strains in terms of resistance to salinity.

[0092] The results of the aniline resistance experiment are shown in Table 4 and Figure 3

[0093] Table 4 Nitrification rate data for aniline resistance experiment

[0094]

[0095] At aniline content of 20 mg / L, strain AA-1 still had 321 mg NH4 + - 0.5 N / L / h nitrification rate, while the rest of the strains had only 100 or less nitrification rate. It can be seen that strain AA-1 was superior to the rest of the strains in terms of resistance to salinity.

[0096] The results of the phenol resistance experiment are shown in Table 5 and Figure 4

[0097] Table 5 Nitrification rate data for phenol resistance experiment

[0098]

[0099] At phenol content of 20 mg / L, strain AA-1 still had 360 mg NH4 + - 0.5 N / L / h nitrification rate, while the rest of the strains had only 100 or less nitrification rate. It can be seen that strain AA-1 was superior to the rest of the strains in terms of resistance to salinity.

[0100] The results of the toxic metal resistance experiment are shown in Table 6 and Figure 5

[0101] Table 6 Nitrification rate data for toxic metal resistance experiment​​​​

[0102]

[0103] It can be seen that when the toxic metal content is Cd 2+ (0.3mg / L), Hg 2+ (0.03mg / L), the strain AA-1 still has a nitrification rate of 206mgNH4 + -N / L / h, while the rest of the strains have no rate. It can be seen that the resistance of strain AA-1 to toxic metals is better than that of the rest of the strains.

[0104] Example 3: Small-scale test of ammonia nitrogen reduction in a sewage plant by European nitrosomonas SZG-AOB-004 (AA-1)

[0105] A small-scale test of ammonia nitrogen reduction in a biological environmental protection sewage plant was carried out. The experimental water sample was provided by the sewage plant. The type of wastewater was electroplating wastewater. The pollutants in this type of wastewater were various metal ions, including chromium, copper, nickel, lead, gold, silver, cadmium, iron, etc. Among them, the concentration of chromium ions was about 100mg / L, the concentration of nickel ions was about 100mg / L, the concentration of copper ions was about 80mg / L, the concentration of zinc ions was about 80mg / L, the concentration of cadmium ions was about 30mg / L, and the concentration of cyanide complex metal ions was about 50mg / L. The nitrification capacity of the system was poor, and the ammonia nitrogen concentration of the biochemical effluent could not meet the first level A standard (ammonia nitrogen ≤5mg / L) of the "Urban Sewage Treatment Plant Pollutant Discharge Standard" (GB18918-2002). The following is the change of ammonia nitrogen in the small-scale test process. The control group is the untreated water sample taken from the sewage plant site, and the experimental group is the water sample added with AA-1 bacterial agent. The ammonia nitrogen change is shown in Table 7 and Figure 6 .

[0106] Table 7 Ammonia nitrogen change and degradation rate data

[0107]

[0108] From the experimental data and the ammonia nitrogen concentration trend chart, it can be seen that the ammonia nitrogen concentration of the control group continues to rise because organic nitrogen is continuously converted into ammonia nitrogen. After adding the bacterial agent in the experimental group, the ammonia nitrogen concentration shows a downward trend after 68h. The ammonia nitrogen removal rate reaches more than 90% at 122h, and the ammonia nitrogen concentration is 2.58mg / L at 144h. The effluent ammonia nitrogen meets the standard.

[0109] Example 4: Small-scale test of ammonia nitrogen reduction in a chemical plant by European nitrosomonas SZG-AOB-004 (AA-1)

[0110] A certain chemical company ammonia nitrogen reduction test, the company wastewater type for ethylene industrial wastewater, water quantity is large, organic pollutants composition is complex, mainly has organic amine, sulfur, phenol, even exist ethylene oxide, acetonitrile and other toxic and harmful substances, wherein phenol content is about 20mg / L, aniline content is about 5mg / L. Because the biochemical system is impacted, although the ammonia nitrogen concentration in water sample is not high, but because there are many harmful substances lead to the difficulty of nitrification. The following is the change of ammonia nitrogen degradation in the process of test, wherein the control group is the untreated water sample sampled from the project site, the experimental group is added with AA-1 bacterial agent, ammonia nitrogen change is shown in table 8 and Figure 7

[0111] Table 8 ammonia nitrogen change data

[0112]

[0113] From the experimental data and ammonia nitrogen concentration trend chart, it can be seen that the ammonia nitrogen concentration of the control group is maintained at about 15mg / L, while the experimental group after adding bacterial agent, ammonia nitrogen concentration appears a downward trend after 24h, ammonia nitrogen is reduced to 2mg / L at 168h, and the effluent ammonia nitrogen is up to standard.

[0114] The above only describes the preferred embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.​

Claims

1. An ammonia oxidizing bacteria, characterized in that: The ammonia-oxidizing bacteria is Nitrosomonas europaea SZG-AOB-004, which is classified as Nitrosomonas europaea SZG-AOB-004, and the preservation number is CCTCC NO: M 20251507.

2. The ammonia-oxidizing bacteria of claim 1 is applied to degrade ammonia nitrogen in wastewater.

3. Use according to claim 2, wherein: The ammonia nitrogen content in the wastewater is ≤ 35 mg / L.

4. Use according to claim 2, wherein: The alkalinity in the wastewater is ≤ 30000 ppm.

5. The use according to claim 2, characterized in that: The salinity in the wastewater is ≤ 30000 ppm.

6. The use according to claim 2, characterized in that: The content of benzene series in the wastewater is ≤ 20 mg / L.

7. Use according to claim 6, wherein: The benzene series comprises at least one of phenol and aniline.

8. The use according to claim 2, characterized in that: The wastewater contains heavy metal ions Cd 2+ and Hg 2+ .

9. Use according to claim 8, wherein: The content of said Cd 2+ is < 0.3 mg / L, the content of said Hg 2+ is < 0.03 mg / L.

10. A microbial inoculant for degrading ammonia nitrogen in wastewater, characterized by, The ammonia-oxidizing bacteria of claim 1 is contained.

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